During the maintenance of bone marrow-derived mesenchymal stem cells (BMMSCs), suspended cells are discarded normally. We noted the osteogenic potential of these cells to be like that of anchorage-dependent BMMSCs. Therefore, we characterized suspended BMMSCs from rabbit bone marrow by bioengineering and applied the suspended BMMSCs to double-canaled dental implants inserted into rabbits. After primary isolation of BMMSCs, we collected the suspended cells during primary culture on the third day. The cells were transferred and maintained on an extracellular-matrix-coated culture plate. The cells were characterized and compared with BMMSCs by colony-forming-unit fibroblast (CFU-f) and cell proliferation assay, fluorescence-activated cell sorter (FACS), in vitro multipotency, and reverse transcription polymerase chain reaction (RT-PCR). We also analyzed the osteogenic potential of cells mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and transplanted into immunocompromised mice. We compared the viability and proliferation of the suspended BMMSCs and BMMSCs on the titanium implant surface and observed cell morphology. Then, the cells mixed with HA/TCP were applied to the double-canaled implants during installation into rabbit tibia. Four weeks later, we analyzed bone formation inside the canal by histomorphometry. The suspended cells showed higher CFU-f on the extracellular matrix (ECM)-coated culture plate and similar results of proliferation capacity compared with BMMSCs. The cells also showed osteogenic, adipogenic, and chondrogenic ability. The suspended cells showed levels of attachment survival and proliferation on the surfaces of titanium implant discs to be higher than or similar to those of BMMSCs. The suspended cells as well as BMMSCs showed stronger bone formation ability in both upper and lower canals of the implants compared with controls on double-canaled implants inserted into rabbit tibia. In this study, we showed that suspended cells after primary BMMSC isolation have bone regeneration capacity like that of BMMSCs, not only in vitro but also in vivo. ECM was valuable for propagation of MSCs for cell-based bone regeneration. Therefore, the suspended cells could also be useful tools for bone regeneration after implant surgery.