Background: Thrombospondin-1 (TSP-1) is a multifunctional glycoprotein involved in various physiological processes, including tissue repair and the regulation of angiogenesis. However, its role in bone regeneration remains unclear. This study aims to investigate the role of TSP-1 in promoting bone healing in a rat fracture model, with a particular focus on its effects on angiogenesis and the recruitment of mesenchymal stem cells (MSCs).

Methods: A stable femoral fracture model was established in rat. The experimental rat received treatment of TSP-1, MSCs, or a combination of both, and bone healing was assessed 2 weeks post-surgery. Micro-computed tomography (micro-CT) was used to evaluate bone regeneration by analyzing bone mineralization and trabecular parameters. Immunohistochemistry was performed to detect angiogenesis and bone formation markers (Cluster of Differentiation 105 (CD105), Cluster of Differentiation 31 (CD31), Bone Morphogenetic Protein-2 (BMP-2)). To investigate the mechanism by which TSP-1 promotes angiogenesis, 5-ethynyl-2′-deoxyuridine staining (EdU) staining was used to assess endothelial cell proliferation, and Western blotting was conducted to measure the protein expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF). A tube formation assay was used to evaluate the effect of TSP-1 on endothelial cell tube formation. Additionally, MSCs were co-cultured with TSP-1 for 7 days, and Transwell migration assays were performed to evaluate MSC migration. Tube formation assays were also used to assess vascular differentiation potential of MSCs. Immunofluorescence staining was performed to detect the expression of endothelial cell markers CD31, vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 (VEGFR2) in MSCs.

Results: Two weeks post-surgery, the TSP-1 and MSC groups exhibited significantly more callus, denser trabecular bone, and higher mineralization levels compared to the model group (p < 0.05). The TSP-1+MSC group showed superior fracture healing, bone mineralization, trabecular thickness, and bone density compared to the single-treatment TSP-1 and MSC groups (p < 0.05). Immunohistochemical analysis revealed that, compared to the model group, the expression of CD105, CD31, and BMP-2 was significantly increased in the TSP-1 and MSC groups (p < 0.05), with the TSP-1+MSC group showing higher expression levels than the single treatment groups (p < 0.05). Hematoxylin-eosin staining results showed that in the TSP-1 and MSC groups, fibrous tissue was gradually replaced by bone tissue, with mature bone formation observed. The TSP-1+MSC group exhibited more mature bone tissue. In vitro, TSP-1 treatment significantly promoted endothelial cell proliferation and tube formation, with higher protein expression levels of COX-2 and VEGF detected as compared to the control group (p < 0.05). Additionally, TSP-1 significantly enhanced MSC migration and tube formation, and upregulated the expression of endothelial cell markers CD31, VEGFR1, and VEGFR2 in MSCs (p < 0.05).

Conclusions: Both TSP-1 and MSCs, whether used alone or in combination, significantly promoted bone formation and angiogenesis during the fracture healing process. The combined treatment with TSP-1 and MSC showed the most significant effects, suggesting a synergistic role in accelerating fracture repair. These results provide new evidence for the potential applications of TSP-1 and MSCs in bone fracture healing and vascular regeneration.