The document discusses regenerative endodontics, which involves biologically based procedures aimed at replacing damaged dental tissues, particularly the pulp-dentin complex. It highlights the importance of stem cells, growth factors, and scaffolds in tissue engineering, along with historical advancements in regenerative techniques. The document also examines the challenges and advantages of using regenerative endodontic therapy compared to traditional root canal treatments.

2. Dr. A. YOGHA PADHMA
1ST YEAR POSTGRADUATE
DEPT OF CONSERVATIVE DENTISTRY

5. Two roads diverged in a wood, I took
the one less travelled by,
And that has made all the difference.
- Robert Frost

9. Each year approx $400 billion is spent treating people suffering some type of
tissues loss.
This includes
20000 organ transplants,
5,00000 joint replacement, and
millions of dental and oral craniofacial procedures, ranging from tooth
restorations to major reconstruction of facial soft and mineralized tissues.

10. 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

11. 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)

12. 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.

13. HISTORY

14. Nygaard-Ostby 1961
Use of a
revascularization
procedure for
regeneration of the
pulp-dentin complex in
immature teeth with
pulpal necrosis
Rule DC 1966
Use of double
antibiotic paste
Hoshino 1993
Use of triple antibiotic
paste
Iwaya 2001
Evoked intracanal
bleeding step
Branchs & Trope 2004
Case reports on
immature mandibular
premolars

15. REPAIR VS REGENERATION
Repair
Healing occurs because remaining damaged
tissue is vital
Repair is defined as the process when healing
takes place by proliferation of connective
tissue elements resulting in fibrosis and
scarring.
Regeneration
The damaged tissue is completely necrotic.
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

16. OBJECTIVES
 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.

17. GOALS OF REGENERATIVE
ENDODONTIC PROCEDURE

18. WHAT IS THE BIOLOGICAL
BASIS FOR REGENERATIVE
ENDODONTIC THERAPY???
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.

20. • 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

21. 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.

23. 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.

24. 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

26. THE THREE KEY ELEMENTS OF
TISSUE REGENERATION ARE
GROWTH
FACTORS
STEM
CELLS SCAFFOLD
COMPONENTS OF REGENERATIVE
ENDODONTICS

27. 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

28. KEY ELEMENTS

29. STEM CELLS
STEM CELLS

30. Undifferentiated cells which
have the capability to produce
cells of the same type or more
differentiated cells

31. CLASSIFICATION

32. BASIS OF
ORIGIN
• EMBRYONIC
• SOMATIC/ADULT/POSTNATAL/MESENCHYMAL
BASIS OF
SOURCE
• AUTOLOGOUS
• ALLOGENIC
• XENOGENIC
• SYNGENIC
BASIS OF
POTENCY
• TOTIPOTENT
• PLURIPOTENT
• MULTIPOTENT
• UNIPOTENT

33. DENTAL STEM CELLS

34. STEM CELLS FROM
HUMAN EXFOLIATED
DECIDUOUS TEETH
(SHED)
DENTAL PULP STEM
CELLS (DPSC)
STEM CELLS FROM
APICAL PAPILLA
(SCAP)
STEM CELLS
FROM DENTAL
FOLLICLE (DFSC)
PERIODONTAL
LIGAMENT
SEM CELLS
(PDLSC)
BONE
MARROW
DERIVED
MESENCHY
MAL STEM
CELLS
(BMSC)

36. STEM CELLS FROM HUMAN
EXFOLIATED DECIDUOUS
TEETH

37. Isolated in 2003 by MIURA et al.,
Have a higher proliferation rate than stem
cells from permanent teeth
In vivo SHED cells can induce bone or
dentin formation but failed to produce
dentin-pulp complex - Immature
High plasticity since they could
differentiate into Neurons, Adipocytes,
Osteoblasts and Odontoblasts

38. ADVANTAGES
Shed banking is more economical
when compared to cord blood and
may be complementary to cord cell
banking
Can be retrieved
from a tissue that
is disposable and
readily accessible.
Only Primary incisors
and Canines

39. 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
Painless stem cell collection
with minimal invasion because
they are retrieved from a tissue
that is disposable and easily
accessible

40. DENTAL PULP STEM CELLS

41. Invitro – DPSC’s
produce sporadic
but densely calcified
nodules & invivo –
With biodegradable
scaffolds forms
dentin pulp like
tissues with irregular
shape
Discovered in the
wisdom tooth in
2000 by Gronthos et
al., - Isolated from
Ectomesenchymal
stem cells from
dental pulp of
extracted wisdom
teeth
Population of
stem cells- DPSC /
Odontoblastoid
cells – Synthesize
& secrete dentin
matrix like
odontoblast cells
they replace

42. Unknown where the cells are
recruited from - The cell rich
subodontoblastic layer of
perivascular cells / Immature
mesenchymal cells and
fibroblasts
1.Differentiation potential for
Odontoblastic, Adipogenic &
Neural cytotypes
2. Putative immune-
suppressive activity – Adv in
allogenic stem cell
transplantation

43. Isolated for first time from
permanent third molars in 2000 by
Gronthos et al.,
Invitro – Produce sporadic
but densely calcified
nodules
Invivo - With
biodegradable Scaffolds
Forms Dentin –pulp like
tissue with an irregular
shape
Ming Yan et al., -
DPSC’s –
Reconstruction of
dentin pulp
complex and
biotooth

44. STEM CELLS FROM APICAL
PAPILLAE

45. Source of primary
odontoblasts involved
in development of root
dentine
Positive for
telomerase
activity -
Embryonic stem
cells – Immature
source of cells
STRO-1 POSITIVE
CELLS, Faster
proliferation, Greater
number of population
doublings, Increased
capacity for invivo
dentine regeneration

46. 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.

47. COMPARISON BETWEEN STEM CELLS
FROM APICAL PAPILLAE AND DENTAL
PULP STEM CELLS

48. PERIODONTAL LIGAMENT
STEM CELLS

49. Isolated & Characterized
by Seo et al., in 2004
Differentiates into
cementoblats-like
cells, Adipocytes &
Collagen-forming
cells invitro capacity
to generate a
cementum/PDL like
structure invivo
More proliferative
Longer lifespan
Higher number of
population doublings
in vitro

50. STEM CELLS FROM DENTAL
FOLLICLE

51. Multipotent tissue
based on its ability to
generate cementum,
bone & PDL from
ectomesenchymal
fibrous tissue
1st isolated from
follicle of impacted
third molars

52. STEM
CELLS

53. STEM CELL
IDENTIFICATION

54. Staining the cells with specific antibody
markers and using a flow cytometer, in a
process called Fluorescent antibody cell
sorting (FACS)
Immunomagnetic bead selection
Immunohistochemical staining
Physiological & histological criteria,
including phenotype (appearance),
chemotaxis, Proliferation, Differentiation
& mineralizing activity

55. STEM CELL ISOLATION

56. 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.

57. 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.

58. STORAGE OF STEM
CELLS

59. 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 -1960c .
 In a vial 1.5 ml of freezing medium is optimum for 1–2 × 106 cells.

60. 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

61. GROWTH FACTORS

62. Growth factors are polypeptides which have the ability
to bond to specific receptors on the target cells and
modulate or facilitate certain activities like migration,
proliferation, differentiation and apoptosis.
Growth factors are extracellular secreted
proteins that bind to cell receptors and
modulate cellular activity eg by regulating
the rate of proliferation, including
differentiation into another cell type, or by
stimulating cells to synthesize mineralizable
matrices

63. Platelet derived growth factors
Bone morphogenetic proteins (BMPs)
Vascular endothelial growth factor
Fibroblast growth factors
Transforming growth factor b
Insulin like growth factor

64. Colony stimulating growth factors
Epidermal growth factor
Insulin like growth factor I or II
Interleukins IL- 1 to 13
Nerve growth factor
Stromal cell derived growth factor

65. REPAIR & REGENERATION PDGF, TGF, BMP, VEGF.FGF, IGF
ANGIOGENESIS FGF2, PDGF, VEGF
NEURONAL GROWTH NGF
DIFFERENTIATION TGF2, PDGF, FGF2, BMP
2,4,7,11,IGF, NGF
PROLIFERATION FGF2, SD-1, TGFß1, VEGF, PDGF
CHEMOTAXIS SDF-1, TGFß1, PDGF, FGF2

68. SCAFFOLDSSCAFFOLDS

69. Three dimensional porous solid biomaterials that provides a
physio-chemical & biological three-dimensional micro
environment for cell growth and differentiation, promoting cell
adhesion, and migration.
Serves as a carrier for morphogens in cell and in protein
therapy.

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

71. IDEAL REQUIREMENTS

72. CLASSIFICATION

73. BASED ON DEGRABILITY
OF MATRICES
BASED ON FORM
 Biodegradable scaffolds
 Permanent or
biostatable scaffolds
 Solid blocks
 Sheets
 Porous sponges
 Hydrogels (Injectable
scaffolds)
BASED ON PRESENCE
OR ABSENCE OF CELLS
 Cell free scaffolds
 Scaffolds seeded with
stem cells

74. BIOLOGICAL/NATURAL
SCAFFOLDS
• Platelet rich plasma
• Platelet rich fibrin
• Collagen
• Chitosan
• Glycosaminoglycans
Hyaluronic acid
• Demineralized or native
dentin matrix
• Blood clot
• Silk
ARTIFICIAL/SYNTHETIC
SCAFFOLDS
• Polymers – PLA (OPLA)
• PGA
• PLGA
• PCL
• PLLA
• Bioceramics – HA
• ß-TCP
• BCP
• Bioactive glass – Silicate
• Borate
• Borosilicate
• Naturally derived – Fibrin
• Collagen
• HYA sponge
• Amniotic membrane
• Polysaccharides

75. CRYSTALLOGRAPHIC STRUCTURE & CHEMICAL COMPOSITION SIMILAR
TO HARD TISSUE
CELL BINDING
MOTIFS
PRESENT
MOST ABUNDANT POLYMER
AFTER CELLULOSE &
RESEMBLES
GLYCOSAMINOGLYCANS OF
ECM
CONTAINS CALCIUM
CROSSLINKING
SMART SCAFFOLD
REPITITIVE
PROTEIN
SEQUENCE
COMBINATION OF
CHITOSAN & SILK
PROTEIN
CONTAINS GROWTH
FACTORS
THERMALLY
REVERSABLE
GELS FORMED
BIO DEGRADABLE ALIPHATIC
POLYMER
TITANIUM DIOXIDE
(E-171)
BIOCOMPATIBILITY
USED TO REGENERATE SMALL DIAMETER BLOOD VESSELS
NUMEROUS CONFIGURATION
POSSIBBLE
S
BIODEGRADABLE ALIPHATIC
POLYESTER
FASTER RESORPTION
THAN HYDROXYAPATITE
SLOW DEGRADATION
SCAFFOLD
S
RESEMBLES
EXTRA
CELLULAR
MATRIX

76. 52
Affect Pulp Tissue Regeneration .
BIOLOGICAL/
NATURAL
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

77. PLATELET RICH PLASMA
 Autologous first generation platelet
concentrate with a rich sourcs of growth
factors.
 Introduced in 1997 by Whitman
 Potential substitute scaffold
 Easy to prepare, rich in growth factors, 3D
fibrin matrix that helps to entrap growth
factors.

78.  Platelet concentration – Exceeds 1 million/mL
– 5 times more than that of normal platelet
 It is a concentrated suspension of different
growth factors like PDGF, TGF-b, IGF, VEGF,
epidermal growth factor, epithelial cell factor
 Released via degranulation of alpha granules
& stimulate bone & soft tissue healing
 Disadvantage – Drawing blood in young
patients, need special equipment & reagents
to prepare, increased cost of treatment.

80. PLATELET RICH FIBRIN
 Second generation platelet concentrate
 Developed by Choukroun et al., in 2001
 Resorbable fibrin matrix enriched with
platelets and leukocytes.
 Rich source of growth factors – VEGF,
IGF – Slowly released

81.  Blood is drawn & collected into test tubes
without an anticoagulant centrifuged
instantaneously.
 Tabletop centrifuge – 10 min @ 3000 rpm for
12min @ 2700 rpm.
 Resultant product –
Acellular PPP @ peak level
PRF clot @ intermediate level
Red fraction of RBC’S @ base level

83. COLLAGEN
 Paticularly type 1
 Used alone or in combination with growth
factors or chitosan
 Higher degree of odontoblastic
differentiation, Expression of osteonectin,
dentin sialophosphoprotein, dentin matrix
protein.

84. CHITOSAN
 Produced commercially by deacetylation of chitin,
which is a structural element in exoskeleton of
crustaceans (Crabs & Shrimps).
 Formation of pores in scaffolds – Influencing
mechanical & biological properties
 Advantage – Nontoxic, Easily absorbable, Antibacterial
activity, increases alkaline phosphate activity, shows
fibroblast & odontoblastic proliferation.
 Disadvantages – Low strength & inconsistency
behavior with seeded cells

85. GLYCOSAMINOGLYCANS
 Hyaluronic acid – One of the glycosaminoglycans in
ECM & plays an important role in maintaining
morphologic organization by preserving extracellular
spaces.
 Advantages – Helps in differentiation of dental
mesenchymal cells to odontoblasts, contributes to
formation of dentin matrix & dental pulp, biocompatible,
bioactive, non immunogenic
 Disadvantages - Water soluble, degrades rapidly by
enzyme hyaluronidase – Overcome by cross linking &
modification of HA.

86. BLOOD CLOT
 First practiced by Ostby and resulted in a
growth of granulation tissues, fibrous tissues
or cementum-like tissues into the root canals.
 Tissues were not able to grow in empty
spaces with the absence of suitable scaffolds
- Blood clot yields good scaffolds & fills
intracanal spaces
 Example : Revascularization

87. SILK
 Biocompatible
 Ability to support attachement,
differentiation, proliferation
 Disadvantage – Formation of
osteodentin, complete degradation
occurs after 2 years

89. 57
ARTIFICIAL
SCAFFOLD
S
• 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).
• Application were first suggested by Vacanti et al..

90.  PGA: Enhanced the growth of new blood
vessels & odontogenic differentiation of
human fibroblasts.
 PLLA: Poly-L-lactic acid polymer
Similarity to DP – Human dermal
micro vascular endothelial cells
 PLA (OPLA): Aliphatic polyester, more
hydrophobic than PGA, attachable to root
canal dentin.

91.  PLGA : Dentin-like & Pulp-like tissue
regenerated after 3-4 months
 PCL: Slowly degrading polymer
Used alone or in combination with HA

96. POTENTIAL
TECHNOLOGIES
Root canal
revascularizat
ion
Post natal
stem cell
therapy
Pulp
implantation
Scaffold
implantation
Injectable
Scaffold
delivery
3D Cell
printing
Gene delivery

100. ROOT CANAL
REVASCULARIZATION

101. Studies prove that the same can be
achieved for an immature or mature
permanent tooth with periapical
periodontitis or abscess by means
of throughly disinfecting the canal
followed by inducing bleeding and
establishing blood clot into the root
canal system via over
instrumentation supplemented by
good coronal seal
It was presumed
that the
formation of a
blood clot in the
sterile root canal
system creates a
scaffold of fibrin
that entraps stem
cells capable of
initiating new
tissue
development.

102. ETIOLOGICAL FACTORS
FOR REP

103. MEDICAMENTS USED IN REGENERATIVE
ENDODONTIC PROCEDURES

106. 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) for several
weeks.
Banchs F, Trope M. Revascularization of immature permanent teeth with apical
periodontitis: new treatment protocol? J Endod 2004;30:196 –200.

107. This particular combination of antibiotics effectively disinfects
root canal Systems and increases revascularization of avulsed
and necrotic teeth suggesting that this is a critical step in
revascularization.
For example, 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

108. This finding suggests that revascularization of necrotic pulps with
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.
It is not clear that the regenerated tissue’s phenotype resembles
dental pulp.

110. ADVANTAGES

111. DISADVANTAGES

112. FACTORS THAT AFFECT
THE RESULTS OF
REVASCULARIZATION

113. Presence of bacteria mounts host
immune defences and diverts the tissue
healing process towards repair

116. CAVITATION-GENERATING
DEVICES (SONIC OR
ULTRASONIC)

117. PHOTON-INDUCED
PHOTOACOUSTIC
STREAMING

118. MULTISONIC DISINFECTION
SYSTEM

119. NANOPARTICLES

120. PHOTODYNAMIC
THERAPY

121. However, several concerns need to be addressed in prospective research.
First, the case reports of a blood clot having the capacity to regenerate pulp
tissue are exciting, but caution is required, because the source of the
regenerated tissue has not been identified.
Animal studies and more clinical studies are required to investigate the
potential of this technique before it can be recommended for general use in
patients.
Generally, tissue engineering does not rely on blood clot formation, because
the concentration and composition of cells trapped in the fibrin clot is
unpredictable.

122. On the other hand, some aspects of this approach may be useful; plasma-
derived fibrin clots are being used for development as scaffolds in several
studies
Second, enlargement of the apical foramen is necessary to promote
vascularizaton and to maintain initial cell viability via nutrient diffusion.
Related to this point, cells must have an available supply of oxygen;
Llames SG, Del Rio M, Larcher F, et al. Human plasma as a dermal
scaffold for the generation of a completely autologous bioengineered skin.
Transplantation 2004;77:350 –5.

125. 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.

126. TYPE IV: CALCIFICATION
(OBLITERATION) OF ROOT
DEVELOPMENT WITHOUT
APICAL CLOSURE.

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

128. OTHER 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

129. Authors Year Important findings
Ding et al 2009 Case selection is the most important part of this treatment approach because
it requires stringent follow-up.
Cotti et al 2008 Concentrations of NaOCl ranging from 2.5% to 6% have been used for
irrigation and provides favorable results.Kim et al 2010
Galler et al 2011 A final irrigation with EDTA provides more optimal conditions for cellular
differentiation, tissue formation, and regeneration.
Trevino et al 2011 CHX is detrimental to stem cells of the apical papilla. It is not recommended.
Cohenca et al 2010 EndoVac system (Discus Dental, Culver City, CA) can be used without the
necessity of intracanal medication (especially triantibiotic paste) to prevent
further discoloration.
Ding et al 2009 If Ca(OH)2 is used as an intracanal medicament, its placement should be
restricted to the coronal half of the tooth to achieve greater increase in the
dentinal wall thickness.
Petrino et al 2010 If bleeding after root canal disinfection fails, clinicians should consider using
an anesthetic without a vasoconstrictor when trying to induce bleeding.
Ding et al 2009 For the formation of a blood clot, the tooth should be left for 15 minutes. The
blood clot acts as a suitable barrier for the placement of MTA (ProRoot MTA,
Dentsply Maillefer, Tulsa, OK, USA).
Petrino et al 2010 It is recommended to place the coronal edge of the MTA (ProRoot MTA,
Dentsply Maillefer, Tulsa, OK, USA) 1–2 mm apical to the CEJ to allow new
tissue growth into the root canal.

130. IS THERE A DIFFERENCE BETWEEN
PULP REVASCULARISATION
AND
PULP REGENERATION ?????
PULP REVASCULARISATION IS
THE RE-INTRODUCTION OF
VASCULARITY IN THE ROOT
CANAL SYSTEM
PULP REGENERATION IS THE RE-
POPULATION OF ODONTOBLASTS
THAT ALIGN ON DENTIN SURFACE
PULP
REVASCULARISATION =
INDUCTION OF
ANGIOGENESIS
PULP REGENERATION =
PULP
REVASCULARISATION +
RESTORATION OF
FUNCTIONAL
ODONTOBLASTS AND
NERVE FIBRES

131. POSTNATAL STEM CELL
THERAPY

132.  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.

133.  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.
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.

134. 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.
A solution fort this later issue may
be to apply the cells together with
a fibrin clot or other scaffold
material

135. In general, scaffolds, cells, and bioactive signaling molecules are needed to
induce stem cell differentiation into a dental tissue type.

136. PULP IMPLANTATION

137. 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.

138. 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.

140. A scaffold should
contain growth factors
to aid stem cell
proliferation and
differentiation,
and Antibiotics to
prevent any bacterial in-
growth in the canal
systems.
This can be
accomplished using a
porous polymer scaffold
seeded with pulp stem
cells
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.

141. 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

142. INJECTABLE SCAFFOLD
DELIVERY

144.  Hydrogels are injectable scaffolds that can be
delivered by a syringe.
 Non invasive & easy to deliver into root canal
systems.
 Promotes pulp regeneration by providing a
substrate for cell proliferation and
differentiation into an organized tissue
structure
 Early stage of development

148. 3 DIMENSIONAL CELL
PRINTING

149.  A three dimensional printer – Recreate the precise shape & size of
the pulp tissue in a particular tooth.
 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
.

150.  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.

151.  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.

152. GENE THERAPY

153.  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. Specific mineralizing genes
can be transferred into the root canal with the help of carrier( Vector).
viral or nonviral
vectors deliver genes for growth
factors, morphogens,
transcription factors, and
extracellular
matrix molecules
Target cell
populations

154. 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

155. 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.

157. Once a tissue engineered pulp has been implanted, it is
not ethical to remove functioning tissues to conduct a
histological analysis.
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
Therefore, it will not
be possible to
histologically
investigate
mineralizing
odontoblastoid cell
functioning or nerve
innervation.

159. CHAIR SIDE TREATMENT
PROTOCOLS

160. AAE
CLINICAL CONSIDERATIONS
FOR
A
REGENERATIVE PROCEDURE

162. The success of pulp revascularization treatment
depends on three elements:
•root canal disinfection,
•the presence of a scaffold
(blood clot),
•hermetic coronary filling.

163. at the first treatment visit, the lower
concentration of sodium hypochlorite
(1.5 %) followed by saline or 17 % EDTA
is recommended as chemical irrigants
based on the survival of SCAP and level of
dentin sialophosphoprotein expression
ACCORDING TO THE AAE
GUIDELINE

164. (It has been demonstrated that bleeding
evoked by overinstrumenting the canals
beyond the apical foramen can deliver
the mesenchymal stem cells from
periapical tissues to the root canal
space in both immature and mature
teeth.
(to wash out the intracanal medicament
but also to promote the release of
fossilized biological factors within
dentin matrix)

165.  Inflammation might also be
considered as an early integral
stage of reparative or
regenerative processes
Evoked bleeding is
considered the influx
of stem cells and
supply of growth
factors, which will
initiate the
differentiation of
stem cells into
endothelial cells as a
critical biological
event during the
early regenerative
processes
INFLAMMATION
CHALLENGE OR
OPPORTUNITY?

166. BIOMIMETIC
MICROENVIRONMENTS
FOR
REGERERATIVE
ENDODONTICS

167.  To regenerate the function & form of the pulp-dentin
complex, the construction of the biomimetic
microenvironment is a key factor.
 Cells respond differently to physicochemical &
mechanical properties of the microenvironment.
 The interaction between cells, and the ECM controls
differentiation, migration, and proliferation as well as
tissue modelling.
 For this reason an ECM mimicking
microenvironment has been designed by
incorporating various moieties and features derived
from ECM.

168.  Biomimetic environments such as ECM
microenvironments through Peptide
amphiphiles (PA), cell homing, stem cells and
through growth factors have been developed.
 ECM proteins carry problems for clinical
applications including undesirable immune
responses, higher risks for infection, variety in
biological sources &increased clinical costs.
 To overcome such limitations, small peptides
sequences derived from ECM proteins have
been utilized such as Gly-Arg-Gly-Asp-Ser
(GRGS).

169. DESIGN OF BIOMIMETIC
APPROACH

170. ENGINEERED NANO-SCALE
SCAFFOLD

171. REGENERATED PULP-
DENTIN TISSUE WITH
CLOSED ROOT APEX

173. PUBLISHED
REGENERATIVE
ENDODONTIC
CASE REPORTS/
SERIES SINCE 2001

182. EVIDENCE BASED TRILEVEL
OUTCOME ASSESSMENT
PYRAMID

183. CHALLENGES AND
FUTURE

184. REFERENCES

185.  Grossman., 2014: (In) Grossman’s Endodontic Practice. 13th ed.
Regenerative Endodontics., Wolters Kluwer Health, 230-236.
 Cohen., 2015: (In) Cohen’s Pathways of the Pulp. 11thed.
Regenerative Endodontics., Elsevier, India., 602-617.
 T.Nivethithan et al. Stem cells in regenerative endodontics.
International Journal of Pharmaceutical Sciences And Research
2017;90(14) :1-6
 Louis M.Lin et al. A review of regenerative endodontics:current
protocols and future directions .J Istanbul Univ Fac Dent
2017;51(3) : S41-S51
 T.Nivethithan et al. Stem cells in regenerative endodontics.
International Journal of Pharmaceutical Sciences And Research
2017;8(5) :1972-1977
 Thouseef ch et al. Scaffolds in regenerative endodontics:A review
2017; Int.J.Adv.Res.5(9) :415-423

186.  Marc Llaquet et al. Regenerative endodontic procedures: a review
of the literature and a case report of an immature central incisor
2017;Giornale Italiano di Endodontia;31 :65-72
 Saaid Ayesh Alshehadat et al. Scaffolds for dental pulp tissue
regeneration:A review 2016;International Dental & Medical
Journal of Advanced Research;2 :1-12
 Qian Zeng et al.Release of growth factors into root canal by
irrigations in regenerative endodontics .JOE 2016;42(12) :1761-
1765
 Kinjal M.Gathani et al.Scaffolds in regenerative endodontics:A
review.Dent Res J(Isfahan)2016;13(5) :379-386
 Sagar N.Kaushik et al.Biomimetic microenvironments for
regenerative endodontics. Biomaterials Research 2016;20(14) :2-
12
 Sahng G.Kim.Infection and Pulp Regeneration.Dent.J.2016;4 :2-
13

187.  Shreya Sharma et al.A comparative evaluation of natural and
artificial scaffolds in regenerative endodontics:A clinical
study.Saudi Endodontic Journal 2016;6(1) :10-15
 Tarek Mohamed A,Saoud et al.Regeneration and Repair in
Endodontics-A Special Issue of the Regenerative Endodontics-A
New Era in Clinical Endodontics.Dent.J.2016;4(3) :2-15
 Anibal Diogenes et al.Regenerative endodontics-A Way
Forward.JADA 2016;147(5) :372-380
 Anil Chandra et al.Scaffold based Regenerative
Endodontics:Present & Future.sjodr 2016;1(2) :37-41
 Bin-Na Lee et al.A review of the regenerative endodontic
treatment procedure.Restor Dent Endod 2015;40(3) :179-187
 Pankaj Gupta et al.Regenerative Endodontics:An Evidence
Based Review.J Cont Med A Dent ;3(1) :12-19

188.  Hacer Askel et al,Recent considerations in regenerative endodontic
treatment approaches.Journal of Dental Sciences 2014;9 :207-213
 Mamta Dali et al.Regenerative endodontics:Changes,chances,and
challenges of revascularization in pediatric dentistry.SRM Journal of
Research in Dental Sciences 2014;5(3) :186-189
 Kenan Cantekin et al.Regenerative endodontic treatment
(revascularization) for necrotic immature premolar.Journal of Pediatric
Dentistry 2014;2(2) :78-81
 Sayesh Vemuri et al.Root canal revascularization via blood clotting in
regenerative endodontics:Essentials and expectations.Journal of
Dr.NTR University of Health Sciences 2013;2(4) :235-238
 Anibel Diogenes et al.An update on clinical regenerative
endodontics;Endodontic Topics 2013;28 :2-23
 American association of endodontics.Scope of
Endodontics:Regenerative Endodontics 2013

189.  Sahng G.Kim et al.Effects of Growth Factors on Dental
Stem/ProgenitorCells.Dent Clin North Am 2012;56(3) :563-575
 Rashmi Bansal et al.Regenerative endodontics:A state of the
art.Indian Journal of Dental Research 2011;22(1) :122-131
 Franklin Garcia-Godoy et al.Recommendatons for using
regenerative endodontic procedures in permanent immature
traumatized teeth.Dental Traumatology 2011; 1-9
 Vinicius Rosa et al.Regenerative endodontics in light of the
stem cell paradigm.International Dental Journal 2011;61(1) :23-
28
 Rita Chandki et al.From stem to roots:Tissue engineering in
endodontics.J Clin Exp Dent 2012;4(1) :e66-e71
 Peter E,Murray et al.Regenerative Endodontics:A Review of
Current Status and a Call for Action.JOE 2007;33(4) :377-390