Interaction Between Endothelial Progenitor Cells and Bone Marrow Mesenchymal Stem Cells Enhances Osteogenic Differentiation via Activation of the Wnt/β-Catenin Signaling Pathway

Abstract

Background: Recent studies have shown that endothelial progenitor cells (EPCs) and bone marrow stromal cells (BMSCs) may interact functionally through the secretion of factors or direct cell-to-cell contact. This interaction is crucial for regulating their migration, proliferation, and differentiation. Particularly during osteogenic differentiation, the Wnt/β-catenin signaling pathway has been proven to be an important regulatory mechanism. This study aimed to elucidate the mechanisms underlying the promotion of BMSCs differentiation by EPCs of bone marrow, providing new insights for the clinical treatment of osteonecrosis of the femoral head (ONFH). Methods: Rabbit BMSCs and EPCs were isolated, and immunofluorescence staining was employed to confirm markers of both cell types. The experimental group involved the co-culture of EPCs and BMSCs, while the control group consisted of BMSCs cultured independently. Osteogenic differentiation was assessed by alkaline phosphatase and Alizarin Red staining. The expression levels of Bone Morphogenetic Protein 2 (BMP2), Osteocalcin (OCN), Osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and β-catenin were measured using Western blotting and qualitative reverse transcription polymerase chain reaction (qRT-PCR). Results: Positive expression levels of differentiation 29 (CD29) and CD90 confirmed successful BMSC isolation, while kinase insert domain receptor (KDR) and CD31 positivity confirmed EPC isolation. The experimental group exhibited a significant increase in alkaline phosphatase activity and mineralized nodules compared the control. EPCs significantly upregulated the expression of osteogenic-associated proteins BMP2, OCN, OPN, and RUNX2 in BMSCs (p < 0.01). Notably, the gene and protein expression of Wnt/β-catenin signaling pathway-related molecules were upregulated in the experimental group compared to the control (p < 0.01). siRNA-mediated silencing of the β-catenin in BMSCs resulted in the downregulation of osteogenic-related genes (BMP2, OPN, OCN, and RUNX2) and reduced β-catenin expression (p < 0.001). Additionally, osteogenic-related proteins (BMP2, OCN, OPN, and RUNX2) were downregulated (p < 0.05, p < 0.01, p < 0.001), accompanied by reduced β-catenin synthesis (p < 0.01). Treatment with 4-Ethyl-5-methyl-5,6-dihydro-[1,3]dioxolo[4,5-j]phenanthridine (HLY78) reversed the inhibitory effect of siRNA on β-catenin, promoting BMSC osteogenic differentiation. Conclusions: These findings emphasize the role of the EPC-BMSC interaction and the Wnt/β-catenin signaling pathway in enhancing osteogenic differentiation. Our study provides novel insights into the potential clinical use of EPCs for ONFH treatment.