Porcine Lung-Derived Extracellular Matrix Hydrogel Properties Are Dependent on Pepsin Digestion Time

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)in vitro. 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. Impact statement Extracellular matrix (ECM) hydrogels, which offer unique material advantages, have been developed to model the ECMin vitro, to improve tissue-engineered and biomanufactured scaffolds, and directly adapted as clinical therapiesin vivo. Standardized enzymatic digestion protocols have supported the rapid adoption of ECM hydrogels, however, contribution of specific digestion parameters has been overlooked. Using pig lung decellularized ECM as a model system, this research demonstrates digestion time-dependent effects on the physical, mechanical, and biological properties of the resulting hydrogels. Importantly, this highlights the possibility that similar evaluations could yield significant and impactful performance benefits if applied for individual tissues and applications.