Tissue Engineering
classified according to their ability to
INTRODUCTION
Tooth engineering is a promising new
therapeutic approach that seeks to replace
the missing tooth with a bioengineered one
to restore the damaged dental tissue.1
Regenerative endodontics is a biological
procedure designed to replace the diseased,
missing, traumatized tissue including dentin
and root structures as well as cells of pulp-
dentin complex.
The first definition of tissue engineering was
said to be given by Langer and Vacanti who
stated it to be “an interdisciplinary field that
applies the principles of engineering and life
sciences toward the development of
biological substitutes that restore, maintain,
or improve tissue function or a whole
organ.”
The general aim of regenerative dentistry is
to use stem cells, tissue engineering, gene
therapy, and/or bioactive molecules to
replace and repair dental tissues damaged by
dental diseases, trauma, and cancer, or
missing teeth due to congenital
malformation.2
Tissue engineering employs use of three
materials:
1. Stem cells/progenitor cells: these
cells are capable of differentiating
into specialized cells by responding
to Morphogens. They can be
differentiate as totipotent,
pluripotent, or multipotent cells.
2. Morphogens/signaling molecules:
these are extracellularly secreted
signals governing morphogenesis
during epithelial-mesenchymal
interactions. The morphogenetic
signaling networks include the five
major classes of evolutionarily
conserved genes: bone morphogenic
proteins (BMPs), fibroblast growth
factors (FSFs), wingless- and int-
related proteins (Wnts), Hedgehog
proteins (Hhs), and tumor necrotic
factor (TNF).
3. Scaffold/matrix: provides a
physicochemical and biological
three-dimensional micro-
environment for cell growth and
differentiation, promoting cell
adhesion, and migration. Natural
polymers such as collagen and
glycosaminoglycans offer good
biocompatibility. Synthetic materials
like poly lactic acid (PLA), poly
glycolic acid (PGA) can also be
used.3,4,5
These approaches to repair and regeneration
depend on the basic principles of tissue
engineering, namely the interaction of a
scaffold/matrix with cells and signaling
molecules, albeit in the guise of natural
wound healing events. The use of a tooth
slice model in which a poly (L-lactic acid)
scaffold seeded with stem cells was applied
to the pulp space allowed the regeneration of
pulp tissue with physiological-like
appearance.6,7 The study model exploited
the acidic nature of the poly (L-lactic acid)
scaffold to locally release bioactive
signaling molecules from the dentin matrix
of the tooth slice highlighting the potential
importance of this endogenous “fossilized”
reservoir of bioactive molecules in dentin,
which may contribute to the signaling
wound healing events.8
The principles of regenerative medicine can
be applied to endodontic tissue engineering.
Regenerative endodontics comprises of
research in adult stem cells, growth factors,
organ-tissue culture, and tissue engineering
materials (Fig. 1). Often these disciplines are
combined, rather than used individually to
create regenerative therapies.9
This review provides a brief overview of the
potential types of regenerative endodontic
therapies. Several major areas of research
that might have application in the
development of regenerative endodontic
techniques include:
 Root canal revascularization via
blood clotting
 Postnatal stem cell therapy
 Pulp implantation
 Scaffold implantation
 Injectable scaffold delivery
 Three-dimensional cell printing
 Gene delivery
STEM CELLS
Stem cells also known as “progenitor or
precursor” cells are defined as clonogenic
cells capable of both self-renewal and multi-
lineage differentiation.11 Stem cells have
manifold applications and have contributed
to the establishment of regenerative
medicine. Regenerative medicine is the
process of replacing or regenerating human
cells, tissues or organs for therapeutic
applications.12 Major breakthrough in dental
history was achieved in the year 2000 when
Gronthos et al. identified and isolated
odontogenic progenitor population in adult
dental pulp.13 These cells were referred to as
dental pulp stem cells (DPSCs).10
DENTAL STEM CELLS:
1. Dental pulp stem cells (DPSCs):
mesenchymal type of stem cells
inside dental pulp. They have
osteogenic and chondrogenic
potential in vitro and can
differentiate into dentin, in vivo and
also differentiate into dentin-pulp-
like complex. They are putative
candidate for dental tissue
engineering due to easy surgical
access to collection site, low
morbidity after extraction, they can
generate much more typical dentin
tissues within a short period and can
 be safely cryopreserved and
recombined with many scaffolds. 21
2. Stem cells from human exfoliated
deciduous teeth (SHED): Miura et
al. confirmed that SHED were able
to differentiate into a variety of cell
types to a greater extent than DPSCs,
including osteoblast-like,
odontoblast-like cells, adipocytes,
and neural cells.15 The main task of
these cells seems to be the formation
of mineralized tissue, which can be
used to enhance orofacial bone
regeneration.10 Primary incisors and
canines with no pathology and at
least one third of root left can be
used for SHED banking. Primary
molar roots are not recommended for
sampling as they take longer time to
resorb, which may result in an
obliterated pulp chamber that
contains no pulp, and thus, no stem
cells.10,16
3. Stem cells from apical papilla
(SCAP): Mesenchymal stem cells
residing in the apical papilla of
permanent teeth with immature roots
are known as SCAP. They are
capable of forming odontoblast-like
cells, producing dentin in vivo, and
are likely cell source of primary
odontoblast for the formation of root
dentin. SCAP support apexogenesis,
which can occur in infected
immature permanent teeth with
periradicular periodontitis or abscess.
They can survive pulp necrosis
because of their proximity to the
periapical tissue vasculature. Hence,
even after endodontic disinfection,
SCAP can generate primary
odontoblasts, which complete root
formation under the influence of the
surviving epithelial root sheath of
Hertwig. 10,17
4. Periodontal ligament stem cells
(PDLSCs): Seo et al.18 described the
presence of multipotent postnatal
stem cells in the human periodontal
ligament. When transplanted into
rodents, PDLSCs had the capacity to
generate a cementum/periodontal
ligament-like structure and
contributed to periodontal tissue
repair. These cells can also be
isolated from cryopreserved
periodontal ligaments while retaining
their stem cell characteristics,
including single-colony strain
generation, cementum/periodontal
ligament-like tissue regeneration,
expression of MSC surface markers,
multipotent differentiation and hence
providing a ready source of
MSCs.10,19
Previous studies have conducted
comprehensive phenotypic analysis of ex
vivo expanded dental pulp, periodontal,
cementum, and bone marrow-derived
stromal cell populations demonstrating a
common expression pattern profile for a
variety of antigens associated with
endothelium (CD106, CD146),
perivascular tissue (α-smooth muscle
actin, CD146, 3G5),
bone/dentin/cementum (BMPs, alkaline
phosphatase, Type-I collagen,
osteonectin, osteopontin, osteocalcin,
bone sialoprotein) and fibroblasts (Type-
 III collagen) (Table 1)20
EFFICACY OF DENTAL STEM
CELLS:
Identification of human dental derived
mesenchymal stem cells (MSC):
Bone marrow stromal stem cells (BMSSC)
have previously been identified by their
capacity to form adherent colonies,
morphologically similar to fibroblasts
(colony forming unit-fibroblastic, CFU-F),
when plated at low-cell densities in the
presence of media supplemented with
mitogenic growth factors or serum.24,25,26
Recently, other mesenchymal stem cell
populations derived from third molard
(DPSCs) have been identified, exfoliated
deciduous teeth 9SHED), and adult
periodontal ligament (PDLSCs), by their
ability to generate clonogenic adherent cell
clusters when plated under the same growth
conditions as described for BMSCCs.
21,22,23,27
Growth potential of human dental-derived
MSC:
Cloning experiments indicated that the
majority of individually isolated colonies
(>80%) failed to proliferated beyond 20
populations doublings for all MSC types
tested.21,22,23,27,28 Proliferation studies using
BrdU labeling of multi-colony-derived
DPSCs, SHED and PDLSC cell cultures
exhibited higher rates of proliferation,
approximately 30,50, and 30% when
compared to the growth of cultured
BMSCCs, respectively. 21,22,23,27,28
Ongoing studies indicate that DOSC, SHED,
and PDLSC maintain a higher growth
potential beyond 100 population doublings,
in contrast to BMSCC that begin to undergo
cellular senescence at approximately 50
population dublings. The finite life-span of
post-natal mesenchymal stem cell
populations has previously been correlated
with a decline in their growth potential in
vitro. Research efforts are continuing to
unravel the mechanisms of how telomerase
activity regulates the growth of MSC. It is
anticipated that these studies will help
develop strategies to genetically manipulate
ex vivo expanded cells, in order to enhance
and regulate the growth potential of
expanded BMSSC, DPSC, SHED, and
PDLSC for potential clinical applications.20
HARVESTING TEETH CREATED
BY TISSUE ENGINEERING:
The characterization of dental mesenchymal
differentiation into pulp, dentin, and enamel
has highlighted some developmental
processes that may be used by tissue-
engineering therapies to create synthetic
teeth. The development of tissue-
engineering science has recently allowed
new tissues such as small intestine to be
created, after seeding stem cells onto
biodegradable polymer scaffolds.30 The use
of this tissue-engineering technique has
allowed the formation of tooth crowns from
stem cells taken from porcine third molars.31
After the cell/polymer constructs have been
formed using in vitro tissue culture, these
are implanted into a suitable host animal to
provide a vascular blood supply to support
the growth and development of higher-
ordered tissue structures.32 However, adult
human pulp stem cells have not yet used
these techniques to create human teeth by
tissue engineering.
POTENTIAL TECHNIQUES FOR
REGENERATIVE ENDODONTICS:
There are several major areas of research
that might have application in the
development of regenerative endodontic
techniques.9
These techniques are:9
a. Root canal revascularization via
blood clotting
b. Post natal stem cell therapy
c. Pulp implantation
d. Scaffold implantation
e. Injectable scaffold delivery
f. Three-dimensional cell printing
g. Gene delivery

REGENERATIVE
ENDODONTICS

The pulp is an organ known to have

tremendous reparative/regenerative abilities.
The bridge of reparative/regenerative dentin
that directly bonds to reactionary and
primary dentin around the exposed site of
the pulp can be more useful protection from
physic-chemical and bacterial irritation than
any restorative material.33,4 Regenerative
endodontics endeavors to use a tissue
engineering approach.
GENE THERAPY:

Gene therapy is recently used as a means of

delivering genes for growth factors,
Morphogens, transcription factors,
extracellular matrix molecules locally to
somatic cells of individuals with a resulting
therapeutic effect. The gene can stimulate or
induce a natural biological process by
expressing a molecules involved in
regenerative response for the tissue of
interest.34 Precise delivery and efficient
transfer of genes into target tissue cells,
prompt assessment of gene expression at
required times and appropriate levels and the
minimilization of undesirable systemic
toxicity and are essential for successful gene
therapy. Both an in vivo and ex vivo
approach can be used for gene therapy.4

Table 1: 4

In vivo methods: (Fig. 1A)

Add gene in vivo to induce new function.

Ex: angiogenesis
Ex vivo methods: (Fig. 1B)
Culture cells ex vivo, add gene, transplant
back to host. Ex: re-growth of cartilage and
bone
Genes can stimulate or induce a natural
biological process by expressing a molecule
involved in regenerative response for the
tissue of interest.36 Precise delivery and
efficient transfer of genes into target tissue
cells, prompt assessment of gene expression
at required times and appropriate levels
and/or minimization of undesirable systemic
toxicity are essential prerequisites for
successful gene therapy. Either viral or non-
viral vectors are used to enable the cellular
uptake and expression of genes. Viral
vectors are genetically altered to eliminate
their disease-causing ability. The viruses can
replicate genes of interest together with their
own genome, through the use of host cell
genetic machinery.35
Non-viral techniques involve either
electroporation or ultrasound method for
gene delivery. Ultrasound-mediated gene
delivery is found to be successful both in
vivo and in vitro but electroporation method
is found successful only in vitro. This may
be because of the lack of erythrocytes in the
plasma clot due to thermal changes during
electroporation in vivo. In the in
vivo approach, the gene is delivered
systematically into the bloodstream or
locally to target tissues by injection or
inhalation. In this approach, the healing
potential of pulp tissue is enhanced by genes
inducing dentin directly applied on the
exposed amputated dental pulp. The ex vivo
approach involves genetic manipulation of
cells in vitro, which are subsequently
transplanted to the regeneration site. The ex
vivo gene therapy stimulates reparative
RECENT PROGRESS ON
DENTAL STEM CELL
RESEARCH
Stem cell biology has become an important
field for the understanding of tissue
regeneration, although much knowledge in
this area has been from the in vitro studies.
In general, stem cells are defined by having
two major properties:
1. They are capable of self-renewal
2. When they divide, some daughter
cells give rise to cells that eventually
become differentiated cells.
dentin formation more optimally and rapidly
in comparison to in vivo gene therapy.35
An ongoing challenge in the field is to
isolate progenitor stem cells with well-
defined cell markers from both dental pulp
tissue and PDL and to expand these cells
types in vitro. Current research in several
centers is focusing on the isolation of
universal human stem cells with defined
markers that potentially could be used to
circumvent immune histocompatibility
barriers. Such cells may ultimately allow the
development of stem cells for use as shuttle
vectors in cellular/gene therapy. (see Box 2).
To date, four types of human dental stem
cells have been isolated and characterized:
a) Dental pulp stem cells (DPSCs), b) Stem
cells from exfoliated deciduous teeth
(SHED), c) Stem cells from apical papilla
(SCAP), and d) Periodontal ligament stem
cells (PDLSCs). Among them, all except
SHED are from permanent teeth. DPSCs
and SHED are from the pulp and SCAP is
from the pulp precursor tissue, apical
papilla. These ex vivo expanded cells can
differentiate into odontoblast-like cells and
produce dentin-like tissue in both in vitro
and in vivo study systems. 38
When grown in cultures and induced under
specific conditions, DPSCs and SHED can
differentiate into neuronal and adipogenic
cells in addition to dentinogenic cells.39,40
SCAP together with PDLSCs are able to
form a root-like structure when seeded onto
the hydroxyapatite-based scaffold and
implanted in pig jaws. These dental stem
cells may potentially be utilized for dental
tissue regeneration, i.e., pulp/dentin and
periodontal ligament. 41-43
The indentification of these dental stem cells
provided us a better understanding of the
biology of pulp and periodontal ligament
tissues and their regenerative potential after
tissue damage. For example, the observation
of severely infected pulp in immature teeth
capable of undergoing complete root
maturation after proper disinfection
procedures may be explained by the
possibility that SCAP somehow survive
from infection.44,45
DRAWBACKS OF
REGENERATIVE ENDODONTIC
TREATMENTS:
The most common drawbacks observed are:
1. Discoloration: due to use of
minocycline in the triple antibiotic
paste, when minocycline comes in
contact with coronal dentinal walls
during treatment procedure.
2. Treatment period: the required time
for disinfection of the root canal
space with triple antibiotic paste or
calcium hydroxide and increased
number of clinical sessions are other
drawbacks.
3. Poor root development: in some
studies, the outcome of regenerative
endodontic treatments of necrotic
immature teeth was lower than ideal,
including absence of increase in root
length, absence of increase in root
wall thickness, or lack of formation
of tooth apex. Formation of hard-
tissue barrier inside the canal
between the coronal MTA plug and
the root apex is another reported
unfavorable outcome.
4. Root canal calcification/obliteration:
in a case series study by Chen et al,
complete root canal
calcification/obliteration happened in
4 of 20 cases within an average
follow-up time of 16 months.
5. Insufficient bleeding: some authors
have reported failure to induce
bleeding. A recent study revealed
that mesenchymal stem cells are
delivered into root canal space after
bleeding induction in human teeth, a
phenomenon that did not happen in
the absence of blood clot inside the
disinfected root canal space. In
addition, it is assumed that the blood
clot formed inside the root canal
space after disinfection contains
platelet-derived growth factors that
serves as a protein-rich scaffold.