Previous studies have suggested the usefulness of a new coating method-namely, the forming of a thin hydroxyapatite (HA) layer on commercially pure titanium (cpTi) by anodization and hydrothermal treatment-for use as a dental root implant material. In vivo and in vitro studies confirmed that an HA layer on cpTi (HA/cpTi) implants showed good compatibility with bone tissue, rat bone marrow stromal (RBM) cells, and immune cells. The aim of the present investigation was to further characterize the in vitro early cellular behavior of RBM cells on HA/cpTi implants. Therefore, in this study we performed surface analysis, analysis of cell initial attachment, and analysis of cell morphology and the cytoskeleton. Drops of distilled water or cell culture medium showed smaller contact angles with HA/cpTi than with cpTi. RBM cells were cultured for 30, 60, and 120 min on HA/cpTi and cpTi, and the level of cell adhesion was shown to increase with time on both substrates. However, cell adhesion on HA/cpTi was significantly higher than on cpTi at 60 and 120 min. Especially at 120 min, when compared with cpTi, the cell morphology on the surface of HA/cpTi not only adopted a flattened and spreading form, but also extended filopodium-like processes with irregular edges that were intimately adapted to the surface of the HA microcrystals. The cytoskeleton on HA/cpTi showed well-formed actin filaments that were parallel to each other and the long axis of RBM cells. The actin filaments of RBM cells on the HA/cpTi surface were localized to the periphery (corresponding to the edge of the filopodium-like processes) well after 120 min. This suggests that actin filaments of RBM cells need to be anchored at the HA/cpTi surface and the numerous HA microcrystals precipitated on the HA/cpTi surface. These findings were similar to the scanning electron microscopic morphology. The peripheral anchorage provide sufficient strength of attachment to allow recognization of actin filaments upon HA/cpTi. The surface of HA/cpTi was more hydrophilic and exhibited markedly improved wettability compared to untreated cpTi, and higher levels of early cell attachment were observed on surfaces after anodization and hydrothermal treatment than on surfaces with untreated cpTi. The results of in vitro experiments suggest that this new method for forming a thin HA layer on the surface of cpTi could be useful to ensure excellent cellular behavior on implant surfaces. The characterization of cell morphology on the thin HA layer formed by anodization and hydrothermal treatment on cpTi implant material suggests that physicochemical or biological conditioning of the implant surface involves implant surface topography.

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All dental root implants come in contact with the oral epithelium, and many complex factors are found to arise in this region. In order to perform a successful dental root implantation, it is necessary to clarify the interaction of the dental root implant material with the host defense mechanisms involved in the specific and nonspecific immune responses to many antigens in oral bacteria and their components. Recently, focusing on developing the dental root implant, the Nikon Corporation improved the surface characteristics of pure titanium even further by developing a hydroxyapatite (HA) layer formed on an anodic titanium oxide film containing Ca and P via hydrothermal treatment (SA treatment). However, since little is known about the effect of SA-treated pure titanium (HA/Ti) on the defense mechanisms of the oral membrane epithelium, we investigated (1) the in vitro proliferation of murine splenic B lymphocytes on the surface of HA/Ti in the presence of three lipopolysaccharide (LPS) concentrations and (2) interleukin-1alpha (IL-1alpha) production by the reaction of human peripheral blood mononuclear cells (PBM cells) on the surface of HA/Ti under the same concentrations. After culture, murine splenic lymphocytes were measured by uptake of 3H-thymidine, and cytokine release (IL-1alpha) from PBM cells was measured by ELISA. Results showed that HA/Ti had hardly any effect on the LPS-induced proliferation of B lymphocytes and IL-1alpha production. In vitro investigations of the effects of HA/Ti on the LPS-induced proliferation of murine splenic B lymphocytes and IL-1alpha from PBM cells might be a useful way of elucidating the defense mechanism between implants and the oral epithelium.

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