|Year : 2016 | Volume
| Issue : 1 | Page : 4-10
Revascularization of immature necrotic teeth with platelet-rich fibrin and blood clot
AR Prabhakar, N Shantha Rani, Chandrashekar Yavagal
Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India
|Date of Web Publication||21-Jul-2016|
N Shantha Rani
Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere - 577 004, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Endodontic treatment of immature permanent teeth with necrotic pulp, with or without apical pathosis, poses several clinical challenges. Regenerative endodontics is a promising alternative for the treatment of such immature necrotic permanent teeth. Aim: To assess the regenerative potential of young permanent teeth with necrotic pulps with platelet-rich fibrin (PRF) and blood clot using radiographic and clinical methods. Settings and Design: This was an experimental, in vivo study comparing the two groups. Materials and Methods: Immature necrotic permanent teeth (n = 14) in patients who belonged to the age group of 10-12 years were selected and divided into two groups. Group 1 was treated by conventional revascularization with blood clot and Group 2 with revascularization using PRF. Follow-up was done for 6 months. Standardized radiographs were analyzed for the periradicular healing and apical closure, and clinical analysis was performed using mobility index as compared to baseline values. Statistical Analysis Used: Results were analyzed using Friedman test, Wilcoxon test, and Mann-Whitney U-test. Results: There was increased periradicular healing and apical closure at 3 months and 6 months in Group 2 (PRF) when compared to Group 1 (conventional). Conclusions: Revascularization with PRF procedure leads to better and faster healing, reduced radiolucency, enhanced apical closure, and reduction in mobility of necrotic immature teeth.
Keywords: Blood clot, immature necrotic teeth, open apex, platelet-rich fibrin, revascularization
|How to cite this article:|
Prabhakar A R, Rani N S, Yavagal C. Revascularization of immature necrotic teeth with platelet-rich fibrin and blood clot. Int J Oral Health Sci 2016;6:4-10
|How to cite this URL:|
Prabhakar A R, Rani N S, Yavagal C. Revascularization of immature necrotic teeth with platelet-rich fibrin and blood clot. Int J Oral Health Sci [serial online] 2016 [cited 2019 Sep 17];6:4-10. Available from: http://www.ijohsjournal.org/text.asp?2016/6/1/4/186657
| Introduction|| |
Endodontic management of necrotic immature permanent teeth with open apices is a challenge for pediatric dentists because of the presence of thin dentinal walls and lack of apical constriction against which an obturation could be placed. ,
Traditionally, the clinical procedure for managing a necrotic immature tooth was called "apexification," and it involved the placement of calcium hydroxide as an intracanal medicament to eliminate the intraradicular infection as well as to stimulate the calcification at the apex. However, it called for multiple visits and a long duration before the root filling could be accomplished. 
Recently, single visit apexification with mineral trioxide aggregate (MTA) gained interest, as it was able to create an artificial barrier against which the obturating material could be compacted. The fact that this procedure could be completed in a single appointment posed a major advantage.  However, none of these "apexification" techniques contributed to continued root maturation thus predisposing them to fractures which further supported the critics of the technique who question the very selection of such an intervention. ,
A novel concept of revascularization of immature necrotic teeth was introduced by Nygaard Ostby in the 1960s, only to be reinforced further by the studies of Rule and Winter, who documented root development and apical barrier formation even in nonvital teeth in children. In 1972, Ham et al. showed the apical closure of immature pulpless teeth in monkeys.  In 2001, Iwaya et al. and in 2004, Banchs and Trope, further reiterated the advantages of this treatment modality, which resulted in a normal development of the entire root that was evident even radiographically. 
Regenerative procedures have thus proved to be an exciting alternative over the traditional methods. The usual method of achieving revascularization is by inducing bleeding into the pulp canal space by mechanically irritating the periapical tissues. The blood clot thus formed acts as a matrix for the in-growth of new tissues into the pulp canal. However, this procedure will cause discomfort to the patient during the process of mechanical irritation of periapical tissues.  Choukroun's platelet-rich fibrin (PRF) is a second-generation platelet concentrate, which is totally autologous and contains platelets, growth factors, and cytokines that might increase the healing potential of both soft and hard tissues with absolutely no discomfort or irritation. 
However, there is paucity of evidence to categorically recommend one technique over the other. Hence, this study was designed to evaluate and compare the revascularization achieved through these two modalities, namely conventional blood clot and PRF.
| Materials and Methods|| |
Ethical clearance for this study was obtained from the Institutional Review Board of Bapuji Dental College and Hospital, Davangere.
This is an experimental, in vivo study.
This study was carried out at the Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere.
Teeth with necrotic pulps, as a result of trauma or carious exposures, with signs and/or symptoms of periapical pathology and teeth with immature apex, either tubular or blunderbuss in patients who belonged to the age group of 10-12 years were included in this study.  Patients with systemic illness and teeth with vertical or horizontal root fractures were excluded from the study.
- Group 1 - Revascularization of immature necrotic teeth with blood clot (conventional)
- Group 2 - Revascularization of immature necrotic teeth with PRF.
Fourteen patients were selected as per the above inclusion criteria. Informed consent was taken from the accompanying parent/guardian for conducting the procedure. Assent was obtained from all the patients. The teeth were evaluated clinically and radiographically to determine the preoperative status with regard to sensitivity, tooth mobility, and presence or absence of sinus tract.
Under rubber dam isolation, access preparation was done on necrotic immature permanent teeth of both the groups. Minimal instrumentation was done, and the canal was irrigated with 10 ml of 2.5% NaOCl. Canal was then dried with paper points. Triple antibiotic paste in equal proportions of ciprofloxacin, metronidazole, and minocycline was ground and mixed with sterile saline to achieve a thick paste like consistency. This antibiotic mixture was placed in the canal using an amalgam carrier and packed with large endodontic pluggers. The access cavity was sealed with Cavit (3M ESPE, Germany). Patients were recalled after 2 weeks, and the tooth was examined for sensitivity to percussion and palpation. Under rubber dam isolation, triple antibiotic paste was removed from the canal using irrigation with 2.5% sodium hypochlorite. Canals were dried again, and one of the following procedures was carried out. 
Under local anesthesia, the apical tissue was stimulated with a no. 20 K-file to induce bleeding. Bleeding was controlled at a level just below the cementoenamel junction (CEJ). A blood clot was formed in the canal about 15 min after stimulation. MTA (Angelus Industria de Produtos Odontologicos S/A, Brasil) was applied over the blood clot, followed by a moist cotton pellet.
In Group 2, instead of blood clot, a PRF membrane was pushed with hand plugger to 1 mm beyond the confines of working length and at a level just below CEJ. MTA was then placed directly over the PRF clot, followed by moist cotton pellet. 
In both the experimental groups, Cavit was placed temporarily over the cotton pellet. After 72 h, permanent restoration was placed with composite resin. Patients were recalled after 3 and 6 months for postoperative checkup. 
Preparation of platelet-rich fibrin
A volume of 5 ml of whole blood was collected from the median cubital vein of the patient. Blood was subjected to centrifugation at 2400 rpm for 12 min for the preparation of PRF utilizing Choukroun's method [Figure 1] and [Figure 2]. 
After 3 and 6 months, the treated teeth were observed for mobility which could be elicited using the butt ends of two mirror handles, one on the buccal aspect and one on the lingual aspect of the tooth.
According to Miller, the following scoring method was used:
- Score 0 - Normal physiologic mobility
- Score 1 - Mobility <1 mm
- Score 2 - Mobility >1 mm in horizontal direction
- Score 3 - Mobility >1 mm in horizontal direction with vertical depressability. 
After 3 and 6 months, standard radiographs were taken for all teeth included in this study. Each individual root was taken as a unit for assessment [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9] and [Figure 10].
|Figure 3: Radiograph showing immature necrotic upper left central incisor with open apex|
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|Figure 8: Immediate postrevascularization with platelet-rich fibrin method|
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|Figure 10: After 6 months follow-up. Radiograph showing increased root length and apical closure|
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- For periradicular healing, the following scoring method was used:
- Score 0 - No radiological changes seen when compared to preoperative radiograph
- Score 1 - Slight decrease in radiolucency seen in periapical region
- Score 2 - Complete reduction of radiolucency seen in periapical region.
- For Apical closure
- Score 0 - No changes were seen when compared to preoperative radiograph
- Score 1 - Partial apical closure
- Score 2 - Complete apical closure.
| Results|| |
- Intergroup comparison for periradicular healing, apical closure, and mobility was done by Friedman test and Wilcoxon sign rank test as a post hoc
- Mann-Whitney U-test was used for pair-wise comparisons
- Statistical analysis was done using Statistical Package for the Social Sciences SPSS (IBM SPSS statistics for windows, version 22.0, IBM corp, Armonk, NY).
Statistical analysis showed no significant difference between both the groups at baseline and 6 months, but there was statistically significant difference found at 3 months in both the groups [Table 1].
|Table 1: Comparison of experimental groups for periradicular healing at different time intervals|
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However, median values of Group 2 (PRF) were >Group 1 (conventional) at 3 months and 6 months [Graph 1].
Statistical analysis showed no significant difference between both the groups at baseline, 3 months, and at 6 months [Table 2].
|Table 2: Comparison of experimental groups for apical closure at different time intervals|
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However, median values of Group 2 (PRF) were >Group 1 (conventional) at 3 months and 6 months [Graph 1].
Statistical analysis showed no significant difference between both the groups at baseline, 3 months, and at 6 months [Table 3].
|Table 3: Comparison of experimental groups for mobility at different time intervals|
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| Discussion|| |
Pulpal necrosis of an immature tooth due to caries or trauma could arrest further root development, leaving the tooth with thin fragile canal walls and open apices.  In the past, such teeth were treated with long-term calcium hydroxide and/or MTA in an attempt to induce apexification. However, these procedures resulted in reduced root strength, thin dentinal walls, and no further root development, thus posing a challenge to the clinician. 
Regeneration of tissues is an emerging and an exciting area of dentistry which has seen rapid progress in the recent past. The ultimate goal of regenerative endodontics is to create a platform for maturation of nonvital immature tooth.  The root maturation may be attributed to many mechanisms. One of the possible mechanisms postulates that the remnants of Hertwig's epithelial root sheath or cell rests of malassez are sufficiently resistant to periapical infections and thus remain at the apex in a vital state.  These are capable of stimulating various stem cells such as stem cells of the apical papilla, periodontal ligament stem cells, and multipotent pulp stem cells (dental pulp stem cell [DPSC]) to differentiate into bone or dentin-forming cells which help in the normal root maturation. 
Another possible mechanism of root development could be attributed to the stem cells from the apical papilla or the bone marrow. Instrumentation beyond the apex of the root canals will induce bleeding which can further transplant mesenchymal stem cells from the bone into the canal lumen. These cells have extensive proliferating capacity. 
The third possible mechanism could be that the blood clot itself, being a rich source of growth factors, could play an important role in regeneration. The growth factors involved include platelet-derived growth factor, vascular endothelial growth factor, platelet-derived epithelial growth factor, and tissue growth factor. These factors can stimulate differentiation, growth, and maturation of fibroblasts, odontoblasts, and cementoblasts from immature, undifferentiated mesenchymal cells of the newly formed tissue matrix.  In the present study, young patients were selected because of their greater healing capacity and thus more stem cell regenerative potential. 
The triad of regenerative endodontics includes disinfection of root canals, placement of a matrix or scaffold conducive to cellular proliferation, and a tight bacterial seal for the access opening.  In the present study, results showed a significant difference in the periradicular healing at 3 months in both the groups. This indicates a favorable healing response to the proposed treatment, which was initiated by canal disinfection via triple antibiotic paste, the combination of ciprofloxacin, metronidazole, and minocycline, mixed with saline as recommended by Hoshino et al. 
Scaffolds are used in regenerative procedures to provide a framework through which cells could vascularize the necrosed pulp again. Using a stable blood clot can act as a scaffold in immature teeth as suggested by many studies.  In this study, we used sterile # 20 k file into the apical tissues to initiate bleeding into the canal. Bleeding was controlled by using a sterile cotton pellet. After the clot got formed, it was carefully touched with the butt-end of a large paper point to confirm the stability and then it was covered with MTA. The blood clot inside the root canal space provides a protein-rich scaffold that contains platelet-derived growth factors and mesenchymal stem cells. 
Like previous case reports, we used PRF as a scaffold in group II, and it showed marginally better results compared to group 1 [Graph 1]. However, PRF has many advantages over traditionally prepared platelet-rich plasma. Its main advantages include the ease of preparation, avoid the biochemical handling of blood, and require only single centrifugation cycle, which makes this preparation strictly autologous. It has been documented that PRF has a very significant sustained yet slow release of various growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF) extending up to 28 days. This could have led to the idea of using PRF as a matrix or scaffold for pulp regeneration. 
These growth factors can stimulate scaffold remodeling, proliferation of undifferentiated mesenchymal stem cells such as DPSCs and promote angiogenesis. Moreover, the natural fibrin of PRF also protects the growth factors from proteolysis. PRF clot traps all important circulating immune cells and different cytokines that act against infection. Most importantly, unlike PRP, PRF by Choukroun's technique does not dissolve quickly after application. 
In the present study, MTA was packed directly over PRF and blood clot so as to obtain a tight seal. This can be justified by the fact that MTA is known to be resistant to bacterial leakage, and it has a remarkable biocompatibility. MTA is also able to set in a moist environment such as the bleeding area of the tooth,  and MTA itself provides signaling molecules for the maturation of stem cells.  The limitation of this study is that it has been documented on a comparatively slender sample size. Further long-term studies with more extensive samples are needed to establish the superiority of PRF over conventional blood clot, as far as the long-term success of the regenerative procedures is concerned.
| Conclusions|| |
Within in the limitation of this study, revascularization with PRF showed marginally better results than conventional revascularization with blood clot. However, further long-term clinical trials are necessary to concretely establish the findings that were noted in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
[Table 1], [Table 2], [Table 3]