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Background and Objectives: The duration of bone turnover is critical, and different time points help in identifying the optimal endpoint of treatment duration. However, investigating the combination of xenograft and stem cells to allow tissue regeneration within an ideal time duration remains an under-investigated topic. The current study aimed to assess the impact of equine-derived xenograft bone blocks in assisting the human periodontal ligament stem cells (PDLSCs) to demonstrate osteogenic differentiation (collagen type 1 expression and calcium deposition) within an osteogenic growth media. Materials and Methods: Human PDLSCs were acquired commercially and seeded onto xenograft bone blocks. After the 14th and 21st day of culture, enzyme-linked immunoassay (ELISA) was utilized for the detection and quantification of levels of collagen type I, while the mineralization assessment (deposition of calcium) was conducted by staining the PDLSCs with Alizarin Red S (ARS). The statistical comparison between the means and standard deviations of study groups were evaluated using analysis of variance (ANOVA). Results: ELISA assessment revealed an upsurge in the expression of collagen type I for PDLSCs cultured with xenograft after 14 and 21 days compared to the controls (intergroup comparisons significant at p < 0.05). Similar findings were obtained for mineralization assessment and on ARS staining. PDLSCs cultured with xenograft bone blocks presented an increased deposition of calcium compared to their control counterparts (intergroup comparisons significant at p < 0.05). Conclusions: PDLSCs embedded in xenograft bone blocks inside an osteogenic growth medium demonstrated greater osteogenic differentiation potential after 14 and 21 days. This superior osteogenic differentiation capability was evident by increased collagen type I expression and more significant calcium deposition at the 14th and 21st days after culture.

To evaluate the ability of osteogenic culture media in comparison with regular growth culture media in enhancing the osteoblastic cell differentiation of human periodontal ligament stem cells (hPDLSCs).Confocal laser microscope analysis showed positive staining for new bone cells with an increased signal intensity when samples were cultured in osteogenic culture media compared with regular culture media. These findings indicate the effect of the active ingredients of the osteogenic culture media in enhancing the osteogenic differentiation hPDLSC. Scanning electron microscopy images validated the osteogenic differentiation showing a flattened, polygonal morphology with multiple extending cytoplasmic processes of new cells.Xenograft bone blocks are biocompatible scaffold for the osteogenic differentiation of seeded hPDLSCs. Osteogenic culture media enhances and increases the osteogenic differentiation of hPDLSCs into new bone cells more than regular growth culture media. Periodontal ligament stem cells are a predictable biological input as a cell-based tissue-engineered construct and biologically acceptable when it is cultured in a suitable growth media that mimics the intended environment.Consideration of the clinical use of equine bone blocks and periodontal ligament stem cells in a suitable biological environment as a potential new option for bone regeneration techniques.