Tideglusib-Preactivated Osteoblasts Encapsulated in Ceramic Particle-Reinforced GelMA Hydrogel for Enhanced Critical-Size Bone Defect Regeneration.

内容摘要

Critical-size bone defects remain a clinical challenge due to limited intrinsic bone regeneration. This study developed a composite scaffold composed of osteoinductive biphasic calcium phosphate (BCP) ceramic particles and gelatin methacryloyl (GelMA) hydrogel, loaded with calvarial osteoblasts (OBs) pretreated with the glycogen synthase kinase-3β (GSK-3β) inhibitor Tideglusib on surface of BCP particles, and evaluated its bone regenerative efficacy. BCP particles were characterized by X-ray diffraction and scanning electron microscopy. GelMA was synthesized and characterized by FTIR and 1H-NMR spectroscopy. Mechanical properties were assessed by compression testing. OBs were treated with Tideglusib to determine optimal concentration via CCK-8, scratch assays, qPCR, ALP and Alizarin Red S staining. Tideglusib-pretreated OBs seeded on BCP particles were evaluated for viability, proliferation, and osteogenic gene expression (COL-I, ALP). In vivo, 3.5 mm rat femoral defects were used to assess the bone regeneration potential of T-OB/BCP particle/GelMA (GPCT) composites in comparison with other groups by micro-CT and histology at 4 and 8 weeks. BCP (200-300 μm, HA/β-TCP ~30:70) incorporation enhanced GelMA compressive modulus ~4-fold. In vitro, 1 μM Tideglusib optimally activated Wnt/β-catenin signaling and promoted OB proliferation, migration, and osteogenic differentiation, with sustained effects after drug removal. OBs on BCP particles showed time-dependent upregulation of osteogenic genes, and Tideglusib pretreatment further elevated this expression. In vivo, micro-CT revealed significantly higher BV/TV, Tb.N, Tb.Th, and BMD in the GPCT group versus others. Histology showed more mature bone with numerous osteocytes, partial degradation of BCP particles, and new bone area fraction >70% in GPCT. The composite BCP particle/GelMA scaffold with Tideglusib-pretreated OBs provides mechanical reinforcement and sustained osteogenic stimulation, effectively accelerating critical-size bone defect regeneration. This ex vivo pharmacological priming strategy represents a promising translational approach for bone tissue engineering. Why was the study done? Repairing large bone defects remains a major clinic challenge. Current treatments, such as taking bone from another body part, cause additional injury and are limited in supply. A leading direction involves designing combined three elements: a supportive scaffold, bone-forming cells, and molecular signals that instruct cells to build new tissue. This study explored whether a scaffold that physically supports new bone growth—while delivering bone-forming cells temporarily “primed” with a bone-stimulating drug—could effectively repair large bone defects. What did the researchers do and find? We created a composite made of a gel reinforced with ceramic particles that naturally encourage bone growth. Bone-forming cells were briefly treated outside the body with a drug called Tideglusib, which activates a key bone-building pathway. The drug was then removed, leaving cells in a primed state without long-term drug exposure. This composite was implanted into a critical-sized leg bone defect in rats—a wound too large to heal on its own. After eight weeks, over 70% of the defect was filled with mature bone in treated animals, while untreated defects remained largely hollow. What do these results mean? This approach—combining a reinforced scaffold, bone-friendly particles, and temporarily boosted cells—can robustly regenerate bone in severe injuries. By briefly priming cells before implantation, we capture the benefits of a powerful bone-building drug while avoiding risks of long-term exposure. This strategy provides a foundation for developing next-generation bone graft for patients with severe bone loss.