力学刺激提高生物3D打印软骨构建物基质的形成  

Mechanical stimulation enhances matrix formation of three-dimensional bioprinted cartilage constructs

作　　者：孙可欣 曾今实 李佳 蒋海越 刘霞 Sun Kexin;Zeng Jinshi;Li Jia;Jiang Haiyue;Liu Xia(Research Center of Plastic Surgery Hospital,Chinese Academy of Medical Science&Peking Union Medical College,Beijing 100144,China)

机构地区：[1]中国医学科学院北京协和医学院整形外科医院研究中心,北京市100144

出　　处：《中国组织工程研究》2023年第21期3293-3299,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81871575),项目负责人:刘霞;中国医学科学院医学与健康科技创新工程(2021-I2M-1-052),任务三项目负责人:蒋海越。

摘　　要：背景:基质分泌不均匀、力学强度不足是影响组织工程构建物在体内形成效果的重要因素,力学刺激是促进细胞外基质分泌的有效手段。目的:探索3D生物打印复合支架在力学加压环境下的生物学表现。方法:采用3D生物打印技术制备软骨细胞-甲基丙烯酰胺基明胶复合支架,活死染色观察细胞存活情况。将复合支架置于力学加压生物反应器中进行加压培养,以6孔板中不加压培养的复合支架为对照,活死染色观察细胞存活情况,组织学染色观察复合支架体外软骨形成情况,qRT-PCR检测成软骨相关基因mRNA的相对表达量。将加压与不加压复合支架分别植入裸鼠皮下,5周后取材,组织学观察软骨形成情况。结果与结论:①复合支架外观呈现清晰的网格状结构,体外培养1,4,7 d时,支架形态稳定、结构清晰,细胞存活率均在90%以上;②培养2周后,加压组复合支架中的细胞存活率低于不加压组(P<0.05);苏木精-伊红染色显示两组复合支架均有明显的软骨陷窝结构,细胞在材料中分布较均匀,加压组复合支架的材料间空隙内有更多新生软骨组织形成;番红O染色显示两组均可见红色软骨基质形成,加压组细胞周基质染色更深;Ⅰ型胶原免疫组织化学染色显示,加压组着色更为明显;加压组弹性蛋白、Ⅱ型胶原的mRNA表达高于不加压组(P<0.05);③植入裸鼠皮下5周后,苏木精-伊红染色显示加压组软骨组织形成更为均一,软骨细胞大小均匀,陷窝结构明显;④结果表明,虽然体外加压刺激会引发3D生物打印软骨细胞-甲基丙烯酰胺基明胶复合支架中的细胞死亡,但同时会促进存活细胞向支架空隙间长入,提高其软骨基质相关基因的表达,促进成软骨能力。BACKGROUND:Uneven secretion of matrix and insufficient mechanical strength are important factors affecting the formation effect of tissue engineering constructs in vivo.Mechanical stimulation is an effective means to promote the secretion of extracellular matrix.OBJECTIVE:To explore the biological performance of 3D bioprinted composite scaffolds under mechanical stimulation.METHODS:The chondrocyte-gelatin methacryloyl composite scaffold was prepared and printed by 3D bioprinting technology.Cell survival was observed by live/dead cell staining.The composite scaffolds were placed in a mechanical pressurized bioreactor for pressurized culture.The composite scaffolds cultured in a 6-well plate without pressurization were used as the control.Live/dead cell staining was used to observe the cell survival.Histological staining was used to observe the in vitro cartilage of the composite scaffolds.The relative expression levels of cartilage-related genes were detected by real-time quantitative PCR.The composite scaffolds were implanted into nude mice for 5 weeks in vitro with or without mechanical stimulation,and the cartilage formation was observed by histological staining.RESULTS AND CONCLUSION:(1)The appearance of the composite scaffolds showed a clear grid-like structure.When cultured for 1,4,and 7 days in vitro,the scaffolds had stable morphology and clear structure,and the cell viability was above 90%.(2)After 2 weeks of culture,the cell survival rate in the composite scaffold in the pressurized group was lower than that in the unpressurized group(P<0.05).Hematoxylin-eosin staining exhibited that composite scaffolds of the two groups had obvious cartilage lacuna structure,and the cells were distributed evenly in the materials.In the pressurized group,more new cartilage tissue was formed in the gap of the composite scaffold.Safranin O staining showed that red cartilage matrix was formed in both groups;the stained pericellular matrix was deeper in the pressurized group.Type Ⅰ collagen immunohistochemical staining sho

关 键 词：3D生物打印 力学刺激 软骨细胞 甲基丙烯酰胺基明胶 组织工程软骨 复合支架 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R68