肌肉脱细胞基质制备双重交联可注射水凝胶用于促进肌母细胞增殖和成肌分化  

A dual-crosslinked injectable hydrogel derived from muscular decellularized matrix promoting myoblasts proliferation and myogenic differentiation

作　　者：赵少华 郝晓亮 菅炎鹏 王一公 刘伟杰 邵欣慰 樊俊 徐松山 ZHAO Shaohua;HAO Xiaoliang;JIAN Yanpeng;WANG Yigong;LIU Weijie;SHAO Xinwei;FAN Jun;XU Songshan(Department of Plastic Surgery,Xuchang Central Hospital Affiliated to Henan University of Science and Technology,Xuchang Henan,461000,P.R.China;Department of Thyroid and Breast Diagnosis and Treatment Center,Weifang Hospital of Traditional Chinese Medicine,Weifang Shandong,261000,P.R.China;Department of Spine and Spinal Cord Surgery,Xuchang Central Hospital Affiliated to Henan University of Science and Technology,Xuchang Henan,461000,P.R.China)

机构地区：[1]河南科技大学附属许昌市中心医院整形美容科,河南许昌461000 [2]潍坊市中医院甲状腺乳腺外科,山东潍坊261000 [3]河南科技大学附属许昌市中心医院脊柱脊髓科,河南许昌461000

出　　处：《中国修复重建外科杂志》2023年第12期1514-1522,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：2020年河南省部共建青年项目(SBGJ202003054)。

摘　　要：目的探讨基于肌肉脱细胞基质(acellular musclar matrix,AMM)制备双重交联可注射水凝胶用于促进肌母细胞增殖和成肌分化的可行性。方法先将透明质酸通过高碘酸钠氧化处理并甲基化制备甲基丙烯酰胺氧化透明质酸(methacrylamidated oxidized hyaluronic acid,MOHA)水凝胶;再采用洗涤酶法获得AMM,并经氨基活化以制备氨基化AMM(aminated AMM,AAMM)。将MOHA水凝胶和AAMM通过席夫碱反应和紫外光交联,制备一种双重交联可注射的MOHA/AAMM水凝胶。采用傅立叶变换红外吸收光谱仪(Fourier transform infrared spectroscopy,FTIR)分别对MOHA、AAMM和MOHA/AAMM水凝胶样品进行表征;通过手动注射和紫外光交联分别检测MOHA/AAMM水凝胶的可注射性和成胶性能;采用力学测试检测水凝胶的流变性能和杨氏模量,并将水凝胶浸没于PBS中15 d检测其降解性能。通过免疫荧光染色和ELISA法检测水凝胶的活性成分。将MOHA和MOHA/AAMM水凝胶分别与C2C12肌母细胞体外共培养9 d,通过活/死细胞染色和细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测水凝胶促进肌母细胞的增殖作用,通过免疫荧光染色和定量聚合酶链反应(real time quantitative polymerase chain reaction,RT-qPCR)检测水凝胶促进肌母细胞的成肌分化能力。结果FTIR图谱示MOHA/AAMM水凝胶成功制备,且表现出良好的可注射性和成胶能力。相较于MOHA水凝胶,MOHA/AAMM水凝胶具有更高的黏度和杨氏模量,更缓慢的降解速率,并且含有更多胶原蛋白(包括Ⅰ型胶原和Ⅲ型胶原)和生物活性因子(包括EGF、FGF-2、VEGF和IGF-1),差异有统计学意义(P<0.05)。活/死细胞染色和CCK-8检测示,MOHA/AAMM水凝胶中C2C12肌母细胞随培养时间延长活细胞明显增多且死细胞减少,各时间点与MOHA组比较差异有统计学意义(P<0.05)。免疫荧光染色和RT-qPCR检测示,相较于MOHA水凝胶,MOHA/AAMM水凝胶中IGF-1沉积量和成肌相关基因(肌原蛋白、肌钙蛋Objective To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix(AMM)for promoting myoblasts proliferation and myogenic differentiation.Methods Firstly,hyaluronic acid was oxidized with NaIO4 and methylated to prepare methacrylamidated oxidized hyaluronic acid(MOHA).Then,AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM(AAMM).MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel.Fourier transform infrared spectroscopy(FTIR)was used to characterize MOHA,AAMM,and MOHA/AAMM hydrogels.The injectability of MOHA/AAMM hydrogel were evaluated by manual injection,and the gelation performance was assessed by UV crosslinking.The rheological properties and Young’s modulus of the hydrogel were examined through mechanical tests.The degradation rate of the hydrogel was assessed by immersing it in PBS.The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits.The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8(CCK-8)assays after co-culturing with C2C12 myoblasts for 9 days.The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction(RTqPCR).Results FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel.The hydrogel exhibited good injectability and gelation ability.Compared to MOHA hydrogel,MOHA/AAMM hydrogel exhibited higher viscosity and Young’s modulus,a reduced degradation rate,and contained a higher amount of collagen(including collagen typeⅠand collagen type Ⅲ)as well as bioactive factors(including epidermal growth factor,fibroblast growth factor 2,vascular endothelial growth factor,and insulin-like growth factor 1).The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time,there

关 键 词：水凝胶 肌肉脱细胞基质 细胞增殖 成肌分化 肌肉组织工程 

分 类 号：R318.08[医药卫生—生物医学工程]