Year : 2009 | Volume
: 20 | Issue : 4 | Page : 442--447
Effects of platelet-rich plasma on healing of alveolar socket: Split-mouth histological and histometric evaluation in Cebus apella monkeys
Roberto S Pessoa, Sergio R Oliveira, Helder HM Menezes, Denildo de Magalhaes
Department of Periodontology, Faculty of Odontology, Federal University Of Uberlāndia, Brazil
Roberto S Pessoa
Department of Periodontology, Faculty of Odontology, Federal University Of Uberlāndia
Context: The prediction of implant treatment is directly influenced by the quality of the remaining bone after tooth extraction.
Aims : The purpose of this experimental study was to, histologically and histometrically, evaluate the bone repair process in the central areas of extraction sockets filled with platelet-rich plasma.
Materials and Methods: Four young adult male Cebus apella monkeys were used. The extraction of both right and left inferior second premolars was accomplished. After extraction, in one of the extraction sockets, coagulum was maintained while in the other it was removed; the alveolus was dried with gauze compress and filled up with platelet concentrate. For PRP production, Sonnleitner«SQ»s protocol was followed. The specimens for histological and histometric assessment were obtained in 30, 90, 120 and 180 days intervals.
Results: In 30 days new bone formation was intense in both experimental and control sockets and no significant differences were observed between the two groups. After 90 days of the extraction, while the control group showed signs of decrease in osteogenesis, in the experimental unit, the process of bone formation and fibroblast-like cell proliferation remained intense. After 120 days, the PRP treated socket was occupied by large trabeculae of bone. After 180 days, the control unit was occupied mostly with bone marrow. The experimental unit remained occupied with large amounts of bone tissue.
Conclusions: It was possible to conclude that bone repair was enhanced by the use of platelet- rich plasma in alveolar sockets.
|How to cite this article:|
Pessoa RS, Oliveira SR, Menezes HH, de Magalhaes D. Effects of platelet-rich plasma on healing of alveolar socket: Split-mouth histological and histometric evaluation in Cebus apella monkeys.Indian J Dent Res 2009;20:442-447
|How to cite this URL:|
Pessoa RS, Oliveira SR, Menezes HH, de Magalhaes D. Effects of platelet-rich plasma on healing of alveolar socket: Split-mouth histological and histometric evaluation in Cebus apella monkeys. Indian J Dent Res [serial online] 2009 [cited 2021 Jun 20 ];20:442-447
Available from: https://www.ijdr.in/text.asp?2009/20/4/442/59448
Osseo-integrated implants have been used for the replacement of lost teeth in a variety of clinical situations with high success rates.  However, despite the protocol used for implant installation and loading, studies have shown that the prediction of this modality of treatment is directly influenced by the quality of the remaining bone after tooth extraction. ,
In this way, techniques and material have been studied and developed for the maintenance of bone quality and quantity after tooth extraction. One of the most recent is the Platelet-rich Plasma (PRP) which has been used to enhance damaged or lost buccal tissues healing process. ,,,[ 7] Some researchers contributed to diffuse the idea that the PRP placed in dental alveolus and in association with autogenous and xenogenous grafts produced higher levels of bone maturation and density. ,,, In a case controlled study, Anitua  included 20 patients who underwent tooth extraction and posterior implants placement. Ten patients had their extraction sockets treated with PRP. The remaining 10 received the same surgical procedures, but extraction sockets were maintained by coagulum. After 10 to 16 weeks, at the time of implant placement, Anitua removed biopsies from the extraction sockets with a trephine drill. From histological analysis of samples, he concluded that the PRP was able to stimulate a greater bone formation and maturation.
However, more recent studies questioned the effectiveness of PRP to stimulate bone repair. ,,, These studies
conclude that PRP does not seem to enhance new bone formation.
Therefore, considering the contradictions of research results, the purpose of this experimental study was to histologically and histometrically evaluate the healing process in the central areas of extraction sockets filled with platelet-rich plasma.
Materials and Methods
Four young adult (above 4 years of age) male Cebus apella monkeys, with weight ranging from 2 to 2.5 kg, were included in the experiment. The animals were maintained in the Vivarium of the Faculty of Veterinary Medicine of Federal University of Uberlândia and were fed with vegetables (carrots, beetroot, cabbage), fruits (papaya, orange, apple, banana) and fresh water, as specified by the Brazilian Environmental and Natural Resources Institute (IBAMA). The animals were maintained in individual cages during the entire experimental period, under constant veterinary surveillance. This research was approved by the Ethical Committee for Animal Experimentations of Federal University of Uberlândia.
Before each surgical procedure, the monkeys fasted 12 hours for solids and six hours for liquids. Then, they were anesthetized with ketamine hydrochloride (five per cent 10 mg/kg) in association with acepromazine maleate (one per cent 0.2 mg/kg) and midazolam (50 mg 0.2 mg/kg).
The monkeys were cleansed with Povidone- iodine surgical scrub and scaled with ultrasonic cleaner under extreme refrigeration. For PRP produce, two vacurettes of citrate blood containing 5 ml each were drawn from the animals. The vacurettes were centrifuged and Sonnleitner's protocol  was followed until the concentrated platelet was obtained.
In the extraction areas a terminal infiltration anesthesia was administered with a solution of prilocaine hydrochloride three per cent and felypressin 0.03UI/ml. Extraction of the second premolars was subsequently performed on both sides of the mandible. Besides, the impressions of surgical areas were taken and acrylic guides confectioned, to orientate biopsy removal.
Extraction sockets were randomly distributed in treated and non-treated groups. In treated groups, the alveolus was dried with gauze compresses and then filled up with platelet concentrate. In non-treated groups, the coagulum was maintained. All animals received this kind of treatment. Then the soft tissue was coapted and extraction sockets closed for healing by first intention.
In the immediate post-operative period, the antibiotic cephazolin sodium 25 mg/kg and the anti-inflammatory/analgesic flunixin meglumine 50 mg, 2.2 mg /kg were administered. Healing following teeth extractions was uneventful. No area exhibited clinical signs of inflammation.
The animals were divided into four different periods, one monkey per period. Biopsies were taken only once, from each animal, at 30, 90, 120 and 180 days intervals. All pre- operative, operative and post-operative procedures with the exception of those for PRP preparation were repeated. A linear incision was made at the top of the alveolar ridge and a full thickness mucosal flap was raised. Extraction socket samples, with a 2.0 mm diameter and a 7 mm of intra- bone depth, were removed using a trephine drill (3i Implant Innovations, Palm Beach Gardens, FL, USA), under intense irrigation with physiologic saline solution.
Routine laboratory procedures were performed  for the preparation of histological serial sections, parallel to the long axis of the fragment. Both histological and histometric assessment of samples were accomplished.
The histological evaluation of the biopsy sample retrieved from the healing sockets revealed the following results:
Healing after 30 days
After 30 days of wound healing, new bone formation appeared to be intense in both experimental and control sockets. The woven bone trabeculae were lined by bone-forming cells (osteoblasts) indicating that bone formation was in progress in most areas of the socket. Connective tissue characterized by the presence of vascular structures and fibroblasts was encountered in large amounts in the PRP socket. Collagen fibers and hematopoietic cells were also present [Figure 1] and [Figure 2].
Healing after 90 days
In the PRP treated socket, the central portion was occupied by large trabeculae of bone delimiting small bone marrow areas. The bone marrow areas were characterized by the presence of a large amount of morphotypes of fibroblast- like cells, collagen fibers, vessels, and a small number of both adipocytes and hematopoietic cells. In the alveolar ridge region, dense connective tissue was present [Figure 3]. In the control socket, a hard tissue bridge was newly formed in the alveolar ridge region. Most of the socket was occupied by bone marrow containing adipocytes, vessels and hematopoietic cells [Figure 4]. In both sockets, the hard tissue had the character of woven bone.
Healing after 120 days
In the PRP-treated socket, the central area remained occupied by large bone trabeculae. However, the bone marrow at this time was characterized by the presence of adipocytes, loose connective tissue, blood vessels and hematopoietic cells. Besides, a newly formed hard tissue bridge was present in the alveolar ridge area, nevertheless, this bridge was discontinuous and a dense connective tissue was present [Figure 5]. In the control group, there were no significant differences between the structures observed in 90 and 120 intervals [Figure 6].
The hard tissue of the control socket was mainly composed of woven bone. On the other hand, the woven bone in PRP treated socket was often combined with both parallel- fibered and lamellar bone.
After 180 days
Neither groups showed significant changes compared to the previous period. However, the hard tissue in both groups had mainly the character of lamellar bone [Figure 7] and [Figure 8].
[Table 1] shows the percentage of bone in all the analyzed samples. The bones in the alveolar ridge region (cortical bone) and deeper portions of the socket (cancellous bone) were assessed separately. However, at a 30-day interval, as the alveolar ridge region was not yet occupied by mineralized tissue, only the total bone area was given.
Control sockets presented the highest area of bone in the alveolar ridge region for all periods. However, PRP sockets showed major bone formation in the deeper portion of the sockets.
The platelet-rich plasma (PRP) has been recommended for increasing the rate of bone deposition and quality of sites for future implant placement. ,,,, The therapeutic strategy of PRP is based on the release of multiple growth and differentiation factors upon platelet activation.  These factors are critical in regulation and stimulation of the wound healing by regulating cellular processes such as mitogenesis, chemotaxis, differentiation, and metabolism.  For this reason, the growth factors contained and released from PRP should enhance and accelerate soft tissue healing and the process of regenerating bone. Nevertheless, controversial results have been reported and some authors questioned the effectiveness of the PRP to enhance the quality of newly formed bone. ,,
In the current experiment, the structures observed in both groups at a 30-day interval were compatible with the initial stage of alveolar repair. ,, The proliferation of connective tissue elements was higher in the PRP-treated socket. However, no significantly differences were found in bone amounts, between experimental and control groups.
At subsequent intervals, the control sockets showed signs of decrease in osteogenesis. A hard tissue bridge was formed at the alveolar ridge region and the deeper regions of the socket became mostly occupied with bone marrow. These observations were in agreement with the findings reported by Evian et al.  and Ohta et al.,  who studied the bone repair in extractions sockets of humans and Macaca fusaca, respectively. In another experimental study in dogs, Cardaropolis et al.  stated that the mineralized bone that occupied 88% of the socket volume at 30 day interval, decreased to only 15% at 180 day interval, while the portion occupied by bone marrow increased to 85% in the same period. The authors argued that these changes happened once the extraction site was exposed to a minimal load and there was apparently no obvious demand for mineralized tissue.
Otherwise, at the same interval in the PRP-treated socket, the process of bone formation and fibroblast-like cells proliferation remained intense. With respect to these observations, it seemed that the effect of PRP on the remaining periodontal ligament on the sockets walls was essential. According to Lin et al.,  the periodontal ligament remnants are the primary origin of the osteoblasts which form the bone during the socket healing. These authors demonstrated that, after tooth extraction, numerous periodontal ligament fibroblasts of the periodontal ligament remnant actively migrated into the coagulum, proliferating and forming dense connective tissue. Then, some of these fibroblasts begin to differentiate into osteoblasts, starting the bone formation. The study of Carvalho et al.  also corroborate with this conclusion. They histologically assessed the influence of alveolar curetting on the chronology of extraction sockets healing in rats. The results showed a delay in the repair process in all observed periods in the experimental group.
In this study, after tooth extraction, the periodontal ligament attached to the bone in the sockets walls was left undisturbed. In this case, the physiological healing process may have been affected by the PRP growth factors, which include large amounts of Platelet-derived Growth Factor AB (PDGF-AB), Transforming Growth Factor Beta 1 (TGF-â1), Insulin-like Growth Factor I (IGF-I); and Platelet-derived Growth Factor BB (PDGF-BB) and Transforming Growth Factor Beta 2 into smaller quantities.  Matsuda et al.  reported from in vitro studies that PDGF in association with TGF-â1 and IGF-I can strongly stimulate the mitogenesis, chemotaxis and matrix synthesis of fibroblasts obtained from the coagulum of healing tooth sockets. Other authors showed, by different animals' experiments, that the PRP growth factors used in guided tissue regeneration (GTR) of periodontal bone defects stimulated higher dense connective tissue formation in initial stage of repair, comparing with GTR alone. , Then, during the remodeling stage, this connective tissue mineralized and transformed into bone or cementum, depending on the localization of the healing site. A similar process should probably happen in the PRP-treated extraction sockets, extending the osteogenic stage and resulting in the formation of large bone trabeculae in the deeper portion of the alveolus.
The action of PRP on the fibroblasts can also be seen in the alveolar ridge areas of the experimental group, in which formed a discontinuous bone bridge encroached by dense connective tissue of probably gingival origin. This finding is in agreement with the research results that showed the PDGF-AB, PDGF-BB, TGF-β1 and IGF-I as a potent mitogenic and chemotaxic agents for gingival fibroblasts. ,,, Both PDGF-AB and TGF-β1 also stimulate the collagen synthesis, while the IGF-I slightly enhanced the synthesis of some proteins., On the contrary, the control sockets presented a continuous large hard tissue bridge in the alveolar ridge area.
In the current study, the histological analysis of the sample sections enabled an overview of the various phases of soft and hard tissue formation. Although the limited number of animals did not permit statistics evidences, these findings indicate that the alveolar repair process was modified by the use of PRP. Thus, the monkeys of this experiment were distributed in 30, 90, 120 and 180 days intervals, with the aim of better understand the events involved in the alveolar repairing using PRP.
To our knowledge, the study accomplished by Anitua  is the only one on the effects of PRP on the alveolar repair process. The results of the present study, are in accordance with Anitua's findings, which showed more mature bone, with better organized trabeculae and greater bone regeneration in extraction sockets treated with PRP. However, Anitua reported that each alveolus of his experiment was carefully curetted after tooth extraction, which makes it difficult to assume that the action mechanisms of PRP were the same in both experiments. Nevertheless, the actions of PRP growth factors are very complex and the same result could be achieved by a different pathway.
Although there are limitations in the methodology used, it was possible to conclude that the alveolar healing process, after the use of platelet-rich plasma, had different histological characteristics from the control group, resulting in an enhancement of the bone repair.
The authors wish to thank Prof. Dr. Cirilo Antônio de Paula Lima, Veterinary Anesthetist of Faculty of Veterinary Medicine of Federal University of Uberlândia, for assistance with the monkeys; to Prof. Dr. Paula Dechichi, for help in histological analysis; and Mr. Stephen Mclnerney, for text review.
|1||Romeo E, Chiapasco M, Ghisolfi M, Vogel G. Long-term clinical effectiveness of oral implants in the treatment of partial edentulism. Seven-year life Table analysis of a prospective study with ITI dental implants system used for single-tooth restorations. Clin Oral Implants Res 2002;13:133-43.|
|2||Jaffin RA, Berman CL. The excessive loss of Branemark fixtures in type IV bone: A 5-year analysis. J Periodontol 1991;62:2-4.|
|3||Weng D, Jacobson Z, Tarnow D, Hürzeler MB, Faehn O, Sanavi F, et al. A prospective multicenter clinical trial of 3i machined-surface implants: Results after 6 years of follow-up. Int J Oral Maxillofac Implants 2003;18:417-23.|
|4||Anitua E. Plasma rich in growth factors: Preliminary results of use in the preparation of future site for implants. Int J Oral Maxillofac Implants 1999;14:529-35.|
|5||Garg AK. The use of platelet-rich plasma to enhance the success of bone grafts around dental implants. Dent Implantol Update 2000;11:17-21.|
|6||Lind M. Growth factor stimulation of bone healing. Effects on osteoblasts, osteomies and implants fixation. Acta Orthop Scand Suppl 1998;283:2-37.|
|7||Marx RE, Carlson ER, Eeichtaedt RM, Shimnele SR, Strauss JE, Geogerff K. Platelet-rich plasma: Growth factor enhancement for bone grafts. J Oral Maxillofac Surg 1998;85:638-46.|
|8||Camargo PM, Lekovic V, Weinlander M, Divnic-Resnik T, Pavlovic M, Kenney EB. 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;37:300.|
|9||Kassolis LD, Rosen PS, Reynolds MA. Alveolar ridge and sinus augmentation utilizing platelet-rich plasma in combination with freeze-dried bone allograft: Case series. J Periodontol 2000;71:297-300.|
|10||Choi BH, Im CJ, Huh JY, Suh JJ, Lee SH. Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. Int J Oral Maxillofac Surg 2004;33:56-9.|
|11||Danesh-Meyer MJ, Filstein MR, Shanaman R. Histological evaluation of sinus augmentation using platelet rich plasma (PRP): A case series. J Int Acad Periodontol 2001;3:48-56.|
|12||Jekse N, Tangl S, Gilli R, Berghold A, Lorenzoni M, Eskici A, et al. Influence of PRP on autogenous sinus grafts. An experimental study on sheep. Clin Oral Implant Res 2003;14:578-83.|
|13||Sonnleitner D, Huemer P, Sullivan DY. A simplified technique for producing platelet-rich plasma and platelet concentrate for intraoral bone grafting techniques: A technical note. Int J Oral Maxillofac Implants 2000;15:879-82.|
|14||Morse A. Formic acid-sodium citrate decalcification and butyl alcohol dehydration of teeth and bone sectioning in paraffin. J Dent Res 1945;24:143.|
|15||Marx RE, Garg AK. Bone grafts physiology with use of platelet-rich plasma and hiperbaric oxigen. The Sinus Bone Graft. Chicago: Quitessense 1999:183-9. |
|16||Cardaropoli G, Araújo M, Lindhe J. Dynamics of bone tissue formation in tooth extraction sites. An experimental study in dogs. J Clin Periodontol 2003;30:809-18.|
|17||Evian CI, Rosenberg ES, Coslet JG, Corn H. The osteogenic activity of bone removed from healing extraction sockets in humans. J Periodontol 1982;53:81-5.|
|18||Park JB, Matsuura M, Han KY, Norderyd O, Lin WL, Genco RJ, et al. Periodontal regeneration in class III furcation defects of beagle dogs using guided tissue regenerative therapy with platelet-derived growth factor. J Periodontol 1995;66:462-77.|
|19||Ohta Y, et al. Comparative changes in microvascular and bone during healing of implants and extraction sites. J Oral Implant 1993;3:184-98.|
|20||Lin WL, McCulloch CAG, Cho ME. Differentiation of periodontal ligament fibroblasts into osteoblasts during socket healing after tooth extraction in the rat. Anat Rec 1994;240:492-506.|
|21||Carvalho PSP, Okamoto T, Carvalho ACP. The influence of intra-alveolar curettage on wound healing after tooth extraction. A histological study in rats. J Nihon Univ Sch Dent 1982;24:28-34. |
|22||Weibrich G, Kleis WK, Kunz-Kostomanolakis M, Loos AH, Wagner W. Correlation of platelet concentration in platelet-rich plasma to the extraction method, age, sex, and platelet count of the donor. Int J Oral Maxillofac Implants 2001;16:693-9.|
|23||Matsuda N, Lin WL, Kumar NM, Cho MI, Genco RJ. Mitogenese chemotactic and synthetic responses of rat periodontal ligament fibroblastic cells to polypeptide growth factor in vitro. J Periodontol 1992;63:515-25.|
|24||Giannobile WV. Periodontal regeneration: Potential role of bone morphogenetic proteins. J Periodontal Res 1996;29:225-35.|
|25||Bartold PM, Raben A. Growth factor modulation of fibroblasts in simulated wound healing. J Periodontal Res 1996;31:205-16.|
|26||Dennison DK, Vallone DR, Pinero GJ, Rittman B, Caffesse RG. Differential effect of TGF-b1 and PDGF on proliferation of periodontal ligament cells and gingival fibroblasts. J Periodontol 1994;65:641-8.|
|27||Marcopoulou CE, Vavouraki HN, Dereka XE, Vrotsos IA. Proliferative effect of growth factors TGF-beta1, PDGF-BB and rhBMP-2 on human gingival fibroblasts and periodontal ligament cells. J Int Acad Periodontol 2003;5:63-70.|