可注射性同种异体工程化软骨的构建

Construction of allogeneic tissue engineered cartilage using injectable biomaterial

目的 研究在有免疫力的动物体内形成同种异体工程化软骨的可行性 ;并验证本实验使用的生物材料聚氧化乙烯丙烯凝胶 (Copolymer gel of ethylene and propylene oxide)在组织工程研究中应用的可行性 .方法 无菌条件下取新生兔关节软骨 ,经 型胶原酶消化 ,将软骨细胞和生物材料复合成细胞浓度为 5× 10 7m l- 1的复合物并移植于兔皮下 ,同时设立单独注射细胞和材料两个对照组 .分别于 4,6 ,8,12 wk取材行大体观察 ,石蜡切片 HE染色 ,Safranin O染色 ,Masson's三色染色 ,了解软骨形成情况 .结果 2 wk后即可于皮下触及新生结节 ,4wk后取材即有基质分泌及胶原形成 .于 6 wk左右软骨接近成熟 ,生物材料基本吸收 .8,12 wk软骨基本接近正常软骨 .实验中未见明显的免疫排斥反应 .而对照组未见软骨形成 .结论 该实验使用的生物材料具有良好的生物相容性 ,可降解性 ,无细胞毒 ,是较理想的组织工程生物材料 ;应用该实验的方法可在有免疫力的动物体内形成同种异体工程化软骨 ,这为临床某些美容和重建手术提供了一种微创而可塑型的可能的手段 .

AIM To study the feasibility of the formation of allogeneic tissue engineered cartilage in immunocompetent animal and to verify that the material used in our experiment has the ideal features as tissue engineering biomaterial. METHODS Fresh newborn rabbits' articular cartilages were obtained under sterile condition (<6 h after death) and incubated in the sterile 3 g·l -1 type Ⅱ collagenase solution. After digestion of 8~12 h, the solution was filtered through a 150 micron nylon mesh and centrifuged, then the chondrocytes were washed twice with phosphate buffered saline (PBS) and mixed with the biomaterial to create a final cell density of 5×10 7 ml -1 . The resulting cell biomaterial admixture was injected subcutaneously 0.3 ml each point, and the control groups were injected with only the biomaterial or the suspension of chondrocyte with the density of 5×10 7 ml -1 . After 4, 6, 8 and 12 weeks, the neocartilages were harvested for analysis. RESULTS We touched the new nodes subcutaneously after 2 wk. In the sections of the samples harvested after 4 wk, we found that the matrix was secreted and the collagen formed. After 6 wk, the neocartilages were mature and the biomaterial was almost completely degraded. In the experiment groups we observed no obvious immune rejection response. On the contrary, in the control groups there were no neocartilage formed. CONCLUSION Biomaterial used in our experiment is a relatively ideal tissue engineering biomaterial. It's feasible to form allogeneic tissue engineered cartilage in an immunocompetent animal that provides an alternative minimum traumatic,malleable and injectable means for some clinic plastic and reconstructive operation.