Elucidating Mechanisms of Gelation, Fiber Assembly, and Stability of Injectable Decellularized Extracellular Matrix Biomaterials.

Decellularized extracellular matrix (dECM)-based biomaterials have been widely used for their applications in tissue engineering. In particular, pepsin digestion of dECM can be used to generate injectable forms, including ECM hydrogels as well as an intravascularly infusible ECM (iECM). However, fundamental materials characterization of these materials has been limited, and thus little is known about what exactly drives gelation of ECM hydrogels or the conditions for fibril assembly and growth. With this study, we sought to answer a fundamental question on how these materials assemble or gel, as well as a translational question on what storage conditions are suitable for these materials. Here, we used second-harmonic generation and transmission electron microscopy to investigate the mechanism of gelation for ECM hydrogels and the nanofibril assembly of the iECM. Overall, these microscopies revealed the origin and morphology of self-assembly and that type I collagen lateral and longitudinal growth drives ECM hydrogel formation. On the contrary, the iECM preserved the same mechanism for nanofiber assembly without gelation. In terms of translation, ensuring the stability after rehydration is critical for therapeutic injection timing since changes in the material could impact both safety and efficacy. Via microscopy in conjunction with bulk material characterization, we found that dECM formulations are best kept at 4°C for a maximum of 24 h after rehydration in order to maintain their original properties. Overall, this work provides evidence for the type I collagen directed self-assembly within heterogeneous, injectable, decellularized ECM biomaterials and also determines clinically relevant material storage conditions.