endodontics

1. Regenerative
Endodontics & Minimally
Invasive Endodontics
Dr. Carlos T. Capitan II

2. CONTENTS
• Introduction
• Components of Regenerative Endodontics

3. INTRODUCTION
• Goals :
 Primary goal- elimination of symptoms and the evidence of
bone healing.
 Secondary goal-increased root length or wall thickness.
 Tertiary goal-positive response to vitality tests.
• Regenerative endodontics are biologically based procedures
designed to replace damaged structures ,including dentin and
root structures, as well as cells of pulp-dentin complex.
• Concept : Normal, sterile granulation tissue developed in the root
canal for revascularization will stimulate the cementoblasts/
undifferentiated mesenchymal cells (periapex) and lead to calcific
material formation at periapex or lateral dentinal walls.

4. INTRODUCTION
Regenerative endodontics is an exciting and developing field in the
treatment of immature teeth with infected root canals that has been
described as a “paradigm shift” in the management of these teeth and
can result in continued root maturation and apical closure
Traditional approaches of calcium hydroxide apexification and apical
barrier techniques with mineral trioxide aggregate (MTA) have been
used in the treatment of immature teeth with pulp necrosis though
generally there is no further root development so the roots remain thin
and fragile with a higher risk of fracture and tooth loss.
Recently, it has been suggested that regenerative endodontic protocols
(REPs) that utilize endogenous stem cells that are introduced in the
canal by lacerating the periapical tissues to fill the canal with blood
should be used for the treatment of immature teeth with pulp necrosis.

5. INTRODUCTION
Even in teeth with complete root formation, the periradicular tissues
comprise mes- enchymal stem cells that can be delivered into the
canal.
However, it is uncertain whether formation of a pulp-like tissue is
biologically possible after pulp necrosis in mature teeth. The tissue
formed inside the root canal after revitalization procedures contains
elements of pulp tissue (fibroblasts, connective tissue, blood vessels,
collagen), but other cell types are missing, among them notably
odontoblasts, whereas non-targeted cell types or tissue such as
osteoblasts and cementum may be present. Thus revitalization
procedures may not so much give healing by regeneration but rather
healing by repair.
(Chrepa, V. et al. (2015) Delivery of apical mesenchymal stem cells into root canals of mature teeth. J Dent Res 94: 1653-165)/
Martin, G. et al. (2013) Histological findings of revascularized/revitalized immature permanent molar with apical periodontitis using platelet-rich plasma. J
Endod 39: 138-144 & Wang, X. et al. (2010) Histologic characterization of regenerated tissues in canal space after the revitalization/ revascularization
procedure of immature dog teeth with apical periodontitis.J Endod 36: 56-63)

6. INTRODUCTION
Stem cells of the apical papilla in immature teeth is another source of
cells that can proliferate, differentiate, and form odontoblasts and pulp
tissue.
These cells may cause regeneration of the pulp after revitalization
procedures. In teeth with incomplete root formation and pulp necro-
sis, revitalization can be performed as an alternative treatment to
apexification, where provocation of bleeding into the canal flushes in
the respective cells from the apical papilla.
((Huang, G.T. et al. (2008) The hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. J Endod 34:645-651/
Lovelace, T.W. et al. (2011) Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative
endodontic procedure. JEndod 37: 133-138)

7. Terminology
A number of terms have been adopted in the literature with
regenerative endodontics, revascularization and revitalization being
the most commonly used.
The term ‘revascularization’ is well established in the endodontic
literature and relates to the reestablishment of vascularity in the pulp
space after traumatic injuries that sever the blood supply to the pulp of
immature teeth. Earlier reports on this new technique showed renewed
root maturation in infected immature teeth and described the
introduction of a blood clot into the root canal as “revascularization”
Regenerative endodontics implies that further root maturation
results in reestablishment of the pulp dentine complex. Many studies
show that this is not the case with a variety of tissues such as dentine,
cementum, periodontal ligament, bone, osteoid and possibly pulp
being the tissue found in treated teeth with regenerative endodontic
protocols, suggesting ‘repair’ rather than ‘regeneration.

8. Terminology
‘Revitalization’ has been suggested as it describes non-specific
vital tissue rather than just blood vessels as implied by the term
‘revascularization’.
The term regenerative endodontic procedures (REPs) has been
widely adopted and refers to all procedures that aim to attain
organized repair of the dental pulp and include future therapies
yet to evolve in the field of regenerative endodontics
Regeneration is defined as reconstitution of damaged tissue by
tissue similar to original tissue and restoration of biological
functions
Repair is replacement of damaged tissue by tissue different from
original tissue and loss of biological functions.The dental pulp has a
limited potential of regeneration.

9. In regenerative endodontic procedures, many growth factors
embedded in the dentin matrix are released into the canal space after
EDTA . These growth factors have been shown to be able to signal
pulp stem cells to differentiate into odontoblast-like cells and produce
reparative dentin However, the mesenchymal stem cells introduced
into the canal space during regenerative endodontic procedures do
not appear to be able to differentiate into odontoblast-like cells and
produce the dentin-pulp complex in many animal and human studies
The term ’regeneration’ refers to the recreation of the original
architecture, form, and function of a specific tissue type; ’repair’
denotes healing by formation of a tissue that has partially lost the
biological function of the original tissue. The distinction between
repair and regeneration is often difficult in the clinical setting. Moreover,
as biological processes, the two overlap in most situations. A complex
fracture of the long bones will typically heal by virtually complete
regeneration of the bone itself, but the surface wound will heal by
fibrous tissue formation and a skin scar.
Terminology

10. History
The promise and potential of regenerative endodontic therapies in
necrotic teeth was first explored by Nygaard-Östby in 1961 who
investigated the potential for repair when bleeding was induced by over-
instrumentation beyond the apex prior to partial root filling of the canal
with limited success.
Forty years later, in 2001 Iwaya et al. reported a case using a
procedure termed ‘revascularization’, on an infected necrotic
immature premolar tooth that showed continued root maturation and
thickening of root canal walls with mineralized tissue. Subsequent
case reports also demonstrated the efficacy of this technique primarily
in premolar teeth when using a triple-antibiotic paste or calcium
hydroxide.
Further reports demonstrated successful outcomes in traumatized
central incisor teeth. A number of authors have described
regenerative endodontic techniques as a “paradigm shift” in the
treatment of immature teeth with necrotic pulps.

11. Histologic characterization of tissues formed
in the canal space
Many histological studies of regenerative endodontic procedures of
immature teeth with necrotic pulps and apical periodontitis have
been investigated in animal models and humans and show that the
tissues formed in the canal space were mineralized tissue similar to
cementum and bone, and fibrous connective tissue resembling
periodontal ligament.
Histologically, regenerative endodontic procedures of human immature
permanent teeth with necrotic pulp are considered a reparative and not a
regenerative process .

12. Histologic characterization of tissues formed
in the canal space
Radiographic thickening of the canal walls, and continued root
maturation of immature permanent teeth with necrotic pulps after
regenerative endodontic procedures should not be regarded as a
regeneration of dentin-pulp complex without histological
confirmation.
If the primary goal of regenerative endodontic procedures is
elimination of clinical symptom/sign and resolution of apical
periodontitis , then repair, although not an ideal wound healing, is not
a treatment failure.

13. Principles of Regeneration and Repair
Root canal treatment can prevent tooth loss by eliminating pulpal and
periradicular disease; however, it is not a biology-based approach.
Advances in pulp biology and tissue engineering challenge the
traditional concept of replacing lost pulp with inert synthetic materials.
Regenerative strategies aim at the production of new vital tissue that
resembles the original in architecture, structure and function.
As for the dental pulp, it has been known for decades that this tissue
possesses regenerative capacity, and dentistry has been pioneering
regenerative approaches with the use of agents such as calcium
hydroxide to promote healing after pulp-capping procedures . Such
therapeutic measures are mainly empirical, but as we unravel the
biological basis and start to comprehend the mechanisms underlying
regeneration and repair, regenerative medicine offers great scope also
for the field of endodontics.
(Herrmann, B. (1928) Ein weiterer Beitrag zur Frage der Pulpenbehandlung. Zahnartzliche Rundschau 37: 1327-1376)

14. Pulp and Periapical
Regeneration Processes
Periradicular bone possesses the capability to regenerate fully after
removal of the inflammatory trigger. Healing of bony lesions is a major
goal of endodontic therapy and it occurs predictably if the clinician
succeeds in eliminating or sufficiently reducing bacterial activity
within the root canal system. Thus, the connective tissues of the
periapical bony structures have an innate capacity, with an adequate
supply of multipotent cells, for complete regeneration of the
periodontal ligament and the alveolar bone in the right circumstance.
Pulp tissue can also regenerate; however, it remains a challenge to
elucidate exactly under which circumstances this may take place. It is
important to identify the cell sources that may be activated for
regeneration. Remaining islands of vital pulp can form the point of
origin for regenerated pulpal tissue (Saoud, T.M. et al. (2015) Histological observations of pulpal replacement
tissue in immature dog teeth after revascularization of infected pulps. Dent Traumatol 31: 243-249).

15. Vital Pulp Therapy
Measures such as indirect and direct pulp capping or pulpotomy aim at
maintaining tooth vitality and supporting the pulp's inherent capability
of regeneration and repair. In either case, the pulp will respond with the
formation of tertiary dentine. The generation of tertiary dentine is an
active defense mechanism to create a mineralized barrier that separates
the tissue from the site of injury; as such, it is a measurable parameter of
healing.
Tertiary dentine can be either reactionary or reparative. During tooth
development, odontoblasts secrete primary dentine at a rate of 4-8 pm
per day, but they adopt a resting state after the completion of root
formation where secretion is reduced to about 0.5 pm per day.
Mild stimulation induces an upregulation of odontoblast activity, where
primary odonto- blasts increase their secretory activity to the original
level, leading to rapid deposition of reactionary dentine, which displays
a tubular structure.
(Kawasaki, K., Tanaka, S., and Ishikawa & T. (1979 Massler, M. and Schour, I. (1946) / Couve, E. (1986) Ultrastructural changes during the life cycle of human
odontoblasts. Arch Oral Biol 31: 643-651 & Simon, S. et al. (2009) Molecular characterization of young and mature odontoblasts. Bone 45: 693-703/ Cooper, P.R. et
al. (2010) Inflammation- regeneration interplay in the dentine-pulp complex. J. Dent 38: 687-697 &Smith, A.J. et al. (1995) Reactionary dentinogenesis. Int J Dev Biol
39: 273-280 )

16. With increasing intensity of the stimulus and delayed intervention, healing
will more likely take place as repair. If the original odontoblast layer is lost,
e.g. after intense stimulation or pulp exposure, it can be replaced after
differentiation of stem cells into secondary odontoblasts [16, 22]. These cells
deposit reparative dentine, a mineralized matrix that may not exhibit the
characteristic tubular structure, but resemble bony tissue and often display
cellular inclusions [33]. Cooper, P.R. et al. (2010) Inflammation-
regeneration interplay in the dentine-pulp complex.
(J. Dent 38: 687-697 & Dimitrova-Nakov, S. et al. (2014), Pulp stem cells: implication in reparative dentin formation. J Endod 40, S13-8 & Goldberg, M. et al. (2001)
Application of bioactive molecules in pulp-capping situations. Adv Dent Res 15: 91-95)
After therapeutic intervention, reversible inflammatory processes
within the pulp tissue are expected to heal by regeneration.

17. It remains unclear whether osteodentine formation is particular to the
cell source, which is not comprised of original odontoblasts, or to the
intensity of stimulation, which leads to hasty deposition of a
mineralized tissue that is less organized.
Furthermore, tertiary dentine is also characterized by increased
peritubular dentine deposition, a vital process which has to be
differentiated from intra-tubular-calcifications due to physicochemical
precipitation of mineral crystals. Thus, the structure of tertiary dentine
is highly variable.
(Smith, A.J. et al. (1995) Reactionary dentinogenesis. Int J Dev Biol 39: 273-280 & Baume, L.J. (1980) The biology of pulp and dentine. A historic, terminologic-
taxonomic, histologic-biochemical, embryonic and clinical survey. Monogr Oral Sci 8: 1-220(

18. Bioactive Materials
Bioactive materials have long been used to induce tertiary dentine formation and to
support healing of the dentine pulp complex. A material is termed bioactive if it
exerts a positive influence on vital tissues and elicits a desirable biological response
at the interface. The response can be indirect, through antibacterial activity, or
direct, by interaction with adjacent cells, e.g. stimulation of proliferation,
differentiation, and/or biomineralization.
(Hench, L.L. and West, J.K. (1996) Biological application of bioactive glasses. Life Chemistry Reports 13: 187-241/ Herrmann, B. (1920) Kalziumhydroxid als Mittel zum
Behandeln und Füllen von Zahnwurzelkanalen. Dissertation, Würzburg./ Herrmann, B. (1928) Ein weiterer Beitrag zur Frage der Pulpenbehandlung. Zahnartzliche
Rundschau 37: 1327-1376
Calcium hydroxide was introduced in root canal therapy nearly a hundred years
ago. and studies with this material in contact with the pulp have demonstrated that
it enables the tissue to remain vital and to form a mineralized barrier.
Calcium hydroxide has been extensively used in endodontics and dental
traumatology, and it has been the material of choice for vital pulp therapy for several
decades.
Application of calcium hydroxide thus has several effects that enable
healing by regeneration.

19. Bioactive Materials
Due to its high alkalinity, it not only exerts antibacterial and antifungal activity , but
induces necrosis of adjacent cell layers and an inflammatory reaction in the
underlying tissue. The proinflammatory cytokines and chemokines are responsible
in recruiting immune cells, which promote healing by clearance of the injury site,
and stem cells, which differentiate into secondary odontoblasts.
(Hench, L.L. and West, J.K. (1996) Biological application of bioactive glasses. Life Chemistry Reports 13: 187-241/ . Tomson, P.L. et al. (2017) Growth factor release from
dentine matrix by pulp- capping agents promotes pulp tissue repair- associated events. Int Endod J 50: 281-292./ Al-Hezaimi, K. et al. (2011) Histomorphometric and
micro-computed tomography analysis of pulpal response to three different pulp capping materials. J Endod 37: 507-512/ Franz, F.E., Holz, J., and Baume, L.J. (1984)
Ultrastructure (SEM) of dentine bridging in the human dental pulp. J Biol Buccale 12: 239-246)
Histological analysis after pulp capping shows a superficial layer of tissue debris
beneath the calcium hydroxide and an adjacent, mineralized barrier. This barrier
may display a tubular structure continuous with the original dentin, only with
fewer and more curved tubules, but more often it presents as an amorphous and
atubular calcified tissue with cellular inclusions.
Furthermore, calcium hydroxide releases growth and differentiation factors that
are bound in the dentin matrix, which affect and modulate cellular behavior. The
use of calcium hydroxide for direct pulp capping has been shown to result in the
formation of a mineralized barrier , its thickness increasing with longer
postoperative periods.

20. Bioactive Materials
Negative Properties of Calcium Hydroxide:
● include high solubility
● low mechanical stability
● the mineralized barrier formed may be porous and exhibit
tunnel defects
● calcium hydroxide appeared to be inferior regarding the
thickness of newly formed mineralized tissue compared to
Portland-cement-based materials such as mineral trioxide
aggregate (MTA)

21. Bioactive Materials
Due to these drawbacks, calcium hydroxide is progressively displaced by
hydraulic calcium silicate cements. These cements form a stable calcium-
silicate-hydrate matrix; calcium hydroxide and calcium ions are side
products of this reaction and create the bioactive effects .
Besides increased stability of these materials compared to calcium
hydroxide, hydraulic calcium silicate cements offer a wide range of
applications in endodontics due to their capability to seal and disinfect
and to induce dentinogenic or osteogenic mineralization. Several studies
provide evidence that the use of hydraulic calcium silicate cements results
in a less distinct necrotic zone, hyperemia, and inflammatory reaction
compared to calcium hydroxide, to a more homogenous and solid layer of
tertiary den- tin, and to a superior clinical performance regarding failure
rates and pulp vitality.
(Aeinehchi, M. et al. (2003) Mineral trioxide aggregate (MTA) and calcium hydroxide as pulp-capping agents in human teeth: a preliminary report. Int Endod J 36:
225-231/ Al-Hezaimi, K. et al. (2011) Histomorphometric and micro-computed tomography analysis of pulpal response to three different pulp capping materials. J
Endod 37: 507-512)
Similar to calcium hydroxide, MTA solubilizes dentin matrix proteins,
which is likely to contribute to the material's bioactivity.

22. Bioactive Materials
MTA solubilizes dentin matrix proteins [91], which is likely to contribute
to the material's bioactivity. Besides increased stability of these materials
compared to calcium hydroxide, hydraulic calcium silicate cements offer
a wide range of applications in endodontics due to their capability to
seal and disinfect and to induce dentinogenic or osteogenic
mineralization.
(Aeinehchi, M. et al. (2003) Mineral trioxide aggregate (MTA) and calcium hydroxide as pulp-capping agents in human teeth: a preliminary report. Int Endod J
36: 225-231/ Hilton, T.J., Ferracane, J.L., and Mancl, L. (2013) Comparison of CaOH with MTA for direct pulp capping: a PBRN randomized clinical trial. J Dent.
Res 92, 16S-22S/ Nowicka, A. et al. (2013) Response of human dental pulp capped with biodentine and mineral trioxide aggregate. J Endod 39: 743-747 / Tabarsi,
B. et al. (2010) A comparative study of dental pulp response to several pulpotomy agents. Int Endod J 43: 565-571)
Several studies provide evidence that the use of hydraulic calcium
silicate cements results in a less distinct necrotic zone, hyperemia, and
inflammatory reaction compared to calcium hydroxide, to a more
homogenous and solid layer of tertiary den- tin, and to a superior
clinical performance regarding failure rates and pulp vitality.

23. Cell Types Involved in Pulp Healing
Among the cells that constitute the pulp tissue, the odontoblasts are
the first target for external stimuli due to their peripheral localization
in the dental pulp and their extension into dentine.
(Rezzani, R., Stacchiotti, A., and Rodella, L.F. (2012) Morphological and biochemical studies on aging and autophagy. Ageing Res Rev 11: 10-31 & Terman, A. et
al. (2010) Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial- lysosomal axis theory of aging. Antioxid Redox Signal 12: 503-535)
Odontoblasts are post-mitotic cells, i.e. they are not replaced during
the life of the organism under physiological conditions. As such, they
share certain features with neurons and myocardiocytes as static cell
populations . Stimuli include thermal variations and biomechanical
forces, but also molecular products derived from microorganisms.

24. Tertiary Dentine
An essential feature of pulpal defense is the formation of tertiary dentine.
Reactionary dentine has to be distinguished from reparative dentine, as
they arise from two different populations of cells and thus their genesis
and nature are distinct.
(Smith, A.J. et al. (1994) Odontoblast stimulation in ferrets by dentine matrix components. Arch Oral Biol 39: 13-22, Smith, A.J. et al. (1995) Reactionary
dentinogenesis. Int J Dev Biol 39: 273-280. & Ten Cate, A.A. (1994) Oral Histology: Development, Structure and Function. St. Louis, MI: Mosby)
Reactionary dentinogenesis refers to the secretion of a tertiary dentine
matrix by surviving post-mitotic odontoblasts, which increase their
secretory activity in response to an appropriate stimulus. Implantation
of dentin extracellular matrix components into unexposed cavities in
ferret teeth leads to a localized stimulation of reactionary dentine . This
can mainly be attributed to the presence of transforming growth factor
p-1 (TGF p-1), the most abundant growth factor in the dentine matrix,
which is known to markedly upregulate odontoblast secretory activity .

25. Tertiary Dentine
In contrast, reparative dentinogenesis is a more complex biological process.
Stronger stimuli will lead to the death of the odontoblasts, but if conditions are
favorable, a new generation of odontoblast-like cells may differentiate from stem
or precursor cells within the pulp.
Additionally, secretion of matrix at the mineralization front and
along the odontoblast process leads to a progressive increase in
thickness of the peritubular dentin, and the gradual occlusion of the
dentinal tubules by centripetal deposition of calcium phosphate
crystals leads to sclerosis and thus a decreased permeability of
dentine.
In carious lesions, the demineralization of dentine induced by
bacterial acids and the subsequent solubilization of bioactive
molecules, in particular TGFp-1, is considered responsible for
initiating the stimulatory effect on the odontoblasts and thus the
main cause of reactionary dentine formation.
(Smith, A.J. et al. (1994) Odontoblast stimulation in ferrets by dentine matrix components. Arch Oral Biol 39: 13-22, Smith, A.J. et al. (1995) Reactionary
dentinogenesis. Int J Dev Biol 39: 273-280. & Ten Cate, A.A. (1994) Oral Histology: Development, Structure and Function. St. Louis, MI: Mosby)

26. Stem Cells: Sources and Activation
A major cell source for regeneration or repair is the pool of stem cells that
is present in the dental pulp, papilla and periapical tissues. Stem cells
have been isolated from the pulp of permanent as well as deciduous
teeth and furthermore from the apical papilla of teeth with incomplete
root formation.
(Gronthos, S. et al. (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 97: 13625-13630, Miura, M. et al. (2003)
SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 100: 5807-5812, Huang, G.T. et al. (2008) The hidden treasure in apical papilla:
the potential role in pulp/dentin regeneration and bioroot engineering. J Endod 34:645-651 & Sonoyama, W. et al. (2008) Characterization of the apical papilla and
its residing stem cells from human immature permanent teeth: a pilot study. J Endod 34: 166-171)
Most commonly, stem cells are separated from the population of ex
vivo cultured primary cells based on presence or absence of specific
glycoproteins on the cells' surface. Thus, patterns characteristic for
mesenchymal stem cells can be recognized, and cells can be sorted by
use of specific antibodies.

27. Stem Cells: Sources and Activation
By definition, stem cells are characterized by their capacity for self-
renewal and their ability to differentiate into different cell types. In
general, stem cells are involved in regular tissue turnover to replace aged
cells, and during repair and regeneration.
Stem cells are undifferentiated cells that are capable of differentiating into
various specialized cell types. They can be pluripotent or multipotent in
nature. They are located in stem cell perivascular niche. where they are
kept in an undifferentiated stage, stem cells in the dental pulp remain
quiescent until an insult occurs.
(Shi, S. and Gronthos, S. (2003) Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 18: 696-704 &
Dimitrova-Nakov, S. et al. (2014) Pulp stem cells: implication in reparative dentin formation. J Endod 40, S13-8. )
Stem cells undergo asymmetric division, meaning that one cell gives
rise to an identical cell to keep the pool of stem cells constant,
whereas the second daughter cell enters the path of differentiation.
Chemotactic signaling recruits stem cells to the site of injury, they
leave their niche, migrate, and differentiate into secondary
odontoblasts, cells that can produce a mineralized barrier at the
interface of soft and mineralized tissue.

28. Stem Cells
Thus, stem cells ful-fil an essential role during regeneration and
repair of the dentine pulp complex. See Figures 8.1 and 8.2.
Figure 8.1 Perivascular niche and multipotency of mesenchymal stem cells. (a) Mesenchymal stem
cells (MSC) reside in perivascular niches where they undergo self-renewal and maintain the
surrounding cells or tissue. Under specific signaling conditions, MSC can undergo differentiation
into different lineages. (b) Immunolocalization of the CD146 antigen, an endothelial surface marker,
to blood vessel walls in human dental pulp tissue. (c) Co-localization of blood vessels and
mesenchymal stem cells in dental pulp. Dual immunofluorescence staining showing reactivity of an
antibody to the mesenchymal stem cell marker STRO-1 labeled with Texas red to a blood vessel to
the endothelial marker CD 146 labeled with fluorescein isothiocyanate.
(Oh, M. and Nor, J.E. (2015) The perivascular niche and self-renewal of stem cells. Front Physiol 6, 367 &Shi, S. and Gronthos, S. (2003) Perivascular niche of
postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 18: 696-704)

29. Stem Cells
Figure 8.2 Stem cell types relevant for regenerative processes in
the pulp and periapex. BMSC: bone marrow stem cells; iPAPC:
inflamed periapical progenitor cells; DPSC: dental pulp stem
cells; SHED: stem cells from human exfoliated deciduous teeth;
SCAP: stem cells of the apical papilla.
(Hargreaves, K.M., Diogenes, A., and Teixeira, F.B. (2013) Treatment options:biological basis of regenerative endodontic procedures. Pediatr Dent
35: 129-140)

30. Stem Cells
• According to Diogenes et al., regenerative procedures are all stem cell
based therapies.
TYPES OF STEM CELLS:
● Dental pulp stem cells (DPSC)
● Stem Cells of the Apical Papilla (SCAP)
● Stem cells from Human Exfoliated Deciduous Teeth (SHED)
● Dental Follicle Stem Cells (DFSC)
● Bone marrow stem cells (BMSC)
● Periodontal ligament stem cells (PDLSCs)
● Inflamed periapical progenitor cells (iPAPCs)
● Tooth germ progenitor cells (TGPCs)
● Salivary gland stem cells (SGSCs)
● Gingival mesenchymal stem cells

31. REGENATIVE
ENDODONTICS
• The three key elements of tissue regeneration are:
 Stem cells
 Growth factors
 Scaffold

32. Regenerative endodontic procedures typically have the best
outcomes in kids. This is because their permanent teeth are
growing and maturing. Regenerative endodontic procedures take
advantage of this by further stimulating the regrowth of damaged or
infected pulp and tooth tissue.

33. After dental trauma to immature permanent teeth (IPT), there can be
pulpitis, necrotic, and periapical periodontitis, which will halt further root
development. Traditional endodontic root canal treatments and apexification
cannot revitalize the necrotic pulp to revitalize the tooth to promote further
root development. As a consequence, IPT with thin dentinal walls can be
prone to fracture and if a fracture occurs, the patient will likely suffer the
loss of the tooth. In an attempt to save IPT, there has been a growing
interest among dentists to use regenerative endodontic procedures (REPs)
to revitalize a replace dental pulp to continue root development and
strengthen the dentinal walls to help prevent a subsequent loss of the
tooth.

34. Histologic characterization of tissues formed
in the canal space

35. • Stem cells derived from the dental pulp.
• Dental pulp stem cells (DPSCs) are a promising source of cells
for numerous and varied regenerative medicine applications.
Their natural function in the production of odontoblasts to
create reparative dentin support applications in dentistry in the
regeneration of tooth structures.
• can form pulp like tissue , in future it is possible to replace
infected pulp tissue of a paining tooth with newly generated
pulp like tissue instead of doing RCT ,thus preserving the
vitality of the tooth
• It also has the ability to form bone that is useful
for the osseointegration of dental implants, thus
increasing its success rate .
 Dental Pulp Stem Cells

36. Cell homing: (a) infected root canal; (b) cleaned and prepared root canal; (c)
transplantation of scaffold with growth factors; (d) attraction of stem cells from
perivascular niche.

37. Dental caries and trauma, particularly in childhood, are among the most prevalent teeth
problems, which result in the creation of cavities and probably tooth loss. Thus, novel
regenerative approaches with high efficiency and less toxicity are required.
Stem cell therapy along with the implementation of scaffolds has provided excellent
opportunities in the regeneration of teeth structure. Hyaluronic acid (HA) hydrogels
have enticed great attention in the field of regenerative medicine. The unique chemical
and structural properties of HA and its derivatives have enabled their application in
tissue engineering. Several factors such as the location and type of the lesion, teeth
age, the type of capping materials determine the success rate of pulp therapy. HA
hydrogels have been considered as biocompatible and safe scaffold supports in human
dental cell therapies.

38.  Stem Cells of the Apical Papilla
• A unique population of dental stem cells known as stem cells
from the root apical papilla (SCAP) is located at the tips of
growing tooth roots . The apical papilla tissue is only present
during root development before the tooth erupts into the oral
cavity .

39. Stem Cells from Human Exfoliated Deciduous Teeth (SHED)
• Dental pulp of human exfolliated deciduous teeth contains
multipotent stem cells from Human Exfoliated Deciduous Teeth
(SHED). were identified to be a population of highly
proliferative, clonogenic cells capable of differentiating into a
variety of cell types including neural cells, adipocytes, and
odontoblasts. Thus, exfoliated teeth may be an unexpected
unique resource for stem-cell therapies including autologous
stem-cell transplantation and tissue engineering.
• have higher rate of proliferation.
• have potential to form bone which is useful during
osseointegration of dental implants
• have the potential to repair calvarial defects in
immuno-compromised mice .

40. Dental follicle stem cells (DFSC)
• The dental follicle is a loose ectomesenchyme-derived
connective tissue sac surrounding the enamel organ and the
dental papilla of the developing tooth germ before eruption .
• It is believed to contain progenitors for cementoblasts,PDL and
osteoblasts.
• Dental follicle cells (DFC)form the PDL by differentiating into
PDL fibroblasts that secrete collagen and interact with fibers on
the surfaces of adjacent bone and
cementum.
• Dental follicles contain progenitor cells which have the
capability of differentiating into cementum forming
cells (cementoblasts), osteoblasts of the alveolar bone,
and periodontal ligament fibroblasts.

41. Histologic characterization of tissues formed
in the canal space

42. DENTAL FOLLICLE DURING ODONTOGENESIS
During odontogenesis, the dental follicle emerges at the beginning of the cap stage. The follicle
comprises condensed ectomesenchyme surrounding the enamel organ and dental papilla
(Figure 1). It differs from the dental papilla in the presence and organization of fibrillar
components of the extracellular matrix and its function. The dental follicle is the source of
cementoblasts, osteoblasts, and fibroblasts, which form the periodontal tissue (ie, cementum,
alveolar bone, and periodontal ligament). Conversely, cells from the dental papilla are the source
of the dentin-pulp complex.8 Despite such well-defined features in the components that form the
dental apparatus, a study using murine models and tissue culture suggested the participation of
early formed dental papilla in the development of the dental follicle.
A study also showcased the maintenance of the potential of dental follicle cells to differentiate
into odontoblasts at the cap stage, revealing the power of inductive signals provided by the inner
enamel epithelia and its microenvironment.9

43. Bone marrow stem cells (BMSC)
• Bone marrow-derived cells (BMDCs) have the potential to
engraft into several tissues after injury, but whether they can
become dental tissue-specific progenitor cells under normal
conditions and the relationship of these cells to the tissue-
resident cells are unknown.
• Bone marrow progenitor cells communicate with dental
tissues and become tissue-specific mesenchymal progenitor
cells to maintain tissue homeostasis.

44. Periodontal Ligament Stem Cells (PDLSCs)
• have potentials of regenerating typical cementum and
periodontal ligament like structure .
• tissue of the periodontium made by stem cell can be
used as treatment modality to replace the treatment to
diseased periodontium around teeth so as treatment to
mobility of teeth.
Schematic representation of periodontal tissue engineering. An engineered matrix (left) with
necessary cells and instructive messages seeded in vitro, and then (right) transferred into a
periodontal defect to promote regeneration. Rapid formation of an epithelial seal should be
encouraged to minimize salivary and microbial contamination during wound healing

45. • According to Hargreaves,stem cells like SCAP, DPSCs, iPAPCs,
PDLSCs, BMSCs are most commonly used in regenerative
endodontics.
• These cells have te capacity of differentiating into odontoblast- like
cells facilitating a progressive repopulation of the radicular pulp
space , promoting organized tissue repair ,angiogenesis and
reinnervation.

46. Growth factors
• Biological factors regulate stem cells to form the desirable cell
type.
• They promote the differentiation of mesenchymal stem cells
into odontoblast-like cells.
Platelet derived growth factors
Bone morphogenetic proteins (BMPs)
Transforming growth factor b
Vascular endothelial growth factor
Fibroblast growth factors
Insulin like growth factor
Nerve growth factor
Stromal cell derived growth factor I

47. BONE MORPHOGENETIC PROTEINS (BMPs)
• Comprises a subgroup of the superfamily TGF(Transforming
growth factor b) and are involved in cell proliferation,
differentiation and apoptosis.
• Have strong osteoinductive and chondrogenic effects.
• BMP2,BMP4,BMP7 &BMP11 invoved in mineralization.

48. Scaffold
• A scaffold provides a physiochemical and biological 3-D micro
environment for cell adhesion ,growth differentiation and
migration. It contains the growth factors.
• Functions :
 Supports cell organization and vascularization.
 Aids cell proliferation and differentiation.
 Contains nutrients,antibiotics for improved and faster
tissue development.
• Classification :-
 Natural – collagen,platelet rich plasma ,fibrin,
glycosaminoglycans
 Synthetic – polylactic acid ,polyglycolic acid ,polylactic-
co-glycolic acid

49. REVASCULARIZATIO
N
 Vital pulp cells at the apical end of root canal proliferate into
newly formed matrix and odontoblasts, under the influence
of HERS. Atubular dentin is laid in the apical end and lateral
aspects of dentinal walls leading to apexogenesis and a thus
strengthening and reinforcement of root occurs.
• Multipotent dental pulp stem cells (immature permanent
teeth)from the apical end might be seeded on to the existing
dentinal walls and differentiate into odontoblasts and
deposit tertiary or atubular dentin.
• Revascularization, as defined by Andreasen, is a the
restoration of the vascularity to a tissue or organ.
• Mechanism of revascularization ,according to Shah N :


50. 
 Stem cells in the periodontal ligament can proliferate
and grow into the apical end and within the root canal
and hence deposit hard tissue at apex and lateral walls.-
cementum and Sharpey’s fibers in newly formed
tissues.
Extensive proliferating property of SCAP & bone
marrow- instrumentation beyond confines of root –
bleeding –mesenchymal stem cell transplantation from
bone.

51. Regeneration of Dentin -Pulp complex
• Three strategies to regenerate dentin-pulp complex have been
proposed:
a. Regeneration of entire tooth.
b. Local regeneration of dentin-pulp complex from
amputed dental pulp.
c. Regeneration of dental pulp from apical dental pulp or
peri- apical tissues.
• Growth factors like bone morphogenetic proteins (BMPs) and
fibroblast growth factors(FGFs), stem cells and scaffolds are
essential for tissue engineering to regenerate tissues.
• Stem cells differentiate into specific cells for tissue defects ,
growth factors like BMPs ,induce proliferation of stem cells.
• Scaffolds with extracellular matrix properties support for
cell proliferation ,differentiation and tissue formation.

52. a) 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 in which dental epithelial and mesenchymal cells from
isolated tooth germs cultured in scaffolds ( synthetic polymers
–polylactic-co-glycolic acid ; bio ceramics- hydroxyapatite ,
tricalcium phosphate, calcium carbonate hydroxyappatite)

53. b) Local regeneration of dentin-pulp
complex from amputed dental pulp
• Induction of appropriate pulp wound healing and formation of
new dentin in dentin defects are essential in regeneration of
dentin-pulp complex.
• It was reported that Bone Morphogenetic Protein (BMP), the
active ingredient in INFUSE Bone Graft — rhBMP-2 — is a
manufactured version of a protein already present in the
body that promotes new bone growth. BMP-2 with dentin
powder induced dentinogenesis in dentin cavity with pulp
exposure. Here stem or progenitor cells where 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

54. To conclude, in arrival of several new
and advanced technologies,
ultrasound-facilitated sonoporation
aids as a bonus in therapeutic
dentistry due to its non-invasiveness
and simplicity which has made it
superior to other methods. Literature
indicates that sonoporation makes it
possible to administer drugs into
cells more efficiently and specifically,
suggesting a novel application for
the treatment of oral SCC. It could be
considered as a forthcoming
modality in the therapeutic field of
medicine and dentistry

55. Usage of ultrasound as an imaging technique is still in current use.
However, in 1927, it came up as a therapeutic challenge when it was
documented that ultrasound (ULTS) could produce eternal variations
in biological systems.
Its principle is based on ultrasonic waves where these waves are
formed in the sonoporator that converts the electric energy into
mechanical or vibrational energy. The ultrasound radiation is
transferred from ultrasound machine to the microparticles
suspension and after transferring it efficiently produces cavitation
bubbles.[1] Henceforth, these microbubbles increase transport of
these huge molecules through creation of transient pores in the cell
membrane enabling transport of drugs into the cell.
SONOPORATION
In therapeutic field, absorption of ultrasonic energy leads to heating
of the tissue which can be used in many conditions. Sonoporation
permits the transport of therapeutic compounds noninvasively into
the specific target cells by utilizing ULTS and its contrast agents
(UCAs) which, thereby, improve the cell permeability.

56. Its advantages are it does enhanced drug penetration (of selected
drugs) over passive transport, allows strict control of transdermal
penetration rates, and permits rapid termination of drug delivery, skin
remains intact, therefore low risk of introducing infection, less painful
than injection and in many cases, greater patient satisfaction, not
immunologically sensitizing, and less risk of systemic absorption than
injection
It has certain limitations in that it is time consuming, causes minor
tingling and burning sensation and irritation of tissues at the site of
application.
Sonoporation plays various role in dentistry osteoinduction,
induction of dental pulp stem cell differentiation into odontoblasts,
site-specific gene delivery DNA transfer, local drug delivery, targeted
drug delivery, tumor cell killing, induction of apoptosis, gene
transduction, recurrent aphthous stomatitis, myofascial pain, TMJ
dysfunction, lithotripsy of salivary calculi, bone healing, and
osseointegration.

57. c) Regeneration of dental pulp from apical dental
pulp or peri- apical tissues
• Begins with identication of stem cells in the apical areas of
developing teeth in which root formation is incomplete.
• mesenchymal stem cells in apical papilla (SCAPs) differentiate
into odontoblast-like-cells that participate in pulp wound
healing and regeneration.
• Bone marrow derived mesenchymal stem cells (BMMSC) has
multipotent abilities and undergoes osteogenic
differentiation.
• Periapical tissues include bone marrow and PDL which is the
source BMMSCs. Localization of SCAPs & BMMSCs in the
apical region --- induction for dentin-pulp complex
regeneration

58. CLINICAL PROTOCOL REGENERATIVE ENDODONTICS
INDICATIONS
• Teeth with necrotic pulp and an immature apex.
• Pulp space not needed for post/core ,final restoration
• Patient compliance.
• No allergy to the medicaments to be used.
ROLE OF ANTIBIOTIC PASTE
• Antibiotic pastes are a combination of more than one antibiotic
mixed into a consistency of a paste.
• The triple antibiotic paste-commonly used-
ciprofloxacin,metronidazole, minocycline(1:1:1) in a
macrogol/propyleneglycol vehicle.
• Remains below CEJ and concentration is maintained to
0.1mg/ml and chamber is sealed with dentin bonding agents.

59. Placement of intracanal medicaments
Ca(OH)2 Low conc.triple antibiotic paste
Temp sealing with cavit/IRM/GIC
Recall patient after 1-4 weeks.
FIRST APPOINTMENT REGENERATIVE ENDODONTICS THERAPY
Local anesthesia, Isolation, access cavity preparation
Irrigation with 20ml of 1.5%NaOCl/5 min and saline rinse (20ml/
canal ,5min)
Drying with paper points

60. Assess response to initial treatment.
Signs of symptoms of
infections persists
No signs
/symptoms
Addtnl Rx time with
antimicrobial pastes Alternative antimicrobials to
be considered
SECOND APPOINTMENT –REGENERATIVE ENDODONTIC
THERAPY
Local anesthesia (3%mepivacine without vasoconstrictor)
Irrigation with 20ml of 17% EDTA,drying with paper points
Intracanal bleeding- k file 2mm past apical foramen Blood
in cavity till CEJ, 3-4 mm restorative material
White MTA(mineral trioxide aggregate)/ Ca(OH)2,capping –3-4mm
GIC
Resorbable matrix over clot

61. FOLLOW --UP
• Clinical and radiographic examination
• No pain ,swelling or sinum tract formation.
• Resolution of periradicular radiolucency(6-12months of Rx)
• Increase in width of canal walls (12—24months of Rx)
• Increased root length
• Pulp vitality tests.

62. ADVANTAGES AND
DISADVANTAGES
ADVANTAGES
• Achieve continued root development (root lengthening )and
strengthening due to enforcement of lateral dentin walls with
hard tissue deposition.
• Obturation of canal is not required.
• Splitting of root during lateral condensation avoided.
•After control of infection, completed in a single visit.
DISADVANTAGES
• Discoloration due to minocyclinein antibiotic paste.
• Prolonged treatment peroid compared with MTA apical barrier
technique.

63. POTENTIAL CAUSES OF FAILURE
• Poor root development.
• Insufficient bleeding during procedure.
• Pulp calcifications/obliterations.

64. Minimally invasive endodontics (MIE) is an endodontic technique
that aims to maintain as much of the healthy coronal, cervical, and
radicular tooth structure as possible. Access opening, root canal
cleaning and shaping, and surgical endodontics are all possible
applications for MIE in endodontic treatment.

69. CONCLUSION
• Regenerative endodontics holds promise of restoring pulp-
dentin complex in teeth with immature roots and necrotic
pulps.
• Procedure has advantages than traditional treatment of
increasing root wall thickness as well as root length while
maintaining immune competency.
• Significant scientific hurdles need to be overcome with
continued growth in knowledge and armamentarium.

70. REFERENCES
• Grossman’s endodontic practices-13th edition –Suresh
Chandra,V.Gopikrishna
• Regenerative Endodontics:regeneration or repair-
Stéphane R.J. Simon, DDS, PhD, Phillip L. Tomson PhD-
Journal of Endodontics.
• Regenerative Endodontics-Biological basis of
Regeneration of Dentin-Pulp Complex- Ariane Berdal,
PhD -Journal of Endodontics
• A review of regenerative Endodontics- Current
protocols and future direction- Louis M. Lin and Bill
Kahle- National Library of Medicine