Regenerative endodontics aims to regenerate damaged pulp and root structures through biologically-based procedures. Historically, studies in the 1960s-70s showed blood clots could induce tissue formation in root canals. Current methods include placing stem cells on scaffolds with growth factors in the root canal to regenerate the pulp-dentin complex. Triple antibiotic paste, calcium hydroxide, and MTA are used as antimicrobial medicaments. The protocol involves inducing bleeding into the root canal to form a blood clot which triggers regeneration. The goal is periradicular health and evidence of vital regenerated tissue through radiographic and clinical measures.

1. Regenerative
Endodontics
SANGHAMITRA SUMAN
JR-3

2.  Regenerative Endodontics:
“biologically based procedures designed to replace damaged structures,
including dentin and root structures, as well as cells of the pulp-dentin
complex .”
Cohen 10th edition

3. Historical development of the field
 Nygard –OstbyB: The role of blood clot in endodontic therapy:an
experimental histologic study. Acta Odontol Scand 19:323,1961
 Nygard- Ostby B:Tissue formation in the root canal following pulp
removal. Scand J DENT RES 79:333,1971

4. Potential methods for regenerating an entire tooth
(Nakhara et al.)
The first approach:
seeding appropriate stem cells onto scaffolding materials with the addition of
specific growth factors and/or signalling molecules.
The second approach:
replicating the natural developmental processes of embryonic tooth formation. Artificial
tooth germs are transplanted into the bodies of animal hosts where there is enough blood
flow to support tissue formation.

5. Alternative
Regenerate a functional pulp-dentin complex within a patient's existing permanent
tooth to restore natural functions and neural sensation.
 from a tissue engineering perspective the dental pulp is a comparatively easier
tissue to regenerate
Although either approach may evolve into a method capable of generating
entire teeth, the natural development of permanent human teeth takes years to
complete. Regenerating an entire tooth from a patient's own stem cells may
not be clinically practical.

6. Triad of Regenerative Endodontics
Regenerative
Endodontics
Control of
inflammation
Stem cellBiomaterials
Nadia Chugal, Louis M. Lin, and Bill Kahler

7. The major domains of research required to develop regenerative
endodontic procedures. (Peter E. Murray et al)

8. Stem cells
Stem cells
Adult /Postnatal
Embryonic/Fetal
A stem cell is commonly defined as a cell that has the ability to continuously divide and
produce progeny cells that differentiate into various other types of cells or tissues

9. Adult Stem cells
 Autogenic, Allogenic and Xenogenic stem cells
Type
• Totipotent
• Pluripotent
• Multipotent
Cell plasticity
• Each cell can develop
into a new individual
• Cell can form any cell
type (over 200
• Cell differentiated but
can form a number of
other tissues
Source of stem
cell
• 1-3 days of
embroyonic life
• 5-14 days of
embruonic life
• Fetal tissue, cord
blood, and
postnatal stem cells
including, dental
pulp stem cells

10. Pulp Stem Cells
A small population of competent progenitor stem cells may exist within the dental pulp
throughout life and are called as pulp stem cells, or, in the case of immature teeth, stem
cells from human exfoliated deciduous teeth (SHED)
Sometimes pulp stem cells are called odontoblastoid cells, because these cells appear to
synthesize and secrete dentin matrix like the odontoblast cells they replace.
 Source: undifferentiated mesenchymal cells

11.  One of the most significant obstacles in regenerative endodontics is to obtain stem cells
that will continually divide and produce cells or pulp tissues that can be implanted into
root canal systems
Possibilities:
Development of an autogenous human pulp
stem cell line that is disease- and pathogen-
free.
1. patients do not need to provide their own
cells through a biopsy.
2. pulp tissue constructs can be
premade for quick implantation
when they are needed
Development of a tissue biopsy
transplantation technique (for e.g
using cells from the oral mucosa)

12. Stem cell identification
from a population of mixed
cells
1. fluorescent antibody cell sorting
(FACS)
2. immunomagnetic bead selection,
3. immunohistochemical staining
4. physiological and histological criteria,
including phenotype, chemotaxis,
proliferation, differentiation, and
mineralizing activity.
Identification of
differentiated cell as
odontoblast
1. DSP
2. Specific genes
3. Phenotype

13. 2. Growth factors
 Growth factors are proteins that bind to receptors on the cell and induce cellular
proliferation and/or differentiation
GF + Stem cells = increased prolifereation & differentiation
For e.g.
 TGF Beta and Recombinant human BMP2 stimulates differentiation of adult pulp
stem cells into an odontoblastoid morphology
(Roberts-Clark DJ, Smith AJ. Angiogenic growth factors in human dentine matrix. Arch Oral Biol
2000;45:1013– 6)

14.  Recombinant BMP-2, -4, and -7 has shown to induce formation of reparative dentin in vivo
( Nakashima M, et al. Regulatory role of transforming growth factor-beta, bone morphogenetic protein-2, and protein-4 on gene expression of extracellular
matrix proteins and differentiation of dental pulp cells. Dev Biol 1994;162:18 –28.)
 The application of recombinant human insulin-like growth factor-1 together with collagen has
been found to induce complete dentin bridging and tubular dentin formation .
(Lovschall H, Fejerskov O, Flyvbjerg A. Pulp-capping with recombinant human insulin-like growth factor I (rhIGF-I) in rat molars. Adv Dent Res
2001;15:108 –12)
 This indicates the potential of adding growth factors in regenerative endodontics and also before
pulp capping to stimulate dentin and pulp regeneration.

16. Scaffolds
 For tissue engineering therapy, pulp stem cells must be organized into a 3
dimensional structure that can support cell organization.
 It can be achieved using a porous polymer scaffold seeded with pulp stem cells

17.  Scaffolds are three-dimensional (3D) porous solid biomaterials designed which
1. Provide a spatially correct position of cell location
2. Promote cell-biomaterial interactions, cell adhesion, and matrix deposition
3. Permit sufficient transport of gases, nutrients, and regulatory factors to allow
cell survival, proliferation, and differentiation
4. Biodegrade at a controllable rate that approximates the rate of tissue
regeneration
5. Provoke a minimal degree of inflammation or toxicity in vivo.

18.  Ideal requirements of a scaffold
1. A high porosity and an adequate pore size are necessary to facilitate
cell seeding and diffusion throughout whole structure of both cells and
nutrients
2. Should allow effective transport of nutrients, oxygen, and waste
3. Biodegradability is essential, since scaffolds need to be absorbed by the
surrounding tissues without the necessity of surgical removal.
4. The rate at which degradation occurs has to coincide with the rate of
tissue formation.
5. Should be biocompatible.
6. Should have adequate physical and mechanical strength.

19. Types of scaffolds:
Scaffolds
Natural.
(Derivatives of
extracellular
matrix)
1. Proteolytic
2.polysaccharide
Synthetic
(Polyster)
PLA,PGA,PCL,

21. Attributes of commonly used scaffold

22. Correct delivery of appropriate stem cells and growth factors embedded within
a scaffold
Stem cells Growth factors Scaffold
If one were to inject cells along the entire coronal-apical extent of a root canal system, the
vast majority of cells would be expected to succumb to tissue hypoxia.
One alternative approach would be to inject a cell/scaffold/growth-factor mixture into the
apical 1 mm of the root canal system and then “back-fill” the root canal system with a
scaffold/growth-factor combination.

24. Potential Technologies for Regenerative
Endodontics
1. Root Canal Revascularization via Blood Clotting
2. Postnatal Stem Cell Therapy
3. Pulp Implantation
4. Injectable Scaffold Delivery
5. Gene Therapy
6. Three-Dimensional Cell Printing

25. Root canal revascularization
 Apexification: “method to induce a calcified barrier in a root with an open apex”
apexification does not attempt to regain vital tissue in the canal space. The outcome of an
apexification procedure is establishment of an apical barrier against which an obturating
material may be placed
 Apexogenesis: “a vital pulp therapy procedure performed to encourage continued
physiologic development and formation of the root end.”
Apexogenesis is indicated for teeth in which there has been no loss of vascularity, thus
no need to “revascularize” the canal space

26. A comparison between regenerative endodontic
treatment and other pulp treatment procedures
 In apexification with Ca(OH)2
1. Chances of root fracture and stem cell toxicity.
2. at least 6 months are required to create an apical barrier, and
mulitple visits are needed to replenish calcium hydroxide.
Andreasen JO, Farik B, Munksgaard EC. Long-term calcium hydroxide as a root canal dressing may
increase risk of root fracture. Dent Traumatol. 2002;18:134–137.

27.  MTA is used in the one or two step apexification procedure, and therefore a
fewer number of appointments are needed.
Bose R, Nummikoski P, Hargreaves K. A retrospective evaluation of radiographic outcomes in immature teeth with necrotic
root canal systems treated with regenerative endodontic procedures. J Endod. 2009;35:1343–1349.
In spite of this advantage, apexification with MTA neither strengthens the root
nor induces further root development. As a result, the roots remain thin and
fragile, and hence another treatment approach is needed.

28. Apexification with
calcium hydroxide
Long time span of the entire treatment
Multiple visits
Increased risk of tooth fracture due to long-term application of Ca(OH)2
Apexification with
MTA
One- or two-step apexification
Neither strengthens the root nor promotes further root development
Roots remain thin and fragile
Revascularization
Promotes further root development
Causes reinforcement of dentinal walls by deposition of hard tissue
(strengthening the root against fracture)
the characteristics of three treatment procedures for immature root formation

29. Revascularization Apexification with
MTA
Apexification with
calcium hydroxide
Root width 28.2% 0.00% 1.52%
Root length 14.9% 6.1% 0.4%
The percentage increase in root width and root length after the treatment
procedure
Jeeruphan T, Jantarat J, Yanpiset K, Suwannapan L, Khewsawai P, Hargreaves KM. Mahidol study 1: comparison of
radiographic and survival outcomes of immature teeth treated with either regenerative endodontic or apexification
methods: a retrospective study. J Endod. 2012;38:1330–1336.

30.  Pulp revascularization = induction of angiogenesis in
endodontically-treated root canal
 Pulp regeneration = pulp revascularization + restoration of
functional odontoblasts and/or nerve fibers

31.  “To date, no published clinical trials have fully incorporated the tissue-engineering
concepts. Instead, studies over the last 50 years have focused on revascularization
techniques, which share some features with the principles of regenerative tissue
engineering.” (Cohen 10th edition.)
 The key distinction is that in contrast to the focused delivery of cells/growth
factors/scaffolds employed in tissue-engineering approaches, revascularization focuses
on triggering bleeding into an empty root canal space with the hope that this will trigger
a process similar to the role of the blood clot in triggering wound healing in surgical
procedures

32. Revascularization protocol

33. Case selection
 “This treatment should be considered for the incompletely developed
permanent tooth that has an open apex and is negative to pulpal
responsiveness testing. Although the ultimate goal of this approach is to
develop a tissue engineering–based method of pulpal regeneration in the fully
developed permanent tooth, it should be recognized that current
revascularization protocols have not been developed or evaluated for these
more challenging cases.”
Cohen 10th edition

34. During the first appointment
 Minimal instrumentation by the use of a small file (determine the working length)
 Copious and slow irrigation with 20 ml of NaOCl (lower concentration) followed
by 20 ml of 0.12% to 2% chlorhexidine (CHX), slow irrigatin with closed end
side vented needle kept at the apex .
 The root canal system is then dried with sterile paper points, and the antimicrobial
medicament is delivered into the root canal space.
 The best available evidence supports the use of either a triple antibiotic paste or
Ca(OH)2. Both medicaments have been shown to be effective .
,

35.  The triple antibiotic paste has the advantage of being a very effective antibiotic
combination against odontogenic microorganisms but carries a potential for
minocycline staining of the crown.
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 29:118, 1996
 Alternatively, Ca(OH)2 has the advantage of being widely available and is a commonly
used medicament, but it may be cytotoxic to stem cells.
Lai WH, Chen YH, Chiang CP: Regenerative endodontic treatment for necrotic immature permanent teeth. J Endod 35:160,
2009
 After antimicrobial medicament is placed, the tooth is then sealed with a sterile sponge
and a temporary filling (e.g., Cavit), and the patient is discharged for 3 to 4 weeks.

36. On the second visit:
 patient is evaluated for resolution of any signs or symptoms of an acute infection (e.g.,
swelling, sinus tract, pain, etc.) that may have been present at the first appointment. The
antimicrobial treatment is repeated if resolution has not occurred.
 Since revascularization-induced bleeding will be evoked at this appointment, the tooth
should not be anesthetized with a local anesthetic containing a vasoconstrictor. Instead, 3%
mepivacaine can be used, which will facilitate the ability to trigger bleeding into the root
canal system
 the tooth should be copiously and slowly irrigated with 20 ml NaOCl, together with gentle
agitation with a small hand file to remove the antimicrobial medicament.

37.  After drying the canal system with sterile paper points, a file is placed a few mm beyond the
apical foramen, and the apical tissue is lacerated with bleeding up to 3 mm from the CEJ.
 A small piece of Colla-Plug (resorbable matrix) may be inserted into the root canal system to
serve as a resorbable matrix to restrict the positioning of the MTA.
 About 3 mm of MTA is then placed, followed by a restoration.
 A 12- to 18-month recall should be considered as the earliest time point to conduct the clinical
examination and evaluate continued radiographic improvement in root development.
Bose R, Nummikoski P, Hargreaves K: A retrospective evaluation of radiographic outcomes in immature teeth with necrotic root canal systems
treated with regenerative endodontic procedures. J Endod 35:1343, 2009.

39. Medicaments being used in cases of
revascularization
1. Triple antibiotic paste (1 : 1 : 1 mixture of
ciprofloxacin/metronidazole/minocycline)
2. Ca(OH)2 alone or in combination with antibiotics,
3. Formocresol

40. 1. The triple antibiotic paste produced significantly greater differences in dentinal wall
thickness compared with either the Ca(OH)2 or formocresol groups.
2. The formocresol group showed the smallest improvement in root length and
thickness.
3. Location of Ca(OH)2 placement appeared to be a strong predictor of radiographic
outcome.
4. When Ca(OH)2 placement was restricted to the coronal half of the root canal, the
increase in root wall thickness was 55%, compared to a 3% increase when it was
placed in the apical half of the root canal system. This might be due to residual
Ca(OH)2 having a cytotoxic interaction with stem cells
Bose R, Nummikoski P, Hargreaves K: A retrospective evaluation of radiographic outcomes in immature teeth with necrotic root
canal systems treated with regenerative endodontic procedures. J Endod 35:1343, 2009.

41. Clinical Measures of Treatment Outcome
 The goal of revascularization extends beyond non surgical root canal treatment
 For regeneration not only radiographic evidence of periradicular health but also
radiographic and other clinical evidence of functioning vital tissue in the canal
space is required.
 Radiographic evidence of functioning pulp (or pulp like) tissue would include
continued root growth, both in length and wall thickness. These findings have
been shown in many published cases.

42.  Other measures of the presence of vital, functioning tissue in the
canal space include laser Doppler blood flowmetry, pulp testing
involving heat, cold, and electricity and lack of signs or symptoms.
 “ The ideal clinical outcome is an asymptomatic tooth that does not
require retreatment, but to validate that regenerative endodontic
techniques are truly effective, nonsubjective vitality-assessment
methods are essential”

43. Advantages of revascularization
Traditionally an immature tooth with open apex is treated by apexification:
1. Calcium hydroxide : short-term or long-term use of Ca(OH)2 can reduce root
strength. A large case series using the traditional apexification protocol showed
that a major reason for tooth loss following apexification was root fracture.
Cvek M: Prognosis of luxated non-vital maxillary incisors treated with calcium hydroxide and filled with gutta-percha. A
retrospective clinical study. Endod Dent Traumatol 8:45, 1992
2. MTA : one step apexification , however does not result in further root development.
In contrast in revascularization there is a greater likelihood of increase in root wall
length and thickness.

44.  This approach is technically simple and can be completed using currently
available instruments and medicaments without expensive biotechnology.
 The regeneration of tissue in root canal systems by a patient’s own blood
cells avoids the possibility of immune rejection and pathogen transmission
from replacing the pulp with a tissue engineered construct.

45. LIMITATIONS
 The source of the regenerated tissue has not been identified
 Relys on blood clot formation but the concentration and composition of cells
trapped in the fibrin clot is unpredictable.
 Enlargement of the apical foramen is necessary to promote vascularizaton and to
maintain initial cell viability via nutrient diffusion. It is likely that cells in the
coronal portion of the root canal system either would not survive or would survive
under hypoxic conditions

46. Overview of case reports on revascularization
 To date, only case reports and case series on endodontic revascularization treatment are
available, no randomized controlled clinical trials have been published
 Nearly all reported cases involve patients 8 to 18 years old and teeth with immature
apices
 In nearly every case there was a lack of instrumentation of the dentinal walls
 NaOCl, alone or in combination with other irrigants, was used to disinfect the canal
space.
 In a majority of cases, a combination of triple antibiotic or NaOCl was left in the canal
space for a period of days to weeks,
 In most cases, a blood clot formed in the canal. The formation of a blood clot might
serve as a protein scaffold, permitting three-dimensional ingrowth of tissue.

47.  Nearly all of these reports noted continued thickening of the root walls and
subsequent apical closure
 There was a lack of histology in all these clinical cases, hence radiographic findings
of continued root wall thickness does not necessarily indicate that dentin was formed
 Increased root wall thickness was limited to the midroot and apical root. There has
been no demonstration of increased root thickness in the cervical area.(prone to
fracture in immature teeth with a history of trauma)
 Very few cases reported about the vitality of pulp, and mainly relied on heat test

48.  1. Revascularization occurs most predictably in teeth with open apices.
the immature permanent tooth in general has a very wide apical opening that likely is
conducive to tissue ingrowth.
 2. Instrumentation with NaOCl irrigation is not sufficient to reliably create
the conditions necessary for revascularization of the infected necrotic tooth
(Cvek M, Nord CE, Hollender L. Antimicrobial effect of root canal debridement in teeth with immature
root: a clinical and microbiologic study. Odontol Revy 1976;27:1–10)

49. 3. Placement of Ca(OH)2 in root canal systems prevents revascularization coronal to the
location of the Ca(OH)2 .
(Schroder U, Granath LE. Early reaction of intact human teeth to calcium hydroxide following experimental pulpotomy and its
significance to the development of hard tissue barrier. Odontol Revy 1971;22:379 –95)
 cases treated with Ca(OH)2 display intracanal calcifications that appear to impede
the continued thickening of the dentinal walls of these immature teeth .
Chueh LH, Huang GT. Immature teeth with periradicular periodontitis or abscess undergoing apexogenesis: a paradigm shift. J
Endod 2006;32:1205–13.
 In addition, other investigators have suggested that the use of Ca(OH)2 might kill
any remaining pulpal cells, including stem or progenitor cells known to be present in
dental pulp tissue.
Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002;81:531–5

50. 4. The use of the “3 mix-MP” triple antibiotic paste, developed by Hoshino and colleagues and
consisting of ciprofloxacin, metronidazole, and minocycline, is effective for disinfection of the
infected necrotic tooth, setting the conditions for subsequent revascularization.
(Hoshino E, Kurihara-Ando N, Sato I, et al. In vitro antibacterial susceptibility of bacteria taken from infected root dentine to
a mixture of ciprofloxacin, metronidazole, and minocycline. Int Endod J 1996;29:125–30.)

51. Posssibile newer techniques of
regeneration.

52. Injectable Scaffold Delivery
 In root canal systems a rigid tissue engineered pulp is not required to provide structural
support of the tooth. This will allow it to be administered in a soft three-dimensional
scaffold matrix, such as a polymer hydrogel
 Hydrogels are injectable scaffolds that can be delivered by syringe
 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
 To make hydrogels more practical, research is focusing on making them
photopolymerizable to form rigid structures once they are implanted into the tissue site
Alhadlaq A, Mao JJ. Tissue-engineered osteochondral constructs in the shape of an articular condyle. J Bone Joint Surg Am 2005;87:936 – 44.
158.

53. 2. Postnatal Stem Cell Therapy
 To inject postnatal stem cells into disinfected root canal systems after the apex is
opened.
 Postnatal stem cells can be derived from multiple tissues, including skin, buccal
mucosa, fat, and bone
Obstacles:
1. 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).
2. 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.

54.  Possible approaches
using 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
an allogenic purified pulp stem cell line that is
disease- and pathogen-free;
xenogneic (animal) pulp stem cells that have been
grown in the laboratory.
Although umbilical cord stem cell
collection is advertised primarily to
be used as part of a future medical
therapy, these cells have yet to be
used to engineer any tissue constructs
for regenerative medical therapies
no purified pulp stem cell lines are
presently available
 Difficulty

55.  Advantages
1. autogenous stem cells are relatively
easy to harvest and to deliver by
syringe, and the cells have the
potential to induce new pulp
regeneration.
2. this approach is already used in
regenerative medical applications,
including bone marrow replacement,
and a recent review has described
several potential endodontic
applications
Disadvantages:
1. the cells may have
low survival rates.
2. the cells might
migrate to different
locations within the
body possibly leading
to aberrant patterns of
mineralization
apply the cells
together with a
fibrin clot or
other scaffold
material.
Solution

56. Pulp implantation
 Pulp tissue is grown in the laboratory in sheets and implanted surgically.
 the pulp cells can be grown on biodegradable membrane filters or on sheets
of extracellular matrix proteins such as collagen I or fibronectin . Many
filters will be required to be rolled together to form a 3 dimensional pulp
tissue, which can be implanted into disinfected root canal systems.
Venugopal J, Ramakrishna S. Applications of polymer nanofibers in biomedicine and biotechnology. Appl Biochem
Biotechnol 2005;125:147–58.
 So far, growing dental pulp cells on collagens I and III has not proved to be
successful. but other matrices, including vitronectin and laminin, require
investigation

57. Advantage :
1. cells are relatively easy to grow on filters in the laboratory.
2. aggregated sheets of cells are more stable than dissociated cells administered
by injection into empty root canal systems
Disadvantage :
1. specialized procedures may be required to ensure that the cells properly adhere to root
canal walls.
2. Implantation of sheets of cells may be technically difficult since the filters are very
thin
3. The sheets of cells also lack vascularity
4. concerns over immune responses

58. Three dimensional cell printing
 an ink-jet-like device used to dispense layers of cells suspended in a hydrogel
to recreate the structure of the tooth pulp tissue.
 The three-dimensional cell printing technique can be used to precisely position
cells, placing odontoblastoid around periphery, with fibroblast in the pulp core
supporting a vascular and neural network
Barron JA, Wu P, Ladouceur HD, Ringeisen BR. Biological laser printing: a novel technique for
creating heterogeneous 3-dimensional cell patterns. Biomed Microdevices 2004;6:139 – 47

59. Gene Therapy
 To deliver mineralizing genes into pulp tissue to promote tissue mineralization.
 However, a literature search indicates there has been little or no research in this
field, except for the work of Rutherford .
 He transfected rabbit pulps with cDNA-transfected mouse BMP-7 that failed to
produce a reparative response, suggesting that further research is needed to
optimize the potential of pulp gene therapy
Rutherford RB. BMP-7 gene transfer to inflamed ferret dental pulps. Eur J Oral Sci 2001;109:422– 4

61. CONCLUSION
 Many teeth are not given the opportunity to be sved and instead are extracted,
with subsequent placement of an artificial prosthesis, such as an implant.
 Regenerative endodontic offer an alternative method to save teeth that may have
compromised structural integrity.
 The available case reports of pulp revascularization were generally reported on
young patients and teeth with open apices. However, for regenerative endodontic
procedures to be widely available and predictable, endodontists will have to
depend on tissue engineering therapies to regenerate pulp dentin tissue.

62.  The proposed therapies involving stem cells, growth factors, and
tissue engineering is an enormous challenge, and the future
development of regenerative endodontic procedures will require a
comprehensive research program directed at each of these
components and their application to our patients.

63. THANK YOU