Acoustic Vibration Enhances Osteogenic Differentiation in Dental Mesenchymal Stem Cells Vibration-assisted orthodontic treatment accelerates tooth movement and reduces complications associated with prolonged interventions. While vibration has been shown to enhance osteogenic potential in bone marrow-derived mesenchymal stem cells (MSCs), its effects on dental tissue-derived MSCs remain unclear. This study investigated the impact of acoustic-frequency vibratory stimulation (AFVS) on gingival-tissue-derived MSCs (GT-MSCs) at 20 Hz and 60 Hz under both basal and osteogenic conditions. A custom vibratory platform was developed, and GT-MSCs were assessed for viability, proliferation, and osteogenic differentiation. Resazurin assay, Calcein-AM staining, and vimentin immunohistochemistry were used to evaluate cell viability, proliferation, and morphology, while Alizarin Red staining and calcium accumulation assays measured extracellular matrix mineralization at 7, 14, and 21 days. A Reverse-Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) reaction was performed to quantify osteogenic markers (colagen type I [COL-I], osteopontin [OPN], and alkaline phosphatase [ALP]), and protein expression for COL-I and OPN was confirmed by immunohistochemistry. The results showed that AFVS at 20 Hz and 60 Hz enhanced osteogenic differentiation in GT-MSCs compare with other groups. Extracellular matrix mineralisation increased significantly, with 60 Hz resulting in the highest calcium deposition. Transcript levels of COL-I and OPN were markedly upregulated at 60 Hz, indicating a frequency-dependent response. Cell proliferation was also promoted, with optimal results observed at 60 Hz compare with other groups. These findings highlight the role of mechanical stimulation in enhancing the osteogenic potential of GT-MSCs, suggesting that AFVS is a promising tool for regenerative and orthodontic treatments. This study provides new insights into the frequency-specific effects of vibration, supporting the use of vibration therapy strategies in dental applications.