京尼平交联对大鼠脱细胞脊髓生物学特性的影响

Effect of genipin cross-linking on biological characteristics of rat acellular spinal cord

目的探讨新型生物交联剂京尼平对大鼠脱细胞脊髓生物学特性的影响。方法从成年SD大鼠(体质量200～250 g,雌雄不限)获取大鼠胸段脊髓,采用化学萃取法对大鼠脊髓组织进行脱细胞处理,再用生物交联剂京尼平(5 g/L)进行化学交联。分别采用HE染色和扫描电镜观察京尼平交联前后脱细胞脊髓的微观结构,并进行孔径大小、交联率、含水量、在PBS溶液及胰酶溶液中的稳定性能检测,再将大鼠骨髓间充质干细胞分别与京尼平交联前后的脱细胞脊髓浸提液共培养,采用MTT法在体外评估该支架材料交联前后的细胞毒性情况。结果京尼平交联前后的脱细胞脊髓拥有类似的多孔结构,孔径大小约30μm,最终交联率可达90%,交联后的脱细胞脊髓含水率从(283.4±11.2)%降至(229.7±12.5)%,但其在PBS及胰蛋白酶中的稳定性得到了明显的增强。交联前后的脱细胞脊髓都未观察到明显的细胞毒性。结论京尼平交联的脱细胞脊髓部分生物学特性得到改善,为应用于脊髓损伤修复的组织工程材料提供了一种新的选择。

Objective To investigate in vitro biological characteristics of genipin （GP）-crosslinked acellular spinal cord in order to provide an ideal biomaterial for tissue engineering application of spinal cord injury. Methods Rat spinal cord tissues were decellularized by the chemical extraction method, followed by chemical crosslinking with 5 g/L genipin solution. The micro-structure of the un-crosslinked and GP-crosslinked acellular spinal cords were observed with HE staining and scanning electron microscopy, and the properties of pore size, crosslinking degree, water uptake ratio, and stability in PBS and tripsin were tested as well. The rat bone marrow mesenchymal stem cells were cultured in lixivium of un-crosslinked and GP-crosslinked acellular spinal cord, and then, MTT assay was used to evaluate in vitro cytotoxicity. Results The un-crosslinked and GP-crosslinked acellular spinal cords possessed a similar porous structure with an average pore diameter about 30 μm, the final crosslinking rate was about 90%, the water uptake ratio were decreased from （283.4±11.2）% to （229.7±12.5）% and the stability in PBS and trypsin were significantly enhanced by GP-crosslinking. Furthermore, no obvious cytotoxicity was observed in both group. Conclusion The GP-crosslinked acellular spinal cord is a potential tissue engineering material for spinal cord injury repairing application.