3D-bioprinted osteocytes expressing Wnt7b protect osteoblast differentiation from microgravity

Abstract

Maintaining bone formation in microgravity/weightless environments remains a major challenge. Under weightless conditions, 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 studies that focused on structural scaffolds or generic cell types, a novel bioprinted scaffold consisting of polycaprolactone (supportive) and osteocyte (functional) hydrogels was constructed in this study. Osteocytes overexpressing Wnt7b were co-cultured with bone marrow stromal cells (ST2) in a 3D biomimetic weightless biomicroenvironmental system (3D-BWBM) to assess osteogenic and lipogenic differentiation. The results indicated 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. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed elevated expression of Sost and Mef2c, downregulation of the Wnt target gene Opg, and elevated expression of pro-osteoclastogenic cytokine Rankl and pro-inflammatory cytokines Tnfa and Il1b, 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 six-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 further in vivo studies on osteocytic Wnt7b are warranted to confirm this causal relationship.

Keywords

3D bioprinting / Microgravity / Osteogenic differentiation / Wnt7b / Wnt noncanonical signaling

Funding

This work was supported by the National Natural Science Foundation of China (grant numbers: 82471909, 81672118, and 32101053) and the Chongqing Natural Science Foundation (grant numbers: CSTB2022NSCQ-LZX0048 and CSTB2023NSCQ-MSX0424).

Conflict of interest

The authors declare they have no competing interests.