Kaempferol-immobilized titanium dioxide promotes formation of new bone: effects of loading methods on bone marrow stromal cell differentiation in vivo and in vitro

Shuhei Tsuchiya,1 Keisuke Sugimoto,2 Hisanobu Kamio,2 Kazuto Okabe,1 Kensuke Kuroda,3 Masazumi Okido,3 Hideharu Hibi2 1Department of Oral and Maxillofacial Surgery, Nagoya University Hospital, Nagoya, Japan; 2Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan; 3Institute of Materials and Systems for Sustainability, Nagoya University, Nagoya, Japan Background: Surface modification of titanium dioxide (TiO2) implants promotes bone formation and shortens the osseointegration period. Kaempferol is a flavonoid that has the capacity to promote osteogenic differentiation in bone marrow stromal cells. The aim of this study was to promote bone formation around kaempferol immobilized on TiO2 implants. Methods: There were four experimental groups. Alkali-treated TiO2 samples (implants and discs) were used as a control and immersed in Dulbecco’s phosphate-buffered saline (DPBS) (Al-Ti). For the coprecipitation sample (Al-cK), the control samples were immersed in DPBS containing 50 µg kaempferol/100% ethanol. For the adsorption sample (Al-aK), 50 µg kaempferol/100% ethanol was dropped onto control samples. The surface topography of the TiO2 implants was observed by scanning electron microscopy with energy-dispersive X-ray spectroscopy, and a release assay was performed. For in vitro experiments, rat bone marrow stromal cells (rBMSCs) were cultured on each of the TiO2 samples to analyze cell proliferation, alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. For in vivo experiments, TiO2 implants placed on rat femur bones were analyzed for bone–implant contact by histological methods. Results: Kaempferol was detected on the surface of Al-cK and Al-aK. The results of the in vitro study showed that rBMSCs cultured on Al-cK and Al-aK promoted alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. The in vivo histological analysis revealed that Al-cK and Al-aK stimulated new bone formation around implants. Conclusion: TiO2 implant-immobilized kaempferol may be an effective tool for bone regeneration around dental implants. Keywords: kaempferol, titanium implant, surface treatment, biomateria