【作者】 金哲；

【导师】 吕刚；

【作者基本信息】 中国医科大学 ， 外科学， 2009， 博士

【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CaCl₂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。更多还原

【Abstract】 ObjectiveIt 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.Method1、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.Result1、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.Conclusion1、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更多还原