|Year : 2016 | Volume
| Issue : 1 | Page : 9-15
A comparative evaluation of natural and artificial scaffolds in regenerative endodontics: A clinical study
Shreya Sharma, Neelam Mittal
Department of Conservative Dentistry and Endodontics, Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Web Publication||16-Dec-2015|
Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Aim: To evaluate and compare the regenerative potential of natural autologous scaffolds (blood clot and platelet rich fibrin [PRF]) with artificial scaffolds (commercially available collagen and poly-lactic-co-glycolic acid [PLGA] polymer) in inducing apexogenesis in necrotic immature permanent teeth. Materials and Methods: Necrotic immature permanent maxillary incisors with or without radiographic evidence of periapical lesion were included. Access opening was done under rubber dam isolation. Canal disinfection was done using minimal instrumentation, copious irrigation, and triple antibiotic paste as interappointment medicament for 4 weeks. After 4 weeks, asymptomatic teeth were divided into four groups on the basis of scaffolds used for revascularization procedure: Group I (blood clot); Group II (PRF); Group III (collagen); Group IV (PLGA). The clinical and radiographic evaluations of teeth were done at 6 and 12 months after the procedure and compared with baseline records. Result: Clinically, patients were completely asymptomatic throughout the study period. Radiographically, all cases showed improvement in terms of periapical healing, apical closure, root lengthening, and dentinal wall thickening. PRF and collagen gave better results than blood clot and PLGA in terms of periapical healing, apical closure, and dentinal wall thickening. Conclusion: Revascularization procedure is more effective and conservative over apexification in the management of necrotic immature permanent teeth. This study has shown that PRF and collagen are better scaffolds than blood clot and PLGA for inducing apexogenesis in immature necrotic permanent teeth.
Keywords: Open apex, revascularization, scaffold
|How to cite this article:|
Sharma S, Mittal N. A comparative evaluation of natural and artificial scaffolds in regenerative endodontics: A clinical study. Saudi Endod J 2016;6:9-15
|How to cite this URL:|
Sharma S, Mittal N. A comparative evaluation of natural and artificial scaffolds in regenerative endodontics: A clinical study. Saudi Endod J [serial online] 2016 [cited 2019 Jul 20];6:9-15. Available from: http://www.saudiendodj.com/text.asp?2016/6/1/9/171995
| Introduction|| |
Necrotic immature teeth that have open and divergent apices are not suitable for cleaning and obturation with traditional techniques and materials. Because of their thin dentinal walls, these teeth are susceptible to subsequent fracture even after treatment. Traditionally, teeth with open apex have been treated using apexification procedure with calcium hydroxide , or MTA., This, however, will not lead to root development. Thus, the ideal outcome for a tooth with an immature root and necrotic pulp would be the regeneration of pulp tissue into a canal capable of promoting the continuation of normal root development.
Extrapolating the results of human avulsion case series  and controlled animal studies,, it was hypothesized that disinfection of a necrotic infected immature permanent tooth with apical periodontitis may render it to the same starting point as a necrotic uninfected avulsed immature permanent tooth. Revascularization should be possible for the disinfected canals, just as it is for the uninfected canals in the avulsion scenario., The advantage of regenerative endodontic procedures over apexification procedures is that it allows root thickening and lengthening to continue by the generated vital tissue.
Hargreaves et al. have identified three components contributing to the success of this procedure. They include stem cells that are capable of hard tissue formation, signaling molecules for cellular stimulation, proliferation, and differentiation, and finally, a three-dimensional physical scaffold that can support cell growth and differentiation.
An empty canal space will not support in-growth of new tissue from the periapical area on its own. A scaffold provides the framework for cell growth and differentiation at a local site. Ideally, a scaffold should be porous, biocompatible with the host tissues, the correct shape and form to allow for replacement of the lost tissues, and biodegradable. Various natural and artificial scaffolds have been used to regenerate dentin or dentin-pulp complexes in combination with dental pulp cells. Natural scaffolds offer good biocompatibility and bioactivity, whereas synthetic scaffolds offer more control over the degradation rate and mechanical properties.
In nearly all revascularization case reports, immature permanent human teeth with a clinical diagnosis of pulpal necrosis are disinfected, and bleeding is evoked. Platelet rich fibrin (PRF) is a second generation platelet concentrate, which has a very significant slow sustained release of key growth factors for at least 1-week , and up to 28 days. PRF could stimulate its environment for a significant time during wound healing. This lead to the idea of using PRF membrane for pulp regeneration.
Although blood clot and PRF are natural autologous scaffolds for pulp regeneration, intentional periapical filing to induce the blood clot formation can cause discomfort for the patient. Venous blood drawn from the patients to make PRF can add to their discomfort and can make children less cooperative during the treatment. Hence, commercially available scaffolds like collagen, and polymers such as poly glycolic acid (PGA), polylactic acid (PLA), poly-lactic-co-glycolic acid (PLGA), polycaprolactone (PCL), etc., can be used.
As a scaffold, collagen allows for easy placement of cells and growth factors and allows for replacement with natural tissues after undergoing degradation. Dental pulp–like structures have been successfully generated in mice by transplantation of dental pulp stem cells onto a collagen scaffold. Several synthetic polymers, such as PLA, PGA, PLGA, have been suggested as potential scaffolds for pulp regeneration. These are nontoxic, biodegradable and allow precise manipulation of the physicochemical properties, such as mechanical stiffness, degradation rate, porosity, and microstructure. Huang et al. used PLGA scaffolds seeded with stem cells from apical papilla and dental pulp stem cells in a tooth slice implantation model wherein dentin like tissue and pulp like tissue was regenerated after 3–4 months of subcutaneous implantation of teeth in immunocompromised mice.
The purpose of this clinical study was to evaluate and compare the regenerative potential of blood clot, PRF, collagen and PLGA scaffolds in immature necrotic permanent teeth.
| Materials and Methods|| |
Regenerative endodontic procedure was done in 16 cases of immature necrotic permanent teeth using blood clot, PRF, collagen, and PLGA as four different scaffolds after approval from the ethical committee of the university. Patients, both male and female, belonged to the age group of 10–25 years. Necrotic immature permanent maxillary incisors, due to trauma, with or without radiographic evidence of periapical lesion were selected. Medically compromised patients with systemic conditions that compromise the healing response or cause bleeding tendencies were excluded. Out of the 16 selected cases, 13 had periapical pathology while 3 cases had widening of periodontal ligament space without periapical apthology. Clinically, 9 cases had a history of spontaneous pain, 7 cases had tooth discoloration, 2 had intraoral sinus and 2 were tender on percussion. The detailed treatment protocol was explained to the patient (or parents if the patient was below 14 years of age) and written consent was obtained.
Under rubber dam isolation, access opening was done in teeth with #2 round diamond bur (Endo Access Bur, DENTSPLY Maillefer). Further axial wall extensions were done with safe tip fissure carbide bur (Endo-Z Bur, DENTSPLY Maillefer). Minimal canal instrumentation was done with K–files to remove the necrotic tissue, and the canals were copiously irrigated with 2.5% sodium hypochlorite solution (Nimai Dento India) using a syringe and side vented needle. The triple antibiotic paste was used as the interappointment medicament for 4 weeks, and the access cavity was sealed with temporary restorative material Cavitemp (AMMDENT, Mohali, India).
After 4 weeks, teeth were re-accessed under rubber dam isolation, and the triple antibiotic paste was washed out of the canal using copious amount of 2.5% sodium hypochlorite solution. Canals were dried, and further revascularization procedure was carried out only if the tooth was asymptomatic with no drainage from the canal.
16 cases of immature necrotic permanent maxillary incisors, selected for the study, were randomly divided into four groups with each group containing four cases.
Group I: Blood clot
Under local anesthesia without adrenaline, a sterile 23 gauge needle was passed beyond the confines of the working length and bleeding was induced in the canal. When frank bleeding was evident from the canal, a tight cotton pellet was inserted in the coronal portion of canal and pulp chamber for 7–10 min to induce clot formation in the apical two third of the canal. Access cavity was sealed with glass ionomer cement [Figure 1].
|Figure 1: Radiographs of teeth number 11.21 showing preoperative status (a), status after 6 months (b) and after 12 months (c) of using blood clot as scaffold|
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Group II: Platelet-rich fibrin
Platelet-rich fibrin was prepared by drawing 5 mL of venous blood from the patient in dried glass test tube and immediately centrifuging it at 3000 rpm for 10 min. After centrifugation, three layers were formed in the test tube–base layer of RBCs, top layer of acelluler plasma, and a PRF clot in the middle. This clot was then pressed between two gauge pieces to form a membrane. The tooth was re-accessed in the same manner as described for Group I. PRF was introduced into the canal and carried to the apical part of root canal using endodontic pluggers. Access cavity was sealed with glass ionomer cement [Figure 2].
|Figure 2: Radiographs of teeth number 11.21 showing preoperative status (a), status after 6 months (b) and after 12 months (c) of using platelet-rich fibrin as scaffold|
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Group III: Collagen
The teeth in this group were re-accessed as mentioned for Group I. Blood clot was induced in the root canal as done in Group I. Sterile collagen sponge (Cologenesis Healthcare Pvt. Ltd., India) was then inserted into the root canal and pushed with the help of endodontic pluggers. Access cavity was sealed with glass ionomer cement [Figure 3].
|Figure 3: Radiographs of tooth number 21 showing preoperative status (a), status after 6 months (b) and after 12 months (c) of using collagen as scaffold|
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Group IV: Poly-lactic-co-glycolic acid
The teeth in this group were re-accessed as mentioned for Group I. Blood clot was induced in the root canal as done in Group I. Sterile PLGA crystals (Sigma Aldrich, USA) were introduced into the canal with the help of tweezer and pushed inside with endodontic plugger. Access cavity was sealed with glass ionomer cement [Figure 4].
|Figure 4: Radiographs of tooth number 21 showing preoperative status (a) and after 12 months (b) of using poly-lactic-co-glycolic acid as scaffold|
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Preoperative intra oral periapical radiograph was taken as base line record. The clinical and radiographic evaluations of teeth were done at 6 and 12 months after the procedure and compared with base line records. The clinical and radiographic evaluation was done by two independent observers who were blinded from the groups.
The scoring criteria used for radiographic healing was as follows: No healing/improvement form baseline was denoted by 0, Fair healing/improvement from baseline by 1, Good healing/improvement from baseline by 2 and Excellent healing/improvement from baseline by 3 respectively.
The data were analyzed by one-way ANOVA test of significance and Z-test for proportions and P < 0.05 was considered statistically significant.
| Results|| |
Clinical and radiographic evaluation after 12 months follow-up showed that all the 16 cases showed improvement as compared to the baseline levels.
Clinically, all the groups showed excellent results. Patients were completely asymptomatic throughout the study period with no tenderness to palpation and percussion. The swelling and sinus had resolved completely and did not reappear.
Radiographically, all 16 cases showed improvement in terms of periapical healing, apical closure, root lengthening and dentinal wall thickening.
Platelet-rich fibrin gave best results with 75% cases showing excellent periapical healing. This was followed by Group III (collagen), which showed excellent results in 25% cases. This was followed by Group I (blood clot). Least periapical healing was seen in Group IV (PLGA) with 75% cases showing only fair amount of periapical healing [Table 1]. One-way ANOVA showed statistically significant difference between these groups (P = 0.026). According to Z-test for proportions, difference between Group II and IV (P = 0.028) and between Group III and IV (P = 0.028) was statistically significant. This implies that Group II (PRF) and Group III (collagen) were better than Group IV (PLGA).
Group II (PRF) and Group III (collagen) gave best results, both with 50% cases showing excellent results. This was followed by Group I (blood clot) with 75% cases showing good apical closure. PLGA group was least effective in apical closure with only 50% cases showing good results [Table 2]. There was no statistically significant difference between these groups in terms of apical closure (P = 0.197).
Group I (blood clot) gave best results with 75% cases showing good root lengthening. Group IV (PLGA) was next with 50% cases showing good results. This was followed by Group III (collagen), which showed only 25% cases with good results. PRF (Group II) gave the least effective results with all the cases showing only fair amount of root lengthening [Table 3]. There was no statistically significant difference between these groups in terms of root lengthening (P = 0.168).
Dentinal wall thickening
Group III (collagen) showed best results with 25% cases showing excellent result. PRF group was next with 75% cases showing good wall thickening. This was followed by Group I (blood clot) with 50% cases showing good results. PLGA was the least effective scaffold for this property as only 25% cases showed good dentinal wall thickening [Table 4]. There was no statistically significant difference between these groups in terms of dentinal wall thickening (P = 0.383).
| Discussion|| |
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.
A proper scaffold material is an essential component in the revascularization procedure. It provide sites for stem cell adhesion, to support cell proliferation and differentiation, and thus to promote tissue regeneration. In an in vitro study, Chandrahasa, showed that different chemical composition of scaffolds result in different rates of mature dental pulp cell proliferation.
In this study, four different scaffolds were used-blood clot, PRF, collagen and PLGA. PRF and collagen gave better results than blood clot and PLGA in terms of periapical healing, apical closure and dentinal wall thickening, though these differences were not statistically significant.
Developed in France by Choukroun et al. PRF production protocol attempts to accumulate platelets and released cytokines in a fibrin clot. Granules present in platelet contain many proteins, which may be platelet specific (e.g., beta-thromboglobulins) or nonplatelet specific (fibronectin, thrombospondin, fibrinogen, and other coagulation, growth promoters, fibrinolysis inhibitors, immunoglobulins, etc.). Activation and degranulation is important to release the cytokines (interleukin-1 (IL-1) beta, IL-6, tumor necrosis factor-alpha) and tumor growth factors (TGF beta 1, platelet derived growth factor, vascular endothelial growth factor, epidermal growth factor) that stimulates cell migration and proliferation within the fibrin matrix and thus begins the healing process. Dohan et al. showed that Choukroun's PRF seemed to stimulate simultaneously, in a dose-dependent way, the proliferation of oral bone mesenchymal stem cells and some kind of differentiation characterized by a strong activity of alkaline phosphatase, and the formation of mineralization nodules. In our study, PRF gave best results for the tested parameters. All the cases (100%) showed good or excellent periapical healing and apical closure. 75% cases showed good dentinal wall thickening. The success of PRF as a scaffold can be attributed to the fact that it a strong but flexible membrane with rich quantities of growth factors required for cellular proliferation, differentiation and angiogenesis  available for a long duration., However, in terms of root lengthening, blood clot was better than PRF with all the cases showing only fair amount of root lengthening. This finding could not be explained.
A collagen scaffold was selected because of its ease of placement and similarity to the collagen component of normal human dental pulp. Yamauchi et al. demonstrated that the use of a crosslinked collagen scaffold with bleeding induction significantly increased formation of mineralized tissues in teeth with incomplete root development and periapical periodontitis. Dental pulp–like structures  and complete tooth morphology with rootlike structures  have been obtained from the collagen sponge scaffolds. In this study also, collagen scaffold gave very good results, almost equivalent to that of PRF. 100% cases showed good or excellent periapical healing, 75% cases showed good apical closure, 75% cases showed good or excellent dentinal wall thickening and 25% cases showed good root lengthening. This could be due to the osteoinductive property of collagen. Its resemblance to the natural extracellular matrix also helps in stem cell adhesion, proliferation and differentiation on this scaffold.
The induced bleeding and subsequent formation of blood clot serves as a scaffold and also as a source of stem cells from the granulation tissue, apical papilla, PDL, and/or from the bone marrow and peripheral blood. Human dentin contains several angiogenic growth factors  that can promote tissue regeneration in the root canal space. Blood clot in the disinfected empty root canal space along with growth factors derived from dentinal walls plays the role of a protein-rich scaffold. An animal study has shown that root canals that had a blood clot formed inside them after disinfection had better radiographic treatment outcomes regarding the thickening of root canal walls and apical closure compared with those that did not have a blood clot in the canal space.
However, blood clot makes a weak fibrin mesh as compared to PRF. It may also get disintegrated in the root canal as a result of which there might be no carrier for stem cells to proliferate. Therefore, collagen along with bold clot gave better results than blood clot alone. Blood clot is not a concentrated source of growth factors as PRF. This can also be a reason for PRF and collagen giving better results.
Poly-lactic-co-glycolic acid is the first copolymer mixture to gain approval from Food and Drug Administration. PLGA has been used in engineering bone, liver and cartilage. Bertoldi tested a 50:50 PLGA copolymer and stated that it is free of inflammation effects, seems to stimulate bone growth, and decomposes after 6–8 months. They also showed a strong alkaline phosphatase expression, indicating a probable promotion effect on bone cells. El-Backly et al. showed that 50/50 PLGA has good porosity for osteoconduction and that PLG scaffold may act as a suitable matrix to support dental pulp stem cells and their differentiation to form an organized dentine/pulp-like tissue  hence we used 50:50 PLGA scaffold. Tooth like structures have been obtained by seeding dental pulp stem cells on PLGA scaffolds., In our study, however, only 25% cases showed good periapical healing and dentinal wall thickening. And 50% cases showed good apical closure and root lengthening. In this study, PLGA gave the least impressive results when compared with the other three scaffolds used. This can be attributed to the fact that all previously done studies were animal studies. The effect of PLGA as a scaffold for regenerative endodontics has never been studied in human subjects. Therefore, those results cannot be directly extrapolated for humans. Blood clot, PRF and collagen have constituents of human extracellular matrix while PLGA does not. This can also be a reason for its inferior results as compared to the other three scaffolds.
Continued root development does not appear to be a predictable outcome of immature permanent necrotic teeth after revascularization procedures in humans. A retrospective evaluation of radiographic outcomes in immature teeth with necrotic root canal systems treated with regenerative endodontic procedures indicated that only a certain percentage of cases showed increase in root length. In our study also, only 37.5% cases showed good root lengthening. But, 75% cases showed good apical closure confirming that regenerative endodontics is a successful treatment modality.
| Conclusion|| |
The present study, combined with prior reports on revascularization of the nonvital immature permanent teeth, constitute a growing case series suggesting that this biologically based treatment approach is of particular value in restoring root development and apical closure in these otherwise difficult cases. This pilot study also shows that PRF and collagen are better scaffolds than blood clot and PLGA for revascularization procedure in immature necrotic permanent teeth.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]