小鼠胚胎干细胞向成骨样时段性细胞分化的形态学特点

背景:纳米羟基磷灰石具有良好的生物活性和相容性,在体外可与细胞短时间内形成紧密结合,是骨组织工程可植入性的材料。目的:观察小鼠胚胎干细胞分化为成骨样时段性细胞的形态学特点。方法:取C57小鼠胚胎干细胞进行培养,将P3胚胎干细胞制备拟胚体。通过碱性磷酸酶、免疫组织化学OCT-4、SOX2、NANOG对原代胚胎干细胞进行鉴定;通过茜素红S、Ⅰ型胶原、冯库萨染色对成骨诱导后的细胞(L-维生素C、β-磷酸甘油、地塞米松)进行检测;扫描电镜观察与纳米羟基磷灰石材料复合培养时细胞的形态及增殖情况。结果与结论:拟胚体贴壁后7d,各组碱性磷酸酶表达即发生变化,14d后碱性磷酸酶表达逐渐增加,光镜下可见轮廓清晰大小不等的绿色结节。茜素红S染色镜下可见橙红色、边界清晰和直径大小不等的结节。冯库萨染色14d可见细胞团内有黑色沉淀,21d后黑色沉淀面积增大。胚胎干细胞与纳米羟基磷灰石材料体外复合培养1,3,5,7,10d,细胞大部分呈细胞团样生长。提示在维生素C、β-磷酸甘油、地塞米松共同作用下,有效促进了胚胎干细胞成骨细胞的产生,胚胎干细胞与支架材料复合培养结合程度高,可用来构建组织工程骨。

Time-dependent morphological characteristics of mouse embryonic stem cells differentiating into osteoblasts

BACKGROUND：Nano-hydroxyapatite has good biocompatibility and bioactivity. It can tightly connect with in vitro tissues within a short time. Therefore, nano-hydroxyapatite has been widely used for bone tissue engineering. OBJECTIVE：To investigate the time-dependent morphological characteristics of mouse embryonic stem cells differentiating into osteoblasts. METHODS：P3 embryonic stem cells from C57 mouse were prepared into embryoid bodies. Primary embryonic stem cells were identified by alkaline phosphatase staining and immunohistochemical analysis （oct 4, sox2, nanog）. After osteogenic induction by L-vitamin C, β-phosphoglycerol, and dexamethasone, cells were identified by alizarin red S, collagen Ⅰ, and von Kossa staining. The morphology and proliferation of cells when cultured with nano-hydroxyapatite were observed using scanning electron microscope. RESULTS AND CONCLUSION：The expression of alkaline phosphatase showed no obvious change in each group within 7 days after adherence of embryoid bodies, but increased gradually after 14 days. Green nodules of varying size with clear border could be seen under optical microscope. After Alizarin red S staining, orange red nodules of varying size with clear border could be observed. Black precipitates appeared in the cell mass after von Kossa staining for 14 days, and amplified after 21 days. After co-culture with nano-hydroxyapatite for 1, 3, 5, 7, 10 days, the majority of embryonic stem cells grew in cell mass. These findings suggest that co-application of vitamin C, β-phosphoglycerol and dexamethasone effectively promote embryonic stem cells to differentiate into osteoblasts; embryonic stem cells composited onto scaffold material can be used in bone tissue engineering.