The healing of bone fractures is a well-orchestrated physiological process involving multiple cell types and signaling molecules interacting at the fracture site to replace and repair bone tissue without scar formation. However, when the lesion is too large, normal healing is compromised. These so-called non-union bone fractures, mostly arising due to trauma, tumor resection or disease, represent a major therapeutic challenge for orthopedic and reconstructive surgeons. In this review, we firstly present the current commonly employed surgical strategies comprising auto-, allo-, and xenograft transplantations, as well as synthetic biomaterials. Further to this, we discuss the multiple factors influencing the effectiveness of the reconstructive therapy. One essential parameter is adequate vascularization that ensures the vitality of the bone grafts thereby supporting the regeneration process, however deficient vascularization presents a frequently encountered problem in current management strategies. To address this challenge, vascularized bone grafts, including free or pedicled fibula flaps, or in situ approaches using the Masquelet induced membrane, or the patient's body as a bioreactor, comprise feasible alternatives. Finally, we highlight future directions and novel strategies such as 3D printing and bioprinting which could overcome some of the current challenges in the field of bone defect reconstruction, with the benefit of fabricating personalized and vascularized scaffolds.

Although autografts are considered to be the gold standard treatment for reconstruction of large bone defects resulting from trauma or diseases, donor site morbidity and limited availability restrict their use. Successful bone repair also depends on sufficient vascularization and to address this challenge, novel strategies focus on the development of vascularized biomaterial scaffolds. This pilot study aimed to investigate the feasibility of regenerating large bone defects in sheep using 3D-printed customized calcium phosphate scaffolds with or without surgical vascularization. Pre-operative computed tomography scans were performed to visualize the metatarsus and vasculature and to fabricate customized scaffolds and surgical guides by 3D printing. Critical-sized segmental defects created in the mid-diaphyseal region of the metatarsus were either left empty or treated with the 3D scaffold alone or in combination with an axial vascular pedicle. Bone regeneration was evaluated 1, 2 and 3 months postimplantation. After 3 months, the untreated defect remained non-bridged while the 3D scaffold guided bone regeneration. the presence of the vascular pedicle further enhanced bone formation. Histology confirmed bone growth inside the porous 3D scaffolds with or without vascular pedicle inclusion. Taken together, this pilot study demonstrated the feasibility of precised pre-surgical planning and reconstruction of large bone defects with 3D-printed personalized scaffolds. Bone is a dynamic tissue that possesses the intrinsic capacity to heal within 6-8 weeks after immobilization of a fracture. However, there are some conditions in which bone regeneration is delayed, compromised or beyond the physiological healing potential 1,2. Notably, the successful repair of large bone defects caused by trauma, tumor resection or disease remains a clinical challenge for orthopedic and plastic surgeons and often requires additional treatments. Autologous bone grafting is still considered the gold standard treatment due to its osteoconductive, osteoinductive and osteogenic properties. This procedure necessitates harvesting the patient's own bone and subsequently transplanting it to the defect site. Bone can be taken from several areas e.g., iliac crest or fibula, depending on the severity and amount needed for reconstruction of the defect. Nevertheless, the amount of

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel ‘off-the-shelf’ strategy for GBR.