Long-term tissue engineered periosteum-mediated allograft healing is hindered due to persistent fibrosis and limited allograft remodeling.

Decellularized bone allografts are used in approximately 1/3 of grafting procedures and are preferred in treating critical-size bone defects, as volumetric constraints limit autografts. However, allografts demonstrate high failure rates, with 60 % of allografts failing within 10-years post-implantation. Allograft failure is linked to poor graft integration, which directly results from lack of periosteum, which surrounds bone and is necessary for successful bone healing. Therefore, a tissue-engineered periosteum (TEP) is a promising approach to recapitulate the missing periosteum and promote allograft healing. We have systematically developed an enzymatically degradable poly(ethylene glycol) (PEG) hydrogel with encapsulated mouse mesenchymal stem cells and osteoprogenitor cells, recapitulating key periosteal paracrine signals and producing improvements in bone allograft healing. While successful TEP-mediated allograft healing has been observed, previous studies have been limited to short-term healing (up to 16-weeks), which has yet to enable the observation of TEP-modified allograft healing resolution. To this end, this study extended evaluation of allograft healing in a murine femur defect model up to 12-months post-implantation. TEP-modified allografts demonstrated improvements in key bone healing outcomes, including graft vascularization and bone callus formation, at early time points (up to 9-weeks post-implantation), but improvements in healing outcomes compared to unmodified allografts were lost after 4-months post-implantation. In addition, unmodified allografts displayed incomplete healing up to 12-months post-implantation, with significant fibrotic tissue, incomplete graft remodeling, and inferior biomechanical strength observed. Given these results, future TEP designs should support long-term healing and graft remodeling to promote resolution of TEP-mediated graft healing in a clinically relevant timeline.