Distinctive Roles of Wnt Signaling in Chondrogenic Differentiation of BMSCs under Coupling of Pressure and Platelet-Rich Fibrin

Background: Although newly formed constructs of feasible pressure-preadjusted bone marrow mesenchymal stem cells (BMSCs) and platelet-rich fibrin (PRF) showed biomechanical flexibility and superior capacity for cartilage regeneration, it is still not very clear how BMSCs and seed cells feel mechanical stimuli and convert them into biological signals, and the difference in signal transduction underlying mechanical and chemical cues is also unclear. Methods: To determine whether mechanical stimulation (hydrostatic pressure) and chemical cues (platelet-rich fibrin, PRF) activate canonical or noncanonical Wnt signaling in BMSCs, BMSCs cocultured with PRF were subjected to hydrostatic pressure loading, and the activation of the Wnt signaling molecules and expression of cartilage-associated proteins and genes were determined by western blotting and polymerase chain reaction (PCR). Inhibitors of canonical or noncanonical Wnt signaling, XVX-939 or L690,330, were adopted to investigate the role of Wnt signaling molecules in mechanically promoted chondrogenic differentiation of BMSCs. Results: Hydrostatic pressure of 120 kPa activated both Wnt/beta-catenin signaling and Wnt/Ca2+ signaling, with the the maximum promotion effect at 60 min. PRF exerted no synergistic effect on Wnt/beta-catenin signaling activation. However, the growth factors released by PRF might reverse the promotion effects of pressure on Wnt/Ca2+ signaling. Real-time PCR and Western blotting results showed that pressure could activate the expression of Col-II, Sox9, and aggrecan in BMSCs cocultured with PRF. Blocking experiment found a positive role of Wnt/beta-catenin signaling, and a negative role of Wnt/Ca2+ signaling in chondrogenic differentiation of the BMSCs. Mutual inhibition exists between canonical and noncanonical Wnt signaling in BMSCs under pressure. Conclusion: Wnt signaling participates in the pressure-promoted chondrogenesis of the BMSCs co-cultured with PRF, with canonical and noncanonical pathways playing distinct roles during the process.