Regenerative endodontics_ ENDODONTICS REVIEW

Regenerative endodontics

1. REGENERATIVE ENDODONTICS Dr.Pratik More JR-3

2. CONTENTS 2 Definition Introduction History Objectives Stem cells Growth factors Potential technologies for regenerative endodontics Clinical technique used for Regenerative Endodontic Therapy. Conclusion References

3. INTRODUCTION 3 Millions of teeth are saved each year by root canal therapy, an ideal form of therapy might consist of regenerative therapy. It presents a new era in biological and clinical endodontics RET is based on the concept of tissue engineering technology to regenerate the dentine–pulp complex in the canal space of immature permanent teeth damaged by caries or trauma, thus restoring development of the arrested tooth root.

4. 4 Regenerative endodontics was pioneered by The term ‘regenerative endodontics’ was adopted by ( Murrayet al.2007),based on a tissue engineering concept. Nygaard- Ostby (1961) and Nygarrd-Ostby & Hjortdal (1971) the American Association of Endodontists in 2007 Regenerative endodontics is defined as ‘biologically based procedures designed to replace damaged toothstructures , including dentine and root structures, as well as cells of the pulp–dentine complex’ (COHEN 20 th ed). Kim SG, Malek M, Sigurdsson A, Lin LM, Kahler B. Regenerative endodontics: a comprehensive review. International Endodontic Journal. 2018 Jun 11;51(12):1367–88. HISTORY

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6. GOALS of Regenerative endodontics 6 03 6

7. 7 Regenerative Endodontics: Regenerative endodontic procedures can be defined as biologically based procedures designed to replace damaged structures, including dentin and root structures, as well as cells of the pulp-dentin complex. (Murray et al, 2007) Tissue Engineering: “ An interdisciplinary field that applies the principles of engineering and the life science towards the development of biological substitutes that restore, maintain or improve tissue function. (Langer & Vacanti, 1993)

8. COMPONENTS OF REGENERATIVE ENDODONTICS 8

9. 9 Scaffolds provide a mechanism to deliver the growth factor to the appropriate site, and/or a surface to support the growth of the cells. Stem cells are progenitors for the tissue to be grown. And are capable of both self-renewal and differentiation. Growth factor stimulates the proliferation and/or differentiation of resident stem cells to regenerate the damaged tissue.

10. classification 10

11. SOURCES OF STEM CELL There are four primary sources for embryonic stem cells: Existing stem cell lines Aborted or miscarried embryos Unused In vitro fertilized embryos Cloned embryos Postnatal stem cells have been sourced from- Umbilical cord blood Umbilical cord Bone marrow Peripheral blood Body fat Almost all body tissues including the pulp tissue of teeth. 11

12. TYPES OF STEM CELLS Stem cells of the apical papilla (SCAP) Dental pulp stem cells (DPSCs) Stem cells from human exfoliated deciduous teeth (SHED) Periodontal ligament stem cells (PDLSCs) 5) Bone marrow stem cells (BMSCs) 6) Inflammed periapical progenitor cells ( iPAPCs ) 7) Tooth germ progenitor cells (TGPCs) 8) Dental follicle stem cells (DFSCs) 9) Salivary gland stem cells (SGSCs) 12

13. HUMAN DENTAL STEM CELLS 4 types of human dental stem cells have been isolated and characterized Dental pulp stem cells ( DPSC’s) Stem cells from exfoliated deciduous teeth (SHED’s) Stem cells from apical papilla (SCAP’s) Periodontal ligament stem cells (PDLSC’s) DPSCs & SHEDs are from the pulp, SCAPs from the pulp precursor tissue – the apical papilla & PDLSCs from PDL. 13

14. STEM CELLS FROM HUMAN EXFOLIATED DECIDUOUS TEETH (SHED) Isolated for the first time in 2003 by miura et al. Differentiate into a variety of cell types including neural cells, adipocytes, osteoblast-like and odontoblast-like cells Resulting tissue presented architecture and cellularity that closely resemble those of a physiologic dental pulp- Cordeiro et al (2008) 14

15. DENTAL PULP STEM CELLS(DPSC’s) 1 st identified by Gronthos in 2000 isolated from the human third molars ,exfoliated deciduous teeth, supernumerary teeth, crown fractured teeth that did not require extraction and permanent tooth germs Huang et al (2006) conducted a study to characterize human adult dental pulp cells isolated and cultured in vitro and to examine the cell differentiation potential grown on dentin. It was concluded that isolated human pulp stem cells may differentiate into odontoblasts or dentin in vitro. 15

16. Properties of human dental pulp stem cells: Self-renewal capability, Multilineage differentiation capacity, Clonogenic efficiency of human dental pulp stem cells (DPSCs) DPSCs were capable of forming ectopic dentin and associated pulp tissue in vivo. 16 The stem cell population in the pulp is very small; approximately 1% of the total cells (Smith et al.2005) and the effect of aging reduce the cell pool available to participate in regeneration which reflects the better healing outcomes seen in younger patients .

17.   A new unique population of mesenchymal stem cells (MSCs) residing in the apical papilla of permanent immature teeth, known as stem cells from the apical papilla (SCAP) discovered by Sonoyama et al (2008) apical papilla is distinctive to the pulp in terms of containing less cellular and vascular components than those in the pulp. Cells in the apical papilla proliferated 2- to 3-fold greater than those in the pulp in organ cultures 18 STEM CELLS OF APICAL PAPILLA (SCAP)

18. Stem cells in the apical papilla may also explain a clinical phenomenon described in a number of recent clinical case reports showing that apexogenesis can occur in infected immature permanent teeth with periradicular periodontitis or abscess. It is likely that the SCAP residing in the apical papilla survive such pulp necrosis because of their proximity to the vasculature of the periapical tissues. Therefore, after endodontic disinfection, and under the influence of the surviving epithelial root sheath of hertwig , these cells can generate primary odontoblasts that complete root formation. 19

19. Role of SCAP Helps in continued root formation In pulp healing and regeneration In replantation and transplantation 20

20. GROWTH FACTORS/MORPHOGENS Growth factors/Morphogens are extracellularly secreted signals governing morphogenesis during epithelial-mesenchymal interactions. They are proteins that bind to receptors on the cell and induce cellular proliferation and/or differentiation. Bone morphogenetic protein(BMP), Colony stimulating factor(CSF), Epidermal growth factors(EGF), Fibroblast growth factors, Insulin like growth factors I or II(IGF), Interleukins IL-1 to IL-13, Platelet derived growth factor , Transforming growth factor – alpha,beta , Nerve growth factor 21

21. SCAFFOLDS The role of the scaffold in tissue engineering is to provide a matrix of a specific geometric configuration on which seeded cells may grow to produce the desired tissue or organ.. Provides a biocompatible 3-D structure for cell adhesion & migration These biomaterials can be produced in solid blocks, sheets, porous sponges or foams, or hydrogels 22

22. Requirements of a scaffold Easy cell penetration, distribution, and proliferation; Permeability of the culture medium; In vivo vascularization (once implanted); Conductive for odontoblast-like cells; Adequate mechanical stiffness; Ease of fabrication (including 3-D printing); Ease of handling; Adequate porosity; Biocompatibility; Proper biodegradation (rate and inflammatory response). 23

23. Types of scaffolds 1. Natural scaffolds. 2. Mineral scaffolds 3. Synthetic scaffolds

24. Natural scaffolds: The examples for natural scaffolds are collage, hyaluronic acid, chitosan and chitin . These natural scaffolds have been used in several craniofacial and dental applications. These lack the desired structural rigidity for use in the load bearing region. 2. Mineral scaffolds: These are composed of calcium phosphates in the form of hydroxyapatite or tricalcium phosphate. These scaffolds are brittle and hence, are prone to fracture.

25. 3. Synthetic scaffolds: The most widely used synthetic materials are polymers of polyglycolic acid, polylactic acid and polydioxanone . These scaffolds lack critical cell signaling capabilities and can interfere with new tissue growth.

26. To create a more practical endodontic tissue engineering therapy, pulp stem cells must be organized into a three-dimensional structure that can support cell organization and vascularization . A scaffold should contain growth factors to aid stem cell proliferation and differentiation, leading to improved and faster tissue development. The scaffold may also contain nutrients promoting cell survival and growth and possibly antibiotics to prevent any bacterial in-growth in the canal systems.

27. INJECTABLE SCAFFOLD DELIVERY: Rigid tissue engineered scaffold structures provide excellent support for cells used in bone and other body areas where the engineered tissue is required to provide physical support. However, in root canal systems a tissue engineered pulp is not required to provide structural support of the tooth. This will allow tissue engineered pulp tissue to be administered in a soft three dimensional scaffold matrix , such as a polymer hydrogel.

28. Hydrogels are injectable scaffolds that can be delivered by syringe. Hydrogels have the potential to be non-invasive and easy to deliver into root canal systems. Hydrogel may promote pulp regeneration by providing a substrate for cell proliferation and differentiation into an organized tissue structure. Research is focusing on making them photopolymerizable to form rigid structures once they are implanted into the tissue site.

29. PRF and PRP in regeneration

30. ADVANTAGES OF PRP Platelet concentration 3-5 times more than normal human plasma . PRP can be used for soft and hard tissue augmentation . Rapid wound healing and homeostasis . Promotes cell proliferation and differentiation during osteogenesis . Control local inflammatory response . Stimulate matrix remodeling and angiogenesis . Growth factors are released within 7-14 hrs and rapid formation of vital tissue .

31. DEMERITS OF PRP Concern over the use of bovine thrombin, the fact that bovine thrombin has been associated with development of antibodies to clotting factors V, XI and thrombin, which had occasionally lead to life threatening coagulopathies. Blood clot faster i .e . within 5min. Lack of uniformity in PRP preparation protocol as different platelet concentration has different storage time .

32. PLATELET RICH FIBRIN PRF was used for the first time by Choukroun et al in oral and maxillofacial surgery. This technique did not require to anticoagulants, thrombin and any other jellying factor and it was not anything except a centrifuged blood product without any other additives that was not included the laws about the prohibition of blood born products .

33. PROCEDURE FOR FORMATION OF PRF

34. PRF has been shown to have several advantages over traditionally prepared platelet- rich plasma. Its chief advantages include ease of preparation and lack of biochemical handling of blood, which makes this preparation strictly autologous. Another advantage of using PRF as a scaffold is that it has a trimolecular or equilateral fibrin branch junction which makes its architecture flexible and can support cytokine enmeshment and cellular migration

35. REGENERATIVE ENDODONTICS PROCEDURES 38

36. ROOT CANAL REVASCULARIZATION 39

37. Revascularization is the procedure to reestablish the vitality in a nonvital tooth to allow repair and regeneration of tissues. the goal from an endodontic perspective is to regenerate a pulp-dentin complex that 40

38. Revascularization Protocol Revascularization should be considered for incompletely developed permanent tooth that has an open apex. Kling M et al (1986) reported that the incidence of revascularization was enhanced by 18%, if the apex showed radiographic opening of more than 1.1 mm. Duration of the infection- the longer standing of an infected pulp in immature teeth there is the less survived pulp tissue and stem cells may remain. If no root development can be seen within three months, the more traditional apexification procedures can then be started. 41

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41. Clinical protocol of first appointment 44

42. Second appointment 45

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44. 47 The blood clot acts as a scaffold and source of growth factors to facilitate the regeneration and repair of tissues into the canal. Induction of bleeding to facilitate healing is a common surgical procedure Currently, there is lack of histological evidence showing that blood clot is required for the formation of repaired tissues in the canal space, nor are there systematic clinical studies to show that this approach is significantly better than without it. However, these cases reports at least provide some guidelines as to what extent the healing potential these immature teeth are capable of.

45. 48 Lin, J., Zeng, Q., Wei, X., Zhao, W., Cui, M., Gu , J., … Ling, J. (2017Journal of Endodontics, 43(11), 1821–1827.

46. 49 Advantages

47. 50 Limitations

48. POST NATAL STEM CELL THERAPY The simplest method to administer cells of appropriate regenerative potential is 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. 52

49. A major research obstacle is identification of a postnatal stem cell source capable of differentiating into the diverse cell population found in adult pulp (e.g., fibroblasts, endothelial cells, odontoblasts ). Technical obstacles include the development of methods for harvesting and any necessary ex vivo methods required to purify and/or expand cell numbers sufficiently for regenerative endodontic applications.

50. Advantages Autogenous stem cells are relatively easy to harvest and to deliver by syringe, and the cells have the potential to induce new pulp regeneration. This approach is already used in regenerative medical applications, including bone marrow replacement.

51. Disadvantages The cells may have low survival rates. The cells might migrate to different locations within the body, possibly leading to aberrant patterns of mineralization.

52. Three-dimensional cell printing

53. Creating replacement pulp tissue using a three-dimensional cell printing technique In theory, An ink-jet-like device is used to dispense layers of cells suspended in a hydrogel to recreate the structure of the tooth pulp tissue. (NE Sanjana 2004 et al) The three-dimensional cell printing technique can be used to precisely position cells (J A Barron et al 2005 ), Potential to create tissue constructs that mimic the natural tooth pulp tissue structure The ideal positioning of cells in a tissue engineering construct would include placing odontoblastoid cells around the periphery to maintain and repair dentin, with fibroblasts in the pulp core supporting a network of vascular and nerve cells. Ji S and Guvendiren M (2017) Recent Advances in Bioink Design for 3D Bioprinting of Tissues and Organs. Front. Bioeng. Biotechnol. 5:23.

54. Experimental Approaches The principle of 3D printing can be applied to deliver stem cells, pulp scaffolds, injectable calcium phosphates, growth factors, and for gene therapy in the endodontics (Murray et al., 2007). Various types of calcium phosphate cements have been developed by 3D printing to form porous scaffolds for regeneration of the pulp-dentin complex (Xu et al., 2017). Research has shown that application of 3Dprinted polycaprolactone coated with freeze-dried platelet-rich plasma to the dental pulp cells has an improved osteogenic activity in vitro (Li J. et al., 2017). Anatomically shaped tooth-like tissue has been generated using 3D printed polyepsilon -caprolactone and hydroxyapatite scaffolds (Kim et al., 2010). Also, bioprinting approaches were developed using dentin-derived bionics. For scaffold-free approaches dental pulp cell-derived spheroids have shown promising results for regenerative strategies (Xiao and Tsutsui, 2013; Dissanayaka et al., 2014, 2015; Neunzehn et al., 2014; Janjic et al., ´ 2018).

55. Techniques involving viral or nonviral vectors to deliver genes for growth factors, morphogens, transcription factors, and extracellular matrix molecules into target cell populations, such as the salivary gland (J.Li et al 2004) One use of gene delivery in endodontics would be 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 in 2001. He transfected ferret 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. Shilpashree HS, Sarapur S. Gene therapy in dentistry: a review. The New York state dental journal. 2013;79(5):60-4. Gene therapy essentially consists of introducing specific genetic material into target cells to compensate for abnormal genes or to make a beneficial protein without producing toxic effects on surrounding tissue. GENE THERAPY

56. CHALLENGES IN REGENERATIVE ENDODONTICS

57. Conclusion Stem cells derived from all sources hold immense medical promises. Stem cell therapies have virtually unlimited medical and dental applications . The need of the hour is high quality research coupled with collaboration between basic scientists and the clinicians. A team effort engaging the expertise of the molecular biologists, immunologists, biomaterial scientists, cell biologists, matrix biologists, and practicing dental surgeons is crucial in attaining the desired goal.

58. REFERENCES Pulyodan MK, Paramel Mohan S, Valsan D, Divakar N, Moyin S, Thayyil S. Regenerative Endodontics: A Paradigm Shift in Clinical Endodontics. J Pharm Bioallied Sci . 2020;12( Suppl 1):S20-S26. The Hidden Treasure in Apical Papilla: The Potential Role in Pulp/Dentin Regeneration and BioRoot Engineering JOE — Volume 34, Number 6 ,2008 Regeneration Potential of the Young Permanent Tooth: What Does the Future Hold? JOE — Volume 34, Number 7S Regenerative Endodontics: A Review of Current Status and a Call for Action JOE — Volume 33, Number 4, 2007 Apexogenesis in an Incompletely Developed Permanent Tooth with Pulpal Exposure February 2003 ORAL HEALTH A paradigm shift in endodontic management of immature teeth : Conservation of stem cells for regeneration J Dentistry 2008.

59. Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth J Endod Aug 2008 Differentiation potential of dental papilla, dental pulp, and apical papilla progenitor cells. JOE VOL 36 , NO 5, 2010 Regenerative Endodontics: A Review of Current Status and a Call for Action JOE — Volume 33, Number 4, April 2007 Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J DENT RES 2009, VOL 88 , NO, 9 Library dissertation on regenerative endodontics by Dr. Niti Shah Regenerative endodontics – DCNA July 2012 vol 56 issue 3 Galler KM, Widbiller M, Buchalla W, Eidt A, Hiller KA, Hoffer PC, Schmalz G. EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells. Int Endod J. 2016 Jun;49(6):581-90.