庆大霉素—富血小板凝胶治疗感染性骨缺损的实验研究
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摘要
目的
骨修复是一个复杂的过程,生长因子在骨修复中有着重要的作用。富血小板血浆(PRP)是自体血小板的浓缩体,具有制备简便、价格低廉、自体、无毒、无免疫原性的优点,而且PRP中释放的多种生长因子可能符合人体生长因子的配比,因此PRP被视为较为理想的符合临床应用需要的生长因子来源。
防治创伤后骨与软组织感染是一个较棘手的问题。常规处理是清创术后全身应用广谱抗生素,在开放性骨折中,由于血运障碍、骨与软组织失活,全身应用抗生素时,骨折局部抗生素浓度往往低于血药浓度,疗效较差而副作用大。局部应用抗生素可在局部获得较高药物浓度,同时保持较低的血药浓度,增强杀菌作用而减少抗生素毒副作用。
本研究通过体外实验和动物感染性骨缺损模型初步探讨是否能将PRP与抗生素复合,利用二者的优点,达到促进骨折愈合和抗感染的作用。
方法
1、对比不同离心条件对PRP制备影响,确定PRP理想制备条件。取健康白兔四只,雌雄不限,体重3-3.5Kg。用预置抗凝剂的真空采血管由耳背中央动脉抽血,按照一定条件离心。用全自动血细胞分析仪测定离心液中血小板浓度,找到一个相对低速、短时的离心条件,并使PRP达到5-6倍浓缩率。
2、使用凝血酶-钙剂激活血小板,制备富血小板凝胶和庆大霉素-富血小板凝胶,PBS液室温培养,用ELISA试剂盒测定、比较不同时点培养液中PDGF,TGF量,判定复合抗生素后是否影响PRP生长因子的释放;用紫外分光光度法测定不同时点培养液中庆大霉素含量,判定载药凝胶是否有缓释作用。
3、参照沈霖的慢性骨髓炎造模方法,使用金黄色葡萄球菌建立兔胫骨慢性骨髓炎模型,对以建立的模型手术清创,植入富血小板凝胶或庆大霉素-富血小板凝胶,并同空白对照比较,肉眼观察比较一般状况,X-ray检查,病理学检查,判定骨缺损愈合情况
结果
1、初次离心使红细胞与富含血小板的血浆分离,B4、C2的血小板收集率回收率均＞80%;再次离心使血小板充分沉淀于离心管的底部,上清液为PPP,弃去大部分PPP,得到约0.5mlPRP,K2和K3条件下血小板浓缩率达到六倍以上。
2、PRP在凝血酶和CaCl_2的作用下活化并形成凝胶,释放PGDF和TGF,添加庆大霉素对这两种生长因子的释放没有明显影响。
3、庆大霉素-富血小板凝胶在体外环境下可以持续释放庆大霉素超过两周,第二天释放达到峰值,而后逐渐下降,在第14天,释放浓度仍可以达到16ug/ml。
4、PRG治疗侧的感染性骨缺损在术后六周没有愈合,应有庆大霉素-富血小板凝胶的一侧骨缺损已经基本愈合。
结论
1、二次离心法,初次离心条件:1400rpm,10min,再次离心条件:1400rpm,15min可稳定获得浓缩率大于6倍的PRP,是实验研究PRP时的理想制备条件。
2、富血小板凝胶和庆大霉素-富血小板凝胶的生长因子释放无明显差异;
3、富血小板凝胶具有缓释作用,PDGF、TGF、庆大霉素可持续释放超过7天。
4、庆大霉素-富血小板凝胶能够有效地促进兔胫骨感染性骨缺损愈合。
Objective
It is generally accepted that growth factors have an essential role in the healing process and tissue formation.In fact,all stages of the repair process are controlled by a wide variety of cytokines and growth factors acting locally as regulators of the most basic cell functions,using endocrine,paracrine,autocrine and intracrine mechanisms.
These findings have led to a significant research effort aimed at testing different growth factors and cytokines as therapeutic molecules for the repair or regeneration of a wide range of tissues.The molecules that have been most intensively investigated in orthopedic research include bone morphogenetic proteins(BMPs),transforming growth factor-β(TGF-β),platelct-derived growth factor(PDGF),insulin-like growth factor (IGF),vascular endothelial growth factor(VEGF) and fibroblast growth factors(FGF). But some limiting factors are related to both the mode of growth factor delivery and the requirements for multiple signals to drive the regeneration process to completion.The latter is essential,assuming that no single exogenous agent can mediate,effectively,all aspects needed for tissue repair.Thus,delivery of a wide range of biological mediators is required if complete tissue engineering is to be achieved.Furthermore,the way these growth factors are made available is of paramount importance.Ideally,they should be delivered locally,following specific and distinct kinetics,to mimic,as far as possible, the requirements of the injured tissue during the different regeneration phases in situ.
Platelets contribute to haemostasis by preventing blood loss at sites of vascular injury,and they contain a large number of growth factors and cytokines that have a key role in bone regeneration and soft-tissue maturation.The source of the new preparation, known as platelet-rich plasma(PRP),consists of a limited volume of plasma enriched in platelets,which is obtained from the patient.Once the platelet concentrate is activated by way of thrombin generation with calcium,a three-dimensional and biocompatible fibrin scaffold is formed,and a myriad of growth factors and proteins are released,progressively,to the local environment,contributing to the accelerated postoperative wound healing and tissue repair.Furthermore,this preparation promotes rapid vascularization of the healing tissue and,because it is autologous,it eliminates concerns about immunogenic reactions and disease transmission.Consequently,the use of autologous PRP as a novel therapeutic alternative opens new avenues in other fields such as orthopedics,sports medicine,dentistry,periodontal surgery and plastic and maxillofacial surgery.
Othopedics infection is common in open fracture.Traditonal treatment include debridement and broad-spectrum antibiotics were used after operation,but local ischemia and devitalization of bone and soft tissue occurred when open fracture,drug concentration of local fracture is lower than blood.Local application of antibiotics in fracture wound may be a good options,to enhance bactericidal effect and depress toxic and side-effect of antibiotics.
PRG has a three-dimensional and biocompatible fibrin scaffold,so it may has the ability of delayd release proterty.This study is execute to probe the treatment of infected bone defects with gentamicin-loaded PRG drug delivery system,controlling the infection and speeding bony growth and healing at one stage.
Method
1、To compare the different centrifugate terms of PRP preparation,define the ideal condition,Four healthy white rabbits were used include male and female,body weight 3-3.5Kg.Blood is collected in hard of hearing central artery using BD Vacutainer(9NC 0.109M),and centrugugated according to given terms.Platelet concentration is survey using automatic blood cell analyzer.Term that lead to platelet concentration ratios of 5-6 fold have been difined as ideal term.
2、Sodium citrate and calcium chloride are used to make PRG or Gentamicin-loaded PRG as an anticoagulant and a clot activator.PRG or Gentamicin-loaded PRG are cultured in PBS.PDGF and TGF in the culture solution at day1,2,4,5,7,14 is measured with ELISA Kit.Culture soltion's gentamicin concentration is measured with ultraviolet spectrophotometry.
3、A total of 10 white rabbits were recruited in the study and the model of chronic osteomyelitis of tibia was made by injecting staphylococcus aureus into the bone hole of two legs.PRP and Gentamicin-loaded PRG implanted into the bone defect.The histologic,radiologic and pathology bone changes were evaluated after therapy.
Result
1、Platelet-rich plasma was obtained by double centrifugation of blood,the term of first centrifuge as B4 and C2 has a coefficient of recovery more than 80%;the term of recentrifuge as K2 and K3 has a 6-fold condense than the blood.
2、PRG obtained by activate PRP by thrombin and CaCl2,addition gentamicin added will form Gentamicin-loaded PRG,they have same release curve.
3、Sustained release of gentamicin from Gentamicin-loaded PRG was observed,it reach the peak at two days,and till 14th day has a 16ug/ml release.
4、The infected bone defect healed at six weeks after operation in Gentamicin-loaded PRG side,the other side is not healed which treated with PRG.
Conclusion
1、Double centrifuging,1400 rpm/10 minutes first,the blood sublayer was removed and the platelet rich fraction was spun(1,400 rpm/15 minutes) followed by removal of the supernatant platelet poor serum fraction(PPP),could easily and stably obtain PRP which more than 6-times above baseline of original blood.
2、There are no significant difference of the release of growth factor of PRP and Gentamicin-loaded PRG.
3、PRG has delayed release characteristics,which can sustained release PDGF, TGF and gentamycin more than 7 days.
4、Gentamycin-loaded PRG is effective in preventing and treating chronic osteomyelitis of rabit tibia
骨修复是一个复杂的过程,生长因子在骨修复中有着重要的作用。富血小板血浆(PRP)是自体血小板的浓缩体,具有制备简便、价格低廉、自体、无毒、无免疫原性的优点,而且PRP中释放的多种生长因子可能符合人体生长因子的配比,因此PRP被视为较为理想的符合临床应用需要的生长因子来源。
防治创伤后骨与软组织感染是一个较棘手的问题。常规处理是清创术后全身应用广谱抗生素,在开放性骨折中,由于血运障碍、骨与软组织失活,全身应用抗生素时,骨折局部抗生素浓度往往低于血药浓度,疗效较差而副作用大。局部应用抗生素可在局部获得较高药物浓度,同时保持较低的血药浓度,增强杀菌作用而减少抗生素毒副作用。
本研究通过体外实验和动物感染性骨缺损模型初步探讨是否能将PRP与抗生素复合,利用二者的优点,达到促进骨折愈合和抗感染的作用。
方法
1、对比不同离心条件对PRP制备影响,确定PRP理想制备条件。取健康白兔四只,雌雄不限,体重3-3.5Kg。用预置抗凝剂的真空采血管由耳背中央动脉抽血,按照一定条件离心。用全自动血细胞分析仪测定离心液中血小板浓度,找到一个相对低速、短时的离心条件,并使PRP达到5-6倍浓缩率。
2、使用凝血酶-钙剂激活血小板,制备富血小板凝胶和庆大霉素-富血小板凝胶,PBS液室温培养,用ELISA试剂盒测定、比较不同时点培养液中PDGF,TGF量,判定复合抗生素后是否影响PRP生长因子的释放;用紫外分光光度法测定不同时点培养液中庆大霉素含量,判定载药凝胶是否有缓释作用。
3、参照沈霖的慢性骨髓炎造模方法,使用金黄色葡萄球菌建立兔胫骨慢性骨髓炎模型,对以建立的模型手术清创,植入富血小板凝胶或庆大霉素-富血小板凝胶,并同空白对照比较,肉眼观察比较一般状况,X-ray检查,病理学检查,判定骨缺损愈合情况
结果
1、初次离心使红细胞与富含血小板的血浆分离,B4、C2的血小板收集率回收率均＞80%;再次离心使血小板充分沉淀于离心管的底部,上清液为PPP,弃去大部分PPP,得到约0.5mlPRP,K2和K3条件下血小板浓缩率达到六倍以上。
2、PRP在凝血酶和CaCl_2的作用下活化并形成凝胶,释放PGDF和TGF,添加庆大霉素对这两种生长因子的释放没有明显影响。
3、庆大霉素-富血小板凝胶在体外环境下可以持续释放庆大霉素超过两周,第二天释放达到峰值,而后逐渐下降,在第14天,释放浓度仍可以达到16ug/ml。
4、PRG治疗侧的感染性骨缺损在术后六周没有愈合,应有庆大霉素-富血小板凝胶的一侧骨缺损已经基本愈合。
结论
1、二次离心法,初次离心条件:1400rpm,10min,再次离心条件:1400rpm,15min可稳定获得浓缩率大于6倍的PRP,是实验研究PRP时的理想制备条件。
2、富血小板凝胶和庆大霉素-富血小板凝胶的生长因子释放无明显差异;
3、富血小板凝胶具有缓释作用,PDGF、TGF、庆大霉素可持续释放超过7天。
4、庆大霉素-富血小板凝胶能够有效地促进兔胫骨感染性骨缺损愈合。
Objective
It is generally accepted that growth factors have an essential role in the healing process and tissue formation.In fact,all stages of the repair process are controlled by a wide variety of cytokines and growth factors acting locally as regulators of the most basic cell functions,using endocrine,paracrine,autocrine and intracrine mechanisms.
These findings have led to a significant research effort aimed at testing different growth factors and cytokines as therapeutic molecules for the repair or regeneration of a wide range of tissues.The molecules that have been most intensively investigated in orthopedic research include bone morphogenetic proteins(BMPs),transforming growth factor-β(TGF-β),platelct-derived growth factor(PDGF),insulin-like growth factor (IGF),vascular endothelial growth factor(VEGF) and fibroblast growth factors(FGF). But some limiting factors are related to both the mode of growth factor delivery and the requirements for multiple signals to drive the regeneration process to completion.The latter is essential,assuming that no single exogenous agent can mediate,effectively,all aspects needed for tissue repair.Thus,delivery of a wide range of biological mediators is required if complete tissue engineering is to be achieved.Furthermore,the way these growth factors are made available is of paramount importance.Ideally,they should be delivered locally,following specific and distinct kinetics,to mimic,as far as possible, the requirements of the injured tissue during the different regeneration phases in situ.
Platelets contribute to haemostasis by preventing blood loss at sites of vascular injury,and they contain a large number of growth factors and cytokines that have a key role in bone regeneration and soft-tissue maturation.The source of the new preparation, known as platelet-rich plasma(PRP),consists of a limited volume of plasma enriched in platelets,which is obtained from the patient.Once the platelet concentrate is activated by way of thrombin generation with calcium,a three-dimensional and biocompatible fibrin scaffold is formed,and a myriad of growth factors and proteins are released,progressively,to the local environment,contributing to the accelerated postoperative wound healing and tissue repair.Furthermore,this preparation promotes rapid vascularization of the healing tissue and,because it is autologous,it eliminates concerns about immunogenic reactions and disease transmission.Consequently,the use of autologous PRP as a novel therapeutic alternative opens new avenues in other fields such as orthopedics,sports medicine,dentistry,periodontal surgery and plastic and maxillofacial surgery.
Othopedics infection is common in open fracture.Traditonal treatment include debridement and broad-spectrum antibiotics were used after operation,but local ischemia and devitalization of bone and soft tissue occurred when open fracture,drug concentration of local fracture is lower than blood.Local application of antibiotics in fracture wound may be a good options,to enhance bactericidal effect and depress toxic and side-effect of antibiotics.
PRG has a three-dimensional and biocompatible fibrin scaffold,so it may has the ability of delayd release proterty.This study is execute to probe the treatment of infected bone defects with gentamicin-loaded PRG drug delivery system,controlling the infection and speeding bony growth and healing at one stage.
Method
1、To compare the different centrifugate terms of PRP preparation,define the ideal condition,Four healthy white rabbits were used include male and female,body weight 3-3.5Kg.Blood is collected in hard of hearing central artery using BD Vacutainer(9NC 0.109M),and centrugugated according to given terms.Platelet concentration is survey using automatic blood cell analyzer.Term that lead to platelet concentration ratios of 5-6 fold have been difined as ideal term.
2、Sodium citrate and calcium chloride are used to make PRG or Gentamicin-loaded PRG as an anticoagulant and a clot activator.PRG or Gentamicin-loaded PRG are cultured in PBS.PDGF and TGF in the culture solution at day1,2,4,5,7,14 is measured with ELISA Kit.Culture soltion's gentamicin concentration is measured with ultraviolet spectrophotometry.
3、A total of 10 white rabbits were recruited in the study and the model of chronic osteomyelitis of tibia was made by injecting staphylococcus aureus into the bone hole of two legs.PRP and Gentamicin-loaded PRG implanted into the bone defect.The histologic,radiologic and pathology bone changes were evaluated after therapy.
Result
1、Platelet-rich plasma was obtained by double centrifugation of blood,the term of first centrifuge as B4 and C2 has a coefficient of recovery more than 80%;the term of recentrifuge as K2 and K3 has a 6-fold condense than the blood.
2、PRG obtained by activate PRP by thrombin and CaCl2,addition gentamicin added will form Gentamicin-loaded PRG,they have same release curve.
3、Sustained release of gentamicin from Gentamicin-loaded PRG was observed,it reach the peak at two days,and till 14th day has a 16ug/ml release.
4、The infected bone defect healed at six weeks after operation in Gentamicin-loaded PRG side,the other side is not healed which treated with PRG.
Conclusion
1、Double centrifuging,1400 rpm/10 minutes first,the blood sublayer was removed and the platelet rich fraction was spun(1,400 rpm/15 minutes) followed by removal of the supernatant platelet poor serum fraction(PPP),could easily and stably obtain PRP which more than 6-times above baseline of original blood.
2、There are no significant difference of the release of growth factor of PRP and Gentamicin-loaded PRG.
3、PRG has delayed release characteristics,which can sustained release PDGF, TGF and gentamycin more than 7 days.
4、Gentamycin-loaded PRG is effective in preventing and treating chronic osteomyelitis of rabit tibia
引文
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12 Graziani F,Cei S,Ducci F,Giuca MR,et al.In vitro effects of different concentration of PRP on primary bone and gingival cell lines.Preliminary results.Minerva Stomatol.2005 Jan-Feb; 54(1-2):15-22.English,Italian.
13 Arpornmaeklong P,Kochel M,Depprich R,et al.lnfluence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells.An in vitro study.Int J Oral Maxillofac Surg.2004 Jan;33(l):60-70.
14 Ogino Y,Ayukawa Y,Tsukiyama Y,et al.The effect of platelet-rich plasma on the cellular response of rat bone marrow cells in vitro.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2005 Sep;100(3):302-7.
15 Fennis JP,Stoelinga PJ Jansen JA.Mandibular reconstruction:a hisological an histomorphometric study on the use of autogenous scaffolds,Particulate cortieo-caneellous bone grafts and Platelet rich Plasma in goats.Int J Oral Maxillofae Surg.2004,33(l):48-55.
16 Fujimori T.The effect of platelet-rich plasma combined with autogenous bone graft for bone regeneration in bone defects.Kokubyo Gakkai Zasshi.2005 Mar;72(l):90-7.Japanese.
17 Aghaloo Tl,Moy PK,Freymiller EG.Investigation of platelet-rich plasma in rabbit cranial defects:A pilot study.J Oral Maxillofac Surg.2002 Oct;60(10):1176-81.
18 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 Jan;33(l):56-9.
19 Butterfield KJ,Bennett J,Gronowicz G et al.Effect of Platelet-rich Plasma with autogenous bone graft for maxillary sinus augmentation in a rabbit model.J Oral Maxillofac Surg.2005,63(3):370-376.
20 Kim ES,Park EJ,Choung PH.Platelet concentration and its effete on bone formation in calvarial defects:An experimental study in rabbits.J Prosthet Dent.2001,86(4):428 — 433.
21 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.2004 Jan-Feb;19(l):59-65.
22 Freymiller EG,Aghaloo TL.Platelet-rich plasma:ready or not?J Oral Maxillofac Surg.2004 Apr;62(4):484-8.
23 Jensen TB,Rahbek O,Overgaard S,et al.Platelet rich plasma and fresh frozen bone allograft as enhancement of implant fixation.An experimental study in dogs.J Orthop Res.2004 May; 22(3):653-8.
24 Sanchez AR,Sheridan PJ,Eckert SE,et al.Regenerative potential of platelet-rich plasma added to xenogenic bone grafts in peri-implant defects:a histomorphometric analysis in dogs.J Periodontol.2005 Oct;76(10):1637-44.
25 Grageda E,Lozada JL,Boyne PJ,et al.Bone formation in the maxillary sinus by using platelet-rich plasma:an experimental study in sheep.J Oral Implantol.2005;31(1):2-17.
26 Kovacs K,Velich N,Huszar T,Fenyves B,et al.Histomorphometric and densitometric evaluation of the effects of platelet-rich plasma on the remodeling of beta-tricalcium phosphate in beagle dogs.J Craniofac Surg.2005 Jan;16(l):150-4.
27 Hokugo A,Ozeki M,Kawakami O,et al.Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel.Tissue Eng.2005 Jul-Aug;ll(7-8):1224-33.
28 Pryor ME,Yang J,Polimeni G,Koo KT,et al.Analysis of rat calvana defects implanted with a platelet-rich plasma preparation:radiographic observations.J Periodontol.2005 Aug;76(8):1287-92.
29 Pryor ME,Polimeni G,Koo KT,et aLAnalysis of rat calvaria defects implanted with a platelet-rich plasma preparation:histologic and histometric observations.J Clin Periodontol.2005 Sep;32(9):966-72.
30 Lucarelli E,Fini M,Beccheroni A,et al.Stromal stem cells and platelet-rich plasma improve bone allograft integration.Clin Orthop Relat Res.2005 Jun;(435):62-8.
31 Yamada Y,Hata K,Ueda M.Injectable bone.Clin Calcium.2002 Feb;12(2):228-32.
32 Yamada Y,Ueda M,Naiki T,et al.Tissue-engineered injectable bone regeneration for osseointegrated dental implants.Clin Oral Implants Res.2004 Oct;15(5):589-97.
33 Ito K,Yamada Y,Nagasaka T,et al.Osteogenic potential of injectable tissue-engineered bone:a comparison among autogenous bone,bone substitute (Bio-oss),platelet-rich plasma,and tissue-engineered bone with respect to their mechanical properties and histological findings.J Biomed Mater Res A.2005 Apr 1;73(l):63-72.
34 Tayapongsak P,O'Brien DA,Monteiro CB,Autologous fibrin adhesive in mandibular reconstruction with particulate cancellous bone and marrow.J Oral Maxillofac Surg.1994 Feb; 52(2):161-5;discussion 166.
35 Anitua E.Plasma rich in growth factors:preliminary results of use in the preparation of future sites for implants.Int J Oral Maxillofac Implants.1999 Jul-Aug;14(4):529-35.
36 Kassolis JD,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 Oct;71(10):1654-61.
37 Mazor Z,Peleg M,Garg AK,et al.Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement:patient series study.Implant Dent.2004 Mar;13(l):65-72.
38 Ueda M,Yamada Y,Ozawa R,et al.Clinical case reports of injectable tissue-engineered bone for alveolar augmentation with simultaneous implant placement.Int J Periodontics Restorative Dent.2005 Apr;25(2):129-37.
39 Sammartino G,Tia M,Marenzi G,et al.Use of autologous platelet-rich plasma (PRP) in periodontal defect treatment after extraction of impacted mandibular third molars.J Oral Maxillofac Surg.2005 Jun;63(6):766-70.
40 Shanaman R,Filstein MR,Danesh-Meyer MJ.Localized ridge augmentation using GBR and platelet-rich plasma:case reports.Int J Periodontics Restorative Dent.2001 Aug;21(4):345-55.
41 Froum SJ,Wallace SS,Tarnow DP,Effect of platelet-rich plasma on bone growth and osseointegration in human maxillary sinus grafts:three bilateral case reports.Int J Periodontics Restorative Dent.2002 Feb;22(l):45-53.
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1 Marx RE,Carlson ER,Eichstaedt RM,et al.Platelet-rich plasma:Growth factor enhancement for bone grafts.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.1998 Jun;85(6):638-46.
2 Weibrich G,Kleis WK,Hafner G et al.Growth factor levels in platelet-rich plasma and correlations with donor age,sex,and platelet count.J Craniomaxillofac Surg.2002 Apr;30(2):97-102.
3 Mokan S,Baylink DJ.Bone growth factors.Clin Orthop Relat Res.1991 Feb;(263):30-48.Review.
4 Thomas KA.Vascular endothelial growth factor,a potent and selective angiogenic agent.J Biol Chem.1996 Jan 12;271(2):603-6.Review.No abstract available.
5 Canalis E.Insulin-like growth factors and osteoporosis.Bone.1997 Sep;21(3):215-6.Review.No abstract available.
6 Slater M,Patava J,Kingham K,et al.Involvement of platelets in stimulating osteogenic activity.J Orthop Res.1995 Sep;13(5):655-63.
7 Lucarelli E,Beccheroni A,Donati D,et al.Platelet-derived growth factors enhance proliferation of human stromal stem cells.Biomaterials.2003 Aug;24(18):3095-100.
8 Kanno T,Takahashi T,Tsujisawa T,et al.Platelet-rich plasma enhances human osteoblast-like cell proliferation and differentiation.J Oral Maxillofac Surg.2005 Mar;63(3):362-9.
9 Ferreira CF,Carriel Gomes MC,Filho JS,et al.Platelet-rich plasma influence on human osteoblasts growth.Clin Oral Implants Res.2005 Aug;16(4):456-60.
10 Choi BH,Zhu SJ,Huh JY,et al.Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells:an in vitro study.Int J Oral Maxillofac Surg.2005 Jun;34(4):420-4.Epub 2005 Jan 27.
11 Marx RE,Platelet-rich plasma (PRP):what is PRP and what is not PRP?Implant Dent.2001;10(4):225-8.
12 Graziani F,Cei S,Ducci F,Giuca MR,et al.In vitro effects of different concentration of PRP on primary bone and gingival cell lines.Preliminary results.Minerva Stomatol.2005 Jan-Feb; 54(1-2):15-22.English,Italian.
13 Arpornmaeklong P,Kochel M,Depprich R,et al.lnfluence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells.An in vitro study.Int J Oral Maxillofac Surg.2004 Jan;33(l):60-70.
14 Ogino Y,Ayukawa Y,Tsukiyama Y,et al.The effect of platelet-rich plasma on the cellular response of rat bone marrow cells in vitro.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2005 Sep;100(3):302-7.
15 Fennis JP,Stoelinga PJ Jansen JA.Mandibular reconstruction:a hisological an histomorphometric study on the use of autogenous scaffolds,Particulate cortieo-caneellous bone grafts and Platelet rich Plasma in goats.Int J Oral Maxillofae Surg.2004,33(l):48-55.
16 Fujimori T.The effect of platelet-rich plasma combined with autogenous bone graft for bone regeneration in bone defects.Kokubyo Gakkai Zasshi.2005 Mar;72(l):90-7.Japanese.
17 Aghaloo Tl,Moy PK,Freymiller EG.Investigation of platelet-rich plasma in rabbit cranial defects:A pilot study.J Oral Maxillofac Surg.2002 Oct;60(10):1176-81.
18 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 Jan;33(l):56-9.
19 Butterfield KJ,Bennett J,Gronowicz G et al.Effect of Platelet-rich Plasma with autogenous bone graft for maxillary sinus augmentation in a rabbit model.J Oral Maxillofac Surg.2005,63(3):370-376.
20 Kim ES,Park EJ,Choung PH.Platelet concentration and its effete on bone formation in calvarial defects:An experimental study in rabbits.J Prosthet Dent.2001,86(4):428 — 433.
21 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.2004 Jan-Feb;19(l):59-65.
22 Freymiller EG,Aghaloo TL.Platelet-rich plasma:ready or not?J Oral Maxillofac Surg.2004 Apr;62(4):484-8.
23 Jensen TB,Rahbek O,Overgaard S,et al.Platelet rich plasma and fresh frozen bone allograft as enhancement of implant fixation.An experimental study in dogs.J Orthop Res.2004 May; 22(3):653-8.
24 Sanchez AR,Sheridan PJ,Eckert SE,et al.Regenerative potential of platelet-rich plasma added to xenogenic bone grafts in peri-implant defects:a histomorphometric analysis in dogs.J Periodontol.2005 Oct;76(10):1637-44.
25 Grageda E,Lozada JL,Boyne PJ,et al.Bone formation in the maxillary sinus by using platelet-rich plasma:an experimental study in sheep.J Oral Implantol.2005;31(1):2-17.
26 Kovacs K,Velich N,Huszar T,Fenyves B,et al.Histomorphometric and densitometric evaluation of the effects of platelet-rich plasma on the remodeling of beta-tricalcium phosphate in beagle dogs.J Craniofac Surg.2005 Jan;16(l):150-4.
27 Hokugo A,Ozeki M,Kawakami O,et al.Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel.Tissue Eng.2005 Jul-Aug;ll(7-8):1224-33.
28 Pryor ME,Yang J,Polimeni G,Koo KT,et al.Analysis of rat calvana defects implanted with a platelet-rich plasma preparation:radiographic observations.J Periodontol.2005 Aug;76(8):1287-92.
29 Pryor ME,Polimeni G,Koo KT,et aLAnalysis of rat calvaria defects implanted with a platelet-rich plasma preparation:histologic and histometric observations.J Clin Periodontol.2005 Sep;32(9):966-72.
30 Lucarelli E,Fini M,Beccheroni A,et al.Stromal stem cells and platelet-rich plasma improve bone allograft integration.Clin Orthop Relat Res.2005 Jun;(435):62-8.
31 Yamada Y,Hata K,Ueda M.Injectable bone.Clin Calcium.2002 Feb;12(2):228-32.
32 Yamada Y,Ueda M,Naiki T,et al.Tissue-engineered injectable bone regeneration for osseointegrated dental implants.Clin Oral Implants Res.2004 Oct;15(5):589-97.
33 Ito K,Yamada Y,Nagasaka T,et al.Osteogenic potential of injectable tissue-engineered bone:a comparison among autogenous bone,bone substitute (Bio-oss),platelet-rich plasma,and tissue-engineered bone with respect to their mechanical properties and histological findings.J Biomed Mater Res A.2005 Apr 1;73(l):63-72.
34 Tayapongsak P,O'Brien DA,Monteiro CB,Autologous fibrin adhesive in mandibular reconstruction with particulate cancellous bone and marrow.J Oral Maxillofac Surg.1994 Feb; 52(2):161-5;discussion 166.
35 Anitua E.Plasma rich in growth factors:preliminary results of use in the preparation of future sites for implants.Int J Oral Maxillofac Implants.1999 Jul-Aug;14(4):529-35.
36 Kassolis JD,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 Oct;71(10):1654-61.
37 Mazor Z,Peleg M,Garg AK,et al.Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement:patient series study.Implant Dent.2004 Mar;13(l):65-72.
38 Ueda M,Yamada Y,Ozawa R,et al.Clinical case reports of injectable tissue-engineered bone for alveolar augmentation with simultaneous implant placement.Int J Periodontics Restorative Dent.2005 Apr;25(2):129-37.
39 Sammartino G,Tia M,Marenzi G,et al.Use of autologous platelet-rich plasma (PRP) in periodontal defect treatment after extraction of impacted mandibular third molars.J Oral Maxillofac Surg.2005 Jun;63(6):766-70.
40 Shanaman R,Filstein MR,Danesh-Meyer MJ.Localized ridge augmentation using GBR and platelet-rich plasma:case reports.Int J Periodontics Restorative Dent.2001 Aug;21(4):345-55.
41 Froum SJ,Wallace SS,Tarnow DP,Effect of platelet-rich plasma on bone growth and osseointegration in human maxillary sinus grafts:three bilateral case reports.Int J Periodontics Restorative Dent.2002 Feb;22(l):45-53.