Physical, Mechanical, and Bioactive Properties of Pig Lung-Derived ECM Hydrogels are Dependent on the Duration of Pepsin Digestion

Abstract

Extracellular matrix (ECM) hydrogels derived from decellularized lungs are promising materials for tissue engineering, in the development of clinical therapies, and for modeling the lung ECM in vitro. Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM after enzymatic solubilization and gelation is 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 ECM (dECM) as a model system, significant time-dependent alterations in the protein concentration, turbidity, and gelation potential occurred between 4 and 24 hours of digestion, plateauing at longer digestion times. These results correlated with qualitative SEM 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 translated to 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 greater than 12 and up to 72 hours of digestion. These studies demonstrate that 12hr structured pepsin digest of pig lung dECM provides an optimal balance between desirable physical dECM hydrogel properties and effects on lung cell behaviors.