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To emulate periosteum\u2010mediated healing of live autografts, we have developed a tissue\u2010engineered periosteum (TEP) to improve allograft healing. The TEP comprises cell\u2010degradable 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\u2010localized vascular volume and callus bridging. Therefore, hydrogel biochemical cues were incorporated into TEP to enable cell\u2013matrix 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\u00a0vitro 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\u2010functionalization and GFOGER\u2010functionalization maximized in\u00a0vitro cell infiltration. RGD and GFOGER hydrogels were then investigated in\u00a0vivo as TEP (RGD\u2010TEP and GFOGER\u2010TEP, respectively) to evaluate the effect of hydrogel functionalization on TEP\u2010mediated allograft healing in a murine femur defect model. RGD\u2010 and GFOGER\u2010TEP promoted bone graft healing, with both groups exhibiting a 1.9\u2010fold increase in bone callus volume over unmodified allografts at 3\u2009weeks post\u2010implantation. RGD\u2010TEP promoted more significant bone tissue development, but GFOGER\u2010TEP promoted greater torsional biomechanics over time. The few differences observed between TEP groups suggest hydrogel functionalization has a limited effect on TEP\u2010mediated 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\u2010mediated bone allograft healing.<\/jats:p>","DOI":"10.1002\/jbm.a.37890","type":"journal-article","created":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T06:39:57Z","timestamp":1741761597000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Optimizing Tissue\u2010Engineered Periosteum Biochemical Cues to Hasten Bone Allograft Healing"],"prefix":"10.1002","volume":"113","author":[{"given":"Alyson","family":"March","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering University of Rochester  Rochester New York USA"},{"name":"Center for Musculoskeletal Research University of Rochester Medical Center  Rochester New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Wu","sequence":"additional","affiliation":[{"name":"Institute of Optics University of Rochester  Rochester New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Regine","family":"Choe","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering University of Rochester  Rochester New York USA"},{"name":"Department of Electrical and Computer Engineering University of Rochester  Rochester New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danielle S. 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