Engineering Shelf-Stable Affibody-Functionalized Hydrogels for Controlled Dual Release of Bone Morphogenetic Protein-2 and Interleukin-4

Abstract

Wound healing is a complex and dynamic process involving the coordinated activity of multiple proteins across three stages: inflammation, proliferation, and remodeling. In the event of traumatic bone fractures, aspects of the healing cascade may be disrupted, leading to non-unions and permanent disability. Current clinical strategies for bone fracture repair deliver the osteogenic growth factor bone morphogenetic protein-2 (BMP-2) in supraphysiological doses. However, large doses lead to non-localized release and ectopic bone growth, and a single exogenous protein fails to mimic the multifaceted nature of wound healing. This work addresses these limitations by developing affinity-based protein delivery platforms using affibodies—small protein binders—to independently control the release of the anti-inflammatory cytokine interleukin-4 (IL-4) alongside BMP-2 to modulate inflammation and bone regeneration.High- and low-affinity affibodies specific to BMP-2 or IL-4 were identified from a yeast surface display library via magnetic- and fluorescent-activated cell sorting. When conjugated into polyethylene glycol-maleimide (PEG-Mal) hydrogels, formulations with high-affinity protein-specific affibodies reduced the release of BMP-2 and IL-4. In vitro studies demonstrated that BMP-2-specific affibodies inhibited the osteogenic differentiation of C2C12 myoblasts, while IL-4-specific affibodies modulated macrophage polarization in THP-1 cells. Computational modeling predicted that BMP-2-specific affibodies interfere with BMP-2 cell receptor binding domains. A mathematical model was developed to determine factors impacting protein release from multi-component affibody-conjugated hydrogels. The model predicted that protein-specific affibody affinity and concentration tune protein release. The affibody-conjugated hydrogels were reinforced within polycaprolactone (PCL) tubular scaffolds, enhancing the hydrogels’ mechanical properties and enabling surgical implantation. Scaffold-reinforced affibody-conjugated hydrogels were lyophilized for extended shelf storage. Lyophilized hydrogels retained there mechanical and controlled release properties upon rehydration. In vivo studies in rats demonstrated that subcutaneously implanted affibody-conjugated hydrogels locally retained more BMP-2 over 21 days compared to affibody-free hydrogels. In a 6-mm femoral defect pilot study, bone bridging occurred in 3 out of 4 rats treated with affibody-conjugated hydrogels, compared to 1 out of 3 rats treated without affibodies. These findings underscore the potential of affibody-conjugated hydrogels as versatile platforms for independently controlled protein delivery, offering a promising strategy to emulate natural wound healing cascade and improving bone fracture repair outcomes.