Schneider Margaret C, Barnes Christopher A, Bryant Stephanie J
Department of Chemical and Biological Engineering, University of Colorado, 3415 Colorado Ave, Campus Box 596, Boulder 80309, Colorado.
Biofrontiers Institute, University of Colorado, Boulder, Colorado.
Biotechnol Bioeng. 2017 Sep;114(9):2096-2108. doi: 10.1002/bit.26320. Epub 2017 May 12.
Poly(ethylene glycol) (PEG) hydrogels are highly tunable platforms that are promising cell delivery vehicles for chondrocytes and cartilage tissue engineering. In addition to characterizing the type of extracellular matrix (ECM) that forms, understanding the types of proteins that are secreted by encapsulated cells may be important. Thus, the objectives for this study were to characterize the secretome of chondrocytes encapsulated in PEG hydrogels and determine whether the secretome varies as a function of hydrogel stiffness and culture condition. Bovine chondrocytes were encapsulated in photoclickable PEG hydrogels with a compressive modulus of 8 and 46 kPa and cultured under free swelling or dynamic compressive loading conditions. Cartilage ECM deposition was assessed by biochemical assays and immunohistochemistry. The conditioned medium was analyzed by liquid chromatography-tandem mass spectrometry. Chondrocytes maintained their phenotype within the hydrogels and deposited cartilage-specific ECM that increased over time and included aggrecan and collagens II and VI. Analysis of the secretome revealed a total of 64 proteins, which were largely similar among all experimental conditions. The identified proteins have diverse functions such as biological regulation, response to stress, and collagen fibril organization. Notably, many of the proteins important to the assembly of a collagen-rich cartilage ECM were identified and included collagen types II(α1), VI (α1, α2, and α3), IX (α1), XI (α1 and α2), and biglycan. In addition, many of the other identified proteins have been reported to be present within cell-secreted exosomes. In summary, chondrocytes encapsulated within photoclickable PEG hydrogels secrete many types of proteins that diffuse out of the hydrogel and which have diverse functions, but which are largely preserved across different hydrogel culture environments. Biotechnol. Bioeng. 2017;114: 2096-2108. © 2017 Wiley Periodicals, Inc.
聚乙二醇（PEG）水凝胶是高度可调节的平台，有望成为软骨细胞和软骨组织工程的细胞递送载体。除了表征所形成的细胞外基质（ECM）的类型外，了解被封装细胞分泌的蛋白质类型可能也很重要。因此，本研究的目的是表征封装在PEG水凝胶中的软骨细胞的分泌蛋白组，并确定分泌蛋白组是否随水凝胶硬度和培养条件而变化。将牛软骨细胞封装在压缩模量为8和46 kPa的可光交联PEG水凝胶中，并在自由膨胀或动态压缩加载条件下培养。通过生化分析和免疫组织化学评估软骨ECM沉积。通过液相色谱 - 串联质谱法分析条件培养基。软骨细胞在水凝胶中保持其表型，并沉积随时间增加的软骨特异性ECM，包括聚集蛋白聚糖、II型和VI型胶原蛋白。对分泌蛋白组的分析共鉴定出64种蛋白质，在所有实验条件下这些蛋白质在很大程度上是相似的。所鉴定的蛋白质具有多种功能，如生物调节、应激反应和胶原纤维组织。值得注意的是，鉴定出了许多对富含胶原蛋白的软骨ECM组装很重要的蛋白质，包括II（α1）、VI（α1、α2和α3）、IX（α1）、XI（α1和α2）型胶原蛋白和双糖链蛋白聚糖。此外，许多其他鉴定出的蛋白质已被报道存在于细胞分泌的外泌体中。总之，封装在可光交联PEG水凝胶中的软骨细胞分泌多种类型的蛋白质，这些蛋白质扩散出水凝胶并具有多种功能，但在不同的水凝胶培养环境中基本保持不变。生物技术与生物工程。2017；114：2096 - 2108。©2017威利期刊公司。
