Subject(s)
Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , Actins/genetics , Actins/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Culture Techniques , Cell Hypoxia , Cells, Cultured , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression , Humans , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Myocytes, Smooth Muscle/physiology , Osteocalcin/genetics , Osteocalcin/metabolism , Phenotype , Phosphoproteins/genetics , Phosphoproteins/metabolism , von Willebrand Factor/immunology , von Willebrand Factor/metabolism , CalponinsABSTRACT
Previous studies have demonstrated that oxygen environment is an important determinate factor of cell phenotypes and differentiation, although factors which affect pericellular oxygen concentration (POC) in murine chondrogenic cell culture remain unidentified. Oxygen concentrations in vivo were measured in rabbit musculoskeletal tissues, which were by far hypoxic compared to 20% O(2) (ranging from 2.29 ± 1.16 to 4.36 ± 0.51%). Oxygen concentrations in murine chondrogenic cell (C3H10T1/2) culture medium were monitored in different oxygen concentrations (20% or 5%) in the incubator and in different medium volumes (3,700 or 7,400 µl) within 25-cm(2) flasks. Chondrogenic differentiation was assessed by glycosaminoglycan production with quantitative evaluation of Alcian blue staining in 12-well culture dishes. Expression of chondrogenic genes, aggrecan, and type II collagen α1, was examined by quantitative real-time polymerase chain reaction. Oxygen concentrations in medium decreased accordingly with the depth from medium surface, and POC at Day 6 was 18.99 ± 0.81% in 3,700-µl medium (1,480-µm depth) and 13.26 ± 0.23% in 7,400-µl medium (2,960-µm depth) at 20% O(2) in the incubator, which was 4.96 ± 0.08% (1,480-µm depth) and 2.83 ± 0.42% (2,960-µm depth) at 5% O(2), respectively. The differences of POC compared by medium volume were statistically significant (p = 0.0003 at 20% and p = 0.001 at 5%). Glycosaminoglycan production and aggrecan gene expression were most promoted when cultured in moderately low POC, 1,000 µl (2,960-µm depth) at 20% O(2) and 500 µl (1,480-µm depth) at 5% O(2) in 12-well culture dishes. We demonstrate that medium volume and oxygen concentration in the incubator affect not only POC but also chondrogenic differentiation.
Subject(s)
Cell Culture Techniques , Chondrocytes/metabolism , Chondrogenesis , Culture Media , Oxygen , Aggrecans/biosynthesis , Animals , Cell Differentiation , Cell Hypoxia , Cell Line , Cell Proliferation , Chondrocytes/cytology , Collagen Type II/biosynthesis , Female , Glycosaminoglycans/biosynthesis , Mice , Mice, Inbred C3H , RabbitsABSTRACT
Regarding the pathobiology of rheumatoid arthritis, oxidative stress induced by reactive oxygen species is an important mechanism that underlies destructive and proliferative synovitis. Abundant amounts of reactive oxygen species have been detected in the synovial fluid of inflamed rheumatoid joints. It is reported that drugs that block tumor necrosis factor-α reduce the oxidative stress marker levels in patients with rheumatoid arthritis. In this study, we measured reactive oxygen species using a free radical analytical system in patients with rheumatoid arthritis treated with disease-modifying antirheumatic drugs, tumor necrosis factor-α-blocking drugs (infliximab, etanercept), and an interleukin-6-blocking drug (tocilizumab). The serum level of oxidative stress was drastically low in patients with rheumatoid arthritis treated with tocilizumab, suggesting that interleukin-6 blocking therapy reduces not only joint damage, but also vascular degeneration in patients with rheumatoid arthritis. We believe that such a drastic effect would reduce the incidence of cardiovascular events and mortality in patients with rheumatoid arthritis.
