Regenerative Endodontics

• Introduction
• Concepts
• Triad of Tissue Engineering
• Environmental Influences on Regeneration
• Regenerative Endodontic Procedures
• Regenerative Strategies
• Root Canal Revascularization
• Pulp Implantation
• Clinical Procedure for Regenerative Endodontic Procedures
• Translational Studies
• Conclusion
2

Tissue Engineering Restoring root canals + periodontium
Introduction
Healthy State
Hope for retaining a natural dentition
Chandra S. Grossman’s endodontic practice. Wolters kluwer india Pvt Ltd; 2014.
4

Definitions
• Regenerative endodontics are biologically based procedures designed to replace damaged structures,
including dentin and root structures, as well as cells of the pulp–dentin complex.
• Revascularization, as defined by Andreasen, is the restoration of the vascularity to a tissue or organ.
• Repair is the restoration of tissue continuity without the loss of original architecture and function.
• Revitalization is described as an ingrowth of vital tissue that does not resemble the original lost tissue.
5

Concepts
Interdisciplinary field
Engineering Life Sciences
Development of biological substitutes
Restore, maintain or improve tissue function or a whole organ
follows principles of
Tissue Engineering
Langer & Vacanti
6

Triad of Tissue Engineering
Stem Cells
Scaffolds
Signaling
Molecules
Optimum time and environment
Regenerated Tissue
7

Stem Cells & Progenitor Cells
Stem Cells Undifferentiated cells
Can differentiate into various
specialized cell types
Regenerative endodontic procedures are stem cell based therapies
(Diogenes et al)
Multipotent
Pluripotent
- Can differentiate into a
limited range of cell types
within a particular tissue
- Hematopoietic stem cells
- Can differentiate into any of the
3 germ layers
- Cannot form an entire organism
Totipotent
- Can differentiate into any cell type.
- Cells from early embryos (1-3 days)
- Eg: Zygote and the initial cells that
arise from its first few divisions
Hargreaves KM, Diogenes A, Teixeira FB. Treatment options: biological basis of regenerative endodontic procedures. Pediatric dentistry. 2013 Apr 15;35(2):129-40.
8

Stem Cells / Progenitor Cells
Allogenic Cells
Autologous Cells Xenogeneic Cells Isogenic Cells
Obtained from same person Obtained from same species
Obtained from different species
Animal cells useful in
cardiovascular
implants
Obtained from genetically
identical organisms
Garg N, Garg A. Textbook of endodontics. Boydell & Brewer Ltd; 2010 Nov 26.
9

Stem Cell Niches
Embryonic Development
Migration of cranial neural
crest cells to mesenchyme
of first brachial arch
Ecto-mesenchymal cells
(dental papilla & dental follicle)
Mature tooth contain various
undifferentiated cells in pulp
These cells contain mesenchymal-
stem-cell like properties (MSC)
Lyu P, Li B, Li P, Bi R, Cui C, Zhao Z, Zhou X, Fan Y. Parathyroid hormone 1 receptor signaling in dental mesenchymal stem cells: basic and clinical implications. Frontiers in Cell and Developmental Biology. 2021 Oct 25;9:654715.
Dental Pulp Stem Cells were the
first population of stem cells to
be described
10

Stem Cell Niches
1. Stem cells of the apical papilla (SCAP)
2. Dental pulp stem cells (DPSCs)
3. Inflamed periapical progenitor cells (iPAPCs)
4. Periodontal ligament stem cells (PDLSCs)
5. Bone marrow stem cells (BMSCs)
According to Hargreaves
Microenvironment for stem cells
Hargreaves KM, Diogenes A, Teixeira FB. Treatment options: biological basis of regenerative endodontic procedures. Pediatric dentistry. 2013 Apr 15;35(2):129-40.
Maintains stem cells in quiescent stage 11

Growth Factors
Biological Factors
Stimulates differentiation of
mesenchymal stem cells
Odontoblast like cells
Functions:
To stimulate division of neighboring cells
To stimulate the differentiation of certain cells along a specified pathway
To stimulate Angiogenesis
To serve as chemoattractant for specific cell types
Also called – Signaling Molecules / Morphogens
PGDF
BMP
12

Growth Factors
Recruitment of stem cells Cellular Proliferation Angiogenesis
Matrix Deposition & Tissue Remodeling
Cellular Differentiation
- Chemotactic agents
- Attraction to injury site
- IGF / PDGF
- Signals stem cell proliferation
- VEGF
- Stimulates formation of
blood vessels
- BMP / TGF – β
- Signals stem cells to form into odontoblasts
- Dentin deposition starts
- MMP’s facilitate remodelling of new matrix
13

Growth Factors
Also called – Signaling Molecules / Morphogens
BMPs
• Fibroblast growth factors (FGFs)
• Tumour necrotic factor (TNF)
• Transforming growth factor (TGF)
• Insulin-like growth factor (IGF)
• Epidermal growth factor (EGF)
• Interleukins (IL)
• PDGF
• Nerve growth factor (NGF)
Embryonic Tooth Development
14

Scaffold
3D structure that contains growth factors. It has the following functions:
• Supports cell organization and vascularization
• Aids stem cell proliferation and differentiation
• Leads to improved and faster tissue development
• Contains nutrients to promote cell survival and growth
• May contain antibiotics to prevent bacterial ingrowth in the canal systems
• Mechanical and biological functions
15

Scaffold
CLASSIFICATION
Natural Synthetic
Collagen
Platelet Rich Fibrin
Glycosaminoglycan
Polylactic Acid (PLA)
Polyglycolic Acid (PGA)
Polyethylene Glycol (PEG)
Polycaprolactone (PCL)
16

Scaffold
17
https://www.mdpi.com/2073-4360/15/5/1082#

“Choukrouns PRF”
2001 – Dr. Joseph Choukroun et al introduced the concept of PRF in dentistry as a scaffold material
A second generation autologous platelet concentrate
suspended in a fibrin mesh
Prepared by centrifuging patients own blood for a given time
Acts as an excellent Scaffold in Regenerative Endodontic Procedures
Gupta V, Bains VK, Singh GP, Mathur A, Bains R. Regenerative potential of platelet rich fibrin in dentistry: Literature review. Asian J Oral Health Allied Sci. 2011 Jan;1:23-8. 18

https://youtu.be/4z7uj4I5kCg?s
i=_OhtQMVUUr2ABBwi
20

Second Generation: L-PRF (Leukocyte- and Platelet-Rich Fibrin)
Preparation: Centrifuged at a slow speed (around 2700 rpm for 12
minutes).
Characteristics: Contains leukocytes, which may influence
inflammation and immunity.
Appearance: Yellow, dense fibrin clot with the red blood cells
concentrated at the base.

A-PRF (Advanced Platelet Rich Fibrin)
Preparation: Reduced centrifugation speeds (around 1500 rpm for
14 minutes)
Characteristics: Improved fibrin architecture, increased leukocyte
and growth factor content
Appearance: More porous, superior for cellular migration

i-PRF (Injectable Platelet Rich Fibrin)
Preparation: 1300 RPM for 8 minutes
Characteristics: Fluid form of PRF; can be combined with other
biomaterials
Usage: Suitable for hard-to-reach areas or as an adjunct with bone
graft materials

S-PRF (Solid Platelet Rich Fibrin)
Preparation: general protocol leans towards that of L-PRF or A-PRF
Characteristics: Solid form, but with modifications to optimize the
regenerative potential.
Usage: Ideal for surgical sites requiring stabilization or protection.

Miron and colleagues
Horizontal centrifugation
better than fixed angle
More concentration of platelets,
leukocytes, and monocytes
Fixed angle centrifugation causes trapping
of essential elements along with the sunk
RBC’s in the back of the test tube
https://bioprf.eu/bio-prf-science/ 26

https://bioprf.eu/bio-prf-science/ 27

Choukrouns Box / PRF Box (Process, Nice, France)
Helps in draining excess exudate from the PRF clot
Makes the PRF clot more manageable
Compression plate helps in draining the PRF clot
Production of an economical autologous fibrin
membrane in approximately one minute
Average Cost: 4000 - 5000
https://www.gdcdental.com/products-detail.php?id=1079 28

Environmental Influences on Regnerative events
Rotstein I, Ingle JI, editors. Ingle's endodontics. PMPH USA; 2019 Jun 1.
Microbial penetration & defense responses:
Micro-organisms and their by-products can penetrate dentin matrix and induce cellular responses in pulp
Overtime the microbial load increases, leading to these micro-organisms being found in the pulp
Role of Odontoblasts:
Odontoblasts play a key role in environmental sensing & defense
Odontoblasts have matrix secretory functions
Their lateral processes makes up for an extensive network for facilitating communication within the dentin matrix
29

Environmental Influences on Regnerative events
Microbial Sensing:
Odontoblasts release antimicrobial peptides and various cytokines
Micro-organisms are detected by Pattern Recognition Receptors (PRR) molecules, specifically Toll-Like-Receptors
Intracellular Signalling:
Microbial sensing activates intracellular signaling pathways like NF-kB, p-38 MAP Kinase, and JNK
This results in release of inflammatory mediators, amplifying defense responses and recruiting immune cells
Rotstein I, Ingle JI, editors. Ingle's endodontics. PMPH USA; 2019 Jun 1. 30

Regenerative Endodontic Procedures
Regenerative endodontics is defined as biologically based procedures designed to physiologically replace
damaged tooth structures, including dentin and root structures, as well as pulp dentin complex.
GOALS OF REGENERATIVE PROCEDURES
32

Regenerative Strategies
Regenerative strategies in endodontics are dependent on stem cells, cellular scaffolds, and
signalling molecules
Cell Based Approach Cell Free Approach
• Avoiding the need to directly transplant cells to
injury site
• Focuses on providing a conductive environment
for regeneration
Bioactive Molecules Chemical treatment of tissues
Stem cell transfer from apical tissues
• Transplantation of cells to the site of injury in
the tissue to promote regeneration
• Offers long term success rate
• Requires the use of cells of the highest good
manufacturing practice (GMP) quality
Rotstein I, Ingle JI, editors. Ingle's endodontics. PMPH USA; 2019 Jun 1. 33

Root Canal Revascularization
Revascularization is the procedure to re-establish vitality in a non-vital tooth to allow repair and regeneration of
tissue. It allows further development of root and dentin structure with a better long-term prognosis.
Necrotic teeth with
open apices
Some vital pulp / Hertwig’s Epithelial Root Sheath
Pulp proliferation once
disinfected
Apexification Calcium Hydroxide No continued root formation
No thickening of root canal walls
Risk of root fractures
Garg N, Garg A. Textbook of endodontics. Boydell & Brewer Ltd; 2010 Nov 26. 34

Teeth presenting symptoms of
chronic apical periodontitis
Immature Apex / Blunderbuss canal Wide canal / Parallel walls
Non – Vital on pulp sensibility tests
Garg N, Garg A. Textbook of endodontics. Boydell & Brewer Ltd; 2010 Nov 26. 35

Metronidazole + Ciprofloxacin in Distilled Water
2mm
from
W.L.
Closed
Dressing
Recall in 3-4 weeks
5.25%
36
P/L ratio – 2.5:1
Madhukumar M, Geetha P, Nair KR, Unnikrishnan M. The effects of double antibiotic paste and amoxicillin-clavulanate paste used in endodontic regeneration on microhardness of radicular dentine: an In vitro study. Journal of
Pharmacy & Bioallied Sciences. 2021 Jun;13(Suppl 1):S510.
Conc: 0.1mg/ml

5.25%
Allow blood to reach
3mm below CEJ
Leave for 15 mins
37

Scaffold Regeneration & Repair
Place MTA over the clot and temporarily restore the tooth
After 1 week – Restore tooth with composite
Patients recalled after a minimum of 6 months -1 year
38

Lack of symptoms
Radiographic evidence of
increased root length
Radiographic evidence of increased
root canal thickness
39

Mechanism of Revascularization
Few cells remain at the apical end of root canal
Differentiate into odontoblasts under the organizing influence of Hertwig’s Epithelial Root Sheath
Newly formed odontoblasts lay down Atubular Dentin
Apexogenesis is favored
Dentin deposition at the lateral aspects of the root canal wall
40

DPSC’s present at apical end, aid in Apexogenesis
Might form Tertiary dentin at the lateral aspects of the dentinal wall
Stem cells in the PDL also lay down hard tissue at apical end & lateral walls
Root development attributed to the stem cells of apical papilla
Inducing bleeding causes mesenchymal cells to enter canal and cause regeneration
41

Platelet derived growth factor
Vascular Factor
Tissue Growth Factor
42

Advantages of Revascularization
Short treatment time – 1-2 visits In-expensive Strengthening of lateral root walls
Autologous grafts crosses out immune rejection
Obturation of root canal not necessary – less chances of root fracture
43

Disadvantages of Revascularization
Composition of cells in fibrin clot unreliable - can result in variations in treatment
Crown Discolouration
Allergic reactions to intra-canal-medications
Development of resistant bacterial strains
Entire canal might get calcified resulting in compromised treatment
44

Stem Cells of Dental Origin
Dental Pulp Stem Cells
Periodontal Ligament Stem Cells
Dental Follicle Precursor Cells
Stem Cells from Apical Papilla
46

Dental Pulp Stem Cells
Origin and Potential : Dental Pulp / Wisdom teeth or
shed primary teeth
Mineralized matrix with tubules lined with
odontoblasts and fibrous tissue containing blood
vessels
Multipotent
Capable of inducing Dentinogenesis
Regenerates other tissues like pulp / periodontal ligament 47

Periodontal Ligament Stem Cells
Harvested from enzymatically digested PDL
Can regenerate cementum lined with collagen fibers
similar to Sharpey’s fibers
isolated PDLSCs Expanded
Cultured
Differentiation into
different cell types
48

Stem Cells from Apical Papilla
Origin : Apical papilla in the apices of developing teeth
Multipotent
Apical cell rich zone – between – Apical papilla & Pulp
Can undergo odontoblastic, osteogenic, or
neurogenic differentiation
49

Pulp Implantation
Abbass MM, El-Rashidy AA, Sadek KM, Moshy SE, Radwan IA, Rady D, Dörfer CE, Fawzy El-Sayed KM. Hydrogels and dentin–pulp complex regeneration: from the benchtop to clinical translation. Polymers. 2020 Dec 9;12(12):2935.
Replacement Pulp Therapy
Hydrogel acts as Scaffold for DPSC’s
DPSC’s can be loaded onto the polymeric sheets and placed into canals
DMP-1 expression observed after 28 days
50

Case Report
7 year old – referred to private practice for evaluation of maxillary right central incisor
Avulsion injury 3 days prior to visit
Previous dentist irrigated tooth with CHX and reimplanted it
Dentist stabilized tooth with stainless steel orthodontic wire and referred the
patient
Total Extra Oral Dry Time : 60 minutes
Tenderness to percussion: Positive
EPT/Cold Test: No Response
Hung R, Torabinejad M. Regenerative Endodontic Procedure (REP) in single session with MTAD disinfection on immature avulsion tooth: 4-year control. J Dent Res.;2:1-4.
51

Case Report
Pulpal Diagnosis: Necrotic Pulp
Periapical Diagnosis: Symptomatic Apical Periodontitis
Local Anaesthesia: Lidocaine 2% 1:100,000 epinephrine
Isolation: Rubber Dam
Working Length Determined using Apex Locator and
Radiographic Method
Light Instrumentation + irrigation with 10ml 5.25% NaOCl
5.25%
52

Case Report
1ml of MTAD was placed for 5 mins
Then rinsed with 4ml MTAD and dried
Local Anaesthesia: Lidocaine 2% 1:100,000 epinephrine
Bleeding was induced: pre bent size 25 K-file
– 2mm beyond working length
3-4mm of MTA gently condensed over the clot
Tooth restored with composite
53

Case Report
Patient reported to clinic after 6 months
Radiographically: root apex started closing
Patient reported no pain / no symptoms
After 1 year 6 months Apex completely closed
After 2 years 6 months
After 4 years
Asymptomatic / positive to EPT & Cold test
54

12 year old female
Tooth involved : maxillary right central incisor
History of trauma : lateral luxation injury 2 year ago
Pulpal Diagnosis : Pulpal Necrosis
Radiographic findings : Periapical Abscess w.r.t. 11, thinning of
lateral walls of root canal
Apical Diagnosis : Acute Apical Abscess
Clinical Procedure for REP’s
55

Access opening and working length determination is done
Local Anaesthesia - 2% Lidocaine with 1:100,000 epinephrine
Isolation done with Rubber Dam
56

Slow Irrigation done with 1.5% NaOCl (20ml/5 minutes)
Canals dried with paper points
Canals then rinsed with 17% EDTA (10ml/2 minutes)
Double Antibiotic paste or Calcium Hydroxide placed in the canals (1mg/1ml)
Canals dried with paper points
Access sealed with GIC
Patient recalled after 4 weeks
57

Patient was asymptomatic, no swelling, no sinus tract
Temporary restoration was removed
Calcium hydroxide was removed with saline irrigation
A size 25, precurved K-file was over-instrumented 2mm into the
periapical area to induce bleeding
Final rinse done with 17% EDTA 20ml for 5 minutes
Canals are dried with paper points
Local anaesthesia with 3% Mepivacaine
Isolation done
58

Collaplug™ (Zimmer Dental Inc, Warsaw, Indiana, U.S.A) placed
on top of the blood clot
Cavity sealed with packable composite
Patient revied at 3 months / 6 months / 1 year / 2 years,
then annually for 5 years
Serves as an internal matrix for a 3mm white MTA or Biodentine
Light cured GIC flowed over the MTA/Biodentine
59

Case Report
Dr. M. Torabinejad
60

Regeneration of Mature Permanent Teeth: Scope?
Regenerative endodontic procedures in
mature human teeth resulted in the formation
of fibrous connective tissue containing
bonelike tissue, vascular structures, and
inflammation, which is similar to the findings
in immature teeth.
Regenerative endodontic procedures have
the potential to be used as a treatment
option for mature teeth with large
periapical radiolucencies.
Arslan H, Şahin Y, Topçuoğlu HS, Gündoğdu B. Histologic evaluation of regenerated tissues in the pulp spaces of teeth with mature roots at the time of the regenerative endodontic procedures. Journal of Endodontics. 2019 Nov 1;45(11):1384-9.
Arslan H, Ahmed HM, Şahin Y, Yıldız ED, Gündoğdu EC, Güven Y, Khalilov R. Regenerative endodontic procedures in necrotic mature teeth with periapical radiolucencies: a preliminary randomized clinical study. Journal of endodontics. 2019 Jul 1;45(7):863-72.
61

Regeneration of Mature Permanent Teeth: Scope?
This systematic review found that REPs effectively treat both mature and immature necrotic permanent
teeth. Mature teeth showed better vitality responses than immature teeth with necrotic pulps.
Murad M, Cooper PR. Regenerative endodontic procedures: are they more successful in mature permanent teeth with necrotic pulp than in immature teeth?. Evidence-Based Dentistry. 2023 Aug 4:1-2.
62

Advanced Scaffolds
Collagen/Alginate Composite Scaffold developed
Mimics the shape of gutta percha with great accuracy and reproducibility
Stem cells seeded onto this scaffold differentiated into osteoblasts producing osseous extracellular matrix
Devillard, R., Rémy, M., Kalisky, J., Bourget, J., Kérourédan, O., Siadous, R., Bareille, R., Amédée-Vilamitjana, J., Chassande, O., & Fricain, J. (2017). In vitro assessment of a collagen/alginate composite scaffold for regenerative endodontics. International Endodontic Journal, 50, 48–57.
63

Conclusion
Endodontic regeneration represents a groundbreaking shift in the way we approach dental
pulp diseases. Harnessing the body's innate ability to heal and regenerate, this method not
only offers a biological solution to dental pathologies but also promises improved patient
outcomes and long-term tooth viability. As technology and research continue to advance, the
potential for endodontic regeneration will undoubtedly expand, further bridging the gap
between traditional endodontics and the broader field of regenerative medicine.
64

References
Hargreaves KM, Diogenes A, Teixeira FB. Treatment options: biological basis of regenerative endodontic procedures. Pediatric dentistry. 2013 Apr
15;35(2):129-40.
https://bioprf.eu/bio-prf-science/
Devillard, R., Rémy, M., Kalisky, J., Bourget, J., Kérourédan, O., Siadous, R., Bareille, R., Amédée-Vilamitjana, J., Chassande, O., & Fricain, J. (2017). In vitro
assessment of a collagen/alginate composite scaffold for regenerative endodontics. International Endodontic Journal, 50, 48–57.
Arslan H, Şahin Y, Topçuoğlu HS, Gündoğdu B. Histologic evaluation of regenerated tissues in the pulp spaces of teeth with mature roots at the time of the
regenerative endodontic procedures. Journal of Endodontics. 2019 Nov 1;45(11):1384-9.
Arslan H, Ahmed HM, Şahin Y, Yıldız ED, Gündoğdu EC, Güven Y, Khalilov R. Regenerative endodontic procedures in necrotic mature teeth with periapical
radiolucencies: a preliminary randomized clinical study. Journal of endodontics. 2019 Jul 1;45(7):863-72.
https://www.gdcdental.com/products-detail.php?id=1079
Abbass MMS, El-Rashidy AA, Sadek KM, Moshy SE, Radwan IA, Rady D, Dörfer CE, Fawzy El-Sayed KM. Hydrogels and Dentin–Pulp Complex Regeneration: From the
Benchtop to Clinical Translation. Polymers. 2020; 12(12):2935. https://doi.org/10.3390/polym12122935
65

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