【作者】 冯伟；

【导师】 靳安民；

【作者基本信息】 南方医科大学 ， 骨外科学， 2009， 硕士

【摘要】 由于创伤、感染和肿瘤等造成的骨缺损是骨科临床工作中经常遇到的问题,目前临床上通常通过植入修复材料来治疗,植入修复材料主要应起以下作用:（1）骨传导作用:移植物主要帮助骨形成支架作用,即以移植物为支架,使宿主的血管和细胞进入植入物,形成新骨,移植物坏死被吸收,并逐渐被新骨替代。（2）移植材料除了起支架作用即骨传导作用外还应具有骨诱导作用,只有这样才能起到快速良好的修复骨缺损作用。长期以来植入物常选用新鲜自体骨和异体骨,新鲜自体骨虽是最理想的植骨材料,其本身既具有骨诱导又具有骨传导功能,但其来源有限,且会造成供骨区慢性疼痛、血肿形成、感染等多种并发症;异体骨虽然来源相对充足且可避免手术后给患者带来的疼痛、感染等由于自体取骨带来的并发症,但其存在排异反应及传播HIV、肝炎等疾病可能,可见上述两种方法均存在各自的弊端。因此,人们一直在寻找既有良好骨传导作用又能具有骨诱导作用的人工骨修复替代材料,从而避免应用自体骨及异体骨材料引起的慢性疼痛、感染和排斥、传播疾病的危险。研究背景:19世纪70年代末,羟基磷灰石（Hydroxyapatite,HA）作为新型生物材料问世以来,引起了材料学科和医学界的广泛兴趣。HA因其化学成分和晶体结构与人体骨骼组织的主要无机矿物成分基本相同,引入人体后不会产生排异反应,故其作为骨修复替代材料在国内外广泛应用。其已被动物实验及临床研究证实具有无毒、无刺激性、良好的生物活性、生物相容性及骨传导性、较高的机械强度及化学性质稳定等特点。但因HA的颗粒和脆性较大、缺乏可塑性、体内降解缓慢及抗疲劳破坏强度低,难于被机体完全替代、利用,使其临床应用受到了限制。随着纳米技术的发展,生物性能更佳的纳米复合材料成为研究热点。研究发现天然骨中基本成分是纳米级的HA和Ⅰ型胶原纤维,纳米羟基磷灰石（Nano-hydroxyapatite,Nano-HA）的组成和结构与人体硬组织、骨骼、牙齿中的无机成分相似,且Nano-HA能自固化成型,使用方便,无毒副作用、可以吸收和降解,具有较小直径的纳米颗粒,使其更有利于功能分化良好的成骨细胞分泌骨基质,更有利于新骨形成,在细胞生长的过程中更有利于其功能代谢具有更佳的生物性能能牢固地结合于生活的骨组织中,提供适应新骨沉积的生理基质,引导周围骨组织再生,具有骨引导性和亲骨性,具有优良的生物活性,被认为是一种很理想的组织替换材料。虽其在强度、韧性及骨诱导性方面还不算完美,但至今它仍是诸多学者认可的作为修复骨缺损替代植入的良好支架材料,基于此,我们在本实验中选用了Nano-HA作为骨缺损替代植入的基质材料。聚羟基丁酸戊酸酯（Polyhydroxybutyrate-hydroxyvalerate,PHBV）是聚羟基丁酸酯（Poly hydroxybutyrate,PHB）与羟基戊酸酯（Hydroxyvalerate,HV）的共聚物,质轻,耐腐蚀,孔隙率、孔径皆可控制,其具有良好的材料可塑性、机械强度,生物相容性、生物可降解等特点,且有压电性,可刺激新骨的成长。研究显示PHBV有很好的引导骨组织再生的功能,是一种具有很好可降解性能与生物相容性的高分子材料及较理想的引导骨组织再生的天然材料。聚乙二醇（Polyethylene glycol,PEG）因其分子链的柔顺性,具有突出的理化和生物性能,具有亲水性、在水和有机溶剂中的可溶性、无毒、无抗原性和免疫性。在生物医用材料应用中,PEG或者其高分子量聚合物常被用于修饰材料,研究表明表面活性剂PEG的加入能有效地降低微粒的表面张力,改善制品的团聚性,是目前在嵌段共聚物中应用最为广泛的亲水链端,在亲水链段PEG长度一定的条件下,可以通过改变疏水链段PLA的长度来调节聚合物胶束的物理化学性质。以PHBV为基础,选择高亲水性材料PEG与之共同混合可改善PHBV、HA在应用中亲水性差的局限性,进而改善复合材料的生物降解性能。可见,将上述两种物质引入Nano-HA基质材料中有望改善复合材料的可塑性、降解速度、生物相容性、机械强度及骨引导性等生物学性能。骨形态发生蛋白（Bone morphogeneticprotein,BMP）具有诱导未分化的间充质细胞分化成软骨成骨的能力。活性人工骨中的BMP具有诱导长入材料的间充质细胞向骨系细胞分化,分泌类骨基质继而矿化成骨的能力。我们在复合材料中加入重组人骨形态发生蛋白-2（recombinant human bone morphogenetic protein-2,rhBMP-2）活性物质来增加复合材料的骨诱导性。综上所述,我们在Nano-HA基质材料中加入PHBV、PEG及rhBMP-2等辅助成分制成Nano-HA/PHBV-PEG-rhBMP-2复合材料人工骨,探讨该复合材料能否在修复骨缺损过程中能表现出较单纯Nano-HA更加优良的生物学性能。目的和意义:通过修复新西兰大白兔桡骨骨缺损的实验研究探讨Nano-HA/PHBV-PEG-rhBMP-2复合材料人工骨对骨缺损的修复作用,为临床应用提供理论依据。实验方法:①通过人工合成方式制成Nano-HA/PHBV-PEG-rhBMP-2复合材料人工骨及Nano-HA材料人工骨。②选择50只健康雄性新西兰大白兔,体重1.5～2.0kg,并将其随机分成A、B两组:每组25只,分别将其称重后,用20g/L戊巴比妥纳30mg/kg兔耳缘静脉注射麻醉,常规消毒铺巾,取兔双侧前肢中段外侧切开长约3cm纵行切口,暴露桡骨干中上段,在距桡骨远端2.5cm处用线锯连同骨膜一起锯断桡骨,以同样的方法于第一截骨线近侧1.5cm处再次锯断桡骨,连同骨膜切除两断端间的桡骨,造成双侧桡骨节段性骨缺损模型,庆大霉素反复冲洗后,其中A组左侧植入Nano-HA/PHBV-PEG-rhBMP-2复合材料人工骨,作为实验组;B组左侧植入Nano-HA材料人工骨,作为对照组;A、B两组右侧均不植入任何材料,作为空白对照组,逐层缝合伤口。术后立即肌肉注射青霉素40万U/只,连续3d,不予外固定,分笼喂养。③观察指标:大体观察:术后观察各组动物精神状态、饮食、大小便、活动情况以及伤口有无肿胀、分泌物出现。取材后观察各组成骨及材料降解情况。X射线摄片检查:分别于材料植入术后第2周、4周、8周、12周及16周随机选取2只兔子处死取材,行X射线摄片检查,观察各组动物骨缺损区的骨痂生长、材料降解及骨连接情况。骨密度测量:分别于材料植入术后第4周、8周、12周及16周从各组中随机选取6只兔子处死取材,将实验组及对照组标本置双能X线骨密度仪,测定骨缺损区成骨密度,对测定结果应用统计软件进行分析。生物力学测试:分别于材料植入术后第4周、第8周、第12周、第16周在骨密度测试结束后剔净骨膜及软组织,在MTS-858材料测试机上行三点抗弯试验,测量各组标本抗弯曲强度,对测定结果应用统计学软件进行分析。实验结果:大体观察:术后实验动物无一例死亡,术后动物进食及活动正常,切口无红肿、渗液等炎性反应,切口如期愈合。材料植入后第4～8周,实验组及对照组材料逐渐降解,与骨界面模糊,有明显新骨形成,与宿主骨组织结合紧密,以实验组明显。第12～16周,两组植入材料已基本降解完全,与宿主骨逐渐融合,新生骨进一步增多,骨缺损得到基本修复,仍以实验组明显。空白对照组随着术后时间的延长在桡骨缺损处未见明显新骨形成,无明显骨性连接形成,髓腔封闭,骨端逐渐硬化吸收,骨缺损无修复。X射线检查结果:材料植入后第2～4周:实验组及对照组材料密度较宿主骨密度高,并与宿主骨分界清楚,材料与骨结合部均有少量骨痂形成,其密度较低,以实验组明显。材料植入后8周:实验组材料与宿主骨分界模糊,体积明显缩小,植入材料已与受体断端骨性愈合,皮质骨连续性较好,骨髓腔部分贯通;对照组植入材料与受体断端骨性愈合,但皮质骨连续性较差,骨髓腔尚未贯通。材料植入后12～16周:实验组植入材料逐渐与受体骨骨性愈合成一体,密度逐渐接近,骨皮质基本连接完成,材料基本完全降解,骨髓腔已通,塑形完全,骨缺损修复完成;对照组植入材料逐渐与受体骨骨性愈合成一体,密度逐渐接近,骨皮质连续性较差,材料大部分降解,骨缺损处充满骨痂,骨髓腔基本再通,塑形基本完全,骨缺损处得到基本修复。空白对照组可见骨缺损处有一定的骨膜反应,未见明显新骨形成,无明显骨性连接形成,骨端逐渐硬化吸收,髓腔封闭,骨缺损无修复。骨密度测量结果:材料植入后两组间新生骨密度整体比较有显著性差异（F=78.748,P=0.000）,实验组（均数为0.128 g/cm²）显著高于对照组（均数为0.093g/cm²）,分别在术后4周、8周、12周、16周时两组间比较有显著性差异(t_4w=14.894、t_8w=5.187、t_12w=3.173、t_16w=4.222,P_4w=0.000、P_8w=0.000、P_12w=0.010、P_16w=0.002),实验组均高于对照组。材料植入后不同时间点间新生骨密度整体比较有显著性差异（F=115.337,P=0.000）,实验组组内不同时间点间比较有显著性差异（F=126.060,P=0.000）,对照组组内不同时间点间比较有显著性差异（F=135.585,P=0.000）,结果均为16周＞12周＞8周＞4周。随着术后时间的延长两组间新生骨密度差异无显著变化（F=0.271,P=0.846）。生物力学测定结果:材料植入后两组间桡骨抗弯曲强度整体比较有显著性差异（F=125.543,P=0.000）,实验组（均数为66.131Mpa）显著高于对照组（均数为41.982Mpa）,分别在术后4周、8周、12周、16周时两组间比较有显著性差异(t_4w=3.245、t_8w=5.530、t_12w=7.303、t_16w=5.806,P_4w=0.009、P_8w=0.000、P_12w=0.000、P_16w=0.000),实验组均高于对照组。材料植入后不同时间点间桡骨抗弯曲强度整体比较有显著性差异（F=284.006,P=0.000）,实验组组内不同时间点间比较有显著性差异（F=142.682,P=0.000）,对照组组内不同时间点间比较有显著性差异（F=153.064,P=0.000）,结果均为16周＞12周＞8周＞4周。随着材料植入术后时间的延长两组间桡骨抗弯曲强度差异呈现逐渐显著的趋势（F=7.043,P=0.001）。主要结论:①较大骨缺损形成后,不植入任何替代材料或不进行任何干预处理,骨缺损处将无明显骨性连接形成,骨端将逐渐硬化,髓腔逐渐封闭,骨缺损难以自行修复愈合。②Nano-HA作为修复骨缺损的替代材料植入后能表现出较好的生物相容性、降解性及骨传导性,是作为修复骨缺损的一种良好的替代植入基质材料。③在Nano-HA材料中加入PHBV、PEG及rhBMP-2等辅助成分并人工合成的Nano-HA/PHBV-PEG-rhBMP-2复合材料人工骨,在修复骨缺损过程中能表现出较Nano-HA更加良好的生物学性能,是一种比较优良的修复骨缺损的替代植入复合材料。④本研究为进一步寻找性能更加优良的修复骨缺损的替代植入材料提供了参考。更多还原

【Abstract】 Because of trauma,infection and tumor of the bone defect causing orthopedic clinical work were often encountered in question,usually on the current clinical repair material through the implantation treatment,implant repair material should play a major role in the following:（1）The role of bone conduction:the main graft stent assist the role of bone formation,that was,for the stent graft to the host blood vessels and cells into the implants,the formation of new bone,graft necrosis was absorbed and gradually by the new bone substitute.（2） Except from stent graft that was the role of role of bone conduction should also have a bone-inducing effect,the only way to play a quick good role to repair bone defects.Long-term implants often choose fresh autogenous bone and bone,fresh autogenous bone graft,although the ideal material,both its own and possesses osteoinductive bone conduction function, but its sources were limited,and will cause for the bone area of chronic pain, hematoma formation,infection and other complication;Although the source of bone is relatively abundant and can avoid the post-operative pain to the patients,because of infection,such as autologous bone complications brought about,but the rejection of its existence and the spread of HIV,hepatitis and other diseases possible.Can be seen that the above-mentioned two methods are the drawbacks of their own existence. Therefore,people have been looking for both good at the role of bone conduction as well with bone-inducing effect of artificial bone substitute material to repair,thus avoiding the application of autogenous bone and allograft bone material caused by chronic pain,infection and rejection,the spread of dangerous diseases.Research background:The 19th century,the end of the 70s,as a new type of hydroxyapatite（HA） since biological material,disciplines and material aroused wide interest in the medical profession.HA because of its chemical composition and crystal structure of human bone tissue and the main inorganic mineral composition was basically the same,the introduction of the human body did not produce rejection, so as a substitute for bone repair materials widely used at home and abroad.It had been animal experiments and clinical research had shown that with non-toxic, non-irritating,good bioactivity,biocompatibility and bone conductivity,high mechanical strength and chemical properties of stable.HA particles but also larger and brittle,the lack of plasticity,in vivo degradation of the slow and anti-fatigue damage to the lower intensity and difficult to be a perfect substitute for the body to use its clinical application is limited.With the development of nanotechnology,biological properties of nanocomposit -es better become a research hot spot.Study found that the basic components of natural bone was nanoscale HA and the collagen type I fibers,nano-hydroxyapatite （Nano-HA） of the composition and structure of human hard tissue,bone,teeth of the inorganic constituents similar,and the Nano-HA was able to self-curing molding, easy-to-use,non-toxic side effects,could absorb and degradation,with a smaller diameter of the nano-particles,and make it more conducive to functional well-differentiated osteoblast secretion of bone matrix,was more conducive to new bone formation,in the process of cell growth was more conducive to its function with better metabolic biology performance can be firmly integrated in the life of bone tissue the provision of adapting to the new bone matrix deposition physiological and guide bone tissue regeneration with bone lead and pro-bone,and have excellent biological activity,was considered to be a very ideal replacement organize material. Although its strength,toughness and bone-induced still not perfect,but so far it was still recognized by many scholars as a substitute for bone defect implants good scaffold material.Based on this,our experiments at the Nano-HA was chosen as an alternative to repair bone defects implanted matrix material. Polyhydroxybutyrate-hydroxyvalerate（PHBV） were polyhydroxybutyrate（PHB） and hydroxyvalerate（HV） copolymer,light weight,corrosion resistance,porosity, aperture could be controlled,and it had a good plasticity material,mechanical strength,biocompatibility,biodegradability,etc.,and there was piezoelectricity,could stimulate new bone growth.PHBV studies had shown there was a very good guided bone regeneration function,was a very good performance and biocompatibility of biodegradable polymer material and better guided bone regeneration of natural material.Polyethylene glycol（PEG） because of its molecular chain of supple line, withoutstanding physical and chemical and biological properties,with a hydrophilic, in water and soluble in organic solvent,non-toxic,non-antigenicity and immunity.At the application of biomedical material,PEG or its high molecular weight polymers are often used for modification of material,the study showed that surfactant addition of PEG could effectively reduce the particle surface tension and improve the products of the reunion,were present in the block copolymer material most widely used hydrophilic side chain,in the hydrophilic PEG segment length under certain conditions,you could by changing the hydrophobic segment to adjust the length of PLA polymer micelles of physical and chemical properties.To PHBV based on selection of high hydrophilic PEG material mixture could be improved with common PHBV,HA in the application of the limitations of bad hydrophilic,thus improving biodegradable composite material performance.Could be seen that these two substances the introduction of Nano-HA matrix material was expected to improve the plasticity of composite material,the degradation rate,biocompatibility,mechanical strength and bone biology,such as leading performance.Bone morphogenetic protein （BMP） could induce undifferentiated mesenchymal cells differentiate into osteogenic ability of cartilage.BMP activity in bone in growth material could induce mesenchymal cells to bone cell differentiation,secretion of type of bone matrix and then the ability of osteoblast mineralization.We added in the composite material of recombinant human bone morphogenetic protein-2（rhBMP -2） active material to increase the composite material-induced bone.To sum up,our Nano-HA in the matrix material by adding PHBV,PEG,and rhBMP-2 and other auxiliary components made of Nano-HA / PHBV-PEG-rhBMP-2 composite bone,the composite material to explore the possibility of repair the process of bone defect can be demonstrated more simply Nano-HA more excellent biological properties.Purpose and significance:Through the repair of radial bone defects in New Zealand white rabbits to explore Nano-HA / PHBV-PEG-rhBMP-2 composite artificial bone for repairing bone defects,in order to provide a theoretical basis for clinical application.Experimental Methods:①Through the synthetic methods separately into Nano-HA / PHBV-PEG-rhBMP-2 composite bone and the purely artificial Nano-HA materials.②Select 50 healthy male New Zealand white rabbits,weighing 1.5～2.0 kg,and were randomly divided into A,B two groups:each group 25,respectively, after its weighing,using 20g/L pentobarbital 30mg/kg satisfied rabbit ear fate intravenous anesthesia,conventional disinfection shop towel,take the rabbit with bilateral forelimb mid-lateral incision approximately 3cm longitudinal incision, exposing the radial shaft in the previous paragraph,At 2.5cm from the distal radius fracture department together with the periosteum using wire saw with radial saw,the same way in the first osteotomy line 1.5cm proximal radial saw again,together with periosteal resection between the two ends of the radial bone,resulting in double lateral radial segmental bone defect model,gentamicin after repeated washing,the left side of one of A group Nano-HA/PHBV-PEG-rhBMP-2 composite artificial bone implants,as the experimental group;B group of the left implant Nano-HA artificial bone material,as a control group;A,B were not implanted into the right side of any material,as the blank control group,the wound sutured layer by layer.Immediately after intramuscular injection of penicillin 400,000U each,only row 3d,no external fixation,sub-cage feeding.③Outcome Measures:Gross observation:Postoperative observation of the mental state of animals in each group,catering,toilet,activities and whether the wound swelling,secretions occur.Based on observation of the various components after the bone and material degradation.X-ray radiography examination: Materials were implanted after the first 2 weeks,4 weeks,8 weeks,12 weeks and 16 weeks were randomly selected based on two rabbit executed,the X-ray radiography examination,observation of each group of animal bone defect area the callus growth, material degradation and bone connections.Bone mineral density measurements: Materials were implanted after the first 4 weeks,8 weeks,12 weeks and 16 weeks from each group were randomly selected based on six killed rabbit,the experimental group and control group specimens home dual-energy X-ray absorptiometry,to measure bone mineral density into a bone defect area,application of statistical software to analyze the results on the determination.Biomechanical test:Materials were implanted after the first 4 weeks,8 weeks,12 weeks and 16 weeks bone mineral density test tick the net after the periosteum and soft tissue,at MTS-858 material testing machine up three points bending test specimens measured in each group of anti-bending strength,the results on the determination of the application of statistical software for analysis.Experimental results:The results of gross observation:None of the experimental animals after the death of animals after the normal eating and activity, incision-free swelling,exudate,such as inflammatory response,wound healing on schedule.Material implanted the first 4 to 8 weeks,experimental group and control group gradually material degradation,and bone interface vague,there was obvious new bone formation,combined with the host bone tissue close to the experimental group significantly.12～16 weeks,two groups of material degradation had been basically complete,the gradual integration with the host bone,new bone to further increase access to basic repair of bone defects is still experimental group.With the blank control group after the extension of time at the radial bone defect had no obvious new bone formation,no obvious bony connection formation,the medullary cavity closed end of the gradual hardening of bone absorption,bone defect without repair.The results of radial X-ray examination:The first 2 to 4 weeks after material implanted:Experimental group and control group than the host material density of bone mineral density in high and clear boundaries with the host bone,the department of material and osseointegration had a small amount of bone callus formation,and its low density to the experimental group significantly.8 weeks after implanted of material:Experimental group materials fuzzy boundaries with the host bone volume significantly reduced,implant materials had been with the receptor fragments of bone healing,better continuity of cortical bone,bone marrow cavity through the part;the control group implanted materials with the receptor fragments of bone healing,but poor continuity of cortical bone,bone marrow cavity has not yet been through.12 16 weeks after implantation of material:The experimental group implanted with the receptor gradually material bone healing into one,density gradually near,the basic cortical connections completed basic fully degradable material,bone marrow cavity had endorsed,completely remodeling,bone completion of defect repair;control group implanted with the receptor gradually materials bone healing into one,density gradually near,poor continuity of cortical bone,most of materials degradation,bone defect filled with bone,bone marrow cavity recanalization basic,plastic the basic shape completely,bone defect basic repair.Blank control group,bone defects can be seen there was a certain degree ofperiosteal reaction,no obvious new bone formation, no obvious connection of the formation of bone,bone-side gradually hardening absorption,medullary cavity closed without repair of bone defects.The results of new bone mineral density measurement:After implantation of material,the new bone mineral density between the two groups was significant difference（F =78.748,P = 0.000）,the experimental group（mean 0.128 g/cm²） was significantly higher than the control group（mean 0.093g/cm²）,respectively after 4 weeks,8 weeks,12 weeks,16 weeks compared between the two groups there was significant difference(t_4w= 14.894,t_8w=5.187,t_12w= 3.173,t_16w = 4.222,P_4w = 0.000,P_8w = 0.000,P_12w = 0.010,P_16w = 0.002),and the experimental group were all higher than the control group.Material at different time point after implantation,the new bone mineral density in the overall comparison between the two groups there was significant difference（F=115.337,P=0.000）,the experimental group at different time point was significantly different（F=126.060,P=0.000）,the control group at different time point was significantly different（F=135.585,P=0.000）,and the results were all 16 weeks＞12 weeks＞8 weeks＞4 weeks.As the time extension after implantation of material,the new bone mineral density differences between the two groups had no significant changes（F=0.271,P=0.846）.The results of biomechanical measurement:After implantation of material,the anti-bending strength of radial between the two groups was significant difference （F=125.543,P=0.000）,the experimental group（mean 66.131Mpa） was significantly higher than the control group（mean 41.982Mpa ）,respectively after 4 weeks,8 weeks, 12 weeks,16 weeks compared between the two groups there was significant difference(t_4w=3.245、t_8w=5.530、t_12w=7.303、t_16w=5.806、P_4w=0.009、P_8w=0.000、P_12w=0.000、P_16w=0.000),and the experimental group were all higher than the control group.Material at different time point after implantation,the anti-bending strength of radial in the overall comparison between the two groups there was significant difference（F=284.006,P=0.000）,the experimental group at different time point was significantly different（F=142.682,P=0.000 ）,the control group at different time point was significantly different（F=153.064,P=0.000 ）,and the results were all 16 weeks＞12 weeks＞8 weeks＞4 weeks.As the time extension after implantation of material,the anti-bending strength of radial difference between the two groups of anti-radius bending strength gradually showed significant differences in trends（F=7.043, P=0.001）.Major conclusions:①The formation of large bone defect after implantation is not any substitute material or deal with any intervention,bone defect will be no obvious connection of the formation of bone,bone-side will gradually hardening,gradually closed chamber,it is difficult to repair bone defect healing.②Nano-HA as the repairing bone defect after implantation of alternative material shows good biocompatibility,degradability and bone conduction,as to repair bone defect for a good alternative to implanted matrix material.③Material at Nano-HA by adding PHBV,PEG,and rhBMP-2 and other auxiliary components and synthetic Nano-HA/PHBV-PEG-rhBMP-2 composite bone in repairing bone defect demonstrated in the course of a more Nano-HA better biological performance,is a relatively good substitute for repairing bone defect implanted in composite material.④In this study,in order to further look for performance more excellent alternative to repair bone defect implanted reference material.更多还原