Bridging the gap in pre-market immunological evaluation: a progressive strategy for recombinant collagen medical devices

Abstract Conventional immunotoxicological approaches are often insufficient to comprehensively elucidate the complex immune responses that may arise during the degradation, metabolite generation, and tissue integration of protein-based medical devices. To address this limitation, we established and applied a six-tiered, stepwise immunological evaluation framework in an appropriate murine model, aiming to systematically characterize the immunological risks of proteinaceous biomaterials. This framework sequentially incorporates baseline immune screening, marketed product comparison, interspecies differentiation, dose–response validation, temporal dynamics assessment, and delayed response detection. By integrating multiple dimensions of readouts, including antibody levels (IgG, IgM, IgA), cytokine profiles (IL-2, IL-6, IL-12p70, IFN-γ, TNF-α), complement activation (C3, C4, C5), immune cell phenotyping, and splenic lymphocyte functionality, the framework establishes a structured, quantifiable, and scalable evaluation pathway. Using recombinant human collagen for injectable fillers as a model material, we demonstrated that this strategy effectively distinguishes diverse immune response types and differentiates material-intrinsic effects from interspecies artifacts, thereby confirming its applicability and interpretive strength in immunological safety assessment of protein-based biomaterials. Collectively, the proposed six-tiered evaluation strategy not only overcomes the limitations of traditional single-parameter or short-term assessments, but also provides a scientifically rigorous and systematic methodology that can serve as a versatile tool for both immunological evaluation and design validation of protein-based and other complex biomacromaterials. Graphical Abstract