【作者】 于绍斌；

【导师】 董启榕；

【作者基本信息】 苏州大学 ， 骨外科学， 2008， 博士

【摘要】 第一部分:兔自体和异体髌韧带移植重建前交叉韧带后移植物Ⅲ型胶原表达与生物力学的变化目的研究兔自体和异体髌韧带移植重建前交叉韧带后,移植物中Ⅲ型胶原表达和生物力学性能的变化。方法36只健康成年新西兰白兔,切除双侧前交叉韧带,每只兔随机一侧移植同种异体髌韧带,共36膝,另一侧移植自体髌韧带,共36膝。分别于手术后3周、6周、12周切取移植物,反转录一聚合酶链反应检测Ⅲ型前胶原-mRNA表达,免疫组织化学方法检测Ⅲ型胶原表达,并截取双侧膝关节股骨-移植物-胫骨复合体行生物力学检测。另取6只健康成年新西兰兔不进行前交叉韧带重建,为正常对照。结果正常前交叉韧带及移植前自体和异体髌韧带Ⅲ型胶原表达阴性;移植后各时间点移植物均可见Ⅲ型前胶原-mRNA表达,自体髌韧带术后6周达到高峰,而异体髌韧带手术后12周Ⅲ型前胶原-mRNA表达仍较高;异体髌韧带手术后生物力学性能丢失明显,随着Ⅲ型胶原表达减少,生物力学性能逐渐增加,但未达到正常前交叉韧带水平。结论受体细胞侵入移植物,合成Ⅲ型胶原,可能是移植物体内重建过程中生物力学性能降低的原因之一;异体移植物Ⅲ型胶原合成时间延长于自体移植物;确切了解交叉韧带移植物重建中胶原基质的改变,对于促进移植物术后重建具有重要意义。第二部分:富血小板血浆来源的富生长因子液对兔异体冻干跟腱移植后早期微血管生成的影响目的观察富血小板血浆来源的富生长因子液对兔异体冻干跟腱移植重建前交叉韧带后早期微血管生成的影响。方法12只健康成年新西兰白兔切除双侧前交叉韧带,随机一侧植入经等量生理盐水预处理后的兔异体冻干跟腱(NS组),另一侧植入经血小板血浆来源的富生长因子液预处理后的兔异体冻干跟腱(PRGF组)。2只健康成年白兔为空白组,仅切开一侧膝关节。术后2周、4周、6周对移植物行苏木精-伊红染色和免疫组织化学染色检查, Weidner法定量检测移植物微血管密度。结果PRGF组MVD高于NS组(P<0.05);术后2周、4周、6周,NS组与PRGF组微血管密度分别为2.52±0.45,3.41±0.44,2.57±0.51和3.56±0.81,4.91±0.46,3.01±0.75,两组间各时间点差异均有统计学意义(P<0.01);而且PRGF组在新血管形成的时间、血管形成数量及血管长入肌腱的深度方面均优于NS组。结论富血小板血浆来源的富生长因子液可促进异体冻干跟腱移植重建前交叉韧带后早期微血管生成。第三部分:富生长因子液对兔异体冻干跟腱重建前交叉韧带后胶原表达及生物力学的影响目的观察富血小板血浆来源的富生长因子液对异体冻干跟腱移植重建兔前交叉韧带后移植物胶原变化和生物力学性能的影响。方法40只健康成年新西兰白兔,手术切断双侧ACL,随机一侧植入经血小板血浆来源的富生长因子液预处理后异体冻干跟腱(PRGF组,共40膝),另一侧植入经等量生理盐水预处理的异体冻干跟腱(NS组,共40膝)。手术后3周、6周、12周、24周取关节内移植物进行苏木精-伊红染色观察,免疫荧光共聚焦方法检测Ⅰ型及Ⅲ型胶原变化,并截取双侧膝关节股骨-移植物-胫骨复合体行生物力学检测。另取6只健康白兔不进行前交叉韧带重建,为正常对照组。结果手术后3周移植物被大量炎性细胞覆盖,PRGF组明显少于NS组;宿主细胞逐渐向移植物深层侵润,移植物深层可见血管形成;手术后3周PRGF组及NS组Ⅲ型胶原含量升高,Ⅰ型胶原含量下降,以NS组明显;手术后12周,PRGF组Ⅲ型胶原含量明显减少,而NS组要延迟到手术后24周;随着Ⅲ型胶原含量下降,Ⅰ型胶原含量升高,移植物生物力学性能开始改善,手术后24周移植物生物力学性能PRGF组明显高于NS组。结论富生长因子液可促进兔异体冻干跟腱移植重建前交叉韧带后胶原重塑,加速移植物重建。第四部分:聚对苯二甲酸乙二醇酯材料LARS韧带体内组织学及超微结构研究目的观察聚对苯二甲酸乙二醇酯材料LARS韧带重建兔前交叉韧带后移植物体内组织学转归及超微结构。方法选择12只健康成年新西兰白兔,随机分为A,B两组,其中A组(共9膝),前交叉韧带完全切断后,用体外编织的聚对苯二甲酸乙二醇酯材料LARS韧带重建,并用前交叉韧带残端覆盖移植物;B组(共3膝)前交叉韧带完全切除后单纯应用体外编织的聚对苯二甲酸乙二醇酯材料LARS韧带重建前交叉韧带。分别于手术后1月、3月、6月切取关节内移植物行苏木精-伊红染色及masson染色观察,并应用透射电子显微镜观察移植物超微结构。结果手术后1月,A组关节内LARS韧带纤维束被纤维结缔组织包裹,而B组手术后6月仍不能被纤维结缔组织包裹;手术后3月,A组关节内LARS韧带纤维束包裹的纤维结缔组织为不成熟的胶原纤维组织,不能沿应力分布,另外,A组及B组在骨道内或LARS韧带纤维束之间,存在中等程度的异物反应或炎症反应,以B组明显;手术后6月,A组关节内LARS韧带纤维束之间仍为不成熟的胶原纤维组织,不能沿应力分布;电子显微镜观察见人工纤维束间存在小直径的胶原纤维分布,人工纤维束间的成纤维细胞及骨道内人工纤维束间的成骨细胞增殖活跃,细胞体积及细胞核增大,粗面内质网扩张;A、B组骨道内均存在骨长入,新生编织骨起始于骨道壁,向人工材料的深层长入,但这种骨长入过程缓慢,在骨道内或LARS韧带纤维束之间,异物反应或炎症反应逐渐减轻。结论聚对苯二甲酸乙二醇酯材料LARS韧带生物相容性较好,为永久型韧带设计,仅具有部分支架型韧带的特性;关节内游离部分需用自体组织覆盖;移植后骨道内是否存在有效骨长入仍需进一步观察。更多还原

【Abstract】 PartⅠExpression of TypeⅢCollagen and Biomechanical evaluation of Anterior Cruciate Ligament Reconstruction with Fresh-frozen Patellar tendon allograft and autograftObjective To observe the expression of type III collagen and biomechanical changes of anterior cruciate ligament reconstruction with fresh-frozen patellar tendon allograft and autograft in rabbits. Methods 42 skeletally matured New Zealand white rabbits were used in the experiment. 36 rabbits were removed two sides of ACL .One side was underwent ACL reconstruction with aseptically harvested fresh-frozen patellar tendon allograft, while the other side was reconstructed with patellar tendon autograft.The remained 6 rabbits were classified as control group.At the 3rd week, 6th week, 12th week after surgery,specimens were collected and processed for histological, immunohistochemical observation and the expression degree ofⅢprocollagen mRNA was detected using method of RT-PCR.The complex of femur-graft-tibia was biomechanical analysis.Results After implantation ,the autografts and allografts underwent a gradually heal process including cellular migration, collagen formation and remodeling. The negative expression of type III collagen were obseved in nomal ACL and fresh-frozen patellar tendon allografts and autografts.The amount of typeⅢprocollagen mRNA of patellar tendon autografts reached a maximum at 6 th weeks after the operation,while typeⅢprocollagen mRNA of fresh-frozen patellar tendon allografts at 12th weeks after the operation remain highly. Mechanical properties deteriorated obviously due to a process of the increase of type III collagen in the grafts. Mechanical properties of the grafts can not reach the lever of nomal.Conclusion The results suggested that the increase in type III collagen produced by cells of the extrinsic origin may be one of the mechanisms that induce mechanical deterioration of fresh-frozen allograft and autograft .The synthetized type III collagen were significant in allograft.A more thorough mechanism of changes in the graft matrix after cruciate ligament reconstruction is critical to the development of a successful means of enhancing remodeling after cruciate ligament reconstruction. PartⅡAngiogenic effect of allgraft freeze-dried achilles treated by Plasma rich in Platelet—released Growth Factors in ACL reconstruction at early stageObjective To observe angiogenic effect of allograft freeze-dried achilles treated by PRGF in ACL reconstruction at early stage.Methods 14 rabbits were used in the experiment .12 rabbits were removed two sides of ACL. Allograft freeze-dried achilles treated by PRGF was transplanted into one side knee to substitute the original ACL, while the other side only treated by saline as control. 2 rabbits were classified as sham group.The grafts were harvested 2nd、4th and 6th week from the 12 rabbits, respectively.The groups were compared on the basis of histologic and immunohistochemical staining method . MVD was measured by Weidner method, an indicator of revascularization.Results When compared with PRGF at the same time ,MVD of group PRGF were significant higher than that of group NS (p<0.01) . Moreover, PRGF promoted MVD of PRGF group was significantly greater than that of NS group. MVD of group NS and PRGF was 2.52±0.45,3.41±0.44,2.57±0.51 and 3.56±0.81,4.91±0.46,3.01±0.75 , respectively(p<0.01). The time of neovascular formation and the depth of vascular penetration into the grafts of the group PRGF are superior to those in the group of NS.Conclusion PRGF significantly promoted angiogenesis of the freeze-dried achilles in ACL reconstruction at early stage .PartⅢCollagen expression and biomechanical effect of allograft freeze-dried achillis treated by Plasma rich in Platelet—released Growth Factors in ACL reconstructionObjectiveTo observe the effect of the Achilles allograft treated with PGGF on the biomechanical and collagen expression on the reconstruction of Anterio rcruciate ligament(ACL) in rabbits . Methods 46 rabbits were used in the experiment .40 rabbits were removed two sides of ACL. Allograft freeze-dried achillis treated by PRGF was transplanted into one side knee to substitute the original ACL, while the other side only treated by normal saline as control. 6 rabbits were sham group.The grafts were harvested 3rd、6th、12th and 24 th weeks and compared on the basis of histologic ;The expression of Type III Collagen and TypeⅠCollagen were analysised by confocal laser scanning microscopy (CLSM).The complex of femur-graft-tibia was biomechanical analysis.Results Many inflammatory cells from recipien covered the surface of the graft at 3rd week after operation, however, the PRGF group was less than NS group. Cells gradually migrated into the deeper part of the graft in two groups. At 3 th week after operation, the proportion of Type III Collagen increased and TypeⅠCollagen decreasing in both groups.As the time passed, the proportion of Type III Collagen gradually increased between 3 th -12 th week after operation ,but compared with in PRGF group,the increasing of Type III Collagen is more significantly in NS group . At 12 th week after operation,TypeⅠCollagen gradually increased and Type III Collagen began to decrease.However, the decreasing of Type III Collagen and the increasing of TypeⅠCollagen in PRGF group was faster than that of in NS group. Mechanical properties of both group begin to increase following the decreasing of the proportion of Type III Collagen and the increasing of TypeⅠCollagen.The increasing of mechanical properties in PRGF group was more significantly than that of in NS group. At 24 th week after operation ,the mechanical properties in PRGF group was superior to that of in NS group . Conclusion The biomechanical and collagen remodeling of allograft freeze-dried achillis treated with PRGF could be promoted on the reconstruction of ACL .PartⅣHistology Study of Terylene Polyester Type LARS after reconstruction of anterior cruciate ligament in rabbitsObjective To study histology characteristics and ultrastructure of Terylene Polyester Type LARS artificial ligament after reconstruction of anterior cruciate ligament in rabbits. Methods 12 rabbits were removed one side of ACL. The Terylene Polyester Type LARS was transplanted to substitute the original ACL, and the transplanted Terylene Polyester Type LARS were covered with the remnant of ACL in 9 rabbits(A group), while only Terylene Polyester Type LARS were transplanted in 3 rabbits , which no covering with the remnant of ACL(B group).The grafts were harvested 1st ,3rd and 6th month and compared on the basis of histologic characteristics , and ultrastructural findings and compatibility.Results After 1 th months surgery , the grafts were covered with recipient connective tissues in group A ,but were not covered anything after 6 th months operation in group B; After 3 th months surgery there were irregularly aligned collagen fiber bundles slightly or in some portions, additionally, there was moderate to severe inflammatory cell reaction or foreign body reaction immediately adjacent to the LARS fibers in bone tunnel even in long-term specimens postsurgery. After 6 th months surgery , there were irregularly aligned collagen fiber bundles slightly or in some portions, collagen fiber couldn’t parallel to stress. The tissue inside the LARS ligament as a whole showed no mature ligamentization and was not well functioning. Electron microscopy investigation showed the tissue near LARS fibers was highly cellular with a matrix of frequant, thin collagen fibrils. Among the collagen fiber bundles of the stroma were numerous osteoblasts and fibroblasts that were elongated, with large nuclei and an abundant, granular endoplasmic reculum. Light examination of these specimens revealed marked trabecular bone ingrowth into the periphery of the prosthesis and the incorporation of the LARS fibers into the bone matrix. Inflammatory cell reaction or foreign body reaction began to diminish.Conclusion The results suggest that Terylene Polyester Type LARS could be covered by recipient connective tissues.However, the recipient connective tissues was immaturity and could not develop into fiber parallel to stress. Terylene Polyester Type LARS was designed as a permanent replacement for the anterior cruciate ligament ,and used Terylene Polyester Type LARS in repairig and the reconstruction of cruciate ligament tissue ,however, may act as partly ,更多还原