This document provides an overview of regenerative endodontics, which aims to regenerate dental tissues through biologically-based procedures using stem cells, growth factors, and scaffolds. Key elements for pulp regeneration include reliable stem cell sources, such as dental pulp stem cells, growth factors to stimulate cell proliferation and differentiation, and appropriate scaffolds. Potential regenerative therapies include revascularization, stem cell therapy, scaffold implantation, and gene delivery. Measuring clinical outcomes and further applications are areas of future focus to develop regenerative endodontic therapies.

1. REGENERATIVE
ENDODONTICS

2. INTRODUCTION
DEFINITIONS
OBJECTIVE
KEY ELEMENTS
Stem Cells
Growth Factors
Scaffolds
POTENTIAL TECHNOLOGIES FOR REGENERATIVE ENDODONTICS
Root canal revascularization via blood clotting
Postnatal stem cell therapy
Pulp implantation
Scaffold implantation
Injectable scaffold delivery
Three-dimensional cell printing
Gene delivery
MEASURING CLINICAL OUTCOMES
FUTURE APPLICATION
CONCLUSION
CONTENTS

3. INTRODUCTION
• 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.
• Regenerative endodontics is the creation and delivery of tissues to replace diseased,
missing, and traumatized pulp.
• Providing an overview of the methodological issues IS required to develop potential
regenerative endodontic therapies, AND present a call for action to develop these therapies
for clinical use.

4. The regeneration or replacement of oral tissues affected by inherited disorders, trauma, and
neoplastic or infectious diseases is expected to solve many dental problems with in next 25
years.
As well as ability to stimulate endodontic regeneration
Replace diseased tissue
Vaccinations against virus
Genetically alter diseased pathogens to help eradicate caries and periodontitis
Meet patient demand for tissue engineering therapy.

5. REGENERATIVE ENDODONTICS
Regenerative endodontic procedures can be defined as biologically based procedures
“designed to replace damaged structures”, including dentin and root structures, as well
as cells of the pulp-dentin complex.
TISSUE ENGINEERING
“ an interdisciplinary field that applies the principle of engineering and life sciences
towards the development of biological substitutes that restore, maintain, or improve
tissue function.”
Langer & vacanti tissue engineering, science
1993:260.920-6
DEFINITIONS

6. TISSUE REGENERATION:
Replacement of injured tissue by the same resident cells, or by differentiation of
progenitor/stem cells into tissue committed cells.
STEM CELLS:
Stem cells are defined as clonogenic cells capable of both self renewal and multi-
lineage differentiation.
(Kumar Et Al. 2009, Majno & Joris 2004)

7. MORPHOGENS / SIGNALING MOLECULES
Morphogens or signaling molecules are proteins that bind to receptors on the cell and
induce cellular proliferation and/or differentiation.
SCAFFOLD
Provides a physicochemical and biological three-dimensional micro environment for cell
growth and differentiation, promoting cell adhesion and migration.

8. REPAIR VS REGENERATION
Repair Regeneration
Healing occurs because remaining damaged
tissue is vital
The damaged tissue is completely necrotic.
Repair is defined as the process when healing
takes place by proliferation of connective
tissue elements resulting in fibrosis and
scarring.
Regeneration is defined as the process when
healing takes place by proliferation of
parenchymal cells and usually results in
complete restoration of the original tissues.
generate cementum like, bonelike or
periodontal-like tissues instead of a normal
dental pulp
Closely resembles normal pulp tissue

9. The objectives of regenerative endodontic procedure, are to
Regenerate pulp like tissue, ideally the pulp -dentin complex
Regenerate damaged coronal dentin, such as following a caries exposure
Regenerate resorbed root, cervical or apical dentin.
OBJECTIVE

10. 10
GOALS OF REGENERATIVE PROCEDURES

11. Historically, long-term calcium hydroxide treatment was used to induce apexification of
the immature tooth with pulpal necrosis before placing an obturation material such as
gutta-percha in the root canal system. While the success rate of calcium hydroxide
apexification is reported to be as high as 95%, there are several associated problems.
What is the Biological Basis for Regenerative Endodontic Therapy?

13. • Studies on MTA apexification report that the success rate of the treatment is as high
as 94%.
• Prospective clinical trials comparing MTA apexification to calcium hydroxide
apexification report that the success rate of the former is comparable to or higher than
that of the latter

14. However, neither of the apexification treatments fosters further root development and
immature teeth remain vulnerable to cervical root fractures.
In contrast, regenerative endodontic therapy has the potential for increased root development,
and thus, may confer a better long-term prognosis.
In addition, successful regeneration of the pulp-dentin complex would likely result in vital tissue
capable of mounting an immune response and signaling tissue damage by sensory neurons.

15. The counterargument to the development of
regenerative endodontic procedures
that the pulp in a fully developed tooth plays no major role
in form, function, or esthetics, and
thus its replacement by a filling material in conventional
root canal therapy is the most practical treatment.

16. In terms of esthetics. there is a potential risk that endodontic filling materials and sealers
may discolor the tooth crown.
van der Burgt TP. Plasschaert AJ .Tooth discoloration induced be dental
materials. Oral Surg Oral Med Oral Pathol 1995b,
666-9
A retrospective study of tooth survival times following root canal filling versus
tooth restoration found that although root canal therapy prolonged tooth survival,
the removal of pulp in a compromised tooth may still lead to tooth loss in
comparison with teeth with normal tissues
Caplan DJ, cai J, yin G, While BA Root canal filled versus non-root canal filled
teeth, a retrospective comparison of survival times
J Public Health Dent 2005;65; 9o-6
ARGUMENTS SUPPORTING REGENERATIVE ENDODONTICS

18. KEY ELEMENTS

19. Three major components of pulp regeneration
• A reliable cell source capable of differentiating into
odontoblasts
• Growth factors that are capable of stimulating cellular
proliferation and directing cellular differentiation.
• An appropriate scaffold to promote cell growth and
differentiation

20. Stem Cell
• An undifferentiated cell of a multicellular organism which is capable of giving rise to
indefinitely more cells of the same type, and from which certain other kinds of cell
arise by differentiation.
Stem cells are self renewing
and thus can generate any
tissue for a lifetime unlike
other progenitor cells

21. • The principles of tissue engineering to the development of Regenerative endodontic
procedures requires the correct understanding of stem cell which contribute to the
formation of dental hard and soft tissues.
• Epithelial-mesenchymal interactions : tooth formation

22. • Dental epithelial cells such as ameloblasts and ameloblasts precursors are eliminated
soon after tooth eruption. Therefore, epithelial cells that could be stimulated in vivo to
form enamel are not present in human adult teeth.
• Mesenchymal cells are primarily required for the formation of dental pulp and are
studied widely as compared to the epithelial stem cells.

23. TYPES OF STEM CELLS

24. 24
CLASSIFICATION OF STEM CELLS (GENERAL)

25. 25
CLASSIFICATION OF STEM CELLS BASED ON
PLASTICITY
Stem cell type Cell plasticity Source of stem cell
Totipotent Each cell can develop into
a new individual
Cells from early (1–3 days)
embryos
Pleuripotent Cells can form any (over
200) cell types
Some cells of blastocyst
(5–14 days)
Multipotent Cells differentiated, but
can form a number
of other tissues
Fetal tissue, cord blood,
and postnatal stem cells
including dental pulp stem
cells

26. POST NATAL STEM CELLS
• Postnatal stem cells have been sourced from umbilical cord blood, umbilical cord, bone
marrow, peripheral blood, body fat, and almost all body tissues, including the pulp tissue of
teeth

27. 27
CLASSIFICATION OF DENTAL STEM CELLS

28. FUNDAMENTAL PROPERTIES OF STEM
CELLS
Undifferentiated cells Have not developed into a specialized
cell type
Long- term self- renewal The ability to go through numerous
cycles of cell division while
maintaining the undifferentiated state
Production of progenitor cells Capacity to differentiate into
specialized cell types.
( eg. Odontoblast, osteoblast,
adipocyte, fibroblast)

29. DENTAL PULP STEM CELLS (DPSCS)DENTAL PULP STEM CELLS (DPSCS)
Epithelial-mesenchymal
interactions
Dental papilla cells  functioning
odontoblasts
DPSCs - isolated in 2000 by Gronthos et al.
• Striking ability to regenerate a dentin-pulp-like complex composed of a mineralized matrix of
tubules lined with odontoblasts, and fibrous tissue containing blood vessels in an
arrangement similar to the dentin-pulp complex

31. 31
STEM CELLS FROM THE HUMAN EXFOLIATED DECIDUOUSSTEM CELLS FROM THE HUMAN EXFOLIATED DECIDUOUS
TEETH (SHED)TEETH (SHED)
• Shed - isolated in 2003 by MIURA et al
• Main task of these cells seems to be the formation of mineralized tissue, which can be used
to enhance oro-facial bone regeneration.
• Have a higher proliferation rate than stem cells from permanent teeth
• Generates dentin-pulp like tissue with distinct odontoblast like cells.

32. 32
ADVANTAGES:
•Can be retrieved from a tissue that is disposable and readily accessible.
•SHED BANKING IS MORE ECONOMICAL WHEN COMPARED TO CORD BLOOD AND
MAY BE COMPLEMENTARY TO CORD CELL BANKING
•Ideally suited for young patients at the mixed dentition stage who have suffered pulp
necrosis in immature permanent teeth as a consequence of trauma.
•SHED may also be useful for close relatives of the donor such as grandparents, parents
and siblings
Only primary incisors
and canines

33. 33
STEM CELLS OF APICAL PAPILLA (SCAP)
• SCAP – isolated in 2008 by SONOYAMA et al.
• Scientists have described the physical and histologic characteristics of the dental papilla
located at the apex of developing human permanent teeth and termed this tissue apical papilla
.
• The apical papilla is essential for root development  loosely attached to the apex of the
developing root and can be easily detached with a pair of tweezers.
• SCAP are capable of forming odontoblast-like cells, producing dentin in vivo.

34. 34
• Because of the apical location of the apical
papilla, this tissue may be benefited by its
collateral circulation due to its proximity to the
periapical tissue vasculature, which enables it to
survive during the process of pulp necrosis.
• 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
• Cells are clonogenic and can undergo odontoblastic, adipogenic or neurogenic
differentiation.
• Scap show higher proliferation rates than dpscs.

35. 35
• PDLSCS- isolated in 2004 by SEO et al.
• Present as multipotent postnatal stem cells in
the human PDL
• When transplanted into immunocompromised
rodents, PDLSCS showed the capacity to
generate a cementum/pdl-like structure and
contributed to periodontal tissue repair.
PERIODONTAL LIGAMENT STEM CELLS (PDLSCS)

36. 36
• Using a mini pig model, autologous SCAP and PDLSCS were loaded onto
hydroxyapatite/tricalcium phosphate and gelfoam scaffolds, and implanted into sockets in the
lower jaw, where they formed a bioroot encircled with the periodontal ligament tissue and in a
natural relationship with the surrounding bone.
• Li et al. have reported cementum and periodontal ligament-like tissue formation when PDLSCS
are seeded on bioengineered dentin.

37. INDUCED PLEURIPOTENT CELLS
This technique was developed using a quartet of trascription factors-
OCT3,SOX2,
KLF4 and c-MYC to reprogram somatic cells into pleuripotent cells

38. STEM
CELLS

39. 39
STEM CELL IDENTIFICATIONSTEM CELL IDENTIFICATION

40. 40
STEM CELL ISOLATIONSTEM CELL ISOLATION
• SIZE-SIEVED ISOLATION
Enzymatic digestion of whole dental pulp tissue in solution of 3% collagenase type I for 1 h at
37°C is done. Through process of filtering and seeding, cells with diameter between 3 and 20
μm are obtained for further culture and amplification. Based on this approach, small-sized cell
populations containing a high percent of stem cells can be isolated.
STEM CELL COLONY CULTIVATION
Enzymatic digestion of the dental pulp tissue is done to prepare single cell suspension cells of
which are used for colony formation containing 50 or more cells that is further amplified for
experiments.

41. MAGNETIC ACTIVATED CELL SORTING (MACS)
Is an immune-magnetic method used for separation of stem cell populations based on
their surface antigens (CD271, STRO-1, CD34, CD45, and c-Kit). MACS is technically
simple, inexpensive and capable of handling large numbers of cells but the degree of
stem cell purity is low.
FLUORESCENCE ACTIVATED CELL SORTING (FACS)
Is convenient and efficient method that can effectively isolate stem cells from cell
suspension based on cell size and fluorescence. Demerits of this technique are a
requirement of expensive equipment, highly-skilled personnel, decreased viability of
FACS-sorted cells and this method is not appropriate for processing bulk quantities of
cells.

42. STORAGE OF STEM CELLSSTORAGE OF STEM CELLS
CRYOPRESERVATION :
It is the process of preserving cells or whole tissues by cooling them to sub-zero temperatures.
Cells harvested near end of log phase growth (approximately. 80–90% confluent) are best for
cryopreservation.
 Liquid nitrogen vapour is used to preserve cells at a temperature of -1960
c .
In a vial 1.5 ml of freezing medium is optimum for 1–2 × 106
cells.

43. MAGNETIC FREEZING
This technology is referred to as cells alive system (CAS)
It works on principle of applying a weak magnetic field to water or cell tissue which will
lower the freezing point of that body by up to 6–7°C.
Using CAS, Hiroshima University (first proposed this technology) claims that it can
increase the cell survival rate in teeth to 83%.
 CAS system is a lot cheaper than cryogenics and more reliable

44. MORPHOGENS
• Growth factors are proteins that bind to receptors on the cell and induce cellular proliferation
and/or differentiation.
• Many growth factors are quite versatile, stimulating cellular division in numerous cell types,
while others are more cell specific .
• GROWTH FACTORS  CONTROL STEM CELL ACTIVITY SUCH AS

45. Transforming growth factorbeta (TGF) family cellular signaling for odontoblast
differentiation and stimulation of dentin matrix secretion.
(secreted by odontoblasts and deposited within the dentin matrix, where they remain protected
in an active form through interaction with other components of the dentin matrix.)
The addition of purified dentin protein fractions has stimulated an increase in tertiary dentin
matrix secretion.
Recombinant human BMP2 stimulates differentiation of adult pulp stem cells into an
odontoblastoid morphology in culture.
The similar effects of TGF B1-3 and BMP7 have been demonstrated in cultured tooth slices.
Recombinant BMP-2, -4,and -7 induce formation of reparative dentin in vivo.
The application of recombinant human insulin-like growth factor-1 together with collagen has
been found to induce complete dentin bridging and tubular dentin formation
TO BE USED IN CONJUNCTION WITH STEM CELLS

46. Abbreviation Factor Primary
Source
Activity Usefulness
BMP Bone
morphogenetic
proteins
Bone matrix Differentiation of
osteoblasts and
mineralization of
bone
Make stem cells
synthesize and
secrete mineral matrix
CSF Colony
stimulating
factor
A wide range of cells Stimulate the
proliferation
of specific
pluripotent bone
stem cells
Increase
stem cell numbers
EGF Epidermal growth
factor
Submaxillary glands Proliferation of
mesenchymal,
glial and
epithelial cells
Increase stem cell
numbers
FGF Fibroblast growth
factor
A wide range of cells Proliferation of
many cells
Increase
stem cell numbers
IGF Insulin-like
growth
factor-I or II
I - liver II–variety of
cells
Proliferation of
many cell types
Increase
stem cell numbers

47. IL Interleukins IL-1 to
IL-13
Leukocytes Stimulate the
humoral and
cellular immune
responses
Promotes inflammatory cell
activity
PDGF Platelet-derived
growth factor
Platelets, endothelial
cells,placenta
Proliferation
of connective tissue,
glial
and smooth muscle
cells
Increase stem cell
numbers
TGF-α Transforming
growth factor alpha
Macrophages, brain
cells,and keratinocytes
Important for
normal wound
healing
Induces epithelial and tissue
structure
development
TGF-β Transforming
growth factor-beta
Dentin matrix,
activated
TH1 cells (T-helper)
and
natural killer (NK)
cells
antiinflammatory,
promotes wound
healing, inhibits
macrophage and
lymphocyte
proliferation
Present in dentin matrix
and has been used to
promote
mineralization of pulp
tissue
NGF Nerve growth factor A protein secreted by a
neuron’s target tissue
Critical for the
survival
and maintenance of
sympathetic and
sensory
neurons.
Promotes neuron
outgrowth and neural
cell survival

48. 48
In vivo therapy, where bmps
or bmp genes are directly
applied to the exposed or
amputated pulp.
Ex vivo therapy, which
consists of isolation of
dpscs, their differentiation
into odontoblasts with
recombinant bmps or bmp
genes, and finally their
autogenous transplantation
to regenerate dentin .

49. SCAFFOLDS
Cells are often implanted or 'seeded' into
an artificial structure capable of supporting
three-dimensional tissue formation. These
structures, typically called scaffolds.

50. 50
IDEAL REQUIREMENTS OF A SCAFFOLDIDEAL REQUIREMENTS OF A SCAFFOLD

51. CRYSTALLOGRAPHIC STRUCTURE & CHEMICAL COMPOSITIONCRYSTALLOGRAPHIC STRUCTURE & CHEMICAL COMPOSITION
SIMILAR TO HARD TISSUESIMILAR TO HARD TISSUE
CELL BINDINGCELL BINDING
MOTIFSMOTIFS
PRESENTPRESENT
MOST ABUNDANT POLYMERMOST ABUNDANT POLYMER
AFTER CELLULOSE &AFTER CELLULOSE &
RESEMBLESRESEMBLES
GLYCOSAMINOGLYCANS OFGLYCOSAMINOGLYCANS OF
ECMECM
CONTAINS CALCIUMCONTAINS CALCIUM
CROSSLINKINGCROSSLINKING
SMART SCAFFOLDSMART SCAFFOLD
REPITITIVEREPITITIVE
PROTEINPROTEIN
SEQUENCESEQUENCE
COMBINATION OFCOMBINATION OF
CHITOSAN & SILKCHITOSAN & SILK
PROTEINPROTEIN
CONTAINS GROWTHCONTAINS GROWTH
FACTORSFACTORS
THERMALLYTHERMALLY
REVERSABLEREVERSABLE
GELS FORMEDGELS FORMED
BIO DEGRADABLE ALIPHATIC
POLYMER
TITANIUM DIOXIDE
(E-171)
BIOCOMPATIBILITY
USED TO REGENERATE SMALL DIAMETER BLOOD VESSELS
NUMEROUS CONFIGURATIONS
POSSIBBLE
BIODEGRADABLE ALIPHATIC
POLYESTER
FASTER RESORPTION
THAN
HYDROXYAPATITE
SLOW DEGRADATION
SCAFFOLDS IN DENTISTRY
RESEMBLESRESEMBLES
EXTRAEXTRA
CELLULARCELLULAR
MATRIXMATRIX

52. 52
BIOLOGICAL/NA
TURAL
SCAFFOLDS
• Natural Polymers - Collagen And Glycosaminoglycan.
• Good Biocompatibility And Bioactivity.
• Collagen- Great Tensile Strength To Tissues.
• Allows Easy Placement Of Cells And Growth Factors
• Allows Replacement With Natural Tissues After Undergoing Degradation.
• Disadvantage- Cells In Collagen Matrices Undergo Marked Contraction, Which Might
Affect Pulp Tissue Regeneration .

53. 53
• Clinically Used Biologic Scaffold- Platelet Rich Plasma (PRP)
Properties : Autologous, Easy To Prepare, Rich In Growth Factors, Degrades Overtime, Forms 3D
Fibrin Matrix

55. PLATELET RICH FIBRIN
(CHOUKROUN’S TECHNIQUE)
• Ease of preparation
• Significant slow sustained release of may growth factors like PDGF and TGF β for atleast
1 week upto 28 days
• Does not dissolve quickly after application
• The strong fibrin matrix is slowly remodeled in a similar way to a natural blood clot.

56. •PRF has been shown to have several advantages over traditionally prepared platelet-
rich plasma.
•Its chief advantages include ease of preparation and lack of biochemical handling of
blood, which makes this preparation strictly autologous.
•Another advantage of using PRF as a scaffold is that it has a trimolecular or equilateral
fibrin branch junction which makes its architecture flexible and can support cytokine
enmeshment and cellular migration

57. 57
ARTIFICIAL SCAFFOLDS
• Polymers with controlled physicochemical features such as degradation rate,
microstructure, and mechanical strength.
• Polylactic acid (pla), polyglycolic acid (pga), and their copolymers-poly lactic-co-glycolic
acid (plga).
• Synthetic hydrogels include polyethylene glycol (peg)- based polymers.

58. 58
• Cell surface adhesion peptides, such as arginine, glycine, and aspartic acid added to
improve cell adhesion and matrix synthesis within the three-dimensional network
• Scaffolds containing inorganic compounds such as hydroxyapatite (HA), tricalcium
phosphate (TCP) and calcium polyphosphate (CPP), are used to enhance bone conductivity.
• Micro-cavity-filled scaffolds to enhance cell adhesion.

59. Properties Of Scaffolds
porepore
sizesize
important role in cell
proliferation and cell
distribution throughout
tissue regeneration
optimal  between 200 and 400um
effect on its mechanical
integrity and its ability to
perform under functional
demands.
• Mechanical properties- Tensile Strength and Viscoelasticity.
• Biocompatibility
• Degradation
• 3-D printing technologies  scaffolds can be fabricated into precise geometries with a
wide range of bioactive surfaces- environments conducive to the growth of specific cell
types

60. 60
Steps to create and use tissue constructs

61. NON CELL BASED APPROACHES
• Used when defect is small
• Growth factors are utilized to attract stem cells residing in the remaining pulp
tissue to the defected site and to regenerate the lost part.

62. AN OVERVIEW OF POTENTIAL TECHNOLOGIES FOR
REGENERATIVE ENDODONTICS

63. ROOT CANAL REVASCULARIZATION VIA BLOOD CLOTTING
Several case reports have documented revascularization of necrotic root canal systems
by disinfection followed by establishing bleeding into the canal system via
overinstrumentation.
An important aspect of these cases is the use of intracanal irrigants (NaOCl
and chlorhexdine) with placement of antibiotics (e.g. a mixture of ciprofloxacin,
metronidazole, and minocycline paste- HOSCHINO’S PASTE) for several weeks.
Banchs F, Trope M. Revascularization of immature permanent teeth with apical
periodontitis: new treatment protocol? J Endod 2004;30:196 –200.

64. This particular combination of antibiotics effectively disinfects root canal systems  broad
spectrum
tetracycline enhances the growth of host cells on dentin, not by an antimicrobial action, but
via exposure of embedded collagen fibers or growth factors
•it has been noted that reimplantation of avulsed teeth with an apical opening of
approximately 1.1 mm demonstrate a greater likelihood of revascularization
•fully formed (closed) apices might require instrumentation of the tooth apex to approximately
1 to 2mm in apical diameter to allow systemic bleeding into root canal systems.
•The revascularization method assumes that the root canal 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.

65. Advantages To A Revascularization Approach.
Technically simple  can be completed using currently available instruments and
medicaments without expensive biotechnology.
Regeneration of tissue in root canal systems by a patient’s own blood cells avoids
the possibility of immune rejection and pathogen transmission from replacing the
pulp with a tissue engineered construct.

67. Chen et al immature teeth diagnosed with pulp necrosis and apical
periodontitis may present 5 types of revascularization outcome:
• TYPE I: INCREASED DENTIN
WALL WIDTH AND ROOT-
END DEVELOPMENT.
TYPE II: INSIGNIFICANT
CONTINUED ROOT DEVELOPMENT
ASSOCIATED WITH APICAL
CLOSURE.

68. TYPE IV: CALCIFICATION
(OBLITERATION) OF ROOT
CANAL.
TYPE III: ROOT-END
DEVELOPMENT WITHOUT
APICAL CLOSURE.

69. TYPE V: MINERALIZED TISSUE BARRIER BETWEEN MTA
CERVICAL PLUG AND RADICULAR APEX.

70. DISADVANTAGES OF REVASCULARIZATION:
MAIN : Coronal discoloration  due to minocycline
Can be replaced by CEFACLOR
• Additional enlargement of apical region in mature roots
• In older individuals  lesser stem cells final outcome unpredictable may be
calcified canal

72. • The simplest method to administer cells of appropriate regenerative potential is to inject
postnatal stem cells into disinfected root canal systems.
• Postnatal stem 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 the 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 numbers sufficiently for
regenerative endodontic applications.
POSTNATAL STEM CELL THERAPY

73. Advantages:
 autogenous stem cells are relatively easy to harvest and to deliver by syringe, and the
cells have the potential to induce new pulp regeneration.
 already used in regenerative medical applications, including bone marrow replacement,
and a recent review has described several potential endodontic applications
Nakashima M, Akamine A. The application of tissue engineering to
regeneration of pulp and dentin in endodontics. J Endod 2005;31:711– 8.
One possible approach would be to use dental pulp stem cells derived from autologous
(patient’s own) cells that have been taken from a buccal mucosal biopsy, or umbilical
cord stem cells that have been cryogenically stored after birth.

74. A solution for this latter issue may be to
apply the cells together with a fibrin clot
or other scaffold material.
probability of producing new
functioning pulp tissue by injecting
only stem cells into the pulp chamber,
without a scaffold or signaling
molecules, may be very low.

75. In general, scaffolds, cells, and bioactive signaling molecules are needed to induce
stem cell differentiation into a dental tissue type.
Nakashima M. Bone morphogenetic proteins in dentin regeneration for potential
use in endodontic therapy. Cytokine Growth Factor Rev 2005;16:369 –76.

76. • The majority of in vitro cell cultures
grow as a single monolayer attached
to the base of culture flasks.  2D
• 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.
PULP IMPLANTATION

77. 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
Fukuda J, Khademhosseini A, Yeh J, Engl G, Cheng J, Farokhzad OC,
Langer R. Micropatterned cell co-cultures using layer-by-layer deposition of
extracellular matrix components. Biomaterials 2006;27:1479–86

78. ADVANTAGES
•the cells are relatively easy to grow on filters in the laboratory.
•Moreover, aggregated sheets of cells are more stable than dissociated cells administered.
potential problems associated with the implantation of sheets of cultured pulp tissue is that
DISADVANTAGES
•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 the 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 layers of cells, they are extremely fragile, and this could make
them difficult to place in root canal systems without breakage.

79. • 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 with pulp stem cells
• A scaffold should contain growth factors to aid stem cell proliferation and differentiation,
leading to improved and faster tissue development.
• The scaffold may also contain nutrients promoting cell survival and growth and possibly
antibiotics to prevent any bacterial in-growth in the canal systems.
SCAFFOLD IMPLANTATION
Ching Yuang Huang et al. Mesoporous Calcium Silicate Nanoparticles with
Drug Delivery and Odontogenesis Properties. JOE,2017, 43: 69–76

80. In pulp-exposed teeth, dentin chips have been found to stimulate reparative dentin bridge
formation
Dentin chips may provide a matrix for pulp stem cell attachment and also be a reservoir of
growth factors
Kitasako Y, Shibata S, Pereira PN, Tagami J. Short-term dentin
bridging of mechanically- exposed pulps capped with adhesive resin
systems. Oper Dent 2000; 25:155– 62.

81. The types of scaffold materials available are natural or synthetic, biodegradable or
permanent.
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
Scaffolds may also be constructed from natural materials
collagen or fibrin,
Polysaccharidic materials, like chitosan or glycosaminoglycans (GAGs), have not been well
studied.

83. INJECTABLE SCAFFOLD
DELIVERY
• Rigid tissue engineered scaffold  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
structural support of the tooth”.
• This will allow tissue engineered pulp tissue to be administered in a soft three-dimensional
scaffold matrix, such as a polymer hydrogel.
• Hydrogels are injectable scaffolds that can be delivered by syringe.

84. Hydrogels have the potential to be noninvasive and easy to deliver into 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 to support cell survival
Desgrandchamps F. Biomaterials in functional reconstruction. Curr Opin
Urol 2000;10:201– 6.

86. Despite these advances, hydrogels at 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

87. 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.
The ideal positioning of cells 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 network of vascular and nerve cells.
THREE-DIMENSIONAL CELL PRINTING

88. The three-dimensional cell printing 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.
Theoretically, the disadvantage of using the 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.

89. target cell
populations
GENE THERAPY
All human cells contain a 1million strands 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.
deliver genes for
growth factors, morphogens,
transcription factors, and
extracellular
matrix molecules
viral or nonviral
vectors

90. A literature search indicates there has been little or no research in this field, except for the work of
rutherford et al (2011)
He transfected ferret pulps with cdna-transfected mouse bmp-7 that failed to produce a reparative
response
A literature search indicates there has been little or no research in this field, except for the work of
rutherford et al (2011)
He transfected ferret pulps with cdna-transfected mouse bmp-7 that failed to produce a reparative
response

91. The FDA did approve research into gene therapy involving terminally ill humans, but
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.
Stolberg SG. Trials are halted on gene therapy: child in experiment
falls ill: new setback for research. NY Times 2002;A1, A25.

92. Measuring Appropriate Clinical Outcomes
Once a tissue engineered pulp has been implanted, it is not ethical to remove
functioning tissues to conduct a histological analysis.
Therefore, it will not be possible to histologically investigate mineralizing odontoblastoid
cell functioning or nerve innervation.
Clinicians will have to rely on the noninvasive tests in use today, such as
laser Doppler blood flowmetry in teeth
pulp testing involving heat, cold, and electricity
lack of signs or symptoms.
Magnetic resonance

93. Imaging (MRI) has shown the potential to distinguish between vital and
nonvital tooth pulps
The ideal clinical outcome is a nonsymptomatic tooth that never needs retreatment,
but nonsubjective vitality assessment methods are essential to validate that
regenerative endodontic techniques are truly effective.

94. EXTENDED USES OF DENTAL STEM CELLS

96. CHALLENGES AND FUTURE
DIRECTION
96

97. Regenerative endodontics is one of the most exciting developments in dentistry today
and endodontists are at the forefront of this cutting-edge research.
Endodontists’ knowledge in the fields of pulp biology, dental trauma and tissue
engineering can be applied to deliver biologically based regenerative endodontic
treatment of necrotic immature permanent teeth resulting in continued root
development, increased thickness in the dentinal walls and apical closure.
These developments in regeneration of a functional pulp-dentin complex have a
promising impact on efforts to retain the natural dentition, the ultimate goal of
endodontic treatment.
CONCLUSION