Background: The events of bone formation and osteoblast/titanium (Ti) interactions may be affected by Hedgehog and Notch signalling pathways. Herein, we investigated the effects of modulation of these signalling pathways on osteoblast differentiation caused by the nanostructured Ti (Ti-Nano) generated by H₂SO₄/H₂O₂. Methods: Osteoblasts from newborn rat calvariae were cultured on Ti-Control and Ti-Nano in the presence of the Hedgehog agonist purmorphamine or antagonist cyclopamine and of the Notch antagonist N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or agonist bexarotene. Osteoblast differentiation was evaluated by alkaline phosphatase activity and mineralization, and the expression of Hedgehog and Notch receptors was also evaluated. Results: In general, purmorphamine and DAPT increased while cyclopamine and bexarotene decreased osteoblast differentiation and regulated the receptor expression on both Ti surfaces, with more prominent effects on Ti-Nano. The purmorphamine and DAPT combination exhibited synergistic effects on osteoblast differentiation that was more intense on Ti-Nano. Conclusion: Our results indicated that the Hedgehog and Notch signalling pathways drive osteoblast/Ti interactions more intensely on nanotopography. We also demonstrated that combining Hedgehog activation with Notch inhibition exhibits synergistic effects on osteoblast differentiation, especially on Ti-Nano. The uncovering of these cellular mechanisms contributes to create strategies to control the process of osseointegration based on the development of nanostructured surfaces.

Background: Tissue engineering and cell therapy have been the focus of investigations on how to treat challenging bone defects. This study aimed to produce and characterize a P(VDF-TrFE)/BaTiO₃ scaffold and evaluate the effect of mesenchymal stem cells (MSCs) combined with this scaffold and photobiomodulation (PBM) on bone repair. Methods and results: P(VDF-TrFE)/BaTiO₃ was synthesized using an electrospinning technique and presented physical and chemical properties suitable for bone tissue engineering. This scaffold was implanted in rat calvarial defects (unilateral, 5 mm in diameter) and, 2 weeks post-implantation, MSCs were locally injected into these defects (n = 12/group). Photobiomodulation was then applied immediately, and again 48 and 96 h post-injection. The μCT and histological analyses showed an increment in bone formation, which exhibited a positive correlation with the treatments combined with the scaffold, with MSCs and PBM inducing more bone repair, followed by the scaffold combined with PBM, the scaffold combined with MSCs, and finally the scaffold alone (ANOVA, p ≤ 0.05). Conclusions: The P(VDF-TrFE)/BaTiO₃ scaffold acted synergistically with MSCs and PBM to induce bone repair in rat calvarial defects. These findings emphasize the need to combine a range of techniques to regenerate large bone defects and provide avenues for further investigations on innovative tissue engineering approaches.

Aim: We evaluated the bone repair induced by MSCs from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) injected into rat calvarial defects at two time points. Methods & results: Both cell popula... Plain language summary We evaluated the bone repair induced by cells that can develop into different types of cells (stem cells) derived from fat and spongy tissue inside the large bones and inject...

Bone formation is driven by many signaling molecules including bone morphogenetic protein 9 (BMP-9) and hypoxia-inducible factor 1-alpha (HIF-1α). We demonstrated that cell therapy using mesenchymal stem cells (MSCs) overexpressing BMP-9 (MSCs^+BMP-9) enhances bone formation in calvarial defects. Here, the effect of hypoxia on BMP components and targets of MSCs^+BMP-9 and of these hypoxia-primed cells on osteoblast differentiation and bone repair was evaluated. Hypoxia was induced with cobalt chloride (CoCl₂) in MSCs^+BMP-9, and the expression of BMP components and targets was evaluated. The paracrine effects of hypoxia-primed MSCs^+BMP-9 on cell viability and migration and osteoblast differentiation were evaluated using conditioned medium. The bone formation induced by hypoxia-primed MSCs^+BMP-9 directly injected into rat calvarial defects was also evaluated. The results demonstrated that hypoxia regulated BMP components and targets without affecting BMP-9 amount and that the conditioned medium generated under hypoxia favored cell migration and osteoblast differentiation. Hypoxia-primed MSCs^+BMP-9 did not increase bone repair compared with control MSCs^+BMP-9. Thus, despite the lack of effect of hypoxia on bone formation, the enhancement of cell migration and osteoblast differentiation opens windows for further investigations on approaches to modulate the BMP-9-HIF-1α circuit in the context of cell-based therapies to induce bone regeneration.