生物三维打印类细胞外基质硬度对骨髓间充质干细胞向皮肤附属器细胞分化的影响  

Influence of the stiffness of three-dimensionally bioprinted extracellular matrix analogue on the differentiation of bone mesenchymal stem cells into skin appendage cells

作　　者：恩和吉日嘎拉 张熠杰 李建军[1] 姚斌[1] 宋薇 黄沙[1] 付小兵[1] Enhejirigala null;Zhang Yijie;Li Jianjun;Yao Bin;Song Wei;Huang Sha;Fu Xiaobing(Research Center for Wound Repair and Regeneration,Medical Innovation Research Department,the PLA General Hospital,Beijing 100048,China;Tianjin Medical University,300070)

机构地区：[1]解放军总医院医学创新研究部创伤修复与组织再生研究中心,北京100048 [2]天津医科大学,300070

出　　处：《中华烧伤杂志》2020年第11期1013-1023,共11页Chinese Journal of Burns

基　　金：国家重点研发计划(2017YFC1103300);国家自然科学基金(81571909、81701906);解放军总医院军事医学创新研究项目(CX19026);解放军总医院杰青培育专项(2017-JQPY-002)。

摘　　要：目的观察生物三维打印类细胞外基质(ECM)硬度对骨髓间充质干细胞(BMSC)向皮肤附属器细胞分化的影响。方法(1)分别将1 g海藻酸钠和4 g明胶、3 g海藻酸钠和8 g明胶混匀,混合物分别溶于100 mL超纯水中,配制2种海藻酸钠-明胶复合水凝胶,分别命名为1A4G水凝胶、3A8G水凝胶,用于后续实验。观察2种水凝胶室温下、4℃冷凝15~30 min(冷凝条件下同)后、冷凝且用25 g/L氯化钙溶液交联(交联条件下同)后、冷凝后且用生物三维打印机(三维打印仪器下同)进行三维打印且交联后形态。取2种水凝胶冷凝并交联后,采用杨氏模量测定仪检测杨氏模量(硬度),样本数为3。取2种水凝胶交联并冷冻干燥,用扫描电子显微镜观察其孔隙结构。取2种水凝胶交联并冷冻干燥,用无水乙醇置换法检测孔隙率,样本数为3。(2)从20只1周龄雌雄不限C57BL/6小鼠股骨和胫骨中分离培养BMSC,取第2代细胞进行实验。分别将1.0×107个/mL的BMSC单细胞悬液与1A4G水凝胶、3A8G水凝胶以1∶9的体积比充分混匀,制备载BMSC的1A4G水凝胶、载BMSC的3A8G水凝胶,进行三维打印,1 mL载细胞水凝胶(打印用量下同)打印1块,交联后加入间充质干细胞(MSC)专用培养基培养。根据水凝胶不同,将打印块分为1A4G组和3A8G组。取2组打印块各1块,培养7 d,采用细胞活/死试剂盒计数50倍视野下活、死细胞。取2组打印块各9块,另将9孔用2 mL MSC专用培养基培养的每孔1.0×106个BMSC设为二维培养组。分别于培养1、3、5 d,1A4G组与3A8G组各取3块打印块、二维培养组取3孔细胞,用细胞计数试剂盒8法检测培养液中吸光度值,以此表示细胞增殖活性。(3)同实验(2)制备载BMSC的1A4G水凝胶、载BMSC的3A8G水凝胶各10 mL,分别加入从10只新生1 d雌雄不明C57BL/6小鼠提取的足趾垫匀浆液各0.5 mL混匀进行三维打印及交联,加入MSC专用培养基培养3 d,更换为汗腺专用培养基培养。根据�Objective To observe the influence of the stiffness of three-dimensionally bioprinted extracellular matrix analogue on the differentiation of bone marrow mesenchymal stem cells(BMSCs)into skin appendage cells.Methods(1)Sodium alginate of 1 g and 4 g gelatin,3 g sodium alginate and 8 g gelatin were mixed respectively,and the two mixtures were dissolved in 100 mL ultra-pure water respectively to prepare two sodium alginate-gelatin composite hydrogels,named 1A4G hydrogel and 3A8G hydrogel,which were used in the subsequent experiments.The morphology of the two hydrogels at room temperature,after condensation for 15-30 min at 4℃(the same condensation condition below),after condensation and cross-linking with 25 g/L calcium chloride solution(the same cross-linking condition below),and after condensation and three-dimensional printing with a three-dimensional bioprinter(the same three-dimensional printer below)and cross-linking were observed respectively.Young′s modulus(stiffness)of the two kinds of hydrogels was measured by Young′s modulus tester after condensation and cross-linking(n=3).Two kinds of hydrogels were cross-linked and freeze-dried,and their pore structure was observed by scanning electron microscope.Two hydrogels were cross-linked and freeze-dried,and the porosity was detected by anhydrous ethanol replacement method(n=3).(2)BMSCs were isolated from femur and tibia of 20 C57BL/6 mice(no limitation with sex,born 7 days)and cultured,and the second passage of cells was used for further test.The BMSCs single cell suspension(1.0×107/mL)was mixed with 1A4G hydrogel and 3A8G hydrogel respectively at 1∶9 volume ratio to prepare BMSCs-loaded 1A4G hydrogel and BMSCs-loaded 3A8G hydrogel for three-dimensional printing.One construct was printed with 1 mL cell-loaded hydrogel(the same dosage for printing below).Mesenchymal stem cells(MSCs)specific medium was added after cross-linking,and the printed constructs were divided into 1A4G group and 3A8G group according to the hydrogel.One construct of each group cul

关 键 词：间质干细胞 硬度 生物三维打印 类细胞外基质 三维微环境 皮肤附属器 

分 类 号：R644[医药卫生—外科学] R622+.1[医药卫生—临床医学] R457