1,25(OH)2D3 Enhances the Osteogenic Differentiation Potential of Multilayered Cell Sheets Derived from Human Periodontal Ligament Stem Cells: Associated with OSX and COL1A1 Upregulation

Objective: We aimed to investigate the regulatory effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] pretreatment on the in vitro osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), to evaluate the ectopic osteogenic tissue-forming capacity of the constructed multilayered cell sheets (MUCSs) after subcutaneous implantation in nude mice, and to examine whether these effects are associated with the expression of OSX and COL1A1. Methods: hPDLSCs were isolated and cultured in vitro and were induced in an osteogenic induction medium supplemented with 1,25(OH)2D3 (100 nM). Changes in the transcription factor OSX and in the key osteogenic markers COL1A1 and OCN were assessed by Western blotting and immunofluorescence staining, and the in vitro matrix mineralization capacity was evaluated by Alizarin Red S staining. After the MUCSs were constructed and characterized histologically by scanning electron microscopy (SEM), they were implanted subcutaneously into nude mice. Tissue samples were harvested at weeks 4 and 8 to evaluate the in vivo histological changes and the ectopic osteogenic tissue-forming capacity by Western blotting, H&E staining, and immunohistochemistry. Results: 1,25(OH)2D3 significantly enhanced the matrix mineralization capacity of hPDLSCs and markedly increased the fluorescence intensities and protein expression levels of COL1A1 and OCN; in contrast, its effect on the protein level of the transcription factor OSX was only a mild upregulation. SEM revealed that the MUCSs possessed a dense physical structure with abundant extracellular matrix deposition, features that were further enhanced by 1,25(OH)2D3. At weeks 4 and 8 after transplantation, the protein expression of OSX, COL1A1, and OCN was upregulated to varying degrees in the 1,25(OH)2D3-pretreated group, which also showed a consistent trend toward more abundant osteoid tissue formation on histological staining. At both time points the levels of OSX, COL1A1 and OCN were higher in the two MUCS groups than in the MCS group, whereas the additional effect of 1,25(OH)2D3 on top of multilayer stacking varied between markers and time points and did not show a consistent pattern. Conclusions: 1,25(OH)2D3 effectively enhances the in vitro osteogenic potential of hPDLSCs by promoting the expression of osteogenic matrix proteins, and the MUCSs constructed with this strategy show the potential to support ectopic osteogenic differentiation in vivo. This effect is associated with, but has not yet been proven to be mediated by, the upregulated expression of OSX and COL1A1. This study provides preclinical evidence and a promising optimization strategy for cell sheet-based periodontal bone regeneration.