Effects of the nanotopographic surface structure of commercially pure titanium following anodization-hydrothermal treatment on gene expression and adhesion in gingival epithelial cells.

The long-term stability and maintenance of endosseous implants with anodized-hydrothermally treated commercially pure titanium surfaces and a nanotopographic structure (SA-treated c.p.Ti) depend on the barrier function provided by the interface between the transmucosal portion of the implant surface and the peri-implant epithelium. This study investigated the effects of extracellular and intracellular gene expression in adherent gingival epithelial cells cultured for 1-7 days on SA-treated c.p.Ti implant surfaces compared to anodic oxide (AO) c.p.Ti and c.p.Ti disks. Scanning electron microscopy (SEM) showed filopodium-like extensions bound closely to the nanotopographic structure of SA-treated c.p.Ti at day 7 of culture. Gene expressions of focal adhesion kinase, integrin-α6ß4, and laminin-5 (α3, ß3, γ2) were significantly higher on SA-treated c.p.Ti than on c.p.Ti or AO c.p.Ti after 7 days (P<0.05). Our results confirmed that gingival epithelial cells adhere to SA-treated c.p.Ti as the transmucosal portion of an implant, and that this interaction markedly improves expression of focal adhesion molecules and enhances the epithelial cell phenotype. The cellular gene expression responses driving extracellular and intracellular molecular interactions thus play an important role in maintenance at the interface between SA-treated c.p.Ti implant surfaces and the gingival epithelial cells.

Anodic oxidation and hydrothermal treatment of commercially pure titanium surfaces increases expression of bone morphogenetic protein-2 in the adherent macrophage cell line J774A.1.

The surface property of commercially pure titanium (cpTi) was improved by forming a thin hydroxyapatite (HA) layer by anodic oxidation and hydrothermal treatment (HA/cpTi). We hypothesize that the adhesion of macrophages to HA/cpTi surfaces is important to the process of osseointegration. This study investigates the effect of adhesion of macrophages to HA/cpTi surfaces on the expression of bone morphogenetic protein-2 (BMP-2). The murine macrophage cell line J774A.1 was cultured on HA/cpTi and polished cpTi (S/cpTi). Macrophage cell adhesion was examined by SEM, 0-72 h following plating onto HA/cpTi and S/cpTi. BMP-2 gene expression was examined by RT-PCR analysis. The level of BMP-2 secreted into the supernatant was measured using an ELISA assay. The extent of macrophage adhesion increased with time on both the HA/cpTi and S/cpTi surfaces, with a" higher degree of spreading observed on HA/cpTi than onS/cpTi surfaces after 24 or 72 h. The ratio of BMP-2 mRNA was higher on HA/cpTi than on S/cpTi after 24 h (0.348 vs. 0, p < 0.05) and 72 h (0.584 vs. 0.189, p < 0.05). After 24 h, secretion of BMP-2 was detected in cultures grown on HA/cpTi, but not on S/cpTi. After 72 h, secretion of BMP-2 was detected in cultures grown on S/cpTi, but the levels were higher in cultures grown on HA/cpTi. These findings show that macrophages have the capacity to adhere to HA/cpTi endosseous implants and provide a source of osteoinductive cytokines that may play a key role in the process of osseointegration.

Titanium surface topography alters cell shape and modulates bone morphogenetic protein 2 expression in the J774A.1 macrophage cell line.

Macrophage cytokine expression significantly affects wound healing. Macrophage secretion of transforming growth factor beta 1 (TGFbeta1) and bone morphogenetic proteins (BMP) may affect osteogenesis at endosseous implant surfaces. The aim of this investigation was to determine the effect of commercially pure titanium (cpTi) substrate topography on adherent macrophage osteogenic and osteoinductive cytokine expression. J774A.1 murine macrophage cell adhesion was examined by scanning electron microscopy, 0-72 h following plating onto polished, machined, and grit-blasted cpTi surfaces. TGFbeta1 and BMP-2 gene expression by adherent macrophages was determined by the reverse transcription polymerase chain reaction. Macrophage adhesion increased with time on all surfaces and spreading increased with increasing surface roughness (polished < machined < grit-blasted). BMP-2 expression was not evident for cells adherent to polished cpTi at 24 h. In contrast, BMP-2 expression occurred at 24 h in cells adherent to machined and grit-blasted cpTi. BMP-2 expression was evident on all surfaces at 72 h and was greatest in grit-blasted titanium adherent cells. Increasing concentrations of cytochalasin B (0-50 microM) inhibited macrophage spreading and reduced BMP-2 mRNA expression, suggesting a relationship between cell shape and BMP-2 expression. This was further characterized using anti-beta1 and anti-beta3 integrin antibodies. The anti-beta1 integrin antibodies inhibited adherent macrophage BMP-2 mRNA expression. Anti-beta3 integrin antibody treatment only modestly reduced BMP-2 mRNA expression. Endosseous implant surface topography induced changes in macrophage shape that were associated with changes in BMP-2 expression in J774A.1 mouse macrophage cell line. This first demonstration of BMP-2 expression by cpTi adherent macrophages suggests that the macrophage may contribute surface-specific osteoinductive signals during bone formation at implanted alloplastic surfaces.

Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2.

Bone wound healing requires osteoinductive signals that are attributed to (the) bone morphogenetic proteins (BMPs). The cellular origin of such osteoinductive signals has only been partially elucidated. Because of the central role of the macrophage in cutaneous wound healing, we hypothesized that the macrophage could play a similar role in osseous healing. It was the aim of the present investigation to examine the possible expression of BMP by the macrophage, and to evaluate the contribution of macrophage products to an early step of bone formation modeled in an in vitro culture system. The synthesis of BMP-2 and BMP-6 by cultured human and murine macrophages was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). When human mesenchymal stem cells (hMSCs) were grown in conditioned media from J774A.1 cells, alkaline phosphatase expression increased. This induction was blocked by anti-BMP-2 antibody and by anti-transforming growth factor-beta1 (TGF-beta1) antibody. Modeling of the macrophage expression of osteoinductive signals by potential physiological situations was evaluated by treatments with lipopolysaccharide (LPS) or macrophage chemotactic peptide-1 (MCP-1). Macrophage BMP-2 expression was reduced by proinflammatory LPS stimulation (which was confirmed to induce release of the proinflammatory cytokine, TNF-alpha), and conditioned media from LPS-treated macrophages had no ability to increase alkaline phosphatase activity in hMSCs. This first study of macrophage BMP-2 expression indicates that the macrophage is capable of physiological regulation consistent with a key role in osteoinduction for osseous wound healing.

Anodic oxidation and hydrothermal treatment of titanium results in a surface that causes increased attachment and altered cytoskeletal morphology of rat bone marrow stromal cells in vitro.

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.

The effect on immunocytes of anodic oxide titanium after hydrothermal treatment.

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.