Cytotoxicity testing of methyl and ethyl 2-cyanoacrylate using direct contact assay on osteoblast cell cultures.

PURPOSE: Cyanoacrylate has been used as a commercial tissue adhesive. Recently, ethyl 2-cyanoacrylate has been suggested for the fixation of onlay autogenous bone graft. However, ethyl 2-cyanoacrylate must be biocompatible with bone tissue. This study evaluated the cytotoxicity of cyanoacrylate adhesives using a direct contact assay on human oral osteoblast cells. MATERIALS AND METHODS: Osteoblastic cells derived from human alveolar bone of the mandible were cultured with or without cyanoacrylate. The CA1 group contained methyl 2-cyanoacrylate, the CA2 group contained ethyl 2-cyanoacrylate, and the CA3 group did not contain cyanoacrylate (control). This study investigated cell morphology, which included the inhibition zone, and cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which was measured as optical density. Data from the MTT assay were tested statistically using SigmaStat 3.5. RESULTS: Dead cells found around the CA1- and CA2-treated cells constituted inhibitory zones that varied from 200 to 500 µm. There was no inhibitory zone in the CA3 group. Cell viability evaluated by the MTT assay showed that the CA2 and CA3 optical densities were not significantly different. The CA1 optical densities differed significantly from the CA3 optical densities. CONCLUSIONS: Within the limits of this study, the MTT method supported the conclusion that ethyl 2-cyanoacrylate is biocompatible according to a direct contact assay on human osteoblast cell cultures and suggests its usefulness in bone graft fixation.

Treatment with a growth factor-protein mixture inhibits formation of mineralized nodules in osteogenic cell cultures grown on titanium.

Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. This study aimed to evaluate the effects of a well-defined PRP-like mixture containing platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin [growth factors (GFs) + proteins] on the development of the osteogenic phenotype on titanium (Ti) in vitro. Human alveolar bone-derived osteoblastic cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs + proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs + proteins and 1:10 or 1:100 dilutions of the mixture. Treated human-derived cell cultures exhibited a significantly higher number of cycling cells at days 1 and 4 and of total cells at days 4 and 7, significantly reduced alkaline phosphatase (ALP) activity at days 4, 7, and 10, and no Alizarin red-stained areas (calcium deposits) at day 14, indicating an impairment in osteoblast differentiation. Although the 1:10 and 1:100 dilutions of the mixture restored the proliferative activity of rat-derived osteogenic cells to control levels and promoted a significant increase in ALP activity at day 10 compared with GFs + proteins, mineralized nodule formation was only observed with the 1:100 dilution ( approximately 50% of the control). These results showed that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti.

Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures.

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red-stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria-derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.

Mast Cell Mediators Inhibit Osteoblastic Differentiation and Extracellular Matrix Mineralization.

Mast cells are multifunctional immune cells that participate in many important processes such as defense against pathogens, allergic reactions, and tissue repair. These cells perform their functions through the release of a wide variety of mediators. This release occurs mainly through cross-linking IgE (immunoglobulin E) bound to high affinity IgE receptors by multivalent antigens. The abundance of mast cells in connective tissue, surrounding blood vessels, and their involvement in the early stages of bone repair support the possibility of physiological and pathological interactions between mast cells and osteoblasts. However, the participation of mast cell mediators in osteogenesis is not fully understood. Therefore, the objective of this work was to investigate the role of mast cell mediators in the acquisition of the osteogenic phenotype in vitro. The results show that pooled mast cell mediators can affect proliferation, morphology, and cytoskeleton of osteoblastic cells, and impair the activity and expression of alkaline phosphatase as well as the expression of bone sialoprotein. Also, mast cell mediators inhibit the expression of mRNA for those proteins and inhibit the formation and maturation of calcium nodules and consequently inhibit mineralization. Therefore, mast cell mediators can modulate osteogenesis and are potential therapeutic targets for treatments of bone disorders.

Changes in actin and tubulin expression in osteogenic cells cultured on bioactive glass-based surfaces.

The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass-based materials are due to altered mRNA and protein levels. Primary rat-derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real-time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals.