Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pre-Gels

Abstract

Solubilized gel-forming decellularized extracellular matrix (dECM) is used in a wide range of research and translational applications due to its inherent bioactivity, which can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM.

In this study, pepsin, papain, α-amylase and collagenase were comparatively evaluated for their solubilization potential with porcine liver dECM. Maximum preservation of bioactive components and native dECM properties were used as decisive criteria for further in-depth investigations with emphasis on minimal destruction of protein structure and function to allow physical thermogelation at neutral pH. The solubilized dECM digests and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAGs content, proteomic composition, and growth factor profile.

This study highlights papain as a plant-based enzyme that can serve as a cost-effective and animal-free alternative to pepsin for the efficient solubilization of dECM with higher amounts of retained growth factors. The resulting homogenous dECM solution exhibited preserved thermally triggered gelation properties similar to pepsin digests, while the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. Viability and proliferation of human HepaRG cells was similar on dECM and pure rat tail collagen type I gels. This highly effective plant-derived alternative for dECM solubilization is not just economically attractive, but is particularly interesting when digesting human tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Keywords: dECM, matrisome proteomics, digestion, fibrillogenesis, hydrogel