Synergy of engineered gelatin methacrylate-based porous microspheres and multicellular assembly to promote osteogenesis and angiogenesis in bone tissue reconstruction.

One of the key challenges in bone defects treatment is providing adequate and stable blood supply during new tissue regeneration. Mesenchymal stem cells (MSCs) and endothelial cells (ECs) have great potential to promote osteogenesis and angiogenesis during bone defect repair through paracrine effects, but their therapeutic efficacy depends on effective cellular assembly and delivery. In this work, we developed various microspheres with different pore sizes for multi-cellular delivery to enhance the angiogenic and osteogenic capability via combining microfluidic and gradient freeze-drying techniques. The particle and pore size of fabricated porous gelatin methacrylate (GelMA)-based hydrogel microspheres (PGMS) could be controllable through adjusting the freezing time of hydrogel microspheres, the range of particles and pores size are 150-250 μm and 10-100 μm with different freezing time from 0 min to 30 min. The optimized particle size (200.8 ± 14.2 μm) and pore size (11.2 ± 1.9 μm) were explored to promote cell assemble, adhesion, growth, and proliferation in the PGMS. Furthermore, the co-assembly and delivery of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) on the PGMS was achieved and an optimal cellular ratio of BMSCs to HUVECs (20:2) was established for co-culturing of them to achieve optimal paracrine effects, further promoting osteogenic differentiation and angiogenesis. Finally, results from both in vitro and in vivo experiments showed that the developed PGMS with co-assembly of BMSCs to HUVECs contributed to accelerate bone regeneration and vascularization process daringly, exhibited great potential in vascularized bone tissue reconstruction.