BMP-2明胶/壳聚糖水凝胶缓释系统复合羟基磷灰石/二氧化锆泡沫陶瓷与诱导多能干细胞来源MSCs的体外研究

In vitro study of bone morphogenetic protein 2 gelatin/chitosan hydrogel sustainedrelease system composite hydroxyapatite/zirconium dioxide foam ceramics and induced pluripotent stem cells derived mesenchymal stem cells

目的构建BMP-2明胶/壳聚糖水凝胶缓释系统,与诱导多能干细胞(induced pluripotent stem cells,iPS)来源MSCs复合种植至羟基磷灰石(hydroxyapatite,HA)/二氧化锆(zirconium dioxide,ZrO2)生物多孔泡沫陶瓷材料,体外共培养,探索缓释系统对iPS-MSCs成骨分化的作用。方法运用油包水相溶液制备BMP-2明胶/壳聚糖水凝胶微球,检测微球的药物包封率、载药率和体外缓释速率。建立HA/ZrO2多孔生物泡沫陶瓷材料复合iPS-MSCs及BMP-2明胶/壳聚糖水凝胶缓释系统共培养体系,作为实验组;以未复合BMP-2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。两组培养3、7、10、14 d,检测细胞的ALP分泌量,RT-PCR检测核心结合因子α1(core binding factorα1,Cbfa1)、Ⅰ型胶原和锌指结构转录因子(Osterix,OSX)基因表达水平;培养14 d时行免疫组织化学染色观察Ⅰ型胶原表达,并通过扫描电镜观察细胞爬行及黏附状态。结果 BMP-2明胶/壳聚糖水凝胶缓释系统具有较好的药物包封率及载药率,可延长BMP-2的活性时间。共培养体系体外培养各时间点实验组ALP分泌量及Cbfa1、Ⅰ型胶原、OSX基因相对表达量均显著高于对照组,差异有统计学意义(P<0.05)。免疫组织化学染色观察示,实验组荧光数量明显多于对照组,即Ⅰ型胶原表达水平高于对照组;细胞能较均匀地分布于材料上,细胞形态良好。扫描电镜观察示缓释系统能较好地黏附于细胞之间。结论 iPS-MSCs具有促成骨分化能力,在BMP-2明胶/壳聚糖水凝胶缓释系统作用下其促成骨能力显著增强。iPS-MSCs与缓释系统结合后能良好黏附于材料上,且细胞活性较好。

Objective To construct bone morphogenetic protein 2(BMP-2) gelatin/chitosan hydrogel sustainedrelease system, co-implant with induced pluripotent stem cells(iPS) derived mesenchymal stem cells(MSCs) to hydroxyapatite(HA)/zirconium dioxide(ZrO2) bio porous ceramic foam, co-culture in vitro, and to explore the effect of sustained-release system on osteogenic differentiation of iPS-MSCs. Methods BMP-2 gelatin/chitosan hydrogel microspheres were prepared by water-in-oil solution. Drug encapsulation efficiency, drug loading, and in vitro sustained release rate of the microspheres were tested. HA/ZrO2 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 s caffold complex without BMP-2 composite gelatin/chitosan hydrogel sustained release system as control group. After3, 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. Results 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 in vitro co-culture system(P<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. Conclusion iPS-MSCs have the ability of osteogenic differentiation,which is significantly enhanced by BMP-2 gelatin/chitosan hydrogel sustained-release system. The combination of iPSMSCs and sustained-release system can adhere to the materials well, and the cell activity is better.