regeneration of dental pulp: a myth or hype

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ENDODONTOLOGYENDODONTOLOGYENDODONTOLOGYENDODONTOLOGYENDODONTOLOGY

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Regeneration of dental pulp : A myth or hype

Sureshchandra B #Roma M #

# Dept. of Conservative Dentistry and Endodontics, A.J Institute of Dental Sciences, Mangalore, Karnataka, India.

ABSTRACTTooth loss compromises human oral health. Although several prosthetic methods, such as artificial denture and dental

implants, are clinical therapies to tooth loss problems, they are thought to have safety and usage time issues. Recently,

tooth tissue engineering has attracted more and more attention. Stem cell based tissue engineering is thought to be a

promising way to replace the missing tooth. Scientific advances in the creation of restorative biomaterials, in-vitro cell

culture technology, tissue grafting, tissue engineering molecular biology and human genome project provides the

basis for the introduction of new technologies in dentistry. The purpose of this article is to highlight the biological

procedures to develop the regenerative endodontic procedures.

Key words: Stem cells, revascularization, Growth factors, Regenerative Endodontics

IntroductionDuring the last 50 years we have realized that

science is the fuel for the engine of technology.

Scientific discoveries from cellular, developmental,

and molecular biology have truly revolutionized our

collective understanding of biological processes,

human genetic variations, the continuity of

evolution, and the etiology and pathogenesis of

thousands of human diseases and disorders. This

enormous accumulation of scientific discovery

(theory, principles, concepts, and facts) provides the

fuel for the ‘clinical research and translation

revolution’ of the 21st century.

The porous material to serve as the matrix to

facilitate the regeneration must have certain pore

characteristics, chemical compositions, and

mechanical properties. An approach has been to

employ materials that serve as analogues of the

extracellular matrix of the tissues to be regenerated.

For selected indications in which the supply of

endogenous precursor cells has been compromised

by disease or prior surgical procedures, it may be

necessary to seed the matrix, prior to implantation

with exogenous cells to have it serve as a delivery

vehicle for the growth of differentiation factors.

Therapeutic intervention with recombinant

growth factors also provides possibilities for control

of cell activity during repair. Harnessing both

endogenous and exogenous sources of growth

factors can provide exciting opportunities for novel

biological approaches to dental tissue repair and

the blueprint for the regeneration of the tooth. These

approaches offer significant potential for improved

clinical management of dental disease and

maintenance of tooth vitality.

STEM CELL THERAPY

The greater plasticity of the embryonic stem

cells makes these cells more valuable among

researchers for developing new therapies. However,

Review Article

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the legal limitations and the ethical debate related

to the use of embryonic stem cells must be resolved

before the great potential of donated embryonic

stem cells can be used to regenerate diseased,

damaged, and missing tissues as a part of future

medical treatments. There are four primary sources

for embryonic stem cells:

· Existing stem cell lines

· Aborted or miscarried embryos

· Unused In vitro fertilized embryos

· Cloned embryos

· Body fat

· Almost all body tissues including the pulp tissue

of teeth.

Stem cells are often categorized according to their

source as: [10]

i. Autologus postnatal stem cells – The most

practical clinical application of a stem cell therapy

would be to use a patient’s own donor cells. These

cells are obtained from the same individual to whom

they will be implanted. Bone marrow harvesting of

a patient’s own stem cells and their reimplantation

back to the same patient represents one clinical

application of autologus postnatal stem cells.

Advantages:

· Most practical

· Readily available

· No immunogenicity

· Least expensive

· Avoids legal and ethical concerns

Disadvantages:

· May have reduced plasticity

· Postoperative sequelae, such as donor site infection

· May take time in isolation from mixed tissues

· In some cases donor cells may not be available

e.g in very ill or elderly patients

ii. Allogenic postnatal stem cells – These cells

are obtained from the donor of same species.

Examples of donor allogenic cells include blood

cells used for blood transfusion, bone marrow cells

used for a bone marrow transplant and donated egg

cells used for in vitro -transplantation. These

donated cells are often stored in a cell bank, to be

used by patients requiring them.;

The sourcing of embryonic stem cells is

controversial and is surrounded by ethical and legal

issues, which reduces the attractiveness of these

cells for developing new therapies. This explains

why many researchers are now focusing attention

on developing stem cell therapy using postnatal

stem cells donated by patients themselves or their

close relatives. Postnatal stem cell therapy was

launched in 1968, when the first allogenic bone

marrow transplant was successfully used.Postnatal

stem cells have been sourced from:

· Umbilical cord blood

· Umbilical cord

· Bone marrow

· Peripheral blood

SURESHCHANDRA B, ROMA M

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Preexisting cell lines and cell organ cultures :

The use of preexisting cell lines and cell

organ cultures removes the problem of harvesting

cells from the patient and waiting weeks for

replacement tissue to form in cell organ-tissue

cultures. However, the most serious disadvantages

of using preexisting cell lines from donors to treat

patients are the risk of immune rejection and

pathogenic transmission. The FDA has approved

several companies producing skin for burn victims

using donated dermal fibroblasts. The same

technology may be applied to replace pulp tissues

after root canal therapy, but it has not yet been

evaluated and published.

iii.Xenogenic cells – These are isolated from

individuals of another species. Pig tooth pulp cells

have been transplanted into mice, and these have

formed tooth crown structures. This suggests it is

feasible to accomplish the reverse therapy,

eventually using donated animal pulp stem cells to

create tooth tissues in humans. In particular, animal

cells have been used quite extensively in

experiments aimed at the construction of

cardiovascular implants. The harvesting of cells from

donor animals removes most of the legal and ethical

issues, associated with sourcing cells from other

humans. However, many problems remain, such

as the high potential for immune rejection and

pathogenic transmission from the donor animal to

human recipient. The future use of xenogenic cells

is uncertain, and largely depends on the success of

the other available stem cell therapies. If the use of

allogenic and autologous pulp stem tissue

regeneration is disappointing, then the use of

xenogenic endodontic cells remains a viable option

for developing an endodontic regeneration therapy.

Heterotropic stem cells for tissue engineering

The marrow is at centre stage for future

technological developments in tissue engineering,

not only as organ in which at least two types of

stem cells (Hemopoeitic stem cells [HSCs] and

skeletal stem cells [SSCs]) reside, but also as the

organ in which progenitors for a number of distant

tissues can be found. SSCs can perhaps give rise to

neurons or glia, purified mouse HSCs can

regenerate liver cells, and cells able to regenerate

bone are also found in blood. Perhaps what we

have referred to as the HSC is in itself much more-

a true multipotent stem cell with transgermal

potentials, normally devoted to haematopoiesis as

a result of local cues.

Marrow cells offer the advantage of being

easily harvested and cultured from an adult

organism, and the HSC can be isolated and purified

ex vivo. Although most of these applications are a

long way from any immediate clinical use, these

studies raise basic scientific issues that are far from

being settled or rationalized, similar, for example,

to the issue of ‘plasticity’ of potential stem cells.

They provide insight on how different the scene of

tissue engineering could be in the relatively near

future. Beyond theoretical considerations, and

pending further experimental proof where needed,

the existence of heterotropic and pleiotropic stem

cells in the bone marrow has obvious practical

implications for the future of stem cell therapy that

should not be missed.

For endodontic regeneration, the most

promising cells are autologus postnatal dental stem

cells because they are less chances for immune

rejection. [30] They show more striking odontogenic

capability as compared to non-dental stem cell

REGENERATION OF DENTAL PULP : A MYTH OR HYPE

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population like the bone marrow stromal stem cells.

Various sources of postnatal dental stem cells are:

· Permanent teeth – Dental pulp stem cells (DPSC):

derived from third molar.[32]

· Deciduous teeth – Stem cells from Human

Exfoliated Deciduous teeth (SHED): stem cells

present within the pulp tissue of deciduous teeth.[24]

· Periodontal ligament – Periodontal Ligament Stem

Cells (PDLSC).[14]

· Stem cells from Apical Papilla (SCAP).[3]

· Stem cells from the supernumerary tooth –

Mesiodens.[8]

· Stem cells from extracted teeth for orthodontic

purposes.[19]

· Dental follicle progenitor cells.[2]

· Stem cells from human natal dental pulp

(h NDP).[1]

Stem cell treatments

Medical researchers believe that stem cell

therapy has the potential to dramatically change the

treatment of human disease. A number of adult stem

cell therapies already exist, particularly bone

marrow transplants that are used to treat leukemia.

In the future, medical researchers anticipate being

able to use technologies derived from stem cell

research to treat a wider variety of diseases including

cancer, Parkinson’s disease, spinal cord injuries,

Amyotrophic lateral sclerosis, multiple sclerosis,

and muscle damage, amongst a number of other

impairments and conditions. However, there still

exists a great deal of social and scientific uncertainty

surrounding stem cell research, which could

possibly be overcome through public debate and

future research, and further education of the public.

Stem cells, however, are already used

extensively in research, and some scientists do not

see cell therapy as the first goal of the research, but

see the investigation of stem cells as a goal worthy

in itself.

BONE MORPHOGENIC PROTEINS

Bone Morphogenetic Proteins (BMPs) form a

unique group of proteins within the Transforming

Growth Factor beta (TGF-â) superfamily. Bone

Morphogenetic Proteins (BMPs) are a group of

growth factors and cytokines known for their ability

to induce the formation of bone and cartilage. BMPs

were first identified by Urist in 1965 when

demineralized bone matrix implanted in ectopic sites

in rats was found to induce bone formation. In 1938

Levander reported that there must be some

stimulating agent which originated from bone and

possibly a substance which was soluble in lymph

tissue. The inducing substance, i.e., Bone

Morphorgenic Protein acting upon a responding

cell, i.e., undifferentiated mesenchymal cell to

become progenitor cell.[26]

BMP exists in the bone matrix (Sampath and

Reddi 1983; Muthukumaran et al 1985), in

Osteosarcoma tissue (Takoaka et al 1980), in dentin

Matrix (Butler et al 1977; Conover and urist 1979;


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Kawai and Urist 1989) and in wound tissue after

tooth extraction (Bessho et al 1990).

BMP known as an osteoinductive factor has

been isolated from dentin and bone. It is reported

to induce reparative dentin formation in contrast to

endochondral bone formation in other tissues.

Crude BMP has desirable properties as a pulp

capping agent in-vivo, complete absorption of BMP,

no formation of necrotic layer at the contact site

such as seen on application of commonly used Ca

(OH)2.

Stimulatory effect of dentin Bone Morphogenic

Protein was more than bone Bone Morphogenic

Protein for reparative dentinogenesis. So there is a

difference in the source.

For the delivery ideally, the carrier for Bone

Morphogenic Protein should be,

- Non collagenous

- Immunogenically inert

- Osteoconductive

- Bioabsorbable

- As well as support angiogenesis and subsequent

vascularization.

When Bone Morphogenic Protein was used

without any carrier, a large amount is needed.

Moreover, the purified Bone Morphogenic Protein

was highly soluble in vivo when used without any

carrier. Based on this, Bone Morphogenic Protein

requires an appropriate carrier for clinical use.

Hence experiments using collagen as carriers were

conducted. Type I collagen may be a useful delivery

system for Bone Morphogenic Protein in clinical

use because it was gradually released from the

collagen.

Optimum activity for the Osteogenic Protein I

(Wey et al 1990, Sampath et al 1992) is achieved

by combining them with a carrier molecule and

implantation of the combination as a solid mass.

Action of bone morphogenic proteins :

The principle of induction was described by

Speemann in 1901. Induction is defined as “an

interaction between one (inducing) tissue and

another (responding) tissue as a result of which the

responding tissue undergoes a change in its

direction of differentiation”.[25] Factors influencing

the inductive process are,

- Timing of the response. (Considering both the

exposure time required as well as the time the

inducer is capable of inducing.)

- Location or proximity of competent cells able to

respond.

- Concentration.

In case of Vital Pulp Therapy we have an

inducing substance (BMP) acting upon a responding

cell (an undifferentiated mesenchymal cell) to

become an osteoprogenitor cell capable of forming

reparative dentin.

Types of bone morphogenic proteins:

Originally, seven such proteins were

discovered. Of these, six (BMP2 through BMP7)

belong to the Transforming growth factor beta

superfamily of proteins. BMP1 is a metalloprotease.

Since then, thirteen more BMPs have been

discovered, bringing the total to twenty.

OF the 9 BMP (Bone Morphogenic Protein),

8, i.e., BMP – 2 through BMP-9 are related to one

another. Also due to their amino acid sequences

BMP-2 through BMP-9 are classified as belonging


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to the Transforming Growth Factor- (TGF-b)

superfamily.

BMP-1, because of its amino acid sequences,

cannot be classified as belonging to the TGF-b

superfamily. It is not capable of inducing bone

formation.

Like the other members of the TGF-b super

family they are derived from precursor Polypeptide

chains ranging in size from 396-513 amino acids.

Bone Morphogenic Proteins are divided into

2 groups. BMP-2 and BMP-4 form one group having

92% identical amino acid. BMP-5 through BMP-9

forms a second group having 82% identical amino

acid. These 2 groups have 59% homology with one

another and only 45% homology with BMP-3.

BMP-7 and BMP-8 are also known as Osteogenic

Protein OP-1 and OP-2 respectively.

BMPs interact with specific receptors on the

cell surface, referred to as bone morphogenetic

protein receptors (BMPRs).

Signal transduction through BMPRs results in

mobilization of members of the SMAD family of

proteins. The signalling pathways involving BMPs,

BMPRs and SMADs are important in the

development of the heart, central nervous system,

and cartilage, as well as post-natal bone

development.

They have an important role during embryonic

development on the embryonic patterning and early

skeletal formation. As such, disruption of BMP

signalling can affect the body plan of the developing

embryo. For example, BMP-4 and its inhibitors

noggin and chordin help regulate polarity of the

embryo (i.e. back to front patterning).

SCAFFOLDS

Material to be used for the fabrication of

matrices to engineer tissue in-vivo must have the

microstructure and chemical composition required

for normal cell growth and function. For bone

regeneration, a material possessing similar physical,

chemical and mechanical properties is desirable

since all of these properties will influence normal

bone cell growth and function.[9]

A majority of craniomaxillofacial reconstructive

procedures are performed to replace or construct

missing or damaged skeletal structures. These

operations require the harvesting of bone or soft

tissue from distant donor sites. The donor site

operation often results in greater morbidity than the

primary reconstructive procedure and there may

not be adequate quantities of bone available for

harvesting in children. Furthermore, there is

unpredictable loss of bone graft volume during the

remodeling process. One tissue engineering is based

on harvesting progenitor or stem cells, expanding

and then, differentiating them into cells that have

potential to form new tissue (e.g. bone) or organ

(e.g. tooth). The harvested cells are seeded on

scaffolds. These scaffolds are fabricated in the


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laboratory to resemble the structure of the desired

tissue or organ to be replaced. Much of the current

tissue engineering research is directed toward the

areas of cell manipulation, isolation, expansion and

differentiation) and scaffold design (biomaterials,

design. and fabrication).

Roles in the Regenerative Process

A matrix can play several roles during the

process of regeneration in vivo:[15]

1. It can structurally reinforce the detective site

so as to maintain the shape of the defect and prevent

distortion of surrounding tissue. For example, cysts

that form in the subchondral bone underlying the

articulating surfaces of joints can lead to collapse

of the joint surface.

2. The matrix can serve as a barrier to the in-

growth of surrounding tissue that may impede the

process of regeneration. The concept of guided

tissue regeneration is based in part on the prevention

of overlying gingival tissue from collapsing into the

periodontal defect.

3. The matrix can serve as a scaffold for

migration and proliferation of cells in vivo or for

cells seeded in vitro.

4. The matrix can serve as an insoluble

regulator of cell function through interaction, with

certain integrins and other cell receptors.

Biomaterials for bone tissue engineering

The role of scaffold in tissue engineering is to

provide a matrix of a specific geometric

configuration on which seeded cells may grow to

produce the desired tissue or organ. The physical

and the chemical characteristics of a scaffold play a

significant role in the proliferation and tissue in-

growth. Biomaterials used as scaffolds for bone

tissue engineering are broadly classified as:

· Naturally derived- Advantages include ability to

support cellular invasion and proliferation.

· Synthetic materials - Offer ease of processing and

mechanical strength

They may be classified as

• Ceramics

• Polymers

These biomaterials may be produced in various

forms

· Solid blocks

· Sheets

· Porous sponges

· Porous Scaffold design

· Hydrogels

Scaffolds in regenerative Endodontics

To create a more practical endodontic tissue

engineering therapy, pulp stem cells must be

organized into three-dimensional structure that can

support cell organization and vascularisation. This

is accomplished using a porous polymer scaffold

seeded with pulp stem cells. A scaffold should

contain growth factors to aid stem cell proliferation


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and differentiation, leading to improved and faster

tissue development. The scaffolds may also contain

nutrients promoting cell survival and growth, and

possibly antibiotics to prevent any bacterial in-

growth in the canal systems. The engineering of

nanoscaffolds may be useful in the delivery of

pharmaceutical drugs to specific tissues. In addition,

the scaffold may exert essential mechanical and

biological functions needed by replacement tissues.

In pulp–exposed teeth, dentin chips have been

found to stimulate reparative bridge formation.

Dentin chips may provide a matrix for pulp stem

cell attachment and also a reservoir for growth

factors. The natural reparative activity of pulp stem

cells in response to dentin chips provide some

support for the use of the scaffolds to regenerate

the pulp–dentin complex.

To achieve the goal of pulp tissue

reconstruction, scaffolds must meet some

requirements:

1. Biodegradability is essential, since scaffolds

need to be absorbed by the surrounding tissues

without the necessity of- surgical removal.

2. A high porosity and an adequate pore size

are necessary to facilitate cell seeding and diffusion

throughout the whole structure of both cells and

nutrients.

3. The rate at which the degradation occurs

has to coincide as much as possible with the rate

of tissue formation; this means while the cells are

fabricating their own natural matrix structure around

themselves, the scaffold is able to provide structural

integrity within the body, and it will eventually break

down, leaving the newly- formed tissue that will

take over the mechanical load.

Most of the scaffold materials used in tissue

engineering has had a long history of use in

medicine as bioresorbable sutures and as meshes

used in wound dressings. The scaffold materials

available are:

1) Biodegradable or Permanent

2) Natural - derivatives of the extracellular matrix,

protein materials such as collagen or fibrin, and

polysaccharide materials, like chitosan or

glycosaminoglycans (GAGS),

Synthetic - common polyester materials that

degrade within the body such as polylactic acid

(PLA), polyglycolic acid (PGA), and

polycaprolactone (PCL).

New technologies for scaffold fabrication

a) Solid Freeform Fabrication: New scaffold

fabrication techniques are being developed such as

Solid Freedom Fabrication (SFF). Products are

designed on a computer screen as 3-D models with

information from CT or MRI scans. Ideally, after

implantation, a construct is organized into normal

healthy tissue as the scaffold degrades. The goal of

this technology is to fabricate with accurate patient-

specific macrostructure (3-D shape) and

microstructure (porosity and interconnected

channels) for ideal nutrient flow and tissue vascular

in–growth.


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One SFF technique, the 3-D printing

technology, is a manufacturing process that creates

parts directly from a computer model used in the

production of a complex 3-D scaffold. The parts

are built by spreading a layer of powder repetitively

and selectively joining the powder in the layer

through the inkjet printing of a binder material.

Moreover, using multiple feeds it becomes possible

to manufacture scaffolds with various architectural

qualities that can maintain multiple cell types on

each layer, thus closely mimicking the anatomic

features of a tissue or organ. Tissue engineering bone

using this technique demonstrates ability of bone

formation in vitro using porous PLGA/ TCP

composite scaffolds.

b) Smart scaffolds: the future: One of the basic

roles of a scaffold in bone tissue engineering is to

act as a carrier for cells and to maintain the space

and create environment in which the cells can

proliferate and produce the desired bone matrix.

Transplanted cells often lose the desired function

upon transfer from the in vitro culture system to the

in vivo recipient site. To address these problems,

scaffolds with the ability to deliver biochemical

factors at a predetermined rate for a definitive time

period are being developed. These smart scaffolds

have the advantage of-being able to:

1. Promote early capillary invasion.

2. Maintain cell activity and desired phenotype.

3. Induce osteoblastic differentiation of existing

progenitor cells in recipient tissue.

These smart materials may revolutionize tissue

engineering research because controlled release of

biochemical and growth factors from a scaffold may

enhance cell penetration, proliferation,

differentiation, and bone matrix production and

improve vascularization of grafts.[11]

APPLICATION OF TISSUE ENGINEERING IN

ENDODONTICS

Regenerative Endodontics

Millions of teeth are saved each year by root

canal therapy. Although current treatment

modalities offer high levels of success for many

conditions, an ideal form of therapy might consist

of regenerative approaches in which diseased or

necrotic pulp tissues are removed and replaced with

healthy pulp tissue to revitalize teeth. [8]

Regenerative endodontics is the creation and

delivery of tissues to replace diseased, missing and

traumatized pulp. These techniques will possibly

involve some combination of disinfection or

debridement of infected root canal system with

apical enlargement to permit revascularization and

use of adult stem cells, scaffolds, and growth factors.

Patient demand is staggering both in scope and cost,

because tissue engineering therapy offers the

possibility of restoring natural function instead of

surgical placement of an artificial prosthesis.[8]

The potential for pulp-tissue regeneration from

implanted stem cells has yet to be tested in animals

and clinical trials. Extensive clinical trials to evaluate

efficacy and safety lie ahead before it is likely the

Food and Drug Administration will approve

regenerative endodontic procedures.


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Several major areas of’ researches have been

identified that might have application in

development of regenerative endodontic techniques.

These techniques are:

· Root canal revascularization via blood clotting

· Postnatal stem cell therapy

· Pulp implantation

· Scaffold implantation

· Injectable scaffold delivery

· Three-dimensional cell printing

· Gene delivery

These regenerative endodontic techniques are

based on the basic tissue engineering principles

already described and include specific

consideration of cells, growth factors and scaffolds.

1) Root Canal Revascularization via Blood Clotting

Several case reports have documented

revascularization of’ necrotic root canal system

disinfection followed by establishing bleeding into

the canal system via overinstrumentation. An

important aspect of these cases is the use of

intracanal irrigants -with the placement of antibiotics

for several weeks. This particular combination of

antibiotics effectively disinfects root canal systems

and increases revascularization of avulsed and

necrotic teeth, suggesting that this is a critical step

in revascularization.Although these case reports are

largely from teeth with incomplete apical closures,

it been noted that reimplantation of avulsed teeth

with an apical opening of approximately, 1.1mm

demonstrate a greater likelihood of

revascularization. This finding suggests that

revascularization of necrotic pulps with fully formed

apices might require instrumentation of the tooth

apex to approximately 1 to 2 mm in apical diameter

to allow systemic bleeding into the root canal

systems.

The revascularization method assumes that

pulp space has been disinfected and that the

formation of a blood clot yields a matrix (e.g. fibrin)

that traps cells capable of initiating new tissue

formation. It is not clear that the regenerated tissue’s

phenotype resembles dental pulp; however, case

reports published to date do demonstrate continued

root formation and restoration of a positive response

to thermal pulp testing.

There are several advantages to a

revascularization approach. First, it is technically

simple and can be completed using currently

available instruments and medicaments without

expensive biotechnology. Second, the regeneration

of tissue in root canal systems by a patient’s own blood

cells avoids the possibility of’ immune rejection and

pathogenic transmission from replacing the pulp with

a tissue engineered construct.


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However, several concerns need to be

addressed in prospective research. First, case

reports of a blood clot having the capacity to

regenerate pulp tissue are exciting, but the caution

is required, because the source of regenerated tissue

has not been identified. Animal studies and more

clinical studies are required to investigate the

potential of this technique before it can be

recommended for general use in patients. Generally,

tissue engineering does not rely blood clot

formation, because the concentration and

composition of cells trapped in the fibrin clot is

unpredictable. This is a critical limitation because

tissue engineering is founded on the delivery of

effective concentrations and compositions of cells

to restore function. Second, enlargement of the

apical foramen is necessary to promote

vascularization and to maintain initial cell viability

via nutrient diffusion. Related to this point, cells

must have an available supply of oxygen; therefore,

it is likely that cells in the coronal portion of the

root canal system either would not survive or would

survive under hypoxic conditions before

angiogenesis.

2) Postnatal stem cell therapy

The simplest method to administer cells of

appropriate regenerative potential is to inject

postnatal stem cells into disinfected root canal

systems after the apex is opened. Postnatal cells can

be derived from multiple tissues, including skin,

buccal mucosa, fat, and bone. A major research

obstacle is identification of a postnatal stem cell

source capable of differentiating into diverse cell

population found in adult pulp (e.g., fibroblasts,

endothelial cells, odontoblasts). Technical obstacles

include the development of methods for harvesting

and any necessary ex vivo methods required to

purify and/or expand cell number sufficiently for

regenerative endodontic procedures.

One possible approach would be to use dental

pulp stem cells derived from autologous (patient’s

own) cells that have been taken from buccal mucosa

biopsy, or umbilical cord stem cells that have been

cryogenically stored after birth; an allogenic purified

pulp stem cell line that is disease- and pathogen-

free; or xenogenic (animal) pulp stem cells that have

been grown in the laboratory. It is important to note

that no purified stem cell lines are presently

available, and that the mucosal tissues have not yet

been evaluated for stem cell therapy.

There are several advantages to an approach

using postnatal stem cells. First, autologous, stem

cells are relatively easy to harvest and to deliver by

syringe, and the cells have the potential to induce

new pulp regeneration. Second, this approach is

already used in regenerative medical applications,

including bone marrow replacement.

However, there are several disadvantages to a

deliver method of injecting cells. First, the cells have

low survival rates. Second, the cells might migrate

to different locations within the body, possibly

leading to aberrant patterns of mineralization. A

solution for this latter issue may be to apply the

cells together with a fibrin clot or other scaffold

material. This would help to position and maintain

cell localization. Therefore, the probability of

producing new functioning pulp tissue by injecting

only stems cells into the pulp chamber, without a

scaffold or signaling molecules may be very low.

Instead, pulp regeneration must consider all three

elements (cells, growth factors, and scaffold) to

maximize potential for success.


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3) Pulp implantation

The majority of in vitro cell cultures grow as a

single monolayer attached to the base of culture

flasks. However some stem cells do not survive

unless they are grown on top of a layer of feeder

cells. In all these cases, the stem cells are grown in

two dimensions. In theory, to take two-dimensional

cell cultures and make them three-dimensional, the

pulp cells can be grown on biodegradable

membrane filters. Many filters will be required to

be rolled together to form a three-dimensional pulp

tissue, which can be implanted into disinfected root

canal systems.

laboratory. Moreover, aggregated sheets of cells are

more stable than dissociated cells administered by

injection into empty root canal systems. The

potential problems associated with the implantation

of sheets of cultured pulp tissue is that specialized

procedures may be required to ensure that the cells

properly adhere to root canal walls. Sheets of cells

lack vascularity, so only the apical portion of canal

systems would receive these cellular constructs,

with coronal canal systems filled with scaffolds

capable of supporting cellular proliferation. Because

the filters are very thin layer of cells, they extremely

fragile, and this could make them difficult to place

in root canal systems without breakage.

In pulp implantation, replacement pulp tissue

is transplanted into cleaned and shaped root canal

systems. The source of pulp tissue may be a purified

pulp stem cell line that is disease or pathogen-free,

or is created from cells taken from a biopsy, that

has been grown in the laboratory. The cultured pulp

tissue is grown in sheets in vitro on biodegradable

polymer nanofibers or on sheets of extracellular

matrix proteins such as collagen I or fibronectin.

So far, growing dental pulp cells on collagens I and

III has not yet proved to be successful, but other

matrices, including vitronectin and laminin, require

investigation.

The advantages of this delivery system are that

the cells are relatively easy to grow on filters in the

Ultra structure of a human tooth with implanted pulp (in purple)created from stem cells and a scaffold in the laboratory(Roxana et al. Medicine in Evolution,Nr.2008;4:11-22)

4) Scaffold implantation

To create a more practical endodontic tissue

engineering therapy, pulp stem cells must be

organized into a three-dimensional structure that

can support cell organization and vascularization.

This can be accomplished using a porous polymer

scaffold seeded pulp stem cells. A scaffold should

contain growth factors to aid in stem cell

proliferation and differentiation, leading to

improved and faster tissue development. The

scaffold may also contain nutrients promoting cell


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survival and growths, and possibly antibiotics to,

prevent any bacterial in-growth in the canal systems.

In addition, the scaffold may exert essential

mechanical and biological functions needed by

replacement tissue.[12]

To achieve the goal of pulp tissue

reconstruction, scaffolds must meet some specific

requirements. Biodegradability is essential, since

scaffolds need to be absorbed by the surrounding

tissues without the necessity of surgical removal. A

high porosity & an adequate pore size are necessary

to facilitate cell seeding and diffusion throughout

whole structure of both cells and nutrients. The rate

at which degradation occurs has to coincide as

much as possible with the rate of tissue formation;

this means that while cells arc fabricating their own

natural matrix structure around themselves, the

scaffold is able to provide structural integrity within

the body, and it will eventually break down, leaving

newly formed tissue that will take over the

mechanical load.

The principle drawbacks are related to the

difficulties of obtaining high porosity and regular

pore size. This has led researchers to concentrate

efforts to engineer scaffolds at nanostructural level

to modify cellular interactions with the scaffold.

Some proteic materials have not been well studied.

However, early results are promising in terms of

supporting cell survival and function, although some

immune reactions to these types of materials may

threaten their future use as part of regenerative

medicine.

5) Injectable scaffold delivery

Rigid tissue engineered scaffold structures

provide excellent support for cells used in bone and

other body areas where the engineered tissue is

required to provide physical support. However, in

root canal systems a tissue engineered pulp is not

required to provide structure support of the tooth.

This will allow tissue engineered pulp tissue to be

administered in soft three-dimensional scaffold

matrix, such as a polymer hydrogel. Hydrogels are

injectable scaffolds that can be delivered by syringe.

Hydrogels have the potential to be noninvasive and

easy to deliver into the root canal systems. In theory,

the hydrogel may promote pulp regeneration by

providing a substrate for cell proliferation and

differentiation into an organized tissue structure. Past

problems with hydrogels included limited control

over tissue formation and development, but

advances in formulation have dramatically improved

their ability support cell survival. Despite these

advances, hydrogels are at an early stage of research,

and this type of delivery system, although promising,

has yet to be proven to be functional in vivo. To

make hydrogels more practical, research is focusing

on making them photopolymerizable to form rigid

structures once they are implanted into the tissue site.

6) 3-D cell printing

The final approach for creating replacement

pulp tissue may be to create it using a three

dimensional cell printing technique. In theory, an

ink-jet like device is used to dispense layers of cells

suspended in a hydrogel to recreate the structure of

the tooth pulp tissue. This technique can be used

to precisely position cells and this method has the

potential to create tissue constructs that mimic the

natural tooth pulp tissue structure. The ideal

positioning of is in a tissue engineering construct

would include placing odontoblastoid cells around

the periphery to maintain and repair dentin, with

fibroblasts in the pulp core supporting a work of

vascular and nerve cells.


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Theoretically, the disadvantage of using three-

dimensional cell printing technique is that careful

orientation of the pulp tissue construct according

to its apical and coronal asymmetry would be

required during placement into cleaned and shaped

root canal systems. However, early research has

yet to show that three-dimensional cell printing can

create functional tissue in vivo.[12]

7) Gene therapy

The year 2003 marked a major milestone in

the realm of genetics and molecular biology. That

year marked the 50th anniversary of the discovery

of the double-helical structure of DNA by Watson

and Crick. On April 14th, 2003, 20 sequencing

centers in five different countries declared the

human genome project complete. This milestone

will make possible new medical treatments

involving gene therapy. All human cells contain a

1-m strand of DNA containing 3 billion base pairs,

with the sole exception of nonnucleated cells, such

as red blood cells. The DNA contains genetic

sequences (genes) that control cell activity and

function; one of the most well known genes is p53.

New techniques involving viral or nonviral

vectors can deliver genes for growth factors,

morphogens, transcription factors, and extracellular

matrix molecules into target cell populations, such

as the salivary gland. Viral vectors are modified to

avoid the possibility of causing disease, but still

retain the capacity for infection. Several viruses have

been genetically modified to deliver genes, including

retroviruses, adenovirus, adeno-associated virus,

herpes simplex virus, and lentivirus. Nonviral gene

delivery systems include -plasmids, peptides, gene

guns, DNA-ligand complexes, electroporstion,

sonoporation, and cationic liposomes. The choice

of delivery system depends on the accessibility and

physiological characteristics of the target cell

population.[18]

One use of gene delivery in endodontics would

be to deliver mineralizing genes into pulp tissue to

promote tissue mineralization. Rutherford

transfected ferret pulps with cDNA-transfected

mouse BMP-7 that failed to produce a reparative

response, suggesting that further research is needed

to optimize the potential of pulp gene therapy.

Moreover the potentially serious health hazards

exist with the use of gene therapy; these arise from

the use of the vector (gene transfer) system, rather

than the genes expressed the FDA did approve

research into gene therapy involving terminally ill

humans, but the approval was withdrawn in 2003

after a 9-year-old boy receiving gene therapy was

found to have developed tumors in different parts

of his body. Researchers must learn how to

accurately control gene therapy and make it very

cell specific to develop a gene therapy that is safe

to be used clinically. Because of the apparent high

risk of health hazards, the development of gene

therapy accomplish endodontic treatment seems


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very unlikely in the near future. Gene therapy is a

relatively new field, and evidence is lacking to

demonstrate that this therapy has the potential to

rescue necrotic pulp. At this time, the potential

benefits and disadvantages are largely theoretical.

Gene therapy by BMP’s

- In vivo

Half life of BMP’s as recombinant proteins is

limiting. Therefore gene therapy is a potential

alternative to conquer disadvantages of protein

therapy. Recombinant adenovirus containing BMP

7 gene induced only a small amount of poorly

organized dentin after direct transduction in

experimentally inflamed pulp. In vivo gene therapy

does not have much effect on reparative dentin

formation in case of severe inflammation.

- Ex- vivo

The transplantation of cultured dermal

fibroblasts transduced with BMP-7 using a

recombinant adenovirus, induced reparative dentin

formation in the exposed pulp with reversible

pulpitis. The great potency of BMP genes to provoke

differentiation of pulp stem cells, even if’ in

reversible pulpitis, demonstrates the utility of ex vivo

gene therapy in reparative / regenerative dentin

formation for clinical endodontic treatment.[5]

Barriers to be addressed to permit introduction of

regenerative endodontics

- Disinfection & shaping of root canals in a fashion

to permit regenerative endodontics.

Chemomechanical debridement - cleaning and

shaping root canals

Irrigants – 6% sodium hypochlorite and 2%

chlorhexidine gluconate and alteratives

Medicaments - Ca(OH )2, triple antibiotics, MT

AD and alternatives

- Creation of replacement pulp-dentin tissues

Pulp revascularization by apex instrumentation

Stem cells; allogenic, autologous, xenogenic,

umbilical cord sources

Growth factors; BMP-2, -4, -7; TGF-â 1, â2, â3

among others

Gene therapy; identification of mineralizing genes

Tissue engineering; cell culture, scaffolds,

hydrogels

- Delivery of replacement pulp-dentin tissues

Injection site

Surgical implantation methods

- Dental restorative materials

Improve the quality of sealing between restorative

materials and dentin

Ensure long-term sealing to prevent recurrent

pulpitis

- Measuring appropriate clinical outcomes

Vascular blood flow

Mineralizing odontoblastoid cells

Intact afferent innervations

Lack of signs or symptoms


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Conclusions :Scientific advances in the creation of restorative

biomaterials, in vitro cell culture technology, tissue

grafting, tissue engineering, molecular biology, and

the human genome project provide the basis for

the introduction of new technologies into dentistry.

This is intended to facilitate the development of stem

cell therapy for use with established therapeutic

modalities to restore and regenerate oral tissues.

Teeth have been shown to mineralize in response

to injury for many decades, but only in recent years

has the position of the stem cells been localized

around blood vessels. The cells have been identified

as myofibroblastoid pericytes. The ability to control

the differentiation and proliferation of these cells is

being examined to create stem cell therapies that

can solve dental problems more effectively than

current treatment regimens.

Furthermore, pulp regeneration will be used

as the basis for tissue engineering to radically alter

restorative dentistry and the prognosis of restored

teeth. To avoid the need for operative dentistry, DNA

vaccines may be used to arrest or prevent the

development of caries lesions. Restorative materials

may contain a “cocktail” of growth factors, delivered

in a slow-release vehicle to regenerate replacement

dentin from intra-coronal pulp matrix. In cases of

partially decayed or fractured teeth, pluripotent cells

may be implanted to regenerate tooth structure.

Eventually, non-restorable or lost and missing teeth

might be replaced by artificial implants of tooth

tissues grown synthetically in an in vitro culture.

For regenerative endodontic procedures to be

widely available and predictable, the endodontists

will have to depend on tissue engineering therapies

to regenerate pulp- dentin tissues. One of the most

challenging aspects of developing a regenerative

endodontic therapy is to understand how the various

component procedures can be optimized and

integrated to produce the outcome of a regenerated

pulp-dentin complex.

References :1. Karaoz E, Dogan BN, Aksoy A, Cacar C, Ayhan S, et al.Isolation and in-vitro Characterization of dental pulp stemcells from natal teeth. Histochem Cell Biol 2010;133:95-112.

2. Nedel F, André Dde A, de Oliveira IO, Cordeiro MM,Casagrande L, Tarquinio SB, Nor JE, Demarco FF. Stem Cells:Therapeutic Potential In Dentistry. J Contemp Dent Pract2009;10(4):90-96.

3. George T-J Huang, Wataru Sonoyama, Yi Liu, He Liu,Songlin Wang, Songtao Shi . The Hidden Treasure in ApicalPapilla: The Potential Role in Pulp/ Dentin Regeneration andBioroot Engineering. J Endod. 2008;34(6):645-51.

4. George T-J Huang. A paradigm shift in endodonticmanagement of immature teeth. Conservation of stem cellsfor regeneration. J Dent. 2008; 36(6):379-386.

5. Hedge Mithra, Naik Siddharth, Soni Garima. TissueEngineering – A Review. Indian Dentist Research and Review2008;2(7): 8-14.

6. Roxana Oancea, Liliana Vasile, Cristian Oancea, SavaRosianu Ruxandra, Daniela Jumanca, Ramona AminaPopovici. Morphological Assay Regarding Behavior of DentalPulp Stem Cells in Glucose Enriched Medium. Medicine inEvolution, Nr. 2008;4:11-22.

7. Trope M. Regenerative Potential of Dental Pulp. J Endod.2008; 34(7 Suppl):s13- s17.

8. Huang AH, Chen YK, Lin LM, Shieh TY, Chan AW. Isolationand Characterization of dental pulp stem cells from asupernumerary tooth. J Oral Pathol Med. 2008;39:571-4.

9. Mauth C, Huwig A, Graf-Hausner U, Roulet J-F. RestorativeApplications for Dental Pulp Therapy. Topics in TissueEngineering 2007;3:1-32.

10. Murray M, Garcia-Godoy F and Hargreaves KM.Regenerative Endodontics : A Review of Current Status and acall for Action. J Endod. 2007;33(4):377-390.

11. Franklin Garcia-Godoy. Tissue Engineering. Dent Clin NAm 2006;50(2):xiii–xiv.

12. Pamela C Yelick, Joseph P Vacanti. Bioengineered TeethFrom Tooth Bud Cells. Dent Clin N Am 2006;50(2):191-203.


regeneration of dental pulp: a myth or hype

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