骨髓间充质干细胞-海螵蛸复合支架的细胞毒性评估

文题释义：骨髓间充质干细胞复合海螵蛸支架：以中药海螵蛸为载体支架，骨髓间充质干细胞为种子细胞，二者共同培养构建复合生物支架，经过各项生物安全性测评后应用于骨缺损的治疗。制作过程为：选用第3代骨髓间充质干细胞，经0.25%胰酶消化制成细胞悬液，将细胞浓度调整为5×10⁸ L^-1，用移液枪将骨髓间充质干细胞悬液缓慢逐滴滴加于24孔板中的海螵蛸上，200 μL/孔，分2次缓慢接种，尽量不使细胞从材料上溢出，置于培养箱中培养4 h，待细胞充分黏附于海螵蛸支架材料后，缓慢逐滴加入培养液700 μL，将支架与细胞复合培养8 d，两三天换液1次。细胞毒性评估：是采用受试物与细胞共同培养的方法，对样品进行毒理学风险评估。生物材料应用于体内后，可能与体内细胞的细胞膜、细胞器、蛋白质合成、DNA合成等相互作用，产生细胞毒性。根据ISO10993-5标准，在复合支架应用于临床治疗前，需通过复合材料浸提液试验以测定其对细胞活性、功能、遗传的影响。  摘要背景：作为骨组织工程的修复材料，应具有良好的生物相容性及降解吸收性，许多学者在此方面也做了较深入的研究，但中药复合细胞生物支架的研究较少。目的：参照《医疗器械生物学评价标准》，对兔骨髓间充质干细胞-海螵蛸生物复合支架进行体外细胞毒性检测，评估复合支架的毒性等级，为临床应用生物支架提供实验依据。方法：依照ISO标准按“材料面积∶浸提介质体积=3-6 cm²∶1 mL”制作骨髓间充质干细胞-海螵蛸生物复合支架材料浸提液。制备L-929细胞悬液，将细胞浓度调整至1×10⁷ L^-1进行接种培养，设置阳性对照组(含苯酚的DMEM培养液)、实验组(材料浸提液)、阴性对照组(新鲜DMEM培养液)。培养24，48，72 h采用MTT法检测L-929细胞吸光度值，计算各组细胞的相对增殖率，评估复合支架毒性等级。结果与结论：实验组、阴性对照组、阳性对照组吸光度值在不同时间点不完全相同(P=0.000 < 0.01)，各时间点内比较，实验组与阴性对照组吸光度值均明显高于阳性对照组(P < 0.01)；兔骨髓间充质干细胞-海螵蛸生物支架细胞毒性为1级。结果说明兔骨髓间充质干细胞-海螵蛸生物支架无明显毒性作用，符合生物材料应用要求。  ORCID: 0000-0002-0003-6228(彭雅) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Cytotoxicity assessment of bone marrow mesenchymal stem cells-cuttlebone composite scaffold

BACKGROUND:Repair materials for bone tissue engineering should hold good biocompatibility and degradability.There are various related studies,but the Chinese medicine composite cellular bioscaffolds are little reported.OBJECTIVE:To detect the in vitro cytotoxicity of rabbit bone marrow mesenchymal stem cells-cuttlebone bioscaffold based on the Biological Evaluation of Medical Device,and assess its cytotoxicity level in order to provide the theoretical support for its clinical application.METHODS:Bone marrow mesenchymal stem cells-cuttlebone bioscaffold extract was prepared according to an ISO standard—material area:extraction medium volume=3-6 cm2:1 mL.L-929 cell suspension was prepared,and the cells were then cultured with a density of 1×107/L.There were three groups:positive group(DMEM medium containing phenol),experimental group(material extract),and negative group(DMEM culture medium).The absorbance value of L-929 cells was detected by MTT assay after 24,48 and 72 hours of culture.The relative proliferation rate of cells was then calculated and the toxicity level was valued in each group.RESULTS AND CONCLUSION:The absorbance values in the experimental,negative and positive groups were not exactly same at different time points(P=0.000<0.01).The absorbance values in the experimental and negative groups were significantly higher than those in the positive group(P<0.01).The cytotoxicity of bone marrow mesenchymal stem cells-cuttlebone bioscaffold was grade 1.To conclude,the bone marrow mesenchymal stem cells-cuttlebone bioscaffold has no obvious toxic effects,and meets the requirements of biomaterial application.