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Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate

机译：使用荧光肽底物直接测量3D细胞微环境中的基质金属蛋白酶活性

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Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated degradation in these materials has remained challenging. There are 20+ MMP family members whose activity is regulated in space and time by a number of biochemical and biophysical cues. Thus, the typical approach of characterizing cleavage of degradable moieties in solution with recombinant enzymes does not easily translate to three dimensional cell-mediated matrix remodeling. To address this challenge, we report here the synthesis of a cell-laden hydrogel matrix functionalized with a fluorogenic peptide substrate to provide real-time, quantitative monitoring of global MMP activity. Using this system, stimulation of MMP activity was observed with growth factor treatment in mammary epithelial cells and compared to classical zymography results. Further, the effect of biophysical cues on MMP activity of human mesenchymal stem cells was also investigated where more rigid hydrogels were observed to increase MMP activity. The regulation of MMP activity by these biochemical and biophysical cues highlights the need for in situ, real time measurement of hydrogel degradation, and use of these functionalized hydrogels will aid in future rational design of degradable synthetic hydrogels for in vitro cell studies and tissue engineering applications.
机译：将可降解部分结合到合成水凝胶中极大地提高了这些三维矩阵在体外细胞培养以及组织工程应用中的实用性。引入可降解性的常用方法是包含对基质金属蛋白酶（MMP）裂解敏感的寡肽，从而实现细胞介导的重塑和在材料中迁移。尽管这种策略很有效，但是在这些材料中表征和测量细胞介导的降解仍然具有挑战性。有20多个MMP家族成员，其活动在空间和时间上受到许多生化和生物物理线索的调节。因此，表征用重组酶在溶液中裂解可降解部分的典型方法不容易转化为三维细胞介导的基质重塑。为了应对这一挑战，我们在这里报告了用荧光肽底物功能化的载有细胞的水凝胶基质的合成，以提供实时，定量的全球MMP活性监测。使用该系统，在乳腺上皮细胞中用生长因子治疗观察到了MMP活性的刺激，并与经典酶谱学结果进行了比较。此外，还研究了生物物理线索对人间充质干细胞MMP活性的影响，其中观察到更多的刚性水凝胶可增加MMP活性。这些生化和生物物理线索对MMP活性的调节凸显了对原位，实时测量水凝胶降解的需求，这些功能化水凝胶的使用将有助于未来可降解合成水凝胶的合理设计，以用于体外细胞研究和组织工程应用。

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