Xing Qi, Yates Keegan, Tahtinen Mitchell, Shearier Emily, Qian Zichen, Zhao Feng
Department of Biomedical Engineering, Michigan Technological University , Houghton, Michigan.
Tissue Eng Part C Methods. 2015 Jan;21(1):77-87. doi: 10.1089/ten.tec.2013.0666.
The application of cell-derived extracellular matrix (ECM) in tissue engineering has gained increasing interest because it can provide a naturally occurring, complex set of physiologically functional signals for cell growth. The ECM scaffolds produced from decellularized fibroblast cell sheets contain high amounts of ECM substances, such as collagen, elastin, and glycosaminoglycans. They can serve as cell adhesion sites and mechanically strong supports for tissue-engineered constructs. An efficient method that can largely remove cellular materials while maintaining minimal disruption of ECM ultrastructure and content during the decellularization process is critical. In this study, three decellularization methods were investigated: high concentration (0.5 wt%) of sodium dodecyl sulfate (SDS), low concentration (0.05 wt%) of SDS, and freeze-thaw cycling method. They were compared by characterization of ECM preservation, mechanical properties, in vitro immune response, and cell repopulation ability of the resulted ECM scaffolds. The results demonstrated that the high SDS treatment could efficiently remove around 90% of DNA from the cell sheet, but significantly compromised their ECM content and mechanical strength. The elastic and viscous modulus of the ECM decreased around 80% and 62%, respectively, after the high SDS treatment. The freeze-thaw cycling method maintained the ECM structure as well as the mechanical strength, but also preserved a large amount of cellular components in the ECM scaffold. Around 88% of DNA was left in the ECM after the freeze-thaw treatment. In vitro inflammatory tests suggested that the amount of DNA fragments in ECM scaffolds does not cause a significantly different immune response. All three ECM scaffolds showed comparable ability to support in vitro cell repopulation. The ECM scaffolds possess great potential to be selectively used in different tissue engineering applications according to the practical requirement.
细胞衍生的细胞外基质（ECM）在组织工程中的应用越来越受到关注，因为它可以为细胞生长提供一组天然存在的、复杂的生理功能信号。由脱细胞成纤维细胞片产生的ECM支架含有大量的ECM物质，如胶原蛋白、弹性蛋白和糖胺聚糖。它们可以作为细胞粘附位点，并为组织工程构建体提供机械强度高的支撑。一种能够在脱细胞过程中大量去除细胞物质，同时保持ECM超微结构和含量最小程度破坏的有效方法至关重要。在本研究中，研究了三种脱细胞方法：高浓度（0.5 wt%）的十二烷基硫酸钠（SDS）、低浓度（0.05 wt%）的SDS和冻融循环法。通过对所得ECM支架的ECM保存、力学性能、体外免疫反应和细胞再填充能力的表征对它们进行了比较。结果表明，高SDS处理可以有效地从细胞片中去除约90%的DNA，但显著损害了它们的ECM含量和机械强度。高SDS处理后，ECM的弹性模量和粘性模量分别下降了约80%和62%。冻融循环法保持了ECM结构以及机械强度，但也在ECM支架中保留了大量细胞成分。冻融处理后，约88%的DNA留在了ECM中。体外炎症测试表明，ECM支架中的DNA片段数量不会引起显著不同的免疫反应。所有三种ECM支架在支持体外细胞再填充方面表现出相当的能力。根据实际需求，ECM支架具有在不同组织工程应用中选择性使用的巨大潜力。
