Differentiation of bone marrow mesenchymal stem cells modulated by Bushen Tian Sui decoction via the TGF-β1-Smad2/3 signaling pathway: In vitro evidence and potential clinical application in delayed fracture healing

Background: Bone-related disorders pose significant challenges in clinical practice. Bone marrow mesenchymal stem cells (BMSCs), as multipotent stem cells, play a pivotal role in bone regeneration. The TGF-β1-Smad2/3 signaling pathway is a well-recognized regulator of BMSC osteogenic differentiation. Traditional Chinese medicine (TCM), such as Bushen Tian Sui decoction (BSTSD), has shown potential in enhancing bone health; however, its molecular mechanisms remain poorly understood.

Objective: Investigating the effects and underlying mechanisms of BSTSD on the osteogenic differentiation of BMSCs.

Materials and methods: The impact of BSTSD on BMSCs was comprehensively analyzed using Cell Counting Kit-8 (CCK8), quantitative polymerase chain reaction (qPCR), western blot (WB), and immunofluorescence assays.

Results: CCK8 results revealed the highest optical density (OD) values in the BSTSD + activator group at 24, 48, and 72 hours, indicating enhanced cell proliferation. qPCR analysis showed significantly increased expression levels of TGF-β1, Smad2, Smad3, SOX9, and RUNX2 in the BSTSD + activator group, suggesting a synergistic effect in promoting osteogenic and chondrogenic differentiation. WB results demonstrated elevated phosphorylation levels of Smad2 and Smad3 (p-Smad2, p-Smad3) in the BSTSD + activator group, while total Smad2 and Smad3 protein levels remained consistent among groups. Immunofluorescence assays confirmed the highest fluorescence intensity, positive area ratio, and cell count containing Smad2 and Smad3 proteins in the BSTSD + activator group, validating the synergistic effect of BSTSD and TGF-β1.

Conclusion: BSTSD exhibits promising effects on BMSC differentiation and bone regeneration, mediated through the TGF-β1-Smad2/3 signaling pathway.