Collagen with simvastatin promotes cell metabolism in osteoblast-like SaOS-2 cells.

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.

Periodontal disease is a major oral health problem. Over the years, various treatment modalities have been tried with varying success to correct periodontal attachment and alveolar bone loss resulting from this disease. The most desirable outcome of such procedures is regeneration of the periodontal tissues lost because of disease. Regenerative therapy requires the introduction of an agent, which not only hampers tissue destruction but also enhances the regenerative capabilities of the periodontal tissues.[1] Pharmacologic agents offer great promise in this direction. Pharmacologic compounds, which have been shown to affect bone growth, could offer a safe and cost-effective alternative to this problem.[2] Simvastatin (SMV), which is used for the treatment of hypercholesterolemia, is a universally accepted and relatively inexpensive drug. Its long-term systemic administration in humans has been shown to result in increased bone mineral density.[3]

SMV is a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase) which is a rate-limiting enzyme of the cholesterol synthesis pathway.[3] Since cholesterol is the main product of the mevalonate pathway, statins are used orally to treat hypercholesterolemia and hyperlipidemia. The safety profile of statins is well documented.[4]

SMV, a synthetic statin, has a number of pleiotropic effects as well. In addition to its anti-resorptive actions, it has been found to exert anabolic effects on bone. These effects have been elucidated in the form of increased bone mineral density in diabetes mellitus patients who were administered statins systemically for the correction of increased cholesterol levels.[5] Furthermore, systemic administration of SMV is found to be associated with a reduced risk of tooth loss in patients diagnosed with chronic periodontitis as observed by a retrospective analysis over a 7-year period.[6] The anabolic effects on bone have been attributed mainly to an upregulation of bone morphogenetic protein 2 (BMP-2) by SMV and other members of the statin family.[7] The biologically significant anti-inflammatory and antioxidant properties of SMV are the other pleiotropic effects of interest from a periodontal therapeutic standpoint.[8]

However, periodontal therapy necessitates a focused effect in specific defects, suggesting the importance of local application of this drug. A number of studies have concentrated on the effects of locally administered SMV on bone formation.[9-11] It has also been observed that application of this agent to a culture of human periodontal ligament cells enhances their proliferation and metabolism.[12] Therefore, SMV could play a significant role as a therapeutic agent in the treatment of periodontal disease. The successful use of SMV to promote new bone formation depends on its local concentration and appropriate delivery system. Among the various delivery systems used, collagen membrane is generally regarded as a non-toxic, non-allergic, and non-irritating material, and is used as a sustained release vehicle for therapeutic drugs.

So far, no studies concerning the combined effect of SMV along with collagen membrane on SaOS-2 cells have been done. Therefore, in this study, we investigated collagen-incorporated SMV on SaOS-2 cells, especially focusing on cell metabolism and also to standardize the dosage of SMV to be incorporated into the collagen membrane.

Materials and Methods

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].

Figure 1

Simvastatin at different concentrations (0.5, 1, 1.5, 2 mg)

Cell culture

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 37°C 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.

24-well plates were selected and 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 5×5 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, 5×104 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.

MTT assay

The cell metabolism assay was carried out in accordance with what was proposed by Mosmann.[13] Briefly, the membranes were retrieved from the wells after 24 h; 100 μl of 0.5% (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) (MTT) was added and then incubated at 37°C for 4 h. After incubation, the MTT containing medium was removed from the plate and 100 μl 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.[13]

Statistical analysis

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. A value of P<0.05 was considered as the level of significance.

Results

Effect of SMV incorporated barrier membrane on cell metabolism (MTT assay)

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 (48.43±1.32) 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 (32.64±3.89), 1 mg of SMV (37.64±6.49), and 2 mg of SMV (30.23±2.67) 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].

Figure 2

Dose-dependent effect of SMV on cell metabolism (MTT assay) in human osteoblast-like SaOS-2 cells. Osteoblastic cells were cultured for 24 h with different concentrations of SMV. Values are expressed as mean ± SD (*significant P<0.05)

Discussion

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.[13] 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,[14] In previous studies,[1516] 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. After 24 h, SMV slightly enhanced metabolic activity, but not significantly. Other studies demonstrated that SMV induced osteoblastic differentiation and mineralization in MC3T3-E3 osteoblasts and bone marrow stromal cells in vitro.[1718] Therefore, we used this concentration (1.5 mg of SMV) for further determination of alkaline phosphatase (ALP) activity, osteopontin, and BMP-2.

Conclusion

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.