【摘要】 目的:探讨重组人源化Ⅰ型胶原蛋白微球(rCol-MSs)对骨髓间充质干细胞(BMSCs)成骨分化的促进作用及其潜在应用价值。方法:采用乳化-交联法制备rCol-MSs,并通过扫描电镜(SEM)、X射线衍射(XRD)、粒度仪及傅里叶变换红外光谱仪(FTIR)进行系统表征观察。将BMSCs分别与rCol-MSs共培养(rCol-MSs组)或常规二维培养(对照组)。采用CCK-8检测细胞增殖，活/死细胞荧光染色评价细胞存活；通过碱性磷酸酶(ALP)活性定量检测及染色、茜素红S染色及矿化结节定量分析成骨分化能力；采用实时荧光定量PCR检测Runt相关转录因子2(RUNX2)、ALP、Ⅰ型胶原蛋白α1链(COL1a1)及骨钙素(OCN)等成骨相关基因mRNA表达水平。结果:rCol-MSs呈均匀球形，平均粒径约5μm,表面光滑，具有典型的胶原特征峰。与对照组相比，rCol-MSs组未显示明显细胞毒性，细胞增殖活性无显著性差异(P>0.05),第14天ALP活性显著升高(P<0.001),第21天矿化结节面积增加约26倍(P<0.001);RUNX2、ALP及COL1a1 mRNA表达水平均在成骨诱导第7天显著上调(P<0.05)。结论:rCol-MSs具有良好的生物相容性，可显著促进BMSCs的成骨分化，在骨组织工程尤其是牙槽骨再生修复领域具有较好的应用潜力。更多还原

【Abstract】 Objective: To investigate the effects of recombinant humanized type Ⅰ collagen microspheres(rCol-MSs) on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells(BMSCs) and to evaluate their potential application in bone tissue engineering. Methods: The rCol-MSs were fabricated and systematically characterized, including morphology observation by scanning electron microscopy(SEM), structural analysis by X-ray diffraction(XRD), particle size and distribution measurement, and functional group identification by Fourier transform infrared spectroscopy(FTIR). In vitro experiments were divided into the rCol-MSs group and the control group(routine culture condition). BMSCs were co-cultured with rCol-MSs to construct a cell-material interaction system. Cell proliferation was evaluated by CCK-8 assay, and Live/Dead staining was performed to assess cytocompatibility. Alkaline phosphatase(ALP) staining and activity assays, as well as Alizarin Red S staining, were used to evaluate osteogenic mineralization. The expression of osteogenesis-related genes, including runt-related transcription factor 2(RUNX2), ALP, collagen typeⅠalpha 1 chain(COL1a1), and osteocalcin(OCN), were detected by real-time quantitative RT-qPCR. Results: The prepared rCol-MSs exhibited a uniform spherical morphology with an average particle size of approximately 5 μm and characteristic collagen-specific absorption bands in FTIR spectra. Compared with the control group, rCol-MSs showed no evident cytotoxicity, with no significant difference in cell proliferation activity(P>0.05). ALP activity was significantly increased on day 14(P<0.001), and the mineralized nodule area was approximately 26-fold higher on day 21(P<0.001). Furthermore, the mRNA expression levels of RUNX2, ALP, and COL1a1 were significantly upregulated during day 7 of osteogenic induction(P<0.05). Conclusions: Recombinant humanized type Ⅰ collagen microspheres can significantly promote the osteogenic differentiation of BMSCs and show potential application in alveolar bone regeneration following tooth extraction.更多还原