【作者】 姜伟；

【导师】 汤逊；

【作者基本信息】 昆明医学院 ， 外科学， 2010， 硕士

【摘要】 目的本研究以活性生物陶瓷双相磷酸钙(Biphasic calcium phosphate,BCP)为基材,通过复合纳米羟基磷灰石(Nano-Hydroxyapatite,Nano-HA)和壳聚糖(Chitosan,Cs),制备出纳米羟基磷灰石/壳聚糖/双相磷酸钙复合材料(Nano-HA/Cs/BCP),再负载重组人类骨形成蛋白2(Recombinant human bonemorphogenetic protein 2,rhBMP-2),制备出重组人类骨形成蛋白2/纳米羟基磷灰石/壳聚糖/双相磷酸钙复合材料(rhBMP-2/Nano-HA/Cs/BCP),拟通过建立兔竖脊肌肌袋包埋模型,评价两者植入动物体内后对局部组织、血液系统、肝肾脏器影响方面的生物相容性,以及观察16周内两种材料异位诱导成骨情况,从而探讨其异位成骨能力、骨诱导性和骨传导性,进而指导下一步的研究。方法48只日本大耳白兔,随机分成A组和B组,每组24只,分别形成竖脊肌肌袋,并在A组中包埋Nano-HA/Cs/BCP材料(A组材料),B组中包埋rhBMP-2/Nano-HA/Cs/BCP材料(B组材料)。术前每组随机选6只兔子,查血液常规,C反应蛋白(CRP),肝、肾功能指标作为正常参考值;术后2、4、8、16周,每组随机选6只兔子作相同检测,对同期两组之间、组内术后与其术前以及术后组内两两之间的检测结果进行比较并统计学分析。术后观察两组实验动物全身及切口愈合情况;4、8、12、16周分别摄X线片进行包埋材料的影像学观察,并处死兔子,每组6只,获取肝脏及肾脏标本、包埋材料与周围组织标本进行大体观察和组织学观察。结果1.实验室检查两组兔子各时期所行实验室检查结果,在同期两组之间比较、组内术后与其术前比较,以及术后组内两两比较差异均无统计学意义(P＞0.05)。2.大体观察两组兔子术后饮食、活动均正常,切口愈合良好,术后各时间点见肝脏、肾脏无明显异常。A组(Nano-HA/Cs/BCP):整个观察周期内,材料与肌肉之间结合紧密,未见明显的纤维囊壁形成;肌肉组织正常,未见充血水肿,无感染、坏死;未见任何软骨及骨生成。4周时,材料表面不平整,孔隙增宽,有少量血管及软组织长入孔隙,并分布在材料外周,中心未见组织分布;8周时,材料孔隙中长入大量血管和软组织,孔隙进一步增宽;12、16周时,材料表面覆盖新生血管网,外周及中心的孔隙中分布大量血管及软组织。B组(rhBMP-2/Nano-HA/Cs/BCP):整个观察周期内,材料与肌肉之间结合紧密,未见明显的纤维囊壁形成;肌肉组织正常,未见充血水肿,无感染、坏死。4周时,材料表面及外周孔隙中长入大量血管及软组织,中心分布较少;8周时,材料表面覆盖大量血管及软组织,材料孔隙增大、增宽,孔隙中血管分布密集;12周时,材料边缘见碎裂的颗粒,内部孔隙明显增宽,被大量新生血管和软组织填充;16周时,材料边缘见少量骨样组织,质地变硬。3.组织学观察A组(Nano-HA/Cs/BCP):4周时,材料与肌肉组织界面、肌肉内和材料外周见成纤维细胞、毛细血管和胶原纤维,以及炎性细胞浸润,以淋巴细胞和巨噬细胞为主,肌肉组织较正常稍萎缩;8周时,材料与肌肉组织界面形成薄层纤维膜,包括致密结缔组织和少量成纤维细胞,材料内部有大量血管和胶原纤维,见少量淋巴细胞和巨噬细胞浸润,肌肉组织无明显异常;12周时,纤维膜明显变薄,材料外周及内部孔隙中形成大量血管和胶原纤维,材料内部见巨噬细胞及成纤维细胞;16周时,纤维膜变得不完整,且较12周更薄,材料内主要形成成熟的胶原纤维,偶见炎性细胞;整个观察周期中见残存材料逐渐减少,16周时大部分材料仍存在,并且未见软骨母细胞、成骨细胞、软骨及骨形成。B组(rhBMP-2/Nano-HA/Cs/BCP):各观察周期肌肉组织未见明显异常;4周时,材料与肌肉组织界面形成一层菲薄的不完整纤维膜,肌肉内有成纤维细胞、毛细血管和疏松结缔组织,见淋巴细胞和巨噬细胞浸润,并有胶原纤维和血管长入材料表面和外周孔隙中;8周时,纤维膜无明显变化,肌肉内纤维增生不明显,见少量炎细胞浸润,以巨噬细胞为主,材料内部孔隙中形成丰富的微血管和胶原纤维;12周时,纤维膜明显变薄,周围有脂肪组织,大量胶原纤维和微血管分布在材料表面,材料外周见透明软骨形成,并见软骨母细胞、软骨陷窝及软骨细胞;16周时,材料边缘见少量骨组织形成,材料外周可见少量类骨质,其周围微血管和胶原纤维丰富。4.影像学观察术后4、8、12、16周X线片见两组材料轮廓均无明显改变,8、12、16周时见两种材料内部逐渐出现透亮区,材料密度逐渐减低;16周时,B组材料边缘局部出现密度增高影。结论1、两种复合材料植入兔体内16周,未引起明显的局部组织坏死、局部炎症反应及排斥反应,血液中主要血液成分,炎性指标,肝、肾功能指标与其术前比较差异无统计学意义,肝、肾组织观察未见明显异常,说明观察周期内两种材料及其降解产物对局部组织、血液系统及肝肾脏器影响方面表现出良好的生物相容性。2、B组材料(rhBMP-2/Nano-HA/Cs/BCP)包埋于兔竖脊肌内16周时见少量骨形成,说明A组材料(Nano-HA/Cs/BCP)负载较低剂量的rhBMP-2(10μg)后表现出一定的成骨能力和骨传导性,rhBMP-2发挥了一定的骨诱导作用。3、A组材料(Nano-HA/Cs/BCP)包埋于兔竖脊肌16周内未见骨形成,说明其在观察周期内未表现出骨诱导性和异位成骨能力。4、对于材料及其降解产物对局部组织和机体的远期影响,仍需进一步研究以全面评价复合材料的体内生物相容性。5、A组材料(Nano-HA/Cs/BCP)作为理想的rhBMP-2载体,尚需改进和优化。更多还原

【Abstract】 ObjectiveIn this study,using biphasic calcium phosphate of biological activity ceramics (Biphasic calcium phosphate,BCP) as the substrate,through compounding nano-hydroxyapatite(Nano-Hydroxyapatite,Nano-HA) and chitosan(Chitosan,Cs) to make out nano hydroxyapatite/chitosan/biphasic calcium phosphate composite material(Nano-HA/Cs/BCP),again loading recombinant human bone morphogenetic protein 2(Recombinant human bone morphogenetic protein 2,rhBMP-2) to make out recombinant human bone morphogenetic protein 2/nano-hydroxyapatite/chitosan / biphasic calcium phosphate composite material(rhBMP-2/Nano-HA/Cs/BCP).Then building muscular bag model in the erector spinae of rabbit,to evaluate the internal biocompatibility of the two composite materials,mainly on the influence of the blood system,liver and kidney,and local organization;furthermore,to observe ectopic bone formation in 16 weeks,to find out the ectopic osteogenesisoste,osteoconductivity and oinductivity of them,and thus to guide future research.Methods48 Japanese white rabbits were randomly divided into A group and B group,24 rabbits each group,embedded Nano-HA/Cs/BCP material(A material) into muscular bag of erector spinae formed in group A,and embedded rhBMP-2/Nano-HA/ Cs/BCP material(B material) in group B.Before operation,6 rabbits in each group were randomly selected to take routine blood test,detect C-reactive protein(CRP),liver and kidney function in the blood,which results used as normal reference value; 2,4,8and16 weeks after operation,rabbits were inspected the same way with preoperation,then compared and analyzed by statistics the results of two groups on the same period,preoperation and postoperation of each group.And the wound healing and general condition of all the experimental animals were observed after operation;4,8,12,16 weeks,postoperative X-rays were taken to observe the embedded material,and 6 rabbits were killed every time to obtain the liver and kidney specimens,and specimens of materials together with surrounding tissue for gross and optical microscope observation.Results1.Laboratory ExaminationAll the results of laboratory examination compared and analyzed by statistics between two groups in the same period,and between preoperative and postoperative each group showed no significant difference(P＞0.05).2.Gross ObservationThe diets and activities of rabbits after operation were normal;the wound healed in normal;and the liver and kidney had no obviously abnormal.A group:during the whole observation period,materials were closely integrated with the surrounding muscle tissue;the muscle had no congestion and edema,no infection and necrosis;and the interface between muscle and material revealed no significant fiber wall formation;surrounding and internal material had no cartilage and bone formation.4 weeks after operation,the surface of material was not smooth, pores enlarged,and a small amount of blood vessels and soft tissue grew into the pores which distributed in the peripheral material.8 weeks after operation,the surface and internal material were covered with blood vessels and soft tissue,pores enlarged significantly.12 and 16 weeks after operation,the surface of material was covered by new vascular,and the internal pores were filled with a large number of blood vessels and soft tissue.B group:during the whole observation period,,materials were closely integrated with the surrounding muscle tissue;the muscle had no congestion and edema,no infection and necrosis;and the interface between muscle and material revealed no significant fiber wall formation.4 week after operation,a large number of blood vessels and soft tissues grew into the surface and peripheral pores of material,and less was distributed in center of materials.8 weeks after operation,a large number of blood vessels and soft tissue were distributed in the increasing surface pores of the material;and the internal pores of material were filled by intensive blood vessels;the number of pore was increased,and the size was enlarged.12 weeks after operation, the edge of the materials appeared crushed powder;internal porosity increased significantly and were full of new blood vessels and soft tissue.16 weeks after operation,the edge of the materials had a small amount of bone,and the texture was hard.3.Histological ObservationLiver and kidney:both of them were normal.A group:4 weeks after operation,fibroblasts,new blood vessels and fibrous tissue were observed in the muscle and interface;muscle cells were slightly shrinking, and the main inflammatory cell were lymphocytes and macrophages.8 weeks after operation,the interface formed a thin layer of fiber membrane,including a small amount of dense connective tissue and fibroblasts;a large number of blood vessels and fibrous tissue were in the material;and a small amount of macrophage was found in the material.12 weeks after operation,the fiber membrane in the interface became thinner;the internal and peripheral pores of materials were distributed by a large number of blood vessels and fibrous tissue;and the macrophages and fibroblasts were in the material;16 weeks after operation,fiber membrane was thinne and became incomplete,and the enlarged pores were filled with blood vessels and fibrous tissue.no bone,osteoblasts and chondroblastoma were found in the whole experiment.B group:muscle tissues were normal during the 16 weeks.4 weeks after operation,the interface formed a thin and incomplete layer of fiber membrane, fibroblast,angiogenesis and loose connective tissue were in the muscle;a small amount of lymphocytes and macrophage were found;fibrous tissue and blood vessels grew into the peripheral and surface pores.8 weeks after operation,fiber membrane had no significant changes,and fiber proliferation in the muscle decreased;there were no significant infiltration of inflammatory cell;a large number of capillaries and collagen fibers were in the material;12 weeks after operation,the fiber membrane became significantly thinner,a lot of fibrous tissue and blood vessels grew into the surface pores,no fibrosis were seen in the muscle,a small amount of cartilage formed in the peripheral materials,and cartilage lacuna,and chondroblastoma were found;16 weeks after operation,the edge of materials formed a small amount of osteoid and bone.4.Imaging Observation4,8,12 and16 weeks after operation,X-ray showed no significant change in the two material profile,but in the two materials had gradual emergence of translucent and the material density reduced gradually;16 weeks after operation,local marginal area of B material appeared high density shadow.Conlusions1.Two kinds of composite material implanted in rabbits for 16 weeks did not cause significant local tissue necrosis,local inflammatory reaction and rejection;the major blood components,inflammatory index,liver and kidney function index analyzed by statistics between the relults of preoperation and postoperation,showed no statistical significance;and observation of liver and kidneyed had no obvious abnormalities;all results above indicated two materials showed good biocompatibility in the blood system,local tissue,liver and kidney.2.B material(rhBMP-2/Nano-HA/Cs/BCP) embedded in the erector spinae of rabbit formed a small amount of bone in the16th week,which indicated A material loaded low dose of rhBMP-2(10μg) showed certain osteoconductivity and osteogenesis,and rhBMP-2 showed certain osteoinductivity.3.A material(Nano-HA/Cs/BCP) embedded in the erector spinae of rabbit showed no bone formation during 16 weeks,which indicated the material did not show osteoinductivity and osteogenesis during the time.4.In order to fully evaluate the in vivo biocompatibility of the composite materials,the long-term effects of the material and its degradation products on the local tissue and the whole body still need further study.5.A material(Nano-HA/Cs/BCP) as an ideal carrier of rhBMP-2 still need to improve and optimize.更多还原