羽毛角蛋白水凝胶-纳米级蒙脱石复合水凝胶对骨髓间充质干细胞生物学行为的影响及成骨性能的初探

口腔生物医学 ›› 2023, Vol. 14 ›› Issue (2): 105-112.

羽毛角蛋白水凝胶-纳米级蒙脱石复合水凝胶对骨髓间充质干细胞生物学行为的影响及成骨性能的初探

柯越,张筱岚,马艳霞,于金华*

南京医科大学附属口腔医院牙体牙髓科，江苏省口腔疾病研究重点实验室，江苏省口腔转化医学工程研究中心

收稿日期:2023-02-06 修回日期:2023-05-18 出版日期:2023-06-25 发布日期:2023-07-31
通讯作者: 于金华 E-mail:yujinhua@njmu.edu.cn
基金资助:
国家自然科学基金面上项目;国家自然科学基金面上项目

Feather keratin-montmorillonite nanocomposite hydrogel on the biological behavior and osteogenic properties of bone marrow mesenchymal stem cell

1. Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Jiangsu Province Key Laboratory of Oral Diseases, Jiangsu Province Engineering Research Center of Stomatological Translational Medicine
2.

Received:2023-02-06 Revised:2023-05-18 Online:2023-06-25 Published:2023-07-31

摘要/Abstract

摘要： 目的：探讨羽毛角蛋白水凝胶与蒙脱石（MMT）复合改性后对骨髓间充质干细胞（BMSCs）的生物学行为及成骨性能的影响。方法：通过改良Shindai法制备羽毛角蛋白，将羽毛角蛋白与MMT混合制备羽毛角蛋白-MMT复合水凝胶。根据MMT/羽毛角蛋白水凝胶质量比例（w/w）分为0%、1%、3%三个组。结合考马斯亮蓝染色和傅里叶红外光谱鉴定所提取的羽毛角蛋白，利用扫描电镜观察羽毛角蛋白-MMT复合水凝胶的表面形态。加入使用含羽毛角蛋白-MMT复合水凝胶浸提液的培养基培养BMSCs，通过CCK-8实验探究其生物安全性，通过碱性磷酸酶（ALP）试剂盒检测其对BMSCs的ALP活性的影响，通过茜素红染色观察其对BMSCs的细胞外基质矿化的影响，通过Western blot检测其对BMSCs的BMP/SMAD信号通路相关蛋白表达水平的影响。结果：结合考马斯亮蓝染色和傅里叶红外光谱证明顺利提取羽毛角蛋白，羽毛角蛋白水凝胶具有天然孔隙，当加入MMT后其形态结构会发生一定程度的改变，当MMT的比例达到3%时，复合水凝胶支架的孔径明显变小。CCK-8实验结果表明，羽毛角蛋白+3%MMT复合水凝胶的浸提液可能对BMSCs的生长有抑制作用。羽毛角蛋白+1%MMT组的ALP染色和茜素红染色明显强于其他两组，说明羽毛角蛋白+1%MMT组的浸提液对BMSCs成骨分化有促进作用。与其他两组比较，羽毛角蛋白+1%MMT组的BMSCs骨形成蛋白-2（BMP‐2）、Wnt3a、磷酸化细胞信号转导分子SMAD 1/5/8（p-SAMD 1/5/8）、Runt相关转录因子2（RUNX2）、骨钙素（OCN）和Ⅰ型胶原蛋白（COL-1）蛋白表达较高（P＜0.01）。结论：羽毛角蛋白-MMT复合水凝胶可通过BMP/SMAD信号通路促进BMSCs成骨向分化。

Abstract: Objective:To clarify the effects of feather keratin-montmorillonite hydrogels on the biological behavior and osteogenic properties of BMSCs. Methods:Keratins were extracted from chicken feathers by the modified Shindai method. The feather keratin-montmorillonite hydrogel was prepared by mixing the montmorillonite and feather keratin by mass ratio（0%, 1%, 3%). The feather keratin was identified and confirmed as coomassie brilliant blue staining and Fourier transform infrared spectroscopy. The morphology of feather keratin-montmorillonite hydrogels was shown by SEM. BMSCs were cultured at the leach liquor of feather keratin-montmorillonite hydrogels in the medium. The cytotoxicity was measured by CCK-8 assay. The BMSCs of ALP activity was also quantified using an ALP assay kit. Mineralization of the extracellular matrix of BMSCs is investigated by alizarin red staining. The expressions of related proteins in BMP/SMAD signaling pathway were detected by Western blot. Results:The results of coomassie brilliant blue staining and Fourier transform infrared spectroscopy demonstrate that we successfully extracted feather keratin from chicken feather. The pores present in feather keratin hydrogels were well-formed and interconnected. The addition of morillonite changes their structure. When 3%MMT was added, the pore size of feather keratin-montmorillonite hydrogel was significantly reduced. The results of the CCK-8 proliferation experiment showed that the leach liquors of the feather keratin+3%MMT group had inhibitory effects on the growth of the cells. The feather keratin+1%MMT group expressed the highest level of ALP activity, ALP staining, and alizarin red staining, and the data indicate that the leach liquors of the feather keratin+1%MMT group may promote the osteogenic differentiation of BMSCs. Protein expression of BMP-2, Wnt3a, p-SMAD1/5/8, RUNX2, OCN, and COL-1 was significantly higher in feather keratin+1%MMT group（P＜0.01）. Conclusions:The feather keratin-montmorillonite hydrogels can potentially promote osteogenic differentiation through BMP/SMAD signaling pathway.

柯越张筱岚马艳霞于金华. 羽毛角蛋白水凝胶-纳米级蒙脱石复合水凝胶对骨髓间充质干细胞生物学行为的影响及成骨性能的初探[J]. 口腔生物医学, 2023, 14(2): 105-112.