Regenerative endodontic procedures for the treatment of necrotic

regenerative Endodontics

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3. Regenerative endodontic procedures for the treatment of necrotic mature teeth -A preliminary randomized clinical trial    International Endodontic Journal, January 2022 AUTHORS : Ahmed Youssef, Magdy Ali, Amr ElBolok, Reham Hassan KEYWORDS : blood clot, mature necrotic teeth, periapical healing, platelet-rich fibrin, pulp sensibility, regenerative endodontic procedures Guided by Dr. K. MADHU VARMA PROFESSOR Presentation by Dr. M. JAGAPATHI BABU I MDS

4. fails to restore pulp tissue vitality loss of protective mechanisms increases the brittleness of the tooth (Gupta et al., 2015) Discoloration and loss of natural translucency of teeth (Ahmed & Abbott, 2012) Mature permanent teeth with necrotic pulps have long been treated with conventional root canal therapy, INTRODUCTION

5. REGENERATIVE ENDODONTIC PROCEDURES (REPs) : ‘Biologically based procedures designed to replace damaged structures, including dentin and root structures, and cells of the pulp–dentin complex’ (Murray et al., 2007).

6. TISSUE ENGINEERING TRIAD STEM CELLS GROWTH FACTORS SCAFFOLD Biological events of REP are the interplay of tissue engineering traid : (Bohl et al., 1998; Hargreaves et al., 2008; Sedgley & Botero, 2012)

7. In REPs, blood clots (BCs), which are formed in an adequately disinfected root canal space by intentionally provoking periapical tissue bleeding, create a 3D-scaffold that entraps undifferentiated stem cells and supports the growth of new tissue (Murray et al., 2007).

8. Drawbacks of BC induction : Procedure is traumatic and may cause discomfort for the patients. Induction of periapical bleeding into the canal space is not always attainable. ( Nosrat et al., 2012)

9. Difficult to control the bleeding volume to the desired level. Precise placement of mineral trioxide aggregate (MTA) over the BC to the desired level is a technique-sensitive procedure ( Petrino et al., 2010)

10. To overcome the disadvantages; Platelet-rich fibrin (PRF), a second-generation platelet concentrate. Autologous high-density fibrin clot packed with an increased concentration of growth factors acts as a biological scaffold (Dohan et al., 2006; Huang et al., 2010).

11. REPs are highly successful in immature permanent teeth with necrotic pulps (Diogenes et al., 2016). Advantages : Resolution of clinical signs and symptoms and periapical healing An increase in the root length, which encourages the thickening of the canal walls and subsequent apical closure (Diogenes et al., 2016).

12. REPs have recently been suggested for treating mature permanent teeth with closed and fully formed apices ( Digka et al., 2020; Glynis et al., 2021). Case reports of mature teeth treated with REPs have revealed favourable outcomes in the resolution of signs, symptoms and in the healing of apical periodontitis Abou Samra et al., 2018; Paryani & Kim, 2013; Saoud, Martin, et al., 2016; Saoud et al., 2014; Shah & Logani , 2012

13. In a histologic study, Arslan, Şahin , et al. (2019) revealed that the ingrowth of vital tissue (fibrous connective and bone-like substance combined with vascular-like structures) within the root canal system is achievable after REPs in mature teeth. This tissue contribute to the re-establishment of the innate immune system, which could control root canal system reinfection (He et al., 2017).

14. AIM; To assess the radiographic healing and the possibility of regaining pulp sensibility in mature permanent teeth with necrotic pulps that were treated using two REPs: revitalization and a PRF-based technique. NULL HYPOTHESIS; No statistically significant difference would be found between the tested groups in terms of periapical healing and regaining sensibility.

15. APPROVAL ; Ethical Committee of the Faculty of Dentistry at Minia University (Minia, Egypt; approval no. 58/306). A single-blinded randomized controlled pilot trial two-arm parallel groups with an allocation ratio of 1:1 Conducted over a period of 1  year

16. The study followed the Consolidated Standards of Reporting Trials (CONSORT) guidelines Preferred Reporting Items for Randomized Trials in Endodontics guidelines ( Nagendrababu et al., 2020)

17. INCLUSION CRITERIA ; Patients aged 18–40 years without any known allergic reactions or systemic disease who required RCT of a nonvital single-rooted maxillary anterior tooth with a mature root. Periapical lesion with a periapical index (PAI) score ≥3, according to the classification of Ørstavik et al. (1986). A diagnosis was based on history, clinical and radiographic examination findings. Sensibility testing was conducted by using an electric pulp tester

18. EXCLUSION CRITERIA : Pregnant women Patients with generalized chronic periodontitis Patients with non-restorable teeth Previous root canal treated teeth Developmental anomalies Teeth with external or internal resorption ( Nageh et al., 2018; Rizk et al., 2019)

19. Informed consent form in Arabic or in English. A detailed explanation of treatment procedures, possible outcomes, complications, and follow-up period.

20. AAE Clinical Considerations for a Regenerative Procedure Revised ,6-8-16

21. RANDOMIZATION AND BLINDING; Computer sequence generation (www.random.org), which provided a table for groups I and II with randomized participant numbers (n  = 10) in each group. Diagnosis and implementation of the random allocation sequence were performed by Magdy Ali

22. The operator ( Ahmed Youssef ) and the patient could not be blinded. The assigned treatment protocol was revealed to the patient by drawing blood from the patient's forearm. Data collectors ( Reham  Hassan and Amr  ElBolok ) were blinded.

23. PRE-OPERATIVE ASSESSMENT; To evaluate the size of the periapical lesion, a preoperative radiograph was taken using the standardized paralleling technique with #2 periapical film, which was mounted on a custom-made anterior bite block connected to the X-ray tube via an adapter ring. Numerical readings of pulp sensibility were recorded using an electric pulp tester.

24. a) Construction of the stent on prefabricated cast b) Radiographic stent c) Periapical film mounted on an anterior bite block with radiographic stent

25. After drying and isolating the examined tooth, a small piece of toothpaste was applied to the tooth. A lip clip was placed and the test was conducted. The average of three readings was recorded for each tooth at an interval of 5 min.

26. TREATMENT PROCEDURES (INTERVENTIONS); First visit: Chlorhexidine mouthwash 1 Carpule of mepivacaine 3%[ infiltration technique] Rubber dam isolation , a straight-line access cavity Working length [ Electronic apex locator ]

27. Intraoral periapical radiograph [confirmation] Canal preparation up to a size #60 K-file During instrumentation, 1.5% NaOCl using side- vented irrigation needles 2mm from the W.L

28. Final irrigation ; 20 ml 1.5% NaOCl, followed by the same amount of 17% EDTA for 1  min each Dentine bonding agent applied to the access cavity wall, coronal third of the canal Root canal received calcium hydroxide intracanal medication, access cavity was sealed with dry sterile cotton pellets and GIC

29. SECOND VISIT; Local infiltration anesthesia without a vasoconstrictor Ca(OH)2 was removed by irrigation with 5ml sterile saline followed by 20 ml of 17% EDTA activated by using an E1 Irrisonic ultrasonic tip [1 Min.] Final irrigation was administered using 5ml saline, and the canals were dried with suitably sized paper points.

30. GROUP I [Blood clot (n = 10)] : Bleeding was evoked in the canal by over-instrumentation using a sterile #25–#35 K-file 2–3 mm beyond the apex Bleeding was controlled at a level just below the CEJ by using a dry cotton pellet canals were left for 7 min to allow for BC formation

31. White MTA was placed 3 mm below the CEJ, followed by a moist cotton pellet and a temporary filling to allow for the complete setting of the MTA temporary filling was replaced by a glass–ionomer cement base, resin composite restoration after 2 days to seal the access cavity

32. GROUP II Platelet‑rich fibrin (n = 10) ; Preparation of PRF Platelet-rich fibrin was prepared, based on Choukroun's technique (Dohan & Choukroun, 2007), by drawing blood from the patient's forearm (right median cubital vein) into a test tube without an anticoagulant It was centrifuged immediately using a tabletop centrifuge at 400 g for 10 min. The product contained three layers: a top layer of acellular platelet-poor plasma, a middle layer of PRF and a bottom layer of red blood corpuscles

33. The PRF was segregated and squeezed to form a membrane. After the bleeding was evoked in the canal as in group I The freshly prepared PRF membrane was fragmented and placed incrementally in the canal using a hand plugger and a finger spreader size 40 up to the level of the CEJ A 3-mm-thick layer of white MTA was placed directly over the PRF matrix. To reduce inter-operator variability all procedures were performed by the same operator (A.A.Y.), who was trained to perform REPs using both techniques

34. POSTOPERATIVE EVALUATION Radiographic assessment; [6th and 12thmonth visits] Standardized radiographs Periapical status was assessed using the PAI score ( Ørstavik et al., 1986). All radiographs were scanned using a flat-bed scanner ( Scanmaker II, Microtek International Inc.) and stored on a computer for evaluation.

35. Before the evaluation, 20 reference radiographs were used to calibrate the investigator. All PAI scores were remeasured by the same examiner 1 week after the first measurement. Intra-observer reproducibility was verified, based on intraclass correlation coefficients (ICCs)

36. Assessment of pulp sensibility ; [6 months and 12 months ] Numerical readings of pulp sensibility were recorded after by using an electric pulp tester in the same manner that was used preoperatively.

37. STATISTICAL ANALYSIS The PAI score showed a nonparametric distribution. The Wilcoxon signed-rank test was used to compare follow-up periods within each group, followed by multiple comparisons with Dunn–Bonferroni correction. The Mann–Whitney test was used to compare the tested groups for the PAI data.

38. Pulp sensibility data after EPs showed a parametric distribution. Repeated measure analysis of variance (ANOVA) was used to investigate the effect of different tested groups and follow-up periods on pulp sensibility after REPs, using SPSS (version 23; IBM).

39. RESULTS

40. RADIOGRAPHIC EVALUATION  

41. The results showed no statistically significant differences between the groups in terms of preoperative radiographic lesion score (p = .579). There was a significant increase in peri-radicular healing in both groups at 6 and 12 months compared to baseline [p<0.01]. After 12  months, there was no significant difference in the PAI scores between the two groups

42. Based on the electric pulp test manufacturer's instructions, 1 – 39 ; a tooth with vital pulp 40 -- 79 ; part of the tooth nerve is nonvital 80 -- a tooth with necrotic nonvital pulp PULP SENSIBILITY EVALUATION ;

43. The results showed a decrease in the mean readings of the electrical pulp tester during the follow-up period. Readings of tooth sensibility showed that, after 6 and 12 months, there was no statistically significant difference between the BC and PRF groups. Within each group, a statistically significant difference was found between the preoperative reading and each of the 6 and 12 months follow-up periods.

44. DISCUSSION ; Findings in this study indicated that REPs using the BC technique or PRF technique can be a treatment option for mature teeth with a necrotic pulp. Regeneration of a diseased pulp was once thought to be impossible. first successful revitalization case - Iwaya et al. (2001) , followed by the report of Banchs and Trope (2004)

45. REPs in mature teeth have been recently suggested as an attempt to overcome the drawbacks of RCT lack of sensation and immune mechanisms, increased susceptibility to root fracture of treated teeth ( Nageh et al., 2018) reinfection due to coronal leakage or microleakage ( Dammaschke et al., 2003).

46. Use of regenerative endodontics in the treatment of mature permanent teeth holds promise for more complicated cases such as root fracture perforating root resorption avulsion (Saoud, Mistry, et al., 2016).

47. REPs may contribute to the re-establishment of the innate immune system, which could control root canal system reinfection (He et al., 2017), and may lead to regaining tooth sensibility Possible proprioceptive defense mechanisms of the pulp, which provide an alarm system against any tissue injury and protect the pulp from further damage (Lin et al., 2021).

48. Differences in the REPs between immature and mature teeth is the mechanical preparation phase, which is avoided in immature teeth to prevent further weakening of the thin dentine walls (American Association of Endodontics [AAE], 2016). Regaining pulp sensibility in necrotic permanent fully mature teeth using REPs has been a controversial subject among scientists (El- Kateb et al., 2020)

49. The presence of stem cells using the evoked bleeding technique in mature permanent teeth was confirmed by Chrepa et al. (2015) Intracanal blood samples ---- quantitative reverse transcription- polymerase chain reaction .

50. NaOCl (1.5%) was used with the aid of side-vented needles to avoid apical extrusion of NaOCl into the periapical tissue (AAE, 2016; Ng et al., 2011). Higher concentrations of NaOCl were cytotoxic to stem cells in the apical tissue and decreased odontoblastic differentiation.

51. EDTA conditioning was linked to the release of growth factors embedded in dentine ( Galler et al., 2011). In addition, it was found to promote cell proliferation, migration, and odontoblastic differentiation (Kawamura et al., 2016). EDTA (17%) was used for 1 min after NaOCl to moderate the adverse effects of NaOCl.

52. Increasing growth factors improves cellular viability and migration, and it acts as chemoattractant and differentiation factor for MSCs from the apical papilla or periapical tissues ( Chrepa et al., 2015) EDTA activation with ultrasonics was used because it appears to increase the transforming growth factor-beta 1 (TGF-β) release from dentine ( Aksel et al., 2020; Widbiller et al., 2017).

53. In the second visit, 3% mepivacaine without a vasoconstrictor was used to facilitate bleeding from the periapical tissues into the canal system. The apex was violated by over instrumentation using K-file #20–#35 to allow the migration of MSCs into the root canal space from the periapical area (Hargreaves et al., 2013).

54. The placement of MTA after REPs upregulates various cytokines and biological markers such as interleukin (IL)-1 α, IL-1 β, IL-4, IL-6, IL-8, alkaline phosphatase, bone sialoprotein, osteopontin , and bone morphogenetic protein 2 (BMP-2) ( Rudagi & Rudagi , 2012; Torabinejad & Parirokh , 2010). To avoid discolouration caused by MTA, a dentine bonding agent was applied to the access cavity walls and coronal third of the canal ( Shokouhinejad et al., 2017)

55. Radiographic outcomes in the current study showed a marked decrease in PAI scores of the periapical lesions in both groups 6 months and 12 months after a REP. This finding might be due to process of natural wound healing which started with the induction of bleeding from periapical tissues as the first phase of the wound healing process (Holland et al., 2017).

56. The other possible explanation seems to be antimicrobial clearance , which was enhanced by the induction of periapical bleeding during REPs as it brings components of the innate and adaptive immune system such as cytokines, complement components, Immunoglobulins, phagocytes and antimicrobial peptides, which allow the localization and opsonization of bacteria, to facilitate phagocytosis(Saoud, Martin, et al., 2016).

57. REPs promotes bone repair, as reported in studies of immature permanent teeth ( Ezeldeen et al., 2015). Our results confirm previous findings by Arslan, Ahmed, et al. (2019) and El- Kateb et al. (2020), which revealed a reduction in the size of the periapical lesions in mature permeant teeth 12  months after REPs.

58. In this study, mean PAI score decreased at the end of 12 months in both groups, when compared to the 6-month scores. Furthermore, the periapical lesion healing response in the PRF group was better than that in the BC group at the end of 6 months and 12 months.

59. In a meta-analysis, clinical efficacy of the revascularization of immature teeth using BC, platelet-rich plasma (PRP) and PRF techniques show that the periapical lesion healing response is 88.9% for BC, 100% for PRP and 100% for PRF (p > .05), and that apical closure occurred more frequently after PRP and PRF than with BC (p < .0011) (Murray, 2018).

60. The PRF, a second-generation platelet concentrate prepared without adding anticoagulants (Dohan et al., 2010), when used as a scaffold in REPs, allows the release of various growth factors such as platelet-derived growth factor (PDGF), TGF-β, basic fibroblast growth factor ( bFGF ) and vascular endothelial growth factor (VEGF) for up to 28 days(He et al., 2009; Naik et al., 2013).

61. These growth factors direct stem cell migration, proliferation and differentiation; supplement angiogenesis; and have an important role in the self-regulation of inflammatory and infectious phenomena (Toffler et al., 2009).

62. Kim et al,(2010) showed that multiple cytokines, bFGF , VEGF or PDGF, alone or in combination with basal set of nerve growth factor and bone morphogenetic protein-7, could induce cell homing, angiogenesis and mineralized tissue formation with regeneration of dental pulp-like tissue in the root canals of mature human teeth after in vivo ectopic implantation in mice.

63. PRF forms a fibrin network which protects the growth factors from proteolysis and traps essential circulating immune cells and cytokines that act against infection (Jadhav et al., 2015). The results of the current study showed that after 1 year of follow-up, two (20%) patients in the BC group and five (50%) patients in the PRF group regained tooth sensibility.

64. This finding is in agreement with the finding of Nageh et al. (2018) in which 60% of their patients treated with the PRF-based technique regained tooth sensibility at the end of 12 months. Similar findings were observed in the study by Arslan, Ahmed, et al. (2019) and El- Kateb et al. (2020) in which 50% and 77% respectively, of mature teeth responded positively to electric pulp testing (EPT) after 1  year of REP.

65. However, the results of the present study were inconsistent with the results of the case series by Saoud, Martin, et al. (2016), who reported a negative response to sensibility testing after 26 months in mature teeth. case series by Bakhtiar et al. (2017), who reported that four immature teeth with necrotic pulp responded negatively to EPT after 12 months of PRF revascularization treatment.

66. Percentage of patients who regained tooth sensibility PRF Group >> BC Group Growth factors in PRF --- Neurogenesis GROWTH FACTORS FUNCTION TGF-β Stimulates neurite outgrowth and regulates differentiation of Schwann cells (SCs) PDGF Induces SC proliferation, differentiation and formation of myelin Insulin growth factor 1  Supports the extension of nerve fibres and suppresses apoptosis in motor, sensory and sympathetic neurons bFGF , Facilitates neuroprotection and SC regeneration

67. The use of PRF can add to the time and complexity of the procedure as it requires a venous blood draw from the arm of the patient. Unlike PRP, PRF can be easily prepared without an anticoagulant added to the blood and requires only one centrifugation cycle.

68. A statistically significant difference was found in our study between EPT readings at baseline and 6 months in both groups. The EPT readings significantly decreased after a 1-year follow-up, which suggested that a longer follow-up period may be required to detect the gain of nerve function and regain sensitivity after REPs in mature teeth (El- Kateb et al., 2020).

69. It must be emphasized that a positive response to pulp testing is not a definitive indicator of the regeneration of pulp tissue in the canal space, and the absence of a pulp response does not imply a lack of vitality. The regenerated vital pulp-like tissue may have an immature innervation system that requires a longer time for the development of a more organized nerve-like tissue (Nakashima et al., 2017).

70. By using MRI, a recent study by El- Kateb et al. (2020) confirmed the presence of vital pulp tissue after REPs in vivo and revealed no statistically significant difference when comparing the baseline MRI signal intensity of the normal contralateral teeth and that of teeth 12 months after treatment with REPs (p > .05). In immature teeth, the continuation of root development indicates the existence of vital tissues in the canal space, which is not the case in mature teeth.

71. Song et al. (2017) reported that the prevalence of intracanal calcification associated with revascularization of immature teeth was 62.1% (18/29 patients) 27.8% (5/18 patients) ------ calcific barriers 72.2% (13/18 patients) ------ canal obliteration Lei et al. (2015) confirmed nervous tissue regeneration by using immunohistochemistry in an extracted immature permanent premolar tooth treated with REPs via BC.

72. LIMITATIONS : low number of participants short follow-up period however, future clinical directions will include extended monitoring and follow-up

73. The accumulation of evidence regarding the successful outcome of REPs in treating mature teeth with necrotic pulp using different protocols could contribute to the development of a specific position statement and recommendations similar to the available ESE position statement ( Galler et al., 2016) for immature teeth revascularization procedures

74. CONCLUSION; The findings of this preliminary trial indicate the potential for using REPs, such as revitalization or PRF-based techniques, as treatment options for mature teeth with necrotic pulp. A higher level of evidence is required however, with appropriately sized prospective clinical trials and longer follow-ups to conclusively validate the different outcomes of REPs.

75. CRITICAL APPRAISAL:

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