Timosaponin B- II Enhances Osteogenic Differentiation of Human Periodontal Ligament Stem Cells via PI3K/AKT/GSK3β Signaling Pathway.

BACKGROUND

This investigation aims to elucidate the effects of Timosaponin B-II (TB-II) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) through both in vitro experiments and an in vivo orthodontic tooth movement model utilizing rats. The primary objective is to clarify the mechanisms by which TB-II influences the remodeling of periodontal tissue under biomechanical stress, thereby providing insights into its potential role in reducing relapses after orthodontic tooth movement.

METHODS

hPDLSCs were isolated and characterized via flow cytometry and multilineage differentiation assays (osteogenic and adipogenic induction). The impact of TB-II on the expression levels of osteogenic genes and proteins, including runt-related transcription factor-2 (RUNX-2), alkaline phosphatase (ALP), and collagen type 1 (COL-1), was evaluated through quantitative real-time PCR (qRT-PCR) and Western blotting. Alizarin Red Staining (ARS) was utilized to assess the formation of mineralized nodules. Additionally, the involvement of the phosphatidylinositol - 3 - kinase (PI3K)/ protein kinase B(AKT)/ glycogen synthase kinase - 3β(GSK3β) signaling pathway in TB-II-mediated osteogenesis was explored using pharmacological inhibitors (LY294002 for PI3K/AKT and CHIR-99021 for GSK3β). Western blot analysis identified key osteogenic markers (GSK3β, p-GSK3β, AKT, p-AKT) in treated cells. For in vivo validation, eighteen male Wistar rats were randomly divided into TB-II-treated and saline-control groups. Micro-computed tomography (micro-CT) evaluated tooth movement and alveolar bone structural changes. Histological assessment included hematoxylin-eosin (HE) staining, Masson trichrome staining, and tartaric-resistant acid phosphatase (TRAP) staining to analyze periodontal tissue morphology. Immunohistochemical (IHC) analysis assessed osteogenic markers (RUNX-2, ALP, COL-1) and the osteoclastogenic regulator RANKL to evaluate tissue remodeling. All statistical analyses were performed using GraphPad Prism 8. Comparisons between groups were conducted via one-way/two-way ANOVA with Tukey's post-hoc test. Values of p < 0.05 were regarded as statistically significant.

RESULTS

In vitro studies revealed that TB-II at 20 μM significantly enhanced the proliferation, ALP activity, and mineralized nodule formation of hPDLSCs, accompanied by markedly elevated expression of RUNX-2, ALP, COL-1, p-AKT and p-GSK3β. Pharmacological inhibition of the PI3K/AKT pathway via LY294002 abolished TB-II's osteogenic effects, while treatment with CHIR99021 indicated that GSK3β activity was downstream and regulated by the PI3K/AKT signaling axis. In vivo, TB-II administration in a rat orthodontic tooth movement (OTM) model upregulated RUNX-2, ALP, and COL-1 expression on the tension side of tooth roots, while simultaneously reducing TRAP + osteoclast numbers and inhibiting RANKL expression.

CONCLUSION

TB-II stimulates the proliferation and osteogenic maturation of hPDLSCs in vitro by activation of the PI3K/AKT/GSK3β signaling axis. In vivo investigations using OTM model further demonstrate that TB-II enhances periodontal tissue regeneration. Collectively, these results highlight the therapeutic potential of TB-II in preventing relapses following OTM, positioning it as a viable candidate for clinical strategies aimed at stabilizing orthodontic outcomes.