REVIEW
Stem Cells in Regenerative Endodontics: A Review
Parul Mehta
ABSTRACT
Regenerative Endodontics is expected to become the next big
treatment revolution. It is the creation and delivery of tissues to
replace a diseased, missing and traumatized pulp dentin
complex, in addition to tooth supporting tissues. It employs
dental stem cells for this which is especially important because
they are a type of mesenchymal stem cells that produce more
neurotrophic factors than other cell types. These cells have
demonstrated the ability to protect and repair neural tissue in
diseases like spinal cord injury and stroke and hence can also be
used for development of treatment solutions for such diseases.
Dental stem cells are also easy to procure and preserve for use in
future therapies. This article takes you through the source,
methods of delivery and outcome of using these stem cells in
regenerative endodontics. It also highlights the scope of the
treatment in the present day and age.
Key words : Regenerative Endodontics, Dental stem cells
Introduction
Stem cells are unique cells which give rise to specialized
tissue. They have a capacity for self renewal and give
rise to one and at times many differentiated cell types.
In mature tissues, these cells play a major role in
homeostasis and cell repair. Five types of dental stem
cells have been established. These are dental pulp stem
cells, stem cells from apical papilla, stem cells from
exfoliated deciduous teeth, periodontal ligament stem
cells and dental follicle progenitor cells. These can
differentiate into odontoblasts, adipocytes, neuron like
cells, glial cells, osteoblasts, chondrocytes,
melanocytes, myotubes and endothelial cells.1
Embryonic Stem Cells-By the blast stage of embryonic
development, a specialized compartment in the inner
cell mass begins to take shape. Cells within this inner
cell mass have been termed embryonic stem
cells(ESC).Thus Embryonic stem cells (ESCs) are derived
from embryos that are 2–11 days old called blastocysts.
They are best grown from supernumerary embryos
obtained from in vitro fertilization centers. Animal
studies have demonstrated that when placed IJCDinto• JUNE,
a 2012
developing embryo,ESCs have the capacity © 2012to
formof Contemporary
Int. Journal
obtained from in vitro fertilization centers. Animal
studies have demonstrated that when placed into a
developing embryo, ESCs have the capacity to form
every type of cell found in the body.2 This potentially
unlimited source of cells could one day relieve the
heavy burden on tissue replacement by whole organ
transplantation. However among obstacles that stand in
the way are difficulty in manipulating the cells to
reproducibility and predictably differentiate them into
deigned tissues.
Adult Stem Cells-were thought to have a rather
restricted potential for generating new tissue. However
recent studies have shown in addition to have
generated the derivatives of the blood system stem
cells within the bone marrow of an adult,they can also
give rise to neuron like cells within the brain and
muscle. Adult stem cells are found in most adult tissues.
They are multipotent – capable of differentiating into
more than one cell type but not all cell types.
Depending on their origin, adult stem cells can be
further classified as hemopoetic stem cells (HSCs) and
mesenchymal stem cells (MSCs). HSCs are obtained
either from cord blood or peripheral blood. MSCs are
those that originate from the mesoderm layer of the
fetus and in the adult reside in a variety of tissues such
as the bone marrow stem cells (BMSCc), limbal stem
cells, hepatic stem cells, dermal stem cells, etc.
Stem cells have also been isolated from orofacial tissues
which include adult tooth pulp tissue, pulp tissue of
deciduous teeth, periodontal ligament, apical papilla,
and buccal mucosa. Gronthos et al. have isolated stem
cells from adult human dental pulp (DPSCs) that exhibit
a similar immunophenotype to bone marrow stem
cells.3 Stem cells from human exfoliated deciduous
teeth (SHED) represent a unique population of
multipotent stem cells that are easily accessible and are
more immature in the cell hierarchy than the adult pulp
stem cells. Using a similar methodology, multipotent
stem cells from the human periodontal ligament
(PDLCs) have also been described.Recently, a new
population of mesenchymal stem cells (MSCs) residing
in the apical papilla of incompletely developed teeth
• 3(2) have been isolated and demonstrated in elegant
(SCAP) 66
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papilla of incompletely developed teeth (SCAP) have
been isolated and demonstrated in elegant studies.3,4
Bone Formation in Vivo-The majority of work in this
area has focused on the ability of BMSC to differentiate
into bone.This invitro expanded BMSCs may be a rich
source of fibronectin.The disadvantage of this
technique is that implantation of sheets of cells may be
technically difficult.The sheets are very thin and fragile
so research is needed to develop reliable implantation
techniques.
Cell-based therapy
Adult stem cells have been used in pilot studies as
potential cell-based therapy for various diseases. The
following stem cell characteristics make them good
candidates for cell-based therapy.
1. Potential to be harvested from patients
2. High capacity of cell proliferation in culture
3. Ease of manipulation to replace existing
nonfunctional genes via gene splicing methods
4. Ability to migrate to host target tissues
(homing)
5. Ability to integrate into host tissues and interact
with the surrounding tissues4
Process of cell-based therapy
Stem cells derived from either peripheral blood, cord
blood, bone marrow, or any adult tissue transported in
the right medium to the laboratory is centrifuged,
trypsinized, and propagated under ideal conditions and
stored in the master cell bank (MCB). The MCB is
further passaged to yield colonies of stem cells, given
the right inductive signals using appropriate growth
factors to allow them to differentiate into required cell
types. These are injected or implanted into a patient as
cell-based therapy. Homing will ensure that the stem
cells reach the site of injury/tissues.
Today, the two common methods of cell delivery are
intravenous injection (direct delivery of cells) and cell
encapsulation systems (indirect delivery of cells using a
carrier). The cell encapsulation approach uses a
biocompatible, biodegradable material construct that is
seeded with cells and implanted into defects in order to
regenerate the lost tissue.
Research Priorities for developing regenerative
endodontic techniques-
1. Improved methods for disinfecting and shaping root
canal systems-The majority of available evidence
suggests that necrotic and infected tooth pulp does not
67 IJCD • JUNE, 2012 • 3(2)
© 2012 Int. Journal of Contemporary Dentistry
heal.It will be necessary to disinfect root canal systems
to remove infected hard and soft tissues before using
regenerating endodontic treatment. Chlorhexidine
gluconate has been studied for it’s various properties
such as antimicrobial activity and biocompatibility with
the objective of evaluating it as an alternative to
sodium hypochlorite.
2.Smear Layer Removal-The presence of smear layer on
the root canal wall is thought to inhibit the adherence
of implanted pulp stem cells, potentially causing
regenerative endodontic treatment to fail.The smear
layer is 1-5micrometre layer of denatured cutting debris
produced on instrumented cavity surfaces and is
composed of dentin, odontoblastic processes, inorganic
contaminants and microorganisms. Chemical chelating
agents are used to remove the smear layer of which the
most popular - 17% of ethylenediaminetetraaceticacid
(EDTA) is used as a final flush. Among other solutions
investigated is MTAD which is an aqueous solution of
3% doxycycline, 4.25% citric acid, .5% polysorbate
and80% detergent.This biocompatible intracanal
irrigant is commercially available as a two part set,that
is mixed on demand.6
Engineering a functional pulp tissue-The success of
regenerative endodontic therapy is dependent on the
ability of researchers to create a technique that will
allow clinicians to create a functional pulp tissue, within
cleaned and shaped root canal systems. The source of
pulp tissue may be from root canal revascularization,
stem cell therapy or pulp implantation involving surgical
implantation of synthetic pulp tissue grown in the
laboratory.7 Pulp tissue regeneration involves either
delivery of autologous/allogenic stem cells into the root
canals or implantation of the pulp that is grown in the
laboratory using stem cells. Both these techniques will
have certain advantages and limitations that need
further research.
Scaffold Implantation –To create a more practical
tissue engineering therapy,pulp cells must be organized
into a three dimensional structure that can support cell
organization and vascularization.This can be
accompalished by using a porous polymer scaffold
seeded with pulp stem cells.A scaffold should contain
growth factors to aid stem cell differentiation and
proliferation.The scaffold may also contain nutrients
promoting cell survival and growth and possibly
antibiotics to prevent any bacterial ingrowth in the
canal systems.Dentin chips may provide a matrix for
pulp stem cell attachment and also be a reservoir of
growth factors.To acheive the goal of pulp tissue
reconstruction,scaffolds must meet some specific
requirements.Biodegradability is essential and high
porosity and adequate pore size are necessary to
facilitate cell seeding and diffusion through the whole
structure.5The rate at which degradation occurs has to
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The following table shows potential regenerative root canal techniques-
Technique Advantage Disadvantage

1.Root canal -Lowest risk of immune -Minimal case reports

revascularization-open up
tooth apex to allow bleeding
into rootcanal.
2.Stem cell therapy-
autologous or allogenic stem
cells are delivered to teeth
via injectable matrix.
3.Scaffold implant-pulp cells
are seeded onto a 3D scaffold
made of polymers and
surgically implanted.
4. 3D cell printing-inkjet like
devices dispense layers of
cells in a hydrogel which is
surgically implanted.
5.Injectable scaffolds-
polymerizable hydrogels
alone or containing cell
suspension are delivered by
injection.
rejection published till date
-Lowest risk of pathogen -Potential risk of necrosis if
transmission tissue becomes infected.
-Quick easy delivery -Low cell survival
-Least painful -Cells do not produce new
-Cells are easy to harvest functioning pulp
-High risk of complications
-Structure supports cell -Low cell survival after
organization implantation
-Bone materials may promote -Must be engineered to fit
vascularization. root canals precisely
-Multiple cell types can be -Must be engineered to fit
precisely positioned root canals precisely
-Early stage result has yet to
prove functional invivo
-Easy delivery -Limited control over tissue
-May promote regeneration formation
by providing substitute for -Low cell survival
extracellular matrix -Early stage research has yet
to be proven

coincide with as much as possible with the rate of tissue
formation.
The types of scaffold materials available are natural or
synthetic. The synthetic materials include polylactic
acid(PLA), polyglycolic acid(PGA) and
polycaprolactone(PCL) which are all common polyester
materials that degrade within the human
body.Synthetic polymers are found to be more
conductive and show less contraction as compared to
natural materials.6,8
Injectable Scaffold Delivery-This will allow tissue
engineered pulp tissue to be administered in a three
dimensional scaffold matrix such as a polymer hydrogel.
Hydrogels are injectable scaffolds that can be delivered
by syringe. They have the potential to be non invasive
and easy to deliver into root canal systems. Past
problems with hydrogels included limited control over
tissue formation and development but advances in
formulation have dramatically improved their ability to
support cell survival. However they are still at an early
stage in research. To make hydrogels more practical,
research is focussing on making them
photopolymerizable, to form rigid structures once they
are implanted into the tissue site. 6,8
Delivery of Regenerative Endodontic procedures-A
promising cellular source for regenerative endodontics
is autologous stem cells from oral mucosa. The oral
mucosa cells are readily available as a source of oral
cells which avoids the problem of patients being
required to store umbilical cord blood or third molar
immediately after extraction. It also avoids the need for
bone biopsies. The oral mucosa cells may be maintained
using invitro cell culture with antibiotics to remove
infection. The cells may then be seeded in the apical
1/3mm of a tissue engineering scaffold with the
remaining coronal 15+mm containing a cellular scaffold
that supports cell growth and vascularization. This
tissue construct may involve an injectable slurry of
(hydrogel+cells+growth factors).This two layer method
is fairly easy to accomplish. Moreover by seeding cells
only in the apical region, there is reduced demand for a
large number of cells derived from the host. Instead
most of the cellular proliferation would occur naturally
in the patients. This proposal delivery method would
help avoid immune and infection issues with the use of
an allogenic pulp stem cell line.
Measuring appropriate clinical outcomes-Once a tissue
engineered pulp has been implanted ,it is not ethical to
remove functional tissue to conduct a histological
analysis. Clinicians will have to rely on non invasive tests
available such as laser doppler blood flowmetry in
teeth,pulp testing involving heat,cold and electricity and
lack of signs and symptoms. Magnetic resonance
imaging has shown the potential to distinguish between
vital and non vital pulps but the machines are very
expensive and must be reduced in price to become
widespread.The ideal clinical outcome is a non
symptomatic tooth that never needs retreatment but
nonspecific vitality assessment methods are essential to
validate that regenerative endodontic techniques are
truly effective.

IJCD • JUNE, 2012 • 3(2) 68

© 2012 Int. Journal of Contemporary Dentistry
 REVIEW
Scope- Denver dentists Dr James De Lapp, Dr Candace
Lapp and Dr Sarah Parsons are partnering with “Stem
Save”,a company that offers dental patients a chance to
bank their valuable stem cells for use in future
regenerative therapies. Stem cells found in teeth are
extracted by Denver dentists and cryopreserved which
enable patients to recover and save these powerful
cells.It has been found stem cells in teeth replicate
faster than stem cells from other tissues. At
present,there are more than 78 clinical trials involving
stem cell treatments underway.9
5. Ma PX, Choi JW: Biodegradable polymer scaffolds
with well defined interconnected spherical pore
network. Tissue Eng. 2001;7:23–33.
6. MurrayPE, Hargreaves KM : Regenerative
Endodontics: A review of current status and a call for
action,Journal of Endodontics 2007;33(4):377-390.
7. Nakashima M,Akamine A:The application of tissue
engineering to regeneration of pulp and dentin in
endodontics,Journal of Endodontics 2005;31:711-718.

Not all teeth are eligible for stem cell preservation.The 8.Nakashima M:Tissue engineering in endodontics,
tooth needs to have a healthy pulp,needs to have an Australian Endodontic Journal,2005;31(3):111-113.
intact blood supply and be free from infection or
9.Seo B, Miura M, Gronthos S, Bartold PM, Batouli S,
cavities. Deciduous teeth may be the best source of
Brahim J, et al: Investigation of multipotent postnatal
stem cells.The incisors that have begun to loosen or
stem cells from human periodontal ligament. Lancet.
baby canine teeth appear to be the best candidates.
2004;364:149–55.
Wisdom teeth between the ages of 16-20 may be a very
good source. The pulp at this stage is large and the
potential for viable cells high.

About the Author

Conclusion
Tissue engineering using the triad of dental pulp stem
cells, morphogens, and scaffolds may provide an 1. Dr. Parul Mehta BDS MDS
innovative approach for treatment of dental diseases
and endodontic regenerative procedures for which the Clinical Practitioner
challenges are substantial but the benefits
groundbreaking. Each one of the regenerative I-302 Som Vihar,
techniques have their advantages as well as R.K Puram,
disadvantages. However for regenerative endodontic New Delhi.
procedures to be widely available and predictable,
endodontics will have to depend on tissue engineering
therapies to regenerate pulp dentin tissue. Better
understanding of cell interactions and growth and how
the various components can be optimized and
integrated can make endodontic tissue engineering a Address for Correspondence
reality in the future.

Dr. Parul Mehta BDS MDS

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© 2012 Int. Journal of Contemporary Dentistry