Review Article
Regenerative endodontics—Creating new horizons
Harnoor Dhillon, Mamta Kaushik, Roshni Sharma
Department of Conservative Dentistry and Endodontics, Army College of Dental Sciences, Secunderabad, India
Received 13 April 2015; revised 5 September 2015; accepted 18 November 2015
Published online 24 December 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33587
Abstract: Trauma to the dental pulp, physical or microbio-
logic, can lead to inflammation of the pulp followed by
necrosis. The current treatment modality for such cases is
non-surgical root canal treatment. The damaged tissue is
extirpated and the root canal system prepared. It is then
obturated with an inert material such a gutta percha. In spite
of advances in techniques and materials, 10%–15% of the
cases may end in failure of treatment. Regenerative endodon-
tics combines principles of endodontics, cell biology, and tis-
sue engineering to provide an ideal treatment for inflamed
and necrotic pulp. It utilizes mesenchymal stem cells, growth
factors, and organ tissue culture to provide treatment. Poten-
tial treatment modalities include induction of blood clot for
How to cite this article: Dhillon H, Kaushik M, Sharma R. 2016. Regenerative endodontics—Creating new horizons. J Biomed
Mater Res Part B 2016:104B:676–685.
INTRODUCTION
The dental pulp is the central vascular tissue in the tooth
enclosed by rigid enamel and dentin. It is derived from
cephalic neural crest cells. Histologically, the pulp consists of
odontoblasts which produce dentin, numerous fibroblasts, and
undifferentiated mesenchymal cells also called perivascular
cells since they surround blood vessels. The matrix is made of
type I and II collagen present in a randomly dispersed manner
with increased density around blood vessels and nerves. The
amorphous ground substance consists of glycoproteins and
proteoglycans. The dental pulp has a complex architecture. An
outer layer of odontoblasts lines the dentin, followed by a cell
poor zone consisting of few blood vessels, unmyelinated nerve
fibers and cytoplasmic processes of fibroblasts. This lies above
a cell rich zone consisting mainly of fibroblasts and finally, the
central pulp resembling a connective tissue. The pulp has a
single source of blood supply via the root apex and lacks col-
lateral circulation. Innervation of the pulp is via myelinated
and unmyelinated nerve fibers. Dental Caries or decay is a
destructive disease of the mineralized structures of a tooth
which can ultimately infect the dental pulp. This condition
requires endodontic treatment which is essentially concerned
with preparation of the root canals followed by obturation
Correspondence to: H. Dhillon; e-mail: simply.hd@gmail.com
676
pulp revascularization, scaffold aided regeneration, and pulp
implantation. Although in its infancy, successful treatment of
damaged pulp tissue has been performed using principles of
regenerative endodontics. This field is dynamic and exciting
with the ability to shape the future of endodontics. This arti-
cle highlights the fundamental concepts, protocol for treat-
ment, and possible avenues for research in regenerative
endodontics. V C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part
B: Appl Biomater, 104B: 676–685, 2016.
Key Words: revascularization, pulp regeneration, tissue engi-
neering, endodontics, dentin formation
with an inert material (Figure 1). Although the techniques
show considerable success (78%–98%),1 long-term prognosis
of these teeth is compromised due to thinning of dentinal
walls during preparation. Also, in immature teeth, pulpal
necrosis due to trauma or infection may lead to arrested root
development. Hence, the focus of modern day endodontics is
shifting toward the regeneration of necrosed pulp tissue
instead of repair using conventional non-surgical root canal
treatment especially in immature teeth.
Regenerative endodontic procedures are defined as bio-
logically based procedures designed to replace damaged
dentin and cells of the pulp–dentin complex.2 Regenerative
endodontics currently has two major concepts: guided tis-
sue regeneration and tissue engineering.3
Guided tissue regeneration involves tissue regeneration
along a blood clot and is also known as revascularization.
€
This concept was introduced by Nygaard-Ostby in the year
19614 and is commonly used by clinicians. Tissue engineer-
ing with stem cells is still evolving. Both these concepts
merge toward the same goal—inducing physiological pulp
formation by activation of stem cells.
Apart from the above mentioned approaches, three
dimensional cell printing and gene therapy have also been
C 2015 WILEY PERIODICALS, INC.
V

FIGURE 1. Conventional root canal treatment and proposed technique of tissue engineering.
described as potential treatment options.2 Lasers are also
being explored as a treatment modality.
HISTORY
Regenerative endodontic procedures were first used in the
1950s when a case of vital pulp amputation using calcium
hydroxide was reported by Dr. B W Hermann.5 Although the
practice of regenerative procedures has gained importance
and popularity in the last 15 years, the foundation of the
modern day procedures was laid by Nygaard-Ostby B. who
observed the blood clot as essential to pulpal regeneration.4
To verify the hypothesis, 17 patients with pulpally damaged
or necrosed teeth were treated by disinfection of the root
canal, enlargement of the apical foramina, and induction of
bleeding into the canal. They were then restored coronal to
the induced blood clot with calcium hydroxide. This was fol-
lowed up for 17 days to 3.5 years. These treated teeth were
then extracted for histologic examination. All treated teeth
showed a similar response. Signs and symptoms resolved in
nearly 2 weeks, no pathosis were observed for necrotic
teeth and some cases showed radiographic closure of the
foramen at the root apex. Histologic examination revealed
that the tissue which had grown into the canal space was a
connective tissue along with islands of mineralization
embedded in it. Since the dental pulp is also a connective
tissue, this study substantiated the possibility of endodontic
regeneration.
However, exaggerated presence of undesirable cemento-
blasts (which lay cementum) but fewer desirable odonto-
blasts (which lay dentin), called for a better protocol.
Another study published during the same period by Rule
and Winter6 reported disinfection of root canals with the
use of polyantibiotic mixes given between appointments.
The investigators used three different formulations but did
not purposefully evoke intracanal bleeding in this study.
They instrumented canals short of the vital tissue deter-
mined by visualization and pain perceived upon instrumen-
tation. Continued root development and an absence of signs
of disease was evident. In another study published by
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Nygaard-Ostby the use of antibiotics for disinfection was
combined with intentional intracanal bleeding.7 Root devel-
opment and resolution of symptoms were observed in
treated teeth. Histologic analysis verified the presence of
connective tissue in 80% of the teeth and cellular cemen-
tum in 51.4% of the teeth.
Modern day regenerative endodontics revived when a
report published by Iwaya et al.8 in the Journal of Dental
Traumatology demonstrated successful response of treated
tooth to vitality tests as well as root development following
regenerative intervention. This study utilized a double anti-
biotic paste consisting of ciprofloxacin and metronidazole
along with repeated debridement to regenerate pulpal
tissue.
Banch and Trope9 developed a triple antibiotic paste
consisting of ciprofloxacin, metronidazole, and minocycline
placed in the canal for 28 days, after debridement with
sodium hypochlorite (5.25%). At the second visit, the antibi-
otic paste was removed with saline, bleeding was induced,
and a filling was placed coronal to the formed clot. The
treatment demonstrated root development and affirmative
response to vitality testing. Although a standardized proce-
dure for regenerative endodontics is lacking, the treatment
modality described in the above study is the method of
choice for most endodontists.
MODERN DAY PRACTICES AND RESEARCH
Revascularization—guided tissue regeneration
A standardized treatment protocol for revascularization is
recommended by Diogenes et al.10 based on clinical and
pre-clinical studies. According to this, the first appointment
consists of access to the root canal and irrigation with
sodium hypochlorite and saline. The canal is dried for place-
ment of a calcium hydroxide dressing as an intracanal
medicament. The second appointment scheduled after 2–4
weeks begins with examination of the treated tooth. In case
of absence of sensitivity to percussion or presence of a
sinus or swelling, the treatment carried out in the first
appointment is repeated. Here, a clinician may elect to use
677
 TABLE I. Characteristics of Stem Cells Derived From Teeth, Apical Papilla, and PDL
Cell Type Source
Dental pulp stem cells (DPSCs) Dental pulp
Stem cells of apical papilla (SCAP) Apical papilla of
immature teeth
Stem cells from exfoliated human Exfoliated
deciduous teeth (SHED) deciduous teeth
Periodontal ligament stem Periodontal
cells (PDLSCs) ligament
the triple antibiotic paste and place it in the canal. If symp-
toms are absent, bleeding is induced in the canal so that
blood fills the canal up to the cemento-enamel junction.
After clot formation, a collagen matrix is placed in the canal
above the clot to aid placement of mineral trioxide aggre-
gate (MTA), an endodontic cement composed of tricalcic sili-
cate, tricalcic alluminate, and bismuth oxide. This is covered
with a layer of glass ionomer cement, a restorative material
which bonds chemically to tooth structure, followed by res-
toration with a composite resin. Follow-up is advised after 3
and 6 months and yearly thereafter for a period of 4 years.
However, evidence in the form of clinical trials is lacking in
support of the proposed protocol. Also, criteria for success
of treatment have not been defined.
Retrospective studies11,12 offer evidence that treatment
with regenerative procedures provides better root thickness
and length than conventional root canal treatment. In 2011,
an analysis of the induced bleed found high levels of CD73
and CD105 markers compared with systemic levels indicat-
ing the presence of mesenchymal stem cells.13 This sug-
gested the role of tissue engineering in regenerative
endodontics. The standard technique for pulp regeneration,
however, needs to be refined. The blood clot formed has an
unpredictable concentration of entrapped stem cells, antibi-
otic paste can harm stem cells and irrigants such as sodium
hypochlorite degrade dentin derived proteins essential for
odontoblastic differentiation.14 The invaginate resembles
periapical tissue rather than the pulp histologically, with
bone like mineralized structures while a cementum like tis-
sue increases thickness of the root.15,16
Tissue engineering
In light of the facts mentioned above, pulpal regeneration
presents an interesting and relatively new avenue for appli-
cation of tissue engineering strategies.
Tissue engineering has three major components: (A)
Scaffolds; (B) Morphogens; and (C) Cell therapy. These have
been used in various studies singularly as well as in combi-
nation in order to regenerate the pulp dentin complex.
Although no clinical trials have been conducted, studies on
animal models so far show considerable progress.
Stem cells (Table I). During the 1970s, Freidstein et al. first
isolated stem cells from bone marrow and characterized
678 DHILLON, KAUSHIK, AND SHARMA
Mineralization
Potential Differentiation Potential
Can produce Odontogenic, neurogenic, myogenic,
mineralized matrix adipogenic, osteogenic
Better than DPSCs Odontogenic, osteogenic, adipogenic
Better than DPSCs Odontogenic, osteogenic, neurogenic
Produce mineralized Odontogenic, adipogenic
matrix only in
special media
them as self-renewing, fibroblastoid cells that could form
colonies on plastic and when transplanted subcutaneously,
gave rise to bone and reconstitute a hematopoietic microen-
vironment.17–19 These cells were later termed mesenchymal
stem cells (MSCs)20 and compose most of the stem cells
found in the orofacial region.21
Stem cells from the dental pulp were identified by Gron-
thos et al. in 200022 based on their ability to regenerate a
pulp dentin like complex. These were eventually termed as
dental pulp stem cells (DPSCs). Although numerous stem
cells have been identified in the oral region, the DPSCs,
Stem Cells of the Apical Papilla (SCAP), Stem Cells of Human
Exfoliated Deciduous teeth (SHED), and Periodontal Liga-
ment Stem Cells (PDLSCs) are believed to have increased
potential for regeneration of pulpal tissue.
DPSCs are multipotent cells with an ability to differenti-
ate into adipocytes, osteoblasts, melanocytes, myoblasts and
endothelial cells, produce mineralized tissue, and demon-
strate neurogenic potential.23–26 Dental pulp cells are known
to produce neurotrophic factors and even rescue motoneur-
ons after spinal cord injury.27 They are capable of retaining
their regenerative potential after cryopreservation28,29 which
renders them storable with minimal processing.29
SCAPs in the apical papilla of immature teeth have been
postulated to differentiate into cells responsible for contin-
ued root development in pulpally damaged immature teeth
with a retained apical papilla.30,31 They demonstrated osteo-
genic and adipogenic potential32 and presence of STRO-1
(human MSC marker), CD34 (marker for stem cell adhe-
sion), and CD146 (marker for human hematopoetic stem
cells), similar to DPSCs. Both cell types possess an ability
for cellular migration, organization, and mineralization but
SCAPs have a higher rate of proliferation and mineralization
potential when compared with DPSCs.33
Stem cells isolated from the remnant pulp of exfoliated
deciduous teeth (SHED) in 2003 are derived from a readily
accessible source and have a higher proliferation rate and
mineralization potential compared with DPSCs supported by
the comparative expression levels of inflammatory cyto-
kines, Col I (Collagen I marker) and PCNA (proliferating cell
nuclear antigen) in both teeth.34,35 They also demonstrate a
higher osteoinductive capacity in vivo and have a higher
neurogenic potential compared with DPSCs.34 On cryopre-
servation, SHED retained their regenerative potential similar
REGENERATIVE ENDODONTICS

to fresh SHED cells. Their therapeutic efficacy was evaluated
by implanting these cells into systemic lupus erythematosus
(SLE) models of mice. Their transplantation improved SLE-
like disorders.36 These cells need to be extracted at the
mixed dentition stage and preserved for use in adults.
Cells isolated from the periodontal ligament displayed
osteogenic and differentiation potential in vitro.37,38 Later,
stem cells isolated by Seo et al. in 200439 from the peri-
odontal ligament exhibited mesenchymal stem cell markers
and demonstrated the potential to regenerate into
cementoblast-like cells, adipocytes, and collagen forming
cells. They are present in all age groups but their regenera-
tive potential, migration, and proliferation capacity
decreases with age.40 PDLSCs when co cultured with DPSCs
show an upregulation of dentin sialoprotein (DSP), exclusive
marker for dentin, and dentin sialophosphoprotein (DSPP),
a precursor for DSP.41 They, however, do not have a capacity
to give rise to mineralized tissues unless grown with scaf-
folds containing tricalcium phosphate/hydroxyapatite (TCP/
HA) or in a medium conditioned with Apical tooth germ
cells.42,43
Adipose tissue derived stem cells (ADSCs) present a
more practical, alternative source of mesenchymal stem
cells for the clinical scenario compared with the dental
pulp.44 The expression of DSPP in these cells, enhanced
expression of genes related to mineralization and early
odontogenic marker genes indicate odontogenic potential.45
They have the potential to give rise to a dental bud-like
structure in vitro.46 They can regenerate same amount of
pulp tissue as DPSCs but have a lower potential to generate
a mineralized matrix.47,48 Further studies are required to
characterize the potential of ADSCs for regeneration of the
lost pulp.
Morphogens. Morphogens are extracellularly secreted sig-
nals that govern morphogenesis during epithelial–mesen-
chymal interactions.49 They play a vital role in controlling
stem cell activity by mechanisms that increase proliferation,
induce differentiation into diverse lineages or stimulate
them to secrete mineralized matrix.50,51 They play an
important role in the formation and repair of the dentin
pulp complex and are found in the dentin matrix post devel-
opment.52 Therapeutic intervention with recombinant
growth factors during repair may provide an avenue for tis-
sue repair and regeneration.53
The Transforming Growth Factors (TGF) b are impli-
cated as important for odontoblast differentiation and den-
tin matrix secretion.2 TGF-beta1 is present in the dentin
matrix in an active form and plays a crucial role in odonto-
blast differentiation, dentin matrix secretion, tooth develop-
ment, and tissue repair. It has been detected in association
with betaglycan, decorin, and latency associated peptide
(LAP) which regulate the availability and biological activity
of this growth factor.54 Another member of the TGF b fam-
ily, bone morphogenic proteins (BMPs) play an important
role in the pulp biology. During ameloblast differentiation,
BMP4 and BMP5 are expressed and BMP2, BMP4, BMP6,
BMP7, and Gdf11 during odontoblast differentiation.55,56
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During terminal differentiation of odontoblasts, increased
expression of BMP2 is seen.57 It is also required for differ-
entiation of SHED and DPSCs into odontoblasts.14,58 Apart
from these factors, bone sialoprotein has also been observed
to stimulate pulp cells to differentiate into matrix secreting
cells at the site of injury.59,60 The importance of growth fac-
tors is highlighted by studies which demonstrated that the
morphology of dentin produced is related to the morphogen
used for induction.61,62
Angiogenesis is critical to the development and survival
of the regenerated pulp. Examination of the dentin matrix
shows high concentration of platelet derived growth factor
(PDGF-AB), vascular endothelial growth factor (VEGF), fibro-
blast growth factor (FGF), placenta growth factor (PIGF),
and low concentration of epidermal growth factor (EGF).63
Pulp fibroblasts in vitro express numerous angiogenic fac-
tors whose levels can be altered by neuropeptides.64 Addi-
tion of VEGF to cultured tooth slices increased microvessel
density.65 Human dental pulp stem cells also show remark-
able angiogenic potential.66,67 Inhibition of miR-424 gene
and induction of SIRT 1 gene promotes endothelial differen-
tiation in DPSCs and can aid pulp repair and regenera-
tion.68,69 However, mechanisms involved in the initiation
and development of the pulp tissue angiogenesis require
further elucidation.70 Although in vivo regeneration of pulp
like tissue in teeth with angiogenic markers has been
done,65,71 establishment of vasculature through the apical
foramen, the only means of blood supply to a formed tooth,
needs to be addressed.
Studies show that dental pulp stem cells have consider-
able neurogenic potential. Neuron and synapse like mor-
phology has also been seen.72–74 Activators of PKC and
cAMP pathways and basic fibroblast growth factor positively
influence neural differentiation of DPSCs.75–77 This aspect of
pulp tissue regeneration requires further exploration.
Morphogens for neurogenesis, angiogenesis, and odonto-
genesis also need to be evaluated when used in combina-
tion, to generate the pulp as a whole. Adequate provision of
desired morphogens for the desired time can probably be
controlled with the use of an appropriate scaffold.
Scaffolds. Scaffolds are three dimensional structures that
serve as a template aiding development of tissue by provid-
ing regulatory molecules and mechanical support. Ideally, a
scaffold should mimic the structure of the extracellular
matrix (ECM) and provide optimum conditions for every
event in tissue generation, should be biocompatible and bio-
degradable.78,79 Natural scaffolds can be derived from con-
stituents of the ECM such as GAGs, chitosan, collagen,
polysaccharides, etc.80–82 The blood clot developed during
revascularization and dentin chips (a reservoir of growth
factors as well) aid in stem cell attachment.11,83,84 Synthetic
scaffolds are generally made from degradable polyesters
such as polylactic acid (PLA), polyglycolic acid (PGA), or
polycaprolactone (PCL).85,86
Platelet rich plasma (PRP) and platelet rich fibrin (PRF)
have been stated as potential ideal scaffolds in human teeth.
They demonstrate the potential to resolve clinical
679
symptoms, periapical pathosis, and aid root develop-
ment.87–90 On histologic examination, tissue formed in a
tooth treated with platelet rich plasma was determined to
be pulp-like tissue.91 In a recent study, PRP was positively
shown to create a scaffold for regenerative endodontic treat-
ment but no significant difference was observed between
PRP and conventional blood clot (BC) scaffold.92
Techniques using nanotechnology for development of
scaffolds offer better control over mechanical and physical
properties of the scaffolds and can mimic the environment
provided by ECM. They stimulate cell proliferation, stem cell
differentiation, and activate cell signaling pathways by
chemical and mechanical stimuli.78,79 They are thus gaining
popularity in regenerative techniques used in endodontics.
Nanofibrous scaffolds can be processed by electospinning,
molecular phase assembly (seen in hydrogels) or thermally
induced phase separation which are believed to best mimic
the structure of the ECM.79
Electrospinning involves application of an electric field
to a polymer solution to create fibers with diameters in the
nanometer to micrometer range. Electrospun scaffolds offer
great control with respect to fiber diameter, morphology,
and alignment.93 The polymer solutions can be incorporated
with bioactive particles and therapeutic substances. These
fibers have been successfully used to deliver controlled
amount of antibiotics to the root canal so that damage to
stem cells is minimal while disinfection is achieved. The
release of drugs in such cases is at a lower concentration
than in pastes as reinforced by analysis through high-
performance liquid chromatography.94–96 An electrospun
R
nanocomposite scaffold made from polydioxanone (PDS IIV)
and halloysite nanotubes (HNTs) has been demonstrated as
a highly biocompatible scaffold that can provide attachment
to pulp derived cells making them good candidates for mak-
ing bioactive scaffolds.97
Hydrogel polymers provide an interesting means of fab-
ricating a matrix by injection into the root canal system.
They use the process of molecular assembly for scaffold
generation. Molecular self-assembly generates scaffolds
which have a molecular arrangement made using noncova-
lent forces.80,98 They have the advantage of being broken
down into constituent molecules by the body. Nanofibers
assembled in this way have an advantage of being injected
as solutions and form gels that can be used to encapsulate
cells.99–101 They provide easy delivery of constituents of
stem cell therapy into the root canal. Growth factors incor-
porated into hydrogels have been demonstrated to effec-
tively reach target tissues. Incorporation of fibroblast
growth factors (FGF) into gelatin has led to regeneration of
components of the pulp–dentin complex.99 A promising scaf-
fold seems to be a self-assembling hydrogel made of 16-mer
peptides in aqueous solutions called PuramatrixTM. It poly-
merizes when it interacts with a physiological environment
and forms a biodegradable scaffold.100,101 It has been shown
to support proliferation and differentiation of DPSCs in vitro
for at least 3 weeks100 and that of SHED in vivo in animal
models.101 Although hydrogels present the most clinically
suitable option as injectable scaffolds, their applications are
680 DHILLON, KAUSHIK, AND SHARMA
limited by lack of control over the pore size, maintenance in
the whole canal and tissue formation.
Thermally induced phase separation (TIPS) is a tech-
nique that uses solvents with low melting points that can
sublime. The solvent rich phase is separated from the sol-
vent poor phase and is then used to make porous scaffolds.
It can be used in conjunction with other techniques to gen-
erate three dimensional porous scaffolds which provides
optimal delivery of nutrients for cell proliferation, angiogen-
esis, and tissue regeneration.79
Techniques for tissue regeneration used in animal
models
Regeneration of the dental pulp presents challenges such as
establishment of vasculature and nerve supply through the
apical foramen, development of the pulpal architecture and
sensory, nutritive, formative, and protective functions. The
studies’ done so far have used two main approaches: one
utilizing the tooth slice as a scaffold and another which uti-
lizes the entire tooth or a tooth root for regeneration of the
pulp.
A technique developed by Cordeiro et al. has been sug-
gested to demonstrate pulp tissue regeneration is the tooth
slice/scaffold model which is based on the tooth slice organ
culture by Sloan et al.102 It utilizes dentin slices as scaffolds
which are seeded with stem cells. These are implanted into
immunodeficient mice to help regenerate pulp tissues.
Regeneration of tissue structurally similar to the pulp has
been demonstrated.65,103,104 One study on 2.5 mm thick sli-
ces combined collagen scaffolds with DPSCs and Dentin
Matrix Protein (DMP1) transplanted in immunodeficient
mice showed development of pulp like tissue in the group
with all three components rather than others where, either
or both stem cells and growth factors were missing.105
Another experiment utilized slices with a canal depth of 5–
6 mm detected pulp-like and mineralized tissue in the canal
space along with odontoblast-like cells when transplanted
in the SCID mouse models. This study used DPSCs and
SCAP with a poly-D,L lactide and glycolide scaffold.106 These
experiments were performed on 1–2.5 mm tooth slices.
Such studies do not adequately address the generation of
large amount of tissue to be deposited in fully formed or
incompletely developed roots or the re-establishment of pul-
pal configuration.
The second technique utilizes full length root models to
regenerate the pulp in vivo in animal models. Kim et al.71
studied collagen scaffolds in combination with FGF, PDGF,
VEGF, Nerve Growth Factor (NGF), and BMP 7 delivered to
endodontically treated tooth roots implanted subcutane-
ously in mice for 3 weeks. This study utilized cell homing
instead of cell delivery for tissue generation as an economi-
cal option to cell transplantation. Successful re-
cellularization along the entire root canal, generation of
mineralized tissue resembling secondary dentin, and revas-
cularization of the canal space with presence of erythrocytes
and blood vessel-like structures was revealed. In a study by
Rosa et al.,101 the roots of premolar teeth were divided into
two groups. One was injected with SHED in a hydrogel
REGENERATIVE ENDODONTICS

FIGURE 2. Schematic representation of tissue engineering technique in regenerative endodontics.
scaffold (PuramatrixTM) and second provided recombinant
human collagen as a scaffold. The growth of vital pulp-like
tissue was seen in both the groups. However, odontoblast
differentiation markers were expressed faster in the hydro-
gel scaffold than in collagen scaffolds. Dentin deposition
was seen along the complete length of the root canal dem-
onstrated by tetracycline staining. This study successfully
demonstrated regeneration of a pulp-like connective tissue
and hard tissues along the length of the canal. This study
serves as a platform for development of newer and effective
approaches in pulp regeneration which would involve a
combined effort between material scientists, biologists, and
dentists. These studies make it possible to regenerate the
volume of the pulp. However, they fail to address the chal-
lenges of gaining a vascular supply solely via the root apex
and establishment of pulpal architecture and function.
An ideal, clinically possible approach toward dental pulp
regeneration in cases of necrotic or non-vital pulp will per-
haps need a combination of endodontic procedures and tis-
sue engineering. Gaining access to the pulp chamber
followed by cleaning and shaping of the root canal will pre-
pare the tooth for reception of the scaffold. Injection of a
biodegradable scaffold with embedded medicaments and
cytokines which are released over a period of time in tan-
dem with development of tissue, may aid in de novo genera-
tion of functional pulp tissue (Figure 2).
Gene therapy
Gene therapy offers a novel approach toward the healing
and regeneration of dental pulp tissue. Genes are located in
the form of a genetic sequence in the DNA of nucleated cells
that control cell activity and function. Vectors are used for
delivery of required sequences to target tissues. These
sequences may belong to morphogens, ECM components, or
may be transcription factors.2 Although viral vectors are a
highly efficient mode of gene transfer compared with non-
viral vectors like plasmids, peptides, electroporation, sono-
poration, and so forth, they pose a risk of infection.
Rutherford encoded the sequence of BMP7 gene into a
recombinant adenovirus and was able to induce reparative
dentinogenesis in vitro but failed to do so in vivo.107 Naka-
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shima et al. published a report wherein a three-dimensional
pellet culture was electrotransfected with growth/differen-
tiation factor 11(Gdf11). After 10 days, markers of odonto-
blast differentiation had a higher expression in transduced
pellets than control. Based on this finding an in vivo investi-
gation was done on a canine. The transplantation of trans-
fected cells onto amputated pulp successfully induced
reparative dentin formation.108 The number of studies
involving gene therapy as a means of endodontic tissue
regeneration is limited. In case of necrotic pulps, gene ther-
apy will have to be used in combination with stem cell ther-
apy for treatment. In a study by Yang et al.,109 chitosan/
collagen scaffolds were loaded with a plasmid encoding
gene for BMP7. DPSCs were seeded into these scaffolds and
were evaluated in vitro and in vivo. The cells were success-
fully transfected and secretion of BMP7 was observed until
day 24. They also displayed better odontoblast differentia-
tion and proliferation properties than non-transduced cells.
In vivo, transfected cells lasted up to 4 weeks and showed
upregulated expression of DSPP.
Lasers
Low level laser irradiation has been demonstrated to
increase proliferation of mesenchymal cells in vitro.110 A
study conducted on bone marrow stem cells demonstrated
a positive effect on BMSC proliferation, growth factor secre-
tion, and myogenic differentiation.111 Research shows low
intensity laser therapy aids and accelerates pulp healing
and dentin formation in damaged pulps.112–116 The result-
ant dentin formed by these therapies was in the residual
pulp chamber akin to formation of tertiary dentin which
occurs during mild pulpal inflammation. The formed defect
was not filled by de novo dentin formation.
In different studies, DPSCs did not demonstrate any
increase in proliferation of the cells or in the secretion of
mineralized tissue117 but SHED subject to low intensity
laser irradiation phototherapy show increased proliferation
and mineralization of dental pulp constructs when a laser
with an optimum energy density is used.118 A recent study,
however, found increased cell proliferation and bone sialo-
protein expression in lased groups of DPSCs.119 An attempt
681
at regeneration of injured pulpal tissue using the Nd:YAG
showed formation of osteodentin and odontoblast-like
cells.120 Dental pulp cells can be guided using endothelial
cells ablated with a Nd:YAG laser in vitro.121 Another study
done by Arany et al.122 established that a non-ionizing, low
power laser can be used to activate the TGF-b1 which dif-
ferentiates human dental stem cells in vitro and increases
dentin regeneration in vivo, in rat models. The studies pro-
vide inconclusive results about enhanced regeneration
potential of pulpal stem cells with lasers. Since the studies
mentioned use lasers with different parameters, perhaps
definite criteria need to be set to ensure success. This
potential therapy needs further research to explore its
potential as a cost effective means of stimulating tissue
regeneration singularly as well as in combination with tis-
sue engineering techniques.
CONCLUSION
Regenerative endodontics is a dynamic field which presents
a new exciting and potentially ideal treatment for damaged
and necrotic teeth. However, the current clinical protocols
are not standardized and clinical trials in this field are lack-
ing. Research in regenerative endodontic therapy using vari-
ous growth factors, stem cells, and scaffolds also need to be
evaluated in combination with other aspects of translational
medicine such as lasers and gene therapy. Combined with
the concepts of tissue engineering, this field offers a wide
range of treatment options which are yet to be explored.
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