Chai Le, Quan Renfu, Hu Jintao, Huang Xiaolong, Lü Jianlan, Zhang Can, Qiu Rui, Cai Bingbing
Zhejiang University of Traditional Chinese Medicine, Hangzhou Zhejiang, 310053, P.R.China.
Department of Spine Surgery, Jiangnan Hospital of Zhejiang Chinese Medicine College, Hangzhou Zhejiang, 311200,
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2019 Feb 15;33(2):252-258. doi: 10.7507/1002-1892.201809060.
To construct bone morphogenetic protein 2 (BMP-2) gelatin/chitosan hydrogel sustained-release system, co-implant with induced pluripotent stem cells (iPS) derived mesenchymal stem cells (MSCs) to hydroxyapatite (HA)/zirconium dioxide (ZrO ) bio porous ceramic foam, co-culture , and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs.
BMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and sustained release rate of the microspheres were tested. HA/ZrO bio porous ceramic foam composite iPS-MSCs and BMP-2 gelatin/chitosan hydrogel sustained release system co-culture system was established as experimental group, and cell scaffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After 3, 7, 10, and 14 days of co-culture in the two groups, ALP secretion of cells was detected; gene expression levels of core binding factor alpha 1 (Cbfa1), collagen type Ⅰ, and Osterix (OSX) were detected by RT-PCR; the expression of collagen type Ⅰ was observed by immunohistochemical staining at 14 days of culture; and cell creep and adhesion were observed by scanning electron microscopy.
BMP-2 gelatin/chitosan hydrogel sustained-release system had better drug encapsulation efficiency and drug loading, and could prolong the activity time of BMP-2. The secretion of ALP and the relative expression of Cbfa1, collagen type Ⅰ, and OSX genes in the experimental group were significantly higher than those in the control group at different time points in the co-culture system ( <0.05). Immunohistochemical staining showed that the amount of fluorescence in the experimental group was significantly more than that in the control group, i.e. the expression level of collagen type Ⅰ was higher than that in the control group. The cells could be more evenly distributed on the materials, and the cell morphology was good. Scanning electron microscopy showed that the sustained-release system could adhere to cells well.
iPS-MSCs have the ability of osteogenic differentiation, which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPS-MSCs and sustained-release system can adhere to the materials well, and the cell activity is better.
构建骨形态发生蛋白2（BMP-2）明胶/壳聚糖水凝胶缓释系统，与诱导多能干细胞（iPS）来源的间充质干细胞（MSCs）共同植入羟基磷灰石（HA）/二氧化锆（ZrO₂）生物多孔陶瓷泡沫中进行共培养，探讨缓释系统对iPS-MSCs成骨分化的影响。
采用油包水法制备BMP-2明胶/壳聚糖水凝胶微球。检测微球的药物包封率、载药量及缓释率。建立HA/ZrO₂生物多孔陶瓷泡沫复合iPS-MSCs与BMP-2明胶/壳聚糖水凝胶缓释系统共培养体系作为实验组，以不含BMP-2复合明胶/壳聚糖水凝胶缓释系统的细胞支架复合物作为对照组。两组共培养第3、7、10和14天后，检测细胞碱性磷酸酶（ALP）分泌情况；采用逆转录-聚合酶链反应（RT-PCR）检测核心结合因子α1（Cbfa1）、Ⅰ型胶原和osterix（OSX）基因表达水平；培养14天时采用免疫组织化学染色观察Ⅰ型胶原表达情况；采用扫描电子显微镜观察细胞爬行及黏附情况。
BMP-2明胶/壳聚糖水凝胶缓释系统具有较好的药物包封率和载药量，可延长BMP-2的活性时间。共培养体系中不同时间点实验组细胞ALP分泌量及Cbfa1、Ⅰ型胶原和OSX基因相对表达量均显著高于对照组（P<0.05）。免疫组织化学染色显示实验组荧光量显著多于对照组，即Ⅰ型胶原表达水平高于对照组。细胞在材料上分布更均匀，细胞形态良好。扫描电子显微镜显示缓释系统与细胞黏附良好。
iPS-MSCs具有成骨分化能力，BMP-2明胶/壳聚糖水凝胶缓释系统可显著增强其成骨分化能力。iPS-MSCs与缓释系统联合能较好地黏附于材料，细胞活性良好。
