Effect of MC3T3-E1osteoblast-like cell differentiation state on response to titanium surface roughness and exogenous arachidonic acid

Osseointegration is influenced by titanium surface roughness and the inflammatory state of the osteotomy wound site during healing. Arachidonic acid (AA) and its metabolites (prostanoids, leukotrienes, etc.) regulate inflammation and have been shown to influence osteoblast proliferation (cell number) and differentiation (alkaline phosphate and calcium deposition in the matrix). Non-steroidal anti-inflammatory drugs are frequently used following surgery and may affect osseointegration by modulating prostaglandin production and hence osteoblast behavior.;The purpose of this study was to investigate the effects of exogenous AA and Ti surface roughness on MC3T3-E1 osteoblast-like cell proliferation and differentiation.;MC3T3-E1 osteoblast-like cells were cultured in minimum essential medium a (aMEM) containing 10% fetal bovine serum (FBS) and antibiotics (standard media) at 37° C on plastic and smooth and rough Ti. A number of various culture conditions were used to determine how the cells responded to changes in surface roughness and varying doses of AA. The first type of media contained ascorbic acid (50 microg/mL) added to the standard media. Cells were cultured on plastic and smooth and rough Ti for 4, 7, 14, and 21 days and treated with two dose ranges of AA for the last 24 hours of culture; the first was 0, 0.1, 1.0, or 10 nM AA and the second was 0, 10, 25, or 50 microM AA. In addition, an alternative treatment regimen was used which involved continuous AA treatment (0 or 10 microM AA) from day 2 of culture to harvest. The second type of media was an osteogenic medium which consisted of 10 mM beta-glycerophosphate added to the ascorbate-containing media. As before, treatment with AA (0, 10, 25, 50 or 100 microM) was for the last 24 hours of culture or continuously from day 2.;For all cell studies, treatment groups contained an n of 4 and each data point is equivalent to the mean (n = 4) +/- the standard error of the mean (SEM). ANOVA was used to analyze the data and post hoc testing was performed using Student's t-test with Bonferroni correction. P-values of less than 0.05 were considered significant.;Cell proliferation, which was only measured after 24 hours of treatment in ascorbate media, was significantly reduced on all surfaces in 21 day cultures treated with 25 and 50 microM AA versus untreated control surfaces. Other treatment doses and times in culture failed to display any consistent trends.;In contrast, continuous AA treatment in osteogenic media demonstrated a consistent trend. Continuous treatment with AA in osteogenic media led to an inhibitory effect on ALPase for cells cultured on plastic and smooth Ti surfaces at day 4, while day 7 and 14 cultures revealed significantly increased ALPase activity on all 3 surfaces in the presence of AA. The effects of AA on ALPase in cultures on smooth and rough Ti leveled off by day 21, but the effect on plastic remained. Increased differentiation was also apparent when calcium deposition was measured. Continuous AA treatment in osteogenic media led to a statistically significant, dose-dependent increase in calcium accumulation in plastic and smooth Ti cultures at day 21, while cells cultured on rough Ti exhibited a significant increase at 25 microM.;Taken together, the results show that MC3T3-E1 cells display a differential response to AA based on their maturation state in the osteogenic pathway. Continuous treatment of MC3T3-E1 cells with AA in osteogenic media led to increased ALPase activity (days 7, 14) on all surfaces. Continuous treatment with AA for 21 days also increased calcium accumulation on plastic and smooth Ti at all doses. In addition, cultures grown on rough Ti exhibited significantly increased calcium deposition at the 25 microM dose at 21 days. Treatment with AA may promote increased osteoblast differentiation by decreasing proliferation and increasing ALPase activity in the middle phase of osteogenic differentiation and calcium deposition in the matrix in the later phase of differentiation.;As NSAIDs are frequently prescribed following implant placement, this study shows that treatment modalities which affect AA metabolism and may affect osteoblast behavior juxtaposed Ti implants. Modulation of AA metabolism may open the way for improved implant outcomes.