Regenerative bio-scaffolds, widely used for clinical tissue reconstruction and tissue repairs, are functionally diversified and structurally complex decellularized tissue materials (e.g., extracellular matrix, ECM). ECM is naturally cross-linked and can be further selectively cross-linked upon processing. Identification, quantification and bioinformatics functional comparison of all ECM proteins are challenging for regenerative bio-scaffolds. In this study, we have applied proteomic profiling with a two-step sequential trypsinization method, and identified and quantified 300-400 constituent proteins in three commercially available regenerative bio-scaffolds (BioDesign Surgisis, ReGen tissue matrix, and ThormalGEN mesh). These proteins were classified into four categories and 14 subcategories based on their mainly biological function. The main components of regenerative bio-scaffolds were highly abundant ECM structural proteins, and the minor parts of bio-scaffolds were lowly abundant, less cross-linked, functionally more diversified proteins, especially extracellular fluid proteins that were easily solubilized by trypsin. The comparative analysis has revealed large differences in the number, type, abundance and function of identified proteins, as well as the extent of decellularization and cross-linking among regenerative bio-scaffolds. So, the proteomic profiling with a two-step sequential trypsinization method could not only provide the molecular basis to better understand the degradation process of regenerative bio-scaffolds in vivo and different clinical outcomes among various regenerative bio-scaffolds, facilitate the exploration of the response mechanisms in the host's early clinical stages of ECM-induced tissue regeneration that is still poorly understood, but also can be used for optimization of the decellularization and crosslinking process, product characterization and rational design of new ECM products.decellularization, extracellular matrix protein, proteomic analysis, regenerative bio-scaffold
| INTRODUCTIONRegenerative bio-scaffolds, now widely used for human soft tissue repair and regeneration, are the acellular extracellular matrices (ECM) of allogeneic and xenogeneic tissues or organs. These bio-scaffolds are naturally cross-linked, and can be further selectively cross-linked during processing to reduce immunogenicity, enhance stability and improve mechanical strength. The functionally diversified and structurally complex ECM protein networks in regenerative bio-scaffolds not only have excellent structural properties, but also contain many bio-active substances (such as collagen, elastin, proteoglycan, fibronectin, laminin, and a variety of cell growth factors, cytokines, etc.) that are involved in the regulation of cell adhesion, migration, proliferation and differentiation to guide the repairing process and functional