3D bioprinted osteocytes expressing Wnt7b protect osteoblast differentiation from microgravity

Maintaining bone formation in microgravity/weightless environments remains a major challenge. In weightlessness, osteocytes act as mechanosensors to inhibit Wnt canonical signaling and bone formation by secreting sclerostin. This study explores whether osteocytic Wnt7b can counteract microgravity-induced bone loss through Wnt non-canonical signaling. Unlike previous bioprinting focusing on structural scaffolds or generic cell types, a novel bioprinted scaffold consisting of polycaprolactone (supportive) and osteocyte (functional) hydrogels was constructed. Osteocytes overexpressing Wnt7b were co-cultured with bone marrow stromal cells (ST2) in a 3D biomimetic weightless bio-microenvironmental system (3D-BWBM) to assess osteogenic and lipogenic differentiation. The results showed that osteocytic Wnt7b enhanced osteogenic differentiation and mineralization of ST2 cells via the Wnt non-canonical pathway PKCδ, while suppressing the expression of lipogenic markers (Pparg, Cebpa) and adipogenesis. RT-qPCR analysis showed elevated expression of Sost and Mef2C, down-regulation of the Wnt target gene Opg, and elevated expression of pro-osteoclastogenic cytokine RANKL and pro-inflammatory cytokines TNFα and IL-1β, thus validating the microgravity effect. Unlike conventional 2D culture of RCCS™ cells, the 3D hydrogels were printed with tunnels (500 μm) for efficient nutrient/metabolite exchange, resulting in good cell growth, high cell viability (97%), and a 6-fold increase in proliferative activity within 7 days. Wnt7b osteocytes were still able to maintain the osteogenic differentiation of ST2 cells as evidenced by elevated alkaline phosphatase activity, mineralization (1.8-fold increase), and a decrease in osteoblast marker genes (Alpl, Runx2, Col1a1). In conclusion, Wnt7b-PCKδ signaling counteracts microgravity-induced bone loss, and future in vivo osteocytic Wnt7b studies will confirm this causal relationship.