Effect of HNE on osteoblast markers ALPase, OC, and Col I. Human osteoarthritic osteoblasts were incubated with increasing concentrations of HNE for 48 hours and then ALPase activity (a) and Col I protein level (e) were determined in cellular extract as described in Materials and methods. The OC release (c) was determined in culture medium by enzyme-linked immunosorbent assay. For mRNA level, cells were incubated for 4 hours in the absence or presence of indicated concentrations of HNE, total RNA was isolated and reverse-transcribed into cDNA, and ALPase (b), OC (d), and Col I (f) were quantified using real-time polymerase chain reaction. All experiments were performed in triplicate, and negative controls without template RNA were included in each experiment as indicated in Materials and methods. mRNA levels were normalised to those of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA. Data are means ± standard error of the mean of n = 3 and expressed as a percentage of untreated cells. Statistics: Student unpaired t test; *p < 0.05, **p < 0.01. ALPase, alkaline phosphatase; Col I, type I collagen; HNE, 4-hydroxynonenal; OC, osteocalcin.