Optimizing Tissue‐Engineered Periosteum Biochemical Cues to Hasten Bone Allograft Healing

Although allografts remain the gold standard for treating critical-size bone defects, ~60% fail within 10 years of implantation. To emulate periosteum-mediated healing of live autografts, we have developed a tissue-engineered periosteum (TEP) to improve allograft healing. The TEP comprises cell-degradable poly(ethylene glycol) hydrogels encapsulating mouse mesenchymal stem cells and osteoprogenitor cells to mimic the periosteal cell population. Despite improvements in allograft healing, several limitations were observed using the TEP, specifically the modulation of host tissue infiltration and remodeling to support graft-localized vascular volume and callus bridging. Therefore, hydrogel biochemical cues were incorporated into TEP to enable cell-matrix interactions and remodeling critical for tissue infiltration. Adhesive peptide functionalization (RGD, YIGSR, and GFOGER) and enzymatic degradation rate (GPQGIWGQ, IPESLRAG, and VPLSLYSG) were screened using an in vitro 3D cell spheroid assay and design of experiments (DOE) to identify hydrogels that best supported tissue infiltration and integration. DOE analysis of various adhesive peptide combinations was used to optimize functionalization, revealing that individual RGD-functionalization and GFOGER-functionalization maximized in vitro cell infiltration. RGD and GFOGER hydrogels were then investigated in vivo as TEP (RGD-TEP and GFOGER-TEP, respectively) to evaluate the effect of hydrogel functionalization on TEP-mediated allograft healing in a murine femur defect model. RGD- and GFOGER-TEP promoted bone graft healing, with both groups exhibiting a 1.9-fold increase in bone callus volume over unmodified allografts at 3 weeks post-implantation. RGD-TEP promoted more significant bone tissue development, but GFOGER-TEP promoted greater torsional biomechanics over time. The few differences observed between TEP groups suggest hydrogel functionalization has a limited effect on TEP-mediated healing, with cell delivery via the TEP enough to improve bone regeneration. Future studies aim to investigate additional adhesive peptides with diverse combinations to identify potential synergies between adhesive peptides to promote TEP-mediated bone allograft healing.