3D打印脱细胞真皮基质皮肤复合支架及其与血管内皮细胞的体外生物相容性研究  

3D printing of decellularized dermal matrix-GelMA scaffolds and its biocompatibility on vascular endothelial cells in vitro

作　　者：张晨晨 沈谦[2] 李恭驰[3] 邝莉雯 杜烨 王知 王晓蓉 潘银根[5] 李炳辉[1,4] Zhang Chenchen;Shen Qian;Li Gongchi;Kuang Liwen;Du Ye;Wang Zhi;Wang Xiaorong;Pan Yingen;Li Binghui(Department of Wound Repair,Liyuan Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430077,China;School of Foreign Languages,Zhongnan University of Economics and Law,Wuhan 430073,China;Department of Hand Surgery,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China;Hubei Clinical Research Centre for Chronic Trauma and Diabetic Foot Medicine,Wuhan 430077,China;Department of Plastic and Reconstructive Surgery,Qidong People’s Hospital,Qidong 226200,China)

机构地区：[1]华中科技大学同济医学院附属梨园医院创面修复科,武汉430077 [2]中南财经政法大学外国语学院,武汉430073 [3]华中科技大学同济医学院附属协和医院手外科,武汉430022 [4]湖北省慢性创面及糖尿病足医学临床研究中心,武汉430077 [5]江苏省启东市人民医院整形美容科,226200

出　　处：《中华实验外科杂志》2022年第12期2349-2352,共4页Chinese Journal of Experimental Surgery

基　　金：国家重大疾病多学科合作诊疗能力建设项目(国卫办医函[2019]542号);湖北省重点研发计划项目(2020BCB029);湖北省自然科学基金面上项目(2020CFB696);湖北省科技创新平台专项资助建设项目(2018BCC340)。

摘　　要：目的:利用3D生物打印技术将脱细胞真皮基质(ADM)及甲基丙烯酸酯化明胶(GelMA)混合制备成不同规格的皮肤复合支架,评价其生物相容性及对人脐静脉血管内皮细胞(HUVECs)生物学行为的影响。方法:将0%、0.75%、1.50%的ADM与GelMA混合配置成光敏性生物墨水,利用3D生物打印技术分别制备A组:单纯GelMA支架、B组:0.75%ADM/GelMA支架及C组:1.5%ADM/GelMA支架,扫描电镜观察其形态结构。通过CCK-8实验方法于第1、2、3天检测细胞增殖;将HUVECs与其共培养,通过Live/Dead染色观察皮肤支架表面细胞生长状态;使用Transwell小室实验检测皮肤支架对HUVECs迁移的影响;两组间比较采用非配对t检验,多组间比较采用单因素方差分析。结果:扫描电镜结果显示,各组皮肤复合支架均为十字交叉网格结构立体结构,且孔隙均匀,C组比A和B两组微观孔隙更加密集,孔隙之间的连通性更好。细胞计数试剂盒(CCK-8)实验结果显示,与阴性对照组(0 d:0.18±0.03;1 d:0.39±0.02;2 d:0.8±0.03;3 d:1.21±0.06)比较,在不同时间A组(0 d:0.19±0.04;1 d:0.39±0.03;2 d:0.75±0.03;3 d:1.19±0.06)细胞活力均无明显变化(F=1.37,P>0.05),而B组(0 d:0.21±0.05;1 d:0.6±0.04;2 d:1.15±0.04;3 d:1.72±0.05)和C组(0 d:0.22±0.06;1 d:0.59±0.03;2 d:1.18±0.05;3 d:1.87±0.02)细胞活力显著提高(F=22.85、26.76,P<0.05)。与A组比较,B组和C组在各时间点细胞活力显著提高(F=24.22、28.13,P<0.05)。Live/Dead实验染色结果显示,细胞在各组皮肤支架表面均能较好地黏附与生长,与A组(2.47±0.15)荧光强度比较,B组(4.63±0.35)和C组(11.98±0.31)荧光强度显著增高(t=10.00、48.26,P<0.05),并且C组荧光强度明显高于B组(t=27.45,P<0.05)。Transwell小室实验结果显示,与A组(38.67±6.56)比较,B组(134±17.59)和C组(251.33±20.13)的血管内皮细胞迁移数显著增多(t=8.375、17.37,P<0.05)。结论:1.5%ADM/GelMA的皮肤支架具有较好的生物相容性和促进血管内皮细胞迁移�Objective To prepare decellularized dermal matrix(ADM)-GelMA skin scaffolds of different kinds with ADM and methacrylated gelatin(GelMA)by 3D bioprinting technology,and to evaluate their biocompatibility and effects on the biological behaviour of human umbilical vein vascular endothelial cells(HUVECs).Methods 0%,0.75%and 1.50%ADM and GelMA were mixed and configured into photosensitive bioink,and Group A:GelMA-only scaffold,Group B:0.75%ADM/GelMA scaffold and Group C:1.5%ADM/GelMA scaffold were prepared using 3D bioprinting technology.The structure was observed by scanning electron microscope.Cell proliferation was detected by cell counting kit-8(CCK-8)method on the 1st,2rd and 3th days.HUVEC were co-cultured with them,and cell growth on the skin scaffold surface was observed by Live/Dead staining.Detection of the effect of skin scaffolds on HUVEC migration using transwell assays.Comparisons between two groups were made using an unpaired t-test,and comparisons between multiple groups were made using a one-way ANOVA.Results The results of scanning electron microscopy showed that the skin scaffolds in all groups had a crossed lattice structure with uniform pores,while group C had more dense microscopic pores and better connectivity between pores than groups A and B.The results of the CCK-8 experiment showed that compared with the negative control group(0 d:0.18±0.03;1 d:0.39±0.02;2 d:0.8±0.03;3 d:1.21±0.06),there was no significant change in cell viability in group A(0 d:0.19±0.04;1 d:0.39±0.03;2 d:0.75±0.03;3 d:1.19±0.06)at different times(F=1.37,P>0.05),while cell viability in groups B(0 d:0.21±0.05;1 d:0.6±0.04;2 d:1.15±0.04;3 d:1.72±0.05)and C(0 d:0.22±0.06;1 d:0.59±0.03;2 d:1.18±0.05;3 d:1.87±0.02)was significantly increased(F=22.85,26.76,P<0.05).Cell viability was significantly higher in groups B and C compared to group A at all time points(F=24.22,28.13,P<0.05).Compared to group A(2.47±0.15),the fluorescence intensity of group B(4.63±0.35)and group C(11.98±0.31)was significantly higher(t=10

关 键 词：3D生物打印 脱细胞真皮基质 皮肤支架 血管内皮细胞 

分 类 号：R318.08[医药卫生—生物医学工程]