胶原-生物衍生骨材料与兔成骨细胞的相容性及其体外附着特点

Histocompatibility and in vitro adhesion of collagen decorated bio-derived bone with rabbit osteoblasts

目的:观察胶原-生物衍生骨复合材料与兔成骨细胞的相容性及体外附着规律,为进一步的体内实验提供参考数据。方法:实验于2003-10/2005-02在华西医科大学组织工程实验室进行。①材料制备:新鲜捐献骨(人),经脱脂、脱蛋白等工艺制成单纯生物衍生骨材料,以Ⅰ型胶原通过真空吸附法修饰单纯生物衍生骨材料表面,构建出胶原生物衍生骨材料。②实验方法:将体外培养的兔骨膜成骨细胞分别复合于单纯生物衍生骨材料和胶原生物衍生骨材料,共同培养7d。③观察指标:分别于培养第1,3,5和7天取材,扫描电镜观察成骨细胞形态及附着情况;用紫外/荧光/可见光高效分析仪测定成骨细胞产生的碱性磷酸酶活性;MTT检测成骨细胞增殖情况;用流式细胞仪检测兔成骨细胞的细胞周期、DNA含量及倍体水平。结果:①扫描电镜结果:胶原生物衍生骨材料组成骨细胞黏附和增殖优于生物衍生骨材料组。②碱性磷酸酶活性:生物衍生骨材料组低于胶原生物衍生骨材料组(0.019±0.003,0.038±0.004,P<0.05)。③细胞增殖情况:在培养第1,3,5,7天胶原生物衍生骨材料组成骨细胞A值均高于生物衍生骨材料组(P<0.05)。④流式细胞检测生物衍生骨材料、胶原生物衍生骨材料对兔成骨细胞的细胞周期影响不大,各组细胞皆为正常的二倍体细胞,未见异倍体细胞形成,胶原修饰后无细胞毒性。结论:以单纯生物衍生骨作为载体,其表面经胶原修饰后的复合材料与成骨细胞有良好的细胞相容性,无细胞毒性。

AIM：To observe the histocompatibility and in vitro adhesion of collagen decorated bio-derived bone and rabbit osteoblast, and provide the references for in vivo experiments. METHODS： The experiment was carded out in the Laboratory of Tissue Engineering, West China University of Medical Sciences from October 2003 to February 2005. ①The donor bones were degreased, deproteinated and calcined to form bio-derived bone, in which the surface was covered with type-I collagen by vacant adsorption method②The in vitro cultured rabbit osteoblasts were compounded to native bio-derived bone and collagen bio-derived bone, respectively, followed by the culture for 7 days.③Samples were obtained at 1, 3, 5, 7 days of culture, the morphology and adhesion of osteoblasts were observed under scanning electron microscope; alkaline phosphatase activities （APA） of osteoblasts were assayed by UV/fluorescence/visible high-efficient analyzer; the proliferation of osteoblasts was detected by MTT method; the cell cycle, DNA content and ploid level of rabbit osteoblasts were tested by flow cytometer.RESULTS： ①The osteoblasts on the collagen loaded bio-derived bone adhered and grew better than those on native bio-derived bone. ②APA was lower on native bio-derived bone materials than on collagen loaded bio-derived bone materials （0.019±0.003, 0.038±0.004, P 〈 0.05）.③At 1, 3, 5, 7 days of culture, the A value of osteoblasts was increased on collagen loaded bio-derived bone materials compared with bio-derived bone materials （P 〈 0.05）.④Cell cycle was not influenced by bio-dedved materials and collagen loaded bio-derived bone materials. No heteroploid cells appeared and there was no cytotoxicity after collagen covering. CONCLUSION： After the surface of the bio-derived bone vector is covered with collagen, the compound materials have a good histocompatibility with the rabbit osteoblasts. No cytotoxicity appears.