Effects of nitric oxide synthase inhibitors on osteoclast-like cell formation

Ahn, Seung Kyu; Kim, Jung Kun; Cha, Kyung Suk

Abstract

Orthodontic tooth movement in response to orthodontic force results from actions of osteoclasts and osteoblasts in the cell level. Convincing evidence has now been provided to support the view that osteoclasts are derived from mononuclear cells that originate in the bone marrow or other hematopoietic organs and they migrate to the bones via vascular routes. Nitric oxide(NO), which accounts for the biological properties of endothelium-derived relaxmg factor(EDRF), is the endogenous stimulator of soluble guanylate cylase. The discovery of the formation of nitric oxide(NO) from L-arginine in mammalian tissues and its biologioal roles has, in the last 7 years, thrown new light onto many areas of research. Data from experiments in vitro showed that N-metyl-L-arginine(L-NMA) and L-nitro-L-arginine(L-NAME) are competitive inhibitors of nitric oxide synthase. This study suggest that the multinucleated cells in our culture have characteristics of osteoclasts and that the potential bone cell activity of nitric oxide in vitro may be mediated in part by stimulation of marrow mononuclear cells to form osteoclast-like cells. Bone marrow cells were obtained from tibia of 19-days old chick embryo. After sacrifice, tibia was quickly dissected and the bone were then split to expose the medullary bone. The cells were attached for 4 hours and the nonadherent cells were collected. Marrow cells were cultured in 96-well plate in medium 199. To examine the number of TRAP-positive multinucleated cells(MNCs), 10(-8) M Vit-D3 and various concentration of L-NMA and L-NAME were added at the beginning of cultures and with each medium change. After 7 days of culture, tartrate-resistant acid phosphatase(TRAP) staining was performed for microscopic evaluation. Cells having more than three nuclei per cell were counted as MNCs. The observed results were as follows; 1. 1,25-dihydroxyvitamine D3 stimulated the osteoclast-like multinucleated cells in cultures of chick embryo bone marrow. 2. Nitric oxide synthase inhibitors(NOSI ; N-NMA, N-NAME) stimulated the osteoclast-like cells in cultures of chick embry bone marrow. 3. 1,25-dihydroxyvltamine D3 and nitric oxide synthase inhibitors did not appear to have additive effect on the generation of TRAP-positive MNCs. These results suggest that nitric oxide synthase inhibitors may stimulate the osteoclast-like multinucleated cell formation and fusion in cultures of chick bone marrow.