friedhelm heinemann* times after augmentation of trial...today, teeth replacement with...

Luigi Canullo*Friedhelm Heinemann*Tomasz GedrangeReiner BiffarChristiane Kunert-Keil

Histological evaluation at differenttimes after augmentation ofextraction sites grafted with amagnesium-enriched hydroxyapatite:double-blinded randomized controlledtrial

Authors’ affiliations:Luigi Canullo*, Private Practice, Rome, ItalyFriedhelm Heinemann*, Reiner Biffar, Departmentof Prosthodontics, Gerostomatolgy andBiomaterials, University Medicine of Greifswald,Greifswald, GermanyTomasz Gedrange, Deptartment of Orthodontics,Technische Universitat Dresden, Dresden,GermanyChristiane Kunert-Keil, Department ofOrthodontics, University Medicine of Greifswald,Greifswald, Germany

Corresponding author:Dr Luigi CanulloVia Nizza, 4500198 Rome, ItalyTel.: +39 06 8411980Fax: +39 06 8411980e-mail: [email protected]

Key words: bone augmentation, graft material, histomorphometry, hydroxyapatite

Abstract

Objectives: To histologically analyze the early angiogenesis–osteogenesis interplay in post-

extraction sockets augmented with magnesium-enriched hydroxyapatite (Mg-enriched HA).

Material and Methods: Ten post-extraction sites underwent post-extraction ridge preservation

procedure. According to randomization, sites were divided into two balanced groups and bone

specimens were collected 2 or 4 months after surgery. Sections were stained with hematoxylin/

eosin, Masson–Goldner trichrome, and tartrate-resistant acid phosphatase (TRAP), respectively.

Furthermore, indirect immunohistochemistry was performed using alkaline phosphatase, CD34 and

caveolin-1 antibodies. Mean values and standard deviations were calculated for each outcome

variable. Data were then compared using one-way ANOVA test. P < 0.05 was considered

statistically significant.

Results: Histomorphometric analysis presented 15.0% (±3.5) regenerated bone after 2 months of

healing. After 4 months, regenerated bone increased 5.1-fold up to 77.4% (± 8.6) (P < 0.001). On

the contrary, vessels and capillary reduced from 645 (±33) to 255 (± 94) (caveolin-1 expression,

P = 0.008). These findings were confirmed by CD34 expression (301 ± 95 and 88 (±24), respectively,

at 2 and 4 months (P = 0.046).

Conclusions: Within the limits of the present randomized controlled study, it can be concluded

that Mg-enriched HA is a suitable material for socket preservation and ensures early angiogenesis

and early osteogenesis.

Today, teeth replacement with implant-sup-

ported prostheses is a predictable option of

therapy. However, nowadays, esthetic

demand requires a prosthetically driven

approach. Thus, there is a high interest in

minimizing tissue resorption after tooth

extraction and maintain the contour of the

alveolar crest. In fact, it is well known that

the alveolar crest reduces in volume after

tooth extraction, especially during the first

6 months and mostly on its buccal wall (Sch-

ropp et al. 2003).

It has been demonstrated that augmenta-

tion material does not prevent, it only

reduces resorption process especially at the

buccal wall (Caneva et al. 2011). Using post-

extraction ridge preservation procedure, 85%

of the initial ridge dimensions could be

preserved, allowing a correct implant place-

ment (Cornelini et al. 2004; Chen et al.

2007). On the other hand, immediate

implant placement without bone grafting

was shown not to prevent this process

(Caneva et al. 2011).

New generation of hydroxyapatites (HAs),

so-called biomimetic scaffolds, simulates

bone structure not only from a chemical

point of view but also microscopically, recon-

structing micropores and their interconnec-

tions. Investigation of the healing of a nano-

crystalline synthetic HA demonstrated

sprouting vessels from pre-existent host ves-

sels enter the granules. Later, they form an

intergranular network that probably trans-

ports osteoblastic precursor cells into the

granules (Gotz et al. 2008). This may be

*These authors contributed equally to this work and areconsidered as joint first authors.

Date:Accepted 30 July 2012

To cite this article:Canullo L, Heinemann F, Gedrange T, Biffar R, Kunert-KeilC. Histological evaluation at different times afteraugmentation of extraction sites grafted with a magnesium-enriched hydroxyapatite: double-blinded randomizedcontrolled trial.Clin. Oral Impl. Res. 24, 2013, 398–406doi: 10.1111/clr.12035

398 © 2012 John Wiley & Sons A/S

relevant for osteoconductive and probably

also osteoinductive features leading to

early osteogenesis and subsequent remodel-

ing of the newly formed bone (Gotz et al.

2010).

Within the graft material category of HAs,

partial substitution of HA with magnesium

(Mg-enriched) results in a non-stoichiometric

structure. Because this kind of material is

chemically and stoichiometrically similar to

bone mineral matrix and the process of

sintering is missing in production process,

Mg-enriched HA is completely resorbed after

6–12 months (Trombelli et al. 2010).

It was shown that autogenous bone biop-

sies provided higher vital over comparable

total bone levels than Mg-enriched HA after

5 months of healing, but Mg-enriched HA

grafts revealed higher expression of certain

specific markers of osteoblast differentiation

and bone formation, associated with a lower

osteoclastogenic potential (Crespi et al.

2009c). Further, it was proven for socket pres-

ervation procedure that, 3 months after

placement, Mg-enriched HA could reduce

bone resorption more than calcium sulfate

and control group. Coincidently, Mg-enriched

HA revealed clearly more residual material

and less vital bone than calcium sulfate and

control groups (Crespi et al. 2009b). After 2–

3 months, implants have been inserted, and

thereafter, bone resorption was neglectable

up to 24 months (Crespi et al. 2009a; Sisti

et al. 2012).

No study has been leaded so far regarding

the transition from 3-month results with

histologically residual material in less vital

bone with clinically reduced resorption and

long-term histologically complete resorption

with clinically proven stability of augmented

areas. Investigations on immunohistochem-

istry of Mg-enriched HA osteogenesis are

missing as well. This gave reason to focus

on the angiogenesis–osteogenesis interplay.

Early angiogenesis and osteogenesis are

determinants for establishment of healthy

natural bone formation in augmented

regions. Angiogenesis, in fact, means the

ingrowth of new vessels from pre-existent

ones in the local bone and is caused by acti-

vation of endothelial and vessel wall stem

cells (Adams & Alitalo 2007). It is consid-

ered to be the most important process for

healing and osteogenesis of implanted sub-

stitutes (Santos & Reis 2010; Gotz et al.

2011). Only thereafter, the new bone takes

part in vital biological remodeling process

with surrounding earlier bone to ensure

long-term bone stability (Chiapasco et al.

2006).

In the present trial, traditional histologic

stainings and immunohistologic antibodies

were adopted for evaluation of vessels and

bone formation. Additionally, caveolin-1

antibody was added, for the first time in a

study on bone regeneration in dental field.

The protein caveolin-1 is a principle compo-

nent of caveolae, which are subcompart-

ments of plasma membrane in vessels and

capillaries (Rothberg et al. 1992). For these

reasons, caveolin-1 antibody was demon-

strated to be ideal as a marker for quantity

and quality of vessels (Sawada et al. 2007;

Hada et al. 2012). Moreover, caveolin-1 has

been identified to interact with variety of cel-

lular proteins, and it plays a role in different

metabolism and disease processes (Tamaskar

& Zhou 2008; Le Lay et al. 2009). Further-

more, it takes function in bone metabolism

(Sawada et al. 2007; Hada et al. 2012), and its

indirect analysis allows for identification of

osteoclasts and osteoblasts. (Gerger et al.

2004).

Aim of this study was to histologically and

immunohistochemically analyze the early

vascularization and the osteogenesis–angio-

genesis interplay of the post-extraction sites

after augmentation, using a magnesium-

enriched HA (SINTlife®; Finceramica, Faenza,

Italy).

This nano-structured hydroxyapatite pre-

sents in fact chemical and morphologic prop-

erties close to natural bone. Its porosity was

demonstrated to reach 90% of the volume,

with macro-pores ranging between 2 and

5 lm and pores of interconnection ranging

between 0.8 and 0.2 lm (Fig. 1a,b). Addition-

ally, partial substitution of HA with magne-

sium (Mg) results in a non-stoichiometric

structure, and because it is more similar to

bone mineral matrix, it was demonstrated to

be completely resorbed after 6–12 months

(Trombelli 2010).

Materials and methods

This study was performed following the prin-

ciples outlined in the Declaration of Helsinki

in 2008 on experimentation involving human

subjects and was approved by the ethical

committee of the University of Greifswald

(Reg-Nr.: BB 13/11a). The design of this study

was a randomized controlled prospective clin-

ical trial.

Patient selection

Any patients requiring at least one tooth

extraction in the maxillary premolar area

who were at least 18 years old and able to

sign an informed consent form were eligible

for inclusion in this study.

They were enrolled in the study according

to specific exclusion and inclusion criteria

(Table 1). Ten patients were selected for this

study that included a 4-month follow-up after

extraction. All were asked to sign a written

informed consent form. Preliminary screen-

ing was performed on the basis of clinical

examination and intraoral radiography. Then,

according to examinations, patients under-

went scaling, root planning, oral hygiene

instruction or any periodontal treatment nec-

essary to provide an oral environment more

favorable to wound healing. All patients

received prophylactic antibiotic therapy

(amoxicillin and clavulanic acid 2 g 1 h

before tooth extraction and 1 g twice a day

for 4 days postoperatively). Patients were

informed about the procedure but were

blinded regarding the group they belonged.

Ten patients (six women and four men)

were recruited. The mean age was 58.4

(±13.6) years.

Treatment

The whole treatment followed standard pro-

tocols for ridge preservation (Sisti et al.

(a) (b)

Fig. 1. (a) SEM image of an Mg-enriched HAP granule (magnification 850x). (b) Particular of the previous image:

nano-pores can be noted (magnification 17.330x)

© 2012 John Wiley & Sons A/S 399 | Clin. Oral Impl. Res. 24, 2013 / 398–406

Canullo et al �Bone augmentation using Mg-eHA

2012). The teeth were extracted using a flap-

less, minimally invasive technique. A peri-

odontal probe was used to measure the

distance between the buccal gingival margin

and the bone wall. In all patients, the soft

and hard tissues were curetted, and Mg-

enriched HA granules (SINTlife, 600–900 lm;

Finceramica) were mixed with blood and

grafted into the socket up to 3 mm apical to

the soft tissue margin. A collagen disk (Gin-

gistat; GABA-Vebas, Milan, Italy) was used

to fill up the alveolar socket, and a custom-

made provisional fixed partial denture (FPD)

was cemented to the adjacent teeth. The pro-

visional FPD included an ovate pontic to sta-

bilize the graft and support buccal soft

tissues. Oral hygiene at the surgical site was

limited to soft brushing for the first 2 weeks.

Regular brushing in the rest of the mouth

and rinsing twice daily with 0.12% chlorhex-

idine were prescribed for 2 weeks. During the

first month after surgery, patients’ follow-up

was performed every week.

Randomization procedure

According to the randomization, augmented

extraction sockets underwent reopening at 2

(Group 1) or 4 months (Group 2). Sites were

assigned to groups according to predefined

randomization tables. A balanced random

permuted block approach, ensuring that, at

any point in a trial, roughly equal numbers

of participants are allocated to all groups,

was used to prepare the randomization tables

to prevent an unequal balance between the

two treatments. To reduce the chance of

unfavorable splits between the two groups in

terms of key prognostic factors, the randomi-

zation process took into account the follow-

ing variables: patient gender, age, biotype,

and site location in dental arch. Assignment

was performed using a sealed envelope.

Biopsy

According to the randomization (either 2 or

4 months after augmentation), the provi-

sional FPD was removed and a re-entry pro-

cedure was performed. In both groups,

preoperative antibiotic prophylaxis was car-

ried out, and after anesthesia, a full-thickness

flap was raised. Using a small trephine (inter-

nal diameter 2.0 mm, external diameter 2.8;

Hu-Friedy, Rockwell Chicago, IL, USA), a

bone specimen was collected before implant

placement. A 13 mm in length and 4.25 mm

in diameter implant (Premium; Sweden &

Martina, Due Carrare, Padua, Italy) was

inserted. Single interrupted 5.0 monofilament

sutures were used for flap adaptation in both

groups. Ten to 14 days later, the sutures were

removed. Two to 3 months after implant

placement, definitive restorations were

seated.

Histological processing

The specimens taken from the test sites were

immersed in 4% buffered formalin for

1 week at room temperature and then decal-

cified for 1 week in ethylenediaminetetraace-

tic acid (EDTA). After decalcification, bone

cores were rinsed with running water for

24 h; routinely dehydrated in a series of

increasing concentrations of ethanol (50%,

70%, 80%, 90%, 96%, and 100%); placed in

xylol for 12 h; and then embedded in paraf-

fin. Serial longitudinal sections of about

5 lm were stained with hematoxylin/eosin

(H.E., to recognize different tissue types) and

Masson–Goldner trichrome (to differentiate

collagen, graft and bone tissues). Addition-

ally, to identify osteoclast-like cells, selected

tissue sections were stained to demonstrate

TRAP as described by Gerber et al. (2006).

A blind histological evaluation of all sec-

tions stained was undertaken by two inde-

pendent investigators. The slices were

observed and photographed under a Nikon

light microscope (Eclipse E600) equipped

with a calibrated digital camera (DXM1200;

Nikon, Tokyo, Japan). Histomorphometric

measurements were performed on images at

a magnification of 109 using image analysis

software (Image J, NIH, v. 1.61, http://rsb.

info.nih.gov/nih-image/). The percentage of

mineralized tissue, connective tissue, and

residual graft material was calculated for all

sections.

Immunohistochemistry

Selected sections from the middle parts of

the series were deparaffinized, rehydrated,

and rinsed for 10 min in Tris-buffered saline

(TBS). Once rinsed, sections were pretreated

with PBS containing 1% bovine serum albu-

min for 20 min at RT and digested with

0.4% pepsin (Roche, Mannheim, Germany)

for 10 min at 37°C. Following further rinsing,

sections were incubated with the primary

antibodies in a humid chamber. Staining was

performed following the instructions for the

ABC-systems (Vectastain ABC Kit; Vector

Laboratories, Burlingame, CA, USA). Bound

antibodies were visualized using a New

Fuchsine alkaline phosphatase substrate pro-

tocol or diaminobenzidine. Sections were

counter stained in hematoxylin and then

cover-slipped. As control for the specificity of

the caveolin-1 antibody, antigen-pre-incu-

bated antibodies were used as shown recently

(Kunert-Keil et al. 2011). For negative con-

trols, the primary antibody was replaced by

PBS and PBS used in the same way. Details

and incubation protocols are available for

consultation in Table 2.

Quantitative and qualitative analysis ofcapillary-like structures

A simultaneous blind test was conducted in

identical conditions (researchers, equipment,

and chemicals) to determine the level of

antigen. From each section, at random, five

Table 1. Patient and study site inclusion and exclusion criteria

Patients inclusion criteria Patients exclusion criteria

Patients with need for tooth extraction andfollowing implant placement. Furthermore,patients with missing teeth and lack of hardand/or soft tissues and desire for implantprosthodontic treatment

All absolute contraindication for implant treatmentsuch as pregnancy or severe poor general health,for exampleSevere renal or liver diseaseHistory of a radiotherapy in the head regionChemotherapy at the time of surgical procedureNon-compensated diabetes mellitus symptoms of amaxillary sinus diseaseActive periodontal diseasePoor oral hygiene

Table 2. Details and incubation protocols of the used antibodies

Antibody Isotype ProducerIncubationprotocol For staining of

Alkalinephosphatase

Rabbitpolyclonal

Quartett (Berlin,Germany)

Ready to use,on, 4°C

Osteoblast cells

Caveolin-1 Rabbitpolyclonal

Dianova (Hamburg,Germany)

1 : 1000, on, 4°C Blood vessels,osteoblasts,osteoclasts

CD34 Rabbitmonoclonal

Epitomics (Burlingame,CA, USA)

1 : 100, 30 min, RT Endothelial cells

On, overnight; RT, room temperature.

400 | Clin. Oral Impl. Res. 24, 2013 / 398–406 © 2012 John Wiley & Sons A/S


digital pictures of different parts of the tissue

were taken (2009 magnification, 3CCD color

camera, Hitachi HV-C20M; Hitachi Denshi

Ltd, Tokyo, Japan, and Axiolab; Carl Zeiss,

Gottingen, Germany). For standardization of

the measurement, the optical density of

white background color was attuned to 250

in each picture. For all sections, mean optical

densities and the quantity of capillary-like

structures were assessed using KSRun soft-

ware (imaging system KS400, release 3.0;

Zeiss, Vision GmbH, Munich, Germany)

(Gerger et al. 2004; Kunert-Keil et al. 2011).

The criteria for a capillary-like structure were

central lumina surrounded by caveolin-1 or

CD34 positively stained cells.

Statistical analysis

Mean values and standard deviations were

calculated for each outcome variable. For

each group, obtained values were compared

using nonparametric Mann–Whitney U-test.

P < 0.05 was considered statistically signifi-

cant.

Statistical analysis was performed using

the SPSS version 20 software (IBM Corpora-

tion, Somers, NY, USA).

Results

At the end of the study, no dropout occurred.

According to the randomization, five biopsies

were grafted at 2 months (Group 1), the other

five at 4 months (Group 2).

All surgical interventions and postopera-

tive healing period were without any serious

complication or side effect for all patients. In

the first postoperative day, some patients

showed a moderate swelling without experi-

encing pain. After 1 week, no inflammation

symptom was detectable.

Histology

According to the decalcification process of the

bone graft material, sections showed

Mg-enriched HA granules as amorphous or

granular bodies of different sizes. In all

biopsies, histological results demonstrated no

inflammatory cell or foreign body reaction after

2 and 4 months of healing, respectively. The

bone formation and remodeling could be cate-

gorized into four different osteogenous trans-

formation stages (I–IV), as described formerly

byGerber et al. (2006) andGotz et al. (2008).

Stage 1: Two of five cases in Group 1 were

classified in the first stage of healing. They

presented non-degraded granules with

smooth surface, mostly covered by a dense

connective tissue and capillaries (Fig. 2a,b).

Stage 2: Three of five cases in Group 1

were classified in the second stage of heal-

ing. Samples presented well-vascularized

connective tissue filling the spaces

between the grafted material residues. Fur-

thermore, osteoid protrusions extended

into the bone graft material. These protru-

sions appeared green with Masson–Goldner

staining and indicate the occurrence of a

collagenous matrix. Some of the woven

bone trabeculae were interconnected with

each other (Fig. 3a,b).

Stage 3: Two of five cases in Group 2 were

classified in the third stage of healing.

Samples presented increased bone graft

material degradation and remodeling of

woven into lamellar bone. Large areas of

newly formed bone appeared mineralized

(Fig. 4a,b).

Stage 4: Three of five cases in Group 2

were classified in the fourth stage of

healing. Samples presented residuals of

woven bone within lamellar bone. Two

of five biopsy specimens appeared to be

nearly completely ossified. Small mate-

rial residues were sometimes visible

(Fig. 5a,b).

Alkaline phosphatase

Stage 1: Strong staining of alkaline phos-

phatase (AP) was found in fibroblasts of

the connective tissue as well as in the

peripheral area of granules (Fig. 2c).

Stage 2: Alkaline phosphatase–positive

cells were found at the surface of granules.

Furthermore, strong AP staining was

detected in the peripheral area of granules

(Fig. 3c).

(a) (b)

(c) (d)

(e) (f)

Fig. 2. Stage I/Case 5; 2 month after graft material insertion. Non-degraded granules covered by connective tissue.

(a) Masson–Goldner, (b) hemalaun/eosin staining, (c) alkaline phosphastase immunoreactivity, (d) Tartrate-resistant

acid phosphatase staining, (e) caveolin-1 as well as (f) CD34 immunoreactivity. rm, residual material; v, blood

vessel; arrowhead, osteoblast cell; asterix, osteoclast cell. Bars represent 100 lm. Magnification: 920.



Stage 3: AP staining was detected in the

peripheral area of granules, osteoblast cells

at the surface of woven bone, fibroblast

cells and in osteocytes of the newly formed

bone (Fig. 4c).

Stage 4: AP-positive cells were still visible

in the connective tissue and around blood

vessels. However, the staining intensity

decreased (Fig. 5c).

TRAP staining

Stage 1: Osteoclast-like cells as well as

mononuclear cells were TRAP stained.

These TRAP-positive cells appeared at the

surface of the granules and were also visi-

ble in the connective tissue, mainly around

vessels (Fig. 2d).

Stage 2: TRAP-positive osteoclast-like cells

appeared at the surface of the granules. In

addition, mononuclear cells stained with

TRAP were visible in the connective tissue

(Fig. 3d).

Stage 3: The amount of TRAP-positive

mono- and multinuclear cells decreased.

However, on the surfaces of newly

formed bone and around residual material,

TRAP-stained cells were still detectable

(Fig. 4d).

Stage 4: The number of TRAP-positive

cells was even more reduced as in stage 3

(Fig. 5d).

Caveolin-1 and CD34

Stage 1: Strong caveolin-1 staining was

found in endothelial cells as well as the

whole vessel wall, including smooth mus-

cle cells. Furthermore, osteoblast and

osteoclast cells showed strong expression

of caveolin-1, whereas fibroblast cells of

the connective tissue were mainly

unstained (Fig. 2e). Only walls of vessel-

like structures resulted strongly positive to

CD34 antibodies (Fig. 2f).

Stage 2: As shown in stage 1, strong caveo-

lin-1 and CD34 expression was found in

endothelial cells as well as in the whole

vessel wall (Fig. 3e,f). In contrast, osteo-

blast and osteoclast cells showed weak

expression of caveolin-1 (Fig. 3e).

Stage 3: No change in caveolin-1 and CD34

expressing cells was found at these stages

compared to stages 1 and 2, whereas the

staining intensity of the cells decreased

(Fig. 4e,f).

Stage 4: As shown for stage 3, caveolin-1

and CD34 expressing cells were detectable.

However, the staining intensity of caveo-

lin-1 seemed to be increased in osteoblast

cells on the surface of woven bone

(Fig. 5e). The expression level of CD34 was

even more reduced as in stage 3 (Fig. 5f).

Histomorphometry

Histomorphometric analysis, as shown in

Table 3, presented a regenerated bone mean

value of 15.0% after 2 months of healing.

After 4 months of healing, a 5.1-fold

increased bone mean value was observed

(77.4%; P = 0.016). At the same time, 21.7%

and 11.6% of graft material was still visible

on average respectively in Groups 1 and 2.

On average, connective tissue/marrow spaces

occupied 63.3% of the bi-optical test sections

after 2 months of healing. This area was sig-

nificantly reduced to 11% (P = 0.016) after

4 months (Table 3).

Computerized analysis of the staining

intensity (optical density) of caveolin-1 in

capillary-like structures is shown in Fig. 6a.

A significant decrease in caveolin-1 protein

expression was found comparing Group 1 and

2 even not considering osteogenous transfor-

mation stages (2 vs. 4 months: 645 ± 66 vs.

256 ± 188; P = 0.029). Quantitation of the

CD34 expression confirmed these results.

The CD34 expression was also significantly

reduced 4 months after material insertion

compared to 2 months of bone healing (2 vs.

4 months, 301 ± 190 vs. 88 ± 54, P = 0.016,

Fig. 6b and Table 3).

Discussion

From a theoretical point of view, there are

several reasons to consider preservation of

the alveolar ridge immediately following

tooth extraction. The first is to stabilize the

(a) (b)

(c) (d)

(e) (f)

Fig. 3. Stage II/Case 6; 4 month after graft material insertion. Granules with early osteogenesis. (a) Masson–Gold-

ner, (b) hemalaun/eosin staining, (c) alkaline phosphastase immunoreactivity, (d) tartrate-resistant acid phosphatase

staining, (e) caveolin-1 as well as (f) CD34 immunoreactivity. rm, residual material; o, osteoid deposition; arrow-

head, osteoblast cell; asterix, osteoclast cell. Bars represent 100 lm. Magnification: 920.



coagulum within the socket and minimize

possible shrinkage of the hard tissues.

The second is to provide a scaffold for the

in-growth of cellular and vascular compo-

nents for new bone regeneration.

Several studies were conducted to test

techniques and materials able to contrast

physiologic resorption process in post-extrac-

tion sites. Available data provide controver-

sial outcomes; however, literature confirmed

that post-extraction alveolar crest preserva-

tion procedures can result in better quantita-

tive regeneration with no histological

qualitative difference compared to sites

healed without any graft.

In fact, according to Jung et al. (2004), graft

materials seem not able to modify resorption

pattern of post-extraction socket. Further-

more, some grafts (bovine bone matrix) are

supposed to lessen healing process and bone

neo-formation (Becker et al. 1998; Araujo &

Lindhe 2009; Araujo et al. 2009). On the

other hand, in post-extraction sockets with

buccal bone wall defects, the same graft cov-

ered by collagen membrane seemed to be

effective from a quantitative point of view

even if histologic data showed ongoing heal-

ing process after 7 months (Carmagnola et al.

2003).

In the present randomized controlled clini-

cal trial, changes of the extraction sites at

different times (2 and 4 months) after aug-

mentation using a Mg-enriched HA were his-

tologically analyzed. In Group 1, 8 weeks

after tooth extraction, bone biopsies were

composed of a low-density trabecular bone

with few interspersed graft particles. After

16 weeks of healing (Group 2), newly formed

bone appeared to have filled any defect:

hydroxyapatite particles became well incor-

porated into new bone formation creating a

dense cancellous network.

At the end of the study, the histomorpho-

metric analysis points out the rare bone

regeneration after two and impressing ossifi-

cation in comparison after 4 months.

To explain the histologic outcomes of

Group 2 compared to Group 1, osteoconduc-

tive properties of new generation HAs might

be advocated. In fact, these properties were

recently demonstrated in animal (Ripamonti

et al. 2008, 2009) and human studies (Gotz

et al. 2008). The hydroxyapatite nano-poros-

ity was demonstrated to allow bone matrix

proteins to adhere and promote differentia-

tion of osteoblast precursor cells (Gotz et al.

2008).

These data seem to be in accordance with

results reported by Gotz et al. (2008) and Ca-

nullo & Dellavia (2009). Although in sinus

lift and GBR procedures, it was demonstrated

that sites grafted with nano-structured HAs

showed new bone regeneration even in the

early stage (after 3 months).

Histomorphometric data after 4 months

from the present study were confirmed by

recently published study on preservation

technique of crestal bone after extraction

grafted with the same hydroxyapatite (Crespi

et al. 2011). However, similar results in

terms of bone formation, presence of graft

particles and connective tissue were found in

the same study also using porcine bone sub-

stitute. The similar biologic behavior might

highlight once again the importance of the

scaffold properties performed by graft materi-

als. In sinus lift procedure, the same Mg-

enriched HA as in the present study showed

after 5 months 29.7% vital bone vs. 78.4%

vital bone for autogenous bone graft

(P < 0.05), while overall bone volume was

nearly the same (Crespi et al. 2009a,b,c).

Coincidentally, Mg-enriched HA revealed

clearly more residual material and less vital

bone than calcium sulfate (Crespi et al.

2009b). This dissimilarity can be explained

by the different vertical height where the

biopsy was taken. In fact, in case of calcium

sulfate, almost 2.5 mm of radiographic verti-

cal bone resorption was demonstrated. On

the other hand, Mg-enriched HAP showed

<0.5 mm of radiographic vertical bone resorp-

tion.

In the present study, together with tradi-

tional histology and histomorphometry, Mg-

enriched HAP effectiveness in post-extraction

procedures was measured analyzing the

angiogenesis–osteogenesis interplay.

In fact, experimental studies investigating

the properties of some synthetic substitute

(a) (b)

(c) (d)

(e) (f)

Fig. 4. Stage III/Case 3; 2 month after graft material insertion. Increased material degradation and remodeling into

newly woven, mineralized bone. (a) Masson–Goldner, (b) hemalaun/eosin staining, (c) alkaline phosphastase immu-

noreactivity, (d) tartrate-resistant acid phosphatase staining, (e) caveolin-1 as well as (f) CD34 immunoreactivity.

rm, residual material; asterix, osteoclast cell. Bars represent 100 lm. Magnification: 920.



materials have already demonstrated the

importance of angiogenesis from the host tis-

sue that is closely related to new bone forma-

tion (Kakudo et al. 2006).

Using indirect immunohistochemistry and

enzymatic analysis, in fact, high expression

of alkaline phosphatase (AP), TRAP, and

CD34 (vessel density) was found in early

stages of osteogenous transformation. On the

other hand, the amount of vessel-like struc-

tures was significantly reduced 4 months

after grafting material insertion. Further-

more, a decrease in the expression of AP and

TRAP was also detectable. The reduction of

AP expression is based on the progressive

osteogenesis with subsequent reduction of

pre-osteoblasts, osteoblasts, and young osteo-

cytes. These data allow to speculate that

Mg-enriched HA is a suitable material for

post-extraction ridge preservation, ensuring

early angiogenesis followed by early osteo-

genesis.

These results are definitely substantiated

by caveolin-1 evaluation. As reported in liter-

ature (Frank et al. 2003), caveolin-1 could be

detected in endothelial cells as well as in the

whole vessel wall, including smooth muscle

cells. Furthermore, osteoblast and osteoclast

cells showed strong expression of caveoline-

1, whereas fibroblast cells of the connective

tissue were mainly unstained (Solomon et al.

2000; Lofthouse et al. 2001; Sawada et al.

2007; Hada et al. 2012). Particularly, caveo-

lin-1 is strongly abundant in endothelial cells

regulating functions as angiogenesis, vascular

permeability, and transcytosis (Frank et al.

2003). In a recent study on caveolin-1-defi-

cient mice, angiogenesis was found to be

markedly reduced in comparison with control

mice (Woodman et al. 2003). Furthermore,

caveolin-1 knockout mice have increased

bone size and stiffness, because caveolin-1

deficiency leads to increased osteoblast differ-

entiation as shown by cell studies from the

same group (Rubin et al. 2007). Thus, the

caveolin-1 antibody can be used for the anal-

ysis of osteoblasts, osteoclasts as well as

blood vessels.

(a) (b)

(c) (d)

(e) (f)

Fig. 5. Stage IV/Case 7; 4 month after graft material insertion. Progressed osteogenesis. Material residues (star)

within mature lamellar bone. (a) Masson–Goldner, (b) hemalaun/eosin staining, (c) alkaline phosphastase immuno-

reactivity, (d) tartrate-resistant acid phosphatase staining, (e) caveolin-1 as well as (f) CD34 immunoreactivity.

Arrowhead: osteoblasts. Bars represent 100 lm. Magnification: 920.

Table 3. Histomorphometric analyses of biopsies

Histologic data

Healing period

2 months 4 months P-value

Regenerated bone (%) 15.1 ± 16.7 77.4 ± 14.9 0.016Graft material (%) 21.7 ± 15.4 11.6 ± 15.7 0.556Connective tissue/marrow space (%) 63.3 ± 22.4 11.0 ± 6.9 0.016Cav1 645.1 ± 65.9 256.4 ± 188.2 0.029CD34 301.2 ± 190.8 87.6 ± 53.6 0.016

(a)

(b)

Fig. 6. Caveolin-1 (a) and CD34 (b) protein levels 2 and

4 month after mHA insertion. The mean optical densi-

ties (MOD) ± SEM are given in all cases for n = 5 biop-

sies. Stars indicate significant differences: *P < 0.05;

**P < 0.01 (one-way ANOVA test).



In the present study, focusing on the angio-

genesis–osteogenesis interplay, strong expres-

sion of caveolin-1 was found in pre-

osteoblasts, osteoblasts, and osteoclasts:

while staining density showed 645 ± 65

after 2 months for blood vessels, the expres-

sion of caveolin-1 in capillary-like structures

decreased to 256 ± 188 after 4 months.

Results of angiogenesis–osteogenesis inter-

play and analysis of AP and CD34 as well as

TRAP staining allow to conclude that, from

an histologic point of view, blood vessel den-

sity developed in opposite to osteogenesis

and that high vascularization after 2 months

could provide a highly accelerated ossifica-

tion of the augmented region, confirming tra-

ditional histology and histomorphometry.

From a clinical point of view, it may be

concluded that Mg-enriched HA is a suit-

able material for ridge preservation and

ensures early angiogenesis and osteogenesis,

suggesting that implant placement could be

appropriate even after 2 months. However,

at this early stage, immediate loading could

be risky. On the other hand, at 4 months,

the regenerated bone could bear loading

stress.

Limitation of the study was represented by

the small patient sample size, which was

antagonized by a strict randomization proto-

col. However, more data are needed, and

especially, influence of local soft tissue and

genetic terms (healing bone pattern) has to be

investigated.

Acknowledgements: This study was

self-supported. The authors highly

appreciated the skills and commitment of Dr

Audrenn Gautier for language suggestions

and Dr Giuliano Iannello for statistical

supervision. Authors would like to thank Dr

S. Lucke and I. Pieper for their excellent

technical assistance.

Conflict of interest

The authors declare that there are no con-

flicts of interest.

References

Adams, R.H. & Alitalo, K. (2007) Molecular regu-

lation of angiogenesis and lymphangiogenesis.

Nature Reviews Molecular Cell Biology 8:

464–478.

Araujo, M., Linder, E. & Lindhe, J. (2009) Effect of a

xenograft on early bone formation in extraction

sockets: an experimental study in dog. Clinical

Oral Implant Research 20: 1–6.

Araujo, M.G. & Lindhe, J. (2009) Ridge preservation

with the use of Bio-Oss collagen: a 6-month

study in the dog. Clinical Oral Implant Research

20: 433–440.

Becker, W., Clokie, C., Sennerby, L., Urist, M.R. &

Becker, B.E. (1998) Histologic findings after

implantation and evaluation of different grafting

materials and titanium micro screws into extrac-

tion sockets: case reports. Journal of Periodontol-

ogy69: 414–421.

Caneva, M., Botticelli, D., Stellini, E., Souza, S.L.,

Salata, L.A. & Lang, N.P. (2011) Magnesium-

enriched hydroxyapatite at immediate implants: a

histomorphometric study in dogs. Clinical Oral

Implant Research 22: 512–517.

Canullo, L. & Dellavia, C. (2009) Sinus lift using a

nanocrystalline hydrxyapatite silicea gel in

severely resorbed maxillae: histological prelimin-

ary study. Clinical Implant Dental Related

Research 11 (Suppl. 1): e7–13.

Carmagnola, D., Adriaens, P. & Berglundh, T.

(2003) Healing of human extraction sockets filled

with Bio Oss. Clinical Oral Implant Research 14:

137–143.

Chen, S.T., Darby, I.B. & Reynolds, E.C. (2007) A

prospective clinical study of non-submerged

immediate implants. Clinical outcomes and

esthetic results. Clinical Oral Implants Research

18: 552–562.

Chiapasco, M., Zaniboni, M. & Boisco, M. (2006)

Augmentation procedures for the rehabilitation of

deficient edentulous ridges with oral implants.

Clinical Oral Implant Research 17: 136–159.

Cornelini, R., Cangini, F., Martuscelli, G. & Wenn-

stro ¨m, J. (2004) Deproteinized bovine bone and

biodegradable barrier membrane to support heal-

ing following immediate placement of transmu-

cosal implants: a short-term controller clinical

trial. The International Journal of Periodontics

and Restorative Dentistry 24: 555–563.

Crespi, R., Cappare, P. & Gherlone, E. (2009a) Den-

tal implants placed in extraction sites grafted

with different bone substitutes: radiographic eval-

uation at 24 months. Journal of Periodontology

80: 1616–1621.

Crespi, R., Cappare, P. & Gherlone, E. (2009b) Mag-

nesium-enriched hydroxyapatite compared to cal-

cium sulfate in the healing of human extraction

sockets: radiographic and histomorphometric

evaluation at 3 months. Journal of Periodontol-

ogy 80: 210–218.

Crespi, R., Cappare, P. & Gherlone, E. (2011) Com-

parison of magnesium-enriched hydroxyapatite

and porcine bone in human extraction socket

healing: a histologic and histomorphometric eval-

uation. International Journal of Oral & Maxillo-

facial Implants 26: 1057–1062.

Crespi, R., Mariani, E., Benasciutti, E., Cappare, P.,

Cenci, S. & Gherlone, E. (2009c) Magnesium-

enriched hydroxyapatite versus autologous bone in

maxillary sinus grafting: combining histomorph-

ometry with osteoblast gene expression profiles ex

vivo. Journal of Periodontology 80: 586–593.

Frank, P.G., Woodman, S.E., Park, D.S. & Lisanti,

M.P. (2003) Caveolin, caveolae, and endothelial

cell function. Arteriosclerosis, Thrombosis, and

Vascular Biology 23: 1161–1168.

Gerber, T., Holzhuter, G., Gotz, W., Bienengraber,

V., Henkel, K.O. & Rumpel, E. (2006) Nanostruc-

turing of biomaterials – a pathway to bone graft-

ing substitute. European Journal of Trauma 32:

132–140.

Gerger, A., Bergthaler, P. & Smolle, J. (2004) An

automated method for the quantification and

fractal analysis of immunostaining. Cellular

Oncology 26: 125–134.

Gotz, W., Gerber, T., Michel, B., Lossdorfer, S.,

Henkel, K.O. & Heinemann, F. (2008) Immuno-

histochemical characterization of nanocrystalline

hydroxyapatite silica gel (NanoBone(r)) osteogene-

sis: a study on biopsies from human jaws. Clini-

cal Oral Implant Research 19: 1016–1026.

Gotz, W., Lenz, S., Reichert, C., Henkel, K.O., Bien-

engraber, V., Pernicka, L., Gundlach, K.K., Gredes,

T., Gerber, T., Gedrange, T. & Heinemann, F.

(2010) A preliminary study in osteoinduction by a

nano-crystalline hydroxyapatite in the mini pig.

Folia Histochemica Et Cytobiologica 48: 589–596.

Gotz, W., Reichert, C., Canullo, L., Jager, A. & He-

inemann, F. (2011) Coupling of osteogenesis and

angiogenesis in bone substitute healing – a brief

overview. Annals of Anatomy 194: 171–173.

Hada, N., Okayasu, M., Ito, J., Nakayachi, M., Ha-

yashida, C., Kaneda, T., Uchida, N., Muramatsu,

T., Koike, C., Masuhara, M., Sato, T. & Hakeda,

Y. (2012) Receptor activator of NF-kappaB ligand-

dependent expression of caveolin-1 in osteoclast

precursors, and high dependency of osteoclasto-

genesis on exogenous lipoprotein. Bone 50:

226–236.

Jung, R.E., Siegenthaler, D.W. & Hammerle, C.H.

(2004) Postextraction tissue management: a soft

tissue punch technique. International Journal of

Periodontics & Restorative Dentistry 24:

545–553.

Kakudo, N., Kusumoto, K., Wang, Y.B., Iguchi, Y.

& Ogawa, Y. (2006) Immunolocalization of vascu-

lar endothelial growth factor on intramuscular

ectopic osteoinduction by bone morphogenetic

protein-2. Life Science 79: 1847–1855.

Kunert-Keil, C., Gredes, T., Lucke, S., Morgenstern,

S., Mielczarek, A., Sporniak-Tutak, K., Gedrange,

T. & Spassov, A. (2011) Caveolin-1, caveolin-3

and VEGF expression in the masticatory muscles

of mdx mice. Folia Histochemica Et Cytobiolog-

ica 49: 291–298.

Le Lay, S., Blouin, C.M., Hajduch, E. & Dugail, I.

(2009) Filling up adipocytes with lipids. Lessons

from caveolin-1 deficiency. Biochimica et Bio-

physica Acta 1791: 514–518.

Lofthouse, R.A., Davis, J.R., Frondoza, C.G., Jin-

nah, R.H., Hungerford, D.S. & Hare, J.M. (2001)

Identification of caveolae and detection of caveo-

lin in normal human osteoblasts. The Journal of

Bone & Joint Surgery (British Volume) 83:

124–129.



Ripamonti, U., Crooks, J., Khoali, L. & Roden, L.

(2009) The induction of bone formation by coral-

derived calcium carbonate/hydroxyapatite con-

structs. Biomaterials 30: 1428–1439.

Ripamonti, U., Richter, P.W., Nilen, R.W. & Ren-

ton, L. (2008) The induction of bone formation by

smart biphasic hydroxyapatite tricalcium phos-

phate biomimetic matrices in the non-human pri-

mate Papio ursinus. Journal of Cellular and

Molecular Medicine 12: 2609–2621.

Rothberg, K.G., Heuser, J.E., Donzell, W.C., Ying,

Y.S., Glenney, J.R. & Anderson, R.G. (1992) Cave-

olin, a protein component of caveolae membrane

coats. Cell 68: 673–682.

Rubin, J., Schwartz, Z., Boyan, B.D., Fan, X., Case,

N., Sen, B., Drab, M., Smith, D., Aleman, M.,

Wong, K.L., Yao, H., Jo, H. & Gross, T.S. (2007)

Caveolin-1 knockout mice have increased bone

size and stiffness. Journal of Bone and Mineral

Research 22: 1408–1418.

Santos, M.I. & Reis, R.L. (2010) Vascularization in

bone tissue engineering: physiology, current strat-

egies, major hurdles and future challenges. Mac-

romolecular Bioscience 10: 12–27.

Sawada, N., Taketani, Y., Amizuka, N., Ichikawa,

M., Ogawa, C., Nomoto, K., Nashiki, K., Sato, T.,

Arai, H., Isshiki, M., Segawa, H., Yamamoto, H.,

Miyamoto, K. & Takeda, E. (2007) Caveolin-1 in

extracellular matrix vesicles secreted from osteo-

blasts. Bone 41: 52–58.

Schropp, L., Wenzel, A., Kostopoulos, L. & Karring,

T. (2003) Bone healing and soft tissue contour

changes following single-tooth extraction: a clini-

cal and radiographic 12-month prospective study.

International Journal of Periodontics & Restor-

ative Dentistry 23: 313–323.

Sisti, A., Canullo, L., Mottola, M.P., Covani, U.,

Barone, A. & Botticelli, D. (2012) Clinical

evaluation of a ridge augmentation procedure

for the severely resorbed alveolar socket: multi-

center randomized controlled trial, preliminary

results. Clinical Oral Implant Research 23:

526–535.

Solomon, K.R., Danciu, T.E., Adolphson, L.D.,

Hecht, L.E. & Hauschka, P.V. (2000) Caveolin-

enriched membrane signaling complexes in

human and murine osteoblasts. Journal of Bone

& Mineral Research 15: 2380–2390.

Tamaskar, I. & Zhou, M. (2008) Clinical implica-

tions of caveolins in malignancy and their poten-

tial as therapeutic targets. Current Oncology

Reports 10: 101–106.

Trombelli, L., Penolazzi, L., Torreggiani, E., Farina,

R., Lambertini, E., Vecchiatini, R. & Piva, R.

(2010) Effect of hydroxyapatite-based biomaterials

on human osteoblast phenotype. Minerva Stoma-

tologica 59: 103–115.

Woodman, S.E., Ashton, A.W., Schubert, W., Lee,

H., Williams, T.M., Medina, F.A., Wyckoff, J.B.,

Combs, T.P. & Lisanti, M.P. (2003) Caveolin-1

knockout mice show an impaired angiogenic

response to exogenous stimuli. American Journal

of Pathology 162: 2059–2068.

Supporting Information

Additional Supporting Information may be

found in the online version of this article:

Table S1. CONSORT checklist.

Please note: Wiley-Blackwell are not respon-

sible for the content or functionality of any

supporting materials supplied by the authors.

Any queries (other than missing material)

should be directed to the corresponding author

for the article.



friedhelm heinemann* times after augmentation of trial...today, teeth replacement with...

Documents