smv at doses of 0.5 , 1 , 1.5 , and 2 mg was incorporated into the barrier membrane and was then exposed to osteoblastic cell line ( saos2 ) . the dose of smv , which expressed maximum cell metabolism , was assessed [ figure 1 ] . simvastatin at different concentrations ( 0.5 , 1 , 1.5 , 2 mg ) an saos-2 cell line was procured from the national center for cell sciences ( nccs ) , pune , india . the cells were cultured in a humidified atmosphere ( 95% air , 5% co2 ) at 37c in dulbecco 's modified eagle 's medium ( dmem ; biochrome , berlin , germany ) containing 10 ml of penicillin / streptomycin solution and 1 ml of amphotericin b supplemented with 10% fetal bovine serum ( fbs ; biochrome ) . upon reaching confluence , cells between the passages 7 and 10 were tested for viability using tryptophan blue and were used for the cell metabolism assay . four different dosages ( 0.5 , 1 , 1.5 , 2 mg ) and four specimens in each dosage ( n=16 ) were trimmed to an approximate size of 55 mm , placed on the floor of 24-well micro culture dishes , and immersed in a serum - free cell culture medium for 1 h. thereafter , this medium was replaced by the medium supplemented with serum . using a micropipette , 510 cells / ml of human osteoblast - like cells ( saos-2 ) suspended in dmem supplemented with 10% fbs were seeded on the surface of the collagen membrane . the cell metabolism assay was carried out in accordance with what was proposed by mosmann . briefly , the membranes were retrieved from the wells after 24 h ; 100 l of 0.5% ( 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide ) ( mtt ) was added and then incubated at 37c for 4 h. after incubation , the mtt containing medium was removed from the plate and 100 l of solubilizing solution consisting of 20% sodium lauryl sulfate in 50% dimethylformamide was added to each well to dissolve the formazan crystals formed from the tetrazolium salts . the optical density ( od ) of the colored complex formed was read at a wavelength of 650 nm using a spectrophotometer ( molecular devices , sunnyvale , ca , usa ) . the number of viable cells adhering to the membranes ( directly proportional to od ) was calculated based on the readings obtained from the spectrophotometer . the results obtained were analyzed statistically and comparisons were made within each group using one - way analysis of variance ( anova ) followed by a bonferroni test . an saos-2 cell line was procured from the national center for cell sciences ( nccs ) , pune , india . the cells were cultured in a humidified atmosphere ( 95% air , 5% co2 ) at 37c in dulbecco 's modified eagle 's medium ( dmem ; biochrome , berlin , germany ) containing 10 ml of penicillin / streptomycin solution and 1 ml of amphotericin b supplemented with 10% fetal bovine serum ( fbs ; biochrome ) . upon reaching confluence , the cells were detached using trypsin  ethylenediaminetetraacetic acid ( edta ) solution . cells between the passages 7 and 10 were tested for viability using tryptophan blue and were used for the cell metabolism assay . four different dosages ( 0.5 , 1 , 1.5 , 2 mg ) and four specimens in each dosage ( n=16 ) were trimmed to an approximate size of 55 mm , placed on the floor of 24-well micro culture dishes , and immersed in a serum - free cell culture medium for 1 h. thereafter , this medium was replaced by the medium supplemented with serum . using a micropipette , 510 cells / ml of human osteoblast - like cells ( saos-2 ) suspended in dmem supplemented with 10% fbs were seeded on the surface of the collagen membrane . the cell metabolism assay was carried out in accordance with what was proposed by mosmann . briefly , the membranes were retrieved from the wells after 24 h ; 100 l of 0.5% ( 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide ) ( mtt ) was added and then incubated at 37c for 4 h. after incubation , the mtt containing medium was removed from the plate and 100 l of solubilizing solution consisting of 20% sodium lauryl sulfate in 50% dimethylformamide was added to each well to dissolve the formazan crystals formed from the tetrazolium salts . the optical density ( od ) of the colored complex formed was read at a wavelength of 650 nm using a spectrophotometer ( molecular devices , sunnyvale , ca , usa ) . the number of viable cells adhering to the membranes ( directly proportional to od ) was calculated based on the readings obtained from the spectrophotometer . the results obtained were analyzed statistically and comparisons were made within each group using one - way analysis of variance ( anova ) followed by a bonferroni test . during the experimental period , there was no evidence of toxic residues from any one of the membranes and no signs of bacterial or fungal contamination on the well chamber . data were represented as the mean  standard deviation . among the four different dosages tested , 1.5 mg of smv ( 48.431.32 ) incorporated collagen membrane showed increased cell metabolism at a 24-h time period , which was significantly higher ( p<0.05 ) when compared with all other dosages . although 0.5 mg of smv ( 32.643.89 ) , 1 mg of smv ( 37.646.49 ) , and 2 mg of smv ( 30.232.67 ) showed increased cell metabolism , the result was not statically significant when compared to 1.5 mg of smv . at 72 h , no further changes occurred in the cell metabolism assay [ figure 2 ] . dose - dependent effect of smv on cell metabolism ( mtt assay ) in human osteoblast - like saos-2 cells . during the experimental period , there was no evidence of toxic residues from any one of the membranes and no signs of bacterial or fungal contamination on the well chamber . data were represented as the mean  standard deviation . among the four different dosages tested , 1.5 mg of smv ( 48.431.32 ) incorporated collagen membrane showed increased cell metabolism at a 24-h time period , which was significantly higher ( p<0.05 ) when compared with all other dosages . although 0.5 mg of smv ( 32.643.89 ) , 1 mg of smv ( 37.646.49 ) , and 2 mg of smv ( 30.232.67 ) showed increased cell metabolism , the result was not statically significant when compared to 1.5 mg of smv . at 72 h , no further changes occurred in the cell metabolism assay [ figure 2 ] . dose - dependent effect of smv on cell metabolism ( mtt assay ) in human osteoblast - like saos-2 cells . in this study , the stimulatory effect of statins on osteoblastic cells has been shown . due to the importance of regenerative therapy in periodontal management , we investigated the effect of smv incorporated collagen membrane on osteoblast - like saos-2 cells in vitro . in this study , we measured the cell metabolism by mtt assay . this assay determines the activity of mitochondrial dehydrogenases and correlates with cell number and cell viability . an mtt assay was used in our study in preference to h and e staining as the number of viable osteoblasts adhering to the membrane could be assessed accurately based on the degree of absorbance registered on the spectrophotometer . to our knowledge , this is the first study that has examined the effect of smv incorporated barrier membrane in osteoblast - like saos-2 cells . smv was chosen among the various hmg - coa reductase inhibitors because different statins have been reported to have a varying effects on the bone , with lovastatin and pravastatin exhibiting the least effect and statins such as smv , atrovastatin , and cerivastatin exerting greater effects , in previous studies , the effect of smv on cell proliferation and metabolism has been shown to vary according to the cell type , type of statins , and their concentration . in the present study , smv enhanced the cell metabolism of human osteoblast - like saos-2 cells at 24 h and the maximum effect was obtained at a concentration of 1.5 mg of smv . other studies demonstrated that smv induced osteoblastic differentiation and mineralization in mc3t3-e3 osteoblasts and bone marrow stromal cells in vitro . therefore , we used this concentration ( 1.5 mg of smv ) for further determination of alkaline phosphatase ( alp ) activity , osteopontin , and bmp-2 . these results indicate the beneficial effect of collagen with smv on human osteoblast - like saos-2 cells , suggesting its potential as a drug for periodontal regenerative therapy . however , long - term studies using different vehicles and concentrations of smv should be carried out to affirm the observations of our study .
background : simvastatin ( smv ) is one of the cholesterol - lowering pharmacological drugs . 
 recent studies demonstrate that it has a bone stimulatory effect . 
 the present study was designed to investigate the effect of smv along with collagen membrane on osteoblast - like saos-2 cells and also to standardize the dosage of smv to be incorporated into the collagen membrane to achieve regeneration.materials and methods : smv at doses of 0.5 , 1 , 1.5 , and 2 mg was incorporated into the collagen membrane and cell metabolism was assessed by ( 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide ) ( mtt ) assay for 24 h.results:smv enhanced cell metabolism dose dependently at 24-h time and the maximum effect was obtained at a concentration of 1.5 mg of smv.conclusion:these results indicate that collagen with 1.5 mg smv exhibits positive effect on cell metabolism of human osteoblast - like saos-2 cells .