CHIR99021-Treated Osteocytes with Wnt Activation in 3D-Printed Module Form an Osteogenic Microenvironment for Enhanced Osteogenesis and Vasculogenesis

Finding a bone implant that has high bioactivity that can safely drive stem cell differentiation and simulate a real in vivo microenvironment is a challenge for bone tissue engineering. Osteocytes significantly regulate bone cell fate, and Wnt-activated osteocytes can reversely regulate bone formation by regulating bone anabolism, which may improve the biological activity of bone implants. To achieve a safe application, we used the Wnt agonist CHIR99021 (C91) to treat MLO-Y4 for 24 h, in a co-culture with ST2 for 3 days after withdrawal. We found that the expression of Runx2 and Osx increased, promoted osteogenic differentiation, and inhibited adipogenic differentiation in the ST2 cells, and these effects were eliminated by the triptonide. Therefore, we hypothesized that C91-treated osteocytes form an osteogenic microenvironment (COOME). Subsequently, we constructed a bio-instructive 3D printing system to verify the function of COOME in 3D modules that mimic the in vivo environment. Within PCI3D, COOME increased the survival and proliferation rates to as high as 92% after 7 days and promoted ST2 cell differentiation and mineralization. Simultaneously, we found that the COOME-conditioned medium also had the same effects. Therefore, COOME promotes ST2 cell osteogenic differentiation both directly and indirectly. It also promotes HUVEC migration and tube formation, which can be explained by the high expression of Vegf. Altogether, these results indicate that COOME, combined with our independently developed 3D printing system, can overcome the poor cell survival and bioactivity of orthopedic implants and provide a new method for clinical bone defect repair.