Department of Biomedical Engineering, Illinois Institute of Technology, Chicago, IL, USA.
Department of Chemical and Biological Engineering, Illinois Institute of Technology, Chicago, IL, USA.
J Mater Chem B. 2020 Mar 25;8(12):2454-2465. doi: 10.1039/c9tb02356a.
Tissue response to intestinal injury or disease releases pro-inflammatory host stress signals triggering microbial shift to pathogenic phenotypes. One such phenotype is increased protease production resulting in collagen degradation and activation of host matrix metalloproteinases contributing to tissue breakdown. We have shown that surgical injury depletes local intestinal phosphate concentration triggering bacterial virulence and that polyphosphate replenishment attenuates virulence and collagenolytic activity. Mechanistic studies of bacterial and host protease expression contributing to tissue breakdown are difficult to achieve in vivo necessitating the development of novel in vitro tissue models. Common techniques for screening in vitro protease activity, including gelatin zymography or fluorogenic protease-sensitive substrate kits, do not readily translate to 3D matrix degradation. Here, we report the application of an in vitro assay in which collagenolytic pathogens are cultured in the presence of a proteolytically degradable poly(ethylene) glycol scaffold and a non-degradable phosphate and/or polyphosphate nanocomposite hydrogel matrix. This in vitro platform enables quantification of pathogen-induced matrix degradation and screening of sustained release of phosphate-based therapeutic efficacy in attenuating protease expression. To evaluate matrix degradation as a function of bacterial enzyme levels secreted, we also present a novel method to quantify hydrogel degradation. This method involves staining protease-sensitive hydrogels with Sirius red dye to correlate absorbance of the degraded gel solution with hydrogel weight. This assay enables continuous monitoring and greater accuracy of hydrogel degradation kinetics compared to gravimetric measurements. Combined, the proposed in vitro platform and the presented degradation assay provide a novel strategy for screening efficacy of therapeutics in attenuating bacterial protease-induced matrix degradation.
组织对肠道损伤或疾病的反应会释放促炎的宿主应激信号，从而触发微生物向致病性表型转变。其中一种表型是蛋白酶产量增加，导致胶原降解和宿主基质金属蛋白酶激活，从而导致组织破坏。我们已经表明，手术损伤会耗尽局部肠道磷酸盐浓度，引发细菌毒力，而多磷酸盐的补充可以减弱毒力和胶原酶活性。体内研究细菌和宿主蛋白酶表达对组织破坏的机制非常困难，因此需要开发新的体外组织模型。常用的体外蛋白酶活性筛选技术，包括明胶酶谱法或荧光蛋白酶敏感底物试剂盒，不易转化为 3D 基质降解。在这里，我们报告了一种体外测定方法的应用，该方法是在存在可被蛋白酶降解的聚（乙二醇）支架和不可降解的磷酸盐和/或多磷酸盐纳米复合水凝胶基质的情况下，培养胶原酶病原体。这种体外平台可以定量病原体诱导的基质降解，并筛选基于磷酸盐的治疗药物持续释放以减轻蛋白酶表达的疗效。为了评估基质降解作为细菌酶分泌水平的函数，我们还提出了一种定量水凝胶降解的新方法。该方法涉及用 Sirius red 染料对蛋白酶敏感的水凝胶进行染色，以将降解凝胶溶液的吸光度与水凝胶的重量相关联。与重量测量相比，该测定法可以更连续地监测和更准确地测定水凝胶降解动力学。总之，提出的体外平台和提出的降解测定法为筛选治疗药物减轻细菌蛋白酶诱导的基质降解的疗效提供了一种新策略。
