组织工程骺板软骨修复幼兔骺板损伤的实验研究
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摘要
骺板损伤后骨桥形成是引起儿童肢体短缩及成角畸形的常见原因之一,现有的治疗方法主要是骨桥切除后植入脂肪等填充物,不同程度地恢复受累肢体的纵向生长能力及矫正部分成角畸形。这些治疗方法不满意的主要原因可能是填充物仅能通过机械阻隔作用,防止骨桥形成,不具备生长能力。因此,寻找具有生长能力的填充物成为研究重点之一。近年来组织工程技术快速发展,组织工程骺板软骨的构建已初步显示出近期效果,本研究试图通过构建天然骺板软骨获得远期疗效。
目的:通过体外细胞培养技术,观察比较不同部位骺板软骨细胞的生长特性,确定生长潜力最大的取材部位;以牛关节软骨细胞外基质(cattlearticular cartilage extracellular matrix CACECM)为支架,复合同种异体骺板软骨细胞体外构建组织工程骺板软骨;将组织工程骺板软骨植入幼兔股骨远端的骺板缺损区,观察其是否可以有效地防止骨桥形成。
方法:(1)体外分离培养骺板软骨细胞,通过大体及组织学观察细胞生长特性;通过生长曲线等比较不同部位骺板软骨细胞生长潜力。
(2)CACECM复合骺板软骨细胞后,光镜、电镜、染色等方面观察细胞在CACECM上的生长情况,确定CACECM对骺板软骨细胞的影响;体内异位植入CACECM,不同的时间取出,大体及染色情况下观察CACECM自行降解时间、局部反应等;复合物植入兔背部肌肉中,不同的时间取出组织块观察是否异位成软骨、降解时间、排异反应等。
(3)将复合骺板细胞的CACECM体外培养后植入幼兔股骨远端骺板缺损区,不同的时间点观察股骨生长及畸形发生情况。
结果:(1)2周龄兔股骨远端及胫骨近端的骺板软骨细胞生长特性及单位体积细胞产量等方面优于髂骨、肱骨远端及尺桡骨近端的骺板细胞,可优先作为骺板组织工程的种子细胞;骺板软骨细胞体外培养不要超过三代,以保持良好的生长潜力。
(2)CACECM组织相容性较好;骺板软骨细胞可以在CACECM支架上不断增殖生长,分泌基质;CACECM可以在体内不断降解,不存在明显的排异反应及组织残留;CACECM复合骺板软骨细胞在体外可以继续生长,体内可形成类软骨样组织、分泌糖胺聚糖(glycosaminoglycans,GAG)及Ⅱ型胶原。
(3)以CACECM作为支架的组织工程骺板软骨在4周、8周、12周及16周均能有效地防止股骨成角和短缩畸形,而单独植入CACECM则不能阻止其畸形的发生。
本文证明异体组织工程骺板软骨在骺板早闭的治疗中,预防骨桥形成及促进损伤骺板生长是有作用的,组织工程骺板软骨在治疗骺板早闭方面有以下几个优点:①不存在填充骺板软骨时的供体短缺的问题;②与脂肪、硅胶、肌肉等相比,它具有一定生长能力;③比单纯细胞移植的免疫源性小;④比填塞骺板软骨更容易通过渗透获得营养。
Growth arrest in the epiphyseal plate during childhood often causes both periarticular deformities and limb length discrepancy,leading to compartmental osteoarthrosis and gait disturbance or spinal disorders,respectively.Traditional treatments for bony bridge include packing an interpositional material such as fat and bone cement after removal of the bone bar.It can partly remedy the angulation deformity and recover the ability of longitudinal growth but got no satisfactory results.The main reason is that the interpositional materials can only restrain the form of bone bridge in virtue of passive barriers,but growth-ability.The interpositional materials with growth ability has become one of the important research point consequently.With the rapid progress of Tissue-engineered technique,the tissue-engineered epiphyseal cartilage has a good short-term effect. Therefore we construct a novel natural epiphyseal plate intent to recove the ability of longitudinal growth,so as to get a satisfactory long-term effect.
Objective:Observed and compared the growth characteristics of cultured physeal chondrocytes by technique of cell culture in vitro to find the right place with the great growth potential;Cultured the engineered epiphyseal growth plate in vitro by using cattle articular cartilage extracellular matrix as cell scaffold seeded with epiphyseal chondrocytes;Implanted the engineered tissue into the lateral distal defects of growth plate of New Zealand rabbit femur to observe whether it can prevent the growth deformity of not.
Method:(1)After cultivate the epiphyseal chondrocytes in vitro,we observed the growth characteristics of cultured physeal chondrocytes by light microscope and staining;Compared the growth potential of chondrocytes from different postion by growth curve to destermined the better one.
(2)It is determined that the cattle articular cartilage extracellular matrix whether have a influence on epiphyseal chondrocytes by light microscope、electron microscope、staining technique after seeded epiphyseal chondrocytes into cattle articular cartilage extracellular matrix.To observe the degradation time and rejection by transplant the complex and cattle articular cartilage extracellular matrix into muscle of rabbit's back.
(3)Transplanted the complex into the lateral distal defects of growth plate of rabbit femur to observed the growth and deformity in different time.
Results:(1)The epiphyseal chondrocyte from femur and tibia of 2 weeks rabbit is better than the others in characteristics and output of unit volume.It's better not to use the chondroeytes after 3 passages in preparing the engineered tissue,otherwise they will lost the optimal growth potential.
(2)The histocompatibility of cattle articular cartilage extracellular matrix is good.CACECM will degradate in vivo generally with no rejection and tissue left.It can provide a three dimensional space for epiphyseal chondrocytes to grow.While seeding in epiphyseal chondrocytes,it would keeping grow in vitro and form similar cartilage tissue in vivo.
(3)The engineered epiphyseal tissue can prevent the formation of the femur deformity in different time.While the implantation of CACECM scaffold alone couldn't.
It is proved in this paper that the engineered epiphyseal tissue can prevent the formation of bony bridge and promote the growth of the injured growth plate in treatment of premature arrest of growth plate.There are several good quality as follow:①donator will not shortage.②the engineered epiphyseal tissue has a certain ability of growth.③little immunogenicity than implantation chondrocytes alone.④It could get nutrition more easier in implant prophase due to the loose tissue structure.
目的:通过体外细胞培养技术,观察比较不同部位骺板软骨细胞的生长特性,确定生长潜力最大的取材部位;以牛关节软骨细胞外基质(cattlearticular cartilage extracellular matrix CACECM)为支架,复合同种异体骺板软骨细胞体外构建组织工程骺板软骨;将组织工程骺板软骨植入幼兔股骨远端的骺板缺损区,观察其是否可以有效地防止骨桥形成。
方法:(1)体外分离培养骺板软骨细胞,通过大体及组织学观察细胞生长特性;通过生长曲线等比较不同部位骺板软骨细胞生长潜力。
(2)CACECM复合骺板软骨细胞后,光镜、电镜、染色等方面观察细胞在CACECM上的生长情况,确定CACECM对骺板软骨细胞的影响;体内异位植入CACECM,不同的时间取出,大体及染色情况下观察CACECM自行降解时间、局部反应等;复合物植入兔背部肌肉中,不同的时间取出组织块观察是否异位成软骨、降解时间、排异反应等。
(3)将复合骺板细胞的CACECM体外培养后植入幼兔股骨远端骺板缺损区,不同的时间点观察股骨生长及畸形发生情况。
结果:(1)2周龄兔股骨远端及胫骨近端的骺板软骨细胞生长特性及单位体积细胞产量等方面优于髂骨、肱骨远端及尺桡骨近端的骺板细胞,可优先作为骺板组织工程的种子细胞;骺板软骨细胞体外培养不要超过三代,以保持良好的生长潜力。
(2)CACECM组织相容性较好;骺板软骨细胞可以在CACECM支架上不断增殖生长,分泌基质;CACECM可以在体内不断降解,不存在明显的排异反应及组织残留;CACECM复合骺板软骨细胞在体外可以继续生长,体内可形成类软骨样组织、分泌糖胺聚糖(glycosaminoglycans,GAG)及Ⅱ型胶原。
(3)以CACECM作为支架的组织工程骺板软骨在4周、8周、12周及16周均能有效地防止股骨成角和短缩畸形,而单独植入CACECM则不能阻止其畸形的发生。
本文证明异体组织工程骺板软骨在骺板早闭的治疗中,预防骨桥形成及促进损伤骺板生长是有作用的,组织工程骺板软骨在治疗骺板早闭方面有以下几个优点:①不存在填充骺板软骨时的供体短缺的问题;②与脂肪、硅胶、肌肉等相比,它具有一定生长能力;③比单纯细胞移植的免疫源性小;④比填塞骺板软骨更容易通过渗透获得营养。
Growth arrest in the epiphyseal plate during childhood often causes both periarticular deformities and limb length discrepancy,leading to compartmental osteoarthrosis and gait disturbance or spinal disorders,respectively.Traditional treatments for bony bridge include packing an interpositional material such as fat and bone cement after removal of the bone bar.It can partly remedy the angulation deformity and recover the ability of longitudinal growth but got no satisfactory results.The main reason is that the interpositional materials can only restrain the form of bone bridge in virtue of passive barriers,but growth-ability.The interpositional materials with growth ability has become one of the important research point consequently.With the rapid progress of Tissue-engineered technique,the tissue-engineered epiphyseal cartilage has a good short-term effect. Therefore we construct a novel natural epiphyseal plate intent to recove the ability of longitudinal growth,so as to get a satisfactory long-term effect.
Objective:Observed and compared the growth characteristics of cultured physeal chondrocytes by technique of cell culture in vitro to find the right place with the great growth potential;Cultured the engineered epiphyseal growth plate in vitro by using cattle articular cartilage extracellular matrix as cell scaffold seeded with epiphyseal chondrocytes;Implanted the engineered tissue into the lateral distal defects of growth plate of New Zealand rabbit femur to observe whether it can prevent the growth deformity of not.
Method:(1)After cultivate the epiphyseal chondrocytes in vitro,we observed the growth characteristics of cultured physeal chondrocytes by light microscope and staining;Compared the growth potential of chondrocytes from different postion by growth curve to destermined the better one.
(2)It is determined that the cattle articular cartilage extracellular matrix whether have a influence on epiphyseal chondrocytes by light microscope、electron microscope、staining technique after seeded epiphyseal chondrocytes into cattle articular cartilage extracellular matrix.To observe the degradation time and rejection by transplant the complex and cattle articular cartilage extracellular matrix into muscle of rabbit's back.
(3)Transplanted the complex into the lateral distal defects of growth plate of rabbit femur to observed the growth and deformity in different time.
Results:(1)The epiphyseal chondrocyte from femur and tibia of 2 weeks rabbit is better than the others in characteristics and output of unit volume.It's better not to use the chondroeytes after 3 passages in preparing the engineered tissue,otherwise they will lost the optimal growth potential.
(2)The histocompatibility of cattle articular cartilage extracellular matrix is good.CACECM will degradate in vivo generally with no rejection and tissue left.It can provide a three dimensional space for epiphyseal chondrocytes to grow.While seeding in epiphyseal chondrocytes,it would keeping grow in vitro and form similar cartilage tissue in vivo.
(3)The engineered epiphyseal tissue can prevent the formation of the femur deformity in different time.While the implantation of CACECM scaffold alone couldn't.
It is proved in this paper that the engineered epiphyseal tissue can prevent the formation of bony bridge and promote the growth of the injured growth plate in treatment of premature arrest of growth plate.There are several good quality as follow:①donator will not shortage.②the engineered epiphyseal tissue has a certain ability of growth.③little immunogenicity than implantation chondrocytes alone.④It could get nutrition more easier in implant prophase due to the loose tissue structure.
引文
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