HA／β-TCA 复合材料与同种异体骨体外兔脂肪干细胞生物相容性的对比实验

摘要

Objective To study the compatibility of the rabbit adipose stem cells with two scaffolds osteo-genesis matrixes hydroxyapatite/β-tricalcium phosphate (HA /β-TCA)and allogeneic bone composite in vitro,and to provide theoretical basis for the further experiments on scaffold materials of bone tissue engi-neering.Methods The adipose tissue,obtained from the groin fat of 4-month-old New Zealand rabbits, was digested with collagenase I and the primary rabbit adipose stem cells (rADSCs)were isolated, cultured and sub-cultured.Then the osteogenesis of the sub-cultured rADSCs were inducted with a medium containing HA/β-TCA and allogeneic bone composite,respectively.Alkaline phosphatase (ALP)staining and alizarin red staining were performed to identify the differentiation of rADSCs;and the adhesion and the proliferate of rADSCs were investigated by scanning electron microscope.Results The 90% cell fusion were taken place 1 week for the primarily rADSCs and 3 days for the sub-cultured rADSCs,indicating a good post-inducing proliferation for the sub-cultured rADSCs.After induction of differentiation for 15 days,the second-generation rADSCs showed the clumps in the center of the cells as demonstrated by eosin staining,indicating the calcification of rADSCs.In addition,light brown and dark brown particles were seen in the cytoplasm of the rADSCs with ALP staining.The OD values of ALP had no statistical differ-ence between the HA/β-TCA and allograft composite at 3,6,and 9 days post co-culture rADSCs with the 2 scaffolds.At the 11 days of co-culture,the rADSCs showed polygonal cell protrusions,which adhered to the scaffold and extended to the surrounding area.The adjacent rADSCs closely linked to form a plate net-structure.The great amount of external matrix was secreted from the cell surface,which filled into the gaps of the scaffold matrix.The growth of cells on the both scaffold was profound and maintained uniform distribution evenly.Conclution HA/β-TCA composite bone tissue engineering scaffold has a comparable biocompatibility for rADSCs compared to the allogeneic bone matrix and provide a potential good resource for bone tissue engineering.%目的：探讨羟基磷灰石／β-磷酸三钙（hydroxyapatite／tricalcium phosphate，HA／β-TCA）复合材料与同种异体骨体外兔脂肪干细胞（rADSCs）生物相容性，为骨组织工程选择支架材料提供实验基础。方法取4月龄新西兰大白兔腹股沟区脂肪，Ⅰ型胶原酶消化、分离、培养及传代原细胞，第2代 rADSCs 消化后，加入成骨培养液中诱导分化，分别行碱性磷酸酶染色、茜素红染色以鉴定分化结果，扩增后分别与 HA／β-TCA 及同种异体骨复合培养，通过扫描电子显微镜、碱性磷酸酶方法检测 rADSCs 在 HA／β-TCA 及同种异体骨表面的粘附能力、增殖活力，评价 rADSCs 与 HA／β-TCA 及同种异体2种材料的生物相容性，并比较分析。结果原代细胞培养1 w 后约90％的融合，传代后平均3 d 即可达到90％以上的融合。第2代 rADSCs 分化诱导后细胞增殖良好，分化诱导15 d后行茜红素染色，细胞结节中央染色为红色团块，形成片状钙化影；碱性磷酸酶染色可见细胞浆内呈浅棕色至棕褐色颗粒。接种培养后的第3天、第6天、第9天，同种异体骨与复合材料各时间点的吸光度（OD）值差异无统计学意义。rADSCs 在2种支架材料培养11 d 后，电镜观察可见有多角形细胞伸出、突起，粘附于材料上和空隙中，并向四围伸展生长，邻近细胞紧密链接，形成拉网状结构。细胞表面分泌很多细胞外基质，填充于支架材料的孔隙中。细胞在2种支架材料上生长良好，增殖明显并保持均匀分布。结论rADSCs 与 HA／β-TCA 生物相容性不弱于同种异体骨，HA／β-TCA 复合材料为骨组织工程支架提供了良好来源。