T HE ROLE OF NITRIC OXIDE ON THE PROLIFERATION OF A HUMAN OSTEOBLAST CELL L INE S TIMULATED WITH HYDROXYAPATITE Wihaskoro Sosroseno, DDS, PhD; Erwan Sugiatno, DDS, MS, PhD; Abdul Rani Samsudin, BDS; Mohd. Fikri Ibrahim, BSc The aim of the present study was to test the hypothesis that the proliferation of a human osteoblast cell line (HOS cells) stimulated with hydroxyapatite (HA) may be regulated by nitric oxide (NO). The cells were cultured on the surface of HA. Medium or cells alone were used as controls. L-arginine, D- arginine, 7-NI (an nNOS inhibitor), L-NIL (an iNOS inhibitor), L-NIO (an eNOS inhibitor) or carboxy PTIO, a NO scavenger, was added in the HA-exposed cell cultures. The cells were also precoated with anti-human integrin aV antibody. The levels of nitrite were determined spectrophotometrically. Cell proliferation was assessed by colorimetric assay. The results showed increased nitrite production and cell proliferation by HA-stimulated HOS cells up to day 3 of cultures. Anti-integrin aV antibody, L-NIO, or carboxy PTIO suppressed, but L-arginine enhanced, nitrite production and cell proliferation of HA- stimulated HOS cells. The results of the present study suggest, therefore, that interaction between HA and HOS cell surface integrin aV molecule may activate eNOS to catalyze NO production which, in turn, may regulate the cell proliferation in an autocrine fashion. Key Words: HOS cells, hydroxyapatite, nitric oxide, osteoblasts, proliferation INTRODUCTION H ydroxyapatite (HA) is known as a ceramic material widely used for or- thopaedic and dental implants, since this biomaterial has the ability to stimulate osteoblast functions in vitro and in vivo. 1–3 However, the exact mechanisms by which HA induces osteoblast func- tions remain unclear. Previous studies indicated that osteoblasts via surface integrin molecules initially attach and spread on protein-coated HA. 4,5 Signal transduction pathways generated by osteoblast inte- grin-HA interaction may also induce the production of cytokines which may, in turn, regulate cell prolifera- tion and differentiation. 6–8 Nitric oxide (NO) is a gaseous molecule generated from L-arginine under catalization of nitric oxide synthase (NOS), and it plays a crucial role on the nervous, cardiovascular, qatalization, and immune system. 9 Three isoforms of NOS, ie, neural NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3), are recognized. 10 Human osteoblasts have been shown to express all NOS isoforms. 11 However, others found that osteo- blasts expressed eNOS and iNOS molecules only. 12,13 Proinflammatory cytokines and bacterial lipopolysac- charides increased iNOS expression, 11,12,14 whereas the eNOS activities could be induced by stimulators such as estradiol, 15 estrogen, 16 and fluid shear stress. 17 These Wihaskoro Sosroseno, DDS, PhD, is at AIMST University, Malaysia, and the School of Dental Sciences, Universiti Sains Malaysia, Malaysia. Address correspondence to Dr Sosroseno at School of Dentistry, AIMST University, Amanjaya Campus, Sungai Petani 08000, Kedah Darul Aman, Malaysia. (e-mail: wsosroseno@yahoo.com) Erwan Sugiatno, DDS, MS, PhD, is with the Faculty of Dentistry, Gadjah Mada University, Indonesia, and the School of Dental Sciences, Universiti Sains Malaysia, Malaysia. Abdul Rani Samsudin, BDS, is with the College of Dentistry, University of Sharjah, Sharjah, The United Arab Emirate, and the School of Dental Sciences, Universiti Sains Malaysia, Malaysia. Mohd. Fikri Ibrahim, BSc, is at the School of Dental Sciences, Universiti Sains Malaysia, Malaysia. 196 Vol. XXXIV/No. Four/2008 RESEARCH Downloaded from http://meridian.allenpress.com/joi/article-pdf/34/4/196/2034599/0_910_1.pdf by guest on 02 December 2021