Engineering bone-forming biohybrid sheets through the integration of melt electrowritten membranes and cartilaginous microspheroids.

Bone fractures are one of the most common traumatic large-organ injuries and although many fractures can heal on their own, 2-12% of fractures are slow healing or do not heal (non-unions). Autologous grafts are currently used for treatment of non-unions but are associated with limited healthy bone tissue. Tissue engineered cell-based products have promise for an alternative treatment method. It was previously demonstrated that cartilaginous microspheroids of periosteum-derived cells could be assembled into scaffold-free constructs and heal murine critically-sized long bone defects (non-unions). However, the handleability of such scaffold-free implants can be compromised when scaling-up. In this work, cartilaginous spheroids were combined with melt electrowritten (MEW) meshes to create an engineered cell-based implant, able to induce in vivo bone formation. MEW polycaprolactone meshes were tailored to contain pores (116 ± 28 µm) of a size that captured microspheroids (180 ± 15 µm). Periosteum-derived microspheroids pre-cultured for 4 days, were seeded on MEW meshes and gene expression analysis demonstrated up-regulation of chondrogenic (SOX9, COL2) and prehypertrophic (VEGF) gene markers after 14 days, creating a biohybrid sheet. When implanted subcutaneously (4 weeks), the biohybrid sheets mineralized (23 ± 3% MV/TV) and formed bone and bone marrow. Bone formation was also observed when implanted in a murine critically-sized long bone defect, though a high variation between samples was detected. The high versatility of this biofabrication approach lies in the possibility to tailor the scaffolds to shape and dimensions corresponding to the large bone defects and the individual patient using robust bone forming building blocks. These strategies are instrumental in the development of personalized regenerative therapies with predictive clinical outcomes. STATEMENT OF SIGNIFICANCE: Successful treatments for healing of large long bone defects are still limited and 2-12% of fractures do not heal properly. We combined a novel biofabrication technique: melt electrowriting (MEW), with robust biology: bone forming cartilaginous spheroids to create biohybrid sheets able to form bone upon implantation. MEW enabled the fabrication of scaffolds with micrometer-sized fibers in defined patterns which allowed the capturing of and merging with cartilaginous spheroids which had the potency to mature into bone via the developmental process of endochondral ossification. The present study contributes to the rapidly growing field of "Biofabrication with Spheroid and Organoid Materials'' and demonstrates design considerations that are of great importance for biofabrication of functional tissues through the assembly of cellular spheroids.