It has to be noted that without a pre-treatment with anti-integrin-1 or anti-integrin-3 antibodies, mRNA expression of ALP was also decreased in cultures on both types of substrates, when day 1 is compared to day 3. anti-integrin-1 or anti-integrin-3 antibodies. Furthermore, after a pre-treatment with either antibody, mRNA expression of integrin-3 and ALP was decreased, on both types of substrate. In conclusion, osteoblast-like cells have the ability to compensate to great extent for the blocking strategy as applied here. Still, integrin-1 and 3 seem to play different roles in attachment, proliferation, and differentiation Cethromycin of osteoblast-like cells, and responses on CaP-coated substrates differ to non coated Ti. Furthermore, the influence on ALP expression suggests involvement of both integrin subunits in signal transduction for cellular differentiation. == Introduction == For implants used in orthopedic or dental surgery the implant-to-bone contact is influenced by the characteristics of the implant, like the surface structure and chemical composition. Regarding Cethromycin the latter, calcium phosphate (CaP) coatings have shown to favor the bone response in vivo [1,2]. However, the mechanism responsible for the improved osteogenic response to CaP coatings is still not clear. Studying the mechanism in vivo is complicated by many factors. Still, cell culture models can be designed to study (parts of) the underlying mechanisms. In vitro, it is known that cells attach to extracellular matrix (ECM) proteins, which are adsorbed to the surface of the material. Specific transmembrane proteins, called integrins, connect the cells to the ECM proteins. These integrins consist of an – and a -unit. Osteoblasts generally express integrin subunits 1, 2, 3, 4, 5, 6, v, 1, 3, and 5 [3]. In Table1, Cethromycin an overview is listed of combinations of integrin subunits and their protein ligands [4]. Important ECM proteins for the attachment Cethromycin of osteoblasts are fibronectin, collagen type I, and vitronectin. For attachment to fibronectin and collagen type I, osteoblasts can express integrin subunit-1. For attachment to vitronectin, they may use integrin subunit-3 [5]. Once cells have attached, the integrin is also involved in passing information from the ECM to the cell (outside-in-signaling), and from the cell towards the ECM (inside-out-signaling) [6]. These signaling pathways can regulate cellular adhesion, migration, and subsequent behavior to a great extent. == Table 1. == An overview of possible integrin subunits and their protein ligands [2] In this study the importance of integrins in the bone-implant-interface was examined on two different implant materials (either CaP-coated or non coated Ti), with the functional blocking of integrins. We hypothesize that blocking integrin-1 and integrin-3 with specific antibodies will have an inhibitory effect on the initial attachment, and thus will delay proliferation and early differentiation of osteoblast-like cells. Furthermore, Cethromycin we hypothesize that differences in cellular response exist between cultures on CaP coatings and non coated Ti. For this purpose, carbonated hydroxyapatite (HA) coatings were deposited with the electrostatic spray deposition (ESD) technique. Subsequently, anti-integrin-1 and anti-integrin-3 antibodies were added to primary osteoblast-like cells, and PLS1 cells were cultured on both substrates. The initial cellular attachment, proliferation, and cell shape were evaluated. Further, gene expression for integrins 1 and 3, and alkaline phosphatase, was examined with quantitative PCR. Results were compared to cultures without antibodies added to the cells on both materials. == Materials and methods == == Substrates == Disc-shaped commercially pure (cp) Ti substrates with a diameter of 12 mm and a thickness of 1 1.5 mm were cleaned ultrasonically in isopropanol for 5 min. Subsequently, the substrates were not coated, or provided with a CaP coating using the ESD technique. The ESD coating procedure was performed using a vertical ESD set-up (Advanced Surface Technology, Bleiswijk, The Netherlands) with a two component-nozzle, as described previously [7,8]. The solvents for the coating consisted of 6.25 mM Ca(NO3)2 2H2O and 3.5 mM H3PO4in butyl carbitol. The applied voltage was 6.36.4 kV, the flow rate 1 mL per hour per solvent, the deposition time 45 min, and the substrate temperature 350 C. The nozzle-to-substrate distance was 20 mm. The deposited coatings had a thickness of 2 m. After deposition, the substrates were subjected to an additional heat-treatment for 2 h at a temperature of 700 C in air to crystallize the coating. After the heat-treatment, coatings were characterized by X-ray diffraction, Fourier-transform infrared,.