骨骺干细胞复合壳聚糖／纳米羟基磷灰石支架体外培养的实验研究  

Precartilaginous stem cells cultured with chitosan/nano- hydroxyapatite composite scaffold in vitro

作　　者：祁军[1] 陈安民[1] 郭风劲[1] 孙淑珍[2] 张树威[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030 [2]武汉理工大学材料学院

出　　处：《中华创伤骨科杂志》2009年第8期752-756,共5页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金（30571872）

摘　　要：目的探讨免疫磁珠分选的骨骺干细胞（PSCs）经诱导后向软骨细胞方向分化的性能，及其与壳聚糖／纳米羟基磷灰石（CS／nHA）支架材料复合培养构建组织工程化软骨的可行性。方法用显微手术器械剪取新生24h内的清洁级SD大鼠股骨下端骺板两端的LaCroix环处组织，应用免疫磁珠分选系统分离纯化PSCs，免疫荧光染色观察PSCs成纤维细胞生长因子受体-3（FGFR-3）的表达情况。体外培养并诱导PSCs向软骨细胞特性方向分化，免疫细胞化学、甲苯胺兰及番红O染色检测诱导后细胞Ⅱ型胶原及软骨基质的表达情况。以诱导后接种于CS／nHA支架培养的PSCs为实验组，以诱导后未接种支架的细胞为对照组。扫描电镜观察细胞的黏附及形态学改变，MTT法检测细胞增殖情况，阿辛蓝法测定细胞合成的糖胺多糖（GAG）情况。对两组间不同培养时间的吸光度值及GAG进行比较。结果免疫磁珠分选的PSCs体外培养增殖迅速，免疫荧光染色显示FGFR-3阳性表达。诱导后的PSCsII型胶原免疫细胞化学染色、甲苯胺兰染色及番红O染色均呈阳性。细胞接种CS／nHA支架后1、4d，实验组与对照组吸光度值比较差异无统计学意义（P〉0．05），而7d实验组与对照组吸光度值比较差异有统计学意义（t=-2．786，P=0．024）。培养14d后培养液GAG含量为（89．664-6．52）μg／10。个细胞，高于对照组[（78．62±4．63）μg／10。个细胞）]，差异有统计学意义（t=-3．084，P〈0．05）。结论免疫磁珠分选的PSCs经诱导后可向软骨细胞功能方向分化，诱导后的PSCs与CS／nHA分层支架复合有望构建组织工程化软骨。Objective To observe the biological behavior of pre-cartilaginous stem cells （PSCs） cultured with chitosan/nano-hydroxyapatite （CS/nHA） composite scaffold. Methods CS/nHA composite scaffolds were prepared using a combination of sintering and freeze-drying techniques. PSCs were isolated and purified using immunomagnetic microbeads, and induced into chondrogenic differentiation, Immunofluorescence analysis was performed to identify the expression of fibroblast growth factor receptor 3 （FGFR-3）. Collagen Ⅱ was detected by immunocytochemistry, and the extracellular matrix by Toluidine Blue and Safranin O staining. After the cells were seeded onto the CS/nHA composite scaffold, the effects of adhesion and morphological changes were observed by the phase-contrast microscopy and scanning electron microscopy （SEM） . The proliferation behavior of the ceils was analyzed by MTT assay. The glycosaminoglycan （GAG） content synthesized by PSCs in the culture medium was measured by histochemistry of Alcian Blue. Results The PSCs were successfully cultured in vitro, and positive expression of FGFR-3 was observed. The induced cells showed a positive rate of Collagen Ⅱ and positive staining of extracellular matrix, adhered to the surface of scaffold and proliferated well. The differences in GAG content between the experimental and control groups were statistically significant. Conclusion Since PSCs can be effectively isolated and purified by immunomagnetic microbeads and induced into chondrogenic differentiation, the CS/nHA composite scaffold loaded with PSCs may be well used for cartilage tissue engineering.

关 键 词：组织工程 软骨 干细胞 壳聚糖 羟基磷灰石类 

分 类 号：R686[医药卫生—骨科学]