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. 2018 Mar 19:13:1665-1676.
doi: 10.2147/IJN.S150786. eCollection 2018.

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 Hibi  2
Affiliations

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 et al. Int J Nanomedicine. .

Abstract

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: biomaterial; kaempferol; surface treatment; titanium implant.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Sketch map of the loading of bioactive molecules on alkali-treated TiO2. Abbreviations: TiO2, titanium dioxide; EtOH, ethanol; DPBS, Dulbecco’s phosphate-buffered saline; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol.
Figure 2
Figure 2
SEM micrograph of the implant surface. (A) Control TiO2 surface showing typical machined surface topology. (B) After alkali treatment, the surface of Al-Ti shows fine nanometric topology. (C) After alkali treatment and adsorption of kaempferol, the surface of Al-aK shows round-shaped structures. (D) After alkali treatment and co-precipitation of kaempferol, the surface of Al-cK shows round-shaped structures. Note: Magnification: 10,000×. Abbreviations: SEM, scanning electron microscopy; TiO2, titanium dioxide; Al-Ti, alkali-treated TiO2; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol; JEOL, JEOL Ltd.; SEI, secondary electron image; WD, working distance.
Figure 3
Figure 3
Characterization of TiO2 surface using EDS analysis. EDS spectrum of (A) control, (B) Al-Ti, (C) Al-aK, and (D) Al-cK showing each metallic element on the TiO2 surface. Quantification of individual metallic elements shows (E) titanium, (F) carbon, (G) phosphate, and (H) calcium. Notes: Data are expressed as means (n=3) with error bars representing standard deviations; ap<0.05 compared to control; bp<0.05 compared to Al-Ti; cp<0.05 compared to Al-aK; dp<0.05 compared to Al-cK. Abbreviations: EDS, electron-dispersive spectroscopy; TiO2, titanium dioxide; Al-Ti, alkali-treated TiO2; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol; CKα, carbon Kα; TiLI, titanium LI; PKα, phosphate Kα; TiKesc, titanium Kesc; CaKα, calcium Kα; CaKβ, calcium Kβ; TiKα, titanium Kα; TiKβ, titanium Kβ.
Figure 4
Figure 4
Drug-release graph of kaempferol from TiO2 discs in Hanks’ solution for 168 h. Note: Data are expressed as means (n=5) with error bars representing standard deviations. Abbreviations: TiO2, titanium dioxide; Al-Ti, alkali-treated TiO2; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol; h, hours.
Figure 5
Figure 5
Cell proliferation of rBMSCs adhered to the representative samples in growth medium for 1, 3, and 7 days. Notes: Data are expressed as means (n=3) with error bars representing standard deviations; ap<0.05 compared to Al-Ti; bp<0.05 compared to Al-cK. Abbreviations: rBMSC, rat bone marrow stromal cell; TiO2, titanium dioxide; Al-Ti, alkali-treated TiO2; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol.
Figure 6
Figure 6
Normalized ALPase activity with respect to total protein of rBMSCs cultured on the representative experimental groups for 1, 3, and 7 days. Notes: Data are expressed as means (n=3) with error bars representing standard deviations; ap<0.05 compared to control; bp<0.05 compared to Al-Ti. Abbreviations: ALPase, alkaline phosphatase; rBMSC, rat bone marrow stromal cell; TiO2, titanium dioxide; Al-Ti, alkali-treated TiO2; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol.
Figure 7
Figure 7
Calcium deposition of rBMSCs cultured on the representative experimental groups for 1, 7, and 14 days. Calcium deposition quantification using the Alizarin Red S assay. Notes: Data are expressed as means (n=3) with error bars representing standard deviations; ap<0.05 compared to control; bp<0.05 compared to Al-Ti; cp<0.05 compared to Al-cK. Abbreviations: rBMSC, rat bone marrow stromal cell; Al-Ti, alkali-treated TiO2; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol; TiO2, titanium dioxide.
Figure 8
Figure 8
Gene expression of rBMSCs cultured on the representative experimental groups for (A) 1, (B) 3, and (C) 7 days. Notes: Data are expressed as means (n=3) with error bars representing standard deviations; ap<0.05 compared to control; bp<0.05 compared to Al-Ti; cp<0.05 compared to Al-cK. Abbreviations: rBMSC, rat bone marrow stromal cell; Al-Ti, alkali-treated TiO2; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol; TiO2, titanium dioxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Runx2, runt-related transcription factor-2; OCN, osteocalcin; ON, osteonectin; OPN, osteopontin; ALP, alkaline phosphatase; ColI, type I collagen.
Figure 9
Figure 9
Histological analysis around the TiO2 implants in vivo. Bone morphogenesis around TiO2 implants as observed under 100× magnification at 2 weeks after implantation (A–H) and 4 weeks after implantation (I–P). (A, B, I, J) Control; (C, D, K, L) Al-Ti implants; (E, F, M, N) Al-aK implants; and (G, H, O, P) Al-cK implants. Bars indicate 500 µm (A, C, E, G, I, K, M, O) and 100 µm (B, D, F, H, J, L, N, P). (Q) Average histomorphometric values of BIC. Notes: Data are expressed as means (n=3) with error bars representing standard deviations; ap<0.05 compared to control; bp<0.05 compared to Al-Ti. Abbreviations: TiO2, titanium dioxide; Al-Ti, alkali-treated TiO2; Al-aK, alkali-treated adsorption with kaempferol; Al-cK, alkali-treated coprecipitation with kaempferol; BIC, bone–implant contact.
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