Effects of gold and silver nanoparticles in cultured human osteoarthritic chondrocytes.

The aim of our study was to evaluate the effects of gold (Au) and silver (Ag) nanoparticles (NPs) at different concentrations on cultured human osteoarthritic chondrocytes. Cell viability and inducible nitric oxide synthase expression were evaluated by light microscopy. Using transmission electron microscopy (TEM) and field emission gun-based scanning transmission electron microscopy/energy dispersive spectroscopy (FEG-STEM/EDS) allowed us to localize NPs. Gene expression of matrix metalloproteinases 1, 3 and 13 and A disintegrin and metalloproteinase with thrombospondin motifs -4 and -5 were carried out by real-time polymerase chain reaction. A cell viability test indicated a significant dose-dependent cytotoxic effect of both NPs. At concentrations of 160 and 250 µM NP light microscopy showed chondrocytes with signs of apoptosis and an increased presence of inducible nitric oxide synthase. Au-NPs were characterized by FEG-STEM/EDS and TEM analysis localized NPs in cytoplasm and in endocytotic vesicles. On the contrary, the Ag-NPs were undetectable by FEG-STEM/EDS and TEM. Increased gene expression, particularly in matrix metalloproteinase-3, was observed for both NPs (160 µM), but at a concentration of 250 µM the expression of the evaluated genes became lower. Our in vitro studies, although preliminary, suggest that engineered Au and Ag-NPs appear to be harmful for human osteoarthritic chondrocytes in high concentrations (160-250 µM).

In vitro effects of VA441, a new selective cyclooxygenase-2 inhibitor, on human osteoarthritic chondrocytes exposed to IL-1ß.

The aim of this in vitro study was to examine the possible effect of [2-methyl-5-(4-methylsulphonyl)phenyl-1-phenyl-3-(2-n-propyloxyethyl)]-1H-pyrrole (VA441), a new selective cyclooxygenase (COX)-2 inhibitor, on human osteoarthritic (OA) chondrocyte cultivated in the presence or absence of interleukin-1ß (IL-1ß). In particular, we assessed the effects of 1 and 10 µM of VA441, celecoxib, and indomethacin on cell viability, COX-2 and inducible nitric oxide synthase (iNOS) gene expression, prostaglandin E(2) (PGE(2)) production, and nitric oxide (NO) and metalloproteinase-3 (MMP-3) release. Furthermore, we carried out morphological assessment by transmission electron microscopy (TEM). The presence of IL-1ß led to a significant increase in PGE(2), MMP-3, and NO production, as well as a significant increase in gene expression of COX-2 and iNOS. All the drugs tested had a statistically significant inhibitory effect on PGE(2) production and gene expression of COX-2 stimulated by IL-1ß. VA441 and celecoxib significantly suppressed IL-1ß-stimulated MMP-3 and NO and iNOS gene expression in a dose-dependent manner, while indomethacin did not show any significant effect on MMP-3 and NO production or on iNOS gene expression. TEM demonstrated that IL-1ß severely alters the structure of chondrocytes; after co-incubation with VA441 or celecoxib, the cells recovered their ultrastructure. Our data suggest that VA441 and celecoxib may have a beneficial effect on chondrocyte metabolism.

Effects of regenerative radioelectric asymmetric conveyer treatment on human normal and osteoarthritic chondrocytes exposed to IL-1ß. A biochemical and morphological study.

PURPOSE: Osteoarthritis (OA) is a degenerative disease characterized by a progressive loss of articular cartilage extracellular matrix and is due to functional impairments occurring in chondrocytes. In previous works, we highlighted that Regenerative Tissue Optimization (TO-RGN) treatment with radioelectric asymmetric conveyer (REAC) technology influenced the gene expression profiles controlling stem cell differentiation and the pluripotency of human skin-derived fibroblasts in vitro. Since interleukin-1 beta signaling has been implicated in the induction and progression of this disease (through metalloproteinase-3 synthesis and nitric oxide production), we investigated whether REAC TO-RGN might influence the biochemical and morphological changes induced by interleukin-1 beta in normal and OA chondrocytes. METHODS: The induction of metalloproteinase-3 and proteoglycan synthesis was evaluated by a solid-phase enzyme-amplified sensitivity immunoassay, and nitric oxide production was evaluated with the Griess method. Ultrastructural features were observed by transmission electron microscopy. RESULTS: REAC TO-RGN treatment decreased nitric oxide and metalloproteinase-3 production in normal and OA chondrocytes, while inducing an increase in proteoglycan synthesis. OA chondrocytes were more affected by REAC TO-RGN treatment than were normal chondrocytes. Ultrastructural changes confirmed that REAC TO-RGN may counteract the negative effects of interleukin-1 beta incubation. CONCLUSION: The results of this in vitro study suggest that REAC TO-RGN treatment may represent a new, promising approach for the management of OA.

Raloxifene protects cultured human chondrocytes from IL-1ß induced damage: a biochemical and morphological study.

It is well known that estrogens are implicated in the pathogenesis of osteoarthritis. Raloxifene is a selective estrogen receptor modulator used in the treatment of osteoporosis, though little is known about the possible effects of raloxifene on cartilage metabolism. The aim of our study was to evaluate the possible in vitro effects of raloxifene in human osteoarthritis chondrocytes cultivated in the presence or absence of Interleukin-1 beta (IL-1ß) (5 ng/ml). The effects of 0.1 µM and 1 µM of raloxifene in the culture medium were assessed using an immuno-enzymatic method for proteoglycans and metalloproteinase-3 (MMP-3), and the Griess method for nitrite. Gene expression of inducible Nitric Oxide Synthase (iNOS) was detected by real-time PCR. A morphological analysis was performed by transmission electron microscopy (TEM). Cell viability was significantly (P<0.01) reduced by the IL-1ß, and restored to basal levels by raloxifene at both of the concentrations used. The presence of IL-1 ß led to a significant decrease (P<0.01) in proteoglycan levels as well as a significant increase of MMP-3 and NO (P<0.01). When the cells were co-incubated with IL-1ß and raloxifene, a significant and dose-dependent increase in proteoglycans and a reduction of MMP-3 and nitric oxide (NO) were detected. iNOS was noticeably expressed in IL-1ß stimulated chondrocytes, while raloxifene decreased in a very significant manner the gene expression of iNOS at both of the concentrations used. The results of the biochemical evaluation were confirmed by TEM. Our data suggest that raloxifene may have a potential chondroprotective role in osteoarthritis.