The effect of autologous, platelet-rich plasma on the osteoinductive potential of
allogenic demineralized bone matrix. An experimental pilot study in rabbits
Minas LEVENTIS, Efstathios ELEFTHERIADIS, Stavros PAPANIKOLAOU, Panagiota OIKONOMOPOULOU, Helen VAVOURAKI, Lubna KHALDI, Konstantinos TOSIOS, Constantinos ALEXANDRIDIS, Ismene DONTAS
Oral and Maxillofacial Surgery Clinic, Dental School, University of Athens (Head: Professor C. Alexandridis),
Human Tissue Bank, National Centre for Scientific Research "Demokritos" (Head: Dr. Helen Vavouraki),
Laboratory for Experimental Surgery and Surgical Research “N.S. Christeas” (Head: Professor D. Perrea)
and Laboratory for Research of the Musculoskeletal System “Th. Garofalidis” (Head: Assoc. Professor N. Papaioannou), Medical School, University of Athens
Hellenic Archives of Oral & Maxillofacial Surgery (2010) 3, 141-156
SUMMARY: Objective: To evaluate the effect of autologous platelet-rich plasma (PRP) on the ability of allogenic demineralized bone matrix (DBM) to induce bone formation in rabbit intramuscular positions. Materials and methods: Nine New Zealand rabbits were used. Allogenic DBM was produced from bones of 3 healthy rabbits. After blood collection, 0.5cm3 of autologous PRP was prepared for 6 animals. Then, 2 muscle pockets were created, where 250 mg allogenic DBM + PRP (experimental sites) and 250 mg allogenic DBM without PRP (control sites) were randomly implanted. Animals were sacrificed 6 weeks after implantation. Results: Histologic examination revealed uneventful healing in all cases, while remineralization at the periphery of the bone graft particles was a constant finding. In both control and experimental sites fibroblasts and other mesenchymal cells (probably osteoprogenitor cells and preosteoblasts) were observed. The only histological difference was the richer vascularity in the experimental sites. Parameters associated with the breakdown degree of the graft and the production of newly-formed connective tissue were measured and calculated. No statistically significant differences were observed regarding the above parameters. Conclusions: When combined with allogenic DBM, autologous PRP does not enhance the osteoinductive process in rabbit intramuscular sites compaired to the allograft alone, at 6 weeks of observation.
KEY WORDS: PRP, DBM, osteoinduction, bone grafts, bone regeneration.
REFERENCES
Acarturk TO, Hollinger JO: Commercially available demineralized bone matrix compositions to regenerate calvarial critical-sized bone defects. Plast Reconstr Surg 118: 862-73, 2006
Aghaloo TL, Moy PK, Freymiller EG: Investigation of platelet-rich plasma in rabbit cranial defects: A pilot study. J Oral Maxillofac Surg 60: 1176-81, 2002 Aghaloo TL, Moy PK, Freymiller EG: Evaluation of platelet-rich plasma in combination with anorganic bovine bone in the rabbit cranium: a pilot study. Int J Oral Maxillofac Implants 19: 59-65, 2004
Aghaloo TL, Moy PK, Freymiller EG: Evaluation of platelet-rich plasma in combination with freeze-dried bone in the rabbit cranium. A pilot study. Clin Oral Implants Res 16: 250-7, 2005
Bae HW, Zhao L, Kanim LE, Wong P, Delamarter RB, Dawson EG: Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products. Spine 31: 1299-306, 2006
Becker W, Becker BE, Caffesse R: A comparison of demineralized freeze-dried bone and autologous bone to induce bone formation in human extraction sockets. J Periodontol 65: 1128-33, 1994
Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, Szatkowski JP, Park JY, He TC: Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am 85: 1544-52, 2003
Cieslik-Bielecka A, Bielecki T, Gazdzik TS, Cieslik T, Szczepanski T: Improved treatment of mandibular odontogenic cysts with platelet-rich gel. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 105: 423-9, 2008
Ferreira CF, Carriel Gomes MC, Filho JS, Granjeiro JM, Oliveira Simões CM, Magini Rde S: Platelet-rich plasma influence on human osteoblasts growth. Clin Oral Implants Res 16: 456-60, 2005
Freymiller EG, Aghaloo TL: Platelet-rich plasma: ready or not? J Oral Maxillofac Surg 62: 484-8, 2004
Garg AK: Bone: Biology, Harvesting, Grafting for Dental Implants. Quintessence 2004, pp 3-15
Gerard D, Carlson ER, Gotcher JE, Jacobs M: Effects of platelet-rich plasma on the healing of autologous bone grafted mandibular defects in dogs. J Oral Maxillofac Surg 64: 443-51, 2006
Gerard D, Carlson ER, Gotcher JE, Jacobs M: Effects of platelet-rich plasma at the cellular level on healing of autologous bone-grafted mandibular defects in dogs. J Oral Maxillofac Surg 65: 721-7, 2007
Geurs NC, Korostoff JM, Vassilopoulos PJ, Kang TH, Jeffcoat M, Kellar R, Reddy MS: Clinical and histologic assessment of lateral alveolar ridge augmentation using a synthetic long-term bioabsorbable membrane and an allograft. J Periodontol 79: 1133-40, 2008
Haddad AJ, Peel SA, Clokie CM, Sá ndor GK: Closure of rabbit calvarial critical-sized defects using protective composite allogeneic and alloplastic bone substitutes. J Craniofac Surg 17: 926-34, 2006
Han B, Yang Z, Nimni M: Effects of gamma irradiation on osteoinduction associated with demineralized bone matrix. J Orthop Res 26: 75-82, 2008
Harakas NK: Demineralized bone-matrix-induced osteogenesis. Clin Orthop Relat Res 188: 239-51, 1984
Heberer S, Al-Chawaf B, Hildebrand D, Nelson JJ, Nelson K: Histomorphometric analysis of extraction sockets augmented with Bio-Oss Collagen after a 6-week healing period: a prospective study. Clin Oral Implants Res 19: 1219-25, 2008
Hosny M, Sharawy M: Osteoinduction in young and old rats using demineralized bone powder allografts. J Oral Maxillofac Surg 43: 925-31, 1985
Intini G, Andreana S, Buhite RJ, Bobek LA: A comparative analysis of bone formation induced by human demineralized freeze-dried bone and enamel matrix derivative in rat calvaria critical-size bone defects. J Periodontol 79: 1217-24, 2008
Kanno T, Takahashi T, Tsujisawa T, Ariyoshi W, Nishihara T: Plateletrich plasma enhances human osteoblast-like cell proliferation and differentiation. J Oral Maxillofac Surg 63: 362-9, 2005
Kassolis JD, Reynolds MA: Evaluation of the adjunctive benefits of platelet-rich plasma in subantral sinus augmentation. J Craniofac Surg 16: 280-7, 2005 Kim SG, Kim WK, Park JC, Kim HJ: A comparative study of osseointegration of Avana implants in a demineralized freeze-dried bone alone or with platelet-rich plasma. J Oral Maxillofac Surg 60: 1018-25, 2002
Lynch S, Marx R, Nevins M, Wisner-Lynch L: Tissue Engineering. Applications in Oral and Maxillofacial Surgery and Periodontics. 2nd ed. Quintessence 2008, pp 3-36
Markou N, Pepelassi E, Vavouraki H, Stamatakis HC, Nikolopoulos G, Vrotsos I, Tsiklakis K: Treatment of periodontal endosseous defects with platelet-rich plasma alone or in combination with demineralized freeze-dried bone allograft: a comparative clinical trial. J Periodontol 80: 1911-9, 2009
Marx RE: Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg 62: 489-96, 2004
Marx RE, Garg AK: Dental and craniofacial appMarx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR: Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85: 638-46, 1998
Mitruka BM: Reference values in experimental animals and humans. In: Mitruka and Rawnsley: Clinical Biochemical and Haematological Reference Values in Normal Experimental Animals and Normal Humans. 2nd ed. Masson Publishing 1983, pp. 74-6
Mokbel N, Bou Serhal C, Matni G, Naaman N: Healing patterns of critical size bony defects in rat following bone graft. Oral Maxillofac Surg 12: 73-8, 2008 Piattelli A, Scarano A, Corigliano M, Piattelli M: Comparison of bone regeneration with the use of mineralized and demineralized freeze-dried bone allografts: a histological and histochemical study in man. Biomaterials 17: 1127-31, 1996
Raghoebar GM, Schortinghuis J, Liem RS, Ruben JL, van der Wal JE, Vissink A: Does platelet-rich plasma promote remodeling of autologous bone grafts used for augmentation of the maxillary sinus floor? Clin Oral Implants Res 16: 349-56, 2005
Ranly DM, Lohmann CH, Andreacchio D, Boyan BD, Schwartz Z: Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice. J Bone Joint Surg Am 89:139-47, 2007
Reddi AH, Huggins C: Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci USA 69: 1601-5, 1972 Schlegel KA, Zimmermann R, Thorwarth M, Neukam FW, Klongnoi B, Nkenke E, Felszeghy E: Sinus floor elevation using autogenous bone or bone substitute combined with platelet-rich plasma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 104: e15- 25, 2007
Schouten CC, Hartman EH, Spauwen PH, Jansen JA: DBM induced ectopic bone formation in the rat: the importance of surface area. J Mater Sci Mater Med 16: 149-52, 2005
Shanaman R, Filstein MR, Danesh-Meyer MJ: Localized ridge augmentation using GBR and platelet-rich plasma: case reports. Int J Periodontics Restorative Dent 21: 345-55, 2001
Thor A, Franke-Stenport V, Johansson CB, Rasmusson L: Early bone formation in human bone grafts treated with platelet-rich plasma: preliminary histomorphometric results. Int J Oral Maxillofac Surg 36: 1164-71, 2007
Tomoyasu A, Higashio K, Kanomata K, Goto M, Kodaira K, Serizawa H, Suda T, Nakamura A, Nojima J, Fukuda T, Katagiri T: Plateletrich plasma stimulates osteoblastic differentiation in the presence of BMPs. Biochem Biophys Res Commun 361: 62-7, 2007
Urist MR: Bone: formation by autoinduction. Science 150: 893-5, 1965 Weibrich G, Kleis WK: Curasan PRP kit vs. PCCS PRP system. Collection efficiency and platelet counts of two different methods for the preparation of platelet-rich plasma. Clin Oral Implants Res 13: 437-43, 2002
Wiltfang J, Schlegel KA, Schultze-Mosgau S, Nkenke E, Zimmermann R, Kessler P: Sinus floor augmentation with beta-tricalciumphosphate (beta-TCP): does platelet-rich plasma promote its osseous integration and degradation? Clin Oral Implants Res 14: 213-8, 2003
Yokota K, Ishida O, Sunagawa T, Suzuki O, Nakamae A, Ochi M: Platelet-rich plasma accelerated surgical angio-genesis in vascular-implanted necrotic bone: an experimental study in rabbits. Acta Orthop 79: 106-10, 2008
You TM, Choi BH, Li J, Jung JH, Lee HJ, Lee SH, Jeong SM: The effect of platelet-rich plasma on bone healing around implants placed in bone defects treated with Bio-Oss: a pilot study in the dog tibia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 103: e8-12, 2007
Zechner W, Tangl S, Tepper G, Fürst G, Bernhart T, Haas R, Mailath G, Watzek G: Influence of platelet-rich plasma on osseous healing of dental implants: a histologic and histomorphometric study in minipigs. Int J Oral Maxillofac Implants 18: 15-22, 2003
Zhang CQ, Yuan T, Zeng BF. Experimental study of the effect of platelet-rich plasma on osteogenesis in rabbit. Chin Med J (Engl) 117: 1853-5, 2004 Zouloumis L, Papadopoulou A, Iliopoulos M, Tsekos A, Katsaros K, Lazaridis N: Clinical use of Plasma-Rich in Growth Factors in oral and maxillofacial surgery. Hellenic Arch Oral Maxillofac Surg 9: 67-80, 2008
Oral and Maxillofacial Surgery Clinic, Dental School, University of Athens (Head: Professor C. Alexandridis),
Human Tissue Bank, National Centre for Scientific Research "Demokritos" (Head: Dr. Helen Vavouraki),
Laboratory for Experimental Surgery and Surgical Research “N.S. Christeas” (Head: Professor D. Perrea)
and Laboratory for Research of the Musculoskeletal System “Th. Garofalidis” (Head: Assoc. Professor N. Papaioannou), Medical School, University of Athens
Hellenic Archives of Oral & Maxillofacial Surgery (2010) 3, 141-156
SUMMARY: Objective: To evaluate the effect of autologous platelet-rich plasma (PRP) on the ability of allogenic demineralized bone matrix (DBM) to induce bone formation in rabbit intramuscular positions. Materials and methods: Nine New Zealand rabbits were used. Allogenic DBM was produced from bones of 3 healthy rabbits. After blood collection, 0.5cm3 of autologous PRP was prepared for 6 animals. Then, 2 muscle pockets were created, where 250 mg allogenic DBM + PRP (experimental sites) and 250 mg allogenic DBM without PRP (control sites) were randomly implanted. Animals were sacrificed 6 weeks after implantation. Results: Histologic examination revealed uneventful healing in all cases, while remineralization at the periphery of the bone graft particles was a constant finding. In both control and experimental sites fibroblasts and other mesenchymal cells (probably osteoprogenitor cells and preosteoblasts) were observed. The only histological difference was the richer vascularity in the experimental sites. Parameters associated with the breakdown degree of the graft and the production of newly-formed connective tissue were measured and calculated. No statistically significant differences were observed regarding the above parameters. Conclusions: When combined with allogenic DBM, autologous PRP does not enhance the osteoinductive process in rabbit intramuscular sites compaired to the allograft alone, at 6 weeks of observation.
KEY WORDS: PRP, DBM, osteoinduction, bone grafts, bone regeneration.
REFERENCES
Acarturk TO, Hollinger JO: Commercially available demineralized bone matrix compositions to regenerate calvarial critical-sized bone defects. Plast Reconstr Surg 118: 862-73, 2006
Aghaloo TL, Moy PK, Freymiller EG: Investigation of platelet-rich plasma in rabbit cranial defects: A pilot study. J Oral Maxillofac Surg 60: 1176-81, 2002 Aghaloo TL, Moy PK, Freymiller EG: Evaluation of platelet-rich plasma in combination with anorganic bovine bone in the rabbit cranium: a pilot study. Int J Oral Maxillofac Implants 19: 59-65, 2004
Aghaloo TL, Moy PK, Freymiller EG: Evaluation of platelet-rich plasma in combination with freeze-dried bone in the rabbit cranium. A pilot study. Clin Oral Implants Res 16: 250-7, 2005
Bae HW, Zhao L, Kanim LE, Wong P, Delamarter RB, Dawson EG: Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products. Spine 31: 1299-306, 2006
Becker W, Becker BE, Caffesse R: A comparison of demineralized freeze-dried bone and autologous bone to induce bone formation in human extraction sockets. J Periodontol 65: 1128-33, 1994
Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B, Vanichakarn P, Szatkowski JP, Park JY, He TC: Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am 85: 1544-52, 2003
Cieslik-Bielecka A, Bielecki T, Gazdzik TS, Cieslik T, Szczepanski T: Improved treatment of mandibular odontogenic cysts with platelet-rich gel. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 105: 423-9, 2008
Ferreira CF, Carriel Gomes MC, Filho JS, Granjeiro JM, Oliveira Simões CM, Magini Rde S: Platelet-rich plasma influence on human osteoblasts growth. Clin Oral Implants Res 16: 456-60, 2005
Freymiller EG, Aghaloo TL: Platelet-rich plasma: ready or not? J Oral Maxillofac Surg 62: 484-8, 2004
Garg AK: Bone: Biology, Harvesting, Grafting for Dental Implants. Quintessence 2004, pp 3-15
Gerard D, Carlson ER, Gotcher JE, Jacobs M: Effects of platelet-rich plasma on the healing of autologous bone grafted mandibular defects in dogs. J Oral Maxillofac Surg 64: 443-51, 2006
Gerard D, Carlson ER, Gotcher JE, Jacobs M: Effects of platelet-rich plasma at the cellular level on healing of autologous bone-grafted mandibular defects in dogs. J Oral Maxillofac Surg 65: 721-7, 2007
Geurs NC, Korostoff JM, Vassilopoulos PJ, Kang TH, Jeffcoat M, Kellar R, Reddy MS: Clinical and histologic assessment of lateral alveolar ridge augmentation using a synthetic long-term bioabsorbable membrane and an allograft. J Periodontol 79: 1133-40, 2008
Haddad AJ, Peel SA, Clokie CM, Sá ndor GK: Closure of rabbit calvarial critical-sized defects using protective composite allogeneic and alloplastic bone substitutes. J Craniofac Surg 17: 926-34, 2006
Han B, Yang Z, Nimni M: Effects of gamma irradiation on osteoinduction associated with demineralized bone matrix. J Orthop Res 26: 75-82, 2008
Harakas NK: Demineralized bone-matrix-induced osteogenesis. Clin Orthop Relat Res 188: 239-51, 1984
Heberer S, Al-Chawaf B, Hildebrand D, Nelson JJ, Nelson K: Histomorphometric analysis of extraction sockets augmented with Bio-Oss Collagen after a 6-week healing period: a prospective study. Clin Oral Implants Res 19: 1219-25, 2008
Hosny M, Sharawy M: Osteoinduction in young and old rats using demineralized bone powder allografts. J Oral Maxillofac Surg 43: 925-31, 1985
Intini G, Andreana S, Buhite RJ, Bobek LA: A comparative analysis of bone formation induced by human demineralized freeze-dried bone and enamel matrix derivative in rat calvaria critical-size bone defects. J Periodontol 79: 1217-24, 2008
Kanno T, Takahashi T, Tsujisawa T, Ariyoshi W, Nishihara T: Plateletrich plasma enhances human osteoblast-like cell proliferation and differentiation. J Oral Maxillofac Surg 63: 362-9, 2005
Kassolis JD, Reynolds MA: Evaluation of the adjunctive benefits of platelet-rich plasma in subantral sinus augmentation. J Craniofac Surg 16: 280-7, 2005 Kim SG, Kim WK, Park JC, Kim HJ: A comparative study of osseointegration of Avana implants in a demineralized freeze-dried bone alone or with platelet-rich plasma. J Oral Maxillofac Surg 60: 1018-25, 2002
Lynch S, Marx R, Nevins M, Wisner-Lynch L: Tissue Engineering. Applications in Oral and Maxillofacial Surgery and Periodontics. 2nd ed. Quintessence 2008, pp 3-36
Markou N, Pepelassi E, Vavouraki H, Stamatakis HC, Nikolopoulos G, Vrotsos I, Tsiklakis K: Treatment of periodontal endosseous defects with platelet-rich plasma alone or in combination with demineralized freeze-dried bone allograft: a comparative clinical trial. J Periodontol 80: 1911-9, 2009
Marx RE: Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg 62: 489-96, 2004
Marx RE, Garg AK: Dental and craniofacial appMarx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR: Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85: 638-46, 1998
Mitruka BM: Reference values in experimental animals and humans. In: Mitruka and Rawnsley: Clinical Biochemical and Haematological Reference Values in Normal Experimental Animals and Normal Humans. 2nd ed. Masson Publishing 1983, pp. 74-6
Mokbel N, Bou Serhal C, Matni G, Naaman N: Healing patterns of critical size bony defects in rat following bone graft. Oral Maxillofac Surg 12: 73-8, 2008 Piattelli A, Scarano A, Corigliano M, Piattelli M: Comparison of bone regeneration with the use of mineralized and demineralized freeze-dried bone allografts: a histological and histochemical study in man. Biomaterials 17: 1127-31, 1996
Raghoebar GM, Schortinghuis J, Liem RS, Ruben JL, van der Wal JE, Vissink A: Does platelet-rich plasma promote remodeling of autologous bone grafts used for augmentation of the maxillary sinus floor? Clin Oral Implants Res 16: 349-56, 2005
Ranly DM, Lohmann CH, Andreacchio D, Boyan BD, Schwartz Z: Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice. J Bone Joint Surg Am 89:139-47, 2007
Reddi AH, Huggins C: Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci USA 69: 1601-5, 1972 Schlegel KA, Zimmermann R, Thorwarth M, Neukam FW, Klongnoi B, Nkenke E, Felszeghy E: Sinus floor elevation using autogenous bone or bone substitute combined with platelet-rich plasma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 104: e15- 25, 2007
Schouten CC, Hartman EH, Spauwen PH, Jansen JA: DBM induced ectopic bone formation in the rat: the importance of surface area. J Mater Sci Mater Med 16: 149-52, 2005
Shanaman R, Filstein MR, Danesh-Meyer MJ: Localized ridge augmentation using GBR and platelet-rich plasma: case reports. Int J Periodontics Restorative Dent 21: 345-55, 2001
Thor A, Franke-Stenport V, Johansson CB, Rasmusson L: Early bone formation in human bone grafts treated with platelet-rich plasma: preliminary histomorphometric results. Int J Oral Maxillofac Surg 36: 1164-71, 2007
Tomoyasu A, Higashio K, Kanomata K, Goto M, Kodaira K, Serizawa H, Suda T, Nakamura A, Nojima J, Fukuda T, Katagiri T: Plateletrich plasma stimulates osteoblastic differentiation in the presence of BMPs. Biochem Biophys Res Commun 361: 62-7, 2007
Urist MR: Bone: formation by autoinduction. Science 150: 893-5, 1965 Weibrich G, Kleis WK: Curasan PRP kit vs. PCCS PRP system. Collection efficiency and platelet counts of two different methods for the preparation of platelet-rich plasma. Clin Oral Implants Res 13: 437-43, 2002
Wiltfang J, Schlegel KA, Schultze-Mosgau S, Nkenke E, Zimmermann R, Kessler P: Sinus floor augmentation with beta-tricalciumphosphate (beta-TCP): does platelet-rich plasma promote its osseous integration and degradation? Clin Oral Implants Res 14: 213-8, 2003
Yokota K, Ishida O, Sunagawa T, Suzuki O, Nakamae A, Ochi M: Platelet-rich plasma accelerated surgical angio-genesis in vascular-implanted necrotic bone: an experimental study in rabbits. Acta Orthop 79: 106-10, 2008
You TM, Choi BH, Li J, Jung JH, Lee HJ, Lee SH, Jeong SM: The effect of platelet-rich plasma on bone healing around implants placed in bone defects treated with Bio-Oss: a pilot study in the dog tibia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 103: e8-12, 2007
Zechner W, Tangl S, Tepper G, Fürst G, Bernhart T, Haas R, Mailath G, Watzek G: Influence of platelet-rich plasma on osseous healing of dental implants: a histologic and histomorphometric study in minipigs. Int J Oral Maxillofac Implants 18: 15-22, 2003
Zhang CQ, Yuan T, Zeng BF. Experimental study of the effect of platelet-rich plasma on osteogenesis in rabbit. Chin Med J (Engl) 117: 1853-5, 2004 Zouloumis L, Papadopoulou A, Iliopoulos M, Tsekos A, Katsaros K, Lazaridis N: Clinical use of Plasma-Rich in Growth Factors in oral and maxillofacial surgery. Hellenic Arch Oral Maxillofac Surg 9: 67-80, 2008
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