iej.10.2009

REVIEW

Apexification: the beginning of its end

G. T.-J. HuangDepartment of Endodontics, Prosthodontics and Operative Dentistry, College of Dental Surgery, University of Maryland, Baltimore,

MD, USA

Abstract

Huang G.T.-J. Apexification: the beginning of its end. Inter-

national Endodontic Journal, 42, 855–866, 2009.

Apexification is a procedure for treating and preserving

immature permanent teeth that have lost pulp vitality.

It contrasts apexogenesis in terms of its outcome in that

apical maturation and normal root thickness cannot be

obtained. Apexification has been a routine practice for

such teeth for many decades, and despite a literature

replete with discussion, including recent artificial

barrier methods with mineral trioxide aggregate, ulti-

mately there has been no major breakthrough to

improve this treatment. Recently, two new clinical

concepts have emerged. One involves a revitalization

approach to achieve tissue generation and regenera-

tion. In this method, new living tissue is expected to

form in the cleaned canal space, allowing continued

root development in terms of both length and thickness.

The other is the active pursuit of pulp/dentine regen-

eration via tissue engineering technology to implant or

re-grow pulps. Although the technology is still at its

infancy, it has the potential to benefit immature

pulpless teeth by allowing continued growth and

maturation. With this understanding, it may be

predicted that apexification will become less needed in

years to come. This study will overview the recent

concept of pulp revitalization in the treatment of

immature teeth with nonvital pulps and the emerging

research on pulp tissue engineering and regeneration.

Keywords: apexification, calcification, pulp/dentine

tissue regeneration, stem cells.

Received 15 July 2008; accepted 26 February 2009

Introduction

Apexification is a procedure to promote the formation

of an apical barrier to close the open apex of an

immature tooth with a nonvital pulp such that the

filling materials can be contained within the root canal

space (Rafter 2005). The capacity of materials such as

calcium hydroxide [Ca(OH)2] to induce the formation

of this calcific barrier at the apex made apexifica-

tion possible and allowed the preservation of many

compromised, immature teeth with nonvital pulps by

endodontic and restorative means. Clinically, when the

pulpal diagnosis of an immature tooth is nonvital,

apexification is undertaken to close the root-end, but

with an understanding that there will be no more

development of the root in terms of apical maturation

and thickening of its dentine walls.

The clinical decision as to whether to perform

apexogenesis or apexification for immature teeth

appears to be clear cut with the teeth deemed to

contain vital pulp tissue being subject to apexogenesis

and teeth deemed to have nonvital pulp tissue receiving

apexification. However, certain clinical observations

reported recently have broken this clear-cut guideline

by showing that apexogenesis may occur in teeth

which have nonvital pulps (Iwaya et al. 2001, Banchs

& Trope 2004, Chueh & Huang 2006). Moreover, it is

Correspondence: George T.-J. Huang, DDS, MSD, DSc, Depart-

ment of Endodontics, Prosthodontics and Operative Dentistry,

College of Dental Surgery, Dental School, University of

Maryland, 650 West Baltimore St, Baltimore, 21201 MD,

USA (Tel.: +410 706 7680; fax: +410 706 3028; e-mail:

[email protected]).

doi:10.1111/j.1365-2591.2009.01577.x

ª 2009 International Endodontic Journal International Endodontic Journal, 42, 855–866, 2009 855

likely that many clinicians had been treating some

cases by an apexogenesis approach despite apparent

pulp necrosis, but never reporting the outcome. A new

protocol has been suggested in which a haemorrhage is

induced to fill the canal with blood clot as a scaffold to

allow generation of live tissues in the canal space and

continued root formation (length and wall thickness)

(Banchs & Trope 2004, Thibodeau & Trope 2007,

Thibodeau et al. 2007). Instead of using Ca(OH)2 as the

intracanal medicament between visits to disinfect and

to induce apical barrier formation, an antibiotic paste is

used for the purpose of disinfection only (Iwaya et al.

2001, Banchs & Trope 2004). This new protocol of

treatment coincides with the recent concept of regen-

erative medicine which promotes the research and

practice of tissue regeneration (National Institutes of

Health 2006).

On another front, pulp/dentine tissue may be

regenerated using tissue engineering technologies.

Attempts to regenerate pulp tissue have been consid-

ered impossible until recently and major developments

in two basic research, namely tissue engineering and

stem cell biology. Investigations on dental pulp tissue

engineering began in the late 1990s (Mooney et al.

1996, Bohl et al. 1998, Buurma et al. 1999). The

isolation and characterization of dental pulp stem cells

(DPSCs) (Gronthos et al. 2000), stem cells from

exfoliated deciduous teeth (SHED; Miura et al. 2003)

and stem cells from apical papilla (SCAP) (Sonoyama

et al. 2006) has capitalized the possibility for pulp/

dentine regeneration (Huang et al. 2006, 2008,

Murray et al. 2007a, Cordeiro et al. 2008, Prescott

et al. 2008). Because of the wide-open apex of the

immature tooth, vascularization via apical ingrowth of

blood vessels into an engineered construct containing

stem cells may facilitate a successful regeneration of

pulp/dentine within the canal space (Huang et al.

2008).

This study will overview the shifting concept of

treating immature teeth using revitalization rather

than apexification and the current status of pulp tissue

engineering and regeneration. The review will analyse

the fate of apexification as a first-line treatment for

immature teeth with nonvital pulps and how this is

affected by the shifting paradigm of the management

and the coming era of pulp/dentine tissue regenera-

tion. Again, apexification does not allow generation or

regeneration of vital tissues in the canal space

whereas the revitalization or tissue regeneration

approaches provide a new chance for those affected

teeth to regain biological tissue recovery and growth.

From this point of view, it seems inevitable that in the

interest of patients, apexification may become a less-

desirable and less needed clinical treatment in the

foreseeable future.

Apexification

Immature teeth undergoing apexification are usually

disinfected with irrigants including NaOCl, chlorhexi-

dine, EDTA and iodine–potassium iodide (Rafter 2005).

The canal is then filled with Ca(OH)2 paste for the

purpose of further disinfection and induction of an

apical calcific barrier. Ca(OH)2 is antimicrobial because

of its release of hydroxyl ions which can cause damage

to the bacterial cellular components. The best example

is the demonstration of its effect on lipopolysaccharide

(LPS). Ca(OH)2 chemically alters LPS which affects its

various biological properties (Safavi & Nichols 1993,

1994, Barthel et al. 1997, Nelson-Filho et al. 2002,

Jiang et al. 2003).

Filling the root canal is undertaken normally when

the apical calcific barrier is formed. Without the barrier,

there is nothing against which the traditional gutta-

percha filling material can be condensed. Besides the

fact that Ca(OH)2 functions as a potent disinfectant,

early evidence has suggested osteo-inductive properties

(Mitchell & Shankwalker 1958), although it has been

difficult to demonstrate this effect in vitro (Raquel Assed

Bezerra da et al. 2008). It was considered that the high

pH may be a contributing factor for the induction of

hard tissue formation (Javelet et al. 1985). The time

required for apical barrier formation in apexification

using Ca(OH)2 may be considerable, often as long as

20 months and other conditions such as age and

presence of symptoms or periradicular radiolucencies

may affect the time needed to form an apical barrier.

Refreshing the Ca(OH)2 paste usually takes place every

3 months (Rafter 2005). A number of shortcomings

can be summarized for Ca(OH)2 apexification: (i) long

time-span of the entire treatment; (ii) multiple visits

with heavy demands on patients and carers and

inevitable clinical costs; (iii) increased risk of tooth

fracture using Ca(OH)2 as a long-term root canal

dressing (Cvek 1992, Andreasen et al. 2002). These

drawbacks led to the use of mineral trioxide aggregate

(MTA) to fill the apical end without the need for calcific

barrier formation. In comparison to Ca(OH)2, some

data suggest that MTA appears to be more predictable

with consistent hard-tissue formation based on in vivo

studies in dogs (Shabahang et al. 1999). Using MTA for

apexification may shorten the treatment period with

Apexification, end in sight Huang

International Endodontic Journal, 42, 855–866, 2009 ª 2009 International Endodontic Journal856

more favourable results and improved patient compli-

ance (Maroto et al. 2003, El-Meligy & Avery 2006,

Pace et al. 2007). Many authors and clinicians propose

a one-visit apexification protocol with MTA, which

presents a major advantages over traditional Ca(OH)2methods (Witherspoon & Ham 2001, Steinig et al.

2003). This expedient cleaning and shaping of the root

canal system followed by its apical seal with MTA

makes the rapid placement of a bonded restoration

within the root canal possible, which may prevent

potential fractures of immature teeth.

While advances with MTA and bonded restorations

go some way towards a better outcome, ultimately no

apexification method can produce the outcome that

apexogenesis can achieve, i.e. apical maturation with

increased thickness of the root. As noted above, clinical

experience on the outcome of apexified teeth with thin

and weak roots after successful treatment is that they

are highly susceptible to fracture (Cvek 1992,

Katebzadeh et al. 1998). Therefore, alternative ap-

proaches that allow the increase of root thickness

and/or length should be pursued.

A paradigm shift in the managementof immature teeth

Although the standardized clinical approach for apexo-

genesis or apexification has been widely practiced,

some clinicians inevitably modify their treatment

procedures based on their clinical judgement. Some

reported their cases using alternative approaches, with

three appearing to capture great interest from the

endodontic community. The first, reported by Iwaya

et al. (2001) presented an immature mandibular

premolar with a sinus tract and periradicular radiolu-

cency. During canal preparation, they did not instru-

ment to full working length because the patient felt

discomfort on the insertion of instruments. The canal

was mainly irrigated with NaOCl and hydrogen perox-

ide and further disinfected with antibiotic agents

(metronidazole and ciprofloxacin). Thirty-five months

after the completion of these procedures, they observed

complete maturation of the root apex with thickened

root structure. The tooth also responded positively to

electronic pulp testing. After observing the success of

this alternative approach, the same idea was applied to

treatment of a mandibular premolar having a similar

condition but with more extensive periradicular bone

loss. During careful follow-up to 2 years after the

treatment, complete maturation of the root was

observed with a positive response to cold testing

(Banchs & Trope 2004). Chueh & Huang (2006) later

reported four mandibular premolars in a similar clinical

condition that were treated between 1988 and 2000,

all again demonstrating healing and apical maturation.

These reports raised a great response and encouraged

further reports (Thibodeau & Trope 2007, Hargreaves

et al. 2008, Jung et al. 2008). A more conservative

approach and a shifting paradigm for the treatment of

nonvital immature teeth has thus been proposed

(Huang 2008). Furthermore, the Regenerative End-

odontics Committee of the American Association of

Endodontists has initiated a pilot study by encouraging

endodontists to submit their cases to a data base

(http://www.aae.org/members/revascularizationsurvey.

htm). The study is designed to determine the incidence

and predictors of healing of apical periodontitis in cases

considered to have nonvital pulps when treated by

nonconventional, biologically based revitalization

methods. Currently, the success rate of this type of

approach is only available from an animal study model

(Thibodeau et al. 2007) and a pilot clinical study in

humans (Shah et al. 2008). In the animal model, it was

found that after disinfection of the root canals, 43.9%

of the cases had thickened canal walls, 54.9% had

apical closure and 64.6% had no radiographic evidence

of periapical radiolucency or showed improvement/

healing of previous periapical radiolucencies (Thibo-

deau et al. 2007). The clinical pilot study involving

teeth in 14 patients demonstrated 93% resolution of

periradicular radiolucencies, thickening of lateral den-

tinal walls in 57%, and increased root length in 71%.

None of the cases presented with pain, reinfection or

radiographic enlargement of pre-existing periapical

lesions (Shah et al. 2008). However, due the prelimin-

ary nature of the study, the clinical success rates should

be interpreted with caution (Messer 2008).

Regarding the use of Ca(OH)2 versus antimicrobial

paste, it was suggested that the former may not be

suitable if there is remaining vital pulp tissue in the

canal. The direct contact of Ca(OH)2 paste with the

tissue will induce the formation of a layer of calcific

tissue which may occlude the pulp space, therefore

preventing pulp tissue from regeneration (Huang

2008). Another concern is that Ca(OH)2 may damage

the Hertwig’s epithelial root sheath (HERS) and thereby

destroy its ability to induce the nearby undifferentiated

cells to become ododontoblasts (Banchs & Trope 2004).

The effectiveness of a triple-antibiotic regimen to

disinfect root canal space was first tested and verified

by Sato et al. (1996) and the clinical use of the mixture

has shown success in terms of clinical outcome (Sato

Huang Apexification, end in sight


et al. 1996, Banchs & Trope 2004, Jung et al. 2008).

Whether the three antibiotics originally described (i.e.

metronidazole, minocycline and ciprofloxacin) must be

used for this purpose or if other choices may serve this

purpose requires further investigation.

These clinical case reports demonstrate that despite

the formation of periapical abscesses with extensive

periradicular bone resorption as the result of root canal

infection in immature teeth, conservative treatment

may allow roots to increase in length and thickness or

even reach mature form. One explanation is that the

clinical diagnosis of pulp status is inaccurate and that

some of those teeth must have contained vital tissues in

the apical pulp space despite negative pulp testing and

periapical lucencies. It is also acknowledged that there

is a lack of scientific studies on the diagnosis of pulpal

pathology in permanent teeth with open apices (Camp

2008). It has been considered that, to have continued

root development, HERS and the recently identified

tissue, apical papilla, must be functional (Huang et al.

2008). On the other hand, if the pulp, HERS and apical

papilla are completely lost, the root may still gain some

level of thickness by the ingrowth of cementum from

the periapical areas onto the internal root canal dentine

walls. Additionally, this cementum ingrowth is accom-

panied by periodontal ligament (PDL) and bone tissue

(Kling et al. 1986, Andreasen et al. 1995a,b).

The outcome of guided generation

and regeneration approach

The use of the term ‘revascularization’ was adapted by

Iwaya et al. (2001) to describe the clinical healing of

periapical abscesses and continued root formation in

immature teeth with nonvital pulps. Other authors

adapted the term without questioning until Huang &

Lin (2008) considered that ‘revascularization’ did not

encompass the actual healing and repair process that

takes place in these clinical cases (Huang & Lin 2008).

The term ‘revitalization’ used by earlier studies

attempting to revive tissues in the pulp space would

perhaps describe the phenomenon more accurately

(Nevins et al. 1976).

Pulp space filled with regenerated pulp

The ideal situation is that there is surviving pulp and

apical papilla tissue after root canal disinfection.

Continued root formation to its maturity and an

increased thickness of root dentine may then be

anticipated. The dental papilla at the apex contains

stem cells, ‘SCAP’ that have been recently described to

be more robust stem cells than DPSCs (Sonoyama et al.

2006). The SCAP may survive the infection and retain

the capacity to give rise to new odontoblasts influenced

by HERS, allowing new root dentine to form and root

maturation to proceed to completion. It was speculated

that the surviving DPSCs in the remaining vital pulp

may rebuild the lost pulp tissue in the canal and

differentiate into replacement odontoblasts to substitute

for the damaged primary odontoblasts (Sonoyama et al.

2008). Under this circumstance, one may anticipate

the newly formed odontoblasts from SCAP to produce

root dentine that leads to the apical extension of the

root. Additionally, the existing primary odontoblasts

that survived in the residual pulp tissue and perhaps

some new replacement odontoblasts may continue to

lay down dentine on the dentinal walls, causing the

root to increase its thickness (Fig. 1). Whilst this

explanation is conjecture and requires further basic

and clinical investigation, some data on the recovery of

pulp tissue after tooth replantation appear to support

this speculation (Kling et al. 1986, Ritter et al. 2004).

Pulp space filled with periodontal tissues

In cases where the entire pulp, apical papilla tissues

and the HERS are lost, current understanding is that

self-regeneration of pulp and new dentine formation is

unlikely to occur. There is abundant evidence in the

literature demonstrating that when the pulp tissue of

Apical papilla

Epithelial diaphragm Bone

CementumCementum

DentinPulp

PDL

Odontoblasts

?

Figure 1 Hypothetical pulp regeneration from the remaining

recovered pulp. The question mark indicates that the regen-

eration of pulp into the empty pulp space is uncertain at

present.



immature teeth with wide-open apices undergoes

complete necrosis but in a sterile environment, other

tissues are capable of filling the canal space. As shown

in the radiographic images (Fig. 2), the replanted

avulsed immature tooth lost pulp vitality but the pulp

space became occupied by the ingrowth of alveolar

bone from the periapex (Kling et al. 1986). There is a

space separating the ingrown bone and the canal

dentinal walls. If one traces this space, it is apparent

that it is continuous with the PDL space on the external

a b

c d

(a)

(b)

Bone

Bone

Dentin

PDL

PDLPDL

PDL

Bone

CementumCementum

Cementum

Figure 2 Ingrowth of periodontal tissue

into pulp space. (a) Radiographs show-

ing an immature tooth 11 (FDI notation)

with an open apex which was

re-implanted and healed. At recalls, the

ingrowth of bone tissue with the PDL

space and lamina dura is evident

[adapted from Kling et al. (1986) with

permission). (b) Illustration depicting the

ingrown tissues of bone, PDL and

cementum into the canal space.



root surfaces. Lamina dura also appears to have been

established in the ingrown bone occupying the pulp

space. The contents of the pulp space were described by

Holan (1998) as ‘tube-like mineralization’ and follow-

ing histological examination it was interpreted that

secondary dentine and the pulp tissue existed in the

canal space. In fact, this ‘secondary dentine’ was

actually cementum and the ‘pulp tissue’ was PDL.

Careful examination of the characteristics of the ectopic

cementum and PDL in the canal space should be the

basis of further research.

There also seems to have been some degree of

vertical and horizontal extension of the root over time

(Fig. 2). Since the pulp tissue has been entirely lost, it

has not been possible to deposit new dentine, and the

newly acquired calcified tissue has to come from a

tissue source where the cellular components are

capable of proliferating and producing new tissues.

Cementum has the capacity to fulfill this purpose.

Histologically, the hard tissues, bone, cementum and

dentine can usually be distinguished unambiguously

merely for their anatomical location. However, when

ectopic formation of these tissues occurs, discerning

them without specific markers may be difficult. None-

theless, the ingrown hard tissues within the pulp space

have been verified by histological examination, reveal-

ing the deposition of cementum onto the dentine

surface in the canal, extending from the outside surface

of the apex (Nevins et al. 1977, Lieberman & Trow-

bridge 1983). The apical extension of roots resulting

from the apposition of cementum is a normal physio-

logical process. The apposition of calcified cemental

tissue on the internal canal wall also increases the

thickness of the root. A distinct feature of cementum is

its connection with the PDL by Sharpey’s fibres, which

can also be observed in the ingrown tissues in the pulp

space. The ingrowth of periodontal tissue may reach all

the way to the coronal pulp chamber (Nevins et al.

1977, 1978, Ellis et al. 1985, Hitchcock et al. 1985).

Similar results were observed in a dog as a study model

(Thibodeau et al. 2007).

When the pulp space is filled with periodontal tissues,

the situation is totally different from normal because

the pulp space is no longer part of the root canal

system, but part of periapical tissues. If the tooth

becomes reinfected causing destruction of the peri-

odontal tissue in the canal space, the understanding of

a root canal infection to this type of infection cannot be

applied, but perhaps more appropriately that of a

periapical tissue pathosis. It is known that periapical

tissue loss will recover if the source of infection from the

root canal space is eliminated though the establishment

of a biofilm by the invading microbes may complicate

management (de Paz 2007). From this perspective,

disinfection does not have to involve with the aggres-

sive entrance into the canal space, but rather dealing

mainly with the source of infection in the crown.

Currently, there is no case report showing the man-

agement and the outcome of infected canal space that

has been filled with periodontal tissues.

Severe disorganized calcification of the pulp space

Whether the pulp space is filled with regenerated pulp

or periodontal tissues, long-term radiographic observa-

tions demonstrate that the pulp space becomes severely

narrowed or filled with radio-opaque mineralized tissue

over time. Histologically, the mineralizing tissues are

either bone-like or dentine-like (Robertson et al. 1997).

The hard tissues may begin as calcific particles that

have been observed to originate or are closely associ-

ated with blood vessels and perineurium sheaths

(Pashley et al. 2002). Interestingly, these are also the

locations where pulp stem cells are believed to exist (Shi

& Gronthos 2003). Whether these stem cells are

activated by the low-grade inflammation to undergo

osteogenic differentiation is unclear at present. Over

time, these particles merge into larger calcific masses

and obliterate the pulp space (Fig. 3). Although this

calcifying phenomenon within the pulp has been well-

documented, the mechanisms underlying this process

are still elusive.

Prolonged inflammation causes calcification in many

parts of the body, e.g. calcifying tendonitis (Uhthoff

1996). Arthritic joints tend to build osteophytes as a

result of the expanding bone tissue over the damaged

cartilaginous tissues (van der Kraan & van den Berg

2007). Another phenomenon named heterotropic

ossification is characterized by the formation of miner-

alized inclusions within the soft tissues (McCarthy &

Sundaram 2005), e.g. muscles of patients who suffered

from severe trauma to their extremities including

soldiers injured by bomb explosions (Owens et al.

2006). It has been speculated that the causes of such

phenomena include systemic factors and/or local

inflammatory conditions. Stem cells in the muscle have

been investigated for their potential contributory role in

this disease. Deficiencies in osteopontin may lead to

vascular calcification (Giachelli 2005).

There has been an ongoing debate on the relative

benefits of calcified material or gutta-percha filled

canals. From a physiological point of view, calcific



metamorphosis is a degenerative disease. Moreover,

from a technical perspective, calcified canals pose a

challenge if they need treatment. Most of the literature

does not support endodontic intervention in the case of

mineralized obliteration unless periradicular pathoses is

detected or the involved tooth becomes symptomatic

(Robertson et al. 1997, Gopikrishna et al. 2004). Sur-

gical intervention may be the only option to contain

the infection from the periradicular tissues if calcified

canals are not accessible for nonsurgical root canal

treatment.

Progress on pulp/dentine tissue

engineering and regeneration

The potential of pulp tissue to regenerate lost dentine is

well-known. Direct pulp capping therapy to induce

dentinal bridge formation is practiced on the basis of this

understanding. The use of various cement-based mate-

rials such as Ca(OH)2 and MTA is believed to promote

such activity. Long-term success using MTA for direct

pulp capping has been reported recently (Bogen et al.

2008). The application of recombinant growth factors

to the injured site to enhance the regeneration of

dentine has also been investigated (Rutherford & Gu

2000). Cell-based therapy using isolated pulp cells or

DPSCs, with genetic manipulation to express bone

morphogenic proteins, to augment the generation of

new dentine bridge formation is an additional area of

exploration (Rutherford 2001, Iohara et al. 2004).

When dealing with the initial phases of dentine

destruction where there is minimal damage, applying a

complicated biotechnological approach appears imprac-

tical. When the tooth is further damaged, regeneration

of dentine becomes difficult as it needs a healthy pulp

which may be compromised by the disease. Ideally, the

regenerated dentine should not replace the pulp space.

Two types of pulp regeneration can be considered based

on the clinical situations: (i) partial pulp regeneration

and (ii) de novo synthesis of pulp.

It has been observed that pulpal infection and

inflammation is compartmentalized until the entire

pulp tissue undergoes necrosis (Seltzer et al. 1963,

Trowbridge 2002). Before the end stage, the remaining

pulp tissue may be recoverable and help regenerate the

lost portion. To enhance the regeneration, engineered

pulp tissues may be inserted into the pulp space to

facilitate the entire recovery of pulp tissue and the

generation of new dentine. When the entire pulp tissue

is lost, de novo synthesis of pulp must take place to

regenerate the tissue.

Early efforts on pulp regeneration

Regenerating pulp tissue has been a long quest. Ostby

(1961) studied the tissue re-organization in the canal

space filled with blood clot. It was observed that the

tissue formed in the canal was not pulp but granulation

or fibrous tissues and in some cases the ingrowth of

cementum and bone occurred. Similar findings were

observed by Myers & Fountain (1974) in a primate

study using blood clot as a scaffold. The average

generation of soft connective tissue into the canal was

only 0.1–1.0 mm, although the authors mentioned

(a) (b) (c)

Figure 3 Common feature of pulp

undergoing calcific metamorphosis. (a)

Pulp tissue from a tooth which had been

previously restored with old fillings and

a clinical diagnosis of normal pulp

(arrows indicate mineral deposits that

appear to have been associated with

vascular structures) (b, c) Pulp tissues

from teeth diagnosed with irreversible

pulpitis. Arrows indicate heavy mineral

deposits.



that teeth with open apices had a few more millimetres

of ingrowth than those with mature apicies (Myers &

Fountain 1974).

It appears that in a natural situation, regeneration of

pulp cannot occur following total loss of pulp tissue.

Pulp cells have been isolated for various studies for

many decades and they have been shown to have the

capacities to differentiate into mineral forming odonto-

blast-like cells in vitro (Tsukamoto et al. 1992, About

et al. 2000, Couble et al. 2000). However, it was not

until it was demonstrated the formation of ectopic

dentine/pulp-like complex in vivo by isolated pulp cells

that the isolation of odontoblast progenitor cells or pulp

stem cells was truly confirmed (Gronthos et al. 2000).

These cells were termed postnatal DPSCs.

Pulp tissue engineering

Before the isolation of DPSCs, pulp regeneration was

tested using modern tissue engineering concepts by

growing pulp cells onto synthetic polymer scaffolds of

polyglycolic acid (PGA) and in vitro and in vivo analyses

performed (Mooney et al. 1996, Bohl et al. 1998,

Buurma et al. 1999). These approaches are basically

a proof-of-principle to test whether cultured pulp cells

can grow well and produce matrix on PGA, and

whether the engineered pulp can be vascularized using

in vivo study models. This approach reflected the

emphasis on providing a three-dimensional structure

for cells to attach to which simulates the in vivo

environment. Using a tooth slice model, generation of

well-vascularized pulp-like tissue has been reported

(Cordeiro et al. 2008, Prescott et al. 2008).

Issues in cell-based pulp tissue engineering

The following questions must be considered when

attempting to engineer and regenerate pulp tissue:

(i) vascularization: can the angiogenesis from the limited

apical blood supply extend to the coronal end if the

entire pulp is to be regenerated? (ii) New odontoblast

formation: can the new odontoblasts form against the

existing dentinal wall that has been chemically

disinfected during the root canal procedures? (iii) New

dentine formation: can the newly differentiated odonto-

blasts produce new dentine and how much would they

produce? (iv) Cell source: autologous cells are still the

best cell source to avoid potential immune rejection.

However, where can one find the cells needed for pulp

regeneration in the clinical setting? These points will

now be discussed in turn.

Vascularization

While vascularization is a universal issue for an

engineered tissue, it is of special concern for pulp tissue

engineering because of the lack of a collateral source of

blood supply. It was considered that the use of

angiogenic inducing factors such as vascular endothe-

lial growth factor (VEGF) could enhance and accelerate

pulp angiogenesis. Alternatively, the insertion of engi-

neered pulp tissue may have to be separated into

multiple steps to allow progressive vascularization

(Huang et al. 2008). The choice of scaffold and the

source of angiogenic factors have become integrated

issues. Artificial synthetic scaffolds such as co-polymer

of d,l-lactide and glycolide can be fabricated with

impregnated growth factors such as VEGF and/or

platelet-derived growth factor (Sheridan et al. 2000,

Richardson et al. 2001, Peters et al. 2002, Kanematsu

et al. 2004, Stiver et al. 2004, Sun et al. 2005). The

size of apical opening would affect the ingrowth of

blood vessels into the engineered pulp tissue. It is

assumed that the larger the opening, the more likely

that angiogenesis can occur. Immature teeth with open

apices are therefore the best candidates for pulp tissue

regeneration.

It is a misconception to adapt the concept of

engineering/regenerating bone for pulp tissue. Certain

scaffolds that have osteo-inductive or conductive prop-

erties and are suitable for bone regeneration, such as

hydroxyapatite and tricalcium phosphate have been

proposed as scaffolds for pulp regeneration. The

misconception is based on the fact that dentine

production has many aspects similar to bone forma-

tion. However, it is important to recognize the key

differences. An obvious one is the anatomic character-

istics. Bone mass contains compact or trabecular bone

and marrow, whereas dentine and pulp in a tooth have

a rigid anatomic location. When regenerating pulp and

dentine, the dentine should be located peripherally to

the pulp, not within it. Therefore, the scaffold that

carries the cells to regenerate pulp and dentine should

not induce dentine formation randomly within the

regenerated pulp.

New odontoblast formation

To address the question whether new odontoblasts can

form on the existing dentine walls, in vitro experiments

have shown that by seeding DPSCs onto the existing

dentine, some cells transformed into odontoblast-like

cells with a cellular process extending into dentinal

tubules (Huang et al. 2006). A tooth slice model has

been utililzed and seeded SHED onto synthetic scaffolds



of poly-l-lactic acid cast in the pulp chamber of the thin

tooth slice. They observed odontoblast-like cells arising

from the stem cells and localized against the existing

dentine surface in their in vivo study model (Nor 2006,

Cordeiro et al. 2008). From these observations, it

appears that stem cells seeded in the scaffold will be

attracted to the dentinal wall, differentiate into odon-

toblast-like cells and extend their cellular processes into

the dentinal tubules. The mechanism behind this

phenomenon has been speculated to be the released

growth factors such as TGF-b by the dentine, which

attracts and induces the differentiation of odontoblasts

(Huang et al. 2006). Chemical disinfection of the root

canal space may damage these embedded growth

factors. Further investigation is needed to seek for

ways to avoid this potential damage, and positively

promote odontoblast-like colonization.

New dentine formation

The next question is whether these newly formed

odontoblast-like cells will make new dentine. In an

in vivo study model, DPSCs were seeded onto dentine

and the construct implanted into the subcutaneous

space of immunocompromised mice. Deposition of

reparative dentine-like structures by odontoblast-like

cells was observed (Batouli et al. 2003). This finding

suggests the possibility of forming additional new

dentine on existing dentine if new odontoblasts can

emerge. Huang G.T.-J., Shea L.D., Shi S. & Tuan R.S.

(upubl. data) also demonstrated that new dentine-like

or osteodentine structure can deposit onto the existing

dentine throughout the entire canal wall in an in vivo

pulp engineering/regeneration study model.

Cell source

With respect to the cell source, there are several

potential sources to obtain autologous cells for pulp/

dentine tissue regeneration: DPSC, SCAP and SHED.

Immature third molars are one of the best sources for

DPSCs and SCAP. The latter have been shown to be

more potent dental stem cells than DPSCs in terms of

their level of immaturity and potentiality. They give

rise to odontoblast-like cells and make ectopic dentine

in in vivo study models (Sonoyama et al. 2006) . SHED

also produce ectopic dentine in vivo (Miura et al. 2003).

The problem is the availability of this source. Banking

personal teeth for future use appears to be a direction

that must be explored and established to ensure this

availability. Allogenic cells are an alternative and

convenient source. The finding of the immunosuppres-

sive capacity of mesenchymal stem cells to avoid

immuno-rejection provides a great possibility that

allogenic stem cells may be a good source (Pierdome-

nico et al. 2005, Chen et al. 2006). However, in vivo

studies to verify the long-term survival of transplanted

allogenic dental stem cells are lacking.

Prospects

The above analysis points out the potential future fate of

apexification procedures. Such procedures may no

longer be the preferred first option to treat immature

permanent teeth with nonvital pulps. Induced genera-

tion and regeneration of vital tissues in the pulp space

can thicken the root structure leading to a stronger tooth

with a potentially reduced fracture risk. The progress of

pulp/dentine regeneration so far has been promising and

is likely to work in the not so distant future.

There is some concern caused by the uncertainty as

to how pulp regeneration would affect the future of

endodontic practice (Murray et al. 2007b) . One may

anticipate that to feasibly deliver stem cell-based

endodontic therapy for pulp/dentine regeneration in

endodontic practice, an uncomplicated clinical protocol

would need to be established. If not, technology transfer

to the commercial sector would be difficult (Rutherford

2007).

Acknowledgements

This work was supported in part by an Endodontic

Research Grant from the American Association of

Endodontists Foundation (G.T.-J.H.).

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Polymerization stress, flow and dentine bondstrength of two resin-based root canal sealers

S. F. C. Souza1,2, A. C. Bombana3, C. Francci2, F. Goncalves2, C. Castellan2 & R. R. Braga2

1School of Dentistry, Federal University of Maranhao, Sao Luiz, MA; Departments of 2Dental Materials and 3Restorative Dentistry

University of Sao Paulo, Sao Paulo, SP, Brazil

Abstract

Souza SFC, Bombana AC, Francci C, Goncalves F,

Castellan C, Braga RR. Polymerization stress, flow and

dentine bond strength of two resin-based root canal sealers.

International Endodontic Journal, 42, 867–873, 2009.

Aim To compare two resin-based root canal sealers

(AH Plus and dual cure Epiphany) in terms of flow,

polymerization stress and bond strength to dentine.

Methodology Flow was evaluated by measuring the

diameter of uncured discs of sealer (0.5 mL) after 7 min

compression (20N) between two glass plates (n = 5).

Polymerization stress was monitored for 60 min in

1-mm thick discs bonded to two glass rods (Ø = 5 mm)

attached to a universal testing machine (n = 3). Bond

strength was analyzed through micropush-out test

(n = 10) and failure mode was examined with scan-

ning electron microscope (100· and 2500·). Data

were statistically analyzed using the Student’s t-test

(a = 0.05).

Results Polymerization stress was 0.32 ± 0.07 MPa

for Epiphany self-cure, 0.65 ± 0.08 MPa for Epiphany

light-cure and zero for AH Plus (P < 0.05). Flow data

and bond strength values were 30.9 ± 1.1,

28.6 ± 0.7 mm and 6.3 ± 5.3, 17.8 ± 7.5 MPa for

Epiphany and AH Plus, respectively (P < 0.001).

Failure mode was predominantly cohesive in the sealer

for both materials.

Conclusions Epiphany had higher flow and poly-

merization stress and lower bond strength values to

dentine than AH Plus. In view of these findings it can

be implied that AH Plus would provide a better seal.

Keywords: apical gap, flow, micropush-out, poly-

merization stress, root canal sealer.

Received 3 November 2008; accepted 5 March 2009

Introduction

Complete filling of the root canal system with biocom-

patible and dimensionally stable filling materials is an

important factor in achieving endodontic success

(Sjogren et al. 1990). Gutta-percha in combination

with sealers of different chemical compositions has been

widely used in clinical practice. However, filling com-

pletely the root canals system remains a challenge

despite the large number of techniques and materials

available (Schwartz 2006). Adhesive bonding and resin

cements developed for endodontic use have emerged as

a possibility to improve root canal filling (Weis et al.

2004). In 2004, a new adhesive root filling material,

Epiphany� Root Filling System, was patented by

Pentron Clinical Technologies (Wallingford, CT, USA).

This system contains a polyester-based thermoplastic

root canal core material (Resilon; Resilon Research LLC,

Madison, CT, USA), a dual-cure methacrylate-based

sealer and a self-etching primer. This material can

promote hybridization with the dentine substrate and a

chemical bond with Resilon, improving resistance to

bacterial leakage (Shipper et al. 2004, 2005) and root

fracture (Teixeira et al. 2004a) due to a potential resin

monoblock formation (Teixeira et al. 2004b). Neverthe-

less, an ultrastructural evaluation revealed a weak link

between Resilon and dentine (Tay et al. 2005a).

Methacrylate-based sealers shrink during polymeriza-

tion (Ferracane 2005), generating stress within the

material and at the tooth-restoration interface that can

Correspondence: Dra Soraia de Fatima Carvalho Souza,

Faculdade de Odontologia, Universidade Federal do Maranhao

(UFMA), Av. dos Portugueses s/n, Bacanga, Sao Luis,

MA 65085-580, Brazil (Tel.: +55 98 21098575; e-mail:

[email protected]).

doi:10.1111/j.1365-2591.2009.01581.x


lead to gap formation (Carvalho et al. 1996, Braga et al.

2002, De Munck et al. 2005). The magnitude of stress is

influenced by several factors, such as composition and

volume of the material and cavity configuration factor

(factor-C) (Davidson & de Gee 1984, Davidson et al.

1984, Davidson & Feilzer 1997). In composite restora-

tions, the use of low viscosity materials has been

associated with a reduced incidence of marginal gaps

at the tooth/restoration interface (Uno & Asmussen

1991, Peutzfeldt & Asmussen 2004) and better adapta-

tion to cavity walls (Ferdianakis 1998, Fruits et al.

2002). On the other hand, viscosity is directly related to

degree of conversion (Lovell et al. 1999, Sideridou et al.

2002) which, in turn, is a determinant factor on

polymerization stress development (Braga & Ferracane

2002, Stansbury et al. 2005). The high C-factor situa-

tion represented by the filling of root canals may

originate high polymerization stresses (Goracci et al.

2004), exceeding bond strength to root dentine and

causing debonding of the interface for stress relief (Tay

et al. 2005b). Furthermore, resin sealer photoactivation

for immediate coronal sealing hinders the resin viscous

flow and increases stress build-up (Ferracane 2005),

resulting in inappropriate bond strength or gap forma-

tion between sealer and root dentine (Nagas et al. 2007).

The aim of this study was to compare an epoxy- and a

methacrylate-based root canal sealer in terms of several

characteristics involved in apical gap formation. The

null hypothesis was that AH Plus� (Maillefer, Dentsply

Ind. e Com. Ltda., Petropolis, RJ, Brazil) or Epiphany�(Pentron Clinical Technologies, Wallingford, CT, USA)

would show no difference terms of flow, polymerization

stress and dentine bond strength.

Materials and methods

Flow

According to ADA 57 Specification (American National

Standard/American Dental Association, 2000), 0.5 mL

of sealers was mixed and placed using a graduated

syringe, on a glass plate (40 · 40 · 5 mm). After

180 ± 5 s another glass plate was placed on top of the

sealer, followed by load application of 20 N. Then,

10 min after mixing, the load was removed and

maximum and minimum diameters of compressed discs

were measured with a digital caliper with a 0.01 mm

resolution (Mitutoyo MTI Corporation, Tokyo, Japan).

Results were recorded only if both diameters were

uniform and were within 1.0 mm. Flow was calculated

by averaging five specimens.

Polymerization stress

Polymerization stress was determined using an estab-

lished method (Condon & Ferracane 2000, Witzel et al.

2007, Goncalves et al. 2008). One end of two glass rods

(B 5 mm · 13 or 28 mm height) was sand-blasted

with alumina (150–250 lm), silanated (RelyX Ceramic

primer S; 3M ESPE, St Paul, MN, USA) and coated with

a layer of unfilled resin (Adper� Scotchbond Multi-

purpose, bottle 3; 3M ESPE), which was exposed to the

light source with 300 mW cm)2 for 40 s. The non-

treated ends were attached to the opposite fixtures of a

universal testing machine (Model 5565; Instron, Can-

ton, MA, USA), and the distance between the treated

surfaces was adjusted to 1.0 mm. The 28-mm rod was

connected to a crosshead/load cell, whilst the 13-mm

rod was connected to a stainless steel fixture containing

a slot that allowed, when necessary the distal end of the

light-curing guide to contact the rod opposite to the

treated surface which was highly polished. Resin sealer

(19.6 mm3) was inserted between the treated glass

surfaces and formed into a cylinder and excess was

removed. An extensometer (Model 2630–101; Instron)

was attached to the rods in order to monitor specimen

height. The approximation of the glass rods due to

composite shrinkage was registered by the extensom-

eter and caused the crosshead to move in the opposite

direction to restore the initial distance, with 0.01 lmaccuracy. Therefore, the values registered by the load

cell corresponded to the force necessary to maintain

the initial height of the specimen in opposition to

the contraction force exerted by the resin sealer

(Fig. 1).

Three specimens were tested in each experimental

condition at 37 �C, and force development was mon-

itored for 60 min, starting 3 min after mixing. Exper-

imental conditions were AH Plus, Epiphany self-cure

(SC) and Epiphany light-cure (LC). Epiphany-LC was

photoactivated (VIP Junior; BISCO, Schaumburg, IL,

USA) 17 min after mixing with 475 mW cm)2 for 51 s

(24 J cm)2), following manufacturer’s instructions.

Maximum nominal stress (r, in MPa) was calculated

by dividing the maximum contraction force [F (N)] by

the cross-sectional area of the rods (A) as follows:

r ¼ FðNÞAðmm2Þ

Micropush-out bond strengths

Twenty mandibular single-rooted human premolar

teeth with straight root canals, anatomically similar

Physicomechanical properties of endodontic sealers Souza et al.


dimensions, fully developed apices and patency

foramen were collected after patient’s informed consent

had been obtained under a protocol reviewed and

approved by the Ethical Research Committee of Sao

Paulo University (protocol number, 177/05). Teeth

were cleaned and the working length of each root was

established with a size 15 K file (Dentsply Maillefer

Ballaigues, Switzerland) 1.0 mm short of the apical

foramen. Canals were prepared with a crown-down

technique up to size 50 and irrigated with 0.5% NaOCl

after every change of instrument. Five millilitres of 17%

EDTA was used as final rinse to remove canal wall

smear layer. EDTA solution was neutralized with 0.5%

NaOCl and then the canal was rinsed with saline

solution (15 mL) and dried with paper points.

Prepared root canals were randomly (http://www.

random.org) divided into two experimental groups

(n = 10): AH Plus (Dentsply Ind. e Com. Ltda.) and

Epiphany-SC (Pentron Clinical Technologies). Three

disc slices of one-millimetre thick (±0.1 mm) were

obtained after transverse sectioning (Isomet 1000

Precision Saw; Buehler Ltd., Lake Bluff, IL, USA) the

apical 5.0 mm of each root under water cooling. The

thickness of each root slice was measured by means of

a digital caliper (Mitutoyo MTI Corporation, Tokyo,

Japan). The diameters of each apical and cervical slice

were photographed by a digital camera (Q-Color 5;

Olympus America Inc., Center Valley, PA, USA)

attached to a stereomicroscope (SZ61; Olympus Amer-

ica Inc., Miami, FL, USA) and was measured using

Image J software (http://rsb.info.nih.gov/ij/; National

Institute of Health) under 25· magnification. Speci-

mens with noncircular shape were discarded to avoid

nonuniform stress distributions during testing, resulting

in approximately 25 slices per group. Endodontic sealers

were mixed according to manufacturer’s instructions

and used to fill the entire root canal space. Prior to filling

with Epiphany sealer, root canal dentine was etched for

30 s with Epiphany primer. Specimens were stored for

72 h at 37 �C and 100% relative humidity.

For the micropush-out test, a compressive load was

applied to the specimen via a cylindrical stainless steel

punch attached to a universal testing machine (Kratos

Dinamometros, Embu, SP, Brazil). For each specimen, a

punch tip 0.2 mm smaller than its apical diameter was

selected and positioned such that it touched only the

sealer and did not stress the surrounding root canal

walls. The apical aspect of the each specimen was

positioned facing the punch tip. Loading was performed

at a crosshead speed of 0.5 mm min)1 until the sealer

was dislodged from the root slice. Tensile bond strength

of each slice was calculated as the force (N) of failure

divided by the bonded cross-sectional surface area and

expressed in MPa (Patierno et al. 1996).

Failure mode analysis

For scanning electron microscope (SEM) observation

(100· and 2500·, LEO Stereoscan 440, Electron

Microscopy Ltd., Cambridge, UK) micropush-out spec-

imens were cut longitudinally and root segments were

covered with platinum (Coating System MED 020;

BAL-TEC AG, Balzers, Liechtenstein). To estimate the

percentage of free substrate the interface area was

divided into eight segments. This approach, suggested

by Fowler et al. (1992), was used to classify failure

mode as: (‡75%); cohesive within sealer (£25%)

adhesive-cohesive (>25% to <75%).

Statistical analysis

Data from bond strength to dentine, flow and polymer-

ization stress were analyzed using the Student’s t-test.

For the bond strength test each tooth derived one single

value. The level of significance was fixed at 5%.

1

2

3

4

5

Figure 1 Schematic representation of the experimental set-up

used for polymerization stress determination: (1) fixture

conectect to the load cell; (2) long glass rod; (3) short glass

rod; (4) stainless steel fixture with a slot to allow for the

positioning of the light guide in contact with the glass rod; (5)

extensometer.

Souza et al. Physicomechanical properties of endodontic sealers


Results

Table 1 summarizes average and SD of the micropush-

out test and flow of both sealers. Epiphany presented

significantly high flow than AH Plus (P < 0.001). A

significant difference was detected between polymeri-

zation stress for Epiphany-SC (0.32 ± 0.07 MPa) and

Epiphany-LC (0.65 ± 0.08 MPa) as shown in Fig. 2

(P < 0.05). Epiphany-SC started to generate stress

20 min after mixing. Epiphany-LC was photoactivated

after 17 min from the beginning of the test, when an

abrupt increase on polymerization stress curve

occurred. AH Plus revealed zero polymerization stress

values during 60 min, and for this reason was excluded

from statistical analysis.

For the micropush-out test Epiphany-SC had lower

values when compared with AH Plus (P < 0.001).

Failure mode distribution is shown in Fig. 3: 79.2%

cohesive within sealer and 20.8% adhesive for AH Plus,

78.3% cohesive within sealer and 21.7% adhesive-

cohesive for Epiphany-SC.

Discussion

Apical gap formation is influenced by local factors such

as substrate morphology (Wu et al. 1998, Ferrari et al.

2000, Mjor et al. 2001), C-factor (Goracci et al. 2004,

Tay et al. 2005b), and also material-related factors

such as physical properties of sealers (i.e. flow,

polymerization contraction) (Bergmans et al. 2005,

Braga et al. 2005) and bond strength to dentine

(Tagger et al. 2002, Bouillaguet et al. 2003). This

study assessed the possible relationship between flow,

polymerization stress and bond strength of AH Plus and

Epiphany sealers with apical gap formation.

The fact that no stress development was observed for

AH Plus up to 60 min after mixing agrees with the

manufacturer information that states a setting time of

8 h at 37 �C. However, running the polymerization

stress test for such long periods is impractical. Notwith-

standing, this information is interesting for comparative

purposes with the other sealer evaluated. For Epiphany,

polymerization stress tests were performed for both

curing modes: self-cured, relying only on the peroxide-

amine reaction and dual-cured. Epiphany was tested in

SC mode because clinically the light from photoactiva-

tion does not reach the middle or apical root regions

(Hiraishi et al. 2005). The increased polymerization

time in SC mode allows materials to flow in a pre-gel

state, which could provide stress relief at the dentine/

resin interface (Braga et al. 2002, Braga & Ferracane

2004), and be advantageous for this material. However,

polymerization stress when light-curing was used

(Epiphany-LC) doubled when compared with Epiph-

any-SC (Fig. 2; P < 0.05). This finding is related to an

increase in polymerization rate caused by light activa-

tion. Nagas et al. (2007) suggested that a decreased

polymerization time can adversely affect Epiphany bond

strength to dentine. In fact, one could speculate that an

Table 1 Mean values and standard deviations of bond

strength to dentine and flow for AH Plus� and Epiphany�sealers

Groups Micropush-out (MPa) Flow (mm)

AH Plus 17.8 (7.5)a 28.6 (0.7)b

Epiphany 6.3 (5.3)b 30.9 (1.1)a

Different letters on the same column show statistically signif-

icant differences (P < 0.001).

Figure 2 Polymerization stress (MPa) as a function of time (s)

of Epiphany self-cure (SC) and light-cure (LC).

Figure 3 Failure mode distribution for experimental groups

(%).



increased polymerization rate conferred by light activa-

tion can restrict the chances for polymerization stress

release during the pre-gel state (Tay et al. 2005b).

In theory, total bond strength is the sum of the

strengths of resin tags, hybrid layer and surface

adhesion (Pashley et al. 1995). The low viscosity and

hydrophilic nature of resin-based sealers in association

with pressure caused by condensation technique

allowed the sealer to infiltrate into dentinal tubules,

forming long tags and secondary branchings (Bergmans

et al. 2005, Tay et al. 2005a) In this study, both resin

sealers differed in flow (P < 0,001; Table 1), and both of

them exceeded specification 57 of American National

Standard/American Dental Association (2000). Despite

that, Tay et al. (2005a) showed in SEM and Transmis-

sion Electron Microscope (TEM) the loss of integrity at

dentine/Epiphany sealer and gutta-percha/AH Plus

sealer interfaces. These gaps, presumably created by

polymerization contraction forces (Tay et al. 2005b),

suggest that hybrid layer and long tags do not guaran-

tee the absence of gaps (Bergmans et al. 2005).

Bond strength between endodontic cements and

dentine may be an important property to provide a

seal (Tagger et al. 2002). Micropush-out values for

Epiphany were lower than for AH Plus (P < 0.001;

Table 1). Epiphany polymerization stress may have

contributed to its lower bond strength value. The

amount of stress associated with shrinkage may result

in separation of resin-based sealer and dentinal walls,

and consequently, bond strength values of this inter-

face would decrease (Hiraishi et al. 2005). In this study,

bond strength results for Epiphany sealer are compa-

rable with other experiments that showed values

between 0.32 and 3.73 MPa (Gesi et al. 2005, Ungor

et al. 2006, Fisher et al. 2007, Sly et al. 2007, Kaya

et al. 2008, Lawson et al. 2008, Lee et al. 2008)

though towards the high end range. Although filling

the root canal only with the sealer does not accurately

represent the clinical situation, this experimental model

was chosen because it represents a worst case scenario,

as polymerization stress development is directly related

to the volume of shrinking material (Tay et al. 2005b).

Moreover, by not using gutta-percha and resilon cones,

it can be assured that the tested interface is comprised

of sealer and dentine only.

Epiphany-LC was not included in the micropush-out

test because the study was designed to simulate the

clinical conditions found at the apical third of the root

canal, where the effect of light-curing is likely to be zero.

It is reasonable to speculate that, when used in SCmode,

the sealer does not totally polymerize. The incomplete

polymerization can impair cement mechanical proper-

(a) (b)

(c) (d)

Figure 4 Representative scanning electron microscope (SEM) micrographs of failure mode for AH Plus� (a and b) and Epiphany�(c and d): (a) sealer cohesive failure showing dentine surface recovered by a thick organic matrix layer with different sizes fillers; (b)

adhesive failure showing clean dentine surface only with small fillers and dentinal tubules with organic matrix tags; (c) sealer

cohesive failure indicating dentine surface recovered by an organic matrix layer with granular small fillers, and major fillers with a

thin plaque format, and also some empty spaces; (d) cohesive and adhesive failure demonstrating dentine surface covered by

Epiphany primer and some sealers fragments with fillers closing total or partially dentinal tubules (pointer).



ties and chemical stability (Braga et al. 2002). In fact,

failure mode analysis revealed a high incidence of sealer

cohesive failure for Epiphany (Figs 3 and 4).

The integrity loss on dentine/Epiphany interface can

be explained by comparing its bond strength to dentine

with stress generated during the polymerization con-

traction. Apparently, shrinkage stress was high enough

to surpass bond strength (Bouillaguet et al. 2003, Tay

et al. 2005a). The apparently negligible polymerization

stress values determined in the mechanical test (Fig. 2)

might be of a much higher magnitude in the root canal,

where geometric shape and material confinement are

obstacles for stress release. According to Tay et al.

(2005b), C-factor of adhesive bonding root filling

materials in root canals is highly unfavourable, chal-

lenging the concept of total bonding in root canals.

Conclusion

The null hypothesis was rejected for the three variables

analyzed. Epiphany had higher flow, lower bond

strength to dentine and also developed higher poly-

merization stress than AH Plus. Within the limitations

of this laboratory study and in view of the results it can

be speculated that, clinically, a better interfacial sealing

could be expected with AH Plus. The higher bond

strength to dentine obtained with AH Plus can be

partially explained by its lower polymerization stress.

Moreover, its higher viscosity compared with Epiphany

did not seem to impair its bond strength.

Acknowledgements

This study was partially supported by CAPES

(Coordenacao de Aperfeicoamento de Pessoal de Nıvel

Superior) Institutional Qualification Program (PQI no.:

0090/03–4). The authors are grateful to Flavia Rodri-

gues for providing the polymerization stress test diagram.

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Evaluation of the cost-effectiveness of root canaltreatment using conventional approaches versusreplacement with an implant

M. W. Pennington1, C. R. Vernazza2, P. Shackley1,3, N. T. Armstrong1, J. M. Whitworth2 &J. G. Steele2

1Institute of Health and Society; 2School of Dental Sciences, Newcastle University; and 3Sheffield Vascular Institute, University of

Sheffield, Sheffield, UK

Abstract

Pennington MW, Vernazza CR, Shackley P,

Armstrong NT, Whitworth JM, Steele JG. Evaluation of

the cost-effectiveness of root canal treatment using conven-

tional approaches versus replacement with an implant. Inter-

national Endodontic Journal, 42, 874–883, 2009.

Aim To evaluate the cost-effectiveness of root canal

treatment for a maxillary incisor tooth with a pulp

infection, in comparison with extraction and replace-

ment with a bridge, denture or implant supported

restoration.

Methodology A Markov model was built to simu-

late the lifetime path of restorations placed on the

maxillary incisor following the initial treatment deci-

sion. It was assumed that the goal of treatment was

the preservation of a fixed platform support for a

crown without involving the adjacent teeth. Conse-

quently, the model estimates the lifetime costs and the

total longevity of tooth and implant supported crowns

at the maxillary incisor site. The model considers the

initial treatment decisions, and the various subsequent

treatment decisions that might be taken if initial

restorations fail.

Results Root canal treatment extended the life of the

tooth at an additional cost of £5–8 per year of tooth life.

Provision of orthograde re-treatment, if the root canal

treatment fails returns further extension of the expected

life of the tooth at a cost of £12–15 per year. Surgical

re-treatment is not cost-effective; it is cheaper, per year,

to extend the life of the crown by replacement with a

single implant restoration if orthograde endodontic

treatment fails.

Conclusion Modelling the available clinical and cost

data indicates that, root canal treatment is highly cost-

effective as a first line intervention. Orthograde

re-treatment is also cost-effective, if a root treatment

subsequently fails, but surgical re-treatment is not.

Implants may have a role as a third line intervention if

re-treatment fails.

Keywords: cost-effectiveness, decision analysis,

implant, Markov, root canal treatment.

Received 16 September 2008; accepted 17 March 2009

Introduction

Clinical decisions could be consistent and straightfor-

ward, if they were informed by unequivocal evidence,

supported by clear and accepted guidelines, and if the

recommended actions were universally acceptable to

patients and care providers. But few areas of practice

are so clear-cut. Patients are not always equipped with

the information they need to make rational decisions

on their short and long-term care, and healthcare

agencies might equally be ill-equipped to advise on best

actions for the short and long term. As a consequence,

patients may submit to the paternalistic decision-

making of a healthcare professional (Kaba & Sooria-

kumaran 2007) whose priorities may be expected to be

objective, consistent and based on the same values as

their own. But observations from medicine and

dentistry suggest that the decisions of healthcare

Correspondence: Mark W. Pennington, MSc, PhD, Research

Associate, Institute of Health and Society, Newcastle Univer-

sity, 21 Claremont Place, Newcastle upon Tyne NE2 4AA, UK

(Tel.: +44 191 222 3544; fax: +44 191 222 6043; e-mail:

[email protected]).

doi:10.1111/j.1365-2591.2009.01582.x


professionals themselves may be highly variable, even

in the case of relatively simple interventions (Dome-

jean-Orliaguet et al. 2004, Lanning et al. 2005, van der

Sanden et al. 2005, Calnan et al. 2007, Tickle et al.

2007), and influenced by a number of personal,

educational and economic considerations (McColl et al.

1999, Brennan & Spencer 2006).

The picture is complicated further in the case of

complex interventions, and interventions that may

not be the final solution within the lifetime of the

patient. Here, the decision-making process may be

limited to a consideration of the ‘next step’, and

informed by short-term ‘success rates’, assessment of

immediate costs, or of the willingness of the patient to

pay for that individual step. Rarely is the decision-

making process informed by a detailed understanding

of the relative lifespan of alternative interventions or

the ongoing costs, both financial and otherwise

(White et al. 2006, Balevi 2008), which may flow

from a particular treatment decision. Restorative

dental treatments are an example of such an inter-

vention, and if patients faced with treatment decisions,

or healthcare providers stewarding finite resources are

to make properly informed decisions, they must be

presented with information on cost and outcome

which they understand and which accounts for the

long-term.

The uncertainties inherent in modelling the costs of

combinations of interventions over a lifetime require a

fundamentally different approach to the use of evidence

to that, with which most clinicians are comfortable.

Decision analytic modelling provides a rational frame-

work for decision making based on expected costs and

outcomes (Raiffa 1968). Many decision analytic models

are based on Markov modelling, a mathematical means

of investigating stochastic or random events over time

(Sonnenberg & Beck 1993). Such modelling lends itself

well to the study of long-term medical conditions,

defining a clear starting point or condition, and

identifying a number of states into which the individual

may or may not move at defined points in the future.

The probability of remaining in the starting condition

or moving to an alternative state is informed by best

outcome and survival data, and the costs of initial and

future interventions estimated from professional

sources.

Markov models are increasingly used in evaluating

the long-term cost effectiveness of clinical interventions

from the chemoprevention of prostatic cancer to the

management of heart failure (Chan et al. 2008, Svatek

et al. 2008, Takao et al. 2008).

By contrast, the economic models applied to dentistry

have generally been quite simple decision trees (Mil-

eman & van den Hout 2003) or Markov models

(Edwards et al. 1999) extrapolating over a fixed num-

ber of years or the assumed lifetime of a specified

intervention (for example, a dental restoration), rather

than over the lifetime of the patient.

Whilst previous publications have investigated the

costs of dental treatments over a fixed time span

(Bragger et al. 2005), as far as the authors are aware,

this report represents the first attempt to provide a

definitive examination of the cost effectiveness of

common dental interventions and look at all realistic

options that flow from this over the lifetime of a

patient. The starting point of the Markov model is a

common clinical scenario; a damaged and irreversibly

pulpitic maxillary central incisor tooth, where initial

treatment options include root canal treatment and

restoration, or extraction and prosthetic replacement.

The model explores the long-term consequences and

cost effectiveness of initial and subsequent decisions

for individuals at different ages. The question at the

heart of this investigation is whether root canal

treatment and restoration of a damaged maxillary

central incisor is a legitimate and cost-effective inter-

vention over the lifetime of an adult patient, and in

comparison with the alternatives of extraction

followed by either a conventional or an implant-

supported restoration.

Methods

Building the model

For this study, a Markov model was built with TreeAge

decision analysis software (TreeAge Software Inc.,

Williamstown, MA, USA, http://www.treeage.com/

index.htm).

The starting point was a damaged, irreversibly

pulpitic maxillary central incisor in an otherwise

healthy adult male of varying age. The loss of coronal

tooth tissue was defined as sufficient to require resto-

ration with a post-retained crown. Assuming that the

patient requests some treatment to fill the space, and

from this starting position, the patient could occupy

any of the six health states listed below at any given

point in time, until the end of their life:

• Tooth extracted with resin bonded bridge (RBB)

in situ

• Tooth extracted with a conventional bridge (fixed

dental prosthesis, FDP) in situ

Pennington et al. C-E of root canal treatment


• Tooth extracted with removable partial denture

(RPD) in situ

• Tooth root canal treated (RoCT) with a post-

retained crown in situ (there may be repair or replace-

ment of any of the parts of the restoration or root filling

within this state)

• Tooth extracted with an implant-supported single

crown (ISC) in situ (again this could be a first, second or

subsequent restoration)

• An implant in situ prior to abutment connection

(the transitional state during osseointegration assum-

ing there is no immediate loading)

• Death of the patient.

The model calculated the probability of the incidence

of all significant mechanical and biological complica-

tions that might arise in each of these states, over each

6 month period of the patients life, based on existing

evidence (see ‘Outcome data’ later). A repair event or

no event occurring meant that the simulated patient

remained in the same restoration state, whereas

complete failure resulted in transition to a different

state (e.g. the event of root fracture would require

extraction and replacement of the tooth with a pros-

thesis of some description).

The analysis was simplified by modelling the selec-

tion of a bridge or denture prosthesis as a random

parameter based on likely distributions in the UK

population rather than a treatment choice. The simu-

lation terminated when the patient reached 100 years

of age or died (using age-related mortality probabilities–

govt. actuaries dept., life tables 2002–2004, http://

www.gad.gov.uk/). The number of possible pathways

through these various states in a lifetime is clearly

massive. The initial treatment decision and then the

potential subsequent treatments necessitated by failure

of a restoration are captured in the ten major strategies

outlined in Fig. 1. Whilst these cannot capture every

single possibility, they were considered the most likely

10 pathways by consensus of two senior clinical

academics in Restorative Dentistry (JGS and JMW).

Strategy 1 illustrates a decision to extract the

irreversibly pulpitic tooth and to replace it with a

conventional removable or fixed prosthesis, not an

implant. The remaining nine strategies involved either

retaining the tooth by root canal treatment, removing

it and placing an implant or a combination of these.

In comparing each of the 10 major strategies, the

costs and expected outcomes of both the initial treat-

ment strategy (first intervention) and supplementary

interventions (second to fourth intervention) are pre-

dicted. Estimations of cost and treatment longevity are

central to the model. To examine fully the cost-

effectiveness of three initial options (bridge/denture,

implant, orthograde endodontics) the costs which

might follow them are required. Clearly a RoCT is less

expensive than an implant at the point of delivery but

will the implant save money in the long term? To do

this, it was necessary to model at least the second and

third interventions and their costs and outcomes. It is

not known what the patient might or should choose

when the restoration fails, so all of the reasonable

subsequent choices if that happened were considered

and evaluated as different strategies. The strategy of

placing an implant initially was also evaluated. One of

these will be the most cost-effective. It was necessary to

look at all of the likely second and third interventions if

implants were to be given a fair comparison against

RoCT.

Strategy 1st Intervention 2nd Intervention 3rd Intervention 4th Intervention

1 Extraction Bridge/denture

2 One RoCT Orthograde RoCT Bridge/denture

3 RoCT then re-treatment Orthograde RoCT Orthograde RoCT Bridge/denture

4 RoCT then surgery Orthograde RoCT Surgical RoCT Bridge/denture

5 RoCT then Implant Orthograde RoCT First implant Bridge/denture

6 RoCT/Implant/2nd Implant Orthograde RoCT First implant 2nd implant Bridge/denture

7 RoCT/re-treatment/Implant Orthograde RoCT Orthograde RoCT First implant Bridge/denture

8 RoCT/Surgery/Implant Orthograde RoCT Surgical RoCT First implant Bridge/denture

9 Implant First implant Bridge/denture

10 Implant then 2nd implant First implant Second implant Bridge/dentureFigure 1 Sequence of interventions in

the ten treatment strategies.

C-E of root canal treatment Pennington et al.


Cost-effectiveness analysis: data sources

Outcome data

In order to function, the model was parameterized

with information on expected treatment longevity/

failure rates, and likely maintenance needs of different

treatment options. Extensive Searching of MEDLINE,

EMBASE, DARE and Cochrane Library databases (from

inception to June 2006) was undertaken for all

papers with terms including failure, fracture, success,

treatment, re-treatment, replacement, complications,

survival, (meta)analysis and terms describing the

tooth state such as root canal, endodont#, #apical.

This was supplemented by systematically checking

the references of all papers retrieved for further

relevant studies. Meta-analyses were utilized, where

available, otherwise parameters were chosen based on

the size, quality, age and selection criteria of the

study. In the very rare instances where no appropriate

data were available, the expert opinions of two senior

clinical academics in Restorative Dentistry (JGS and

JMW) were sought to define the likely limits of

parameters.

Three meta-analyses were retreived on the survival

of ISCs. The meta-analysis of Branemark implants

(Lindh et al. 1998) was selected to parameterize

implant survival as it differentiates between implant

loss after loading and failure to osseo-integrate. A meta-

analysis of prospective studies (Berglundh et al. 2002)

provided data to parameterize complications in the

implant states. However, the exclusion criteria limited

the paper to a small number of studies. Hence, the

analysis was judged less satisfactory than those

reported by Lindh et al. (1998). The FDP state was

parameterized using the most recent and largest meta-

analysis (Tan et al. 2004). There are fewer reports on

the survival and complication rates for RBBs and no

meta-analyses were retrieved. The available data on

RPDs is minimal. These states were parameterized from

published individual trial or longitudinal studies where

available. The heterogeneity of success criteria in

reports on RoCT has defied meta-analysis to date

(Creugers et al. 1993). Creugers analysis selected only

three papers of which one (Mentink et al. 1993) was by

far the largest, hence this report was prioritized when

parameterizing the post-supported crown states. Rates

of failure of root canal after re-treatment were taken

from a 10-year Swedish study (Sjogren et al. 1990)

whilst rates of treatment failure following surgical

endodontics were based on an evaluation of apical

surgery (Buhler 1988).

Costs

For the purposes of this model, typical staff time and

resource use for each procedure was estimated based

on a UK National Health Service (NHS) secondary care

setting. Staff costs were taken from published reference

costs (Curtis 2006), and costs are in UK 2006 pounds.

The base case analysis for this study assumes that all

implant procedures were carried out by a senior

specialist (consultant) dentist. All of the conventional

dental procedures were costed at more junior specialist

staff (Specialist Registrar or Senior House Officer) rates

reflecting the more routine nature of such interven-

tions. Staff costs were based on mid-band salaries and

included overheads, training costs and administrative

support. Costs and outcomes are discounted at 3.5%

according to NICE guidelines for economic analyses.

Mortality is parameterized using data for UK males

(2002–2004 Government actuaries department). It is

important to note that the costs used are based on

standard data and represent the costs to the NHS, not

the price that may be paid, for example in private

practice where there are a range of additional consid-

erations, such as profit margins and variations in

overhead costs.

Cost-effectiveness analysis: assumptions

In order to develop an economic model such as this, a

number of assumptions need to be made. Where

possible these are supported by published evidence.

The following assumptions were made for this model:

• That the patient retains most of the dentition over

his/her lifetime (Kelly et al. 2000)

• That the longevity of the restoration is proportional

to the lifetime benefit of the restoration to the patient

• ISCs and crowned and root treated teeth provide the

same Oral Health Quality of Life (OHQoL)

• Apical surgery is undertaken alongside orthograde

re-treatment to enhance success rates, and not as a

response to a distinct clinical indication such as a cyst

• RBBs, RPDs and conventional FDPs provide the

same OHQoL, inferior to that of the ISC or crowned

tooth. This assumption infers that the retention of a

tooth unit in the maxillary anterior region in the form

of the original tooth or an implant is preferable to loss

of a fixed platform (natural or artificial) for restora-

tion. Whilst it is acknowledged that this is not

universally the case, this was considered a reasonable

working rule, which was necessary to allow the model

to compare endodontic strategies with implant strat-

egies



• A constant hazard rate is assumed for mechanical

and biological complications following an intervention

• The same hazard rate applies to an event, such as

tooth fracture, in the post-supported crown states

regardless of whether a surgical or nonsurgical end-

odontic re-treatment had occurred. The exception to

this was the rate of root canal treatment failure for

which there was available data (see above)

• Probability of implant loss and peri-implantitis are

independent. These were modelled independently on

the basis of data reported in the literature (Berglundh

et al. 2002)

• Results are presented for UK males only on the

assumption that dental costs and benefits are indepen-

dent of gender. As life expectancy rather than gender

dictates costs, results for females would be similar to

those for a slightly younger cohort of males with the

same life expectancy

The literature consists predominantly of follow-up of

patients treated in dental hospitals, or in specialist

clinics in the case of implants. This may not accurately

reflect outcomes achieved in primary-care settings, but

robust data in these environments are generally lack-

ing. However, sensitivity analysis allowed the cost

variables related to hospital staff costs to be varied (see

below).

Cost effectiveness: ratio calculation

The outcome measure used in the cost-effectiveness

analysis is the total longevity of a fixed platform

supported crown, both root canal treated and post-

crowned natural tooth, and implant supported crowns.

After reviewing the costs and longevity for all ten

strategies and ranking them by cost, strategies that were

clearly less cost effective (those that were ‘dominated’ or

‘extendedly dominated’, see results) were removed and

the rest retained for the calculation of an incremental

cost-effectiveness ratio (ICER). This widely used index of

cost-effectiveness (Drummond et al. 2005) is the addi-

tional financial cost divided by the additional effective-

ness (in this case the prolonged longevity of the crown)

of that strategy over the next cheapest alternative.

Cost-effectiveness analysis: sensitivity analysis

The key parameters (such as costs and survival) are all

estimates and, by definition, likely to be imprecise. To

allow for this, plausible ranges for key parameters (such

as survival of restorations) were estimated by the

academic dental authors, allowing one-way sensitivity

analysis of the model to be undertaken for each of these

parameters. This re-running of the model with different

starting parameters illustrates the impact that the

inevitable inaccuracies might have on the overall

model.

The overall costs of each strategy are clearly a

product of the estimated dental procedure costs. Dental

costs are considerably lower in eastern European

countries but average wages and hence patient budgets

are also likely to be lower. However, varying the costs

of dental wages or implant components will influence

the relative cost-effectiveness of each treatment strat-

egy. The relative effect of decreasing component costs

or increasing dental salaries is likely to be similar –

implant costs will fall relative to alternative restorative

procedures and implant strategies will be more cost-

effective. We simulated three different potential cost

environments to illustrate the impact of higher and

lower wage costs and the impact of lower implant

component costs.

Results

Table 1 shows both the expected total lifetime costs and

the expected longevity of the root canal treated tooth

and/or implant supported crowns for a male aged 35,

55 and 75 years, without inflation. The values have

been ‘discounted’ to take account for change in

perceived value with time, using standard measures

recommended by NICE (http://www.nice.org.uk/

media/F13/6E/ITEM3FINALTAMethodsGuidePostCon-

sultationForBoardCover.pdf) and this partly accounts

for the relatively low monetary values in all strategies.

Crown longevity is the sum of the total lifetimes of root

canal treated tooth and/or implant supported crowns at

that site prior to failure and replacement with a bridge

or denture. It is assumed that if no endodontic or

implant treatment is provided there will still be a need

over the lifetime to fill the space, with a cost

consequence [statistically, unfilled anterior spaces are

very rare in the UK (Kelly et al. 2000)].

The model predicts superior survival of the ISC over a

conventional root canal treated tooth with a post-

crown based on published evidence. After 20 years

around 25% of root canal treated and re-treated teeth

are predicted to have been lost, whereas 10% of first

implants have failed, necessitating a further implant or

replacement with a bridge or denture. Despite improved

longevity, the implant based strategies still require

more interim interventions if a two stage procedure is

assumed.



Figure 2 shows the cost accumulation (discounted)

for each strategy over 65 years for a male aged

35 years. The significantly greater initial outlay on

placing an implant is evident but slightly mitigated by

lower ongoing costs, illustrated by the rather shallow

curve. The ongoing costs of strategies five (RoCT/

Implant) and six (RoCT/Two implants) show the

steepest gradient, due to a combination of relatively

high failure rates of the first treatment (RoCT), and the

high cost of the second treatment (implant).

Cost-effectiveness analysis

The 10 strategies model both the initial intervention

and the possible subsequent interventions required to

maintain a tooth or prosthesis at that site for the

patient’s lifetime. To establish cost effectiveness these

are ranked in order of cost and their longevity

reviewed. When this was done, some strategies were

clearly less cost-effective because they have poorer

longevity but still cost more than others. They are said

to be ‘dominated’. Strategies five (RoCT/Implant), nine

(One Implant) and 10 (Two Implants) were dominated

for patients at all ages analysed (35, 45, 55, 65, 75,

85) and have been excluded.

The remaining strategies are each more effective

than less expensive alternatives, but some are signifi-

cantly more expensive than a comparator but only

marginally more effective. It would not make sense to

choose such a strategy if, by paying only a little more,

we could get a much bigger increase in effectiveness,

hence these strategies are excluded (they are said to be

‘extendedly dominated’). Both strategies involving a

surgical endodontic re-treatment (strategies four and

eight) fell in to this category at each age analysed.

Whilst surgical endodontic re-treatment has a higher

reported success rate than nonsurgical re-treatment in

some studies, this has generally followed endodontic

re-treatment. The overall increase in longevity, relative

to the increased cost, is small. Additional crown years

(longevity) can actually be achieved at a lower cost per

year with implants.

The results of the cost-effectiveness analysis are

shown in Table 2.1 Strategy 1 (No Treatment) is the

least effective and the cheapest, and so this is the

comparator for calculating the ICER for strategy two

0

200

400

600

800

1000

1200

1400

1600

1800

35 45 55 65 75 85 95Age

£

Implant/2nd implant ImplantRoCT/Implant/2nd implant RoCT/ImplantRoCT/Surg/Implant RoCT/re-treat/ImplantRoCT/Surg RoCT/re-treatOne RoCT Extraction

Figure 2 Cumulative costs of each strategy (male age

35 years).

Table 1 Base case results – cost and

total crown longevity for each strategy

Strategy

Male age 35

Cost

(£) Longevity

Male age 55

Cost

(£) Longevity

Male age 75

Cost

(£) Longevity

1 (Extraction) 731 0 649 0 540 0

2 (One RoCT) 805 15.81 717 12.62 597 7.1

3 (RoCT then re-treatment) 828 17.29 730 13.56 601 7.41

4 (RoCT then Surgery) 847 17.51 746 13.66 611 7.43

7 (RoCT/re-treatment/Implant) 1071 21.58 916 15.78 694 8

8 (RoCT/Surgery/Implant) 1079 21.59 924 15.78 701 8

5 (RoCT then Implant) 1113 21.47 967 15.73 736 7.99

6 (RoCT/Implant/2nd Implant) 1140 21.85 983 15.88 741 8.02

9 (Implant) 1623 20.12 1570 14.96 1487 7.74

10 (Implant then 2nd Implant) 1717 21.73 1642 15.83 1527 8.01

1The costs generated by the model are the expected future

costs discounted to the present and not the actual costs faced

by a patient if he/she was to receive each of the interventions

in the strategy. We would expect many patients to die with an

intact root treated tooth, only a proportion will go onto to

receive subsequent interventions and the model presents the

‘average’ costs given the likelihood of failure of restorations

undertaken.



(One RoCT). The comparator for each subsequent

strategy is the next best alternative after excluding

dominated and extendedly dominated options. All the

cost-effective strategies involve initial root treatment.

Strategy 2 is expected to cost £5–8 more per year of

longevity of the root treated tooth than replacement

with a bridge or denture. The table reveals that patients

who would choose othograde re-treatment should the

root canal treatment fail (strategy three) can expect to

extend the longevity of the root treated tooth at a cost

per year of additional life of £11–£15 over and above

the expected cost if a bridge or denture is fitted on

failure of the root treated tooth. Patients who would

choose an implant rather than a bridge or denture

should the re-treatment fail (strategy 2) can expect to

extend the longevity of fixed platform supported crown

at a likely additional cost of £57–241 per year.

Sensitivity analysis

When each of the key parameters was altered over the

limits of likely variation and the models re-run, the

impact on the overall cost-effectiveness of each strategy

was small, and no changes in the overall rankings were

observed.

General diffusion of implant technology is likely to

lead to lower potential component costs and also more

efficient provision by general dentists. The impact of

halving all of the implant component costs, and

re-costing implant procedures at lower professional

rates (£50/hour instead of £87/hour) was examined.

The impact of a higher wage setting (such as the US)

was simulated by costing all procedures using the UK

consultant rate (£87/hour) for dentists and by increas-

ing labwork costs by 50%. The impact of a lower wage

setting was examined by reducing all wage costs

(dentists, assistants and hygienists) to 30% of the UK

estimates and by reducing dental laboratory costs by

50%. Costs and ICERs for each scenario for nondom-

inated strategies are presented for a 55-year-old male in

Table 3. It can be seen that whilst the absolute effect of

higher or lower wage rates on overall costs is marked,

the impact on ICERs is small. Unsurprisingly, lowering

both wage rates and component costs only for implant

procedures leads to a significant reduction in the costs

of implant based strategies, but they are still more

expensive than conventional treatment. Only when

component costs are radically reduced to 10% of the

current costs does an implant strategy (strategy five,

RoCT/Implant) extendedly dominate an endodontic

strategy (strategy three, two RoCTs), in this case for

younger males below the age of 37 years.

Discussion

It is unrealistic to expect most dental restorations to last

for life (Richardson et al. 1999). Although data may be

scarce, one systematic review estimated that 50% of all

routine dental restorations may be anticipated to last

between 10 and 20 years (Downer et al. 1999), whilst

life-expectancy for women is now currently 80 years or

more (http://www.statistics.gov.uk/cci/nugget.asp?id=

168). As our urban populations continue to age and

expectations of dental function and aesthetics continue

to rise, patients, dentists and health planners need to

recognize that the next intervention may not be the

last, particularly in younger patients. Decisions made at

a fixed point in time may set individuals on a pathway

with long-term ramifications.

The example considered in this study was a compro-

mised, irreversibly pulpitic maxillary central incisor,

with the starting expectation that very few would opt

for no treatment at the point of presentation. The

immediate choice facing the theoretical patient is

whether to preserve the tooth by root canal treatment

and a post-retained crown, or whether to have the

tooth extracted and replaced with a prosthesis, includ-

ing the possibility of a single implant. This decision may

be influenced by patient and practitioner-based factors,

including perceptions of ‘success’, the special interests

of the practitioner, and the attitudes and financial

considerations of the patient (Brennan & Spencer 2006,

White et al. 2006). Debates on the merits of individual

treatment decisions are not new and have been

recognized clearly at the endodontic/implant interface,

where strong arguments have been made on both sides

that certain options are more likely to succeed or to be

more economic at the point of delivery (Felton 2005,

Trope 2005). But debates on ‘survival’ and immediate

costs cannot always account for the lifetime implica-

tions, including maintenance and repair, and costs of

replacement after outright failure. A decision analytic

Table 2 Incremental cost-effectiveness ratios (ICERs) for non-

dominated strategies over the age range 35–85

Strategy

ICERs for males aged 35–85 (£)

35 45 55 65 75 85

2 (One RoCT) 5 5 5 6 8 ED

3 (RoCT then re-treatment) 15 15 14 13 11 12

7 (RoCT/re-treatment/Implant) 57 67 84 111 158 241

6 (RoCT/Implant/2nd Implant) 252 383 654 1272 2813 6916

ED-extendedly dominated.



framework combines expected costs and expected

benefits in a manner that aids decision making. In

the absence of data on patient utility, it was assumed

that benefits are proportional to the longevity of a root

canal treated tooth or implant; the presentation of

ICERs guides the decision according to the value placed

on those benefits by the decision maker.

For the clinician, the patient, the commissioner or the

policy maker the model reported here gives a reason-

ably strong guide to the general courses of action that

are likely to be the most cost effective in this relatively

common scenario. It suggests that root treatment in the

first instance is a cost effective strategy, and that the

lifetime costs are relatively low, even compared with

extraction and replacement with a denture or bridge.

Where root treatment fails, in general terms, ortho-

grade re-root treatment is still a reasonably cost

effective approach. The lifetime costs are a little higher,

but still not a great deal higher, than extraction and

bridge or denture placement. Following endodontic

re-treatment with surgery was not cost effective in a

typical presentation, though this does not rule-out the

clinical need for surgery in the event of lesions requiring

a biopsy, or the diagnosis of a lesion unlikely to heal by

orthograde endodontic means. Implant placement is

expensive, and is cost effective in this scenario only after

endodontic treatment has failed twice. It is not cost-

effective as an initial option. Of course these calcula-

tions do not take into account the value that an

individual patient may place on any given treatment.

Markov modelling presents a valuable tool for

examining such complex lifetime events. Central to

the model is a body of survival and outcome data,

which informs the probability of a patient remaining in

a given health state or moving to a new health state at

defined points in time. It allows extrapolation of the

clinical data to estimate the expected costs and

outcomes over the patient’s lifetime. The ICERs com-

bine costs and outcome data in a manner which

facilitates rational decision making at the level of the

individual, the insurer or the state. It would be easy to

misinterpret these findings as some sort of clinical

guidance – they are explicitly not that. The model deals

in probabilities spread across the generality of patients.

Technical or patient issues will tip the balance in favour

of one or other approach to treatment for individual

patients. However, an understanding of costs and cost

effectiveness may help the clinician to advise their

patients about the long term costs of any given course

of action, or to help insurers or health planners to

decide on the basic treatment strategies that give the

best value for money. For example, based on this

evidence, a reasonable starting point for an insurer

may be to provide high quality endodontic treatment,

and perhaps to put a premium on high endodontic

standards, in the first instance rather than funding

implant provision as a first line treatment.

The substantial body of evidence that defined the

current model is available in the on-line Appendix S1.

The literature was unable to provide the very best

quality of evidence on all of the interventions consid-

ered, so the model was informed by the best available

evidence. It is likely that survival of restorations will

vary widely according to patient characteristics and the

skill of the dentist. The evidence for survival of implants

and root treatments was meagre, though of reasonable

quality. The weakest evidence related to the survival of

partial dentures and bridges. This problem is of course

not restricted to Markov modelling, and impacts on any

attempt to conduct dental care on a base of evidence.

Long-term, prospective clinical trials with large sample

sizes and clearly defined outcome criteria are desper-

ately needed (Torabinejad et al. 2007).

The costs incorporated within the current model

were specific to the state funded healthcare system

currently operating in the UK. Clearly salary and

labwork costs vary significantly in different countries

and the impact on overall strategy costs is large.

However, it is the relative costs between strategies

rather than the actual values that are important. The

Table 3 Impact of varying wages and implant component costs on cost-effectiveness (55 years old)

Strategy

Base case Cheaper implants Higher wages Lower wages

Cost (£) ICER (£) Cost (£) ICER (£) Cost (£) ICER (£) Cost (£) ICER (£)

1 (Extraction) 649 649 993 281

2 (One RoCT) 717 5 717 5 1088 8 315 3

3 (RoCT then re -treatment) 730 14 730 14 1103 16 321 7

7 (RoC T/re-treatment/Implant) 916 84 822 41 1242 63 451 59

6 (RoCT/Implant/2nd Implant) 983 654 848 254 1286 437 501 486

ICER, incremental cost-effectiveness ratio.



relative impact of changing wage costs is surprisingly

small. ICERs are changed, but not by an order of

magnitude, and overall ranking of strategies remains

the same. Hence recommendations based on the

calculated ICERs are less susceptible to care costs in

different settings. The sensitivity analysis, which dem-

onstrated the stability of the strategy rankings to

changes in event probabilities and costs, suggests the

findings are robust.

Conclusions

Root canal treatment is an appropriate and cost-

effective intervention to extend the life of a maxillary

incisor tooth with a diseased pulp. Orthograde

re-treatment is also cost-effective, but unless clinically

indicated the benefits of additional apical surgery do

not justify the additional cost. Increased longevity of

the crown can be achieved at a lower cost per year with

an implant. At current costs the role of implants is

limited to a third line intervention if re-treatment fails.

Acknowledgements

The authors are grateful for the advice of Pelham

Barton on the appropriate analysis of sequential

treatment decisions, and the critical review and com-

ments from Rob Anderson. This work was undertaken

by Mark Pennington at Newcastle University without

external financial support.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Appendix S1. A detailed description of the model

including all of the data sources used to parameterise

it.

Please note: Wiley-Blackwell are not responsible for

the content or functionality of any supporting materials

supplied by the authors. Any queries (other than

missing material) should be directed to the correspond-

ing author for the article.



Long-term sealing ability of Resilon apicalroot-end fillings

M. A. A. De Bruyne & R. J. G. De MoorDepartment of Operative Dentistry and Endodontology, Dental School, Ghent University, Ghent University Hospital, Gent, Belgium

Abstract

De Bruyne MAA, De Moor RJG. Long-term sealing ability

of Resilon apical root-end fillings. International Endodontic

Journal, 42, 884–892, 2009.

Aim To evaluate ex vivo the long-term sealing ability

of the SE Resilon Epiphany system as an apical root-end

filling material.

Methodology A total of 60 standardized horizontal

bovine root sections were divided into three groups filled

with either gutta-percha with AH 26, tooth-coloured

mineral trioxide aggregate (MTA) or Resilon pellets with

Epiphany SE, and submitted to capillary flow porometry

at 48 h, 1 and 6 months to assess the minimum, mean

flow and maximum pore diameters. Results of the

different materials and results bymaterial and time were

analysed statistically using nonparametric tests; the

level of significance was set at 0.05.

Results Resilon had smaller pore diameters than

gutta-percha and MTA at 48 h and smaller mean flow

and maximum pore diameters than gutta-percha and

MTA at 1 month. At 6 months Resilon had larger

minimum pore diameters than gutta-percha. Although

not always statistically significant, the minimum,

mean flow and maximum pore diameters of gutta-

percha and MTA diminished with time. This was not

the case for Resilon, where the same parameters

increased.

Conclusions All materials leaked at all times. Resi-

lon performed better than gutta-percha and MTA in the

short-term, but the seal of MTA and gutta-percha

improved over time whereas the seal of Resilon

deteriorated. It is critical to evaluate the performance

of materials in the long-term contrary to most studies

which are short-term.

Keywords: capillary flow porometry, Epiphany,

leakage, Resilon, root-end filling, seal.

Received 14 October 2008; accepted 17 March 2009

Introduction

When orthograde root canal treatment is associated

with post-treatment disease, surgical endodontics may

be indicated. The procedure involves surgical debride-

ment of pathological periradicular tissue, apical root-

end resection, root-end cavity preparation and the

placement of a root-end filling in an attempt to seal the

root canal (Gutmann & Harrison 1994). The root-end

filling should ideally produce a fluid-tight seal that

prevents residual irritants and oral contaminants from

exiting the root canal system and entering the perira-

dicular tissues (Arens et al. 1998).

An ideal root-end filling material would adhere and

adapt to the walls of the root-end preparation, prevent

leakage of micro-organisms and their toxins into the

periradicular tissues, be biocompatible, be insoluble in

tissue fluids and dimensionally stable and remain

unaffected by the presence of moisture (Arens et al.

1998). It is generally accepted that the most fluid-tight

apical seal possible is required for successful periapical

healing (Hirsch et al. 1979). If the seal is not fluid-tight,

microleakage may occur. Leakage of various root-end

filling materials has been investigated widely, mainly

using dye penetration methods. However, there are

certain disadvantages in using the linear measurement

Correspondence: Dr M. A. A. De Bruyne, Department of

Operative Dentistry and Endodontology, Dental School, Ghent

University, Ghent University Hospital, De Pintelaan 185 P8,

9000 Gent, Belgium (Tel.: +32/9/332 58 35; fax: +32/9/332

38 51; e-mail: [email protected]).

doi:10.1111/j.1365-2591.2009.01583.x


of dye penetration, including the destruction of the

specimen, which makes further evaluation of samples

impossible, and the lack of reproducible and compara-

ble results (Schuurs et al. 1993, Wu & Wesselink

1993).

The reported pattern of leakage in endodontics differs

according to the various techniques adopted (Wu et al.

2003). The fluid transport method was first reported by

Greenhill & Pashley (1981) and adapted by Wu et al.

(1993). This method investigates through-and-through

voids and the result when using this technique

indicates the diameter of the void. The dye penetration

method investigates through-and-through as well as

cul-de-sac voids and the result when using this

technique indicates the length of the void rather than

the diameter (Wu et al. 2003).

Capillary flow porometry which was first introduced

in dentistry in 2005 (De Bruyne et al. 2005) is also

used to evaluate through-and-through voids. This

technique is used in membrane and filter media testing

to measure through pores (Jena & Gupta 2002), as does

the fluid transport method. In contrast to the fluid

transport method, which gives an indication on the

diameter of the void, CFP provides exact information on

the diameter of the minimum, mean flow and maxi-

mum pore diameter at its most constricted part. The

method has been approved by the American Society of

Testing and Materials (1999) and was adapted suc-

cessfully in collaboration with VITO (Flemish Institute

for Technological Research, Mol, Belgium) to evaluate

through pores in filled root canals or root sections

(De Bruyne et al. 2005). The method also provides

information on pore distribution.

A variety of substances have been proposed as root-

end filling materials including amalgam, gutta-percha,

zinc oxide–eugenol cements, dentine bonding agents,

glass–ionomer cements, mineral trioxide aggregate

(MTA) and other restorative materials (Gutmann &

Harrison 1994). MTA shows excellent biocompatibility

(De Bruyne & De Moor 2004) and, in spite of the limited

clinical research, is considered by many clinicians as a

standard during apical surgery (Nicholson et al. 1991,

Asrari & Lobner 2003, Pistorius et al. 2003, Sousa

et al. 2004). After the introduction of grey MTA a

tooth-coloured or white MTA was introduced (Matt

et al. 2004, Tselnik et al. 2004). Gutta-percha has been

used frequently as a root-end filling material in the past

and often the filling material is exposed apically when

no root-end filling is placed. The Epiphany endodontic

obturation system (Pentron, Wallingford, CT, USA)

consists of Resilon obturation material available in

points and pellets, and a dual-cure, hydrophilic resin

sealer. The Resilon points or pellets can be processed in

the same way as gutta-percha. Recently, a self-etch (SE)

version of this sealer was introduced. Resilon material

is a formulation of polymers of polyester with fillers and

radiopacifers in a soft resin matrix. The pellets are used

with a delivery system (Obtura-Spartan, Fenton, MO,

USA). The manufacturer claims that after curing the

combination of obturation material and sealer will

create a monoblock in the canal that effectively resists

leakage.

After periradicular surgery, the surface of the root-end

filling is exposed to the periapical environment. Because

of this exposure, decomposition of the material may

occur and the seal of the filling may degrade. In order to

obtain information on the performance of root-end

filling materials on the long-term, the seal of root-end

filling materials should be tested at different intervals

after filling (Wu et al. 1998, De Bruyne et al. 2006).

The purpose of this study was to evaluate the sealing

ability of the SE Resilon-Epiphany system as a root-end

filling material and to compare it with warm gutta-

percha and white MTA in standard bovine root sections

at 48 h and after 1 and 6 months.


Preparation and filling of root sections

Roots of freshly extracted bovine incisors with an

external diameter of approximately 7 mm were selected

and prepared into standardized sections 3 mm high.

The central pulp lumen was drilled to 2.5 mm in

diameter. For this purpose, the sections which were

verified to have a natural internal diameter smaller

than 2.5 mm were fixed in a clamp. A bur of 2.5 mm

in diameter which was secured in a fixed position was

passed once through the lumen.

Sixty of these sections were divided into three

different groups and each group was filled according

to the following scheme:

Group 1: warm gutta-percha (Obtura II, Obtura-

Spartan) and AH 26 (Dentsply De Trey, Konstanz,

Germany) (gutta-percha).

Group 2: Pro-Root MTA Tooth-Colored Formula

(Dentsply Tulsa, Tulsa, OK, USA) (MTA).

Group 3: Resilon pellets (Pentron) (Obtura II delivery

system; Obtura-Spartan) and Epiphany SE (Pentron)

(Resilon).

The root sections were rinsed with physiological

saline solution, dried with paper points and air spray

De Bruyne & De Moor Resilon root-end fillings


and placed on a glass plate on top of a strip of polyester.

All materials were mixed and handled according to the

manufacturer’s instructions and the root sections were

filled. The filling materials were condensed with a

plugger (RCPS 12P; Hu Friedy, Chicago, IL, USA) and

excess material was removed. The root sections were

kept for 24 h at a temperature of 37 �C and 95–100%

relative humidity and then immersed in demineralized

water for 24 h before measurement. After the first

capillary flow measurement at 48 h the root sections

were removed from the capillary flow porometer and

stored in demineralized water at a temperature of

37 �C. They remained under these conditions except

during the follow-up measurements that were under-

taken at 1 and 6 months.

Measurement of capillary flow

Capillary flow porometry (CFP-1200-A; PMI, Ithaca,

NY, USA) provides fully automated through pore anal-

ysis. A wetting liquid (Galwick: 15.9 Dynes cm)1, PMI)

was used to fill the pores of the sample. Because the

wetting liquid’s liquid/solid surface free energy is less

than the solid/gas surface free energy, filling of the pores

is spontaneous, but removal of the liquid from the pores is

not. In order to remove thewetting liquid from pores and

permit gas flow, pressure must be applied to the sample.

The fully wetted sections were fixed in the sample

chamber afterwhich the sample chamberwas sealed.Air

was then allowed to flow into the chamber behind the

sample.When the pressure reaches a point, it overcomes

the capillary action of the fluid within the largest pore

(maximumpore), and the sample’s bubble point pressure

is identified. After determination of the bubble point

pressure, the pressure is increased and the flow is

measured until all pores are empty, and the sample is

considered dry. At this time the smallest or minimum

pore has been identified. Themean flow pore is described

as follows: half of the flow through a dry sample is

through pores having a diameter greater than the mean

flow pore diameter. The other half of the flow is through

pores having a diameter smaller than themean flow pore

diameter. Pressure in CFP ranges from 0 to 200 psi or

1.4 MPa and the pore size range that can be measured

lies between 0.035 and 500 lm. The flow meters detect

the presence of pores by sensing the increase in flow rate

due to emptying of pores. Differential pressures and flow

rates through wet and dry samples are measured.

Application of differential pressure on excess liquid on

the sample causes liquid displacement. Measurement of

the volume of displaced liquid allows computation of

liquid permeability. The pore diameter (D) is derived from

the following equation: D = 4 c cos h/p (c = surface

tension of the wetting liquid, h = contact angle of the

wetting liquid, p = differential pressure required to

displace the wetting liquid from the pore) (Jena & Gupta

2003). All measurements were performed at VITO

(Vlaamse Instelling voor Technologisch Onderzoek or

Flemish Institute for Technological Research).


Results were analysed statistically using nonparametric

tests. Comparisons were made between the leakage

results of the different materials at 48 h, 1 and

6 months using Kruskal–Wallis tests; two by two

analyses were performed by Mann–Whitney U-tests

with Bonferroni correction.

Comparisons between the leakage results of each

material at the specified time intervals were completed

using Friedman tests and two by two comparisons were

carried out by Wilcoxon Signed Ranks tests with

Bonferroni correction. The level of significance was

set at 0.05.

Results

Measurements were obtained for each sample at each

point in time, confirming the presence of through pores

regardless of which root-end filling material was being

tested. Exact values for minimum, mean flow and

maximum pore diameters of each sample were

obtained.

The results of the study are summarized in Tables 1–

3. For reasons of completeness the range and median of

minimum, mean flow and maximum pore diameters of

gutta-percha and MTA as reported in De Bruyne et al.

(2006) are repeated in Tables 1–3.

Leakage results at 48 h, 1 and 6 months

From the Kruskal–Wallis tests and the Mann–Whitney

U-tests with Bonferroni correction the following results

were obtained. At 48 h significant differences between

the minimum (P < 0.001), mean flow (P < 0.001) and

maximum (P < 0.001) pore diameters could be dem-

onstrated.

No significant differences between gutta-percha and

MTA could be demonstrated but there were significant

differences between gutta-percha and Resilon and

between MTA and Resilon for minimum, mean flow

and maximum pore diameters. At 48 h Resilon showed

Resilon root-end fillings De Bruyne & De Moor


smaller pore diameters than gutta-percha and MTA.

The range and median of minimum, mean flow and

maximum pore diameters at 48 h are shown in

Table 1.

At 1 month there was no significant difference

between the minimum pore diameters of the different

materials, but significant differences between the mean

flow (P < 0.001) and maximum (P < 0.001) pore

diameters could be demonstrated. Concerning the

mean flow and maximum pore diameters, no signifi-

cant differences between gutta-percha and MTA could

be demonstrated, but there were significant differences

between gutta-percha and Resilon and between MTA

and Resilon. At 1 month Resilon showed smaller mean

flow and maximum pore diameters than gutta-percha

and MTA. The range and median of minimum, mean

flow and maximum pore diameters at 1 month are

shown in Table 2.

At 6 months a significant difference between the

minimum pore diameters could be demonstrated

(P < 0.05), but there were no significant differences

between the mean flow and maximum pore diameters

of the different materials. Concerning the minimum

pore diameters, there was a significant difference

between gutta-percha and Resilon. No significant

differences between gutta-percha and MTA and

between MTA and Resilon could be demonstrated.

At 6 months Resilon showed larger minimum pore

diameters than gutta-percha. The range and median of

minimum, mean flow and maximum pore diameters at

6 months are shown in Table 3.

Leakage results by material

From the Friedman tests the following results were

obtained. Concerning the minimum pore diameters

there were significant differences between the different

points in time for gutta-percha and MTA, but not for

Resilon. Results of the two by two comparisons are

summarized in Table 4. Statistically significant

Table 1 Range and median of minimum, mean flow and maximum pore diameters by root-end filling material at 48 h (lm)

Group Filling material

Minimum pore diameter

(lm)

Mean flow pore diameter

(lm)

Maximum pore diameter

(lm)

Range Median Range Median Range Median

1 GP + AH 26 0.075–0.355 0.1995 0.141–0.395 0.2630 0.177–1.714 0.4375

2 MTA 0.070–0.258 0.2210 0.183–0.925 0.2760 0.193–1.304 0.4440

3 Resilon 0.082–0.201 0.1165 0.100–0.272 0.1465 0.127–0.433 0.2140

MTA, mineral trioxide aggregate.

Table 2 Range and median of minimum, mean flow and maximum pore diameters by root-end filling material at 1 month (lm)



(lm)


(lm)


(lm)


1 GP + AH 26 0.070–0.362 0.0875 0.106–0.455 0.2730 0.128–0.896 0.4410

2 MTA 0.070–0.330 0.2010 0.152–0.393 0.2880 0.162–0.854 0.4370

3 Resilon 0.069–0.198 0.1175 0.075–0.350 0.1525 0.088–0.432 0.2265


Table 3 Range and median of minimum, mean flow and maximum pore diameters by root-end filling material at 6 months (lm)



(lm)


(lm)


(lm)


1 GP + AH 26 0.069–0.199 0.1060 0.077–0.302 0.1315 0.104–0.418 0.2200

2 MTA 0.069–0.216 0.1055 0.084–0.346 0.1490 0.111–0.818 0.2455

3 Resilon 0.083–0.240 0.1335 0.095–0.340 0.1685 0.106–0.402 0.2380




decreases in size were found between 48 h and

6 months for gutta-percha and MTA and between 1

and 6 months for MTA.

Concerning the mean flow pore diameters there were

significant differences between the different points in

time for gutta-percha and MTA but not for Resilon.

Results of the two by two comparisons are summarized

in Table 4. Statistically significant decreases in size

were found for gutta-percha and MTA between 48 h

and 6 months and between 1 and 6 months.

Concerning the maximum pore diameters there were

significant differences between the different points in

time for gutta-percha but not for MTA and Resilon.

Results of the two by two comparisons are summarized

in Table 4. Statistically significant decreases in size

were found for gutta-percha between 48 h and

6 months and between 1 and 6 months.

Discussion

Capillary flow porometry generates highly reproducible

and accurate data (Gupta & Jena 1999). Therefore,

because of its nondestructive nature and following a

previous study (De Bruyne et al. 2006) CFP was chosen

as the evaluation method for the present study. It

provides, as the first and only method in leakage

research, exact data on pore diameters which can be

compared statistically and gives an indication whether

bacteria or their metabolites will be able to pass

through the sample. This is in contrast to other

methods, which only compare materials without giving

any information on the size of pores. As such, the

method can overcome the problem of limited repro-

ducibility and comparability of conventional methods

for evaluating leakage (Wu & Wesselink 1993). CFP

uses a wetting liquid with a low surface tension such

that pores as small as 0.035 lm can be measured,

which assures the detection of gaps of about 2 lmwhich were already observed between the root dentine

and the Resilon primer. These gaps might be too small

to be detected by, for example, bacterial penetration

models (De-Deus et al. 2007).

The relatively high pressures used during CFP may

be a concern. It needs to be emphasized, however, that

during the present study and during all previous

studies none of the fillings were dislodged. Results from

a pilot study also showed that no statistically significant

differences were evaluated between measurements

when samples were measured multiple times immedi-

ately after each other (De Bruyne 2006). Apart from

this the results from push-out tests revealed that

micropush-out bond strengths of all materials tested

were higher than the pressures used in the present

study (Yan et al. 2006, Sly et al. 2007, Ureyen et al.

2008). This implies that the filling materials used in the

present study will not be damaged during CFP.

As the purpose of the study was to compare root-end

filling materials, standardized root sections were essen-

tial. Because human teeth are too small to be used to

prepare standardized samples that are easy to handle,

fix and evaluate in a reliable way, bovine teeth were

used. As bovine teeth are easy to obtain and as the

sections are large enough to adjust the central pulp

lumen to the exact diameter, standardization is straight-

forward. Consequently cavities of equal size could be

filled with different materials and compared under the

same conditions, although these differ from the clinical

situation. From the study of Nakamichi et al. (1983) it

appeared that no statistically significant difference was

found in adhesion of various materials to human or

bovine dentine. Because of the larger diameter and same

height, the C-factor in the present samples will be lower

Table 4 Summary of significant differ-

ences (marked by an asterisk) between

minimum, mean flow or maximum

pore diameters at 48 h, 1 and 6 months

and for two by two comparisons by

material (> means the pore diameter is

larger at the former than at the latter

measurement)

Root-end

filling

material

Friedman’s

test

Two by two comparisons

48 h vs.

1 month

48 h vs.

6 months

1 month vs.

6 months

GP +

AH 26

Minimum pore diameter *(P < 0.05) >

Mean flow pore diameter *(P < 0.001) > >

Maximum pore diameter *(P < 0.001) > >

MTA Minimum pore diameter *(P < 0.01) > >

Mean flow pore diameter *(P < 0.005) > >


Resilon Minimum pore diameter






than in human teeth which results in less influence

from contraction forces (Tay et al. 2005a).

As the manufacturer claims that after curing the

combination of obturation material and sealer will

create a monoblock that effectively resists leakage, it

seemed that Resilon would be a perfect root-end filling

material. It can be applied easily in the same way as

gutta-percha, and the material sets fast, which is an

advantage during surgery. Because of the similarity to

gutta-percha and because of the fact that MTA is often

considered as a standard of care, both these materials

were selected as controls.

Similar to the results of previous studies performed

with CFP on root-end fillings (De Bruyne et al. 2005),

measurements were obtained for each sample at each

time interval. The average length of bacteria varies

between 0.2 and more than 10 lm, the width between

0.2 and 1.5 lm (Hobot 2002); and their metabolites

are even smaller. Apart from this, one has to keep in

mind the fact that bacteria are not rigid structures but

can alter their outline. Therefore, in general the

maximum pore diameter and the size of bacteria and

their metabolites will be indicative of the possible

leakage along the root-end filling materials. The

minimum and mean flow pore diameters are relevant

in terms of pore size distribution. Looking at the results,

this means that some bacteria and definitely their

metabolites will be able to pass along root end fillings.

At 48 h the minimum, mean flow and maximum

pore diameters were smaller for Resilon than for gutta-

percha and MTA. At 1 month this was not the case for

the minimum pore diameter, but remained so for the

mean flow and maximum pore diameters. At 6 months

the difference for the mean flow pore and maximum

pore diameters had disappeared, whereas at this time

Resilon had larger minimum pore diameters than

gutta-percha. Looking at the tables, although not

always statistically significant, it appears that the

minimum, mean flow and maximum pore diameters

of gutta-percha and MTA diminished in the course of

time, which was not the case for Resilon. For Resilon

there was an increase. As the maximum pore diameter

will determine the eventual seal of the material, this

diameter is of major importance.

Until now improvement of the seal was seen over time

for all materials tested by CFP (De Bruyne et al. 2006);

Resilon seems to act differently. In a short-term study by

Maltezos et al. (2006), which also tested Resilon as a

root-end filling material, the bacterial leakage analysis

(4-week observation) showed that Super-EBA leaked

significantly more than Resilon and that there was no

difference between Resilon and white Pro Root MTA.

This is contrary to the present study where after

1 month Resilon still performed better than MTA. In

contrast to the above, most other studies evaluated root

fillings and not root-end fillings. In a recent study which

evaluated short-term coronal leakage, Epiphany SE

sealer and Resilon as a root filling was compared with

gutta-percha with AH 26 or AH plus sealer using dye

leakage and performed better (Bodrumlu & Tunga

2007a). Shipper et al. (2005) evaluated the prevention

of apical periodontitis in an in vivo dog model and

concluded that the Resilon ‘Monoblock’ System was

associated with less apical periodontitis than gutta-

percha with AH 26, maybe because of its superior

resistance to coronal microleakage. In other studies on

root fillings, Resilon performed better, equal or worse

than gutta-percha (Shipper et al. 2004, Aptekar &

Ginnan 2006, Biggs et al. 2006, Bodrumlu & Tunga

2006, 2007b, von Fraunhofer et al. 2006, Onay et al.

2006, Pitout et al. 2006, Sagsen et al. 2006, Shemesh

et al. 2006, 2007, Stratton et al. 2006, Tunga &

Bodrumlu 2006, Almeida et al. 2007, Baumgartner

et al. 2007, De-Deus et al. 2007, Ishimura et al. 2007,

Paque & Sirtes 2007, Raina et al. 2007, Silveira et al.

2007, Verissimo et al. 2007, Pasqualini et al. 2008),

sometimes depending on the sealer used in combination

with gutta-percha or the leakage assessment method.

Interesting in the context of root-end fillings though is

the fact that Resilon had significantly less leakage than

gutta-percha with Grossman’s cement in moist canals

in a (short-term) study (Zmener et al. 2008). In surgical

circumstances, which often are not ideal, this might be

a major benefit. On the other hand, the biodegradation

of Resilon (polycaprolactone) as mentioned by Tay et al.

(2005b), but contradicted by Trope (2006) might also

be of relevance.

Different from most studies which are short-term,

Paque & Sirtes (2007) performed a long-term study

using a fluid transportation model in which they

showed that initially there was no difference in leakage

between gutta-percha with AH Plus sealer and Resilon/

Epiphany but after 16 months gutta-percha retained its

seal whereas Resilon/Epiphany lost its sealing capacity.

The results of the present study confirmed these long-

term results. Different factors might contribute to this

loss of seal over time. Although the configuration factor

(C-factor) of the specimens in the present study was not

as high as in root canals (Tay et al. 2005a) one or more

bonded areas might pull off or debond in the course of

time. De Munck et al. (2005), in their review on the

durability of adhesion to tooth tissue, reported that



after about 3 months all adhesives exhibited mechan-

ical and morphological evidence of degradation, which

probably will also be true for Epiphany SE. Colour

discharge from the Resilon pellets, according to the

manufacturer only food grade dye ‘leaching out into

the tooth’, was seen in 11 samples at 1 month and in

13 samples at 6 months in the present study. This

might also have contributed to the increased leakage

(Shemesh et al. 2006). As it is not common during

surgery, in the present study no EDTA was used to

remove the smear layer as suggested by the manufac-

turer. Removing the smear layer might positively

influence the results.

This evolution of the seal over time of Resilon is in

contrast to the improvement of seal over time of gutta-

percha and MTA, which has already been discussed

extensively in a former study (De Bruyne et al. 2006).

Changes in these root-end fillings occurred probably at

the interface with the root dentine as the absence of

voids within the materials was confirmed earlier (De

Bruyne et al. 2005). Dimensional changes during time

(Orstavik et al. 2001) and further hydration of MTA

powder (Wu et al. 1998) are factors which might have

contributed to the improvement of their seal.

Conclusion

Irrespective of the root-end filling material, each sample

leaked along the filling material at all times. Resilon

was unable to provide a fluid-tight seal. Whereas in the

present study Resilon performed better than gutta-

percha and MTA in the short-term, the seal of these

materials improved over time whereas the seal of

Resilon deteriorated. As not all materials evolve the

same way, it is important to evaluate on the long-term

basis contrary to most studies which are short-term.

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Sealing ability, water sorption, solubilityand toothbrushing abrasion resistanceof temporary filling materials

C. M. Pieper1, C. H. Zanchi1, S. A. Rodrigues-Junior1, R. R. Moraes2, L. S. Pontes1 & M. Bueno1

1Department of Restorative Dentistry, School of Dentistry, Federal University of Pelotas, Pelotas, Brazil; and 2Department of

Restorative Dentistry, Dental Materials Area, Piracicaba Dental School, State University of Campinas, Campinas, Brazil

Abstract

Pieper CM, Zanchi CH, Rodrigues-Junior SA, Moraes

RR, Pontes LS, Bueno M. Sealing ability, water sorption,

solubility and toothbrushing abrasion resistance of temporary

filling materials. International Endodontic Journal, 42, 893–899,

2009.

Aim To evaluate marginal seal, water sorption, solu-

bility and loss of mass after brushing of several

temporary filling materials.

Methodology For marginal seal, Class I cavities,

including endodontic access preparations, were made in

human molar teeth and restored using one or other of

several temporary filling materials (n = 10): zinc oxide/

calcium sulphate-based cement (Cavit, 3M,ESPE, St.

Paul, MN, USA), zinc oxide/eugenol cement (IRM,

Dentsply Caulk,Milford, DE, USA), glass ionomer cement

(Vidrion R, SSWhite, Rio de Janeiro, RJ, Brazil) or a

dimethacrylate-based filling (Bioplic, Biodinamica,

Londrina, PR, Brazil). Dye penetration was assessed after

thermocycling and immersion in 0.5% basic fuchsine

solution. For water sorption, solubility and loss of mass

analyses, disc-shaped specimens were made. Water

sorption and solubilitywere evaluated bymass alteration

after storage in distilled water for 7 days (n = 7). Loss of

mass was calculated based on the difference of mass after

abrasion with a toothbrush (n = 5), and surfaces were

analysed by SEM. Data of water sorption, solubility and

loss of mass were submitted to anova and Tukey’s test,

and marginal sealing data to Kruskal–Wallis test

(P < 0.05).

Results Statistically significant differences were ob-

served for marginal sealing (P < 0.0001), water sorp-

tion (P < 0.01), solubility (P < 0.01) and loss of mass

(P < 0.05). Bioplic had the best marginal seal. Cavit

had the greatest water sorption and solubility. Vidrion

R and Bioplic had the lowest solubility. Loss of mass

after brushing was higher for Cavit, followed by Bioplic,

IRM and Vidrion R. Cavit and Vidrion R were worn

aggressively by brushing.

Conclusions The resin-based temporary filling Bio-

plic produced the best marginal seal, and was associ-

ated with the lowest water sorption, solubility and loss

of mass.

Keywords: loss of mass, marginal sealing, temporary

filling,toothbrushingabrasion,watersorption/solubility.

Received 13 October 2008; accepted 26 March 2009

Introduction

The outcome of root canal treatment depends, amongst

other factors, upon the sealing capacity of temporary

restorations that prevents bacterial infiltration and

recontamination of the root canal system (Torabinejad

et al. 1990, Ray & Trope 1995, Hommez et al. 2002).

Besides avoiding bacterial percolation, temporary fillings

may help to protect weakened coronal tooth tissue from

fractures when they have adhesive properties (Soares &

Goldberg 2002). Conversely, fillings that expand during

or after setting, due to hygroscopic expansion,may cause

cusp deflection or fractures (Laustsen et al. 2005).

Characteristically, restorative materials undergo deg-

radation in contact with water, such as leaching of

Correspondence: Sinval Adalberto Rodrigues-Junior, Rua

Goncalves Chaves 457, Centro, 96015-560, Pelotas, RS,

Brazil (Tel.: +55 53 3222-6690; e-mail: rodriguesjr2002@

yahoo.com.br).

doi:10.1111/j.1365-2591.2009.01590.x


components that may weaken their structure (Ferra-

cane 2006). In addition, the oral environment is

inhospitable for restorative materials, with extremes

of thermal and mechanical challenges. The mechanical

action of toothbrushing might also abrade the materi-

als (Moraes et al. 2008).

Several temporary filling materials with different

microstructures, compositions and setting mechanisms

are available commercially. Cavit (3M; ESPE, St. Paul,

MN, USA) is a premanipulated eugenol-free material

that sets in contact with moisture, but has given

conflicting marginal sealing results (Naoum & Chan-

dler 2002). Bioplic (Biodinamica, Londrina, PR, Brazil)

is a resin-based material that sets upon light-curing,

characteristically presenting volumetric shrinkage dur-

ing polymerization. This contraction, however, is

usually followed by expansion due to water sorption

(Deveaux et al. 1992), although whether this hygro-

scopic expansion is sufficient to adequately seal the

cavity is still unknown. Conventional glass–ionomer

cements (GIC) are considered suitable materials for

restorations for several reasons: they form a hard

material upon setting, present relatively little or no

exothermic reaction or shrinkage during setting, have

no free monomer in the set matrix, and adhere to tooth

structure (Culbertson 2001). Based on its adhesion

potential, it could be expected that the marginal sealing

produced by GICs is good. Naoum & Chandler (2002)

have concluded that GIC is a satisfactory endodontic

temporary filling, even in the long-term. IRM (Dentsply

Caulk, Milford, DE, USA), a zinc oxide-eugenol (ZOE)

based cement, has been associated with antibacterial

activity (Naoum & Chandler 2002). Together with

Cavit, IRM has been the most used temporary filling in

endodontics (Koagel et al. 2008), even though its

sealing capability has generated conflicting results

(Mayer & Eickholz 1997, Naoum & Chandler 2002,

Zmener et al. 2004, Koagel et al. 2008).

Discrepancies between studies still raise concerns

about the capacity of temporary filling materials with

different compositions to avoid bacterial percolation

that could lead to post-treatment disease. As these

materials have different setting mechanisms, different

reactions with moisture and variable dimensional

stability, there is a potential for them to produce

different marginal sealing abilities. In addition, few

studies have evaluated the in vitro performance of

temporary fillings. Therefore, the aim of this study was

to evaluate the marginal sealing ability, water sorption,

solubility and toothbrushing abrasion resistance of

different filling materials used as temporary restoration

in root filled teeth.

Material and method

Temporary filling materials

Four temporary filling materials with different constit-

uents and setting mechanisms were evaluated: a ZOE-

based cement (IRM; Dentsply Caulk, Milford, DE, USA),

a eugenol-free ZO cement (Cavit; 3M ESPE, St. Paul,

MN, USA), a GIC (Vidrion R; SS White, Rio de Janeiro,

RJ, Brazil), and a resin-based cement (Bioplic; Biodin-

amica, Londrina, PR, Brazil). Table 1 presents the

composition of all materials.

Marginal sealing

Forty unrestored, caries-free human first and second

molar teeth were selected under approval of the institu-

tional Ethics Committee of School of Dentistry/Federal

University of Pelotas (UFPel), Brazil (protocol no. 16/

04). All teeth were examined at 10·magnification, and

those with microcracks were excluded. The teeth were

stored in 0.2% thymol solution for 7 days, after which

the periodontal ligamentwas removedwith a razor blade

Table 1 Temporary filling materialsMaterial Composition Manufacturer Batch no.

Vidrion R Powder: aluminium silicate glass

Liquid: copolymers of polyacrylic,

itaconic and tartaric acids

SS White 6040306

Cavit Zinc oxide, calcium sulphate,

zinc sulphate

3M ESPE 215000

Bioplic Silicium dioxide, dimethacrylates,

inorganic filler

Biodinamica 632/05

IRM Powder: Zinc oxide, polymethyl

methacrylate

Liquid: Eugenol

Dentsply Caulk 679307

Sealing of temporary fillings Pieper et al.


and the teeth cleaned at low-speed with a water-pumice

slurry. They were then stored in saline at 5 �C.Class I endodontic access cavities with standardized

outline were prepared using a handpiece under water-

cooling. The coronal access to the pulp chamber started

with a cylindrical diamond bur no. 1014 (KG Sorensen,

Barueri, SP, Brazil) in enamel, and carbide burs no.

245 (SS White) in dentine. The burs were changed

after 10 preparations. The pulp cavity and the root

canals were rinsed with 1% NaOCl solution in order to

remove debris. Root canals were dried through aspira-

tion and using cotton pellets, and their entrance was

filled with gutta-percha. To standardize the cavity

depth, a periodontal probe was used to assure the

existence of at least 4 mm between the cavity outline

and the entrance of the root canals (Cruz et al. 2002).

Since unrestored, caries-free molar teeth were used, the

dentine surfaces after cavity preparation were sound.

The teeth were randomly assigned into four groups,

defined by the temporary restorative fillings (Table 1).

All materials were manipulated according to the

manufacturers’ specifications. IRM was prepared in a

6-g mL)1 powder/liquid ratio, and inserted and

adapted to the cavity walls with a dental spatula.

Vidrion R was manipulated and inserted with a Centrix

syringe. For Cavit, the cavity was left slightly moist, the

material inserted with a dental spatula and allowed to

set in contact with a moist cotton pellet. Bioplic was

inserted into the cavity, carved and light-cured for 40 s

with a quartz–tungsten–halogen light-curing unit

(Ultralux; Dabi Atlante, Ribeirao Preto, SP, Brazil –

irradiance >400 mW cm)2). The root apices were

sealed with self-cured epoxy resin (Durepox; Alba

Quımica Ind. e Com. Ltda., Sao Paulo, SP, Brazil) and

teeth were covered with two coats of nail polish, except

the restorations and a 1-mm area surrounding them.

After storage in saline for 7 days, at 37 �C, the teethwere submitted to 500 thermal cycles between 5 ± 5

and 55 ± 5 �C, with 30 s dwell time and 3 s interval

time. The teeth were then immersed in 0.5% basic

fuchsine solution for 24 h, at room temperature, and

washed for 24 h in running tap water. Sectioning was

performed bucco-lingually to the long axis of the tooth

using a diamond disc. Two previously calibrated

examiners analysed both sections using a stereomicro-

scope, at 40· magnification, recording the highest

penetration score. Dye penetration was determined

based on the following scores: 0 – no visible dye

penetration at the tooth/filling interface; 1 – dye

penetration limited to the dentine–enamel junction; 2

– dye penetration up to half of the pulp chamber; 3 –

dye penetration over half of the pulp chamber. Data

were submitted to nonparametric Kruskal–Wallis test

(P < 0.05).

Water sorption and solubility

Disc-shaped specimens (n = 7), 6 mm in diameter (D)

and 1 mm in height (h) were prepared for each

material. The GIC specimens were prepared and

allowed to set in the mould with polyester strips for

2 days, in order to avoid dehydration of the material.

All specimens were stored in a desiccator at 37 �C with

silica gel, and were weighed daily to verify mass

stabilization (dry mass, m1), which was represented by

mass variations lower than 0.1 mg in any 24 h

interval. Thereafter, the specimens were stored in

distilled water at 37 �C for 7 days to obtain the mass

after saturation with water (m2).

The specimens were then placed in the desiccator

again, at 37 �C, and reweighed again until a constant

dry mass (m3) was obtained. Weighing was performed

using an analytical balance with 0.1 mg accuracy (AG

200; Gehaka, Sao Paulo, SP, Brazil). The volume (V) of

each specimen was calculated based on the following

equation: V = pR2h, where R is the specimen radius.

Water sorption and solubility, given in lg mm)3, were

calculated as follows: WS = m2 ) m3/V; SL = m1 ) m3/

V. Data were submitted to One-Way Analysis of

Variance and Tukey’s test (P < 0.05).

Toothbrushing abrasion and loss of mass

Five disc-shaped specimens were prepared for each

material following the same procedures previously

described. The specimens were ultrasonically cleaned

(MaxiClean 750; Unique, Indaiatuba, SP, Brazil) in

distilled water for 10 min and dry-stored at 37 �C for

stabilization of specimen mass. The pre-brushing mass

(m1) was obtained by weighing the specimens every

24 h until a constant mass was achieved. The abrasion

test was carried out in a multi-station brushing device.

Each sample was brushed in a different station, using a

soft nylon-bristled toothbrush with a brush-head load

of 200 g. During the brushing cycle, the specimens

were completely immersed in slurry of dentifrice

(Colgate Total, Sao Bernardo do Campo, SP, Brazil)

and distilled water (1 : 2 wt ratio). In total, 5000

strokes (forward and reverse movement) were per-

formed with a frequency of 4 Hz at 37 �C.After testing, the specimens were cleaned with a air/

water spray for 1 min and in a ultrasonic bath for

Pieper et al. Sealing of temporary fillings


10 min. They were then dry-stored at 37 �C to

constant mass (m2). Mass loss, expressed in mg, was

calculated by the difference between m2 and m1. Data

were submitted to One-Way Analysis of Variance and

Tukey’s test (P < 0.05). Representative specimens for

each material before and after brushing were gold-

sputter coated (Denton Vacuum Desk II; Denton

Vacuum, Moorestown, NJ, USA) for observation with

scanning electron microscopy (SEM). Imaging of the

surfaces was performed in secondary electron mode

(JSM-5600LV; Jeol Inc., Peabody, MA, USA) at accel-

erating voltage of 15 kV.

Results

Marginal sealing

Results are shown in Fig. 1. The Kruskal–Wallis test

revealed statistically significant differences between

groups (P < 0.0001). Bioplic produced the best mar-

ginal seal (all specimens with score 0), followed by

Cavit. Vidrion R presented intermediate results, whilst

IRM resulted in the poorest marginal seal (9 out of 10

specimens presenting score 3).

Water sorption and solubility

Results are shown in Fig. 2. Significant differences

occurred between materials for water sorption

(P < 0.01) and solubility (P < 0.01). Both parameters

were significantly higher for Cavit. IRM and Vidrion R

presented similar intermediate values for water sorp-

tion, whilst Bioplic had the lowest values. Significantly

lower solubility was observed for Vidrion R and Bioplic

compared with the other materials (P < 0.05).

Toothbrushing abrasion and loss of mass

Results of loss of mass after toothbrushing are shown in

Fig. 3.One specimen of Vidrion R fractured during the

brushing cycling and was replaced. Significant differ-

ences were observed between materials (P < 0.05).

Loss of mass after brushing was significantly higher for

Cavit (P < 0.05). Bioplic had intermediate loss of mass

values, similar to IRM and to Vidrion R, which had the

lowest loss of mass of all groups. SEM micrographs of

the control and brushed surfaces are shown in Fig 4.

Before abrasion, a relatively smooth surface was

observed for all groups, especially for Bioplic and

IRM. After toothbrushing, all materials had character-

istic worn surfaces, with Cavit and Vidrion R showing

an aggressive wear pattern characterized by extensive

loss of substance for Cavit, and deep grooved scratches

Figure 1 Marginal leakage observed for the different tempo-

rary filling materials. Distinct letters indicate statistical differ-

ences amongst materials (P < 0.05).

Figure 2 Results for water sorption and solubility. Distinct

letters indicate statistical differences amongst materials

(P < 0.05).

Figure 3 Mass loss (mg) of the temporary filling materials

after toothbrushing abrasion. Distinct letters indicate statisti-

cal differences amongst materials (P < 0.05).



for Vidrion R. Bioplic had the least altered surface after

abrasion.

Discussion

The sealing ability of temporary fillings can be

evaluated in several ways (Cruz et al. 2002, Naoum

& Chandler 2002, Balto et al. 2005, Sauaia et al.

2006). According to Raskin et al. (2001), lack of

standardization of the test methods compromises

comparisons and, therefore, the reliability of marginal

sealing results. Methodological aspects of the test

used in the study, namely basic fuchsine as leakage

tracer, the thermocycling protocol and the assess-

ment of dye penetration through sections of the

specimen, have been reported as the most frequent

choices in marginal sealing evaluations (Raskin et al.

2001). In this sense, the test protocol employed

allows comparisons with similar studies, besides being

a rapid way to determine the sealing ability of the

materials used.

Conventional GICs adhere to tooth tissue as a result

of a chelation reaction with calcium (Culbertson 2001).

Therefore, one could expect dye penetration in enamel

to be lower than in dentine, since the former has more

calcium available. However, 9 out of 10 specimens of

Vidrion R group had dye penetration up to the enamel–

dentine junction, which might reflect the effect of the

thermocycling on the interaction between GIC and

enamel and their different coefficients of thermal

expansion. The bond strength of conventional GIC to

tooth tissue is difficult to evaluate, due to the extremely

brittle nature of the cement, which leads to cohesive

failure within the material (Mount 1991). Thus, one

could hypothesize that the tracer percolated through

fracture lines within the cement, close to the tooth/

restoration interface.

Bioplic, a dimethacrylate-based temporary filling,

prevented dye penetration in all the specimens. This

material has the advantage of not requiring etching of

the dental surface or application of an intermediate

bonding material, thus eliminating additional clinical

steps. According to the manufacturer’s information,

Bioplic tends to expand in contact with moisture,

improving its adaptation to the cavity walls. The light-

curing characteristic of Bioplic seems to be an important

factor on its sealing ability, as the contact with the wet

environment occurs after polymerization. In a previous

study, Jenkins et al. (2006) observed considerably high-

er marginal sealing ability for a resin-based light-cured

material in comparison with conventional self-curing

cements and other temporary fillings. Moreover, the

translucency of Bioplic allows the passage of the curing

light through the material, requiring a single light

activation step, even with layers thicker than 2 mm.

The eugenol-free ZO cement Cavit sets in contact

with moisture, and has produced conflicting sealing

results (Uranga et al. 1999, Jenkins et al. 2006, Sauaia

et al. 2006). The hygroscopic properties result in

expansion of the material, potentially sealing the

tooth/filling interface (Cruz et al. 2002, Sauaia et al.

2006), and might explain the absence of interfacial dye

penetration in 70% of the samples. The presence of dye,

though, was observed in the material itself, confirming

BP IR

Before

After

CA VD

Figure 4 Representative SEM micrographs of the temporary filling materials before and after toothbrushing abrasion. A relatively

smooth surface was observed for all groups before abrasion, especially for BP and IR. After toothbrushing, all materials presented

characteristics of worn surfaces, with CA and VD showing an aggressive pattern of wear, characterized by extensive loss of

substance for CA, and deep grooved scratches for VD. BP showed the least altered surface after abrasion.



previous findings (Cruz et al. 2002) and indicating the

possibility of recontamination of the canal by bacterial

infiltration through the material itself.

The sealing ability of the eugenol-based ZOE cement

(IRM) was poor, confirming previous reports (Deveaux

et al. 1999, Balto et al. 2005). Extensive degradation

was observed, with the presence of dye within the body

of the material (Zmener et al. 2004). Studies have

pointed out that stress, such as the one imposed by

thermocycling, promotes a significant degradation of

IRM (Gilles et al. 1975), whilst others indicate that

variations in volume resulting from contraction of the

material and the inhomogeneous mixing process could

partially explain the poor sealing results with this filling

(Deveaux et al. 1999). In addition, it has been reported

that ZOE-based cements may impair the polymerization

of resin composites, and should be avoided when final

restorations of such materials are to be made (Naoum &

Chandler 2002). In contrast, temporary fillings such as

Bioplic and Vidrion R are compatible with resin-based

materials, and theoretically do not need to be com-

pletely removed to execute the final restoration.

Water sorption and solubility were calculated by

weight differences of specimens, and were used as a

measure of the degradation of the fillings (Carvalho

Junior et al. 2003, Ferracane 2006) (Fig. 2). Carvalho

Junior et al. (2003), also determined the sealing ability

of temporary fillings, and recommend that water

sorption and solubility should be minimal. Usually,

the absorption of water precedes events such as

volumetric changes, swelling and softening of the

materials (Ferracane 2006), which may compromise

their microstructure and, as a consequence, the seal

produced by the restoration.

Water uptake is a key factor in the setting mecha-

nism of Cavit. The expansion caused by the water

diffusion is responsible for the sealing of the tooth/

restoration interface, but also allows the swelling of

components from the spaces occupied by water (Ferra-

cane 2006), explaining the high solubility observed for

this material (Fig. 2). The intermediate sorption results

observed with IRM and Vidrion R reflect the cement

nature of these materials, which characteristically

absorb water. IRM had greater solubility than Vidrion

R, confirming the previously reported disintegration

this cement undergoes in contact with moisture. This

process was explained by Wilson & Batchelor (1970) as

eugenol loss of the cement matrix by aqueous leaching,

resulting in microstructural degradation and reduction

of mechanical strength. It is important to highlight that

Vidrion R specimens were dehydrated in order to reach

the first dry mass. Although this procedure does not

mimic the in vivo situation, it is inherent to the test and

might have caused appreciable structural modifications

in the GIC that might have influenced the results.

Resin-based materials have different patterns of

water uptake, depending upon the chemical structure

of the resin (Sideridou et al. 2007), which involves the

hydrophilic nature of the monomers and differences

between the solubility parameter of the monomers and

the solvent (Ferracane 2006). In addition, the cross-

link density of the polymer network is also important,

since it dictates the presence and the amount of

pendant molecules that could be swelled following

water uptake (Ferracane 2006). In this sense, light-

cured materials, such as Bioplic, justify their low water

sorption and solubility by being able to set prior to

contact with moisture.

Brushing simulation was used in the present study to

test the surface wear and degradation of the fillings

under cyclic mechanical challenge (Moraes et al.

2008). The eugenol-free ZO cement underwent consid-

erable disintegration after brushing, as shown by the

substantial loss of mass and the rough surface pattern

observed (Figs 3 and 4). SEM images of the GIC Vidrion

(Fig. 4) also depicted aspects of aggressive wear in the

surface of the cement. Nevertheless, Vidrion had the

lowest mass loss, indicating that the brushing action

might affect only the surface of the material. SEM

images of Bioplic and IRM (Fig. 4) revealed smoother

surfaces after brushing, which indicate a more homo-

geneous wear pattern.

Conclusions

The resin-based light-cured temporary filling material

Bioplic produced the best marginal sealing and was

associated with the lowest water sorption, solubility

and loss of mass in comparison with all other materials.

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A comparative study of image quality and radiationexposure for dental radiographs produced using acharge-coupled device and a phosphor plate system

S. L. Farrier, N. A. Drage, R. G. Newcombe, S. J. Hayes & P. M. H. DummerSchool of Dentistry, Cardiff University, Wales, UK

Abstract

Farrier SL, Drage NA, Newcombe RG, Hayes SJ,

Dummer PMH. A comparative study of image quality and

radiation exposure for dental radiographs produced using a

charge-coupled device and a phosphor plate system. Interna-

tional Endodontic Journal, 42, 900–907, 2009.

Aim To investigate the quality of periapical radio-

graphic images produced by two digital dental radiog-

raphy systems, a charge-coupled device (CCD) and a

photostimulable phosphor (PSP) image plate system,

and to examine the overall radiation exposure when

using these systems in a clinical setting.

Methodology Patients were randomly allocated to

both systems and the resultant radiographs rated for

quality. The expected radiation exposure for an inves-

tigation was calculated.

Results Overall, 98 images were acquired using the

CCD system and 108 with the PSP system. The PSP

system produced significantly higher quality

(P < 0.001) periapical images compared with the

CCD system. The CCD system required significantly

more (P < 0.001) repeat exposures to obtain a diag-

nostic image than the PSP system but at a lower

expected radiation exposure.

Conclusions The image quality was superior using

the phosphor plate system. Although more repeat

radiographs were required using the CCD system, the

images were produced with a lower expected radiation

exposure.

Keywords: dental digital radiography, radiation

doses.

Received 3 February 2009; accepted 1 April 2009

Introduction

Digital radiography is increasingly being used in

clinical practice. Two common systems employed use

either a charge-coupled device based sensor (CCD) or a

photostimulable phosphor (PSP) imaging plate system.

The literature is replete with studies, conducted

ex vivo, comparing the quality of image between CCD

and PSP systems for diagnosis of a specific pathological

condition, either naturally occurring or mechanically

formed (Lim et al. 1996, Borg & Grondahl 1996a, Borg

et al. 2000, Boscolo et al. 2001, de Almeida et al.

2003). Subsequently, various advantages and disad-

vantages of both CCD and PSP systems have been

suggested but results tend to show both systems

comparable in terms of image quality, with neither

significantly superior (Wenzel & Borg 1995, Kang et al.

1996, Velders et al. 1996, Borg et al. 1997, 1998,

Cederberg et al. 1998, Versteeg et al. 1998, Syriopoulos

et al. 2000). However, within the clinical environment

there are many variables that may influence the quality

of the image obtained. Few studies have examined

the effectiveness of either system for diagnostic pur-

poses in vivo (Morner-Svalling et al. 2003) .

It is well documented that the optimum individual

exposure using the CCD system requires a lower

radiation exposure than the PSP systems (van der Stelt

2005), but this does not take into account any repeat

exposures that may be necessary. The aims of the study

were therefore to investigate whether there were any

differences in image quality and radiation exposure

Correspondence: Nicholas Drage, Department of Dental Radi-

ology, University Dental Hospital, Cardiff, CF14 4XY, UK

(Tel.: 029 20766483; fax: 029 20743605; e-mail: nicholas.

[email protected]).

doi:10.1111/j.1365-2591.2009.01593.x


between a CCD and a PSP digital system for periapical

radiography.

Material and methods

The study was approved by the Cardiff & Vale NHS

Trust Research and Development Committee (reference

number 04-DH-3089), and South East Wales Local

Research Ethics Committee. Adult patients, referred to

the Radiology Department at the University Dental

Hospital, Cardiff, UK and requiring periapical radio-

graphs of at least one individual tooth, were recruited

into the study. Written, informed consent was obtained

from each patient, by the principal investigator (SF),

prior to the radiograph being exposed.

Two digital radiography systems were compared: the

Sidexis CCD system (Sirona Dental Systems GmbH,

Bensheim, Germany), and the Vistascan PSP system

(Durr Dental GmbH, Bissingen, Germany). The resolu-

tion for Sidexis CCD system is measured at

<10lp mm)1. For the Vistascan the high resolution

setting was chosen which corresponds to a measured

resolution of 8 lp mm)1 (horizontal) and 10 lp mm)1

(vertical) The sensor sizes used were 31 mm · 41 mm

and 22 mm · 35 mm for the Vistascan PSP system.

For the Sidexis CCD system, the universal sensor which

measured 25.4 mm · 36.8 mm · 6.6 mm (11 mm

over cable insert) and the full size sensor which

measured 29.9 mm · 40.1 mm · 6.8 mm (11.2 mm

over the cable insert) were used. The active area of the

Sidexis CCD system is 26 mm · 34 mm for the full size

sensor and 20 mm · 30 mm for the universal sensor.

Sample size

The primary outcome was identified as image quality

assessment rated on a 3-point scale as described later.

Assuming 70% excellent, 20% satisfactory and 10%

unsatisfactory are the quality assessment scores for one

system, and 50% excellent, 20% satisfactory and 30%

unsatisfactory are the scores for the other, a sample size

of 120 (60 per system) gives a power of 80% to detect

this difference. The intention was to increase the total

sample size to 240 (120 per system) in order to detect a

difference of the order of 15% with a power of 80%. It

was planned to use each system uniformly across six

areas of the dentition, namely incisors and canines,

premolars and molars, in both the maxillary and

mandibular arches. Thus, 40 radiographs were to be

used in each of the six regions, 20 allocated to each

system according to a predetermined concealed ran-

domization scheme. The chief investigator was blind to

the system allocation until it was disclosed in the

clinical setting. Some patients had requests for more

than one tooth to be radiographed. If this was the case,

the same digital system was used for all exposures, but

only one radiograph formed part of this particular

study. This was chosen by the principal investigator,

before meeting the patient and taken first.

Radiological process

Each radiograph was taken by the principal investiga-

tor using the paralleling technique, using an appropri-

ate sensor holder and beam aiming device. The

manufacturers’ instructions regarding exposure factors

were followed for all examinations (Table 1).

If the resultant image was deemed nondiagnostic by

the principal investigator, a repeat exposure was

carried out immediately using the same system. In

the situation that a patient could not tolerate the sensor

and holder, or a repeated intraoral digital image was

again assessed undiagnostic, a conventional film based

intraoral radiograph or an extraoral radiograph was

used to obtain the necessary information. These addi-

tional images were not evaluated in the study.

Evaluation of images

The principal investigator evaluated all images imme-

diately after the exposure on a Fujitsu Siemens com-

puter monitor (Hansol Electronics Inc., Jinchon-Kun

Table 1 Exposure factors for the digital

systemsRegion mA kV

Time of exposure (seconds)

CCD, Sidexis PSP, Vistascan

Maxilla Incisors and canines 7 60 0.05 0.12

Premolars 7 60 0.06 0.16

Molars 7 60 0.06 0.25

Mandible Incisors and canines 7 60 0.05 0.12

Premolars 7 60 0.06 0.16

Molars 7 60 0.08 0.25

CCD, charge-coupled device; PSP, photostimulable storage phosphor.

Farrier et al. Subjective quality assessment of digital intraoral radiographs


Choongbuk, Korea). Each image was assessed in a

systematic fashion, within its own software and

enhanced if necessary, and assigned a Quality Score

(1–3), based on National Radiological Protection Board

(NRPB 2001) guidelines (Table 2).

To assess inter- and intra-observer variability, the set

of images was reviewed and any with particularly

memorable features were excluded. Then 60 images

were selected using a stratified random sampling

scheme. Three observers, the principal investigator,

an experienced endodontic specialist and a maxillofa-

cial radiologist assigned these a Quality Score (1–3),

using the same guidelines and viewing conditions. The

specialist endodontist and radiologist were also allowed

to enhance the images if necessary. Images were

selected equally from both systems, and from all areas

of the mouth. Should images chosen be associated with

a repeat exposure, this second image was also graded in

the same manner. Each observer analysed the images

in the same order and no knowledge of the previous

quality score was available.

Radiation exposure

The observed probability of requiring a repeat radio-

graph and the standard exposure times given for the

different regions for the two systems were considered.

From this both the overall average exposure time and

radiation exposure (surface entrance doses) were cal-

culated, with confidence intervals.


Chi-squared tests were used to compare radiographic

quality scores between the two systems. A 1 degree of

freedom v2 was used for the binary variable indicating

whether a repeat was required, and a 1 df trend

component is reported for analyses relating to the

3-point ordinal radiographic quality score. Confidence

intervals (CIs) for differences between proportions were

calculated using method 10 of Newcombe (Newcombe

1998), and CIs for weighted means of proportions

analogously.

Inter- and intra-observer agreement was assessed

using quadratic weighted kappa for the 3-point scale

(Fleiss & Cohen 1973). For the binary decision as to

whether repeat radiography should be performed,

Scott’s pi was used (Scott 1955, Newcombe 1996),

with CIs calculated by the method of Donner & Eliasziw

(1992).

Proportions of radiographs rated as excellent, accept-

able and unacceptable were compared to NRPB targets

using upper and lower tail probabilities based on

summation of trinomial probabilities generalizing

P-values (Newcombe & Farrier 2008).

Results

Sample

In total, 209 patients were included in the study; 108

subjects were male and 101 female, with ages ranging

from 17 to 90 years. Table 3 shows the number of

radiographs from each of the six areas of the mouth

allocated to each system. Ideally 240 images should

have been obtained. However, it was not possible to

collect the planned numbers of mandibular incisors and

premolars in the time available for the study. In total,

206 images from 206 different patients were assessed

as three patients could not tolerate the digital sensor.

Quality of the original periapical radiographs

Table 3 shows the quality scores assigned to the 206

images obtained by the principal investigator. A 1 df

trend component chi-square indicates a highly signifi-

cant preference for the PSP system (v2 = 26.3,

P < 0.001), with a very large difference commensurate

with what was initially assumed in the power calcula-

tion. Clinically the most relevant dichotomization was

obtained by combining categories 1 and 2, (excellent and

clinically acceptable), in contrast to category 3 (unac-

ceptable, undiagnostic). The proportion of images judged

excellent or acceptable was 94% for PSP and 78% for

CCD. The estimated difference in the proportions unac-

ceptable is 17% with 95% CI from 8% to 27%.

Table 2 Three-point scale for assessment of radiograph quality

Rating Quality Basis

1 Excellent No errors of patient preparation, exposure, positioning, processing or handling

2 Diagnostically acceptable Some errors of patient preparation, exposure, positioning, processing or handling,

but which do not detract from the diagnostic utility of the radiograph

3 Unacceptable Errors of patient preparation, exposure, positioning, processing or handling, which

render the radiograph diagnostically unacceptable

Subjective quality assessment of digital intraoral radiographs Farrier et al.


Repeat required for clinical purposes

Repeat exposures were actually performed for 27 (27%)

of 98 radiographs using the CCD system and 8 (7%) of

108 radiographs using the PSP system. This was

because the clinician in charge of the patients’ care

deemed some of the images unacceptable when SF

deemed them acceptable and vice versa. Nevertheless,

this 20% difference (95% CI 10% to 30%, v2 = 14.8,

P < 0.001) was closely in line with the results shown

in Table 3.

Quality of the repeat periapical radiographs

Analyses for the quality scores for the repeat radio-

graphs and whether a further repeat exposure was

required were restricted to 34 patients (Table 4). The

results of this showed a similar pattern to the original

radiographs but statistical significance was not reached

due to the very small sample size.

Quality scores in relation to area of dentition

Table 3 shows the quality scores of the original

periapical radiograph as graded by the principal inves-

tigator with regard to the area of the dentition. There

were marked differences (>20%) between the two

systems in favour of the PSP system, for all areas of

the dentition. The greatest differences were for maxil-

lary premolars and molars, and mandibular molars.

The observed overall difference in repeat rates

between the CCD and PSP systems needed slight

adjustment to allow for the imbalance in numbers of

radiographs taken in each area of the dentition. In the

maxillary arch the required samples of 20 incisors, 20

premolars and 20 molars were studied. However, in the

mandibular arch, this was not the case and fewer

subjects used the CCD system than the PSP system.

Therefore, a ‘balanced scorecard’, consisting of equal

numbers of radiographs in all areas of the dentition was

used to provide a more accurate representation. The

projected proportion requiring a repeat periapical

radiograph for the CCD system is 27% and for the

PSP system 6.8%, a difference of 20.2% (95% CI 10–

30%, v2 = 14.8, P < 0.001). It is therefore reasonable

to say that the difference between the two systems is

not substantially related to tooth type.

Inter- and intra-observer variability

Weighted kappa for the 3 point scale of quality of the

original images ranged from 0.46–0.72 for inter-

Table 3 Quality scores for the two radiography systems. These show the numbers of radiographs taken for each area of the

dentition, and the quality scores of the original periapical radiographs as assessed by the principal investigator

Radiography system Area of dentition

Number of

radiographs

Quality score

Excellent Acceptable Unacceptable

CCD Maxillary incisors 20 6 (30%) 13 (65%) 1 (50%)

Maxillary premolars 20 7 (35%) 11 (55%) 2 (10%)

Maxillary molars 20 8 (40%) 6 (30%) 6 (30%)

Mandibular incisors 7 3 (43%) 4 (57%) 0

Mandibular premolars 13 6 (46%) 2 (15%) 5 (38%)

Mandibular molars 18 6 (33%) 4 (22%) 8 (44%)

Total 98 36 (37%) 40 (41%) 22 (22%)

PSP Maxillary incisors 20 11 (55%) 7 (35%) 2 (10%)

Maxillary premolars 20 18 (90%) 2 (10%) 0

Maxillary molars 20 14 (70%) 5 (25%) 1 (5%)

Mandibular incisors 10 7 (70%) 3 (30%) 0

Mandibular premolars 18 11 (61%) 6 (33%) 1 (6%)

Mandibular molars 20 16 (80%) 2 (10%) 2 (10%)

Total 108 77 (71%) 25 (23%) 6 (6%)

CCD, charge-coupled device; PSP, photostimulable phosphor.

Table 4 Quality scores of repeat radiographs

Quality score

Radiography system

CCD PSP

Excellent 5 (20%) 5 (63%)

Acceptable 14 (54%) 2 (25%)

Unacceptable 7 (27%) 1 (13%)

Total 26 8

CCD, charge-coupled device; PSP, photostimulable phos-

phor.



observer agreement and was 0.98 for intra-observer

agreement (SF), with broadly similar results for the

repeat radiographs.

For the binary outcome of whether a repeat radio-

graph was judged necessary, Scott’s pi ranged from

0.60 (95% CI 0.30–0.80) to 0.87 (95% CI 0.59–0.96)

for inter-observer agreement and was 1.0 (95% CI

0.76–1.0) for intra-observer agreement.

Performance targets

The 3 point quality scores for each system were

compared to the minimum target for radiographic

quality (‡70% excellent, £10% unsatisfactory) sug-

gested by the National Radiological Protection Board

(NRPB 2001). It is clear that the results obtained for

the CCD system fell short of these targets. Conversely,

the results obtained for the PSP system, with 71%

excellent and only 6% unsatisfactory, were ostensibly

slightly better than the ultimate target performance for

quality.

For the PSP system, the probability of observing

performance as good as, or better than, the observed

proportions of 71% excellent and 6% unacceptable,

assuming that in the underlying population the method

yields exactly 70% excellent and 10% unacceptable

quality of images, may be calculated as a summation of

multinomial probabilities. For these results, an upper

tail probability U = 0.058 was obtained, which repre-

sents the probability that results as good as or better

than those observed would arise if the true population

proportions were 70% excellent, 20% acceptable and

10% unacceptable. A corresponding lower tail proba-

bility of L = 0.64 was obtained, representing the

probability of results no better than those observed.

Both U and L use a mid-P accumulation of tail

probabilities (Lancaster 1949). A high value of L with

a low value of U indicates that the observed results

surpass the standard, whereas a high U and a low L

indicate results that fall short of the standard.

For the CCD system, U > 0.99 and L < 0.0001

which indicated strongly that the performance of this

system fell short of the target.

Radiation exposure

The physical characteristics of the X-ray machine used

(Siemens Heliodent DS, Bensheim, Germany) were

measured by the radiation physicist as part of the

annual radiation safety survey. The 60 kV machine

gave a dose rate at the end of the spacer cone of

5.98 mGy/s. Using this value an entrance doses for the

examinations was derived.

Based on a ‘balanced scorecard’ and disregarding

repeat exposures, the mean time of exposure of the CCD

system was 0.06 s. The expected additional exposure

due to the 27.6% risk of requiring a repeat exposure,

based on all areas of the dentition is 0.0171 s, with a

95% confidence interval of 0.0131–0.0232 s. Taking

account of the original periapical radiograph and a

single repeat if required, the average time for the CCD

system was 0.0771 s with a 95% CI from 0.0731 to

0.0832. This translated into an expected 0.46 mGy

radiation entrance dose, with a 95% CI of 0.44 to

0.50 mGy.

Similarly, for the PSP system the mean exposure time

averaged across the dentition was 0.1767 s. The

expected additional exposure due to the » 7.4% risk

of requiring a repeat was 0.0122 s, with a 95% CI from

0.0077 to 0.0274. Thus, taking into account the

original periapical radiograph and a single repeat if

required, the average exposure time with the PSP

system was 0.1889 s, with a 95% CI from 0.1844 to

0.2041. This translated into a 1.13 mGy expected

radiation surface dose, with a 95% CI of 1.10–

1.22 mGy.

Therefore, the difference in average exposure time

between the CCD and PSP systems based on the

balanced scorecard and taking into account the

original radiograph and repeat radiograph if required,

was estimated to be 0.1889–0.0771 = 0.1118 s, in

favour of the CCD system. The 95% confidence interval

for this difference is from 0.1042–0.1275 s. This

translated into a 0.67 mGy difference in expected

radiation surface dose with a confidence interval of

0.62–0.76 mGy.

Discussion

In this study comparing CCD and PSP systems in the

clinical environment, patients were allocated randomly

to a digital system and stratification methods used so

that each area of the dentition was uniformly exam-

ined. The principal investigator, a qualified dentist, was

responsible for exposing and assessing all the radio-

graphs and was blind to the system allocation until it

was disclosed in the clinical setting to ensure a more

fair comparison.

The subjective image quality rating score as des-

cribed by the NRPB was chosen since it is based on the

diagnostic potential of the image produced (NRPB

2001). This rating system is recommended when



conducting audits based on the quality of radiographs

and is thus familiar and clinically relevant. Since this is

an established grading system used in the UK and by its

very nature is subjective no calibration of the investi-

gators was performed. Each investigator was familiar

with the grading scheme before the study commenced

and deemed whether the radiographs were diagnosti-

cally acceptable independently.

Various studies have been carried out which suggest

that digital equipment may improve the quality of

the image, especially if the contrast and density are

not optimum (Wenzel 1993, Gotfredsen et al. 1996,

Yoshiura et al. 1999, Svanaes et al. 2000, Li 2004).

Therefore, manipulation of the digital images was

allowed by all observers within the appropriate system

software, since this made the study more reminiscent of

the clinical environment. The same monitor was used

to view both types of image so that confounding screen

factors were not introduced. Each image was analysed

within its own software and it is possible that this could

lead to observer bias. An alternative approach would

have been to export the digital images to one common

viewing environment, as was done by Berkhout et al.

(2004), this would have made the observers blind to

the identity of the imaging systems. However, a more

realistic assessment was desired by enabling the

observers to manipulate the image within their own

software so as to simulate a true clinical situation. The

active areas of the CCD sensor and PSP imaging plates

are also different and thus masking the borders would

also have been required for all bias to be eliminated.

This would have resulted in some of the image from the

PSP systems being absent from view, since it has a

larger active pixel area and thus some vital radio-

graphic data could be prevented from being viewed and

this would have had a direct impact on the relevant

quality score.

The overall quality of the PSP system was found to be

significantly better than the quality of the images

produced with the CCD system. This agrees with Borg &

Grondahl (1996b) and Boscolo et al. (2001), but is the

opposite to that reported by de Almeida et al. (2003),

however, these studies were ex vivo and involved

imaging dried specimens with soft tissue equivalents

where control of radiographic positioning is more

consistent and reproducible. In addition, these studies

did not use the NRPB system for grading image quality.

There was a 20% difference between the two systems

in favour of the PSP digital system as to whether a

repeat image was required (95% CI 10–30%,

P < 0.001). This is potentially a very important benefit

since such a difference could have a marked influence

on the patient dose received, the ease at which the

process is carried out by both the patient and clinician

and the time taken for a successful radiological

examination.

Suggested explanations for the improved quality of

the PSP images and the less frequent need for a repeat

exposure can be sensibly combined. The CCD sensor is

far more bulky and stiff than the PSP imaging plates

and has a cable attached; it is also associated with a

larger sensor holder and beam aiming device. The CCD

sensor was found to be more difficult to position than

the PSP imaging plates. Patients susceptible to gagging

also found the CCD more difficult to tolerate than the

PSP sensor.

The active pixel area of the two sensors also differs,

which may be a contributing factor to the image

quality. The larger active image size of the PSP imaging

plates enables more information to be captured and a

greater probability that all the relevant information

required is actually recorded.

Difficulties in positioning CCD sensors have been

reported before (Wenzel & Moystad 2001, Berkhout

et al. 2002). In two surveys of dental practitioners,

there were significant problems with the positioning of

CCD sensors with an increase in the number of CCD

images taken compared with PSP systems (Wenzel &

Moystad 2001, Berkhout et al. 2002). In addition,

when compared to conventional film it has been shown

that there is an increase in the number of unsatisfactory

images with the CCD systems (Berkhout et al. 2003).

The ‘balanced scorecard’ allowed for the imbalance

in numbers throughout the areas of the dentition and

between the two digital systems. This gives a projected

difference of 20.2% between the CCD and PSP systems

in favour of the PSP system regarding whether or not a

repeat radiograph is required.

The CCD system did not reach the suggested NRPB

quality targets. The PSP system performed slightly

better than the ultimate targets. These performance

targets do not take into consideration the radiation

dose received by patients in order to eventually obtain

the correct diagnostic information.

The mean exposure time and radiation surface dose

for the PSP is greater than that for the CCD system by a

factor of 2.45. Therefore, despite the CCD system

requiring more repeat exposures, the radiation received

by the patient is less. In a questionnaire based survey

comparing CCD and PSP systems, CCD systems showed

a larger dose reduction in comparison to PSP imaging

plates (Berkhout et al. 2004). However, the authors



also raised concerns that the radiation reduction may

be less than originally perceived as more CCD exposures

were carried out than PSP exposures. In the context of

the present study, it might have been possible to reduce

the mean exposure time without affecting the quality

for the PSP system, since the sensor has a very wide

exposure latitude; this is an area for further research. In

this study, if conventional radiographs or extraoral

radiographs were required because the patient could

not tolerate the sensor or there had been two failures

using the digital sensor the additional radiation expo-

sure was not included in the calculations.

Another study reported that the dose reduction as a

result of shorter exposure times exceeded the increase

in doses as a result of the greater number of radio-

graphs with both digital systems (Berkhout et al.

2003). However, with the CCD sensors the dose

reduction per exposure was almost cancelled out by

the increase in the number of radiographs taken. These

results are very different to the findings of the present

study.

Conclusion

The PSP Vistascan system produced significantly high-

er quality intraoral periapical images compared with

the CCD Sidexis system. The CCD system did not reach

the set performance targets of ‡70% excellent and

£10% unsatisfactory. There was also a significantly

higher repeat rate using the CCD system compared to

the PSP system. The mean exposure time and radiation

exposure for the PSP system is greater than for the CCD

system.

Acknowledgement

The authors are grateful to Paul Beere and his staff in

the dental radiology department for their help in this

research project.

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Microflora in teeth associated with apicalperiodontitis: a methodological observationalstudy comparing two protocols and threemicroscopy techniques

N. Richardson, N. J. Mordan, J. A. P. Figueiredo, Y-L. Ng & K. GulabivalaUnits of Endodontology & Microscopy, Divisions of Restorative Dental Science and Biomaterials & Tissue Engineering, UCL

Eastman Dental Institute, University College London, London, UK

Abstract

Richardson N, Mordan NJ, Figueiredo JAP, Ng Y-L,

Gulabivala K. Microflora in teeth associated with apical

periodontitis: a methodological observational study comparing

two protocols and three microscopy techniques. International

Endodontic Journal, 42, 908–921, 2009.

Aim The aim of this study was to compare two

protocols to examine bacterial colonization in teeth

associated with chronic apical periodontitis with acute

episodes (ap), using light microscopy (LM), transmis-

sion electron microscopy (TEM) and scanning electron

microscopy (SEM).

Methodology Nine root samples (seven teeth) were

processed using either Eastman Dental Institute (EDI)

(n = 4 teeth/4 roots) or Zurich (n = 3 teeth/5 roots)

protocols. The roots were sectioned longitudinally; one

root portion was viewed with SEM, descriptively

dividing its length into apical, middle and coronal;

semi-thin and ultra-thin transverse sections were

viewed under LM and TEM from each third of the

other root portion. Each root was therefore examined

using all microscopy techniques. Observations of bac-

terial presence, description and distribution within the

root canal lumen and root dentine were systematically

recorded using pre-determined criteria.

Results The Zurich technique gave a more predict-

able division of the root, but the surface was slightly

smeared and demineralization was incomplete. The

Eastman Dental Institute (EDI) approach appeared to

provide better ultrastructural detail. Bacteria were

detected in eight of the nine roots. Bacterial biofilms

were commonly seen adhering to the root canal

surface, containing various cellular morphotypes: rods,

cocci, filaments and spirochaetes. Bacteria were more

evident apically than coronally, associated with the

canal wall but were more commonly evident coronally

than apically within the dentinal tubules. Polymorphs

(PMNs) were found in all the root thirds, especially

apically, often numerous and walling off the bacterial

biofilm from the remaining canal lumen.

Conclusions Both protocols had merits and de-mer-

its. The combination of microscopy techniques offered

complementary views of intra-radicular bacterial colo-

nization. The perception of confinement of the host/

microbial interface at the apical foramen is not entirely

correct; PMNs may be found even in the coronal third

of root canals containing necrotic pulp tissue.

Keywords: intra-radicular bacteria, microscopy, pro-

tocols, root canal.

Received 12 August 2008; accepted 25 March 2009

Introduction

The role of a polymicrobial infection of the root canal

system in apical periodontitis is well established

(Kakehashi et al. 1965, Sundqvist 1976, Fabricius

et al. 1982, Tani-Ishii et al. 1994) but deep insights

into the ecology and physiology of the bacterial

Correspondence: Professor Kishor Gulabivala, Unit of

Endodontology, UCL Eastman Dental Institute, 256 Grays

Inn Road, London WC1X 8LD, UK (Tel.: 020 7915 1033; fax:

020 7915 2371; e-mail: [email protected]).

doi:10.1111/j.1365-2591.2009.01594.x


colonization remain elusive. Much of the current

knowledge of intra-radicular infection stems from

in vivo and ex vivo culture studies of sampled bacteria;

such approaches tend to bias the revealed micro-flora

(Akpata 1976, Kumar et al. 2002). The picture of

bacterial diversity is influenced by many factors,

including growth conditions, sub-culture strategy and

the nature of bacterial identification (Rolph et al. 2001,

Kumar et al. 2002, Munson et al. 2002, Gulabivala

2004). The number of detected and identified taxa per

tooth has increased from 1 to 12 cultured varieties up

to 20 phylotypes using culture-independent tech-

niques, with estimates of actual numbers up to 90

(Rolph et al. 2001, Munson et al. 2002). Whilst, the

known diversity of the microflora has increased with

improved culture techniques and culture-independent

techniques, direct microscopy suggests, as indeed it did

even in the time of Miller (1894) that a proportion of

the flora still remains uncultured. Furthermore, the

process of sampling disturbs insights about the intimate

and intricate relationships between bacteria and their

abiotic environment (Nair 1987).

Microscopically, bacterial strains are evident as cocci,

rods, filamentous or spiral morphotypes and have been

shown in a landmark paper to exist mainly in a biofilm

lining the root canal wall in the root apex (Nair 1987).

This paper provided the first real insight into the

morphological distribution of the root canal flora in the

root apex and its association with the host response.

Study of the excellent photo-micrographs provides

visual evidence to support the predicted ecological

and physical spatial relationships between bacteria

(Sundqvist 1992).

Different microscopy techniques possess different

properties and propensities to reveal the inherent

‘truth’ about the bacterial distribution and its struc-

ture. Light microscopy (LM) remains a useful base-line

technique to provide an overall perspective but lacks

resolution to reveal finer details. In contrast, trans-

mission electron microscopy (TEM) possesses the high

resolution to reveal ultra-structural details, losing

something of the perspective as a trade-off. Hence,

Nair used the approach he described as correlative LM

and electron microscopy studies to decipher both

aspects. Although not using the term ‘biofilm’, he

provided the first real detailed description of the root

canal biofilm within root apices, as it related to the

aetio-pathogenesis of apical periodontitis. The struc-

ture of the microflora within the entire tooth, as it

relates to approaches to treatment has been little

studied.

The validity of the observations made by microscopy

rests on the assumption that the processing stages have

accurately preserved the anatomical structures and

that the imaging system possesses the means and

resolution to highlight the relevant features. Knowl-

edge of imaging principles is essential but empirical

studies are also necessary to reveal the true in situ

potential of microscopy techniques. Distortion of tissues

and translocation of structural components are possible

but need to be minimized or else recognized as artefacts.

Detection of such artefacts may not be straightforward

but is an important element in the critical appraisal of

findings. To this end, the nature of sample fixation and

processing may also influence results.

The aim of this methodological observational study

was to compare different tooth processing protocols and

microscopy (LM, SEM and TEM) techniques to examine

bacterial colonization within the coronal, middle and

apical thirds of roots associated with apical periodon-

titis.


Sample collection and storage

The material for this study consisted of extracted

human teeth with radiographically evident periapical

lesions (and associated acute episodes) and an absence

of periodontal disease or previous pulpal therapy. The

teeth were carefully extracted by General Dental

Practitioners with minimal pumping motion (Kapalas

et al. 2001, 2002) and immersed into tubes containing

3% glutaraldehyde (Agar Scientific, Stanstead, UK) in

0.1 mol L)1 sodium cacodylate (Agar Scientific) after

de-coronation with a sterile diamond bur. The sample

teeth were stored at 4 �C to provide a total fixation

period of 1 week. Informed consent had been obtained

from the patients prior to inclusion in the study pool;

seven teeth meeting the above criteria were selected for

the study.

Processing for microscopy

Two methods of sample processing were used: (i) the

EDI protocol (Vrahopoulos 1989), which involved

demineralization after embedding; and (ii) the Zurich

protocol (Nair 1987), which involved demineralization

before embedding. The seven selected teeth were ran-

domly assigned to the two processing groups; EDI

protocol (n = 4 teeth/4 roots with apical periodontitis)

and Nair protocol (n = 3 teeth/5 roots/4 roots with

Richardson et al. In situ microscopy of endodontic microflora


apical periodontitis). An overview of the key stages in

the two processing protocols is shown in Fig. 1.

EDI protocol

Longitudinal splitting of the roots

The roots were grooved longitudinally using an ultra-

fine diamond disc (Metrodent, Huddersfield, UK) along

the narrowest surface of the root in a fume cupboard

(Labcaire, Clevedon, UK). The root was then firmly

pressed into unset lab putty (Optosil� and Xantopren�;

Heraeus Kulzer, Hanau, Germany) and allowed to set.

Splitting of the root was completed using an osteotome,

exposing the pulp canal space in both sections. The

section containing more hard tissue was used for the

LM and TEM examination, whilst the other half was

used for SEM examination.

Processing for SEM

The root halves allocated for SEM examination were

dehydrated in a graded series of alcohol (20%, 50%,

70%, 90% and 3· 100% for 10 min each), placed in

hexamethyldisilazane (HMDS) (TAAB Laboratories Ltd,

Reading, UK) for 5 min, then removed and left on filter

paper for 2–3 h for the HMDS to evaporate. The

samples were attached to aluminium SEM stubs (Agar

Scientific) using carbon conducting cement (Neubauer

Chemikalien, Munster, Germany) and sputter-coated

with gold/palladium in a Polaron E5000 Sputter

Coater (Quorum Technologies Ltd, Newhaven, UK).

Zurich protocolEDI protocol

Tooth split longitudinallywith an osteotome

Dehydrated

DehydratedSEMprocessing

SEMprocessing

Embeddedin resin

Embeddedin resin

Cut into 1 mmslices & placed

in EDTA

Ultra-thinsectioningfor TEM

Ultra-thinsectioningfor TEM

Semi-thinsectioning

for LM

Semi-thinsectioning

for LM

4 months later, teeth cutlongitudinally with razor blade

Re-dehydrated& re-embedded

Placed in EDTA

Cut into 4sections

Fixed in 3% glutaraldehydein 0.1% sodium cacodylate

Toothcollection

Apical

Coronal

Middle1 mm

Figure 1 Flow chart showing the succession of stages for each processing protocol.

In situ microscopy of endodontic microflora Richardson et al.


The specimens were viewed in a Cambridge Stereoscan

90B (LEO Electron Microscopy Ltd, Cambridge, UK)

operating at 15 kV and digital images were captured

using i-scan2000 software (ISSGroup,Manchester, UK).

Processing for LM and TEM

The root halves allocated for LM and TEM were

dehydrated in a graded series of alcohol (20%, 50%,

70% and 3· 90% for 10 min each) and infiltrated with

LR White resin (The London Resin Company, London,

UK). This was performed in stages as follows: initial

immersion in LR White resin and 90% alcohol (ratio of

1 : 1) for 2 h at 4 �C; immersion in pure fresh LR

White for 30 min at 4 �C; immersion in fresh LR White

overnight (10–12 h) at 4 �C and the following morn-

ing, for 1 h, at 4 �C. The sections were embedded in

tinfoil containers (Buyrite UK Ltd, Aldershot, UK)

containing 20 mL of LR White and 30 lL LR White

accelerator. Air was excluded from the setting process

using parafilm (Agar Scientific) over the exposed resin

mix, which was polymerized for 1 h in the freezer, then

overnight at 4 �C and then removed to warm up to

room temperature.

The embedded roots were sliced transversely using a

high-speed diamond saw (Exact, Aberdeen, UK) into 1-

mm thick sections. The slices were decalcified in

0.15 mol L)1 EDTA in specimen tubes for 3–8 weeks

at room temperature on a tissue rotator at 2 rpm

(TAAB, Rotator type N; Agar Scientific). The EDTA

solution was changed every 2–3 days until the dentine

could be easily cut with a single edge carbon steel razor

blade (Agar Scientific). The slices were dehydrated

again and re-embedded in LR White resin as described

above.

Sectioning for LM

Semi-thin sections of 0.5 and 1 lm were cut with a

Diatome (Diatome AG, Biel, Switzerland) diamond knife

on an ultramicrotome (Reichert UltracutE; Cambridge

Instruments, Cambridge, UK). These were stained with

toluidine blue and used to check sample orientation

before proceeding with LM and TEM. Slides were

viewed on an Olympus BX50 optical microscope

(Olympus, Southall, UK).

Sectioning for TEM

Ultra-thin sections (90–100 nm) were cut using the

same technique, and collected on either carbon-form-

var coated copper 200 mesh grids (Agar Scientific) or

gold 400 mesh grids (Agar Scientific). The sections

were then stained on the grid with 0.4% (w/v) uranyl

acetate in absolute alcohol for 5 min, followed by

5 min in Reynold’s (1963) lead citrate. Sections were

examined on a TEM (100CXII; JEOL, Welwyn Garden

City, UK) operating at 80 kV and images were recorded

onto Kodak 4 EM film (TAAB Laboratories Ltd).

Zurich protocol

Demineralization

The roots were placed in 0.15 mol L)1 EDTA and

0.5% glutaraldehyde (Agar Scientific) in specimen

tubes on a tissue rotator at 2 rpm. Initially, the EDTA

solution was replaced every 2–3 days over 3 months,

and then changed everyday for the remaining

1 month. Progress was checked by carefully inserting

a single-edged carbon steel razor blade (Agar Scien-

tific) into the dentine, taking care not to penetrate to

the root canal.

Longitudinal cutting of the roots

After approximately 4 months in EDTA, the roots were

demineralized sufficiently to allow, gentle, controlled,

longitudinal cutting of the roots. At this point, one-half

of each root was randomly designated for SEM and the

other half for LM and TEM.

The root associated with the periapical lesion was

used from each tooth, except for tooth R6, a molar,

from which all three roots were used for comparison,

although only two were radiographically associated

with periapical lesions (R6 a, b – Table 1).

The root halves designated for SEM were dehydrated

to 100% ethanol, immersed in HMDS and allowed to

dry as for the EDI protocol, handling with greater care

because of the demineralization. Those samples due for

TEM examination were dehydrated to 90% ethanol and

embedded in LR White resin in the same manner as the

initial part of the EDI protocol without the necessity for

demineralization and re-embedding.

Selection of fields of view for both protocols

Scanning electron microscopy

The entire root half was first examined under low

magnification. Then, starting coronally, the root was

examined horizontally millimeter by millimeter, using

the lbar on the image as a guide. At each millimeter

level, the site of examination was magnified to ·5000.This horizontal scanning was repeated at the next

adjacent apical level until the entire root canal had

been traversed. Observations were made on this basis

and representative photographic images were recorded



or when bacterial colonization patterns worthy of note

were discerned.

Light microscopy

For the EDI protocol, 1-lm sections were cut from the

most coronal, middle and most apical slices of the root.

The Zurich protocol involved cutting the whole embed-

ded root into four equal corono-apical portions and

then 1-lm thick sections were cut from the coronal,

apical sections and from either of the two middle

sections (Fig. 1). Stained sections were examined to

verify presence of the canal in the section; upon

confirmation 5–7 sections of either 0.5 or 1 lm were

cut and examined at ·200, ·400 and ·1000 (oil

immersion) magnifications. Representative photo-

graphs were taken at both low and high magnification

for maintaining perspective and obtaining the highest

resolution.

Transmission electron microscopy

The LM findings informed the further sectioning for

TEM for both protocols. Two sections were cut and

examined from the same sites as for the LM sections for

each third of the root. The sections were initially

examined at the lowest magnification for perspective

before zooming in at higher magnifications. Photo-

graphic images were recorded at a number of magni-

fications to illustrate findings.

Comparison between EDI and Zurich protocols

The EDI and Zurich protocols were subjectively com-

pared using the following measures:

1 Ease of processing. This was judged by the ability to

split or section the root in a controlled manner to view

the root canal and its contents, as well as the time

taken for complete processing of the roots;

Table 1 Summary of viewable fields for each protocol (EDI/Zurich) and the presence/absence of bacteria by microscopy technique,

tooth, root and segment

Protocol

Root

no.

Root

portion

Periapical lesion

visible on

radiograph

SEM LM TEM

Lumen

Dentinal

tubules Lumen

Dentinal

tubules Lumen

Dentinal

tubules

EDI

protocol

R1 Coronal 4 · · · · · ·Middle 4 · · · · · ·Apical 4 o o · · · ·

R2 Coronal 4 4 · o o o o

Middle 4 4 4 o o o o

Apical 4 o o 4 · 4 ·R3 Coronal 4 · · o o o o

Middle 4 4 · 4 · – –

Apical 4 · · o o o o

R4 Coronal 4 4 · 4 4 4 4

Middle 4 4 · 4 4 4 4

Apical 4 4 4 4 · 4 ·Zurich

protocol

R5 Coronal 4 4 4 4 4 4 4

Middle 4 4 · 4 4 4 4

Apical 4 4 · 4 · 4 ·R6A Coronal 4 4 · 4 4 4 4

Middle 4 4 · 4 4 – –

Apical 4 4 · 4 · 4 ·R6B Coronal 4 · · 4 4 4 4

Middle 4 · · 4 4 – –

Apical 4 4 · 4 · - –

R6C Coronal · o o 4 4 4 4

Middle · · · 4 4 – –

Apical · · · · · · ·R7 Coronal 4 4 · 4 4 4 4

Middle 4 4 · 4 4 – –

Apical 4 4 · 4 4 4 4

4, bacteria detected; ·, Bacteria not detected; –, insufficient demineralization; o, canal not visible.

R6A, root; from tooth 6; root A.



2 Accuracy of findings. Note was made of actual or

apparent artefacts, distortion or evident bacterial

translocation.

Analysis of findings

Observational data were collected as systematically as

possible to build a coherent picture of the intra-

radicular infection, in particular highlighting any

common, surprising or unusual findings. An attempt

was made to record presence or absence and density of

bacteria as objectively as possible to enable comparison.

Simple descriptive statistics were used to analyse the

findings.

Results

Comparison of processing protocols

The principal difference between the processing for the

two protocols was the length of time to progress from

unfixed sample to SEM/TEM examination. The Zurich

protocol was several weeks longer than the EDI

protocol because of longer decalcification times. How-

ever, once demineralization was complete, the Zurich

protocol allowed more controlled and accurate bisect-

ing of the root, than the less predictable root splitting

required for the EDI protocol. Table 1 summarizes the

viewable fields for each protocol (EDI/Zurich) and the

presence/absence of bacteria by microscopy technique,

tooth, root and segment.

Comparison of techniques by SEM

From each root, one-half was prepared for SEM, four

from the EDI protocol and five from the Zurich

protocol. The tooth structure and root canal contents

observed in samples processed using the two protocols

were similar (Fig. 2) although it was noted that in

some of the Zurich samples the dentine surface had a

‘smeared’ appearance (Fig. 3). As a result of the more

accurate dividing of the root with the Zurich protocol,

there were more root portions, 14 of 15, in which the

root canal was visible as opposed to 10 of 12 with the

EDI protocol. In both of the EDI samples without a

visible canal, this occurred in the important apical

portion.

Translocation of root canal contents as a result of

processing was sometimes observed with both proto-

cols. On the cut (Zurich – Fig. 3) or fractured (EDI –

Fig. 6) dentine surface, this could be clearly discerned

as superficial cells and debris (Fig. 3) but within the

canal this was less easy to identify.

Bacterial cell morphology (rods, cocci and filaments)

was easily distinguished with SEM (Fig. 4), but only at

the sample surface, and the presence of a thick extra-

cellular matrix masked underlying bacteria. It was,

however, possible to discern the relative thickness of

D

D

CC

1 mm

Figure 2 R5 (Zurich protocol) SEM low magnification LS root

showing dentine (D), the root canal and cellular material (CC)

(lbar represents 1 mm).

D

60 μm

Figure 3 R5 (Zurich protocol) SEM showing the smeared

dentine and some translocated RBCs (arrows) (l bar represents

60 lm).



the bacterial layer in some instances where a fortuitous

cut through the thickness of the biofilm revealed the

inner topography (Fig. 5). The appearance of the

bacterial biofilm within the canal seemed similar for

both protocols and the relationship between the bac-

terial biofilm and the canal anatomy was clear (Fig. 5).

The SEM examination detected bacteria within the

canal in seven of nine roots, and 16 of 27 root portions.

In only three roots were bacteria observed in the

dentine tubules, two from the EDI protocol (Fig. 6) and

one from the Zurich protocol, although the slight

smearing of the dentine made examination more

difficult.

Comparison of techniques by LM

Light microscopy provided the best overall perspective

of the root canal, enabling larger areas to be observed

at low magnification (Fig. 7). There was little difference

between the two protocols in terms of the type of

information gained from the samples, providing details

of the structure and distribution of bacterial biofilms

and cells, and also an indication of the bacterial

morphology, although care should always be taken

interpreting cross-sections of cells. It was evident from

the LM observation of all three portions from root R1

that this was, in fact, a vital pulp.

However, a difference between the protocols was

noted, a consequence of the splitting of the roots with

the EDI protocol, in which the whole canal was within

the SEM portion and therefore no canal could be found

in the LM samples. In all the Zurich samples, the lumen

was present in the LM sections whereas in two of the

roots processed by the EDI method there was no visible

lumen in two of the three portions. The dentine tubules

were easily visible in all the LM sections and, in 12 of

23 portions, were observed to contain bacteria, even

30 μm

Figure 4 R4 (EDI protocol) SEM middle section showing

bacteria morphotypes, filaments (F) and cocci (arrows)

(l bar represents 30 lm).

D

B

L

60 μm

Figure 5 R5 (Zurich protocol) SEM apical section through

dentine (D) and biofilm (B) within the canal lumen (L) (l bar

represents 60 lm).

10 μm

Figure 6 R4 (EDI protocol) SEM apical section showing

bacteria (arrows) within the dentine tubules (l bar represents

10 lm).



when the bacterial film was sparse (Fig. 8). In 10 of

these, bacteria were found in the LM sections where

SEM had not found them, and in one, the opposite

occurred.

In some samples, it was observed that polymorphs

(PMNs) and some RBCs formed a layer several cells

thick over the bacterial biofilm (Fig. 9). This was

observed in two teeth and was most prominent in the

apical segments but less so in the middle and coronal

segments. In one root, a second bacterial biofilm

(although less dense) could be observed on the luminal

aspect of the PMN layer (Fig. 9), thus a layer of PMNs

was sandwiched between two bacterial biofilms.

Comparison of technique by TEM

As the same samples were used for both the semi-thick

LM and ultra-thin TEM sections, the reported absence

of a root canal in both was due to inadequate

demineralization. However, in five of the 15 root

portions processed by the Zurich technique, although

the dentine was demineralized sufficiently for the LM

sectioning, it was insufficient for the ultra-thin section-

ing and therefore these were not viewed by TEM. In

four of the five cases, this was a middle portion of the

root (Table 1).

In most cases, TEM provided similar information to

LM except that TEM conferred the considerable advan-

tage over the other techniques in the detail of visual

information available on the cells and bacteria. The

TEM of the biofilm in Figs 10 and 11 showed the close

arrangement and morphology of the cells, including

spirochaetes. Furthermore, the PMNs in this Zurich

processed sample (Fig. 11) appeared to be ‘leached’ of

100 μm

B

Figure 7 R5 (Zurich protocol) light microscopy (LM) apical

section showing the overall view. The canal wall has a thick

biofilm (B) with the luminal part containing some poorly

visible amorphous substance (l bar represents 100 lm).

10 μm

Figure 8 R4 (EDI protocol) LM middle section showing

bacteria (arrows) within the dentine tubules (l bar represents

10 lm).

30 μmm

D

I

B

B

Figure 9 R5 (Zurich protocol) LM apical section showing

bacterial biofilm (B) adherent to the canal surface and walled

in by PMNs and RBCs (I) beyond which there is a further

biofilm (l bar represents 30 lm).



cytoplasmic contents, whereas in EDI samples the

immune cells appeared healthy (Fig. 12).

When LM detected bacteria in the dentinal tubules,

this was confirmed by TEM, except for those samples

that were not sufficiently demineralized. In many of

these samples, there was an apparent attachment of

some bacteria to the collagen (Fig. 13) that must have

been present before demineralization and may indicate

exposed or available collagen epitopes within the

canal.

Summary of observations

The Zurich technique allowed examination of the root

canal in most SEM samples, all LM sections but only

half of the TEM sections. In contrast, for the EDI

technique, most of the canals were visible in the SEM,

but only three-quarters could be used for LM and TEM.

Generally, the correlation between LM and TEM was

good but SEM provided rather different information.

When bacteria were detected in the canal using LM or

TEM, their presence was not always found in the SEM

samples, although this may reflect the use of different

halves of the root canal for each type of technique.

D

Figure 10 R5 (Zurich protocol) transmission electron

microscopy (TEM) apical section showing bacterial biofilm

(B) extending from the canal surface with palisading of the

bacterial cells. The initial attachment to the canal dentine

wall (D) appears to be due to filamentous morphotypes with

coccal forms further out towards the canal lumen (TEM

·5000).

Figure 11 R5 (Zurich protocol) TEM apical section showing

the layer of PMNs (¤) and RBCs (*) covering the bofilm. Note

the loss of cellular contents from the PMNs (TEM ·2700).

C

Figure 13 R4 (EDI protocol) TEM middle section showing the

apparent attachment of a bacterium to the collagen fibres (C)

(TEM ·40 000).

Figure 12 R2 (EDI protocol) TEM coronal section a healthy

inflammatory cell, probably a lymphocyte, within the canal

lumen (TEM ·6700).



Bacteria were detected in eight of the nine roots

examined, including root R6C, apparently not associ-

ated with a periapical lesion, but bacteria were not

found in root R1, which although positive for presence

of periapical lesion, was found to be a vital pulp. The

pooled data from all microscopy techniques (Table 1)

showed that in the bacteria-positive teeth, bacteria

were detected in the canal lumens in all the root

segments except 3 (R3 coronal and apical; R6c apical).

In contrast, they were less frequently detected in the

dentinal tubules, especially in the apical portions, and

detection was more accurate by LM and TEM tech-

niques.

The pattern of bacterial distribution, both in the

canal lumen and on the canal walls, varied enormously

both from root to root and within each root. Contin-

uous biofilms were only evident in teeth with grossly

carious exposures and continuous communication

with the oral environment. The structure, thickness

and morphotypic composition varied considerably. The

bacterial biofilm was mainly evident on the canal wall

with interspersed bacterial aggregates in what seemed

to be residual necrotic pulp tissue. Sometimes bacterial

cells seemed to be present in the canal lumen in

apparent isolation (perhaps planktonic forms). In teeth

with intact pulp chambers, the canal lumen appeared

empty but was filled with some amorphous material

(Fig. 7). In some teeth, the relative abundance of

detectable bacteria was greater coronally, usually

associated with carious crowns. Rarely did the middle

portion have the greater abundance but, in teeth with

intact pulp chambers, the relative abundance of bac-

teria was greater in the apical segments. Each tooth

seemed to have its own variation of infection pattern.

The patterns of colonization of the dentinal tubules

appeared to follow relatively more predictable but

nevertheless variable behaviour. Dentinal tubules usu-

ally appeared to be colonized as a continuation of the

canal wall infection, although the diversity of morpho-

types were more restricted. Other instances showed

variable colonization of adjacent tubules sometimes

with highly dense colonization of the tubules in the

predentine with reducing density of colonization fur-

ther away from the canal lumen into the dentine.

The observation regarding the presence and close

association of PMNs and sometimes RBCs with bac-

terial aggregates and films was not consistent. It was

more prominent in some teeth than others and, where

present, was always observed in the apical portions

and frequently in the middle and coronal portions. In

one tooth, a second bacterial biofilm, although less

dense, was observed on the luminal aspect of the PMN

layer, implying that this was not a result of tooth

preparation.

Discussion

The prime purpose of this study was to evaluate the

utility of different microscopy techniques and protocols

to gain visual insights into the presence, distribution

and structure of bacterial colonization in teeth associ-

ated with apical periodontitis, regardless of the clinical

condition of the tooth; the intention was to use a wide

selection of tooth conditions meeting selection criteria

to evaluate the breadth of morphotypic bacterial

diversity. Many studies on the microflora of infected

roots have used teeth with gross caries, presumably

because of their easier availability (Nair 1987, Baum-

gartner & Falkler 1991, Sen et al. 1995). An additional

clinical parameter in this study was the acute presen-

tation of the selected teeth. A subsidiary but important

aim was to compare two tooth processing protocols.

The importance of this aspect is that the validity of

microscopy observations rest on accurate preservation

of the in situ anatomical structures and relationships.

Distortion of tissues or translocation of structural

components may obscure the ‘truth’, and they cor-

rectly need to be recognized as artefacts. The problem

for the observer is to be able to distinguish real from

artefact without a ‘positive control’. To be able to do so

requires a good appreciation of what is to be expected

based on understanding of biology, familiarity with the

technical aspects of the procedures and critical inter-

pretation. Purely morphological studies are able to give

morphological insight but cannot enable dissection of

the relationships and roles of bacterial species and their

interaction with host cells. Such insights may only be

obtained in the future through in situ labelling studies,

which require the preservation or exposure of target

cell surface, structural or chemical elements (Lam et al.

2000, Tan et al. 2000); the preserving protocols

therefore become important. The purpose of this study

was not to explore the effect of protocols on such cell

surface targets but to evaluate the effect of such

protocols on normal structural viewing, in the first

instance. Studies on in situ hybridization will be

reported separately. Another key factor is that each

microscopy technique requires an independent section;

the same section may not be viewed by all techniques.

Absolute comparison between microscopy techniques is

therefore impossible and relative comparison reliant on

viewing adjacent sections that are thin enough to



represent, more-or-less, the same structures. This was

more easily possible for LM/TEM views than for SEM,

because, of necessity, the opposite halves were viewed

and these could theoretically have different bacterial

colonization, particularly in teeth with intact pulp

chambers.

Some key features of difference between the protocols

bear discussion. Previous microscopy studies have split

their test teeth using a technique similar to the EDI

protocol (Lin & Langeland 1981, Molven et al. 1991,

Sen et al. 1995) but none commented on its inherent

problem of unpredictability; a feature mostly reduced

by practicing on spare rather than sample teeth.

Furthermore, the lack of comment may reflect that

the studies could select from both halves, whereas in

the present study, the portion with the larger canal

component was reserved for LM and TEM, theoreti-

cally compromising that used for SEM. Numerous

approaches have been used to split teeth and alter-

native methods have been reported (Rapp 1985) but

without tested consensus. The Zurich protocol was

favoured for its more predictable cutting of the demin-

eralized root compared with the splitting of mineralized

tissues in the EDI protocol. The predictable cuts in the

Zurich protocol were, however, associated with an

apparently smeared appearance of the dentine, in

contrast to the rougher fractured surface produced by

the EDI protocol (Fig. 3 vs. Fig. 6); the significance of

this feature is unknown although it may affect an

appraisal of dentine tubule content. It should be added

that such a feature was absent in the published

material from the Zurich laboratory and could be a

feature of adaptation in another laboratory.

A further putative advantage of the Zurich protocol

is that the natural morphological relationships and

conjunction of the structures would be less likely to be

disturbed. In contrast, in the EDI protocol, the various

washings of the pre-split and open canal surface prior

to sputter-coating could potentially result in transloca-

tion of ‘loose’ structures such as planktonic bacteria or

pulp debris (Nair 1987, and personal communication)

(Fig. 3). Translocated debris and bacteria are some-

times evident in publications using the SEM (Sen et al.

1995). In general though, the SEM views did not seem

much distorted or different between the protocols in

this study. Furthermore, the enmeshed and matted

appearance of the bacterial biofilm was confirmed by

the different microscopy techniques and appeared to

suggest that at least this feature of the bacterial

colonization remained preserved regardless of the

protocol used.

The careful and slow approach used by Nair (1987)

to demineralize the test specimens is laudable and is

most likely to yield accurate images representing the

‘truth’, nevertheless, within the time constraints

imposed in this study, the slower process resulted in

several middle root segments remaining un-demineral-

ized and therefore un-viewable (Table 1). The counter-

argument against the Zurich protocol was that the

more aggressive demineralization, albeit slower, and

associated long fixation periods, may damage surface

antigens (Hobot & Newman 1991) and probe targets

for in situ hybridization (Binder 1992).

It would be intuitively expected that each microscopy

technique with its own unique characteristics would

yield different perspectives on the objects under scru-

tiny; each hopefully yielding unique accuracy in some

way so that they together complement findings to build

a more accurate overview. The findings of this study

confirm these expectations and potential, whilst at the

same time highlighting the advantages and disadvan-

tages of each microscopy technique.

The SEM, with its propensity for revealing surface

topography was generally useful for deciphering detail

over the entire canal surface, whilst retaining contex-

tual perspective at lower magnifications; this also

enabled the proportion of the surface colonized to be

estimated. The technique was also useful for describing

cell morphotypes but by the same token, surface

coverage with cells or extra-cellular matrices precluded

revelatory insight into biofilm structure and relation-

ships.

The LM provided an excellent overview of the

collective bacterial colonization and its variation from

site to site within the selected section, particularly on

the canal wall. Its main limitation is the level of

magnification and resolution necessary to determine

inter-cellular and cellular-abiotic relationships. Fur-

thermore, morphotypic differentiation was relatively

gross and lacked discriminatory detail.

Transmission electron microscopy was the most

discriminating technique for providing fine detail of

the microflora and its relationship to adjacent struc-

tures, as well as cell-to-cell contacts. Furthermore, the

internal cellular morphology was also most clearly seen

by TEM.

It is evident that correlative studies using LM and

TEM provide the best conjunction, as reported by Nair

(1987). Furthermore, the combination with SEM pro-

vides further insights but the processing required is

different from that for LM and TEM and may be more

prone to distortion of surface detail.



The pictures of bacterial quantity and density were

broadly comparable between the microscopy tech-

niques (Table 1), confirming the utility of using adja-

cent serial sections for LM and TEM. However,

differences were sometimes apparent, both between

sections, and by microscopy technique, the latter

mainly because of SEM sections, which would by

definition have viewed geographically different loca-

tions from those viewed by LM or TEM.

Bacteria were not detected in one root apparently

associated with a periapical lesion, otherwise, a variety

of morphotypes were found in all canal segments

consisting of cocci, rods, filaments, spiral forms and

yeasts. The existence of a periapical lesion associated

with an inflamed but vital pulp is not a novel finding.

The present study found that of all the roots which

could be examined fully, only one had fewer bacteria

apically than coronally. In the other roots (including

those with intact pulp chambers), there was a transi-

tion from the coronal segment to the apical segment, of

greater relative bacterial abundance apically. This

contrasts with other work (Shovelton 1964), where

the sample was also made up of both open and closed

pulp systems. There was a lack of consistency in the

middle root segments, where some roots had fewer

bacteria than either the coronal or apical segments and

others where the bacterial abundance formed a con-

tinuous transition from coronal to apical. The distri-

bution could potentially be explained by abundance of

nutritive sources coronally and apically. A carious

exposure may allow seepage of salivary components

from the coronal aspect, forming a diffusion gradient

towards the apex. Once the bacteria are established

apically, the stimulation of inflammation apically may

then play a part in deriving nutrition from the

inflammatory serum exudate (Khot et al. 2004). The

relative scarcity of bacteria in the middle segment could

be explained by its farthest location from opposing

sources of nutrition (coronal or apical); it being the

lowest point on two opposing gradients. The evidence

of dividing bacterial cells in the middle segments

suggests the presence of sufficient nutrients in this part

of the canal at some point. In some species, such as

staphylococci, divided cells may remain joined for some

time after division.

The patterns of bacterial distribution in the canal

lumen and on canal walls varied. Some teeth had

discontinuous biofilms together with variable density

and layers of cells, whilst others had thick continuous,

dense biofilm layers. The structure, thickness and

morphotypic composition also varied considerably but

the species diversity of the flora may only be speculated

upon without in situ hybridization. Some niches in the

root canal seemed apparently more suited to biofilm

growth than others, although the main bacterial colo-

nization seemed to be on the canal walls; that within the

lumen, in the middle and coronal segments, seemed

more scattered. It is not known whether these bacteria,

apparently ‘floating’ without attachment represent

planktonic phenotypes or are biofilm phenotypes

attached to a ‘surface’ of degrading tissue that is invisible

in the chosen microscopy technique. Each tooth seemed

to have its own variation of infection, corroborating the

findings of various culture and culture-independent

studies (Sundqvist 1976, Rolph et al. 2001, Munson

et al. 2002). The impression in some teeth was that,

indeed this was a nutrient-depleted environment but in

others, the canal system appeared to be nutrient-rich

with active bacterial growth and propagation. It is

possible that acute apical symptoms may be due to such

rapid and proliferative bacterial growth rather than

because of specific species. Associations between species

and acute symptoms although often made, have not

proved fruitful, because the presence of the same species

can be confirmed in asymptomatic teeth. The answers

may lie in strain variation.

Yeast cells were detected in 3/7 (43%) teeth in this

study, a value that fits within the range previously

reported: by microscopy, 8–40% (Molven et al. 1991,

Sen et al. 1995); by culture, 5–55% (Slack 1975, Egan

et al. 2002); and by molecular detection, 21% (Baum-

gartner et al. 2000). Yeasts have been implicated in

failed cases, raising the suggestion that reduction of

bacteria during treatment may allow yeasts to over-

grow and predominate in the low-nutrient environ-

ment (Sundqvist 1992).

Bacterial invasion of dentinal tubules was predom-

inantly seen in the coronal and middle root segments;

in contrast Sen et al. (1995) reported dentinal tubule

invasion in the middle and apical root segments. The

presence of bacteria on inter-tubular dentine casts

some doubt on the SEM findings. The findings in the

apical root segments are consistent with reports of

fewer dentinal tubules in this region (Mjor et al. 2001).

Dentinal colonization was heaviest in the pre-dentine

and mainly confined towards the canal lumen end of

tubules than the cementum; in agreement with some

(Shovelton 1964, Nair 1987) but contradicting others

(Peters et al. 2001). The finding of apparent bacterial

association or attachment to dentine collagen (Fig. 13)

would appear to be in situ confirmation of the sugges-

tion previously made by Love & Jenkinson (2002).



Another interesting finding in the present study was

the presence of PMNs in all thirds of the roots with

necrotic pulps; the finding was particularly surprising

in the coronal segments but would be consistent with

pus exudation into the canal. Nair (1987) had previ-

ously reported PMNs amongst bacteria in the apical

sections of roots associated with apical periodontitis,

but these were described as isolated wandering cells.

Their presence was explained by virtue of chemotactic

signals from intra-canal bacteria. The extensive pres-

ence of PMNs in the root canals of teeth associated with

apical periodontitis (with acute episodes), apparently

strategically attempting to ‘wall off’ the bacterial

biofilm adherent to the canal wall was unexpected

and unique in the endodontic published literature. The

observation which was consistent between LM/TEM

techniques and different teeth and roots, alters the

perception of the root canal ecology in such acute

cases. First, it implies the presence of sufficient moisture

or a water-saturated medium through which they can

propel themselves to such distances into the canal.

Second, there should be sufficient nutrients to allow

them to migrate and survive in such locations (‘tech-

nically’ beyond the viable part of the body), bearing in

mind their short life-span [3–4 days (Taussig 1984)].

Third, it changes the perception of the host/microbial

interface as being confined to the apical foramen.

Clearly, at least one branch of this host/microbial

interaction is capable of extending into the length of the

necrotic, infected canal associated with symptoms.

Given the short life span of PMNs, the growth of a

bacterial biofilm on the luminal aspect of the layer of

PMNs suggests a very dynamic ecological niche in such

a tooth. The relative equality between the protocols, at

least in terms of quality of viewable sections, opens the

doors towards use of microscopy with immuno-label-

ling to further dissect the root canal ecology and the

dynamics of infection. The PMNs would appear to play

an important role in apical periodontitis and perhaps

apical healing.

Conclusions

The Zurich protocol was more predictable than the EDI

protocol in creating longitudinal sections and possibly

bacterial detection by microscopy but the quality of

observed sections seemed equivalent. Each microscopy

technique provided a unique perspective and together

allowed complementary synthesis of the presence and

morphological distribution of bacteria within roots.

Each tooth presented a unique pattern of bacterial

infection but all exhibited bacterial biofilms on canal

walls; 8/9 roots showed bacteria. Bacteria in the canal

lumen were often associated with other structures but

sometimes appeared ‘free-floating’. In general, bacteria

appeared more abundant apically than coronally but

dentinal tubule colonization was more common in

coronal and middle thirds. PMNs were often found

‘walling off’ bacterial biofilm along the entire length of

the root canal wall, although they were in higher

numbers apically. The findings provide interesting

insights into the nature of host/microbial interaction

and the ecology of infected root canals.

Acknowledgement

The authors are grateful to Dr PNR Nair for his

generous advice on the adoption of the Zurich protocol,

as well as on the finer points of microscopy.

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An in vivo experimental model to assess furcallesions as a result of perforation

M. J. B. Silva1, M. V. Caliari3, A. P. R. Sobrinho2, L. Q. Vieira1 & R. M. E. Arantes3

1Departamento de Bioquımica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo

Horizonte, MG, Brazil; 2Departamento de Odontologia Restaurativa, Faculdade de Odontologia, Universidade Federal de Minas

Gerais, Belo Horizonte, MG, Brazil; and 3Departmento de Patologia Geral, Instituto de Ciencias Biologicas, Universidade Federal de

Minas Gerais, Belo Horizonte, MG, Brazil

Abstract

Silva MJB, Caliari MV, Sobrinho APR, Vieira LQ, Arantes

RME. An in vivo experimental model to assess furcal lesions as

a result of perforation. International Endodontic Journal, 42,

922–929, 2009.

Aim To design and validate a rat molar model of

furcal perforation to allow investigation of the biolog-

ical phenomena that follow and to explore its potential

for evaluating repair materials under standardized

conditions.

Methodology Eighteen male Wistar rats were used.

Surgical aseptic procedures were carried out in order to

open the pulp chamber of a first molar tooth. A cavity

was prepared on the floor of the pulp chamber using a

¼ round bur that created a communication between

the furcation and the periodontal tissues. Six animals

for each time point were sacrificed on days 14, 21 and

28 to assay morphological changes at the furcation

region of molars. Maxillary bone was processed,

removed and sectioned. Cellular infiltration, collagen

deposition and bone resorption were assessed by

histological analysis. Cellularity in the lesion area was

determined by morphometric analysis. Data were

analysed using parametric Student’s t-test.

Results A furcal perforation model was standardized

in which both radiological outcome and periodontal

tissue reactions could be assessed through evaluation of

cellularity, osteoclast activity and collagen deposition.

The morphometric analysis revealed a greater number

of cells 21 day post-surgery when compared with

14 days.

Conclusion This animal model was suitable for

radiological and histological evaluation of the processes

that accompany surgical furcal perforation.

Keywords: animal models, biomaterials, furcal per-

forations.

Received 3 September 2008; accepted 14 April 2009

Introduction

Accidental root canal or furcal perforation complicates

the treatment and compromises the outcome of root

canal treatment (Seltzer & Bender 1990, Walton &

Torabinejad 2002). The prognosis of treatment of

perforations is dependent on site, size, setting time of

the repair material, and the efficacy of the material in

termsof to seal (Walton&Torabinejad2002).All of these

factors are related to the ability to prevent or eliminate

bacterial infiltration (Daoudi & Saunders 2002).

The chronic inflammatory reaction after accidental

furcal perforation has been the object of several studies

(Bhaskar & Rappaport 1971, Meister et al. 1979,

Beavers et al. 1986, Seltzer & Bender 1990, Balla et al.

1991, Arens & Torabinejad 1996, Wu et al. 2005,

Vajrabhaya et al. 2006, Al-Daafas & Al-Nazhan

2007,). This inflammatory reaction provokes alveolar

bone damage around the perforation site where bone is

progressively substituted by granulation tissue. The

Correspondence: Rosa Maria Esteves Arantes, MD, PhD,

Laboratorio de Neuro-imunopatologia Experimental, Departa-

mento de Patologia Geral, Instituto de Ciencias Biologicas,

Universidade Federal de Minas Gerais, Av. Antonio Carlos,

6627, 31270-901, Belo Horizonte, MG, Brazil (Tel.:

+5531 3409 2896/2884; fax: +5531 3409 2879; e-mail:

[email protected]).

doi:10.1111/j.1365-2591.2009.01595.x


lack of bone tissue leads to loss of periodontal attach-

ment that can affect tooth stability (Arens & Torabine-

jad 1996, Wu et al. 2005, Vajrabhaya et al. 2006,

Al-Daafas & Al-Nazhan 2007).

Accidental furcal perforation has stimulated evalua-

tion of the immunological responses that occur in the

periodontal tissues and also the materials to seal the

defects. It is believed that an ideal sealingmaterial should

seal the communication between the periodontium and

pulp chamber and, at the same time, be biocompatible so

as to induce bone and cement deposition (Hartwell &

England 1993, Bernabe & Holland 2004). However, so

far no ideal sealing material has been available.

To evaluate the prognosis and the best treatment

choice different animal models have been used. Small

rodents have many advantages as experimental mod-

els: (i) age and genetic rodent background can be well

defined; (ii) better cost benefits; (iii) mouse and rats may

be kept in controlled environments easily; (iv) there is a

wide variety of gene knockout models. The rat bears

much resemblance to man with respect to periodontal

anatomy, development and composition of dental

plaque, histopathology of periodontal lesions, and basic

immunobiology (Klausen 1991). In this context, the

characterization of alternative rodent models for dental

research is important. Therefore, this study aimed at

describing a new model to evaluate the outcome of

furcal perforation including histopathological aspects

using rats as the experimental model.


Animals

Wistar male rats weighing 240–260 g were obtained

from CEBIO (Centro de Bioterismo da UFMG, Belo

Horizonte, MG, Brazil) and kept in a conventional

animal house with barriers and controlled light cycle.

The experimental protocol was approved by the insti-

tutional animal ethical committee (protocol number

097/04, CETEA – UFMG). Six rats were used for each

time point: 14, 21 and 28 days after the surgical

procedure. Three rats were used as controls.

Anaesthesia

All experimental procedures were carried out under

general anaesthesia. Rats were injected intra-muscu-

larly with 100 mg kg)1 of ketamine hydrochloride

(Dopalen, Division Vetbrands Animal Health, Jacareı,

SP, Brazil) and 10 mg kg)1 of Xilazine (Anasedan,

Agribrands do Brasil Ltda, Paulınia, SP, Brazil). The

absence of flick reflex to hindpaw interdigital skin

stretch was documented before the beginning of

surgical procedures.

Surgical procedures

All experimental procedures were carefully performed

under aseptic condition to avoid contamination. The

disinfection of the surgical field was accomplished as

proposed by Moller (1966).

Access to the pulp chamber in the rat molar tooth was

prepared via the occlusal surface using a number 33½

carbide bur (KG Sorensen, Barueri, SP, Brazil) coupled to

a controlled rotation handpiece (Driller, Sao Paulo, SP,

Brazil) under air cooling. Once the pulp was exposed, the

roof of the chamber was removed with an Endo Z bur

(Dentsply Maillefer�, Catanduva, SP, Brazil). Coronal

pulp tissue was removed using an endodontic probe

until the level of root canal orifices. Haemorrhage was

controlled by irrigating the chamber with saline solu-

tion and by applying pressure with sterilized cotton

balls. As soon as bleeding was controlled, the chamber

was irrigated again with saline solution and dried with

cotton pellets. Subsequently, a perforation was created

in the centre of the pulp chamber floor towards the

alveolar bone using a number ¼ carbide drill (KG

Sorensen, Barueri, SP, Brazil). To standardize the depth

of the perforation, a cursor was glued to the drill 1 mm

from its tip; the width was limited to the bur diameter

(0.5 mm). A layer of gutta percha was inserted on the

pulp chamber floor, avoiding contact between the

sealing material and the perforation site. Condensation

of gutta-percha was accomplished with the endodontic

plugger and the excess was removed and contoured

using a Hollemback 3S (SS White, Rio de Janeiro, RJ,

Brazil). The tooth was restored with silver amalgam that

was burnished using Dycal instrument (SS White);

finally, the waste materials was removed using sterilized

cotton balls. Prior to sacrifice the integrity of the

amalgam was checked under an endodontic microscopy

(Alliance microscopia, Sao Paulo, SP, Brazil).

MTA sealing procedure: testing the model

application

To evaluate if it would be feasible to seal the experi-

mental perforation and to analyse the surrounding

periodontium tissues, six animals had their perforations

filled with grey MTA (Angelus�, Londrina, PR, Brazil).

This material was manipulated according to the

Silva et al. Model of furcal perforation


manufacturer’s recommendation and inserted using a

probe. Animals were killed on day 21 post-surgery.

A video animation was designed using the blender

software (Blender Foundation, Amsterdam, the Neth-

erlands) to represent the entire surgical procedure (see

Video Clip S1 of Supporting Information).

Radiographic procedures

Prior to the sacrifice, the animals were anaesthetized

and killed by decapitation on days 14, 21 and 28 after

the procedure. The hemi-maxilla was removed and

radiographs were taken using a dental X-ray unit

(Bioatlant, Ribeirao Preto, SP, Brazil). The optimal

exposure parameters were determined as 0.12 s

(70 kVp, 10 mA). Ekta-speed plus X-ray film (Eastman

Kodak Co, Rochester NY, USA) was placed and fixed

on a wooden surface to avoid movement. Then, the

buccal side of the maxillary bone was placed so that

side faced the machine. To take the image, the X-ray

cone was set perpendicular to the X-ray film and 5 cm

from it.

Histological preparation

The hemi-maxillas were immersed to 10% buffered

formalin for 72 h at room temperature. Demineraliza-

tion was performed for 30 days in EDTA solution (10%

and pH 7.2) (Merck; Darmstadt; Germany). After

demineralization, the samples were washed in running

tap water for 24 h. Restorations were removed from

the access cavities and the tissues were routinely

processed for paraffin embedding. The hemi-maxillas

were embedded with the buccal side of molars facing

the base of the paraffin block. Consecutive (buccal-

lingual direction) sections of 5 lm encompassing the

perforation of the furcation area and both mesial and

distal roots were stained with Haematoxylin/Eosin

(H&E) and Gomori’s Trichome and used to assess

inflammatory infiltration, periodontal ligament organi-

zation and bone resorption.

Morphometric analysis

The inflammatory cells infiltrating the tissue adjacent

to the perforation area was quantified. Images taken

from the furcal areas were used to assay quantitatively

the longitudinal inflammatory cell infiltration that

occurred around the perforation, as shown in Fig. 3.

Quantification of the inflammatory cells in the furca

area was conducted from three images selected from

the same animal. The images were obtained at 40·magnification through a JVC TK-1270-RGB adapted to

a microscope and analysed using KS300 software built

into a Kontron Elektronick/Carl Zeiss image analyzer

(Caliari 1997). An automatic macro recorder assembler

(an algorithm of the KS300 software) was elaborated

for capture, image processing and segmentation, defi-

nition of morphometrical conditions and counts of all

the cells detected in each image. Image processing

techniques were applied. Segmentation permitted the

separation of these nuclei from cytoplasm and other

structures in the section, such as blood vessels and

extracellular space. Consequently, a binary image was

created containing just the nuclei and other spaces

(Pacheco et al. 2008). The nucleus from the cellular

types usually found in the furca area and newly

recruited leukocytes were then counted. It was imper-

ative that only connective tissue, excluding bone and

root dentine were visualized. This approach limited the

acquisition of more than three images. The measure-

ments were made by an observer who was unaware of

the nature of the tissue sample. Three nonoperated rats

were used as reference of normal periodontal cellularity.


Statistical analysis of mean values of cellular numbers

obtained by morphometry was carried out using

parametric Student’s t-test. Statistical significance was

set at P < 0.05.

Results

Clinical aspects

After anaesthesia and prior to sacrifice the animals

were submitted to clinical evaluation. Animals toler-

ated the surgical procedure well and did not have signs

of distress, weight loss or oral swelling. Colour and

texture of the gingival regions were not altered.

Radiographic assessments

Figure 1a represents the nonoperated furcation and its

adjacent tissue. Radiographs were performed to allow

visualization of bone rebsorption. Radiographs were

effective in revealing the three molar teeth as well as

their associated alveolar bone and periodontal ligament

space (Fig. 1b). Radiographic analyses demonstrated

the position of the perforation (Fig. 1c) and, in some

animals, interdental crestal destruction. In perforation

Model of furcal perforation Silva et al.


sites sealed by MTA, radiographs demonstrated that it

extended into the periodontal tissues (Fig. 1d,e).

Histopathology

Figure 2 shows the furcation area from operated

animals on days 14, 21 and 28 post-surgery. On day

14, the periodontal ligament presented significant

alterations in its architecture, showing intense cellular

infiltration and edema, especially close to the perfora-

tion area (Fig. 2a). The following aspects were

observed: mononuclear and polymorphonuclear cells

prevailed in the underlying inflammatory infiltrate;

edema interposition between tissue elements; intense

vascular neoformation (Fig. 2b, inset); cement and root

dentine reabsorption (Fig. 2f); intense collagen and

connective tissue deposition and osteoclastic activity

(Fig. 2f inset). On day 21 post-surgery, the samples

showed chronic mononuclear inflammatory infiltrate

around the perforation area. Collagen deposition was

observed in the surrounding tissue apart from the

perforation area (Fig. 2c). Fig. 2e represents samples

from animals sacrificed 28 days after surgery. It shows

a discrete chronic inflammatory infiltrate (Fig. 2e) and

evidence of periodontal reorganization. In one sample,

small necrotic areas surrounded by polymorphonuclear

cells and debris were observed close to the deep bone

layer, indicating that in some cases the surgical

procedure resulted in bone microabscesses (data not

show).

Figure 2d represents samples from rats that were

killed on day 21 after surgery in which perforations

were sealed by MTA. It demonstrates the presence of

debris inside the perforation sites which could be MTA

residue. The adjacent connective tissue presented a

prevalence of mononuclear cells in the infiltrate, despite

the presence of polymorphonuclear cells in some areas.

Morphometric analysis

As an example of quantitative analysis made possible

by this model, we quantified the inflammatory cells

infiltrating the tissue adjacent to the perforation area.

Images taken from furcal area were used to assay

quantitatively the longitudinal inflammatory cell infil-

tration that occurred around the perforation, as shown

in Fig. 3. It was observed that cell numbers were much

higher in furcal areas of operated animals than in

animals that were not submitted to surgery.

Discussion

It is fundamental to the treatment of a perforation that

the site does not become infected, and that it is sealed

immediately. Prognosis of treatment depends on the

perforation site, size of damage, adjacent periodontal

condition and type of sealing material (Walton &

Torabinejad 2002). The sealing ability of the material

and its possible extrusion into the periodontal area

should also be considered. Furthermore, good visibility

(a) (b) (c)

(d) (e)

Figure 1 Histological aspect of the first left maxillary molar and radiograph aspects of tooth and periodontium tissue.

(a) Panoramic normal histological aspect of the molar, (T, pulp tissue; PF, pulp chamber floor; PL, periodontal ligament; R, root; B,

interradicular bone), H&E, ·40. (b) Radiological aspects of nonoperated group. White arrows mark the three molars. (c) Operated

molar at day 14 post-surgery. White arrow marks the amalgam restoration. Note the gutta-percha material (arrow head). Open

white arrow marks periodontal bone loss. (d) and (e) Operated and MTA-treated molar at day 21 post surgery. The white arrow

represents MTA material sealing the furcal perforation. Note the MTA periodontium extrusion (open white arrow) and alveolar

bone loss (arrow head).



(a) (b)

(c) (d)

(e) (f)

Figure 2 Histological aspects of the rat furcal lesion. (a) Day 14 post-surgery, perforation area showing granulation tissue (arrow).

(b) Higher magnification of (a) inflammatory cells at the periphery of the perforation and disorganization of periodontal ligament

(inset, arrow), the perforation site (arrow) and MTA fragments (arrow heads). (c) and (e) day 21 and 28, respectively showing the

evolution of lesion towards repair (d) MTA-treated perforation at day 21, residues of MTA are present (arrows heads), notice

the granulation tissue at the border of the perforation (arrows). (f) Intense vascular neorformation and deposition of collagen in the

periodontal tissue, cement reabsorption (arrows), and osteoclastic activity (inset, arrow). C, cement; Co, collagen deposition T, pulp

tissue; R, root; Bo, inter root bone D, dentine; P, perforation; Pl, periodontal ligament. (a), (c), (d) and (e) H&E, ·100; (b), H&E,·200; (f) Gomori’s trichome, ·400 and insets, ·400.



of the perforation will help to facilitate the repair

procedure (Daoudi & Saunders 2002). Hence, a model

that simulates as much as possible the human clinical

situation is crucial to the systematic assessment of the

many biological aspects involved in this procedural

accident.

In this study, all efforts were undertaken to repro-

duce the clinical conditions in an experimental model.

Thus, the design of the stainless steel clamp was

modified to increase its ability to completely grasp the

tooth (Sampaio 1967). All surgical procedures were

performed under aseptic conditions, since a rubber dam

could isolate the tooth in a similar fashion to the

practice in humans.

It should be recognized that the inflammatory

response in rodents is different from that found in

humans, however, the rat was selected as a model

because the periodontal anatomy, the development of

plaque and histopathology of the periodontal lesion in

this animal is similar to that found in humans (Klausen

1991, Genco et al. 1998). Other advantages include

knowledge of animal age, genetic background, and the

ethical and economical issues that contraindicate the

choice of large animals for research (Beavers et al.

1986, Balla et al.1991, Arens & Torabinejad 1996,

Bramante et al. 2004). In addition, the rat model

provides good access to the tooth and allows visibility of

the surgical field. As manipulation of genetic and

pharmacological parameters is possible in the rat, the

establishment of this model will allow further studies

on furcal perforation.

Histopathological results demonstrated the effects of

the furcal lesion on the periodontal tissues. An increased

number of polymorphonuclear cells at 14 day after

surgery could be observed, counterbalanced by prevail-

ing mononuclear cells on days 21 and 28. At 28 days,

an increase of collagen deposition and an exuberance of

granulation tissue were observed. Furthermore, mor-

phometric assessments were in agreement with out-

comes observed by previous workers (Balla et al. 1991,

0Time after surgery

Non-operated

14 days

21 days

28 days

50

100

150

200

250

300

Cel

l num

bers

(a) (b) (c)

(d)

Figure 3 (a), (b) and (c) show, respectively, the intensity of periodontal tissue cellularity on days 14, 21 and 28 post-surgery. Also

notice the presence of vascular congestion (small black arrows) and polimorfonuclear cells (encircled) on day 21 (H&E, ·400); (d)Morphometry of furcal lesion periodontal tissue. The graphic shows the average of cell numbers counted in two fields per animal

sample at 400· magnification, at each time point. Data are presented as mean values of counted cells in each animal (n = 3).

• Statistically significant difference (P < 0.05) between data from the non-operated group and the operated group 14 days post-

surgery. •• Statistically significant difference (P < 0.05) between data from the operated groups on days 14 and 21 post-surgery.



Sousa et al. 2004) and showed an increase in inflam-

matory cell numbers around furcal perforations, which

decreased with time. Other histological parameters such

as periodontal ligament thickness, bone resorption area,

collagen deposition could be accessed easily. Further-

more, despite its limitations this model was useful in

accomplishing qualitative and quantitative (by mor-

phometry) assessments.

It is well-known that failure of perforation repairs

may be the result of the absence of a seal (Saunders &

Saunders 1994). Therefore, in this study, perforations

were sealed with MTA and histopatological analysis

was performed. This is an example of the application of

this model to analyse the biocompatibility aspects of

sealing materials. The preliminary results presented

here indicate that the presence of MTA may interfere

positively with lesion progression. The assessment was

taken on day 21 post-surgery, but further and system-

atic investigations are necessary.

The radiographic technique proposed revealed the

anatomical structures of the normal periodontal and

also demonstrated the adaptation of the filling materi-

als, the perforation position and, in some cases, the

destruction of interdental crest of bone. Alveolar bone

mineral density was previously documented in man-

dibular radiographs and the film used was tested in rats

previously (Xiong et al. 2007, Mahl & Fontanella

2008). Although morphometrical evaluation of alveo-

lar bone loss was not documented, correlation between

bone resorption and histological parameters has been

described previously (Waterman et al.1998, Teixeira

et al. 2000, Xiong et al. 2007).

Conclusion

This experimental animal model for furcal perforation is

not totally comparable to human models of furcal

lesions. However, under carefully standardized condi-

tions, this model can be used to simulate the inflamma-

tory process induced iatrogenically in humans during

root canal treatment as an effective tool to study several

aspects of tissue reaction to this specific kind of injury.

Therefore, it is suitable for other studies on the triggering

and control of the inflammatory reaction, as well as on

the search for suitable sealing materials.

Acknowledgements

R.M.E. Arantes and L.Q. Vieira are supported by

research fellowships from Conselho Nacional de Desen-

volvimento Cientifico e Tecnologico (CNPq). This work

was supported by grants from Fundacao de Amparo a

Pesquisa do Estado de Minas Gerais – FAPEMIG (CBB

APQ-1323-3.13/07 and CNPq (grant number

350567/1995-6 and 571093/2008-6). The authors

are indebted to the technicians Vania Aparecida do

Nascimento Silva for histopatological processing.

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Video Clip S1. A video animation was designed

using the blender software (Blender Foundation,

Amsterdam, the Netherlands) to represent the entire

surgical procedure. The video clip is in avi format.








The morphology of the apical foramen in posteriorteeth in a North Indian population

S. Arora & S. TewariDepartment of Operative Dentistry and Endodontics, Government Dental College, Post Graduate Institute of Medical Sciences,

Rohtak, Haryana, India

Abstract

Arora S, Tewari S. The morphology of the apical foramen in

posterior teeth in a North Indian population. International

Endodontic Journal, 42, 930–939, 2009.

Aim To determine the position and shape of the apical

foramina in posterior teeth derived from an Indian

population.

Methodology A total of 800 freshly extracted max-

illary and mandibular premolar and molar teeth from a

native Haryana population were collected. Apices of

teeth were stained with methylene blue and then

examined stereomicroscopically (40·). The following

observations weremade: number of apical foramina; size

and shape of the minor apical foramen; accessory

foramina frequency, and deviation of the minor apical

foramina (frequency and distance) from the apex.

Results The mean maximum and minimum dia-

meter of the minor apical foramina ranged from 0.158

to 0.323 mm. The most common minor apical foramen

shape was oval (81%). Frequency of accessory

foramina was between 2% and 41% for the various

tooth types. The frequency of deviation of the minor

apical foramina from the anatomic apex varied from

43% to 83% and the distance of deviation in all the

teeth was between 0.052 and 2.921 mm.

Conclusions The incidence of oval canals was

higher in this Indian population compared to other

populations. In 92% and 96% of teeth the difference

between the maximum and the minimum diameter of

all foramina was less than or equal to 0.20 and

0.25 mm, respectively. Therefore, four to five instru-

ment sizes larger than the first binding file would have

been necessary to shape the minor apical foramen of

more than 95% of the teeth included in this study to

make them round.

Keywords: anatomical apex, computer-aided stereo-

microscope, major apical foramina, minor apical

foramina.

Received 14 October 2008; accepted 16 April 2009

Introduction

The main objective of root canal treatment is thorough

mechanical and chemical cleansing of the pulp cavity

and its complete filling with an inert material (Kuttler

1958). From the early work of Hess & Zurcher (1925)

to the most recent studies demonstrating anatomic

complexities of the root canal system, it has been

established that a root with a tapering canal and a

single foramen is the exception rather than the rule.

Root canal morphology especially in the apical third is

a critically important factor during conventional root

canal treatment and surgical endodontics.

The apical constriction, when present, is the nar-

rowest part of the root canal, and preparation to this

point should result in a small wound and optimal

healing conditions (Ricucci & Langeland 1998). The

dimension of the apical constriction has been a focus of

debate. The horizontal dimension of the root canal

system is not only more complicated than the vertical

dimension (root canal length or working length) but

also more difficult to investigate. These measurements

could provide clues for the size of master apical file

Correspondence: Dr Sanjay Tewari, Principal and Professor &

Head of Department of Operative Dentistry and Endodontics,

Government Dental College, Post Graduate Institute of Medical

Sciences, Rohtak, Haryana, 124001 India (Tel.: +91 12622

13737; fax: +91 1262213737; e-mail: tewarisanjayrohtak@

yahoo.co.in).

doi:10.1111/j.1365-2591.2009.01597.x


during root canal preparation (Ricucci 1998, Jou et al.

2004) and can have an impact on the selection of the

best instrumentation technique (Cheung et al. 2007).

The clinical philosophy that apical sizes should be

kept as small as possible (Buchanan 2000), rather than

as large as required, disregards the existing scientific

facts. Studies have reported better debridement and

reduced bacterial load with larger apical preparation

(Ram 1977, Chow 1983, Dalton et al. 1998, Siqueira

et al. 1999, Shuping et al. 2000). Dental manufactur-

ers and some individuals are suggesting apical instru-

mentation with rotary instruments of sizes 20, 25 and

30 (Spangberg 2001). However this gives the errone-

ous impression that apical diameters of canals are small

in size; even though the dimensions of the apical

foramina and the apical canal region are reported to be

larger (Morfis et al. 1994, Gani & Visvisian 1999, Wu

et al. 2000, Marroquin et al. 2004).

Wide variations in the dimensions of the apical

constriction have been reported. Cheung et al. (2007)

reported the mean value for the longest and the

shortest diameter of the apical constriction of mesial

and distal canals of C-shaped mandibular second

molars to be 0.26 : 0.15 mm and 0.36 : 0.22 mm,

respectively. Marroquin et al. (2004) reported mean

narrow (0.20 mm) and wide (0.26 mm) diameters of

minor apical foramina in mandibular molars, 0.18–

0.25 mm in the mesio-buccal and distobuccal roots

and 0.22–0.29 mm in the palatal root of the maxillary

molars. Morfis et al. (1994) reported a mean diameter

of 0.26 and 0.39 mm for the mesial and distal canals,

respectively, in mandibular molars (without defining

the exact site of measurement).

Previous studies have frequently demonstrated that

the apical foramen is not always located at the tip of the

root. The frequency of deviation of the major foramen

from the anatomical apex ranged from 46% to 92%

and the mean distance between them ranged from 0.2

to 1.38 mm (Kuttler 1955, Green 1956, 1960, Palmer

et al. 1971, Burch & Hulen 1972, Pineda & Kuttler

1972, Blaskovic-Subat et al. 1992, Morfis et al. 1994,

Marroquin et al. 2004, Cheung et al. 2007). Further-

more, these studies have shown that frequency of

deviation varies in different races. If the foramen

deviates in the buccal/lingual plane, it is difficult to

locate its position using radiographs alone (Schaeffer

et al. 2005). ElAyouti et al. (2001, 2002) also reported

that a seemingly accurate working length ending

radiographically 0–2 mm short of the radiographic

apex can result in overestimation of working length in

51% of the root canals. Therefore, misinterpretation of

dental radiographs may lead to an incorrect determi-

nation of working length and to subsequent complica-

tions of over-instrumentation and overfilling of the root

canal (Seltzer et al. 1971).

The cosmopolitan nature of urban populations

means that endodontists treat an increasing number

of patients of different and mixed racial origin. It is,

therefore, important to be aware of the frequency of

racially determined anatomic variations. A number

of studies have shown different trends in morphology of

roots and canals amongst the different races (Caliskan

et al. 1995, Gulabivala et al. 2001, 2002, Ng et al.

2001, Wasti et al. 2001, Sert & Bayirli 2004, Al-Qudah

& Awawdeh 2006). Additionally, no such morphomet-

ric study has yet been conducted in Indian populations.

In view of conflicting findings regarding the apical

zone and scarce reports on teeth of Indian origin, the

objective of this study was to determine further the

number, shape, diameter of the apical foramina as well

as the incidence of their deviation from the anatomical

apex, the distance between them and the frequency of

accessory foramina in an Indian population.


A total of 800 freshly extracted human permanent

maxillary and mandibular posterior teeth, with com-

pletely formed apices, obtained from a North Indian

(Haryana) population were included. Teeth were col-

lected from a general district hospital attended by local

population. Ethnicity of the population was further

verified from the outpatient records. Teeth were

extracted because of periodontal or pulpal disease.

Following extraction teeth were washed under tap

water, and stored in 5% sodium hypochlorite (Aroma

Agencies, Mumbai, Maharashtra, India) and used

within 6 months of extraction. The teeth were identi-

fied as maxillary or mandibular first and second

premolars, or first and second molars.

One hundred teeth, with intact crowns (for clear

identification), in each group were selected according to

strict inclusion and exclusion criteria as described by

Marroquin et al. (2004). Primary teeth and roots with

fractures, resorption, or underdevelopment (40·mag-

nification) or that had received any previous endodon-

tic treatment were discarded (Marroquin et al. 2004).

Soft tissues around and in the foramen area were

removed with a size 6 K file (Dentsply Maillefer,

Ballaigues, Switzerland) at 40·magnification. The roots

were then placed in methylene blue (Macsen Labora-

tories, Udaipur, Rajasthan, India), washed under

Arora & Tewari Morphology of apical foramina


running water for 10 min, and dried with pressurized

air before examination.

A computer-aided stereomicroscope with 40·magni-

fication (Stereo zoom microscope RSM-9; Radical Sci-

entific Equipments Pvt. Ltd., Ambala Cantt, Haryana,

India) and VideoTesT-Size 5.0 measurement software

(VideoTesT, St Petersburg, Russia) was used. Measure-

ment accuracy was assured through calibration

between a micro scale with 0.1 mm markings and

the software. The measuring dialogue menu was set in

millimetres and adjusted to three decimal places.

On the external root surface, the opening of the root

canal was called the apical foramen (AF) and its

outermost diameter was termed the ‘major apical

foramen’ (Fig. 1). The minor apical foramen (apical

constriction) was considered to be the region of the

apical foramen with the smallest diameter. From the

minor apical foramen the canal widens as it app-

roaches the major apical foramen (Fig. 1). In clinical

practice, the minor apical foramen is a more consistent

anatomical feature (Ponce & Vilar Fernandez 2003)

and is the preferred landmark for the apical end-point

for root canal treatment. The ‘anatomic apex’ was

defined as the most apical root structure (Fig. 1), and

was traced by marking with red ink. These three

anatomical entities could, theoretically, coincide in

one. A foramen was categorized as accessory when its

diameter was narrower than 0.10 mm (Marroquin

et al. 2004).

After initial confirmation of tooth type, the root

morphology of the apical area was examined under

40·magnification. Teeth were oriented until the major

apical foramen was located in the middle of and parallel

to the objective lens. The image of the minor apical

foramen was then captured by regulating the focus.

Here, minor foramen was that part of the apical

foramen with the smallest planar dimension, as

observed by focusing below the major apical foramen

(Cheung et al. 2007). If a root had more than one

apical foramen, then each foramen was focused sepa-

rately parallel to objective lens by changing the

orientation of tooth and individual photographs were

captured.

The following observations were then made:

1. size of minor apical foramen,

2. shape of minor apical foramen,

3. accessory foramina frequency (if found), and

4. deviation of the minor apical foramen (in mm) from

the apex.

Diameters of the minor apical foramen

The widest and narrowest diameters of each minor

apical foramen were measured using the length mea-

suring mode of the software and defined as the

maximum and the minimum diameters, respectively

(Fig. 2).

Shape of the minor apical foramen

A minor apical foramen with a difference greater than

or equal to 0.02 mm between its maximum and

minimum diameters was considered to have an oval

shape. This criterion was established according to

Marroquin et al. (2004) in consideration of the ISO

tolerances for root canal instruments. The shape of the

minor apical foramen was accordingly determined to

Figure 1 Diagrammatic representations of the morphological

features investigated.

Figure 2 Two minor apical foramina and one accessory

foramen (top most) at apex, measurement of maximum and

minimum diameters of each foramen has been made.

Morphology of apical foramina Arora & Tewari


have either a round, oval, or irregular (triangular,

kidney, or irregular) form.

Frequency and distance of deviation of minor apical

foramen from anatomical apex

If the minor apical foramen was not located at the

anatomical root apex, but at a more cervical position

on the long axis of the root, a straight line parallel to

the root from the most apical point of the foramen to a

tangent line at the most apical point of the anatomical

apex was used to determine the distance between the

minor apical foramen and anatomical apex (Marroquin

et al. 2004) (Fig. 3).

The statistical data were arranged by means, max-

imum, minimum and SD.

Results

A total of 2004 foramina were investigated. The

distance between the minor apical foramen and ana-

tomical apex; frequency of accessory foramina; num-

ber, shape and diameter of apical foramina in each root

of maxillary and mandibular premolars and first and

second premolars are shown in Tables 1–5. In tables

S1–S14 (supporting information) the mean values for

minimum and maximum diameters and distance of

deviation are reported separately for each root with

one, two, three, four, and five foramina. In tables S15–

S16 minimum and maximum diameters of accessory

foramina have been reported.

Number of apical foramina

Mandibular teeth

The number of apical foramina in all posterior teeth is

described in Table 1. In the mandibular posterior teeth

the incidence of a single apical foramen ranged from

64% to 81% except in the mesial root of mandibular

first molars (33%). As many as five apical foramina

were observed in 1% teeth in mandibular first and

second premolars and mesial roots of first molars. The

greatest variation from a single apical foramina was

observed in the mesial roots of mandibular first molars

with an incidence of two, three, four, five foramina in

46%, 16%, 4% and 1%, respectively.

Maxillary teeth

In maxillary teeth the greatest variations from a single

apical foramina were observed in both first and second

premolars followed by the mesiobuccal root of first

molars. The incidence of two foramina was 57%, 38%

and 37% andmore than two foramina in 17%, 21% and

9% of teeth, respectively. In maxillary teeth five apical

Figure 3 Method for measuring distances between the minor apical foramen and the anatomic apex. Each foramen was separately

focused parallel to the objective lens and measurements were made on different photographs. AA denotes anatomic apex, traced by

marking with red ink.



foramina were found in maxillary second premolars

only, with an incidence of 3% (Fig. 4). Distobuccal roots

of first and second molars showed simpler anatomy

having a maximum two apical foramina only, and no

main apical foramina in 1% teeth.

Frequency of accessory foramen

There was a higher frequency of accessory foramina in

maxillary premolars followed by mandibular premolars

(Table 2). The highest frequency of accessory foramina

was in maxillary second premolars (41%). Values as

low as 2%, for palatal roots of maxillary second molars

were also obtained.

Deviation of minor apical foramina from the

anatomical apex

Deviation of the minor apical foramina from the

anatomical apex was seen in all teeth but there was

no conclusive pattern of variation (Table 3). Frequency

of deviation of minor apical foramina (Table 3) from

the apex varied from 43% in maxillary first premolars

to 83% in mandibular first premolars.

The highest mean value of the distance between the

minor apical foramen and the anatomical apex was

observed in the mesiobuccal root of maxillary first

molars (0.996 mm). The lowest value of 0.632 mm

was observed for the distobuccal root of maxillary

second molars (Table 3).

Diameters of minor apical foramina

The mean maximum diameter (Table 4) of the minor

apical foramina ranged from 0.230 mm (distobuccal

root of maxillary second molars) to 0.323 mm (distal

root of mandibular second molars). The mean mini-

mum diameter (Table 5) of the minor apical foramina

ranged from 0.158 mm (mandibular second premo-

lars) to 0.227 mm (distal root of mandibular second

molars).

Discussion

A digital stereomicroscope with integrated software

was used to provide accurate measurement of a large

number of teeth. Teeth were oriented until the apical

foramen was located in the middle of and parallel to the

objective lens to allow measurement of the true

dimensions of the minor apical foramen irrespective

of the curvature that a canal follows inside the root.

If the apical foramen was located at a more cervical

position on the long axis of the root, a straight line

parallel to the root from the most apical point of the

foramen to a tangent line at the most apical point of the

anatomical apex was drawn for each foramina. This

determined the true distance not the vertical distance

between the apical foramen and anatomical apex.

However this distance can not be measured in vivo

because of limitations of radiographs in identifying

accurately the foramina on buccal/lingual root surfaces

Table 1 Number of apical foramina by tooth type

Single Two Three Four Five

Mandibular teeth

Mandibular first premolar 70 24 3 2 1

Mandibular second premolar 74 16 6 3 1

Mandibular first molar

Mesial root 33 46 16 4 1

Distal root 75 22 2 1 X

Mandibular second molar

Mesial root 64 33 3 X X

Distal root 81 18 X 1 X

Maxillary teeth

Maxillary first premolar 25 57 13 4 X

Maxillary second premolar 41 38 16 2 3

Maxillary first molar

Mesiobuccal root 54 37 6 3 X

Distobuccal root 91 8 X X X

Palatal root 78 20 2 X X

Maxillary second molar

Mesiobuccal root 61 31 5 3 X

Distobuccal root 80 13 X X X

Palatal root 88 11 1 X X

Table 2 Frequency of accessory foramina by tooth type

0 1 2 3 4 5 Total

Mandibular teeth

Mandibular first premolar 91 7 2 X X X 11

Mandibular second premolar 91 6 0 1 2 X 17

Mandibular first molar

Mesial root 86 13 1 X X X 15

Distal root 96 4 X X X X 4

Mandibular second molar

Mesial root 95 5 X X X X 5

Distal root 94 6 X X X X 6

Maxillary teeth

Maxillary first premolar 87 8 1 3 X 1 24

Maxillary second premolar 72 12 3 3 1 2 41

Maxillary first molar

Mesiobuccal root 92 5 2 1 X X 12

Distobuccal root 95 5 X X X X 5

Palatal root 93 5 2 X X X 9

Maxillary second molar

Mesiobuccal root 86 7 5 X 2 X 25

Distobuccal root 96 4 X X X X 4

Palatal root 98 2 X X X X 2



(Schaeffer et al. 2005), as well as the inability of the

electronic apex locators in locating the canal terminus

with 100% accuracy (Wrbas et al. 2007, ElAyouti et al.

2009).

The frequency of deviation of the minor apical

foramen from the apex (43–83%), distances of the

minor apical foramina from the apex (0.052–

2.921 mm) and mean distance of minor apical foram-

ina from anatomic apex reported in the present study

(0.632–0.996 mm) compare with the literature

(Kuttler 1955, Green 1956, 1960, Palmer et al.

1971, Pineda & Kuttler 1972, Vertucci 1984, Blask-

ovic-Subat et al. 1992, Morfis et al.1994, Marroquin

et al. 2004). The minor differences observed between

various studies may be explained by the different

measuring methods, by the different apical foramen

definitions used and by difference in reference points to

measure the distances. These results demonstrate the

complexity of the apical zone in this Indian population

and the similarity with other parts of the world. Thus,

they suggest that the endodontic principles and prac-

tices being followed in the other parts of the world can

also be applied to this Indian population. The unpre-

dictable nature of the position of the apical constriction

with respect to the radiographic apex further strength-

ens the need of using apex locators rather than relying

on radiographs for canal length determination. These

findings also support the current practice of cutting

3 mm of the root apex (Kim & Kratchman 2006)

during surgical procedures to ensure the removal of

most of the unprepared and unfilled canals.

Unlike most of the previous studies mesial and

mesiobuccal roots of mandibular and maxillary molars

respectively had a higher frequency of single foramen.

This may be interpreted that there was a high tendency

for Type II (two canals merging into one at apex) canals

Table 3 Distance (in mm) and %age

deviation of minor apical foramina from

the apex by tooth type

Minimum Maximum Average SD %Deviation

Mandibular teeth

First premolar 0.112 2.186 0.796 0.415 83

Second premolar 0.052 2.892 0.781 0.463 78

First molar: mesial root 0.154 2.808 0.834 0.496 50

First molar: distal root 0.167 1.617 0.817 0.297 68

Second molar: mesial root 0.127 2.202 0.78 0.465 46

Second molar: distal root 0.065 2.031 0.809 0.322 44

Maxillary teeth

First premolar 0.18 2.03 0.78 0.391 43

Second premolar 0.122 2.523 0.984 0.407 49

First molar: mesiobuccal root 0.176 2.921 0.996 0.676 59

First molar: distobuccal root 0.225 2.224 0.824 0.441 54

First molar: palatal root 0.26 2.455 0.924 0.466 68

Second molar: mesiobuccal root 0.276 2.527 0.992 0.580 59

Second molar: distobuccal root 0.106 1.433 0.632 0.319 49

Second molar: palatal root 0.122 0.873 0.719 0.313 61

Table 4 Maximum diameter (in mm) of

minor apical foramina by tooth typeMinimum Maximum Average SD

Mandibular teeth

First premolar 0.1 1.169 0.256 0.122

Second premolar 0.104 1.45 0.241 0.144

First molar: mesial root 0.102 1.265 0.261 0.153

First molar: distal root 0.116 0.585 0.300 0.104

Second molar: mesial root 0.112 0.793 0.303 0.143

Second molar: distal root 0.122 1.168 0.323 0.142

Maxillary teeth

First premolar 0.106 0.553 0.24 0.092

Second premolar 0.103 1.394 0.254 0.139

First molar: mesiobuccal root 0.1 0.835 0.263 0.125

First molar: distobuccal root 0.103 0.524 0.257 0.082

First molar: palatal root 0.108 0.794 0.320 0.146

Second molar: mesiobuccal root 0.105 0.509 0.244 0.098

Second molar: distobuccal root 0.103 0.43 0.230 0.078

Second molar: palatal root 0.117 0.794 0.309 0.12



in mesial/mesiobuccal roots in this north Indian

population. This anatomy is best treated by preparing

and filling the straighter canal (generally palatal/

lingual) to the apex and the other (buccal) canal to

point of juncture. If both canals are enlarged to the

apex, an ‘hour glass’ preparation results, which leaves

voids in apical third during filling (Vertucci 2005).

The presence of two root canals with two apical

foramina in the palatal root of maxillary molars is

uncommon. However, the present study revealed that

approximately 20% of palatal roots of maxillary first

molars and 11% of maxillary second molars had two

minor apical foramina of similar dimensions. About 2%

and 1% palatal roots of maxillary first and second

molars, respectively, had three foramina. This finding

may indicate the presence of two or more root canals or

one root canal with an apical ramification in the

palatal roots of maxillary molars. Up to five apical

foramina were observed in 0.75% samples in mandib-

ular and maxillary premolars and mesial and mesio-

buccal roots of first molars similar to the study of

Gutierrez & Aguayo (1995). There was a high fre-

quency of accessory foramina in both maxillary and

mandibular premolars amongst all tooth type; support-

ing the findings of Morfis et al. (1994) that maxillary

and mandibular premolars have the most complicated

apical morphologic make up with respect to main

foramina and accessory foramina. A high frequency of

accessory foramina as well as multiple foramina

suggests the extensive branching of the root canal or

the presence of multiple canals at the apex and thus

relates to high incidence of post-treatment apical

periodontitis (Green et al. 1997, Barthel et al. 2004)

due to non negotiation of extra canals by orthograde

instrumentation alone and further suggests scope of

surgical endodontics for management of such teeth.

The mean maximum diameter of the minor apical

foramina ranged from 0.23 to 0.32 mm and the mean

minimum diameter was in the range 0.158–

0.227 mm. These values were in accordance with

Marroquin et al. (2004) (0.20–0.29 mm), Cheung

et al. (2007) (0.32 mm) and Wu et al. (2000) (0.13–

0.46 mm) but lower than those reported by Morfis

et al. (0.418–0.977 mm) and by Gani & Visvisian

(0.332–0.594 mm). These results support that instru-

ment sizes 10 or 15 often do not have contact at the

minor apical foramen but rather encounter resistance

elsewhere because of root canal irregularities or

Table 5 Minimum diameter (in mm) of

minor apical foramina by tooth typeMinimum Maximum Average SD

Mandibular teeth

First premolar 0.064 0.512 0.173 0.074

Second premolar 0.06 0.439 0.158 0.065

First molar: mesial root 0.065 0.495 0.178 0.078

First molar: distal root 0.074 0.544 0.222 0.084

Second molar: mesial root 0.061 0.654 0.198 0.098

Second molar: distal root 0.101 0.581 0.227 0.083

Maxillary teeth

First premolar 0.059 0.493 0.171 0.064

Second premolar 0.032 0.406 0.169 0.068

First molar: mesiobuccal root 0.045 0.428 0.174 0.068

First molar: distobuccal root 0.043 0.398 0.183 0.069

First molar: palatal root 0.073 0.714 0.226 0.108

Second molar: mesiobuccal root 0.045 0.422 0.168 0.07

Second molar: distobuccal root 0.052 0.387 0.171 0.07

Second molar: palatal root 0.065 0.506 0.218 0.087

Figure 4 Photomicrograph of maxillary second premolar

(40·), with five minor apical foramina and one accessory

foramen, identified by regulating the focus.



curvature. So, in this Indian population the first file

that will truly bind at the apex, i.e. initial working

width at working length would correspond to at least a

size 20 file.

The most common shape of the minor apical

foramen was oval. The frequencies were between

79% and 89% for various roots. This prevalence of

oval canals were higher than Marroquin et al. (2004)

and Wu et al. (2000) and lower than Gani & Visvisian

(1999). Other forms of apical foramina such as

triangular, kidney, or irregular forms were observed

in 0.5% of the roots (Fig. 5).

One of the major point of interest when planning this

investigation was its clinical significance when shaping

and cleaning the root canal of this Indian population.

The determination of the first file that binds in the

apical part of the root canal does not allow a reliable

prediction of the appropriate final instrument size

required for complete apical enlargement. The final

instrument size must be large enough to touch all

walls. Because most canals are oval in their cross

sectional shape, the goal should be to make the final

apical instrument size correspond to the largest diam-

eter of the oval to make these canals round. The

difference between the wide and narrow diameters of

the apical constriction region in mandibular premolars

in this population, was less than or equal to 0.15 mm

in 86% teeth therefore enlarging up to three instru-

ment sizes larger than the first binding file in apical

constriction region (FAB) will shape the minor apical

foramen area round only in 86% teeth. This difference

between the wide and narrow diameters in these

premolars was less than or equal to 0.20 mm in 91%

and less than or equal to 0.25 mm in 97% teeth so

shaping up to 4 instrument sizes larger than FAB will

shape apical area in 91% and five instrument size

larger than FAB will shape the apical area in 97% teeth

to a round outline. Similarly, for the rest of the teeth it

was found that up to three instrument sizes larger than

FAB would shape the apical constriction round in only

84% of mandibular molars, 88% of maxillary premo-

lars and 87% of maxillary molars. While four instru-

ment sizes larger than FAB will shape apical

constriction round in 89% of mandibular molars,

93% of maxillary premolars and 94% of maxillary

molars, and five instrument sizes larger than the FAB

will shape apical constriction round in 94% of man-

dibular molars, 97% of maxillary premolars and 96% of

maxillary molars. These findings suggest that in the

absence of any predictable method to measure accurate

working width, enlargement of apical third 4–5 sizes

larger than first file to bind at the apex may ensure

complete involvement of the largest diameter of an oval

canal in more than 95% teeth and will produce a round

shape of apical preparation for proper filling with a

round gutta percha cone.

Conclusions

The incidence of oval canals was higher in this Indian

population (81%) compared to other populations and

occurred in 79–88% of roots depending on tooth type.

In 92% and 96% of teeth the difference between the

maximum and the minimum diameter of all foramina

was less than or equal to 0.20 and 0.25 mm, respec-

tively, therefore four to five instrument sizes larger than

the first binding file would have been necessary to

shape the minor apical foramen of more than 95% of

the teeth included in this study to make them round.

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Table S1. Mandibular First Premolar.

Table S2. Mandibular Second Premolar.

Table S3. Mandibular First Molar Mesial Root.

Table S4. Mandibular First Molar Distal Root.

Table S5. Mandibular Second Molar Mesial Root.

Table S6. Mandibular Second Molar Distal Root.

Table S7. Maxillary First Premolar.

Table S8. Maxillary Second Premolar.

Table S9. Maxillary First Molar (Mesiobuccal Root).

Table S10. Maxillary First Molar (Distobuccal Root).

Table S11. Maxillary First Molar (Palatal Root).

Table S12. Maxillary Second Molar (Mesiobuccal

Root).

Table S13. Maxillary Second Molar ( Distobuccal

Root).

Table S14. Maxillary Second Molar (Palatal Root).

Table S15.Maximum diameter (in mm) of accessory

foramina by tooth type.

Table S16. Minimum diameter (in mm) of accessory

foramina by tooth type.








Carbon dioxide laser irradiation stimulatesmineralization in rat dental pulp cells

Y. Yasuda1, E. Ohtomo1, T. Tsukuba2, K. Okamoto2 & T. Saito1

1Division of Clinical Cariology and Endodontology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences

University of Hokkaido, Hokkaido, Japan; and 2Division of Oral Pathopharmacology, Department of Medical and Dental Sciences,

Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

Abstract

Yasuda Y, Ohtomo E, Tsukuba T, Okamoto K, Saito T.

Carbon dioxide laser irradiation stimulates mineralization in rat

dental pulp cells. International Endodontic Journal, 42, 940–946,

2009.

Aim To examine the effect of carbon dioxide laser

irradiation on mineralization in dental pulp cells.

Methodology Rat dental pulp cells were irradiated

with a carbon dioxide laser at 2 W output power for

20, 40 and 60 s, and were cultured in ascorbic acid

and b-glycerophosphate containing media. Cell viabil-

ity was examined 24 h after laser irradiation by a

modified MTT assay. Alizarin Red S staining was

performed 10 days after laser irradiation. The amounts

of secreted collagen from the cells after irradiation were

quantified following Sirius Red staining. The expression

levels of collagen type I and HSP47, collagen-binding

stress protein, were analysed by real-time PCR. HSP47

protein expression was examined by Western blotting.

Statistical analysis was performed using one-way

analysis of variance (anova) followed by the Tukey’s

multiple comparison test.

Results The cell viability was not affected by laser

irradiation at 2 W for up to 40 s. However, it was

significantly decreased by 20% at 60 s (P < 0.05). The

amount of mineralization after 10 days of irradiation at

2 W for 40 s was significantly increased in comparison

to the other conditions (P < 0.05). The extracellular

collagen production was significantly increased by 73%

on day 2 and 38% on day 4 after laser irradiation

(P < 0.05). Although collagen type I gene expression

was not changed by laser irradiation, HSP47 gene and

protein expression was induced within 12 and 24 h,

respectively.

Conclusions These results suggested that carbon

dioxide laser irradiation stimulated mineralization in

dental pulp cells. The laser irradiation also increased

HSP47 expression but not collagen gene expression.

Keywords: carbon dioxide laser, collagen, dental

pulp cells, heat shock protein 47, mineralization.

Received 28 December 2007; accepted 16 April 2009

Introduction

Reparative dentine forms in the dental pulp in response

to various external stimuli such as caries and abrasion

(Kamal et al. 1997, Lee et al. 2006). Direct pulp

capping with calcium hydroxide has been advocated

to accelerate reparative dentine formation on the

exposed pulp surface. However, calcium hydroxide is

highly alkaline and causes an inflammatory response.

In addition, it has not always been clinically highly

efficacious in the uniform formation of reparative

dentine (Scarano et al. 2003). Recently, the use of

mineral trioxide aggregate (MTA) in direct pulp

capping has been reported (Aeinehchi et al. 2003,

Chacko & Kurikose 2006). MTA is superior to calcium

hydroxide for pulp capping of mechanically exposed

human teeth; however, a variety of histological

responses were still observed (Caicedo et al. 2006).

Furthermore, no data from long-term clinical results

Correspondence: Yoshiyuki Yasuda, DDS, PhD, Division of

Clinical Cariology and Endodontology, Department of Oral

Rehabilitation, School of Dentistry, Health Sciences University

of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido

061-0293, Japan (Tel.: +81 133 23 2841; fax: +81 133 23

1423, e-mail: [email protected]).

doi:10.1111/j.1365-2591.2009.01598.x


are yet available. In an ideal situation, the exposed pulp

surface should be covered promptly with reparative

dentine and the dental pulp should not demonstrate an

inflammatory response.

The application of lasers has expanded into various

fields and has also been frequently used in clinical

dentistry (Pearson & Schuckert 2003, Parker 2007). In

cultured cells, irradiation by low power laser, such as a

diode laser, has been reported to accelerate cell

differentiation and mineralization in calvarial and

dental pulp cells (Ozawa et al. 1998, Ohbayashi et al.

1999, Ueda & Shimizu 2003). On the other hand,

Moritz et al. (1998a,b) reported that the utility of a

high power laser, such as a carbon dioxide laser, is

useful on exposed pulp surfaces in direct pulp cap-

ping experiments. Furthermore, Melcer et al. (1987)

observed that a neo-dentine bridge was formed in the

pulp tissue after carbon dioxide laser irradiation on

teeth, suggesting that this laser is effective in mineral-

ization. However, the mechanism by which carbon

dioxide laser irradiation stimulates mineralization in

direct pulp capping treatment is not fully elucidated.

Laser irradiation may affect the collagen production in

dental pulp cells, because the collagenous network

plays an important role in mineralization (Linde 1989).

Heat shock proteins (HSPs) are induced by stress

from heat and chemical stimuli (Noda et al. 2002).

HSPs have been known to suppress the aggregation of

denatured protein (Guzhova & Margulis 2006). They

are also constitutively expressed in normal cells, and

are associated with important functions such as protein

synthesis and intracellular transport (Eisenberg &

Greene 2007). In particular, HSP47 is a collagen-

specific molecular chaperone. It specifically binds to

collagen and plays an essential role in collagen

production (Masuda et al. 1994, Koide et al. 2002).

The purpose of this study was to examine the effect of

a carbon dioxide laser on mineralization in rat dental

pulp cells. Moreover, the amount of extracellular

secreted collagen and the HSP47 expression levels

were examined to clarify the stimulatory effects of

carbon dioxide laser irradiation on mineralization of

dental pulp cells.


Cells and cell culture conditions

All animal protocols were approved by the Institutional

Animal Care and Use Committee of the Health Sciences

University of Hokkaido, and experiments were carried

out under the control of the University’s Guidelines for

Animal Experimentation. The dental pulp cells were

isolated from incisors of Wistar rats (female, 5-week-

old) as described previously (Yokose et al. 2000). The

cells were cultured in Dulbecco’s modified eagle

medium (DMEM; Sigma, St Louis, MI, USA) supple-

mented with 10% foetal bovine serum (Sigma),

10 000 U mL)1 penicillin (Invitrogen, Grand Island,

NY, USA), and 10 mg mL)1 streptomycin (Invitrogen)

at 37 �C in a humidified atmosphere of 5% carbon

dioxide.

Laser irradiation

A carbon dioxide laser apparatus (Bel Luxar LX-20SP,

Takara, Kyoto, Japan) with a wavelength of 10.6 lmand a power output of 2.0 W (A4 mode, 10 pps,

average power output of 0.3 W) was used. Rat dental

pulp cells (5 · 104 cells per well) were seeded out in

24-well plates and cultured for 24 h, and then serum-

starved for 24 h. After withdrawal of medium, the cells

were irradiated at 2 W output power for 20, 40 and

60 s using the scanning method as applied in clinical

laser irradiation. The tip was moved gradually at a

constant rate, avoiding concentrating laser light on one

site, and the whole area was irradiated. The laser beam

was delivered by a ceramic tip (0.8 mm diameter) with

the distance from the tip of the fibre to the cell layer

being 2 cm (irradiation diameter approximately

2 mm). The total energy of irradiation time of 40 s

was 382.2 J cm)2. Irradiated or nonirradiated (control)

cells were cultured in DMEM containing 50 lg mol L)1

ascorbic acid (AA, Sigma) and 10 m mL)1 b-glycero-phosphate (b-GP, Sigma) for 10 days.

Cell viability assay

Rat dental pulp cells were cultured in DMEM contain-

ing 50 lg mL)1 AA and 10 mmol L)1 b-GP for 24 h

after irradiation or nonirradiation (control). Cell viabil-

ity was determined by a modified MTT assay (WST-8

assay: Dojindo, Kumamoto, Japan), and data are

presented as a percentage of viability values seen under

control culture conditions. The assay is based on the

cleavage of tetrazolium salt WST-8 to formazan by

cellular mitochondrial dehydrogenase. The amount of

the dye generated by activity of dehydrogenase is

directly proportional to the number of living cells. For

the WST-8 assay, a 10-lL quantity of WST-8 dye

solution was added directly to 100 lL of culture

medium per well. The absorbance of the dye was

Yasuda et al. Effect of laser irradiation


measured at 450 nm using a Model 680 microplate

reader (Bio-Rad, Hercules, CA, USA).

Quantitative Alizarin Red S staining

Rat dental pulp cells were cultured as before in

50 lg mL)1 AA and 10 mmol L)1 b-GP-containingmedia for 10 days. Cells were fixed in 70% ice-cold

ethanol for 1 h and rinsed with distilled water. Cells

were stained with 40 mmol L)1 Alizarin Red S (Sigma),

pH 4.2, for 10 min with gentle agitation. Alizarin Red S

staining is specific for calcium deposition. Cells were

rinsed thrice with distilled water and then rinsed with

PBS for 15 min. Dye was extracted from fixed cells by

treatment with 500 lL 10% cetylpyridinium chloride

(Nakarai Tesque., Kyoto, Japan) for 20 min with gentle

agitation. The absorbance of the extracted dye was

measured at 570 nm using a Model 680 microplate

reader. The amount of Alizarin Red S was determined

according to an Arizarin Red S standard curve.

Quantitative analysis of extracellular secreted

collagen

The amount of extracellular secreted collagen was

measured on day 2–10 using the method described by

Ohbayashi et al. (1999). Conditioned media (100 lL)were dispensed into wells of 96 well plates, and plates

were incubated at 37 �C for 24 h until dry. After

rinsing with distilled water, 0.2% Sirius Red (Sigma) in

saturated picric acid (w/v) was placed in each well for

30 min. The plates were washed with 0.5% NaOH. The

eluted stain was then drawn up and down several times

in a pipette and placed into a second plate. Absorbance

was read at 540 nm in a Model 680 microplate reader,

and the amount of extracellular secreted collagen was

estimated from a standard curve.

Real-time PCR

The mRNA expression of collagen type I, HSP47 and

glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

was determined by real-time PCR with rat-specific

primers. Total RNA was extracted using RNeasy

(Qiagen Inc., Chatworth, CA, USA) and was digested

with DNase I (Sigma), according to the manufacturer’s

instructions. Single-strand cDNA was synthesized with

SuperScript II reverse transcriptase (Invitrogen) and

random primers. Real-time PCR was performed on a

volume of 15 lL containing 1.5 lL (50 ng) of cDNA

and 13.5 lL of master mix containing 7.5 lL of mix

(SYBR Green PCR Master Mix, Invitrogen), 0.75 lL of

each primer (10 pmol L)1), and 4.5 lL of diethyl

pyrocarbonate-treated water using an ABI PRISM

7500 Sequence Detection System Thermal Cycler

(Applied Biosystems, Foster City, CA, USA). The

sequences of the rat-specific primers were as follows:

collagen type I, forward 5’-TTGACCCTAACCAAG

GATGC-3’, reverse 5’-CACCCCTTCTGCGTTGTATT-3’;

HSP47, forward 5’-GTGCGCTCCCTCAGTAACTC-3’,

reverse 5’-CCACATCCTTGGTGACCTCT-3’; Control

primers specific for GAPDH were: forward 5’-TCCACC

ACCCTGTTGCTGTA-3’, reverse 5’-ACCACAGTCCAT

GCCATCAC-3’. The program was set at 50 �C for

2 min and 95 �C for 10 min followed by 40 cycles of

denaturation at 95 �C for 15 s and annealing at 60 �Cfor 60 s. SYBR green fluorescence was monitored after

each elongation period. The threshold was set above

the nontemplate control background and within the

linear phase of target gene amplification to calculate

the cycle number at which the transcript was detected

(denoted CT).

Samples were amplified in triplicate, averages were

calculated, and differences in CT data were evaluated by

Sequence Detection Software V1.3. (Applied Biosys-

tems). For each primers set, validation experiments

demonstrated that the efficiencies of target and refer-

ence gene amplification were approximately equal; the

absolute value of the slope of log input amount versus

CT was <0.1. For data analysis, we used the compar-

ative CT method (DDCT method) with the following

formula: DCT = CT (Target))CT (GAPDH). The com-

parative DDCT calculation involved finding the differ-

ence between DCT of irradiated cells and the mean

value of the DCT from the control cells. Fold increase in

the expression of specific mRNA in irradiated cells

compared to control cells was calculated as 2–(DDCT).

The data are expressed as relative quantity (RQ) and

differences are shown in the figures as the expression

ratio of the normalized target gene according to the

software results.

Western blotting

For investigating the expression of HSP47 protein in

dental pulp cells, immunoblot analysis was performed.

The extracts were prepared from irradiated or non-

irradiated cells using lysis buffer [100 mmol L)1 Tris–

HCl (pH 7.2) containing 150 mmol L)1 NaCl,

0.1 mmol L)1 DTT/EDTA, 0.1% Triton X-100]. The

protein concentrations were determined using protein

assay kit (Bio-Rad). Protein (20 lg) was loaded onto

Effect of laser irradiation Yasuda et al.


10% SDS–PAGE gel. After electrophoresis, the SDS–

PAGE separated proteins were transferred to nitrocel-

lulose membrane at 60 V for 2 h. The membrane was

blocked with 10% bovine serum albumin in TBST

[10 mmol L)1 Tris–HCl (pH8.0), 150 mmol L)1 NaCl,

0.05% Tween 20] for 30 min, and incubated with a

1 : 1000 dilution of polyclonal rabbit IgG against

human HSP47 (Stressgen, Ann Arbor, MI, USA) in

TBST for 1 h. Then, the membrane was incubated

with a 1 : 2000 dilution of goat anti-rabbit IgG

conjugated with horseradish peroxidase (Sigma) for

1 h. Horseradish peroxidase activity was detected using

the ECL system (Amersham Biosciences, Piscataway,

NJ, USA).


Statistical analysis was performed with data obtained

from three independent experiments. The data are

expressed as mean ± SD and analysed using one-way

analysis of variance (anova) followed by the Tukey’s

multiple comparison test. Statistical significance was

accepted at P < 0.05.

Results

Rat dental pulp cells were irradiated with a carbon

dioxide laser at 2 W output power for 20, 40, and 60 s.

Thereafter, the cell viability was measured 24 h after

irradiation (Fig. 1a). There was no difference in the cell

viability between the control and the cells which were

irradiated 20 or 40 s. However, it was significantly

decreased by 20% in the cells which were irradiated for

60 s in comparison to the controls (P < 0.05). Next,

the effect of laser irradiation was examined on the

mineralization in dental pulp cells. The cells which

were irradiated for 40 s had a clearly increased number

and total area of calcified nodules stained by Alizarin

Red S (Fig. 1b). In addition, when the mineralization

was determined quantitatively on day 10, the cells with

40-s irradiation had significantly increased the degree

of mineralization in comparison to the other conditions

(P < 0.05). However, no significant differences were

observed between the controls and the cells with 20- or

60-s irradiation (P > 0.05; Fig. 1c).

Next, the culture media were collected every 2 days

up to 10 days and the amount of extracellular secreted

collagen was determined quantitatively after Sirius Red

staining. The amount of secreted collagen significantly

increased after laser irradiation in comparison to the

controls 73% and 38% on day 2 and 4, respectively

(P < 0.05; Fig. 2). However, there was no difference in

comparison to the controls after day 6.

To clarify the mechanism of increased collagen

secretion after irradiation, the effect of laser irradiation

on the expression of collagen type I and HSP47 was

examined by real-time PCR method. There was no

significant difference in the expression of the collagen

type I gene between the irradiated cells and the controls

at any time point (P > 0.05; Fig. 3a). Interestingly, the

expression of the HSP47 gene in the irradiated cells was

significantly increased compared to the controls by

54%, 57% and 24% at 12, 24 and 48 h, respectively

(P < 0.05). In addition, Western blot analysis showed

that HSP47 protein with a molecular weight of 47 kDa

was increased in the cells 24 h after irradiation

compared to that in control cells (Fig. 3b).

Discussion

A carbon dioxide laser has a photothermal effect that is

applied when making incisions in soft tissue and

obtaining haemostasis. It also has a photochemical

0

0

0.1Aliz

arin

red

S(m

g/m

L)

0.2

0.3

0.4

0.5

Control

Control

2 W, 40 s2 W20 s 40 s 60 s

2 W 2 W

Control 2 W20 s 40 s 60 s

2 W 2 WC

ell v

iabi

lity

(% o

f con

trol

)

20

40

60

80

100

*

* * *

(a) (b)

(c)

Figure 1 Effect of laser irradiation on cell viability and miner-

alization of dental pulp cells. (a) Cell viability after carbon

dioxide laser irradiation at 2 W for 20, 40 and 60 s was

analysed by a modified MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) assay. (b) After laser irradiation

at 2 W for 20, 40 and 60 s, rat dental pulp cells were cultured in

DMEM containing AA and b-GP for 10 days. Representative

photographs of Alizarin Red S staining are shown (Original

magnification 200 · ). (c) Quantification of Alizarin Red S

staining. Bar represents mean ± SD (n = 3). The data were

analysed using one-way anova: *P < 0.05 versus control.



effect used in alleviating pain (Posten et al. 2005).

Melcer et al. (1987) reported that neo-dentine bridge

formation was observed in pulp tissue after carbon

dioxide laser irradiation on teeth of dogs and monkeys.

This finding indicates that carbon dioxide laser may be

useful for the induction of mineralization. The current

study showed a decrease of cell viability for irradiation

at 2 W for 60 s using a carbon dioxide laser. In other

words, if the irradiation time is long, then the energy

density is increased even with a low power laser and

injuries to cells occur. Therefore, the effect of carbon

dioxide laser on mineralization, collagen secretion and

HSP47 expression was examined under conditions

(2 W, 40 s) that did not injure the cells.

Collagen, which constitutes almost 90% of the

dentine matrix protein, is synthesized by the odonto-

blasts and secreted into predentine, where collagen

molecules are arranged into fibres. These fibres form

the collagenous network in which the mineral crystals

are deposited (Linde 1989). Furthermore, it has been

reported that collagen type I time-dependently stimu-

lates the expression of osteopontin and alkaline phos-

phatase (ALP), whilst also inducing the differentiation

of bone marrow cells into osteoblasts (Mizuno & Kuboki

2001). The DGEA (Asp-Gly-Glu-Ala) domain of type I

collagen binds with integrin on the cellular membrane.

Differentiation is thought to be promoted through its

binding with integrin (Mizuno et al. 2000). In this

study, carbon dioxide laser irradiation significantly

increased in the secretion of collagen into culture

media on day 2 and 4. This finding suggests that

increased collagen in culture media acted on the

integrin of the cells, and mineralization was thus

02 4 6 8 10

Day

ControlIrradiation

* *

#S

ecre

ted

colla

gen

(μg/

wel

l)

10

20

30

40

Figure 2 The amount of collagen secreted into the culture

media of dental pulp cells. The dental pulp cells were irradiated

at 2 W for 40 s and cultured in DMEM supplemented with AA

and b-GP for 10 days. Conditioned media from control and

irradiated cells were collected every 2 days. The amount of

collagen (lg per well) was measured by the Sirius Red staining

method. Bar represents mean ± SD (n = 3). The data were

analysed using one-way anova. Significantly different from

the control at each time point: *P < 0.05. Significantly

different from the day 2: #P < 0.05.

00 12 24

Control

Irradiation#

##

#

##

##

**

*

# #

48 72

24 h– + Irradiation

HSP47

0 12 24 48 72(h) (h)

Rel

ativ

e qu

ality

(col

lage

n ty

pe I/

GA

PD

H)

Rel

ativ

e qu

antit

y(H

SP

47/G

AP

DH

)

0.5

1.0

1.5

2.0

2.5

2.5

3.0

1.5

2.0

0.5

0

1.0

(a)

(b)

Figure 3 The expression of collagen type I and HSP47 in control and irradiated cells. (a) The dental pulp cells were irradiated at

2 W for 40 s and cultured in DMEM supplemented with AA and b-GP. The mRNA expression of collagen type I and HSP47 was

analysed at the indicated time points by real-time PCR. Bar represents mean ± SD (n = 3). The data were analysed using one-way

anova. Significantly different from the control at each time point: *P < 0.05. Significantly different from time 0: #P < 0.05. (b)

The HSP47 protein expression was examined by Western blotting.



stimulated. Most recently, Lee et al. (2008) reported

that heat stress at 42 �C for 30 min significantly

elevated ALP activity on days 7 and 14 in rat pulp

cells compared to control groups, revealing the possi-

bility that heat stress generated by laser elevated ALP

activity, thereby stimulating mineralization.

HSP47 knockout mice cannot produce collagen with

the correct triple helix. Therefore, they die by 11.5 days

post-coitus due to apoptosis in various tissues and

vascular ruptures because they cannot form collagen

fibres and basement membranes (Nagai et al. 2000). In

addition, HSP47 expression is induced by thermal

stimuli, and its constitutive expression is closely coupled

with the amount of the collagenmatrix. For example, an

increase in the HSP47 expression has been reported in

pulmonary fibrosis in which there is increased produc-

tion of collagen (Razzaque et al. 1998). Therefore,

HSP47 expression, which has a close relationship with

collagen production, was examined. The results clearly

showed that the HSP47 gene was induced by laser

irradiation within 12 h and HSP47 protein was induced

within 24 h. The observation that the expression level

of HSP47 was correlated with the amount of collagen

secretion is consistent with the findings of a previous

study (Razzaque et al. 1998). Although collagen gene

expression was not altered by carbon dioxide laser

irradiation, extracellular collagen secretion did increase.

Regarding this discrepancy, increased HSP47 produc-

tion by laser irradiation have led to efficient assembly of

procollagen molecules prior to their secretion, thereby

promoting extracellular collagen secretion (Lamande &

Bateman 1999).

To date, the studies of the mechanism of minerali-

zation induction by laser have been conducted using a

low power laser. Irradiation by a low power laser on

osteoblasts resulted in increased expression of ALP and

osteocalcin (Ozawa et al. 1998, Ohbayashi et al. 1999,

Ueda & Shimizu 2003). It has been reported that these

increases are one cause of mineralization induction.

Hamajima et al. (2003) indicated that the gene expres-

sion of a bone-inducing factor called osteoglycin

increased by twofold within 2 h when MC3T3-E1

osteoblast-like cells were irradiated by a low power

laser. These findings indicate that the mechanism of

mineralization induction might differ according to the

cells, type of laser, and irradiation conditions.

Conclusion

Carbon dioxide laser irradiation stimulated collagen

production and calcified nodules formation on rat

dental pulp cells. Furthermore, laser irradiation en-

hanced HSP47 gene and protein expressions but not

type I collagen gene expression. Further study will be

needed to elucidate the role of HSP47 on laser-induced

mineralization in dental pulp cells.

Acknowledgements

This work was supported by Grant-in-Aid for Scientific

Research 18659563, and Grant-in-Aid for Young

Scientists (B) 18791407 and 20791390 from the

Japan Society for the Promotion of Science, and by a

Grant from the Research Center, Health Sciences

University of Hokkaido. The authors give special thanks

to Toru Kawamorita (Division of Clinical Cariology

and Endodontology, Department of Oral Rehabilitation,

School of Dentistry, Health Sciences University of

Hokkaido) for his technical assistance in the molecular

biology portion of this study.

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Torsional behaviour of rotary NiTi ProTaperUniversal instruments after multiple clinical use

E. P. Vieira1, R. K. L. Nakagawa1, V. T. L. Buono2 & M. G. A. Bahia1

1Department of Restoration Dentistry, Faculty of Dentistry, Federal University of Minas Gerais, Belo Horizonte-MG, Brazil; and2Department of Metallurgical and Materials Engineering, Engineering School, Federal University of Minas Gerais, Belo Horizonte-

MG, Brazil

Abstract

Vieira EP, Nakagawa RKL, Buono VTL, Bahia MGA.

Torsional behaviour of rotary NiTi ProTaper Universal instru-

ments after multiple clinical use. International Endodontic

Journal, 42, 947–953, 2009.

Aim To assess the influence of multiple clinical uses

on the torsional behaviour of ProTaper Universal

rotary NiTi instruments.

Methodology Root canal treatments were per-

formed on patients using the ProTaper Universal rotary

system to prepare canals. Ten sets of instruments were

used by an experienced endodontist, each set being

used in five molar teeth. After clinical use, S1, S2, F1

and F2 instruments were analysed for damage by

optical and scanning electron microscopy. The used

sets, along with a control group of 10 sets of new

instruments, were then torsion tested based on the ISO

3630-1 specification. Data obtained were subjected to a

one-way analysis of variance (anova) with a = 0.05.

Results The use of the ProTaper Universal rotary

instruments by an experienced endodontist allowed for

the cleaning and shaping of the root canal system of

five molar teeth without fracture. The maximum

torque for instruments S2, F1 and F2, and the angular

deflection at fracture for instruments S2 and F1 were

significantly lower following clinical use. The largest

decrease in maximum torque was 18.6% (P = 0.014)

for S2 instruments. The same maximum percent

decrease was found for angular deflection at fracture

for F1 instruments (P = 0.009).

Conclusions Torsional resistance and angular

deflection of used instruments, as compared to that

of new instruments, were reduced following clinical

use.

Keywords: clinical use, endodontic instruments,

nickel–titanium, ProTaper Universal, torsional resis-

tance.

Received 21 January 2009; accepted 28 April 2009

Introduction

Reasons for the fracture of rotary NiTi instruments

include variations in canal anatomy, such as merging,

curving, re-curving, dilacerating or dividing canals

(Ruddle 2002). In addition, other factors can affect the

fracture resistance of endodontic instruments, such as

size, taper, alloy composition, manufacturing methods,

flexibility and rigidity, instrument shape and direction

of rotation (Hilt et al. 2000). The cross-sectional profile

also has a significant influence on the mechanical

behaviour of NiTi instruments (Schafer et al. 2003,

Melo et al. 2008). The factors affecting the performance

include the depth of the flute, the area of the inner core,

the radial land and the peripheral ground surface

(Gambarini 2005, Xu & Zheng 2006).

The fatigue life of a rotary endodontic instrument is

related to the degree to which it is flexed when placed

in a curved root canal, with greater flexures leading to

a shorter fatigue life expectation (Pruett et al. 1997,

Melo et al. 2002, Bahia & Buono 2005). Torsional

failure occurs when the tip or another part of the

instrument is locked in the canal, whilst the shaft

continues to rotate. If the elastic limit of the metal is

Correspondence: Vicente T. L. Buono, Professor, Department of

Metallurgical and Materials Engineering, Federal University of

Minas Gerais, Rua Espırito Santo 35 room 206, 30160-030,

Belo Horizonte, MG, Brazil (Tel.: +55 31 3409 1859; fax: +55

31 3409 1815; e-mail: [email protected]).

doi:10.1111/j.1365-2591.2009.01602.x


exceeded, the instrument undergoes plastic deforma-

tion, which can be followed by fracture if the load is

high enough (Blum et al. 1999, Gambarini 2000).

Peters et al. (2003) established that torque is corre-

lated not only with apically exerted force, but also with

preoperative canal volume. Hence, the preparation of

narrow and constricted canals can subject rotary NiTi

instruments to higher torsional loads and high-apically

directed forces. The problem of fracture by torsional

overload has been dealt with by determining the

maximum torque at separation for each type of

instrument and then using low-torque endodontic

motors, which can be programmed in such a way as

to avoid the application of torque values higher than

that of each instrument can support without failing.

Nevertheless, this approach does not take into account

the fact that fatigue loads developed during curved root

canal shaping may decrease the torsional resistance of

endodontic instruments. This effect was studied by

various authors (Yared et al. 2003, Ullmann & Peters

2005, Bahia et al. 2006), who reported a reduction in

the maximum torque to failure for all instruments

evaluated.

The reuse of rotary instruments of NiTi is a constant

concern. The cumulative effects of multiple clinical uses

on the incidence of fatigue, deformation and instru-

ment separation have been analysed (Yared et al.

2000, Gambarini 2001, Fife et al. 2004, Bahia &

Buono 2005), with the conclusion that their clinical

reuse progressively reduced their resistance to fatigue.

During canal preparation, especially in curved root

canals in molar teeth, these instruments are submitted

to a high degree of cyclic deformation that may

consume a considerable amount of their fatigue life

(Bahia & Buono 2005).

In a recent study, Vieira et al. (2008) observed that

the flexural fatigue resistance of ProTaper instruments,

used clinically by an experienced endodontist for the

cleaning and shaping of five molars, was reduced up to

52% when compared with that of new instruments.

The present work was undertaken to assess the

influence of multiple clinical uses on the torsional

behaviour of ProTaper Universal rotary NiTi instru-

ments.


Twenty sets of ProTaper Universal instruments (Dents-

ply Maillefer, Ballaigues, Switzerland), type S1, S2, F1

and F2, totalling 88 files, were analysed. They were

divided into two groups: (i) control group (CG), with 10

sets of new instruments tested in torsion until fracture

to establish the mean values of maximum torque and

angular deflection at fracture for each type of instru-

ment and (ii) experimental group (EG), with 10 sets of

instruments, each set used clinically by an endodontist

with experience using the ProTaper Universal system in

five molar teeth to shape between 15 and 20 root

canals. The instruments of the EG were tested subse-

quently in torsion until fracture. The SX and F3

instruments used in the clinical procedures were not

included in the study, since these instruments work

only in the straight portion of the canals (SX) or in

preparation of straight canals (F3).

Direct and angled radiographs of each tooth were

obtained using a paralleling technique to evaluate

anatomy, as well as to determine the canal radius and

angle of curvature, as defined by Pruett et al. (1997),

and its approximate length. The measurement of these

parameters was performed by projecting the radio-

graphic images using a profile projector (Mitutoyo,

Tokyo, Japan) at 10 · magnification. The canal radius

of curvature was measured along the outer canal wall.

After the orifices were located and the canal explored

with sizes 10 and 15 stainless steel K-files (Dentsply

Maillefer), cleaning and shaping of the canals were

completed in accordance with a crowndown technique

recommended by Ruddle (2005). Once a glide path had

been created, the ProTaper Universal shaping instru-

ments were used like a ‘brush’ to laterally and

selectively cut dentine on the outstroke. The prepara-

tion was finished using the ProTaper Universal finish-

ing instruments F1 and F2 in a ‘nonbrushing’ manner.

The clinical protocol was followed with recapitulations

until the working length, established at 0.5 mm of the

canal patency length, could be reached by at least an

F2 instrument, at which point shaping was considered

complete.

A 5.25% sodium hypochlorite solution was used for

irrigation and Rc-prep (Premier Dental Products, Nor-

ristown, PA, USA) was used as a lubricant. The

rotational speed was 300 rpm, applied by an endodon-

tic electric motor (Endo Plus, VK Driller, Sao Paulo, SP,

Brazil), operating at a torque of 5 NÆcm together with a

hand piece of 16 : 1 reduction (W&H 975, Dentalwerk,

Burmoos, Austria).

After use in each patient, the instruments were

washed, ultrasonically cleaned for 5 min in ethanol

and steam autoclave sterilized. The S1, S2, F1 and F2

instruments of the EG were observed by optical

microscopy (Mitutoyo TM, Tokyo, Japan), at 30 ·magnification, to determine the presence of distortion,

Torsional behaviour of clinically used ProTaper Universal Vieira et al.


unwinding defects and macroscopic deformation.

Before torsion testing, three sets of instruments were

randomly selected and examined by scanning electron

microscopy (SEM) (Jeol JSM 6360, Tokyo, Japan) to

assess their surface characteristics.

Torsion testing was based on ISO 3630-1 specifica-

tion, and using a torsion machine (Analogica, Belo

Horizonte, MG, Brazil) was described in Bahia et al.

(2006). The rotation speed was set clockwise to 2 rpm.

The end of the shaft was clamped into a chuck

connected to a reversible geared motor. Three millime-

tres of the instrument’s tip was clamped in another

chuck with brass jaws to prevent sliding. Continuous

recording of torque and angular deflection, as well as

measurements of the maximum torque and angular

deflection to failure, was provided by a specifically

designed computer program.

To determine the statistical significance of differences

in the measured parameters amongst different groups,

data obtained were subjected to a one-way analysis of

variance (anova). Significance was determined at the

95% confidence level.

Results

During the clinical part of the study, none of the

instruments fractured or deformed permanently. The

mean values (and standard deviations) of radius and

angle of curvature characterizing the geometry of the

root canals of the 50 molars instrumented with the 10

sets of files (five molars for each set) were 4.0 mm

(1.7 mm) and 33.1� (11.1�), respectively.The results of the torsion tests are summarized in

Fig. 1, which shows mean values of the maximum

torque and angular deflection at fracture of new

instruments (CG) and of those previously used in the

clinical practice (EG). As is common, torsional resis-

tance increased as the diameter of the instruments

increased, with the mean values of maximum torque

appearing statistically different when instruments in

the CG were compared one to another: S1–S2, S2–F1

and F1–F2. A similar tendency was observed for

angular deflection at fracture in the CG, and statisti-

cally significant differences were found when compar-

ing instruments S1 with S2, and F1 with F2, but not

when comparing S2 with F2 instruments.

The mean values in Fig. 1 indicated that multiple

clinical uses caused a reduction in maximum torque

and angular deflection at fracture of ProTaper Uni-

versal instruments. Comparison between the values of

maximum torque, measured for the same type of

instruments from the Control and EGs, showed that

this tendency was significant for instruments S2

(P = 0.014), F1 (P = 0.007) and F2 (P = 0.006), but

not for S1 (P = 0.475). When a similar analysis was

performed for angular deflection at fracture, statisti-

cally significant reduction in this parameter was found

for S2 (P = 0.003) and F1 (P = 0.009) instruments,

but not for S1 (P = 0.546) and F2 (P = 0.097).

After canal shaping, all instruments examined by

SEM had microcracks and widening of machine

grooves, as well as wear and blunting of the cutting

edges. These surface characteristics were qualitatively

similar in all three sets of randomly selected instru-

ments of the EG. The SEM images shown in Fig. 2

illustrate typical microcracks found in used S2 instru-

ments. The majority of the cracks were transverse to

(a)

(b)

Figure 1 Mean values of maximum torque (a) and angular

deflection at fracture (b) of ProTaper Universal instruments

from the control and experimental groups. Error bars represent

the standard deviations.

Vieira et al. Torsional behaviour of clinically used ProTaper Universal


the cutting edge (Fig. 2a), but longitudinal cracks,

parallel to the long axis of the instrument, were also

observed (Fig. 2b).

Discussion

The torsional behaviour of rotary NiTi endodontic

instruments is affected by a variety of factors, such as

size, taper, design, alloy chemical composition and

thermomechanical processes applied during manufac-

turing (Kuhn & Jordan 2002, Bahia et al. 2005, Miyai

et al. 2006). Nevertheless, there is a strong relationship

between the maximum torque an instrument can

withstand and its diameter (Peters & Barbakow 2002,

Bahia & Buono 2005). It has also been suggested that

the cross-sectional shape of instruments affects the

stress distribution pattern as well as their torsional

properties (Turpin et al. 2000, Berutti et al. 2003, Melo

et al. 2008, Camara et al. 2009, Kim et al. 2009). The

results for the CG depicted in Fig. 1 are thus in

agreement with the general observation that the

maximum torque of endodontic instruments increases

as instrument diameter becomes larger. On the other

hand, measurements of angular deflection at fracture

showed that this parameter does not correlate with

instrument diameter in the same way (Gambarini

2001, Bahia et al. 2006). The results shown in Fig. 1

for new instruments confirm this observation.

In straight root canals, rotary endodontic instru-

ments operate by cutting and removing organic tissue

and debris, experiencing mostly frictional forces, which

run in opposition to their torsional motion. However,

when the instrument rotates inside a curved root canal,

it is bent and thus submitted to tensile–compressive

strain cycles in the region of the canal curvature, in

addition to the torsional restraints. The strain levels

attained by endodontic instruments during this cyclic

loading depend on the root canal and instrument

geometries, being concentrated at the portion of the

instrument positioned in the maximum curvature

region of the root canal (Bahia & Buono 2005, Cheung

& Darvell 2007). These cyclic forms of stress cause

flexural fatigue, involving crack nucleation and

growth. The value of the tensile strain amplitude, eT,on the surface of an instrument of diameter D inserted

into a canal of radius of curvature R can be estimated

by the expression:

eT ¼ D

2R� Dð1Þ

which is valid when the canal radius is measured at the

outer canal wall (Bahia & Buono 2005), as was done in

the present study. Alternatively, when R is measured at

the canal central axis, this expression becomes (Cheung

& Darvell 2007):

eT ¼ D

2Rð2Þ

If the maximum amplitude is assumed to occur at

3 mm from the instrument tip, the region of the

instrument subject to the maximum tensile strain

amplitude is D3. Table 1 shows the values of D3

measured for ProTaper Universal instruments by

Camara et al. (2009) and the corresponding estimated

values of eT, calculated using equation 1 for the

average radius of curvature, 4.0 mm, of the root

canals instrumented in the present study.

Cyclic flexural straining by the amounts shown

in Table 1 would certainly cause damage to the

(a)

(a)

Figure 2 SEM images of the surface of an S2 ProTaper

Universal instrument used for the cleaning and shaping of

five molars showing (a) cracks transversal to the cutting edge

and (b) longitudinal cracks.



instruments. The microcracks exemplified in Fig. 2

constitute evidence of this damage. The presence of

longitudinal cracks, that is, cracks parallel to the long

axis of the file, has previously been described (Peng

et al. 2005, Tripi et al. 2006, Vieira et al. 2008), and is

thought to reflect the direction of the stress on the

surface of the instrument under torsional load. Similar

cracking patterns have been observed on other rotary

NiTi endodontic instruments subjected to cyclic tor-

sional straining (Bahia et al. 2008). During this type of

cyclic deformation, planes with a maximum shear

stress are either perpendicular or parallel to the

longitudinal axis, whilst the normal stress component

on the slip plane is zero. Microscopic investigations

have shown that microcracks nucleate in a slip band

under cyclic torsion and then grow further in a

direction perpendicular to the main stress. In a

cylindrical bar, this direction makes an angle of 45�with the axis of the bar. Consequently, cracks in a

round axle under cyclic torsion grow in the form of a

spiral around its surface (Schijve 2001). The longitu-

dinal appearance of the cracks observed in endodontic

instruments is because of the fact that the instruments

have helical shapes and that the cracks, being rather

small in size, require large magnifications to be

observed (Bahia et al. 2008).

When the torsional resistance of similar instru-

ments belonging to CG and EG was compared, a

tendency for this property to decrease with the clinical

use in five molars was observed for all instruments

analysed (Fig. 1). This tendency was statistically

significant for S2, F1 and F2 instruments. Previous

studies (Yared et al. 2003, Ullmann & Peters 2005,

Bahia et al. 2006) reported that simulated clinical use

lowered the mean values of maximum torque when

compared with that of new instruments. Regarding

the behaviour of angular deflection at fracture, Yared

et al. (2003) and Ullmann & Peters (2005) found no

statistically significant changes in this parameter

between new instruments and those submitted to

simulated clinical use. In the present study, angular

deflection at fracture tended to decrease for the used

instruments (Fig. 1b) and statistically significant

decreases were found for S2 and F1 instruments. This

result confirms previous findings on ProFile instru-

ments submitted to simulated clinical use (Bahia et al.

2006). However, it is important to mention that

angular deflection at fracture has little clinical signif-

icance, because at a typical rotational speed of

300 rpm, one complete revolution of a tip-locked

instrument will occur in one-fifth of a second. Thus,

differences in this parameter will not be perceived in

clinical practice.

The reduction in maximum torque measured in the

present study were, on average, 6%, 19%, 12% and

13% for S1, S2, F1 and F2 ProTaper Universal

instruments, respectively. These results confirmed the

role played by flexural fatigue in the torsional resis-

tance of these instruments. However, in a previous

work (Vieira et al. 2008) considerably higher values

were found for the reduction of flexural fatigue life of

ProTaper instruments clinically employed for the

cleaning and shaping of five molars: 33%, 52%, 45%

and 44% for S1, S2, F1 and F2 instruments, respec-

tively. Taken together, these results indicated that the

cumulative effects of multiple clinical uses on rotary

NiTi endodontic instruments have a stronger influence

on flexural fatigue behaviour than on their torsional

resistance.

Although flexural fatigue appears to have a

cumulative effect on rotary endodontic instruments,

causing weakening over time, clinical studies have

failed to demonstrate the extent of the cumulative

effects of multiple clinical uses on the fatigue resis-

tance of these instruments. For instance, Fife et al.

(2004) did not observe statistically significant differ-

ences when the remaining fatigue life of ProTaper

instruments used in two and four molars were

compared, whilst Vieira et al. (2008) obtained a

similar result after shaping of five and eight molars.

Moreover, simulated clinical use of ProFile instru-

ments up to one of two and three-fourth of their

fatigue life (Bahia et al. 2006) and of ProTaper

instruments up to 30%, 60% and 90% of their

fatigue life (Ullmann & Peters 2005) did not signif-

icantly alter their torsional resistance when the pre-

strained instruments were compared. These results

were interpreted as indicating that crack nucleation

occurs early during flexural fatigue of NiTi rotary

instruments, low-crack growth occupying a large

fraction of their low-cycle fatigue life (Bahia & Buono

2005).

Table 1 Diameter of the ProTaper instruments at 3 mm from

their tip, D3, and corresponding maximum tensile amplitudes,

eT, estimated for the average radius of curvature of 4.0 mm

Instrument D3 (mm)a eT (%)

S1 0.29 3.8

S2 0.35 4.6

F1 0.42 5.5

F2 0.50 6.7

aCamara et al. (2009).



Conclusions

Torsional resistance of used instruments was reduced

by average amounts varying from 6% to 19%, when

compared with that of new instruments. Structural

fatigue took place during the clinical use of the

instruments and, in addition to the usual transversal

cracks generate by flexural fatigue, longitudinal cracks

were also observed on the surface of the used instru-

ments. Comparisons with data on ProTaper instru-

ments indicate that the cumulative effects of multiple

clinical uses on rotary NiTi endodontic instruments

have a stronger influence on flexural fatigue behaviour

than on their torsional resistance.

Acknowledgements

This work was partially supported by Fundacao de

Amparo a Pesquisa do Estado de Minas Gerais –

FAPEMIG, Belo Horizonte, MG, Brazil and Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico –

CNPq, Brasılia, DF, Brazil.

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Erratum

Somma F, Leoni D, Plotino G, Grande NM, Plasschaert A. Root canal morphology of the mesiobuccal root of

maxillary first molars: a micro-computed tomographic analysis. International Endodontic Journal 42, 165–74. 2009.

The above paper did not include the following acknowledgements:

Acknowledgements

The authors express their gratitude to Drs. Rossella Bedini and Drs. Raffaella Pecci and to ISS Italian Superior

Health Institute for the micro CT scan images and reconstructions.

The publisher apologizes for this error.

doi:10.1111/j.1365-2591.2009.01628.x

International Endodontic Journal, 42, 954, 2009 ª 2009 International Endodontic Journal954

iej.10.2009

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