PROJECT SUMMARY Repair of large bone defects remains a significant clinical challenge. While autologous bone graft is still considered the gold standard for most applications, it is limited by morbidity at the donor site and challenges associated with preparing anatomically-shaped grafts from harvested bone. Bone tissue engineering is considered a promising alternative, with two of the most widely-studied tissue engineering approaches being biomaterials-mediated exogenous stem/progenitor cells transplantation (e.g., bone marrow mesenchymal stem cells, BMSCs) and growth factors/hormones delivery (e.g., bone morphogenetic protein, BMPs). These approaches, BMPs in particular, have received widespread attention for their potential therapeutic use as stimulators of bone repair. FDA-approved BMP2 and BMP7, used successfully in the treatment of bone repair, recruits and induces endogenous stem/progenitor cells for osteogenic differentiation. BMP-based therapy, however, has been significantly impeded in clinical practice due to several critical barriers: high dose, high costs, and serious side effects. Accordingly, we aim to: (1) develop a novel 3D nanofibrous (NF) scaffold that can modulate both endogenous BMP and angiogenic signals; and (2) promote bone repair by a functionalized scaffold in a critical-size mouse cranial defect model. Our central hypothesis is that, without the addition of any exogenous cells or growth factors, biomimetic gelatin NF scaffold can improve in situ endogenous bone regeneration. Functionalization of essential components for bone formation will be accomplished via: (1) BMP binding peptide (BBP) for selectively capturing and stimulating endogenous BMPs from osseous injury sites to induce local bone formation; and (2) desferrioxamine (DFO) for mimicking temporary hypoxia, thereby triggering angiogenesis/neovascularization/osteogenesis and reparative cell recruitment. Our previous work demonstrates that a 3D porous, biodegradable NF scaffold is advantageous in tissue regeneration. Additional osteogenic signals (e.g., BMPs/osteoprogenitors), however, are still required and must be supplemented in NF scaffold for bone regeneration. In Aim 1, we will immobilize BBP and DFO to NF scaffold by chemical crosslinking and nanosphere incorporation, respectively. The bioactive functions of modified scaffolds will be studied by measuring BMP-2 binding, and BMP-2/VEGF expression using an in vitro cell culture model. In Aim 2, we will investigate the contributions of immobilized BBP and DFO (alone and synergistically) to bone formation and angiogenesis, using a critical-size mouse cranial defect model. This work will advance knowledge and therapeutic translation by exploring the use of the BBP peptide/DFO immobilized biomaterials for challenged bone repair.