体外构建抗结核性骨组织工程复合体的实验研究  

Establishment of an anti- tuberculosis bone tissue engineering complex in vitro

作　　者：梁求真 折胜利[1] 宋兴华[1] 呼西旦[1] 毕晓娟[1] 罗兰[1] 马艳[1] 蒋岳华[1] 白晶晶[1] 胡玉[1] 

机构地区：[1]新疆医科大学第一附属医院骨科中心,830011

出　　处：《中国矫形外科杂志》2014年第19期1789-1796,共8页Orthopedic Journal of China

基　　金：国家自然科学基金资助项目(编号:81360283)

摘　　要：[目的]基于兔脂肪干细胞（rabbit adipose-derived stem cells,rADSCs）、利福喷丁聚乳酸微球、同种异体部分脱钙骨支架,初步于体外构建抗结核性骨组织工程复合体。[方法]分离、培养rADSCs,经检测细胞周期、表面抗原CD44及分化诱导鉴定干细胞特性。用利福喷丁生长液干预rADSCs,按浓度分为0μg/ml、20μg/ml、40μg/ml及60μg/ml四组,分别绘制细胞生长曲线、检测细胞凋亡及碱性磷酸酶（alkaline phosphatase,ALP）含量。制备利福喷丁微球,计算平均直径、载药率及包封率。构建rADSCs-利福喷丁微球二维复合物并成骨诱导。制备骨支架并构建rADSCs-利福喷丁微球-骨支架三维复合物,计算细胞粘附率;扫描电子显微镜及苏木精-伊红（HE）染色观察复合情况;每3 d更换生长液,检测体外释药特性。[结果]rADSCs呈长梭形生长,G0/G1期占（80.3±3.2）%,表面抗原CD44阳性表达。成骨诱导后ALP、茜素红染色,成脂诱导后油红O染色及成软骨诱导后阿利新蓝染色均为阳性。生长曲线示60μg/ml组第4~12 d增殖能力较其他三组弱（P〈0.01）;凋亡检测示60μg/mL组凋亡率高于其他三组（P〈0.01）;ALP检测示第7、14 d 60μg/ml组ALP含量较其他三组少（P〈0.05）;临界浓度为40μg/ml。微球平均直径为（26.4±3.6）μm,载药率为（40.89±0.63）%,包封率为（75.24±0.18）%。二维复合物成骨诱导后茜素红染色阳性。三维复合物细胞粘附率为（96.94±1.24）%;扫描电镜及HE染色观察复合体相容性良好;体外培养第3、6、39 d累积释药量分别达总量的8.54%、20.12%及93.66%,每个时间点检测的释药浓度均低于临界浓度,但高于最低抑菌浓度。[结论]体外构建的抗结核性骨组织工程复合体具有持续缓慢释放药物的性质,且该释药浓度不影响rADSCs的增殖及成骨活性。[ Objective] We aimed to establish an in vitro anti -tuberculosis bone tissue engineering complex consisting of rabbit adipose -derived stem cells （rADSCs）, rifapentine, polylaetic acid microspheres, and a partially decalcified allograft bone scaffold. [ Methods] Adipose tissue samples were obtained from inguinal fat pads of the rabbit and were digested using type I collagenase. Characteristics of the rADSCs were verified via cell - cycle analysis, anti - CD44 antibody staining, and multipotential differentiation assays. The rADSCs were randomly divided into four groups based on specific drug concentrations of rifapentine, including 0 μg/ml, 20 μg/ml, 40 μg,/ml, and 60 μg/ml. Cell growth curves, cell apoptosis, and alkaline phos- phatase （ALP） activity were used to determine the effects of rifapentine on rADSCs. Rifapentine/polylactic acid microspheres were prepared using a double - emulsion solvent - extraction technique. The microsphere appearance was observed using scanning electron microscopy （ SEM）, and the average diameter was measured. The drug - carrier rate and envelopment rate were deter- mined by UV spectrophotometry. The two -dimensional （2D） rADSCs/rifapentine microspheres complex was established, and the osteogenic differentiation potential was also observed. The three -dimensional （3D） rADSCs/rifapentine microspheres/bone scaffold complex was established, and the cell adhesion rate was calculated. The appearance of 3D complex was observed bySEM and hematoxylin and eosin （HE） staining. The release characteristics were evaluated in vitro, with the released quantity determined every three days, and the mean release rate for each day calculated to determine the total release rate. [ Results] The rADSCs exhibited a spindle - shaped morphology in vitro, approx- imately （80. 3 ± 3.2） % of cells were of the G0/G1 cycle, andcells were positive for CD44 expression. ALP and Alizarin red staining showed positive expression of ALP and the formation of mineralized nodules, and th

关 键 词：骨结核 脂肪干细胞 利福喷丁 骨组织工程 

分 类 号：R529.2[医药卫生—内科学]