生物珊瑚人工骨支架材料生物相容性检测

目的评价生物珊瑚人工骨(BCAB)材料作为骨组织工程支架材料与小鼠胚胎干细胞(MESCs)构建组织工程骨的有效性及材料生物相容性。方法设MESCs与BCAB支架材料混合黏附培养为实验组,单纯MESCs培养为对照组,分别于第2、4、6、8天进行MTT法检测细胞增殖活性,特异性胚胎抗原-1检测细胞对材料的黏附性。于第8日对接种细胞材料片行成骨诱导,诱导培养10 d后行茜素红染色及电镜扫描检测成骨诱导及体外组织工程骨构建情况。取12只大鼠,脊柱左侧皮下植入空白BCAB支架片状材料,右侧植入黏附细胞BCAB片状材料,随机分4、8、12周3组行影像学检查,并取双侧标本行病理切片观察局部炎症反应,四环素标记下荧光显微镜观察成骨情况。第12周组取心、肝、肾病理切片及评估心、肝、肾毒性反应。结果 MTT法检测细胞增殖活性结果显示,在培养2 d和4 d时实验组与对照组间MTT值差异无统计学意义(P>0.05),在6 d及8 d时实验组MTT值明显高于对照组,差异具有统计学意义(P<0.05)。特异性胚胎抗原-1检测证实MESCs对BCAB支架材料具有良好的黏附性,在其三维微孔隙内能较快增殖。茜素红染色及电镜扫描检测证实黏附细胞材料成骨诱导有效,体外组织工程骨构建成功。材料植入局部组织炎症反应轻,空白支架材料于第8周开始降解,12周达初步降解,无异位成骨;黏附细胞支架材料则有明显异位成骨现象,且较对侧空白支架材料降解时效延长。第12周组实验动物心、肝、肾标本病理切片未见异常损害。结论 BCAB支架材料具有良好的生物相容性,其降解周期与新骨重建周期大致相当,是一种良好的骨组织工程支架材料。

Biocompatibility test of biological coral artificial bone as scaffold materials

【Objective】 To test the biocompatibility of biological coral artificial bone(BCAB) and the effect of tissue engineered bone construction using BCAB scaffolds with mouse embryonic stem cells(MESCs).【Methods】 MESCs seeded-scaffold were given as experimental group and axenic cultivation of MESCs served as control group.The cell proliferation was detected by MTT method at 2nd day,4th day,6th day and 8th day respectively,and stage specific embryonic antigen-1(SSEA-1) detection were also performed to observe the cell’s adhesion on scaffold materials in the same time.The MESCs-seeded scaffolds were cultured under the condition of osteogenic induction at 8th-day.Alizarin red staining and SEM observation were performed to test effct of osteogenic induction and construction of tissue engineered bone in vitro.A total of 12 rats undergoing subcutaneous implantation of BCAB scaffolds,cell-seeded scaffolds for right of spine and empty scaffolds for left of spine,were randomly divided into three groups according to the time of specimens extraction: 4 week group,8 week group,12 week group.Each group has undergone x-ray examination and local inflammatory response of scaffolds was measured by histopathology.The condition of osteogenesis was observed by tetracycline labelling.The heart,liver and renal tissue sections of 12 week group were observed.The data was compared and statistically analyzed with SPSS 13.0.【Results】 The differences of MTT detection results between MESCs seeded scaffold group and axenic cultivation group were not significant at 2nd day and 4th day(P >0.05).But it changed at 6th day and 8th day,the MTT detection results of experimental group were higher than control group and there was significant difference between two groups(P >0.05).The results of SSEA-1 detection demonstrated MESCs had good bioadhension to BCAB scaffold and remained good proliferation in 3D-micropore space of BCAB scaffold.The results of alizarin red staining and SEM observation proved the osteogenic induction and construction of tissue engineered bone on MESCs-seeded scaffold were succeeded in vitro.The local inflammatory response of BCAB scaffolds was mild.The empty scaffolds were starting to degrade at 8th week and achieved preliminary degradation at 12th week.No osteogenesis was observed on empty scaffolds,but it was proved on cell-seeded scaffolds by histopathology and tetracycline labelling.Osteogenesis made the degradation periods of cell-seeded scaffolds be longer than empty scaffolds.The heart,liver and renal tissue sections of 12 week group were normal.【Conclusion】 The BCAB has good biocompatibility,the degradation periods of which is almost equal with regeneration cycle of new bone.It presents an effective potential to be a good bone tissue engineering material.