A Comprehensive In Vitro Approach for Assessing the Physiological Degradation of Medical Implants: The Silkothane Arteriovenous Graft Case Study

Current in vitro degradation methodologies for absorbable medical devices often employ nonphysiological enzyme concentrations or bacterial proteases, limiting their predictive value for in vivo performance. Here, a framework for assessing protein‐based device degradation is developed using a three‐layered silk fibroin/polyurethane (Silkothane) Arteriovenous Graft (SAG) as a case study. A mixture of human enzymes targeting silk fibroin, the degradable component of the graft, is selected and their activities balanced by adjusting concentrations, enabling reliable tracking of activity decay and determination of a buffer‐refresh frequency. The enzyme mixture, applied at physiological concentrations, is then used to digest the grafts for up to 26 weeks, with undigested samples serving as controls. Grafts are assessed for morphology, mass loss, mechanical properties, and molecular weight, while buffers are evaluated for morphology, cytotoxicity, and molecular weight. Mass loss measurements are corrected for enzyme adsorption onto the graft, revealing actual enzymatic degradation rates. Results demonstrate minimal degradation with preserved structural integrity, consistent with surface erosion and in qualitative agreement with published and unpublished in vivo data. This framework provides a reproducible, physiologically relevant methodology for assessing protein‐based absorbable devices, in a first step toward bridging regulatory requirements and biological relevance while reducing reliance on animal testing.