Search

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. [ABSTRACT FROM AUTHOR]

Finding and constructing an osteogenic microenvironment similar to natural bone tissue has always been a frontier topic in orthopedics. We found that osteocytes are targeting cells controlling bone anabolism produced by PTH (JBMR 2017, PMID: 27704638), and osteocytes with activated Wnt signaling orchestrate bone formation and resorption (PNAS 2015, PMID: 25605937). However, methods for taking advantage of the leading role of osteocytes in bone regeneration remain unexplored. Herein, we found that the osteocytes with SKL2001-activated Wnt signaling could be an osteogenic microenvironment (SOOME) which upregulates the expression of bone transcription factor Runx2 and Bglap and promotes the differentiation of bone marrow stromal cell ST2 into osteoblasts. Interestingly, 60 μM SKL2001 treatment of osteocytic MLO-Y4 for 24 h maintained Wnt signaling activation for three days after removal, which was sufficient to induce osteoblast differentiation. Triptonide, a Wnt inhibitor, could eliminate this differentiation. Moreover, on day 5, the Wnt signaling naturally decreased to the level of the control group, indicating that this method of Wnt-signaling induction is safe to use. We quickly verified in vivo function of SOOME to a good proximation in 3D bioprinted modules composed of reciprocally printed polycaprolactone bundles (for support) and cell bundles (for bioactivity). In the cell bundles, SOOME stably supported the growth and development of ST2 cells, the 7-day survival rate was as high as 91.6%, and proliferation ability increased linearly. Similarly, SOOME greatly promoted ST2 differentiation and mineralization for 28 days. In addition, SOOME upregulated the expression of angiopoietin 1, promoted endothelial cell migration and angiogenesis, and increased node number and total length of tubes and branches. Finally, we found that the function of SOOME could be realized through the paracrine pathway. This study reveals that osteocytes with Wnt signaling activated by SKL2001 are a safe osteogenic microenvironment. Both SOOME itself and its cell-free culture supernatant can improve bioactivity for osteoblast differentiation, with composite scaffolds especially bearing application value. [ABSTRACT FROM AUTHOR]