A novel bioprinting platform and its applications in bone tissue engineering and vascularization
Keywords: Mechanical Engineering.

Of all the clinical treatments that have been conducted for bone fractures and segmental bone defects, bone grafts are widely considered the most promising. Bone grafts, such as autografts, allografts, and artificial bone grafts, are typically matched to the appropriate clinical scenario based on a variety of factors, but autografts are considered the gold standard. Autografts come with an osteoconductive scaffold, osteoinductive growth factors, and osteogenic cells, all key mechanisms necessary for bone healing. Allografts lack osteogenic cells, and artificial bone grafts only provide an osteoconductive scaffold for mechanical support. However, even autografts have their limitations. They cannot treat critical-sized bone defects - around 4 to 5 cm for humans - due to insufficient oxygen and nutrient delivery after transplantation. Further drawbacks include substantial donor site morbidity and technically demanding surgical technique. These limitations in current treatments motivated us to develop a vascularized tissue engineered graft (TEG) that has an osteoconductive scaffold, osteoinductive growth factors, and osteogenic cells. Delivery of oxygen and nutrients mainly rely on the transmission of blood, so achieving vascularization in our tissue engineered graft was key. We proposed a prevascularized tissue engineered model constructed with three components: a synthetic bone scaffold for mechanical support, a major vessel branch for perfusion, and a cell-laden hydrogel for angiogenesis. To fabricate our design, we developed a custom bioprinting platform, Hybprinter, to integrate three modules: molten material extrusion (MME), digital light-based stereolithography (DLP-SLA), and syringe-based micro-extrusion (SBM). The bone scaffold is 3D printed via MME, the major vessel branch by DLP-SLA, and the cell-laden hydrogel by SBM. Our Hybprinter enables seamless integration between a hard bone scaffold and a soft hydrogel, and this hybrid construct has many potential tissue engineering applications. To form the major vessel branch and the vascular bed, we developed a dual-hydrogel system to combine a sustainable polyethylene glycol dimethacrylate (PEGDMA) channel and a gelatin methacrylate (GelMA) 3D extracellular matrix (ECM). With dense human umbilical vein endothelial cells (HUVECs) layers seeded in between the perfusable channel and the ECM, the HUVECs differentiated and sprouted into the ECM, while the perfusable channel maintained its functionality throughout a 14-day incubation period. We also perfused fluorescent dye to visualize the enhanced diffusion under cell-laden conditions. We demonstrated that the functionally graded scaffold (FGS) printed by Hybprinter can be an improved treatment for early stage osteonecrosis of the femoral head (ONFH). Our bone scaffold insertion at the femoral head resulted in a higher bone in-growth rate than for core decompression, the current standard treatment. In addition, the injection of bone marrow-derived mononuclear cells (BMMCs) to the FGS decrease the osteonecrotic area within the femoral head. The combination of FGS and BMMCs has the potential to improve the clinical outcome for early stage ONFH. In conclusion, we demonstrated a strategy to 3D print a TEG composed of an osteoconductive scaffold, a major vessel branch, and a cell-laden hydrogel. The challenges of manufacturing and vascularization were addressed, and the possible solutions were proposed