Histological Evaluation of Regenerative Endodontic Treatment in Immature Permanent Teeth with Different Scaffolds- a Systematic Review

Transcription

1 Volume 120 No , ISSN: (on-line version) url: Histological Evaluation of Regenerative Endodontic Treatment in Immature Permanent Teeth with Different Scaffolds- a Systematic Review 1 Ahsana. A, 2 Deepa Gurunathan, 3 Deepa Gurunathan, 4 Lakshmi T 1 Post-Graduate Student, Department of Pedodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamilnadu,India. 2 Professor, Department of Pedodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamilnadu, India. 3 Professor,Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamilnadu, India. 4 Associate Professor, Department of Pharmacology, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamilnadu, India. 2 drgdeepa@yahoo.co.in ABSTRACT Regenerative endodontics is a treatment option for immature necrotic permanent tooth. It is an alternative option to specification procedure. The outcomes of the teeth treated with regenerative endodontics have been evaluated clinically and radiographically. The aim of this study was to evaluate and assess the histology of the tissues formed in immature permanent teeth treated with regenerative endodontic treatment using different scaffolds. A systematic literature search was done in pubmed, Cochrane library, google scholar and also through hand search through mid December Only human studies were included and the animal studies were excluded from the study. Articles without histological features are excluded. A total of 80 articles were identified. After duplicate removal, 64 articles were filtered. A total of 10 articles which satisfied the exclusion and inclusion criteria were included in this systematic review. Histological features of the included articles revealed that Cementum like and bone like tissues are seen rather than dentin like tissues are seen in the teeth treatment with revascularization procedure. Intracanal tissues also appeared to be an extension of periapical tissues. Thus to conclude, there is no definitive formation of true pulp-dentin complex following regenerative endodontic treatment using any scaffolds. Key words: Histology, Human teeth, Immature permanent teeth, Regenerative endodontics 549

2 INTRODUCTION Regeneration is defined as the replacement of damaged tissue by the same type of parenchymal cells (Kumar et al. 2009). 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). Regeneration of immature necrotic teeth was introduced by Iwaya et al (Iwaya et al. 2001)and Banchs and Trope (Banchs et al. 2004). Iwaya showed that revascularization procedure could induce thickening of root canal walls and continued root development. The three essential and critical steps of regenerative endodontics includes: adequate disinfection of the root canal, to create a scaffold for stem cells, and coronal sealing of the scaffold with a biocompatible material (Law 2013, Hargreaves et al. 2013, Wigler 2013). A scaffold is a threedimensional replica of the extracellular matrix which provides support to the stem cells (Murray 2007). It should provide (Dr.Lakshmi T and Rajeshkumar S 2018) an environment for the cells to migrate, proliferate, and differentiate (Zhang, Yelick 2010). Scaffolds should ideally be biocompatible, biodegradable, and should degrade without releasing any toxic by-products (Taylor MS et al. 1994, Schopper C et al. 2005) Inducing bleeding into the root canal and using blood clot Trishala A, Lakshmi T and Rajeshkumar S (2018),as a scaffold for the stem cells has been used traditionally in the regenerative endodontic procedures (Bezgin et al. 2014). Recently scaffolds with stem cells and growth factors are introduced and are being used to enhance the outcomes of regeneration treatment (Wang Y et al 2013, Nagy et al. 2014, Altaii et al. 2017) Regeneration is a histological observation and it cannot be determined radiographically (Shimizu et al. 2012). There are various animal studies which describe the histological outcomes of the teeth treated with regenerative endodontics(wang et al. 2010, Yamauchi et al. 2011). The aim of this systematic review was to evaluate the histological outcomes of the different regenerative endodontic procedures in human immature teeth using different scaffolds. MATERIALS AND METHODS We searched electronic databases (Pubmed, COCHRANE library, Google scholar) and hand search and cross references through mid December The PICO (P: immature permanent teeth undergoing regenerative endodontic treatment, I: blood clot scaffold, C: additional or alternative scaffolds or no scaffold, O: histological examination) question 550

3 generated for this systematic review was What are the histological outcomes in human immature permanent teeth treated with regenerative endodontic treatment using blood clot scaffold compared to additional or alternative scaffolds or no scaffold? Inclusion Criteria Studies/ case reports which evaluate the histological features of teeth treated with regenerative endodontic treatment with different or no scaffolds Human studies or case reports Articles published in English language Any irritant and intracanal medicament Any coronal barrier and seal Articles published from 1990 to 2017 Exclusion Criteria Animal studies Articles published in languages other than English Studies without histological assessment The titles and the abstracts of the articles were evaluated as an initial screening. Later, full texts were evaluated to assess the eligibility against the inclusion and exclusion criteria. The selected studies were evaluated following the development of a data extraction table. It captured details like authors, year of publication, journal, tooth number, pretreatment diagnosis, disinfection protocol, scaffold types,coronal seal used, follow up, reason for extraction / RCT and histological outcomes. RESULTS A total of 80 articles were identified. After duplicate removal, 64 articles were filtered. 10 articles were selected which fulfilled the exclusion and inclusion criteria and was used to perform the systematic review. The 10 articles are case reports in which one article reported 2 case cases. Therefore, this systematic review was done on 11 case reports. Characteristics of the included study like tooth number, disinfection protocols, different scaffolds used, coronal seal, reason for extraction/ RCT are summarised in Table

4 Among the 11 case reports, Blood clot was used as scaffold in 8 cases and PRP was used in 2 cases and colla plug was used in 1 case. Table 2 shows hard tissue formed with each scaffold. Dentin like tissue was seen in 1 case. Cementum like tissue was seen in 7 cases. Bone like tissue was seen in 4 cases. Table 3 shows the intra canal soft tissue with each scaffold. Case report by Lin et al, is a case of failed revascularization and the canal was apparently empty without any cells DISCUSSION Regenerative endodontics has been defined as biologically based procedures designed to replace damaged structures, including dentin and root structures, as well as cells of pulp dentin complex (Murray et al. 2007, Hargreaves et al. 2013). Tissue regeneration requires interaction between stem cells and growth factors in a bioactive scaffold, which is referred as tissue engineering triad(langer & Vacanti 2008). In regenerative endodontic treatment, SCAP serves as the source of stem cells. These stem cells can differentiate into odontoblasts (Sonoyama et al. 2008). An ideal scaffold should selectively binds and localizes cells, should contain Growth factors and should undergo biodegradation over time (Hargreves et al. 2008). Blood clot is a protein rich scaffold which contains platelet derived growth factors that act as a signalling molecule(hargreves et al. 2008). In the case report by Torabinejad & Faras 2012 continued apical closure of the root apex and resolution of the periapical lesion was observed after 14 months of treatment. The Patient s chief complaint was presence of pain and sensitivity to cold. The tooth was sensitive to thermal and electric pulp test which was similar to the control tooth. There was no lingering pain after the application of CO 2 ice. Diagnosis of reversible pulpitis and normal periapical tissues was given to the tooth. Root canal treatment was initiated because of Patient s complaint to the guardian and her insistence on performing root canal treatment or extraction of the tooth. Root canal treatment was performed and the pulp tissue was processed for histological examination. In the case report by Shimizu et al. 2012, the patient reported approximately 1 month after treatment by a general dentist. The tooth was asymptomatic and responded erratically to pulp sensibility test. It was not known whether deep pulpotomy or partial pulpectomy was performed. The patients dentist could not be contacted to confirm the treatment done. Clinical diagnosis of irreversible pulpitis with normal periapical tissue was given. Three and a half weeks after the revascularization treatment, the 552

5 patient reported with a fractured crown at the level of alveolar bone. The tooth was extracted and processed for histological examination. In the case report by Shimizu et al. 2013, the tooth had a complicated crown fracture restored with composite resin. Tooth was asymptomatic and a sinus tract was present in the periapical area. Tooth did not respond to the pulp sensibility tests. There was a periapical radiolucent lesion measuring 8*8mm with an open apex. The diagnosis of pulpal necrosis and chronic apical abscess was given. 26 months after revascularization treatment, the patient presented with a horizontal crown fracture at the cervical level. The tooth showed closure of root apex with thickening of canal walls and resolution of periapical lesion. The tooth was extracted and processed for histological examination. In the case report by Becerra et al. 2014, the patient reported with a localised swelling on the mucosa in the mandibular left premolar region. The tooth presented with dens evaginatus with a fractured cusp. The tooth was asymptomatic and showed no response to pulp sensibility tests. There was a large radiolucent lesion in the periapical region with an open apex and narrow canal walls. Diagnosis of pulpal necrosis and a chronic apical abscess with a sinus tract was given. 2 years after the treatment, the tooth was extracted for orthodontic treatment. The tooth showed narrowing of root apex and normal periapical condition. The extracted tooth was processed for histological evaluation. In the case report by Aggarwal et al. 2014, the patient reported with a history of trauma to the anterior teeth 2 years back. The teeth were carious,discoloured and were tender on palpation. There was a localised swelling in the mucosa. The teeth showed no response to pulp sensibility tests. There was periapical radiolucency in both the teeth with incomplete root formation. The diagnosis of pulpal necrosis with acute periapical abscess was given. 3 years after revitalisation treatment, patient reported with sensitivity to hot and cold in one of the teeth. The tooth 11 had secondary caries and showed exaggerated response to cold rest. The radiograph showed root completion and hence root canal treatment was initiated and the pulp tissue was processed for histological evaluation. In the case report by Lei et al. 2015, the patient reported with pain on mastication in the lower right region. The tooth had dens evaginatus with a fractured occlusal tubercle. Localised swelling was seen on the mucosa. The tooth was tender to percussion with class 2 mobility. Pulp sensibility tests showed no response. Diagnosis of pulpal necrosis with symptomatic apical periodontitis was given. 10 months after the revascularization treatment, the tooth was asymptomatic and elicited positive response to cold test and EPT. There was narrowing of apical foramen and thickening of canal walls. The tooth was extracted for orthodontic reason and was processed for histological evaluation. 553

6 Nosrat et al. 2015, reported 2 case reports. Two birooted fully erupted immature first premolar teeth scheduled for extraction for orthodontic treatment from 2 patients were included in this study. Radiographs were taken to confirm absence of caries and restoration and the presence of open apex. 4 months after revascularization treatment radiographs were taken to evaluate root development. In both the teeth root development was evident. Pulp sensibility tests were also performed, but the result was not mentioned in this case report. The teeth were extracted and processed for histological examination. In the case report by Martin et al. 2013, the patient reported with pain on mastication in the lower right back tooth region. The tooth 46 had occlusal carious lesion. The tooth showed no response to pulp sensibility tests. The tooth was tender to percussion. Radiograph revealed large radiolucent lesion in both the roots with incompletely formed. Follow up after 14 months revealed a small periapical radiolucent lesion associated with the mesial root. 2 years after the treatment, the patient reported with oblique fracture of the lingual cusp extending to the alveolar crest level. Radiograph revealed resolution of periapical lesion except widening of PDL around the mesial root. The tooth was extracted and processed for histological evaluation. In the case report by Peng et al. 2017, the patient s case record presented with spontaneous pain and pain on mastication. The tooth had a fracture of dens evaginatus. Thermal test induced pain in the tooth. Diagnosis of symptomatic irreversible pulpitis with symptomatic apical periodontitis was given. Patient s dentist had removed most of the pulp and had placed a cotton pellet. Two days after the initial treatment, patient reported to Peng. The tooth showed no response to pulp sensibility tests. Radiograph revealed peri radicular radiolucency with an open apex. 12 months after revascularization treatment, the tooth was asymptomatic and showed no response to thermal and electric stimuli. A year after, the tooth was extracted for orthodontic treatment and was processed for histological evaluation. Lin et al reported a case of failed revascularization. Tooth was avulsed and replanted with rigid splint by an oral surgeon within 40 minutes of avulsion approximately 4 months before following which the patient developed a localized swelling in the periapical area of tooth about 2 months after tooth replantation. The general dentist performed instrumentation of the canal and irrigated the canal with 2% chlorhexidine, filled the canal with Calasept. On clinical examination the tooth was asymptomatic and had a class I mobility. There was no swelling or sinus tract associated. The tooth was not tender to percussion and palpation. It did not respond to pulp sensibility tests. Periapical radiograph revealed that tooth was an immature permanent tooth and had a localized radiolucent lesion. Diagnosis of previously initiated root canal therapy and asymptomatic apical periodontitis was given. The patient developed symptoms of pain and local swelling at the periapical area approximately 16 months after 554

7 revascularization treatment. Radiograph did not show evidence of increased thickening of the canal walls and continued root development. The tooth was extracted and processed for histological examination. Successful regenerative endodontic treatment reveals radiographic thickening of the root canal walls, narrowing of the root apex and increase in root length (Jeeruphan et al and Nosrat et al. 2011). But the histological findings showed that the treatment outcome was different from the normal dentin and pulp tissue. The tissues inside the root canal appear to be an extension of the tissues in the periapical area. Animal studies also showed that there was lack of formation of pulp and dentin like structures with all scaffold types and that the tissue resembled cementum, PDL, bone in majority of the case (Altaii et al. 2017). Thus, the narrowing of root apex, root lengthening and thickening occurs by deposition of cementum like and bone like tissue and not dentin. Cementum produced by cementoblasts can differentiate from three sources:neural crest derived ectomesenchyme of the dental follicle cells (Sonoyama et al 2007), progenitor/stem cells in the periodontal ligament (Seo et al 2004) or Hertwig s epithelial root sheath (HERS) mesenchymal transformation (Zeichner-David et al 2003). In regenerative endodontic treatment, cementoblast like cells are derived from progenitor/stem cells in PDL. It is not clear whether these cells enter the canal immediately after inducing of bleeding or clot formation. The mechanism by which these cells differentiate into cementoblast like cells is unknown. Therefore, it can be stated that regenerative endodontic treatment promotes repair rather than regeneration, which restores the function and tissue continuity but with distortion of normal architecture (Sonoyama et al 2007). Further investigation is needed to check the effectiveness of usage of alternative scaffolds or adding materials to blood clot scaffold. For practice of evidence based medicine, systematic reviews on randomized controlled trials are of prime importance. But this systematic review was done on case reports since randomized controlled trial on revascularization treatment procedure is not practically possible. CONCLUSION Based on the results, it can be concluded that no substantial difference in the histological outcomes of the teeth treated with regenerative Endodontic treatment using different scaffolds. Though there is a difference in the histological outcomes of teeth treated with regenerative procedures from normal dentin and pulp tissue, this should not prevent the implementation of regeneration procedures in clinical practice. 555

8 REFERENCE [1] Aggarwal, Gaurav & Bogra, Poonam & Singh, S Vijay & Gupta, Saurabh & Kumar Manchanda, Saru & Saini, Nitin. (2014). Regeneration of Human Dental Pulp: A Myth or Reality? A Case Report. Endodontology 26, [2] Altaii M, Kaidonis X, Koblar S, Cathro P, Richards L (2017) Platelet rich plasma and dentine effect on sheep dental pulp cells regeneration/re- vitalisation ability (in vitro). Australian Dental Journal 62, [3] Altaii M, Richards L, Rossi Fedele G (2017). Histological assessment of regenerative endodontic treatment in animal studies with different scaffolds: a systematic review. Dental Traumatology 33, [4] Banchs F, Trope M (2004) Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? Journal of Endodontics 30, [5] Becerra P, Ricucci D, Loghin S, Gibbs JL, Lin LM (2014) Histologic study of a human immature permanent premolar with chronic apical abscess after revascularization/revitalization. Journal of Endodontics 40, [6] Bezgin T, Yılmaz A, Celik B, Sonmez H (2014) Concentrated platelet-rich plasma used in root canal revascularization: 2 case reports. International Endodontic Journal 47, [7] Hargreaves K, Geisler T, Henry M, Wang Y(2008) Regeneration potential of the young permanent tooth: what does the future hold? Journal of Endodontics 34, S51 S56. [8] Hargreaves KM, Diogenes A, Teixeira FB (2013) Treatment options: biological basis of regenerative endodontic procedures. Journal of Endodontics 39, S30-S43. [9] Iwaya S, Ikawa M, Kubota M (2001) Revascularization of an immature permanent tooth with apical periodontitis and sinus tract. Dental Traumatology 17, [10] Jeeruphan T, Jantarat J, Yanpiset K, Suwannapan L, Khewsawai P, Hargreaves KM(2012) Mahidol study 1: comparison of radiographic and survival outcomes of immature teeth treated 556

9 with either regenerative endodontic or apexification methods: a retrospective study. Journal of Endodontics 38, [11] Kumar V, Abbas AK, Fausto N (2009) Robbins and Cotran Pathologic Basis of Disease, 8th ed. Philadelphia: Saunders. [12] Langer R, Vacanti JP(1993) Tissue engineering. Science 2609, [13] Law AS (2013) Considerations for regeneration procedures. Journal of Endodontics 39, S44-S56. [14] Lei L, Chen Y, Zhou R, Huang X, Cai Z (2015) Histologic and immunohistochemical findings of a human immature permanent tooth with apical periodontitis after regenerative endodontic treatment. Journal of Endodontics 41, [15] Lin LM, Rosenberg PA (2011) Repair and regeneration in endodontics. International Endodontic Journal 44, [16] Lin LM, Shimizu E, Gibbs JL, Loghin S, Ricucci D (2014) Histologic and histobacteriologic observations of failed revascularization/revitalization therapy: a case report. Journal of Endodontics 40, [17] Martin G, Ricucci D, Gibbs JL, Lin LM (2013) Histological findings of revascularized/revitalized immature permanent molar with apical periodontitis using platelet rich plasma. Journal of Endodontics 39, [18] Murray PE, Garcia-Godoy F, and Hargreaves KM (2007) Regenerative endodontics: a review of current status and a call for action. Journal of Endodontics 33, [19] Nagy MM, Tawfik HE, Hashem AA, Abu-Seida AM (2014) Regenerative potential of immature permanent teeth with necrotic pulps after different regenerative protocols. Journal of Endodontics 40, [20] Nosrat A, Seifi A, Asgary S (2011) Regenerative endodontic treatment (revascularization) for necrotic immature permanent molars: a review and report of two cases with a new biomaterial. Journal of Endodontics 37, [21] Nosrat A, Kolahdouzan A, Hosseini F, Mehrizi EA, Verma P, Torabinejad M (2015) Histologic outcomes of uninfected human immature teeth treated with regenerative endodontics: 2 case reports. Journal of Endodontics 41,

10 [22] Peng C, Zhao Y, Wang W, Yang Y, Qin M, & Ge L (2017). Histologic findings of a human immature revascularized/regenerated tooth with symptomatic irreversible pulpitis. Journal of Endodontics 43, [23] Schopper C, Ziya-Ghazvini F, Goriwoda W et al (2005) HA/TCP compounding of a porous CaP biomaterial improves bone formation and scaffold degradation a long-term histological study. Journal of Applied Biomaterial 74, [24] Seo B-M, Miura M, Gronthos S, et al (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364, [25] Shimizu E, Jong G, Partridge N, Rosenberg PA, Lin LM (2012) Histologic observation of a human immature permanent tooth with irreversible pulpitis after revascularization/regeneration procedure. Journal of Endodontics 38, [26] Shimizu E, Ricucci D, Albert J et al (2013) Clinical, radiographic, and histological observation of a human immature permanent tooth with chronic apical abscess after revitalization treatment. Journal of Endodontics 39, [27] Sonoyama W, Seo BM, Yamaza T, et al (2007) Human epithelial root sheath cells play crucial roles in cementum formation. Journal of Dental Research 86, [28] Sonoyama W, Liu Y, Yamaza T, et al(2008) Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. Journal of Endodontics 34, [29] Taylor MS, Daniels AU, Andriano KP, Heller J (1994) Six bioabsorbable polymers: in vitro acute toxicity of accumulated degradation products. Journal of Applied Biomaterial 5, [30] Ten Cate R, Bartold PM, Squier CA, Naci A (2003) Repair and regeneration of oral tissues. In: Nanci A, eds. Ten Cate s Oral Histology, 6th edn. St Louis, MO: Mosby; [31] Torabinejad M, Faras H (2012) A clinical and histological report of a tooth with an open apex treated with regenerative endodontics using platelet-rich plasma. Journal of Endodontics 38,

11 [32] Wang X, Thibodeau B, Trope M, Lin LM, Huang GT (2010) Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. Journal of Endodontics 36, [33] Wang Y, Zhao Y, Jia W, Yang J, Ge L (2013) Preliminary study on dental pulp stem cellmediated pulp regeneration in canine immature permanent teeth. Journal of Endodontics 39, [34] Wigler R, Kaufman AY, Lin S, Steinbock N, Hazan-Molina H, Torneck CD(2013) Revascularization: a treatment for permanent teeth with necrotic pulp and incomplete root development. Journal of Endodontics 39, [35] Yamauchi N, Nagaoka H, Yamauchi S, Teixeira FB, Miguez P, Yamauchi M (2011) Immunohistolocal charaterization of newly formed tissues after regenerative procedure in immature dog teeth. Journal of Endodontics 37, [36] Zeichner-David M, Oishi K, Su Z, et al(2003) Role of epithelial root sheath cells in tooth root development. Developmental Dynamics 228, [37] Zhang W, Yelick PC (2010) Vital pulp therapy-current progress of dental pulp regeneration and revascularization. International Journal of Dentistry 2010, [38] Dr.Lakshmi T and Rajeshkumar S (2018) In Vitro Evaluation of Anticariogenic Activity of Acacia Catechu against Selected Microbes, International Research Journal of Multidisciplinary Science & Technology, Vol. 3, No. 3,pp [39] Trishala A, Lakshmi T and Rajeshkumar S (2018), Physicochemical profile of Acacia catechu bark extract An In vitro study, International Research Journal of Multidisciplinary Science & Technology, Vol.3, No. 4,Pp Table 1 : search strategy PubMed Immature teeth OR Necrotic pulp OR Revascularization OR Histology OR Immature tooth OR Pulp necrosis OR Revascularization OR Histological Immature permanent OR Pupal necrosis OR Revitalisation OR Immature necrotic OR Necrosis of pulp OR Revitalization OR 559

12 Open apex OR Open apices Non vital OR Pulp infection OR Infected pulp OR Pulpal infection Regeneration OR Apexogenesis OR Root completion OR Root formation Table 2 : Characteristics of the included studies * in FDI tooth notation NaOCl- Sodium hypochlorite + Triple antibiotic Paste NM- not mentioned Table 3: Histologic characteristics of dentin and associated hard tissue with each scaffold Histologic Blood clot Structures Cementum like Shimizu et al (2012) Becerra et al(2014) Nosrat et al(2015) Peng et al(2017) Blood clot with addition Martin et al(2013) Lei et al(2015) 560

13 Dentin like Bone like Lin et al Peng et al(2017) Nosrat et al(2015) Lin et al(2013) Martin et al(2013) Lei et al(2015) Table 4: Histologic characteristics of intra canal soft tissue with each scaffold Case Reports Torabinejad et al 2012 Martin et al 2013 Shimizu et al 2012 Shimizu et al 2013 Becerra et al 2014 Aggarwal et al 2014 Lei et al 2015 Nosrat et al 2015 Peng et al 2017 Histologic Features Collagen fibres,fibroblasts, blood vessels. No odontoblasts like cells seen Fibrous connective tissue and blood vessels interspersed among mineralized hard tissue Connective tissue enclosed in the mineralzed tissue. No pulp like tissue. Loose connective tissue consisting of spindle shaped fibroblasts or mesenchymal cells, blood vessels. Tissue appears to be an extension of periapical tissue. Odontoblasts like cells are seen. No hard tissue is seen. Fibrous connective tissue characterized by fibroblasts and collagen fibers and similar to that of periodontal ligament tissue. Large cells resembling cementoblasts or fibroblasts are seen. Chronic inflammatory cells are seen Fibrous connective tissue with spindle shaped fibroblasts, chronic inflammatory cells, RBC s are seen. Connective tissue in the periapical region appears to be an extension from peri apical tissue. No HERS or odontoblasts are seen. Fibrous connective tissue consisting of blood vessels, spindle shaped fibroblasts, collag fibers are seen. Connective tissue in the apical region appears to be an extension periodontal ligament. No odontoblasts like cells are seen A fibrotic connective tissue is seen which resembles the tissue of periodontium. No epithelial rests or inflammatory cells are seen. Pulp like tissue is seen with a layer of flattened odontoblasts like cells. This tissue with hypercellular and hypervascular connective tissue appears to be extension of periapical 561

14 Included Eligibility Screening Identification Lin et al (2013) tissue Root canal was apparently empty. No cells were seen. PRISMA Flow Diagram Records identified through database searching (n = 76 ) Additional records identified through other sources (n = 4 ) Records after duplicates removed (n = 64 ) Records screened (n = 64) Records excluded Irrelevant studies(16) Animal studies(34) Full-text articles assessed for eligibility (n =14) Full-text articles excluded No histologic features(4) (n =4 ) Studies included in qualitative synthesis (n =10) 562

15 563

16 564