Abstract
Despite serving as the clinical "gold standard" treatment for critical size bone defects, decellularized allografts suffer from long-term failure rates of ~60% due to the absence of the periosteum. Stem and osteoprogenitor cells within the periosteum orchestrate autograft healing through host cell recruitment, which initiates the regenerative process. To emulate periosteum-mediated healing, tissue engineering approaches have been utilized with mixed outcomes. While vascularization has been widely established as critical for bone regeneration, innervation was recently identified to be spatiotemporally regulated together with vascularization and similarly indispensable to bone healing. Notwithstanding, there are no known approaches that have focused on periosteal matrix cues to coordinate host vessel and/or axon recruitment. Here, we investigated the influence of hydrogel degradation mechanism, i.e. hydrolytic or enzymatic (cell-dictated), on tissue engineered periosteum (TEP)-modified allograft healing, especially host vessel/nerve recruitment and integration. Matrix metalloproteinase (MMP)-degradable hydrogels supported endothelial cell migration from encapsulated spheroids whereas no migration was observed in hydrolytically degradable hydrogels in vitro, which correlated with increased neurovascularization in vivo. Specifically, ~2.45 and 1.84-fold, and ~3.48 and 2.58-fold greater vessel and nerve densities with high levels of vessel and nerve co-localization was observed using MMP degradable TEP (MMP-TEP) -modified allografts versus unmodified and hydrolytically degradable TEP (Hydro-TEP)-modified allografts, respectively, at 3 weeks post-surgery. MMP-TEP-modified allografts exhibited greater longitudinal graft-localized vascularization and endochondral ossification, along with 4-fold and 2-fold greater maximum torques versus unmodified and Hydro-TEP-modified allografts after 9 weeks, respectively, which was comparable to that of autografts. In summary, our results demonstrated that the MMP-TEP coordinated allograft healing via early stage recruitment and support of host neurovasculature.