Pepsin Concentration Affects Structural, Physico-Chemical, Mechanical, and Biological Properties of Decellularized...

Decellularized extracellular matrix (dECM) has emerged as a promising material for tissue engineering and regenerative medicine (TERM), particularly for hydrogel development. Although dECM offers unmatched biochemical complexity compared to collagen type I (Coll-I), the most abundant protein in dECM and widely used in TERM, it is often reported that extraction processing-particularly pepsin digestion-compromises protein integrity and functional performance. Digestion protocols typically use 1 mg/mL pepsin and a 10:1 dECM/pepsin weight ratio, potentially leading to excessive degradation of structural proteins and diminished biological performance. This study investigated the effects of reduced pepsin concentrations (0.75 and 0.5 mg/mL) on structural, physico-chemical, mechanical, and biological features of porcine pericardium dECM hydrogels (10 mg/mL), compared to Coll-I. Compared to the standard digestion protocol, lower pepsin levels significantly improved the preservation of collagen's secondary structure and thermal stability (from 317°C ± 4°C to 324°C ± 1°C). Hydrogels digested with 0.5 mg/mL pepsin exhibited an elastic modulus of 119 ± 12 kPa, 2-3 times higher than standard dECM (65 ± 6 kPa) and Coll-I (41 ± 2 kPa), and a storage modulus of 1.5 ± 0.1 kPa, approximately twice that of the standard dECM (0.8 ± 0.1 kPa) and Coll-I (0.7 ± 0.1 kPa) hydrogels. Human dermal fibroblasts showed enhanced adhesion and over twice the viability on these optimized hydrogels. Despite dECM's intrinsic compositional advantages, its performance can be diminished by harsh digestion. Our findings highlight pepsin concentration as a critical and tunable parameter that governs the mechanical integrity and cellular responses of dECM hydrogels. Optimizing this variable enables the development of more robust, bioactive, and scalable dECM hydrogels tailored for TERM applications.