Xu Gang, Hu Xiantong, Han Liwei, Zhao Yantao, Li Zhonghai
Department of Orthopaedics, First Affiliated Hospital of Dalian Medical University, Dalian, 116011, PR China.
Key Laboratory of Molecular Mechanism for Repair and Remodeling of Orthopaedic Diseases, Liaoning Province, Dalian, 116011, PR China.
J Orthop Translat. 2021 Mar 2;28:74-82. doi: 10.1016/j.jot.2021.02.001. eCollection 2021 May.
Xenograft bone scaffolds have advantages such as mechanical strength, sufficient source and safety. Combined with siRNA properly targeting CKIP-1, a negative regulator of osteogenesis, may contribute to the repair result of calcine bone alone.
Herein, we constructed a novel xenograft bovine bone scaffold namely (DSS)6-liposome/CKIP-1 siRNA/calcine bone, the characteristics of which were investigated by confirming the effect of (DSS)6-liposome, observing the appearance and testing mechanical strength of calcine bone, and observing the combined result of CKIP-1 siRNA by FAM immunofluorescence. In addition, cytotoxicity by CCK-8 and LDH activity of L929 cells and MC3T3-E1 osteoblasts cultured with the scaffold were tested in vitro, primary osteoblasts proliferation, the mRNA expressions of CKIP-1, ALP, COL1-α and OCN, the protein expressions of CKIP-1, BMP-2, COL-1 and Runx2 and calcium nodules were also determined by CCK-8, RT-qPCR, western-blot and Alizarin Red staining in vitro. Then, we successively established the skull defect model for evaluating the repair result of the novel scaffold by HE staining of 2, 4, 8 and 12 weeks, immumohistochemical stainings of 2, 4, 8 and 12 weeks such as ALP, COL-1α and OCN, Mirco-CT scanning of 4 and 12 weeks and the relative parameters and so on in vivo.
It indicated that (DSS)6-liposome/CKIP-1 siRNA/calcine bone could successfully knock down the CKIP-1 mRNA and protein expressions, promote osteoblasts proliferation with the little cytotoxicity in vitro, increase the protein expressions of BMP-2, COL-1 and Runx2 in vitro, increase mRNA expressions of ALP, COL-1α and OCN in vitro and in vivo, and have a better bone defect repair effect with few side effects in rats after 12 weeks.
Our research indicates (DSS)6-liposome/CKIP-1 siRNA/calcine bone could repair skull defects well in rats, and it may lay the foundation of applicating the novel xenograft bone scaffold in the clinical.
These findings provide evidence that (DSS)6- liposome/CKIP-1 siRNA/calcine bone could be used as a novel xenograft bone scaffold for osteogenesis with the good safety.
异种骨支架具有机械强度高、来源充足和安全性好等优点。与适当靶向成骨负调节因子CKIP-1的小干扰RNA（siRNA）联合使用，可能有助于单纯煅烧骨的修复效果。
在此，我们构建了一种新型异种牛骨支架，即（DSS）6-脂质体/CKIP-1 siRNA/煅烧骨，通过确认（DSS）6-脂质体的作用、观察煅烧骨的外观和测试其机械强度以及通过FAM免疫荧光观察CKIP-1 siRNA的结合结果来研究其特性。此外，体外测试了用该支架培养的L929细胞和MC3T3-E1成骨细胞的CCK-8细胞毒性和LDH活性，还通过CCK-8、RT-qPCR、蛋白质免疫印迹法和茜素红染色在体外测定了原代成骨细胞增殖、CKIP-1、碱性磷酸酶（ALP）、I型胶原蛋白（COL1-α）和骨钙素（OCN）的mRNA表达、CKIP-1、骨形态发生蛋白-2（BMP-2）、I型胶原蛋白（COL-1）和Runx2的蛋白质表达以及钙结节。然后，我们依次建立颅骨缺损模型，通过2、4、8和12周的苏木精-伊红（HE）染色、2、4、8和12周的免疫组织化学染色如ALP、COL-1α和OCN、4和12周的微计算机断层扫描（Micro-CT）及其相关参数等来评估新型支架在体内的修复效果。
结果表明，（DSS）6-脂质体/CKIP-1 siRNA/煅烧骨能够成功下调CKIP-1的mRNA和蛋白质表达，在体外以较小的细胞毒性促进成骨细胞增殖，增加体外BMP-2、COL-1和Runx2的蛋白质表达，增加体外和体内ALP、COL-1α和OCN的mRNA表达，并且在12周后对大鼠具有较好的骨缺损修复效果且副作用较少。
我们的研究表明，（DSS）6-脂质体/CKIP-1 siRNA/煅烧骨能够很好地修复大鼠颅骨缺损，这可能为该新型异种骨支架在临床上的应用奠定基础。
这些发现提供了证据，表明（DSS）6-脂质体/CKIP-1 siRNA/煅烧骨可作为一种新型的具有良好安全性的用于成骨的异种骨支架。
Zotero 插件
