Traditionally, the treatment of immature permanent teeth with apical periodontitis is

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1 Histological Findings of Revascularized/Revitalized Immature Permanent Molar with Apical Periodontitis Using Platelet-rich Plasma Gabriela Martin, DDS,* Domenico Ricucci, MD, DDS, Jennifer L. Gibbs, MAS, DDS, PhD, and Louis M. Lin, BDS, DMD, PhD Abstract Introduction: An immature mandibular right first molar (#30) with apical periodontitis of a 9-year-old boy was treated with a revascularization/revitalization procedure using either a mixture of platelet-rich plasma (PRP) and a blood clot or a blood clot alone on the same tooth. Methods: Tooth #30 fractured 2 years and 1 month after the revascularization/revitalization procedure and could not be saved. The tooth was extracted and processed for histologic examination to determine the nature of the tissues that formed in the canals. Results: Clinically, the endodontic treatment of the case was successful based on the resolution of apical periodontitis and the absence of clinical signs and symptoms. Histologically, the tissues formed in the distal and mesial canals were mineralized tissue similar to cementoid/ osteoid tissue and uninflamed fibrous connective tissue regardless of PRP or no PRP treatment. No pulp-like tissue characterized by the presence of odontoblastlike cells polarized along the dentin-like mineralized tissue was observed. Conclusions: The tissues formed in the canals were mineralized tissue and some fibrous connective tissue. No pulp-like tissue characterized by the presence of odontoblast-like cells was observed lining the dentin-like mineralized tissue. (J Endod 2013;39: ) Key Words Apical periodontitis, cementoid/osteoid tissue, immature permanent tooth, platelet-rich plasma, revascularization/revitalization From the *Department of Endodontics, National University of Cordoba, Cordoba, Argentina; Private Practice, Cetraro, Italy; and Department of Endodontics, New York University, New York, New York. Address requests for reprints to Dr Domenico Ricucci, Piazza Calvario, 7, Cetraro (CS), Italy. address: dricucci@libero.it /$ - see front matter Copyright ª 2013 American Association of Endodontists. Traditionally, the treatment of immature permanent teeth with apical periodontitis is accomplished by an apexification procedure to achieve apical closure followed by nonsurgical root canal therapy (1). Since Iwaya et al (2) reported that a revascularization procedure using an antibiotic paste (metronidazole and ciprofloxacin) in immature permanent tooth with apical periodontitis could result in increased thickening of the canal walls, continued root development, and restoration of pulp vitality, revascularization has become a preferable treatment choice over apexification as shown in many recent case series reports (3). In addition to elimination of the root canal infection, which is the most important aspect of the revascularization/revitalization procedure, an additional step regarded necessary is the induction of blood clot formation in the canal by provoking bleeding from the periapical tissue (4, 5). It is hypothesized that the blood clot serves as a matrix for migration of progenitor cells into the canal, possibly from the apical papilla (6). This procedure is similar to inducing bleeding and clot formation in the bony crypt after apical surgery to initiate bone wound healing. Bleeding and clot formation is an initial step of tissue wound healing, which will lead to granulation tissue formation, which is an essential component of wound healing. The purpose of the induction of bleeding into the canal space in the revascularization/revitalization procedure is to recreate the events of tissue wound healing (regeneration or repair). It has also been shown histologically that the outcome of revascularization of immature dog teeth with apical periodontitis either in the presence or absence of a blood clot in the canal space is not statistically significant (7). However, cell migration or movement requires extracellular matrix in the canal space. The nature of the tissues formed in the canals of the revascularized/revitalized immature permanent teeth with apical periodontitis in humans is not currently known because no histologic studies have been reported. However, in animal studies, the tissues formed in the canal of the revascularized immature teeth with apical periodontitis were described as cementoid/osteoid tissue and periodontal ligament like tissue (7 10). Surprisingly, no pulp-like tissue as characterized by the presence of odontoblast-like cells lining the dentin-like mineralized tissue was observed in animal studies. This suggests the possibility that the apical papilla did not survive in apical periodontitis; this is thought to provide a source of the progenitor cells driving root pulp development. Recently, platelet-rich plasma (PRP) was used as a matrix instead of a blood clot in revascularization of the tooth with necrotic pulp and an open apex (11). PRP contains concentrated growth factors such as platelet-derived growth factor, transforming growth factor, vascular endothelial growth factor, epidermal growth factor, and insulin-like growth factor (12, 13). The target cells of the growth factors are not very clear at this point because there are multiple potential targets for each factor (14). PRP has been shown to enhance wound healing if the parenchymal tissue of the organs was not completely destroyed (13, 15). However, it is not known whether PRP could induce tissue regeneration if the parenchymal tissue was completely destroyed. Torabinejad and Faras (16) showed that pulp-like tissue could be generated in a human tooth with necrotic pulp and an open apex by using PRP as a scaffold in regenerative endodontic procedures. The generated tissue was removed from the canal of the tooth 14 months after the revascularization procedure, and histologic examination revealed vital pulp-like connective tissue although with no evidence of 138 Martin et al. JOE Volume 39, Number 1, January 2013

2 odontoblasts and typical overall pulp tissue architecture. Pulp is a loose connective tissue characterized by the presence of highly specialized odontoblasts lining the surface of predentin with cytoplasmic processes extending into the dentinal tubules. This is the first case report describing histologic findings of tissues formed in the canals of human revascularized/revitalized immature permanent tooth #30 with apical periodontitis using either a mixture of a blood clot and PRP or a blood clot alone as a scaffold in the same tooth. The tooth unfortunately had to be extracted because of a fracture, which allowed for the rare opportunity to perform a histologic analysis of the tissues formed in the canals more than 2 years after receiving revascularization/revitalization treatment. Case Report A 9-year-old boy was referred by a general dentist to an endodontist for the treatment of tooth #30. The patient reported pain with mastication. Clinical examination showed that tooth #30 had an extensive occlusal carious lesion. Sensitivity tests (heat, cold, and electric pulp test) of the tooth gave no response. The tooth was tender to percussion. A periapical radiograph of the tooth showed that the carious lesion seemed to approach the mesial pulp horn of the tooth, and a large radiolucent lesion was present at the periapical area of both the mesial and distal roots (Fig. 1A). The root apices were not fully formed although apical resorption of the distal root could not be excluded, and the canal walls appeared to have enough thickness. The clinical diagnosis of tooth #30 was pulp necrosis and symptomatic apical periodontitis. Treatment options and procedures, including apexification and revascularization/ revitalization, were explained to the patient and the patient s parents. They chose the revascularization/revitalization procedure of the tooth for the child, and informed consent was obtained. First Session To make sure that no vital pulp was present in the canal of tooth #30, local anesthesia was not given initially. The tooth was isolated with a rubber dam, and the caries were completely removed. After preparing an adequate access cavity, the pulp chamber and the root canal orifices were irrigated with 10 ml 5.25% NaOCl (Tedequim SRL, Cordoba, Argentina). The canals were explored with a #15 K-file (Dentsply-Maillefer, Ballaigues, Switzerland). When the file reached to the area between the middle and the apical third of the mesial canals, the patient felt painful sensation, indicating vital pulp tissue in the apical portion of the canal. The distal canal could be explored to the apex without any painful sensation of the patient. Local anesthesia (2% lidocaine with 1:100,000 epinephrine) was immediately administered by an inferior alveolar block. The working length was determined radiographically with a K-file at the radiographic apex. To ensure effective delivery of the irrigant and intracanal medication to the apical portion of the canal, the distal canal was instrumented to a #40 K-file and the mesial canals to a #30 K-file at the radiographic apex. The canals were gently irrigated with 10 ml 5.25% NaOCl to avoid forcing irrigant into the periapical tissues and dried with paper points. A mixture of ciprofloxacin (200 mg), metronidazole (500 mg), and minocycline (100 mg) paste was placed into the apical portion of canals with a Lentulo spiral (Dentsply-Maillefer) as intracanal medication. The access cavity was closed with a cotton pellet and Intermediate Restorative Material (IRM) (Dentsply International, Milford, DE). Second Session The patient was scheduled for a second visit after 4 weeks. However, the patient did not keep the appointment and returned for the second visit 5 months later. The tooth was asymptomatic during the entire postoperative period, and the temporary filling was intact. A new periapical radiograph showed that the size of the periapical radiolucent lesion had reduced considerably (Fig. 1B), which indicated that the root canal infection was under control. Local anesthesia was accomplished with 3% mepivacaine without a vasoconstrictor drug. After isolation with a rubber dam, the access cavity was reopened. A #15 K-file was introduced into all canals to ensure the patency of the canals. Some bleeding tissue was observed in the apical portion. The canals were irrigated with 10 ml 5.25% NaOCl to remove the triple antibiotic paste. NaOCl solution remained in the root canals for 5 minutes and was then dried with sterile paper points. A #25 K-file was introduced into the mesial canals and a #35 K-file into the distal canals through the apical foramen to successfully provoke bleeding from the periapical tissue into the canals up to the floor of the pulp chamber. An autologous PRP in a 1-mL insulin syringe with a 30-G needle (Ulti- Care, St Paul, MN) was inserted into the apical portion of the distal canal, and approximately 0.5 ml PRP was injected slowly into the canal. PRP could not be injected into the mesial canals because they were too small. A preparation of white mineral trioxide aggregate (MTA) (Pro- Root MTA; Dentsply Tulsa Dental Specialties, Tulsa, OK) and saline solution was gently placed over the mixture of blood and PRP in the distal canal and over the blood in the mesial canals followed by a moist cotton pellet in the floor of the pulp chamber. The access cavity was closed with IRM (Fig. 1C). The patient was asked to return 1 month later. The tooth was asymptomatic when the patient returned for the third appointment. IRM was removed and replaced by a bonded resin restoration (Filtek Z350 XT; 3M ESPE Dental Products, St Paul, MN). Follow-up Visits Seven months postoperatively, healing of the periapical lesions had improved further. A follow-up radiograph taken 14 months later showed that a small periapical radiolucent lesion was still associated with the mesial root (Fig. 1D). The tooth was asymptomatic. The patient returned after 2 years 1 month complaining of mobility of the coronal portion of the tooth. A clinical examination showed an oblique fracture of the lingual cusps extending to the alveolar crest bone level. A periapical radiograph taken showed that the periapical lesions had healed completely except a slight widening of the PDL space around the mesial root (Fig. 1E). The distal canal appeared to be obliterated by the hard tissue, and the mesial canal space was reduced in size. Treatment options including periodontal surgery and a full crown coverage of tooth #30 or extraction were explained to the patient s parents. The parents decided to have the tooth extracted. At extraction, soft tissue was seen attached to the apices of both the mesial and distal roots (Figs. 1F and G). The extracted tooth was immediately immersed in a 10% neutral buffered formalin solution for histologic processing. Preparation of PRP To obtain ml PRP, 10 ml whole blood was drawn from the patient s arm and equally divided into 4, 2.5-mL tubes. Each tube contained 3.8% sodium citrate (EuroTube; Deltalab, Barcelona, Spain) to prevent blood coagulation. The whole blood in the tube was centrifuged at 1,500 rpm for 30 minutes to separate the precipitated erythrocytes from the PRP suspension. The plasma next to the precipitated erythrocytes, which was rich in platelets, was aspirated into a syringe and placed into 2, 3.6-mL vials (Cryo Tube Vials; Nunc Brand, Roskilde, Denmark). Fibrin coagulation of PRP was initiated by adding calcium chloride (CaCl 2 ) (CaCl 2 = mol/ L; GT Lab, Rosario, Argentina). For 2.5 ml PRP, 1.25 ml CaCl 2 was added into each tube, and the tube was shaken by hand. Fibrin matrix formation occurred within 10 minutes after the addition of JOE Volume 39, Number 1, January 2013 Revascularized/Revitalized Immature Permanent Molar 139

3 CaCl 2. The solution of PRP and CaCl 2 was immediately aspirated into an insulin syringe and injected into the distal canal before PRP became overcoagulated. Tissue Processing Demineralization was performed in an aqueous solution consisting of a mixture of 22.5% (vol/vol) formic acid and 10% (wt/vol) sodium citrate for 3 weeks. The endpoint was determined radiographically. At the end of the demineralization process, the mesial and distal roots were separated from the tooth with a sharp razor blade just beyond the root canal orifices. The roots were then divided longitudinally into 2 equal portions (Fig. 1H and I). The 4 radicular segments were washed in running water for 48 hours, dehydrated in ascending grades of ethanol, cleared in xylene, Figure 1. (A) A preoperative radiograph. (B) A radiograph of the tooth 5 months after triple antibiotic treatment. (C) A postoperative radiograph after revascularization/revitalization. (D) A 14-month follow-up radiograph. (E) A radiograph taken after 2 years 1 month at the time of root fracture. (F and G) Photographs of the distal and mesial roots after tooth extraction. (H and I) Distal and mesial roots separated longitudinally into 2 portions. 140 Martin et al. JOE Volume 39, Number 1, January 2013

4 Figure 2. The distal root. (A) The section passing approximately at the center of the root canal (hematoxylin-eosin; original magnification 16). (B) A detailed view of the apical canal in A (original magnification 50). (C) A detailed view of the upper portion of the canal in A. Coronally to the mineralized tissue, amorphous debris is present (original magnification 50; inset, 400). (D) Magnification of the apical portion of the canal in B (original magnification 100). (E) A highpower view of the area of the root canal wall indicated by the left arrow in D. Few cells housed in lacunae can be seen in the newly formed mineralized tissue, resembling cementocytes or osteocytes (original magnification 400). (F) A high-power view of the area of the root canal wall indicated by the right arrow in D. No cells can be seen in this area (original magnification 400). (G) A detailed view from the apical foramen in A (original magnification 100). (H) A detailed view from the center of the periapical soft tissue (original magnification 100; upper inset, 400; lower inset, 1,000). JOE Volume 39, Number 1, January 2013 Revascularized/Revitalized Immature Permanent Molar 141

5 Figure 3. The mesial root. (A) The section passing approximately at the center of the root (hematoxylin-eosin; original magnification 16). (B) The section taken about 50 sections after that in A (original magnification 16). (C) The section taken after another 50 sections (original magnification 16). (D) A detailed view of the apical canal in A (original magnification 100). (E) A high-power view of the area indicated by the arrow in D (original magnification 400). (F)In another section, the canal is not blocked. The area indicated by the arrow is magnified in the inset (orig magnification 50; inset, 400). (G) A detailed view of the right dentin wall and the apical foraminal area in F (original magnification 100). (H) A high-power view of the area indicated by the arrow in G (original magnification 400). 142 Martin et al. JOE Volume 39, Number 1, January 2013

6 and infiltrated and embedded in paraffin (melting point, 56 C) according to standard procedures. Serial sections of 4 5 mm were cut on a mesiodistal plane until the specimen was exhausted. Occasionally, some sections were lost, but the neighboring sections allowed for the full histologic reconstruction of the root. Every fifth slide was stained with hematoxylin-eosin for screening purposes and the assessment of tissues formed in the canals. Slides were examined under the light microscope. Histologic Observations A mineralized tissue has irregularly filled the canal space up to the coronal MTA plug of both the distal and mesial canals (Figs. 2 and 3). This mineralized tissue occupied the entire apical canal of the distal canal in all the serial sections (Fig. 2A). In the mesial canal, the mineralized tissue was observed filling the apical canal in some sections (Fig. 3A and D). In other sections, the canal lumen appeared narrowed by a cementum-like tissue deposited on the root canal walls, with fibrous connective tissue that could be followed up to the coronal MTA plug (Fig. 3B). In the majority of sections, the mineralized tissue formation was highly irregular. In some areas, the mineralized tissue apparently blocked the canal space, whereas in other areas the canal space was occupied by fibrous connective tissue (Fig. 3C). In the distal canal, the mineralized tissue resembled cementoid/ osteoid tissue with few cementocyte/osteocyte-like cells housed in the lacunae (Fig. 2E). Numerous irregular spaces containing vital connective tissue and blood vessels (Fig. 2C) or necrotic debris and dentin chips (Fig. 2B and D) could be seen within the mineralized tissue in the distal canal. No odontoblast-like cells with long cytoplasmic processes could be observed lining the canal dentin walls. The canal dentin appeared to show direct junction with the newly formed mineralized tissue (Fig. 2E and F). In the apical foraminal area, a layer of cementum-like tissue covered the dentin walls. An uninflamed fibrous connective tissue with spindle-shaped fibroblasts filled the apical foramen (Fig. 2G). The soft tissue attached to the root tips was found to be a fibrous tissue exhibiting scattered chronic inflammatory cells (Fig. 2H). Many elongated amorphous bodies could be observed in his context surrounded by macrophages whose cytoplasm contained tiny particles of foreign materials (Fig. 2H, upper inset). Some multinucleated foreign body cells could also be seen (Fig. 2H, lower inset). The root apex was well formed. No Hertwig epithelial root sheath like cells surrounding the root apex were observed. In the mesial canal, the mineralized tissue filling the apical portion of the canal in some sections showed the characteristics of cellular cementum (Fig. 3D and E). At the apical foraminal area, a layer of cellular cementum also covered the canal walls (Fig. 3F and G). In some sections slightly short of the apical foramen, irregular areas of mineralization surrounded by uninflamed connective tissue could be observed (Fig. 3F). A high-power view revealed that this tissue was dystrophic calcifications with necrotic debris and dentin chips embedded in the mineralized mass (Fig. 3, inset). The apex was well formed. No Hertwig epithelial root sheath like cells surrounding the root apex could be observed; only bundles of fibrous connective tissue were noted (Fig. 3G and H). Discussion In most revascularization protocols involving human immature permanent teeth with apical periodontitis, the induction of bleeding into the canal by irritating the periapical tissues is an essential step (2, 4 6, 17, 18). Bleeding and fibrin clot formation is also the initial stage of wound healing after tissue injury. The blood clot serves as a matrix for adhesion between cell surface integrins and clot fibrin expressing the arginine-glycine-aspartic acid sequence containing protein (19). In addition, the matrix is an important reservoir of growth factors (20). PRP instead of a blood clot has been proposed as a matrix or scaffold in revascularization procedures of human immature permanent tooth with necrotic pulp and an open apex (11). PRP contains more concentrated growth factors than are present in a blood clot (12, 13). It has been shown that PRP likely promotes regeneration of the periodontal tissues in periodontal regenerative therapy (21). In the present case, the addition of PRP to the blood clot did not appear to induce pulp tissue regeneration after revascularization procedures of an immature permanent molar with apical periodontitis. The tissues observed in the distal canal of the revascularized immature tooth using PRP consisted of massive mineralized tissue resembling cellular cementoid tissue, which almost completely obliterated the canal space. However, cellular cementum sometimes can be difficult to differentiate from compact bone histologically (22). Irregular spaces of necrotic tissue debris or dentin chips were surrounded by mineralized tissue. No odontoblast-like cells were observed polarized along the dentin-like mineralized tissue. The fibrous connective tissue in the apical portion of the canal was devoid of inflammation and appeared to be an extension of the periodontal ligament. The cementoid/osteoid tissue deposited in the canal and the apex could be caused by cementoblasts/osteoblasts differentiated from the stem cells in the PDL (23). A foreign body reaction was present in the periapical tissues of the distal root. The foreign bodies could be dentin chips or other materials introduced into the periapical tissues by mechanical instrumentation. When comparing the tissues formed in the distal canal with PRP treatment with the tissues formed in the mesial canals without PRP treatment, the tissues were similar histologically. These tissues are also similar to the tissues formed in the revascularized immature teeth with apical periodontitis in animal studies (7 10). Regeneration is an ideal outcome of wound healing after tissue injury. However, repair is the usual result of postnatal wound healings (24, 25). The dental pulp is one of the components of a tooth. Repair of the pulp by vital tissue is better than replacement of the pulp with biomaterials, gutta-percha, and root canal sealer in an immature permanent tooth with pulp necrosis and apical periodontitis. The revascularization/revitalization procedure provides several advantages over apexification such as increased thickening of the canal walls, continued root development, and restoration of tooth vitality. Based on resolution of the apical periodontitis and the absence of clinical signs and symptoms, the endodontic treatment of the present case is successful (26) regardless of whether the canals are filled with vital mineralized tissue or root canal sealer and gutta-percha. The long-term outcome between apexification and revascularization/revitalization needs to be investigated. Acknowledgments The authors deny any conflicts of interest related to this study. References 1. Rafter M. Apexification: a review. Dent Traumatol 2005;21: Iwaya SI, Ikawa M, Kubota M. Revascularization of an immature permanent tooth with apical periodontitis and sinus tract. Dent Traumatol 2001;17: Bose R, Nummikoski P, Hargreaves K. A retrospective evaluation of radiographic outcomes in immature teeth with necrotic root canal system treated with regenerative endodontic procedures. J Endod 2009;10: Ding RY, Cheung GS, Chen J, et al. Pulp revascularization of immature teeth with apical periodontitis: a clinical study. J Endod 2009;35: Lenzi R, Trope M. Revitalization procedures in two traumatized incisors with different biological outcomes. J Endod 2012;38: JOE Volume 39, Number 1, January 2013 Revascularized/Revitalized Immature Permanent Molar 143

7 6. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod 2004;30: Thibodeau B, Teixeira F, Yamauchi N, et al. Pulp revascularization of immature dog teeth with apical periodontitis. J Endod 2007;33: Bezerra da Silva LA, Nelson-Filho P, Bezerra da Silva RA, et al. Revascularization and periapical repair after endodontic treatment using apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing in dog s teeth with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109: Wang X, Thibodeau B, Trope M, et al. Histological characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod 2010;36: Yamauchi N, Nagaoka H, Yamauchi S, et al. Immunohistological characterization of newly formed tissues after regenerative procedure in immature dog teeth. J Endod 2011;37: Torabinejad M, Turman M. Revitalization of tooth with necrotic pulp and open apex by using platelet-rich plasma. J Endod 2011;37: Eppley BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plast Reconstr Surg 2004;114: Foster TE, Puskas BL, Mandelbaum BR, et al. Platelet-rich plasma: from basic science to clinical applications. Am J Sports Med 2009;37: Werner S, Gross R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83: Lacci KM, Dardik A. Platelet-rich plasma: support for its use in wound healing. Yale J Biol Med 2010;83: Torabinejad M, Faras H. A clinical and histological report of a tooth with an open apex treated with regenerative endodontics using platelet-rich plasma. J Endod 2012;38: Jung IY, Lee SJ, Hargreaves KM. Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series. J Endod 2008;34: Chen MY, Chen KL, Chen CA, et al. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures. Int Endod J 2011;45: Kierszenbaum AL. Histology and Cell Biology. St Louis, MO: Mosby; Taipale J, Keski-Oja J. Growth factors in the extracellular matrix. FASEB J 1997;11: Camargo PM, Lekovic V, Weinlaender M, et al. Platelet-rich plasma and bovine porous bone mineral combined with guided tissue regeneration in the treatment of intrabony defects in humans. J Periodontal Res 2002;37: Bosshardt DD. Are cementoblasts a subpopulation of osteoblasts or a unique phenotype. J Dent Res 2005;84: Seo B-M, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364: Longaker KT, Karyn S, Bouhana BS, et al. Wound healing in the fetus. Possible role for inflammatory macrophages and transforming growth factor-ß isoforms. Wound Repair Regen 1994;2: Bullard KM, Longaker MT, Lorenz HP. Fetal wound healing: current biology. World J Surg 2003;27: Strindberg LZ. The dependence of the results of pulp therapy on certain factors. An analytic study based on radiographic and clinical follow-up examination. Acta Odontol Scand 1956;14(Suppl 21): Martin et al. JOE Volume 39, Number 1, January 2013