Stem cells derived from oral tissue represent a highly attractive alternative source for clinical bone regeneration because they can be collected by non-invasive or minimally invasive procedures. Herein, we describe the human dental stem cells (DSCs) deriving from buccal fat pads (BFP), dental pulp (DP) of impacted teeth, and periodontal ligaments (PDL) to obtain BFPSCs, DPSCs, and PDLSCs, respectively. Cells were purified with selected medium and expanded through passages in stem cell culture medium. Purified cells were characterized for stemness by their growth rate, immunostaining, and multilineage differentiation ability. They showed plastic adherence, expression of stemness-specific markers, and multilineage differentiation potential. Immunocytochemistry analysis confirmed that DPSCs had more osteogenic potential than BFSCs and PDLSCs. Calcium-rich deposits, evaluated by von Kossa and Alizarin red staining, showed greater mineralization when DPSCs were cultured on collagen type I matrix than without collagen. Furthermore, DPSC-seeded collagen type I matrix maintained consistent osteogenesis and boosted mineral formation by 1-2 weeks over that in DPSCs cultured without collagen. Radiographic analysis of DPSC-seeded collagen type I matrix transplanted into rat cranial defects showed significant bone regeneration after 8 weeks. These results suggested that the redundant oral tissue can be used as a source of adult multipotent stem cells for clinical bone regeneration. Triple overlay images with biomarkers (red), nuclei (blue) and bright field morphology of DPSCs. The specifically osteo-differentiation shown by osteocalcin (left) expression and lack of sox9 (right) expressed in the images below which were cultured with collagen matrix, contrast with no collagen matrix group above.