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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
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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
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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.
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