College of Medicine Pulmonary Department, University of Vermont, Burlington, Vermont, USA.
Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia, USA.
Tissue Eng Part C Methods. 2020 Jun;26(6):332-346. doi: 10.1089/ten.TEC.2020.0042. Epub 2020 Jun 9.
Hydrogels derived from decellularized lungs are promising materials for tissue engineering in the development of clinical therapies and for modeling the lung extracellular matrix (ECM) . Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM (dECM) after enzymatic solubilization and gelation are thus of key interest. As the role of enzymatic pepsin digestion in effecting these properties has been understudied, we investigated the digestion time-dependency on key parameters of the resulting ECM hydrogel. Using resolubilized, homogenized decellularized pig lung dECM as a model system, significant time-dependent changes in protein concentration, turbidity, and gelation potential were found to occur between the 4 and 24 h digestion time points, and plateauing with longer digestion times. These results correlated with qualitative scanning electron microscopy images and quantitative analysis of hydrogel interconnectivity and average fiber diameter. Interestingly, the time-dependent changes in the storage modulus tracked with the hydrogel interconnectivity results, while the Young's modulus values were more closely related to average fiber size at each time point. The structural and biochemical alterations correlated with significant changes in metabolic activity of several representative lung cells seeded onto the hydrogels with progressive decreases in cell viability and alterations in morphology observed in cells cultured on hydrogels produced with dECM digested for >12 and up to 72 h of digestion. These studies demonstrate that 12 h pepsin digest of pig lung dECM provides an optimal balance between desirable physical ECM hydrogel properties and effects on lung cell behaviors.
脱细胞肺衍生水凝胶是组织工程中极具前景的材料，可用于临床治疗的开发以及模拟肺细胞外基质 (ECM)。因此，对脱细胞 ECM (dECM) 在酶解和凝胶化后产生的物理、生化、机械和生物学特性进行表征和控制是至关重要的。由于胃蛋白酶消化在这些特性中的作用尚未得到充分研究，因此我们研究了酶解时间对 ECM 水凝胶关键参数的影响。我们使用可再溶解的、均质的脱细胞猪肺 dECM 作为模型系统，发现蛋白质浓度、浊度和凝胶化潜力在 4 至 24 小时的消化时间点之间存在显著的时间依赖性变化，并在更长的消化时间内达到稳定。这些结果与定性扫描电子显微镜图像以及水凝胶连通性和平均纤维直径的定量分析相关。有趣的是，储能模量随水凝胶连通性结果的时间依赖性变化而变化，而杨氏模量值与每个时间点的平均纤维尺寸更密切相关。结构和生化变化与几种代表性肺细胞在水凝胶上的代谢活性的显著变化相关，随着消化时间的延长，细胞活力逐渐降低，在消化时间超过 12 小时至 72 小时的 dECM 消化产物上培养的细胞形态发生改变。这些研究表明，猪肺 dECM 的 12 小时胃蛋白酶消化提供了理想的物理 ECM 水凝胶特性与对肺细胞行为影响之间的平衡。
