EGYPTIAN DENTAL JOURNAL

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1 EGYPTIAN DENTAL JOURNAL Vol. 59, 985:993, January, 2013 I.S.S.N REVASCULARIZATION OF IMMATURE PERMANENT TEETH WITH NECROTIC PULP BY USING PLATELET-RICH PLASMA: CASE REPORT Mohammad Aldakak * ; Mohammad S. Rekab * ; M Bachar Abou Al Shaar ** and Hamad A. Alzoman *** ABSTRACT Objectives: To explore the outcomes of treatment done by using Plasma Rich Protein (PRP) for revascularization a traumatic tooth. Methods: A 14 year old male who was referred to the endodontic clinic to manage an traumatic injury to both maxillary central incisors. Clinical examination showed pulpal exposure and necrotic pulp tissues on both central incisors and the radiographic examination displayed immature roots with open apices for both teeth. Access openings were performed for both teeth, necrotic pulp tissues were removed and canals were irrigated with 5.25% NaOCl and dried with paper points. A triple antibiotic agent consisting of Metronidazole, Minocycline, and Ciprofloxacin was packed into the canals and the teeth were temporized. Three weeks later, blood was withdrawn from the patient for preparation of PRP. The intra-canal antibiotic dressing was removed then irrigated with saline. The PRP was introduced into the root canal systems up to the cement enamel junction levels. A plug of white mineral trioxide aggregate was placed over the PRP gel and the teeth were temporized. A week later, both teeth were free of symptoms, thus permanent filling were placed. Results: After 6 months treatment, there was no sensitivity to percussion or palpation tests. Radiographic examination showed resolution of the periapical lesion, further root development, and continued apical closure. Conclusions: It was concluded that the treatment of immature permanent teeth with necrotic pulp by using PRP as a scaffold, placed in the root canal can create a suitable environment revascularization, resulting in the completion of root maturation. KEYWORDS: pulp, revascularization, regeneration, Plasma rich protein, dental trauma * Department of Endodntics, Faculty of Dentistry,Damascus University, Damascus, Syria ** Department of Preventive Dental Sciences, College of Dentistry, Taibah University, Madinah, Saudi Arabia *** Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia

2 (986) E.D.J. Vol. 59, No. 1 Mohammad Aldakak, et al. INTRODUCTION The presence of an open apex provides significant challenge in the treatment of pulpal injury. When the apex is not closed, routine root canal procedures cannot be performed; the results of treatment are unpredictable. Depending on the vitality of the affected pulp, there are two possible approaches where either apexogenesis (vital pulp therapy) or apexification (necrotic pulp) will take place (Capurro et al.1999, Kleier et al.1991).in immature non vital-teeth the root canal walls diverge apically making preparation of an apical stop impossible. Treatment is therefore targeted towards inducing the formation of an apical buttress of hard tissue and avoiding any overfilling of the canal. The proper approach in this treatment modality is known as apexification and its goal is to make an apical stop (Beer 2006). Many materials have been reported to successfully stimulate apexification. The use of nonsetting Calcium hydroxide Ca (OH) 2 was first reported by Kaiser in 1964, but the technique was popularized by the work of Frank in 1966 (Frank 1966). However, the main disadvantage of this technique is the increased possibility of cervical fracture (Andreasen et al. 2002) as well as the frequent number of clinical visits to complete this procedure. Some investigators have reported the use of tricalcium phosphate as an apical barrier in The material was packed into the apical 2 mms of the canal, against which gutta-percha was compacted. The treatment was completed in one appointment, and radiographic assessment confirmed successful apexification comparable to that achieved with Calcium hydroxide Ca(OH) 2. Also, Calcium hydroxide powder has been used successfully as an apical barrier against packed gutta-percha (Schumacher 1993). One-step apexification by using a material such as MTA has great advantages of decreasing the number of appointments and reducing the clinical time. On the other hand, One-step apexification, does not result in further root development (Ballesio et al. 2006, Felippe et al. 2006). The goal of regenerative procedures is to induce biological replacements of the lost dental tissues. Many of these procedures have emerged from the growing field of tissue engineering (Langer & Vacanti 1993). Over the last several decades, the scope and clinical application of regenerative dental procedures has continuously advanced to include guided tissue regeneration, guided bone regeneration, and distraction osteogenesis (Oh et al. 2009). The application of platelet-rich plasma for guided bone regeneration has been established in several reports. Also, other materials such as Emdogain, recombinant human bone morphogenic protein (rhbmp) platelet-derived growth factor (PDGF) have been used for periodontal tissue regeneration (Heijl et al. 1997, Takayama et al. 2001). Recently, there are some investigations for the potential use of these therapies in the endodontics field. (Huang et al. 2009). Regenerative endodontics has been defined as biologically based procedures designed to replace damaged structures such as dentin, root structures, and cells of the pulp-dentin complex (Murray et al. 2007).Until these days there are two types of approaches to achieve regeneration in immature permanent teeth with necrotic pulp. The first one requires multiple visits, while the second one can be done in a single visit (Shin et al). The first approach can be accomplished by many techniques. In 2001 Iwaya et al have introduced the revascularization of necrotic pulp of an immature mandibular second premolar with periapical involvement in a 13-year-old patient by using calcium hydroxide Ca(OH) 2 (Iwaya et al. 2001). In addition, Banchs et al have suggested using triple antibiotic paste to disinfect the canal and then produce bleeding inside it to form a colt that works

3 REVASCULARIZATION OF IMMATURE PERMANENT TEETH (987) as a scaffold to allow the stem cells migration to the canal to complete the root maturation (Banchs et al. 2004). Later, Shah et al have used a light cotton pellet with formocresol as an interappointment dressing (squeezed dried) to disinfect the canal (shah et al. 2008). Finally Torabinejad et al have produced the platelet rich plasma (PRP) as an intracanal scaffold after using triple antibiotic for disinfection (Torabinejad & Turman 2011). Case Report A 14 -years-old male patient was referred to the clinic of Endodontics and Operative Dentistry at Damascus University s Dental School with a chief complaint of fracture in both maxillary central incisors. Dental History revealed an old traumatic injury to both maxillary central incisors. Initial clinical examination has showed pulpal exposure of both central incisors and intact gingiva. Electric pulp testing (Anthos, Imola-Italy) and cold test (endo ice, Frisco Spray, Ad-Arztbedarf GmbH, Germany) indicated that both teeth were non-vital. also, incomplete roots with open apices were noted radiographicly for both teeth (Figure 1). A final diagnosis was made of having necrotic pulp with open apices. The teeth were isolated by Fig. (1) Initial radiographs showing incomplete root formation with open apices of the upper central incisors. rubber dam and local anesthesia was administrated by infiltration using (Mepecaine, Mepivacaine HCL 3%, Alexandria, Egypt). Access cavities were prepared and the necrotic pulp tissues were removed. Canals were initially irrigated with 5.25 % NaOCl solution. Working lengths were established radiographically and by apex locater. The canals were irrigated to 2 mm shorter than the working length using NaOCl for three minutes followed by saline. This was followed by irrigation with saline then applying EDTA 17 % for one minute to remove the smear layer without damaging the stem cells but giving more efficiency to disinfect the canal (Trevino et al. 2009). The canal irrigation with saline then NaOCl 5.25 % was done in three minutes, followed by irrigating the canal with saline then Chlorhexidine 2 % CHX for five minutes. This is because the in vitro study has reported that root canals treated with 2 % Chlorhexidine had 72 hours of residual antimicrobial activity against Streptococcus mutans which is related to the substantivity effect of this antiseptic solution (White et al. 1997). In addition, it has been reported that interactions between NaOCl and chlorhexidine forms para-chloroaniline, which is known to be a carcinogen. Basrani et al suggested that prior to irrigating with chlorhexidine, it is recommended to wash away the existing NaOCl to diminish the formation of para-chloroaniline (Basrani et al. 2007). Equal proportions of triple antibiotics (Metronidazole & Minocycline & Ciprofloxacin) were mixed with distilled water and packed with large endodontic plugger into the canals and the teeth were temporized with light cured resin reinforced glass ionomer (Rriva Light Cure, SDI, Australia) and the patient was dismissed. The patient came to the endodontic clinic three weeks later without any symptoms. Teeth were asymptomatic to percussion and palpation tests. A 15 ml sample of whole blood was drawn from the patient s right arm for PRP and PRP gel preparation in the following procedure: First, blood was collected in sterile plastic test tubes

4 (988) E.D.J. Vol. 59, No. 1 Mohammad Aldakak, et al. that contained Citrate Phosphate Dextrose-Adenine (CPD-A, HL Haemopack, Thiruvanathapuram, Kerala, India) as an anticoagulant. After that the test tubes were shaken gently to enhance the complete mixing of the blood with the anticoagulant. Tubes were centrifuged using a refrigerated centrifugal machine at 3000 rpm for 10 min, which resulted in the separation of three basic fractions: the bottom red blood cells (RBC), middle PRP and top layer of PPP (Platelet-poor plasma) (Figure 2). Two to three milliliters of the top layer corresponding to the PPP was aspirated with a pipette and collected in a separate sterile plastic tube. This was used to obtain the autologous thrombin at the time of application. The PRP was collected in conjunction with the top 1 2 mm of the RBC fraction because the latter is also rich with newly synthesized platelets (Lekovic, Camargo et al. 2003). quantities from the same blood donor to prepare the gel (Su, Chiang et al. 2004) The antibiotic dressing was removed from the canals and supplementary disinfection of the root canals system was done again as in the first visit with a final flush using 2 % CHX solution. PRP liquid was introduced into the canals space up to the cementoenamel junction level then the PRP gel was placed over by cutting a small piece from PRP gel and applying it by sterilize tweezers in the same level (cementoenamel junction level). A mixture of white mineral trioxide aggregate (MTA) (PRO Root MTA, DENTSPLY, Johnson City, USA) was prepared in a powder/liquid ration according to the manufacturer instructions and placed over the PRP gel using endodontic pluggers. X-ray was taken to check the level of MTA plug (Figure 5), a wet cotton pellet placed and the tooth temporized. After 24 hours, the teeth reopened to check the maximum setting of MTA and then sealed with non eugenol temporary filling (MD-Temp White, Hydraulic Temporary Restorative, META Biomed Co.LTD, Korea). Fig (2) Separation of the three basic fractions after blood centrifugation PRP gel was prepared according to the method described by Su et al (Su, Chiang et al. 2004). Five grams of Beads and 0.3 ml of 10 % CaCl2 were added to PPP (10 ml) to activate coagulation. The mixture was agitated once a minute for 8 10 min at room temperature. The supernatant containing human thrombin (HT) was recovered after an additional 10 min. HT and PRP were mixed in equal Fig (3) PRP gel after preparation

5 REVASCULARIZATION OF IMMATURE PERMANENT TEETH (989) A week later, the teeth were double-sealed with permanent filling materials (3M ESPE, Filtek TM Z250, Universal Restorative, USA). The patient was recalled after six month for clinical and radiographic examination which showed complete root maturation with intact supporting soft tissues without since tract, pain, or swelling in these tissues (Figure 6). Fig (6) Radiographic photo after 6 months DISCUSSION Fig (4) Application of the PRP into the root canals of the upper central incisors Fig (5) Radiographic photo after application the MTA over the PRP Nygaard-Ostby approach was based on the well known role of the blood clot formation in wound healing and involved laceration of the periapical tissues.17 teeth were followed up from 13 days to 3 years prior to tooth extraction and histologic analysis. Both clinical and histologic findings were reported. In general, apical inflammation from the over instrumentation resolved in about two weeks. At the one month follow-up visit, the periodontal ligament healed. After ten months, the periapical bone was still forming. The blood clot in the root canal systems gradually was replaced with granulation tissue, followed by fibrous connective tissue. However, the growth of these tissues was incomplete, and there was an evidence of variable resorption of the dentinal walls along with the deposition of cementum. No newly formed dentin was observed (Nygaard-Ostby 1961, Nygaard- Ostby 1971). The blood clot might serve as a scaffold for the periapical cells including mesenchymal stem cells to migrate into the root canal and eventually induce new tissue formation in the space (Thibodeau et al. 2007). The ingrowth of periodontal tissues may reach up to the coronal pulp chamber (Nevins et al.

6 (990) E.D.J. Vol. 59, No. 1 Mohammad Aldakak, et al. 1977). In a traumatic avulsion case report, blood vessels slowly grow from the apex toward the pulp horn by replacing the necrotic pulp left behind after the avulsion injury (Skoglund et al 1978). It has been reported that teeth with an apical foramen larger than 1.1 mm have the potential for revascularization after replantation (Kling et al. 1986). Theoretically, when a well established connection between the pulp space and the periapical tissues exists, as with young teeth, periapical diseases are more likely present as a result of partially necrotic and infected pulp tissues. Vital pulp may still be present in the most apical part of the canal. If this was the case, successful removal and disinfection of the necrotic infected coronal pulp would still leave vital pulpal cells with the potential to proliferate new pulpal tissues into the coronal pulp space. Thus obturation of the root canal space, with a temporarily medicament or with gutta-percha, would eradicate the possibility for revascularization, thus be counterproductive (Iwaya & Ikawa 2001). There is no single possible mechanism of revascularization of the pulp tissues. It is possible that a few vital pulpal cells remain at the apical end of the root canal (Banchs et al & Heithersay 1970). As the root and pulp develop, the dental papilla is located apical to the developing pulp and is called the apical papilla. Clinically, this is a gelatinous soft tissue, which is easily detached from the root apex. Histologically, it is distinct from the pulp and less vascular and cellular with the two tissues separated by a cell-rich zone (Sonoyama et al. 2006, 2008(. The stem cells from the apical papilla or the bone marrow could also be the trigger for the mechanism of root development (krebsbach et al & Gronthos et al. 2000). Another possible mechanism of continued root development could be due to multipotent dental pulp stem cells (Gronthos et al. 2002). These are present in permanent teeth and might be in abundance in immature teeth. The third possible mechanism could be attributed to the presence of stem cells in the periodontal ligament (Lieberman et al & Nevin et al. 1977). Another possible mechanism could be from the blood clot itself as being a rich source of growth factors and could play an important role in regeneration (Shah et al. 2008). Hargreaves et al have identified three components contributing to the success of this procedure (Hargreaves et al. 2008).They include; (1) stem cells that are capable of formation of hard tissues, (2)signaling molecules for cellular stimulation, proliferation, and differentiation, and finally, (3)a 3-dimensional physical scaffold that can support cell growth and differentiation (Hargreaves et al. 2008). Revascularization of immature teeth with apical periodontitis is predictable if three challenges can be solved; (a) disinfection of the canal; (b) intracanal matrix for tissue in-growth; and (c) a coronal bacterial-tight-seal (Windley, Teixeira et al. 2005). The absence of bacteria is essential for successful revascularization because the new tissues will stop at the level they meet bacteria in the canal space (Myers & Fountain 1974, Yanpiset & Trope 2000). Unlike previous case reports, Ding et al proposed and discussed the importance of using PRP in patients whom it is difficult to produce bleeding in their teeth root canals. (Ding et al. 2009, Freymiller & Aghaloo 2004). Platelet-rich plasma (PRP) has been proposed as scaffold for regenerative endodontic treatment regimens (Hargreaves et al. 2008, Ding et al. 2009). PRP contains growth factors, stimulates collagen production, recruits other cells to the site of injury, produces antiinflammatory agents, initiates vascular ingrowth, induces cell differentiation, controls the local inflammatory response, and improves soft and hard tissue wound healing (Hiremath et al. 2008). Platelets make up a large part of a blood clot. They contain and secrete active growth factors and contain a number of serum proteins including fibrin,

7 REVASCULARIZATION OF IMMATURE PERMANENT TEETH (991) fibronectin, and vitronectin that can act as cell adhesion molecules for osteoconduction. Platelets also serve as a matrix for bone, connective tissue, and epithelial cells through migration in wound healing (Marx 2004). Compared with DPSC (Dental Pulp Stem Cells), SCAP (Stem Cell Apical Papilla) have faster proliferation and greater number of population doublings and increased capacity for in vivo dentine regeneration. SCAP might be the source of primary odontoblasts involved in the development of root dentine, in contrast to DPSC, which are most likely involved in reparative dentine formation (Friedlander et al. 2009). Wang et al found that revitalization approach for managing immature permanent teeth with infected pulp and/ or apical periodontitis allow ingrowth of vital tissue consisting of tissues resembling cementum, PDL and bone. These tissues are not pulp parenchymal tissues. They do not function like pulpal tissues. Therefore, revitalization is not tissue regeneration but wound repair. Pulp tissue may survive the infection, recover, and remain healthy. The clinical case reports showing severe narrowing of the canal space cannot be explained by the present study as the experimental period was very short. Although the revitalization treatment may be more favorable than traditional apexification procedures in terms of having vital tissues including cementum-like tissue deposited on canal walls, the long-term outcome of these new tissues in the canal space needs further investigation (Wang et al. 2010). Based on the first histological evidence in the dental literature, it appears that pulp-like tissue can be generated in a human tooth with the use of PRP as a scaffold in regenerative endodontic procedures (Torabinejad & Faras 2012). CONCLUSIONS On the basis of the present case report with shortterm follow up, it appears that revascularization of non-vital immature teeth by using Platelet-Rich Plasma PRP as a scaffold in the intracanal root is possible and participate in root development, and continued apical closure. REFERENCES 1. Andreasen JO, Farik B, Munksgaard EC (2002) Long-term calcium hydroxide as a root canal dressing may increase risk of root fracture. Dent Traumatol 18, Ballesio I, Marchetti E, Mummolo S, Marzo G (2006) Radiographic appearance of apical closure in apexification: follow-up after 7-13 years. Eur J Paediatr Dent 7, Banchs F, Trope M (2004) Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol?. J Endod 30, Basrani B, Manek S, Sodhi R, Fillery E, Manzur A (2007) Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 33, Beer R et al (2006) Pocket Atlas Of Endodontics, Emergencies with Incomplete Root Formation, Thieme Capurro M, Zmener o (1999) Delayed apical healing after apexification treatment of non-vital immature tooth: a case report, Endod Dent Traumatol 15(5), Chueh L-H, Huang GTJ (2006) Immature teeth wit periradicular 8. periodontitis or abscess undergoing apexogenesis: a paradigm shift. J Endod 32, Ding RY, Cheung GSP, Chen J, et al (2009) Pulp revascularization of immature teeth with apical periodontitis: a clinical study. J Endod 35, Felippe WT, Felippe MC, Rocha MJ (2006) The effect of mineral trioxide aggregate on the apexification and periapical healing of teeth with incomplete root formation. Int Endod J 39, Frank AL (1966) Therapy for the divergent pulpless tooth by continued apical formation. J Am Dent Assoc Freymiller EG, Aghaloo TL (2004) Platelet-rich plasma: ready or not? J Oral MaxillofacSurg 62, Friedlander LT, Cullinan MP, Love RM (2009) Dental stem cells and their potential role in Apexogenesis and Apexification. Int Endod J 42, Gronthos S, Brahim J, Li W, et al (2002) Stem cell properties of human dental pulp stem cells. J Dent Res 81, Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 97,

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