Abstract
Nitric oxide is a free radical which in vivo is solely produced during the conversion of the amino acid arginine into citrulline by nitric oxide synthase enzymes. Recently, the importance of nitric oxide on inflammation and bone metabolism has been investigated. However, the knowledge regarding possible in vitro effects of arginine supplementation on chondrogenic differentiation is limited.
ATDC5, a cell line which is derived from mouse teratocarcinoma cells and which is characterized as chondrogenic cell line, were proliferated in Dulbecco's Modified Eagle Medium (DMEM)/F12 and subsequently differentiated in proliferation medium supplemented with insulin, transferrin and sodium-selenite and where arginine was added in four different concentrations (0, 7.5, 15 and 30 mM). Samples were harvested after 7 or 10 days and were stored at −80 °C for subsequent RNA isolation for qPCR analysis. To determine chondrogenic differentiation, Alcian Blue staining was performed to stain the proteoglycan aggrecan, which is secreted by differentiated ATDC5 cells. All measurements were performed in triplo.
Alcian Blue staining showed a qualitative increase of proteoglycan aggrecan secretion in differentiated ATDC5 cells after treatment with 7 and 15 mM arginine, with additional increased expression of ColII, ColX, Bmp4 and Bmp6. Treatment with 30 mM arginine inhibited chondrogenic differentiation and expression of aforementioned genes, however, Cox-2 and Vegfa gene expression were increased in these samples. Bmp7 was not significantly expressed in any experimental condition.
The obtained results are suggestive for a dose-dependent effect of arginine supplementation on chondrogenic differentiation and associated gene expression, with 7.5 and 15 mM as most optimal concentrations and implications for apoptosis after incubation with 30 mM arginine. A future recommendation would be to investigate the effects of citrulline in a similar experiment, as this shows even more promising results to enhance the nitric oxide metabolism in sepsis and bone healing.
