This document provides an overview of regenerative endodontics and tissue engineering. It discusses definitions of key terms like regeneration, repair, and the regenerative triad of stem cells, growth factors, and scaffolds. The history and objectives of regenerative endodontics are described. Regenerative endodontic procedures aim to physiologically replace damaged tooth structures using principles of tissue engineering. This involves stimulating regeneration of the pulp-dentin complex using the building blocks of stem cells, signaling molecules, and matrices.

1. REGENERATIVE
ENDODONTICS
Presented by: Dr Abhisek Guria
Dept. of Conservative Dentistry
& Endodontics
……Science fiction to Science !!!

2. Contents
• Introduction
• Definitions
• Regenerative endodontics
• Tissue engineering
• Biomimetics
• Revascularization
• Revitalization
• Maturogenesis
• Regeneration
• Repair
• Repair vs regeneration
• History
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3. • Objectives of regenerative endodontics
• Goals
• Primary
• secondary
• Tertiary
• Pinnacle of regenerative goals
• Biological Basis for Regenerative Endodontic Therapy
• The counterarguments
• Supporting arguments
• Treatment modalities for necrosed tooth
• Need for tissue engineering
• Regenerative triad
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4. • Stem Cells
• Definition
• Properties
• Classification
– Basis Of Origin
– Based On Plasticity
– Basis Of Source
• Embryonic Stem Cells
– Early Embryonic Stem Cells
– Blastocyst Embryonic Stem Cell
– Fetal Stem Cells
– Umbilical Cord Stem Cells
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5. • Adult Stem Cells
 Dental Stem Cells
 Progenitor Cells
 Dental Pulp Stem Cell
 Stem Cells From Human Exfoliated Deciduous Teeth
 Stem Cells From Apical Papillae
 Periodontal Ligament Stem Cells
 Stem Cells From Dental Follicle
• Progenitor cells in dentin regeneration and novel therapeutics
• Stem cell identification
• Isolation approaches of DPSCs
1. Size-sieved isolation
2. Colony cultivation
3. Magnetic activated cell sorting
4. Fluorescence activated cell sorting 55/11/2020 9:26 AM DR ABHISEK

6. • Stem Cell Banking
– Collection
– Isolation Protocol
– Preservation / Storage
• Cryo-preservation
• Magnetic Freezing
• Growth Factors Or Morphogens
• Definition
• Properties
• Classification
• Various Growth Factors
– Primary Sources
– Mechanism Of Action
– Uses
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7. • Scaffolds
– Definition
– Properties
– Ideal Characteristics
• Classification
– Based On Degradability Of Matrices
– Based On Presence Or Absence Of Cells
– Based On Form
– Based On Origin
• Biological / Natural Scaffolds
• Platelet Rich Plasma
• Platelet Rich Fibrin
• Collagen
• Chitosan
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8. • Glycosaminoglycans
• Blood Clot
• Silk
• Artificial Scaffold
• Polylactic Acid (Pla)
• Polyglycolic Acid (Pga)
• Poly Lactic-co-glycolic Acid (Plga).
• Steps To Create And Use Tissue Constructs
• Tissue engineering
1. Ex-vivo tissue engineering
2. In-vivo tissue engineering
• general approaches for tissue engineering
• Tissue engineering is a multidisciplinary approach
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9. • Preclinical and clinical accomplishments of tissue engineering
– Conductive (passive) approaches
– Inductive approaches
– Cell transplantation
• Regeneration Of Dentin -Pulp Complex
1. Regeneration Of Entire Tooth.
2.Local Regeneration Of Dentin-pulp Complex From Amputed
Dental Pulp.
3.Regeneration Of Dental Pulp From Apical Dental Pulp Or
Peri- Apical Tissues.
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10. • Potential technologies in Endodontics
1. Root canal revascularization
2. Post natal stem cell therapy
3. Pulp implantation
4. Scaffold implantation
5. Injectable Scaffold delivery
6. 3D Cell printing
7. Gene delivery
1. Root Canal Revascularization Via Blood Clotting
• History
• Case Selection
• Disinfection Protocol
• Causative Factors For Regenerative Endodontic Procedures
• Medicaments Used In REP
• Pulp Revascularization Using Calcium Hydroxide
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11. • Pulp revascularization using a triple antibiotic paste
• Instrumentation
• Irrigation
• Hydrogen peroxide.
• Chlorhexidine
• Sodium hypochlorite
• Iodine
• Ethylene diamine tetra-acetic acid
• Disinfection
• Calcium hydroxide
• Triple antibiotic paste (tap)
 Composition
 Protocol for preparation
 Carrier
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12.  Storage
 Ph
 Limitations Of TAP
• The Tissue Regeneration
• Follow-up And Treatment Outcome
• Unfavorable Outcomes
• Treatment Outcome
• Advantages
• Limitations
• Factors That Affect The Results Of Revascularization
• Advanced Disinfection Techniques
2. Postnatal Stem Cell Therapy
3. Pulp Implantation
4. Scaffold Implantation
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13. 5. Injectable Scaffold Delivery
6. Three-dimensional Cell Printing
7. Gene Therapy
• Challenges And Future Direction
• Conclusion
• References
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14. Introduction
• Goal of modern restorative dentistry
• Need for tissue replacement
• Biologically based endodontic procedure
• Traditional vs regenerative approach
• Regenerative endodontics is the creation and delivery of
tissues to replace diseased, missing and traumatized pulp.
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15. New arrivals
• 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.
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16. Regenerative endodontics
“Biologically-based procedures designed to
physiologically replace damaged tooth
structures, including dentin and root
structures, as well as cells of the pulp-dentin
complex.”
-Cohen 10th edition
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17. 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
It is the science of design and manufacture of new
tissues to replace lost parts because of disease
including cancer and trauma, with the inclusion of
certain ingredients like stem cells, morphogens and
scaffold.
( Nakashima, 2005)
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18. Application of the principles and methods of engineering
and life science toward fundamental understanding of
structure- function relationship in normal and
pathological mammalian tissues and the development of
biological substitute for the repair and regeneration of
tissue or organ function.
( Shalak and Fox, 1988)
Understanding the principle of tissue growth and
applying this to produce functional replacement tissue
for clinical use.
( Mac Arthur and Oreffo)
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19. • Biomimetics
It is the science of reconstructing or mimicking natural process or
tissue, with the expectation that regeneration will follow
• Revascularization
Reestablishment of vascular supply to immature permanent teeth.
(Andreasen JO and Andreasen FM)
• Revitalization
Ingrowth of vital tissue that does not resemble the original lost
tissue.
• Maturogenesis
Physiologic root development, not restricted to the apical segment.
Weisleder et al (2003)
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20. • Repair
Healing of a wound in response to injury in an attempt to
restore normal structure and function.
• Regeneration
It is a biologic process whereby the continuity of the
disrupted or lost tissue is regained by new tissue which
restores structures and function.
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21. 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
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22. History
• Regenerative capability of a living creature was recorded at 330
BC, when Aristotle observed the lizard could grow back the lost tip
of its tail
• Herman (1952) was the first to carry out regenerative endodontic
procedure , when he applied CaOH in vital pulp amputation
• In 1961, Nygaard – Otsby established a blood clot to use as a
scaffold to revascularize tissue within the root canal of teeth
• In 1963 Hematopoietic stem cells giving rise to blood cells were
identified in bone marrow.
• Rule and Winter(1966) documented root development and apical
barrier formation in cases of pulpal necrosis in children.
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23. • Myers and Fountain in 1974 attempted to regenerate dental pulp
with blood clot filled in the canal.
• Concepts of guided tissue and bone regeneration were first
published by Melcher in 1976
• McCulloch reported the presence of progenitor cells in Pdl of
mice in 1985
• Subsequent regenerative dental procedures included GTR/ GBR
procedures and distraction osteogenesis (block et al 1995)
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24. • Emdogain for periodontal tissue regeneration ( Heijl et al 1997)
• Bohl KS (1998) described a tissue engineering approach to dental pulp tissue
replacement utilizing cultured cells seeded upon synthetic extracellular
matrices.
• The application of PRP for bone augmentation ( Kassolis et al 2000)
• In 2004 the isolation and identification of multipotent stem cells in human
Pdl were reported
• Huang et al (2006): isolated human pulp stem cells may differentiate into
odontoblasts on dentin in vitro.
• In January 2011, ADA adapted a new procedure code to allow practionars to
induce apical bleeding into the root canal in immature permanent teeth with
necrotic pulp that have been extripated.
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25. Objectives
1. To Regenerate pulp like tissue, ideally the
pulp -dentin complex
2. Regenerate damaged coronal dentin, such as
following a caries exposure
3. Regenerate resorbed root, cervical or apical
dentin.
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26. Goals
• Elimination of symptoms
• Evidence of bone healingPrimary
• Increasing root wall thickness
• Increased root lengthSecondary
• Positive response to vitality testTertiary
• Histologic confirmation to structural
and functional restoration
Pinnacle of
regenerative goals
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27. Biological Basis for Regenerative Endodontic Therapy
• Long-term calcium hydroxide treatment was
used to induce apexification of the immature
tooth
• Success rate is reported to be as high as 95%
• There are several associated problems.
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More reliable
barrier
formation
Reduction in
treatment time
Lesser visit
Advantage
over CaOH
apexification
MTA
• Success rate of the treatment is as high as 94%

30. • However, neither of the apexification treatments encourage
further root development
• Immature teeth remain vulnerable to cervical root fractures.
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31. In contrast
• Regenerative endodontic therapy has the potential for
increased root development, and thus, may confer a better long-
term prognosis.
• 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.
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32. The counterarguments
• The pulp in a fully developed tooth plays no major role
in form, function, or esthetics
• Thus its replacement by a filling material in
conventional RCT is the most practical treatment.
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33. SUPPORTING ARGUMENTS
• In terms of esthetics, 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 was done.
• They found that although RCT prolonged tooth survival, the
removal of pulp in a compromised tooth may still lead to tooth
loss
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
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35. Need for tissue engineering
• Most tissue cant regenerate when injured or
diseased
• Even tissues that regenerate spontaneously
may not completely do so in large defects (eg
bone)
• Replacement of tissue with permanent implant
is very limited
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36. Scientist have to find solution for
• To avoid cell rejection
• Specialize cell behavior
• Identification for appropriate stem cell
• Expression and regulation of surface proteins
• Longevity of undifferentiated cells
• Ethical issues
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37. Regenerative triad
Stem cell/
Progenitor cell
Signaling
molecule
/Growth
factors
Scaffold/
Extracellular
matrix
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38. STEM CELLS
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39. Stem cell
• Term was proposed by
Alexander Maksimov in 1908
• Undifferentiated cells,
capable of proliferation
and differentiation into
specialized cells
(AAE)
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40. • 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.
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41. PROPERTIES
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Undifferentiated
cells
Have not developed
into a specialized
cell type
Production of progenitor
cells
Capacity to differentiate
into specialized cell types.
( eg. Odontoblast,
osteoblast, adipocyte,
fibroblast)
Long- term self-
renewal
The ability to go
through numerous
cycles of cell division
while maintaining the
undifferentiated state

42. Classification
• Basis of origin
• Embryonic ( from blastocyst)
• Adult/somatic/postnatal/mesenchymal
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44. BASED ON PLASTICITY
Stem cell type Cell plasticity Source of stem cell
Totipotent
Or
Omnipotent
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
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46. • Basis of source
• Autogenic
• Allogenic
• Xenogenic
(synthetic)
• Syngenic / isogenic
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47. Embryonic stem cells
• Early embryonic stem cells
• When the newly fertilized zygote begins to divide
• Production of embryo
• They are totipotent
• Blastocyst embryonic stem cell
• 5 days after fertilization
• Contains 2 layers
• Outer layer of trophoblast- forms placenta
• Inner cell mass become embryo
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48. • Fetal stem cells
• After 8 weeks
• These stem cells responsible for initial development
of all tissue
• Umbilical cord stem cells
• Multipotent in nature
• Can be stored
cryogenically after birth
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49. Adult stem cells
• Post natal
• Present in tissue that have
already developed
• Multipotent
• Typically generates cell
types of tissue, in which
they reside
• New phenomenon-
PLASTICITY – able to
generate cell types of
other tissue
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50. DENTAL STEM CELLS
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51. Progenitor cells
• SC generate intermediate
cell types before they
achieve their fully
differentiated state
• They are known as
precursor or progenitor
cell.
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52. Dental pulp stem cell
• Discovered in the wisdom tooth in 2000 by Gronthos et al.
• Isolated from - Ectomesenchymal stem cells from dental
pulp
Several stem cell niches may exist in mature dental pulp
1. Undifferentiated mesenchymal cells residing in cell rich
zone
2. A perivascular cell population associated with pulp
vasculature
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53. • Characterization
• Express surface markers – CD44, CD106, CD146, 3G5 and
STRO1.
• Express matrix protein associated with mineral tissue
formation- ALP, osteocalcin, osteopontin
• 30% higher proliferation rate than bone marrow stem cell
• Cells expressing neural crest cell marker have been found
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54. STEM CELLS FROM HUMAN EXFOLIATED
DECIDUOUS TEETH
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55. • Isolated in 2003 by MIURA et al
• Higher proliferation rate than stem cells from
permanent teeth
• Can induce bone or dentin formation but failed to
produce dentin-pulp complex – Immature
• High plasticity - can differentiate into Neurons,
Adipocytes, Osteoblasts and Odontoblasts
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56. ADVANTAGES
• Can be retrieved from a tissue that is disposable and readily
accessible.
• SHED banking is more economical when compared to cord blood
banking
• Ideally suited for young patients at the mixed dentition stage
who have suffered pulp necrosis in immature permanent teeth
• Useful for close relatives of the donor such as grandparents,
parents and siblings
• Painless collection with minimal invasion because they are
retrieved from a tissue that is disposable and easily accessible
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57. STEM CELLS FROM APICAL
PAPILLAE
• Source of primary odontoblasts involved
in development of root dentine
• Positive for telomerase activity
• Embryonic stem cells
• STRO-1 POSITIVE CELLS, Faster
proliferation, Greater number of
population doublings, Increased capacity
for in vivo dentine regeneration
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58. • 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
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59. • Complete root formation under the influence
of the surviving HERS
• Can undergo odontoblastic, adipogenic or
neurogenic differentiation.
• Higher proliferation rates than DPSCS
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60. PERIODONTAL LIGAMENT STEM CELLS
• Isolated & Characterized by Seo et al., in
2004
• More proliferative
• Longer lifespan
• Higher number of population doublings in
vitro
• Differentiates into cementoblats-like
cells, Adipocytes & Collagen-forming cells
invitro
• capacity to generate a cementum/PDL like
structure invivo
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61. STEM CELLS FROM DENTAL
FOLLICLE
• 1st isolated from follicle of
impacted third molars
• Multipotent tissue
• Ability to generate
cementum, bone & PDL from
ectomesenchymal fibrous
tissue
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62. Progenitor cells in dentin regeneration and
novel therapeutics
Presence of pulpal inflammation hamper repair process
If injury is severe damage of odontoblast happens
Then cell migration from subodontoblastic cell rich zone happens.
Mechanism is unknown.
During pulp capping procedure reparative dentinogenesis happens
Growth factors present in the dentin matrix direct the process of
reactionary and reparative dentinogenesis.
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63. Recruitment of SC can be achieved through local application of
enriched population of cells
It is done either by harvesting cells from non-autologus teeth or
autologous exfoliated primary teeth.
It gives rise to osteodentin, similar to reparative dentin
Cells from non dental sites have been explored and found to be
capable of differentiation into odontoblast
Local angiogenesis is very important for success of vital pulp therapy.
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64. IDENTIFICATION
ISOLATION
STORAGE
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65. STEM CELL IDENTIFICATION
1. Staining the cells with specific antibody markers -
Fluorescent antibody cell sorting (FACS)
2. Immuno-magnetic bead selection
3. Immuno-histochemical staining
4. Physiological & histological criteria, including
phenotype (appearance), chemotaxis, Proliferation,
Differentiation & mineralizing activity
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66. Isolation approaches of DPSCs
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67. 1. Size-sieved isolation
2. Colony cultivation
3. Magnetic activated cell sorting
4. Fluorescence activated cell sorting
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68. 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.
• cells with diameter between 3 and 20 μm are obtained by
filtering and seeding
• Based on this approach, small-sized cell populations containing
a high percent of stem cells can be isolated.
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69. STEM CELL COLONY CULTIVATION
• Enzymatic digestion of the dental pulp tissue is done
to prepare single cell suspension
• Used for colony formation containing 50 or more cells
that is further amplified for experiments.
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70. MAGNETIC ACTIVATED CELL SORTING
(MACS)
• Is an immune-magnetic method
• Isolated based on their surface antigens (CD271,
STRO-1, CD34, CD45, and c-Kit).
Advantages
• Technically simple
• Inexpensive
• capable of handling large numbers of cells
Disadvantage
• degree of stem cell purity is low.
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71. • First pulp is isolated and single cell suspension is prepared
• Incubated with primary monoclonal antibody against specific
membrane surface molecule and immune magnetic beads
• placed in magnetic particle concentrator
• Cells bound to beads will attach to test tube wall
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72. FLUORESCENCE ACTIVATED CELL
SORTING (FACS)
• Is convenient and efficient
method
• Based on cell size and
fluorescence.
• Cells are stained with fluorescent
dyes, specific to target cell
• Fluorescence of each cell is
quantified as it transects the
laser beam
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73. Demerits
• Requirement of expensive equipment,
• Highly-skilled personnel
• Decreased viability of FACS-sorted cells
• Not appropriate for processing bulk quantities of
cells.
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74. Stem cell banking
1. Collection
2. Isolation
3. Preservation
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75. 1. Collection
• The pulp in exfoliated tooth should have
red color
• If color is gray- likely to be necrotic
• Highly mobile tooth excluded
• Teeth with apical abscess, tumor and cyst
not taken
• Then transferred into a vial containing
PBSS in hypothermic condition –
SUSTENTATION
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76. 2. Isolation protocol
After receiving the sample disinfect with povidone
iodine
Wash with PBSS
Pulp is isolated with forceps or excavator
Digested with collagenase type1 and dispase for 1 hr
at 370c
Filtration to obtain single cell suspension
cultured in MSC medium
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77. • Only epithelial and endothelial cells are required
• If contamination is present
1. Re-trypsinising culture for a short time so that
stromal cells are detached
2. Changing medium 4-6 hrs after sub-culture, because
stromal cells attach earlier.
3. Use fluroscence activated cell sorting.
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79. 3. PRESERVATION / STORAGE
A. CRYO-PRESERVATION
B. MAGNETIC FREEZING
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80. A. CRYOPRESERVATION
• 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 vapor 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.
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81. B. MAGNETIC FREEZING
• Referred to as cells alive system (CAS)
• Mechanism - 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.
• Hiroshima University claims that it can
increase the cell survival rate in teeth to
83%.
• Cheaper than cryogenics and more reliable
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82. GROWTH FACTORS
OR
MORPHOGENS
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83. Definition
• Growth factors are
polypeptides which have the
ability to bind to specific
receptors on the target cells
and modulate or facilitate
certain activities like
migration, proliferation,
differentiation and apoptosis .
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Cohen

84. • 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
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85. Properties
• They may be specific or non-specific .
Control stem cell activity such as
1. Stimulating stem cells to synthesize and secrete
,mineralized matrix.
2. By increasing the rate of proliferation
3. Induce differentiation of the cells into another
tissue type
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86. Classification
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91. SCAFFOLDS
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92. Scaffolds
• A lattice that provides a
framework for stem cells to
grow for pulpal regeneration.
(AAE)
• Three dimensional porous
solid biomaterials that
provides a physio-chemical &
biological 3D micro
environment for cell growth
and differentiation,
promoting cell adhesion, and
migration.
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93. Properties
• Carrier for morphogens in cell and in protein therapy
• Extracellular matrix molecules control the differentiation
of stem cells
• Temporary platform for repairing.
• Surround the cells and provide structural support for
formation and maintenance of tissues and organs.
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95. Ideal characteristics
• a
5/11/2020 9:26 AM DR ABHISEK 95

96. Classification
• Based on degradability of matrices
• Biodegradable scaffolds
• Permanent or biostable scaffolds
5/11/2020 9:26 AM DR ABHISEK 96

97. • Based on presence or absence of cells
• Cell free scaffolds
• Scaffolds seeded with stem cells
5/11/2020 9:26 AM DR ABHISEK 97

98. • Based on form
• Solid blocks
• Sheets
• Porous sponges
• Hydrogels (Injectable scaffolds)
5/11/2020 9:26 AM DR ABHISEK 98

99. Based on origin
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, PLLA, PGA, PLGA,
Bioceramics –
HA, ß-TCP, BCP
• Bioactive glass –
Silicate, Borate, Borosilicate
• Naturally derived –
• Fibrin
• sponge
• Amniotic membrane
• Polysaccharides
5/11/2020 9:26 AM DR ABHISEK 99

100. A. 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
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101. PLATELET RICH PLASMA (PRP)
• Autologous 1st generation platelet concentrate with a
rich sources 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.
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102. • Platelet concentration – Exceeds 1 million/mL
• Different growth factors present, Eg- PDGF, TGF-b, IGF,
VEGF, epidermal growth factor, epithelial cell factor
• Released via degranulation of alpha granules & stimulate bone &
soft tissue healing
• Disadvantage –
1. Drawing blood in young patients
2. need special equipment & reagents to prepare
3. increased cost of treatment.
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103. 5/11/2020 9:26 AM DR ABHISEK 103

104. PLATELET RICH FIBRIN
(CHOUKROUN’S TECHNIQUE)
• 2nd 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 at least 1 week up to 28 days
5/11/2020 9:26 AM DR ABHISEK 104

105. • 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
5/11/2020 9:26 AM DR ABHISEK 105

106. 5/11/2020 9:26 AM DR ABHISEK 106

107. Advantages
• lack of biochemical handling
of blood - autologous.
• it has a trimolecular or
equilateral fibrin branch
junction which makes its
architecture flexible and
can support cytokine
enmeshment and cellular
migration
5/11/2020 9:26 AM DR ABHISEK 107

108. BLOOD CLOT
• First practiced by Ostby
• 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
• Example : Revascularization
5/11/2020 9:26 AM DR ABHISEK 108

109. 5/11/2020 9:26 AM DR ABHISEK 109
Blood clot PRP PRF
Lesser cytokines compared
to PRF & PRP
Lesser cytokines
compared to PRF
Max. concentration of
cytokines
Rate of clot formation
corresponds to inherent
body clotting time
Addition of thrombin for
conversion of fibrinogen
to fibrin in PrP leads to
drastic activation & rapid
polymerization.
Show physiological
polymerization. Allows
the formation of flexible
3D fibrin network that
support cellular migration
Slower healing compared
to PRP & PRF
Slower healing compared
to PRF
Faster and stronger
healing kinetics
Not rich in growth factors
Max release of
morphogens occurs
before actual cell
ingrowth
Releases GF with peak
level reaching at 14 days
Inhibits differentiation
of BMSC
Shows proliferation and
differentiation of BMSC
Fibrin matrix susceptible
to washout in surgical
field
Stronger stable fibrin
matrix

110. COLLAGEN
• Paticularly type 1
• Used alone or in combination
with growth factors or
chitosan
• Advantages
• Higher degree of
odontoblastic differentiation,
• Expression of osteonectin,
DSPP, dentin matrix protein.
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111. CHITOSAN
• Produced by de-acetylation of chitin
• Formation of pores in scaffolds – Influencing
mechanical & biological properties
• Advantages
• Nontoxic, Easily absorbable
• Antibacterial activity
• Increases ALP activity
• shows fibroblast & odontoblastic proliferation.
• Disadvantages
• Low strength & inconsistency behavior with seeded
cells
5/11/2020 9:26 AM DR ABHISEK 111

112. GLYCOSAMINOGLYCANS
• Hyaluronic acid – One of the
glycosaminoglycans in ECM
• Maintains 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
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113. • Disadvantages
• Water soluble
• degrades rapidly by enzyme hyaluronidase – Overcome by
cross linking & modification of HA.
5/11/2020 9:26 AM DR ABHISEK 113

114. SILK
• Biocompatible
• Ability to support
attachement, differentiation,
proliferation
• Disadvantage
• Formation of osteodentin
• complete degradation occurs
after 2 years
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115. B. ARTIFICIAL SCAFFOLD
• First suggested by Vacanti et al.
• Polymers with controlled physicochemical features
such as degradation rate, microstructure, and
mechanical strength.
• Polylactic acid (pla)
• polyglycolic acid (pga)
• poly lactic-co-glycolic acid (plga).
5/11/2020 9:26 AM DR ABHISEK 115

116. • PGA: Enhanced the growth of new blood vessels &
odontogenic differentiation of human fibroblasts.
• PLA (OPLA): Aliphatic polyester, more hydrophobic
than PGA, attachable to root canal dentin.
• PLGA : Dentin-like & Pulp-like tissue regenerated
after 3-4 months
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117. Steps to create and use tissue
constructs
5/11/2020 9:26 AM DR ABHISEK 117

118. TISSUE
ENGINEERING
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119. 1. Ex-vivo tissue engineering
2. In-vivo tissue engineering
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120. A. Ex-vivo tissue engineering
• Involves expansion, differentiation or modification of
progenitor cells in culture
• They become functional tissue through cell- cell
signaling, biomolecule production and formation of
extracellular matrix
• They have potential for integration and further
maturation after implantation
• Requires hervesting of progenitor cells at various
stage of differentiation
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121. Complication faced
• Difficulty in culturing
• Need for cellular patterning and topographic control
• Need for providing a microcirculation for the
development of larger biologically meaningful tissue
• Costly
• Risk of contamination with bacteria or virus
• autologous cells can be immunogenic by prolonged in
vitro culture
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122. B. In-vivo tissue engineering
• Depends on administration of growth factors that
recruit progenitor cells
• Patient acts as his own bioreactor
• Compensate the deficiency in number and function of
progenitor cells, which is seen in in vitro
5/11/2020 9:26 AM DR ABHISEK 122

123. 3 general approaches for tissue engineering
1. Design and grow human tissue invitro for later implantation.
Eg- skin graft
2. Implantation of cell-containing or cell free devices that induce
generation of functional human tissue- signal molicules.
Eg- growth factor used to assist in biomaterial guided tissue
regeneration.
Use of polymer matrix to form cartilage invivo
3. Development of external devices containing human tissue
designed to replace the function of diseases internal tissue.
Eg- repair of bone, muscle, tendon, heart valve substitutes.
5/11/2020 9:26 AM DR ABHISEK 123

124. Tissue engineering is a multidisciplinary approach
• Clinician: biopsy of small sample of tissue containing
cells of interest
• Cell biologist: multiplies cells and maintain their
function.
• Bioengineer : manufactures the tissue, bioreactor and
the material onto which the cells will be placed for
transplantation.
• Lastly the clinician transplants the engineered tissue.
• Polymar scaffold degrades and is remodeled by the
host and transplanted cells resulting in complete
natural tissue
5/11/2020 9:26 AM DR ABHISEK 124

125. Preclinical and clinical accomplishments of
tissue engineering
A.Conductive (passive) approaches
B. Inductive approaches
C. Cell transplantation
5/11/2020 9:26 AM DR ABHISEK 125

126. Conductive (passive) approaches
• Utilize biomaterials in a
passive manner to facilitate
growth of existing tissue.
• Eg – dental implants. GTR
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127. Inductive approach
• It activates cells situated close to the
damaged tissue with specific signals
• Urist first demonstrated that new bone
could be formed at a non-mineralizing
site after implantation of powdered
bone
• This led to isolation of active
ingredients from the bone powder,
cloning of genes encoding this proteins.
• These proteins are BMPs.
5/11/2020 9:26 AM DR ABHISEK 127

128. • An alternative approach involves placing specific
extracellular matrix molecules on a scaffold, support at
the tissue site
• These molecules will have ability to direct the function of
cells already present at that site.
• Eg- a preparation of enamel proteins derived from pigs is
used to promote new bone formation in Pdl defects
5/11/2020 9:26 AM DR ABHISEK 128

129. Cell transplantation
• When the inductive for a specific tissue factors are not known
• when a large tissue mass or organ is needed or when tissue
replacement must be immediate.
• Eg- the greatest success in this era has been the development
of a tissue engineered skin equivalent.
• 250000ft skin tissue can be manufactured from a 1inch sample
of starting tissue
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130. 5/11/2020 9:26 AM DR ABHISEK 130

131. Regeneration of Dentin -Pulp
complex
1. Regeneration of entire tooth.
2. Local regeneration of dentin-pulp complex
from amputed dental pulp.
3. Regeneration of dental pulp from apical
dental pulp or peri- apical tissues.
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132. Regeneration of entire tooth
• Accepted as a model of
organ replacement and
regeneration therapy.
• Tooth germ can be
bioengineered using 3D
organ germ culture
method
• Dental epithelial and
mesenchymal cells from
isolated tooth germs
cultured in scaffolds
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133. b) Local regeneration of dentin-pulp
complex from amputed dental pulp
• BMP-2 with dentin powder induced
dentinogenesis in dentin cavity with
pulp exposure.
• Here stem or progenitor cells were
induced from residual pulp through
the exposure site in the floor of the
cavity.
• Ultrasound mediated delivery of
growth differentiating factor - 11
(GDF-11) in dental pulp stem cells
through sonoporation, induced
reparative dentinogenesis
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134. c) Regeneration of dental pulp from
apical dental pulp or peri- apical tissues
• Identification of stem cells in the apical areas of
developing teeth in which root formation is
incomplete.
• SCAPs differentiate into odontoblast-like-cells that
participate in pulp wound healing and regeneration.
• BMMSC has multipotent abilities and undergoes
osteogenic differentiation.
• Localization of SCAPs & BMMSCs in the apical region
--- induction for dentin-pulp complex regeneration
5/11/2020 9:26 AM DR ABHISEK 134

135. 5/11/2020 9:26 AM 135
POTENTIAL
TECHNOLOGIES
Root canal
revascularization
Post natal
stem cell
therapy
Pulp
implantation
Scaffold
implantation
Injectable
Scaffold
delivery
3D Cell
printing
Gene delivery
DR ABHISEK

136. 1. ROOT CANAL
REVASCULARIZATIO
N VIA BLOOD CLOTTING
5/11/2020 9:26 AM DR ABHISEK 136

137. History
Nygard Ostby , in the early 1960s - new vascularized tissue could
be induced in the apical third of the root canal of endodontically
treated mature teeth with necrotic pulps and apical lesions.
He proposed that through formation of a clot (scaffold), a
vasculature could be established to support growth of new tissue
into the unfilled portion of the root canal.
5/11/2020 9:26 AM DR ABHISEK 137

138. Revascularization with continued root development and continued
deposition of hard tissue has also been shown to occur when
immature teeth were reimplanted after intentional or
traumatically related avulsion
(Cvek M, Cleaton-Jones P, Austin J, et al 1990,. Kling M, Cvek M, Mejare
I 1986).
Skoglund and Tronstad (1981) investigated changes in the root
canal of replanted and auto-transplanted immature teeth in dogs
Result - during the first 6 months there was an ingrowth of
vascularized, cell-rich connective tissue throughout the entire
root canal.
marked reduction After 6 months.
5/11/2020 9:26 AM DR ABHISEK 138

139. Iwaya et al in 2001 - revascularization on a necrotic immature
mandibular 2nd premolar with a chronic apical abscess.
After 30 months - thickening of the root canal walls by
mineralized tissue and continued root development.
Banchs and Trope (2004) - revascularization of a necrotic
immature mandibular second premolar with an open apex and a
large apical lesion.
5/11/2020 9:26 AM DR ABHISEK 139

140. 5/11/2020 9:26 AM DR ABHISEK 140
Jeeruphan et al

141. Feature
Similarity
1. Osteogenic differentiation
2. Dentinogenic differentiation
3. Low adipogenic potential
Difference
1. Cell proliferation: SCAP > DPSCs
2. Number of population doublings:
SCAP > DPSCs
3. Tissue regeneration capacity:
SCAP > DPSCs
5/11/2020 9:26 AM DR ABHISEK 141
Comparison between SCAP and DPSCs
Source of stem cells

142. Outline
5/11/2020 9:26 AM DR ABHISEK 142

143. Case selection:
• Tooth with necrotic pulp and an immature apex.
• aged from 8 - 16 years
• Pulp space not needed for post/core, final
restoration.
• Compliant patient/parent.
• Patients not allergic to medicaments and antibiotics
necessary to complete procedure.
5/11/2020 9:26 AM DR ABHISEK 143

144. Disinfection protocol
• Keep in mind:
• Thin, fragile dentinal walls -
prone to fracture during
instrumentation or
obturation.
• Open apex increases the risk
of extruding material into
the periradicular tissues.
5/11/2020 9:26 AM DR ABHISEK 144

145. • Over instrumentation and dressing
using cytotoxic antiseptics may
remove:
– Pulp tissue that can survive in
the wide, well nourished apical
area
– the cells capable of forming
pulp and dentin.
• Under instrumentation have the
benefit of avoiding a smear layer
that could occlude the dentinal
walls or tubules.
5/11/2020 9:26 AM DR ABHISEK 145

146. MEDICAMENTS USED
5/11/2020 9:26 AM DR ABHISEK 146

147. REVASCULARISATION USING CaOH
LA & Isolation of tooth
Access opening
Irrigation ( Often with 10 ml of 2.5%of NaOCl)×
No instrumentation in root canal
Preparation of calcium hydroxide paste××
Insertion of the paste in the in the coronal part (third or half) of
root canal (with a cotton ball)
Sealing of the access cavity
5/11/2020 9:26 AM DR ABHISEK 147
FIRSTSTEP

148. 5/11/2020 9:26 AM DR ABHISEK 148
SECONDSTEP
2-3weekslater LA without vasoconstrictor×××
Isolation of the tooth
Access opening
Removal of the CaOH paste
Copious irrigation of root canal with NaOCl
Rinsing root canal with sterile water
Drying root canal with paper points
An apical bleeding is caused by irritation of the apical region
with a 15 K-file ××××
Preparation of MTA and placement on the clot to form a
hermetic sealing
Place GIC 2mm
Sealing of the cavity with resin

149. REVASCULARIZATION USING TRIPLE
ANTIBIOTIC PASTE
5/11/2020 9:26 AM DR ABHISEK 149
LA & Isolation of tooth
Disinfection with 10% povidone-iodine
Access opening
Irrigation with 20mL NaOCl (1.25%–5.25%) then with
physiological serum and finally with 2% CHX
No instrumentation in root canal
Drying root canal with paper cones
Insertion of TAP into root canal
Place a cotton ball at the root canal entrance
Sealing of the access cavity with a temporary filling
FIRSTSTEP

150. 5/11/2020 9:26 AM DR ABHISEK 150
SECONDSTEP
(twoorthreeweekslater) LA without vasoconstrictor×
Isolation with a rubber dam
Disinfection with 10% povidone-iodine before opening it××
Access opening
Removal of TAP using irrigation with NaOCl(1.25%–5.25%) then with
physiological serum and finally with 2% CHX×××
An apical bleeding is caused. Blood level must be at the cement-enamel
junction.
Preparation of MTA and its placement on the clot××××
Place GIC 2mm
Sealing of the cavity with a resin composite

151. INSTRUMENTATION.
• 2 types of cells are required to achieve a normal root
development:
– odontoblasts and epithelial cells of HERS.
– They are able to resist inflammation
[N. Shah et al. 2008, A. Nosrat et al 2011]
• No instrumentation procedure remains consistent with vital stem
cells preservation
1515/11/2020 9:26 AM DR ABHISEK

152. IRRIGATION
 3% H2O2
 0.12%-2% CHX - may be detrimental to
the stem cells.
 0.5–5.25% NaOCl
 Povidine-iodine
 17% of EDTA
5/11/2020 9:26 AM DR ABHISEK 152

153. Hydrogen Peroxide
• it has an hemostatic action.
• Acts as antiseptic by release of oxygen
radical.
• Its action is too short and quickly
neutralized by organic debris.
• requires a rinse to reduce pain and
possible postoperative gaseous
emphysema.
5/11/2020 9:26 AM DR ABHISEK 153

154. Chlorhexidine
• 2% CHX
• It has good action on candida and gram+
bacteria by the
• Its positively charged molecules confer
the property of being absorbed by the
dentin walls and thus allow release of
chlorhexidine for at least 2-12 weeks
• Does not have an effective dissolving
action.
5/11/2020 9:26 AM DR ABHISEK 154

155. Sodium Hypochlorite
• It has a solvent action on necrotic
tissue and an antiseptic effect
• 0.5% - 5.25%.
• Cytotoxicity of sodium hypochlorite
is proportional to its concentration.
• 2.5% - best compromise between
efficiency and lack of toxicity
[M. Zehnder, 2006]
5/11/2020 9:26 AM DR ABHISEK 155

156. • Trevino et al (2008) : survival rate of human stem cells of the
apical papilla (SCAP) exposed to 6% NaOCl, followed by 17%
EDTA, 6% NaOCl again, was 74%.
• The needle should be introduced 2 mm short of the apical
foramen
• To minimize emphysema, negative pressure irrigation can be used
Silva et al (2010).
5/11/2020 9:26 AM DR ABHISEK 156

157. Iodine
• Iodine is bactericidal, antifungal,
antiviral, sporicidal and sedative.
• Purulent secretions and blood do not
inactivate it.
• disadvantage - it colors dental
tissues in brown.
5/11/2020 9:26 AM DR ABHISEK 157

158. Ethylene Diamine Tetraacetic Acid
• Better wettability of the irrigator and
a removal of the smear layer.
• 17% of EDTA is often used in cases of
bacterial infection to remove the smear
layer
• And induce a better penetration of the
irrigator and of root canal medications
[B.O. Aktener andU. Bilkay, 1993]
5/11/2020 9:26 AM DR ABHISEK 158

159. • Its chelating effect would allow the release of growth
factors from dentin
• That would stimulate the proliferation of stem cells .
• Ring et al. have compared effects of CHX and hypochlorite
after treatment with EDTA .
• They show that there is no survival stem cell after using a
combination of EDTA and 2% CHX.
• chlorhexidine salts are formed- toxic and prevent cell
adhesion to the canal wall.
5/11/2020 9:26 AM DR ABHISEK 159

160. DISINFECTION
5/11/2020 9:26 AM DR ABHISEK 160

161. CALCIUM HYDROXIDE
• It is a strong base (pH = 12.5–12.8)
• its ionic dissociation in Ca2+ and OH− induce genesis of
hard tissue (apexification, tertiary dentin)
• These OH- ions
– damage the cytoplasmic membrane
– suppress the bacterial enzyme activity
– denature proteins
– damage DNA
– thus inhibit any replication, and inactivate endotoxins.
5/11/2020 9:26 AM DR ABHISEK 161

162. • Ca(OH) increase the expression of some kinases, which are
indicators of proliferation of stem cells from pulp and ligament
• Therefore, used in usual concentrations, it would not be
cytotoxic for stem cells
• However, tricalcium silicates cements, such as MTA, Ca(OH)2, or
Biodentine, have a weakening effect on dentin because of their
pH.
5/11/2020 9:26 AM DR ABHISEK 162

163. • 0.01 mg/mL CaOH for canal disinfection allows
survival of 100% of the apical stem cells.
• Even at 1mg/mL, Ca(OH) give a maximal survival of
stem cells.
• At the same concentration, antibiotics paste only
allows between 33% and 56%cells survival.
[N. B. Ruparel et al 2012]
5/11/2020 9:26 AM DR ABHISEK 163

164. Triple Antibiotic Paste (TAP)
• A combination of antibiotics is essential to cover a
maximum range of action.
• must be used in proper concentration for a balance
between a lower cytotoxicity against stem cells and a
maximum bacterial disinfection.
• An in vitro study has shown that a TAP concentration
of 39𝜇 g/mL would be best for application in
disinfection root canal
[S.Chuensombat, 2013]
5/11/2020 9:26 AM DR ABHISEK 164

165. • mixture paste of 3 antibiotics with propylene glycol
put into root canal at a concentration of 20 𝜇g/mL
decreases bacterial colonies by more than 99%
Hoshino and Takushige; 1998
• Most of the revitalization regeneration procedures
use a triple antibiotic paste, called Hoshino’s paste
5/11/2020 9:26 AM DR ABHISEK 165
Sato I, Ando-Kurihara N, Kota K, Iwaku M, Hoshino E. Sterilization of infected root-canal dentine by topical application of a mixture of
ciprofloxacin, metronidazole and minocycline in situ. Int Endod J 1996;29:118–24.

166. Composition (adapted from Hoshino et al.
1996)
Antibiotics (3 Mix)
• Minocycline 100 mg
• Ciprofloxacin 200 mg
• Metronidazole 500 mg
Carrier (MP)
• Macrogol ointment
• Propylene glycol
5/11/2020 9:26 AM DR ABHISEK 166

167. Protocol for preparation
do not cross- contaminate.
Remove sugar coating from tablets with surgical
blade
crush individually in separate mortars.
Grind each antibiotic to a fine powder.
Combine equal amounts of antibiotics (1:1:1) on mixing
pad.
5/11/2020 9:26 AM DR ABHISEK 167

168. Carrier (MP)
Equal amounts of macrogol ointment and progylene glycol (1:1)
Using clean spatula, mix together on pad.
Result should be opaque.
1:5 (MP : 3 Mix) → creamy consistency.
1:7 (standard mix) → smears easily but does not crumble
If result is flaky or crumbly, then too much 3 mix has been
incorporated.
5/11/2020 9:26 AM DR ABHISEK 168

169. • Video-
5/11/2020 9:26 AM DR ABHISEK 169

170. Storage
5/11/2020 9:26 AM DR ABHISEK 170
• Antibiotics must be kept separately in
moisture-tight porcelain containers.
• Macrogol ointment and propylene
glycol must be stored separately.
• Discard if mixture is transparent
(evidence of moisture contamination)

171. Ph
• Acidic pH of minocycline is not favorable for
cultivation of stem cells
• It facilitate cell permeability of the antibiotic, which
keep long-term cytotoxicity.
• Ciprofloxacin has also an acidic ph.
• Metronidazole - neutral pH and thus it has no
cytotoxicity for stem cells.
• Metronidazole and ciprofloxacin could induce the
formation of fibroblasts .
5/11/2020 9:26 AM DR ABHISEK 171

172. • In 2009, Bose et al. analyzed cases of regenerative
endodontic treatment performed all over the world.
• Use of TAP, CaOH, and formocresol produced
significantly greater increases in root length and
width
• In terms of changes in root width, TAP produced
significantly greater increases in dentin wall
thickness
5/11/2020 9:26 AM DR ABHISEK 172

173. Limitations of TAP
1. Bacterial resistance
[sedgley CM, lee EH. Et al. 2008].
2. A risk of precipitating an allergic reaction
3. Tooth discoloration due to tetracycline
4. minocycline binds to Ca++ ions by chelation and form
insoluble complexes- discoloration
5. persistent sinus tract after using triple antibiotic
paste
Jung et al.
5/11/2020 9:26 AM DR ABHISEK 173

174. Other properties
• Indeed, TAP combined with a saline solution shows
the lowest minimum inhibitory concentration (MIC)
against Enterococcus faecalis (MIC = 77.5 𝜇g/mL).
• Calcium dihydroxide combined with saline is absolutely
not effective against Enterococcus faecalis.
5/11/2020 9:26 AM DR ABHISEK 174

175. Case report by Iwaya et. Al 2001
• Preoperative clinical view showing intraoral fistula at the apical gingiva
in the mesiobuccal side of tooth 46
• Iwaya S, Ikawa M, Kubota M. Revascularization of an immature permanent tooth
with apical periodontitis and sinus tract. Dent Traumatol 2001; 17
5/11/2020 9:26 AM DR ABHISEK 175

176. Preoperative radiograph
of tooth 45. Incomplete
apex formation and
periapical radiolucency
5/11/2020 9:26 AM DR ABHISEK 176
3D CT image of involved tooth and its
periapical legion taken 15 days after the
initial treatment.

177. 5 months after the
application of CaOH. Dentin
bridge formation is observed
5/11/2020 9:26 AM DR ABHISEK 177
30 months postoperative. Completion of
the root apex and increase in the
thickness of the canal wall was revealed.

178. THE TISSUE REGENERATION
• Through studies on animal cuts,
the apposition material-inducing
thickening of root walls may be
of different nature dentin,
cementum, or even bone
• [X.Wang et al. 2010]
• Therefore, this procedure is
not a process of pulp
revascularization but a process
of tissue regeneration.
5/11/2020 9:26 AM DR ABHISEK 178

179. • The inability to obtain sections of
human teeth after revascularization is
a handicap for understanding and
validating this process.
• Only radiographic assessments of in
vivo clinical studies and laser Doppler
flowmetry can give us an idea of
treatment success.
• [H. Strobl et al. 2003].
• cold test also seems to be a good
determinant of success.
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180. • Emi Shimizu et al. 2012 has done histological evaluation of
immature permanent tooth #9 after 3.5 wks of revascularization
procedure
• Result - more than one half of the canal was filled with loose
connective tissue similar to the pulp tissue.
• A layer of flattened odontoblast-like cells lined along the
predentin.
• Layers of epithelial-like cells, similar to the HERS surrounded
the root apex.
• No hard tissue was formed in the canal.
5/11/2020 9:26 AM DR ABHISEK 180
Emi Shimizu, George Jong, Nicola Partridge, Paul A. Rosenberg, and Louis M. Lin, Histologic observation of a human
immature permanent tooth with irreversible pulpitis after revascularization/regeneration procedure(J Endod
2012;38:1293–1297)

181. • Bacerra P et al. 2014 done a histological examination of a revascularized
mandibular premolar which was presented with chronic periapical
abscess
• after 2 yr successful follow up which was extracted for orthodontic
reasons.
• They found that the large canal space filled with fibrous connective
tissue.
• The apical closure was caused by cementum deposition without dentin.
Some cementum-like tissue was formed on the canal dentin walls.
5/11/2020 9:26 AM DR ABHISEK 181
Bacerra P et al Histologic Study of a Human Immature Permanent Premolar with Chronic Apical Abscess after Revascularization/
Revitalization(J Endod 2014;40:133– 139)

182. Single vs multiple visit
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183. • Video
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184. FOLLOW-UP AND TREATMENT
OUTCOME
• resolution of the apical lesion in approx. 6
months
• Root elongation and apical closure, with
thickening of the root canal walls, within
12–24 months
(Neha K, Kansal R, Garg P, et al. 2011, Banchs F, Trope M. 2004;. Iwaya SI,
Ikawa M, Kubota M 2001)
• During the first year, 3-month recalls
should be scheduled, followed by 6-month
recalls unless clinical symptoms develop.
5/11/2020 9:26 AM DR ABHISEK 184

185. • most revascularization were performed on incisors and
premolars of children 8–14 years of age
• (Iwaya SI, Ikawa M, Kubota M 2001Chen MY, Chen KL, Chen CA, et al. 2012 , Huang GT. 2008; Shah N,
Logani A, Bhaskar U, Aggarwal V. 2008).
• Successful revascularization case reports in older ages have also
been published
• (Shah N et al. 2008, Aggarwal V et al.2012).
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186. UNFAVOURABLE OUTCOMES
• Lenzi and Trope 2012 found empty root canal space after
treatment of an immature maxillary central incisor with a
necrotic pulp.
• Nosrat et al. 20 12 showed the absence of vital tissue
inside the root canal space of treated immature maxillary
incisors with necrotic pulps after 6 years.
• Nosrat et al. 2013 presented a case where root maturation
occurred in a maxillary central incisor, even though a
regenerative endodontic procedure resulted in an empty
root canal space.
5/11/2020 9:26 AM DR ABHISEK 186

187. • Even after using tissue engineering strategies,
cementum-like hard tissue was deposited on root
canal walls, and bony islands were found throughout
the root canals.
[Yamauchi N et al . 2011]
• Formation of a hard-tissue barrier inside the canals
between the coronal MTA plug and the root apex
[ Chen MY et al. 2011]
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188. TREATMENT OUTCOME depends upon
(1) chemical disinfection of the canal
without instrumentation
(2) production of a suitable environment for
a scaffold to support tissue ingrowth; and
(3) a tight bacterial seal of the access
opening to prevent the ingress of bacteria.
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189. Advantages
Requires a shorter treatment time- after control of infection, can be
completed in a single visit.
Cost-effective - the number of visits is reduced
Obturation of the canal is not required unlike in calcium hydroxide–
induced apexification
Continued root development (root lengthening) and strengthening of the
root as a result of reinforcement of lateral dentinal walls with deposition
of new dentin/hard tissue.
5/11/2020 9:26 AM DR ABHISEK 189

190. Technically simple
avoids the possibility of immune rejection and pathogen
transmission from replacing the pulp with a tissue
engineered construct.
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191. Limitations
Long-term clinical
results are as yet not
available.
Entire canal might be
calcified, compromising
esthetics and
potentially increasing
the difficulty in future
endodontic procedures
In case post and core
are the final
restorative treatment
plan, revascularization
is not the right
treatment option.
5/11/2020 9:26 AM DR ABHISEK 191

192. Concentration and composition of cells trapped in fibrin clot
is unpredictable, which may lead to variations in treatment
outcomes.
Enlargement of the apical foramen is necessary to
promote vascularization and to maintain initial cell viability
via nutrient diffusion.
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193. FACTORS THATAFFECT THE RESULTS
OF REVASCULARIZATION
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194. Presence of bacteria mounts host immune defenses and
diverts the tissue healing process towards repair
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195. 2. POSTNATAL STEM CELL
THERAPY
• The simplest method to administer stem cells
• Stem cells can be derived from multiple tissues, including
skin, buccal mucosa, fat and bone.
• A major research obstacle - 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
• ex vivo methods required to purify and/or expand cell
sufficiently
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196. Disadvantages
• Cells may have low survival rate.
• The cells might migrate to different location leading to
aberrant patterns of mineralization
• Probability of producing new functioning pulp tissue by
injecting only stem cells without a scaffold or signaling
molecules, may be very low.
5/11/2020 9:26 AM DR ABHISEK 196
Nakashima M. Bone morphogenetic proteins in dentin regeneration for potential use in endodontic therapy.
Cytokine Growth Factor Rev 2005;16:369 –76.

197. 3. PULP IMPLANTATION
• The majority of in vitro cell cultures
grow as a single monolayer attached
to the base of culture flasks- 2D
• In theory, to take 2D cell cultures
and make them 3D 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.
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198. ADVANTAGES
• The cells are relatively easy to grow on filters in the
laboratory.
• aggregated sheets of cells are more stable than
dissociated cells administered.
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199. 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
Because the filters are very thin layers of cells, they are
extremely fragile
Difficult to place in root canal systems without breakage.
5/11/2020 9:26 AM DR ABHISEK 199

200. 4. SCAFFOLD IMPLANTATION
• Using a porous polymer scaffold seeded with pulp
stem cells
• A scaffold should contain growth factors to aid stem
cell proliferation and differentiation
• The scaffold may also contain nutrients and possibly
antibiotics
5/11/2020 9:26 AM DR ABHISEK 200
Ching Yuang Huang et al. Mesoporous Calcium Silicate Nanoparticles with Drug Delivery
and Odontogenesis Properties. JOE,2017, 43: 69–76

201. • 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
5/11/2020 9:26 AM DR ABHISEK 201
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.

202. 5/11/2020 9:26 AM DR ABHISEK 202

203. 5. 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 3D scaffold matrix, such as a
polymer hydrogel.
• Hydrogels are injectable scaffolds that can be delivered
by syringe.
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204. • 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
5/11/2020 9:26 AM DR ABHISEK 204
Desgrandchamps F. Biomaterials in functional reconstruction. Curr Opin
Urol 2000;10:201– 6.

205. 5/11/2020 9:26 AM DR ABHISEK 205

206. • To make hydrogels more practical, research is focusing on
making them photopolymerizable to form rigid structures once
they are implanted into the tissue site
5/11/2020 9:26 AM DR ABHISEK 206

207. 6. 3D CELL PRINTING
• 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.
• Ideally odontoblastoid cells should be placed around
the periphery to maintain and repair dentin, with
fibroblasts in the pulp core supporting a network of
vascular and nerve cells.
5/11/2020 9:26 AM DR ABHISEK 207

208. • precisely position cells
• disadvantage - careful orientation of the pulp tissue
construct according to its apical and coronal
asymmetry would be required during placement
5/11/2020 9:26 AM DR ABHISEK 208

209. • video
5/11/2020 9:26 AM DR ABHISEK 209

210. 7. 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
5/11/2020 9:26 AM DR ABHISEK 210
viral or nonviral
vectors
deliver genes for growth factors,
morphogens, transcription factors, and
extracellularmmatrix molecules
target cell
populations

211. 5/11/2020 9:26 AM DR ABHISEK 211

212. 5/11/2020 9:26 AM DR ABHISEK 212

213. CHALLENGES AND FUTURE DIRECTION
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214. CONCLUSION
• 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.
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215. References
• 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
5/11/2020 9:26 AM DR ABHISEK 215

216. • 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
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217. Thank you
5/11/2020 9:26 AM DR ABHISEK 217