Azide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.

中文翻译：

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通过代谢糖工程获得的化学修饰细胞衍生基质的折衷表征和常用方法的重新评估

含有叠氮化物的细胞衍生的细胞外基质（“clickECMs”）已成为一种令人兴奋的新型生物材料。它们保留了天然细胞外基质 (ECM) 的大量特征，同时提供了能够实现生物正交生物共轭反应的小型非生物官能团。尽管它们很有吸引力，但由于细胞衍生基质 (CDM) 的不溶性和高度复杂性，对其生物分子组成的研究非常具有挑战性。然而，对典型 ECM 特异性生物分子的整体材料组成、组织、定位和分布进行彻底的定性和定量分析对于 CDM 的持续发展和对已开发生物材料的预期功能的理解至关重要。在这项研究中，我们评估了分析复杂 CDM 的常用方法。十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和（免疫）组织化学染色方法与几种显微技术相结合被发现是高度合格的。商业上可用的比色蛋白质分析结果证明提供的 CDM 信息不准确。相比之下，我们通过元素分析确定了 CDM 的氮含量，并使用从匹配的氨基酸组成计算的转换因子将其转换为总蛋白质含量。基于羟脯氨酸含量评估不溶性胶原蛋白的量。Sircol™ 测定法被确定为定量可溶性胶原蛋白的合适方法，而 Blyscan™ 测定法被发现非常适合硫酸化糖胺聚糖 (sGAG) 的定量。最终，我们提出了一系列合适的方法来可靠地表征成纤维细胞衍生的 clickECM 的生物分子组成。