RESEARCH
Effects of commonly used medications on bone tissue mineralisation in SaOS-2 human bone cell line AN IN VITRO STUDY M. Salai, D. Somjen, R. Gigi, O. Yakobson, S. Katzburg, O. Dolkart From Tel-Aviv Sourasky Medical Center, Tel-Aviv University, Tel-Aviv, Israel
M. Salai, MD, Orthopaedic Surgeon, Professor, Chairman of the Orthopaedic Surgery Division R. Gigi, MD, Orthopaedic Surgeon O. Yakobson, PhD, Researcher O. Dolkart, PhD, Director of the Orthopaedic Research Unit Tel-Aviv Sourasky Medical Center, Division of Orthopedic Surgery, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. D. Somjen, PhD, Director of the Endocrinology Research Lab S. Katzburg, PhD, Researcher Tel-Aviv Sourasky Medical Center, Institute of Endocrinology, Metabolism and Hypertension, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. Correspondence should be sent to Dr O. Dolkart; e-mail: [email protected] ©2013 The British Editorial Society of Bone & Joint Surgery doi:10.1302/0301-620X.95B11. 31158 $2.00 Bone Joint J 2013;95-B:1575–80. Received 25 October 2012; Accepted after revision 9 July 2013
We analysed the effects of commonly used medications on human osteoblastic cell activity in vitro, specifically proliferation and tissue mineralisation. A list of medications was retrieved from the records of patients aged > 65 years filed in the database of the largest health maintenance organisation in our country (> two million members). Proliferation and mineralisation assays were performed on the following drugs: rosuvastatin (statin), metformin (antidiabetic), metoprolol (β-blocker), citalopram (selective serotonin reuptake inhibitor [SSRI]), and omeprazole (proton pump inhibitor (PPI)). All tested drugs significantly stimulated DNA synthesis to varying degrees, with rosuvastatin 5 μg/ml being the most effective among them (mean 225% (SD 20)), compared with metformin 10 μg/ml (185% (SD 10)), metoprolol 0.25 μg/ml (190% (SD 20)), citalopram 0.05 μg/ml (150% (SD 10)) and omeprazole 0.001 μg/ml (145% (SD 5)). Metformin and metoprolol (to a small extent) and rosuvastatin (to a much higher extent) inhibited cell mineralisation (85% (SD 5)). Our results indicate the need to evaluate the medications prescribed to patients in terms of their potential action on osteoblasts. Appropriate evaluation and prophylactic treatment (when necessary) might lower the incidence and costs associated with potential medicationinduced osteoporosis. Cite this article: Bone Joint J 2013;95-B:1575–80.
Our knowledge of the influence of medications used by patients who might also suffer from systemic osteoporosis is limited. We investigated the effects of five families of drugs that are widely prescribed to people aged > 65 years. The age-associated diseases that we addressed included hyperlipidaemia, diabetes mellitus, hypertension, mood disorders and peptic-ulcer disease. The effects of the drugs were tested in the SaOS-2 cell line. SaOS-2 (‘sarcoma osteogenic’) is a non-transformed cell line that was derived from the primary osteosarcoma of an 11-year-old Caucasian girl in 1973.1,2 One of the advantages of using the SaOS-2 line is that the cells become fully differentiated in a manner that osteoblastic cells do naturally, making them a valuable model for studying events associated with the late osteoblastic differentiation stage in human cells.3 In this study we evaluated the effects of some of the most commonly administered medications on bone biology using this cell culture model and analysing their influence on cellular activity.
Materials and Methods A list of medications was retrieved from the files of over two million patients aged
VOL. 95-B, No. 11, NOVEMBER 2013
> 65 years stored in the database of the largest Health Maintenance Organisation (HMO) in Israel. We identified the five most common diseases to be hyperlipidaemia, diabetes mellitus, cardiovascular diseases, mood disorders and peptic ulceration. The most common drug that was prescribed to treat each of these five conditions was chosen to be tested. We performed proliferation and mineralisation assays on rosuvastatin4 (statin), metformin5 (antidiabetic), metoprolol6 (β-blocker), citalopram7 (a selective serotonin re-uptake inhibitor (SSRI)) and omeprazole8 (proton pump inhibitor). The range of doses was chosen based on relevant clinical doses. We also performed dose–response analyses in order to identify the dose that induces the most robust osteoblasts proliferative response in vitro (i.e. DNA synthesis). An SaOS-2 human bone cell line was obtained from American Type Culture Collection (ATCC, Manassas, Virginia) and grown in Dulbecco’s Modified Eagle Medium (DMEM) (SigmaAldrich Israel Ltd, Rehovot, Israel) containing 10% fetal bovine serum (FBS), according to the manufacturer’s instructions. Cells were cultured without any stimulatory supplements or vitamins in Falcon flasks (BD Biosciences, San Jose, 1575
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California) in a humidified incubator at 37°C, using a standard mixture of 95% air and 5% CO2. We added 40 IU/ml of penicillin/streptomycin/ampicillin to each medium. As a control, sub-confluent cultured cells were treated with the culture medium alone. Different doses of rosuvastatin, metformin, metoprolol, citalopram and omeprazole were evaluated either for 24 hours for DNA synthesis as previously described,9 or the cells were grown for 21 days in the presence of dexamethasone 10-8M, 50 μg/ml sodium L-ascorbate (vitamin C) and 10 mM β-glycerophosphate. The growth medium was changed on day four of the month-long culture. All of the studied drugs were obtained from Enzo Life Sciences (Farmingdale, New York). All other chemicals were of analytical grade. Assessment of DNA synthesis. After the drugs had been treated for 22 hours, [H] thymidine was added for two hours and its incorporation into DNA was determined as previously described.9 Alizarin red S staining protocol for calcium detection. Alizarin Red S (Sigma Chemicals, St Louis, Missouri) is an anthraquinone derivative used to identify calcium in biological tissues, with which it forms a complex that stains red. After treatment, the cells were fixed with absolute methanol for ten minutes and stained with Alizarin Red S for one minute. The excess dye was washed with double-distilled water (DDW), and the cells were stained with Light Green (Sigma Chemicals) to detect mineralisation. Cultures were observed under a microscope.10 Quantification of SaOS-2 mineralisation. Cells were grown in six-well plates for 21 days, washed with DDW, fixed in 10% formaldehyde for 15 minutes at room temperature, washed again with DDW, and stained with Alizarin Red at 40 mM for 20 minutes at room temperature with gentle shaking. Extra dye was removed by DDW. The cells were observed microscopically and frozen at -20°C.9 In order to quantify the dye, 800 μl of 10% acetic acid was added to each well, incubated at room temperature for 30 minutes with shaking, scraped off the wells and transferred to Eppendorf tubes, vortexed and heated at 85°C for 30 seconds, cooled and centrifuged at 20 000 g for 15 minutes. The supernatant was transferred to new Eppendorf tubes (Axygen Scientific Inc., Union City, California), and 200 μl of 10% ammonium hydroxide was added to each tube to adjust the pH to 4.1 to 4.5, after which 150 μl of the liquid were transferred to 96-well plates and the optical density (OD) at 405 nm was determined.10 Statistical analysis. Statistical analyses were performed using SPSS v21.0 (SPSS Inc., Chicago, Illinois). The values are expressed as mean and standard deviation (SD) for a minimum of five independent repetitions, and a p-value ≤ 0.05 was considered statistically significant. One-way analyses of variance (ANOVA) were used. When significant differences were found they were followed by Tukey’s Honestly Significant Difference test for non-equal sample size for comparisons. When percentages were compared,
before the data were analysed by ANOVA they were subjected to an arcsine of square root-transformation for percentages and ratios to meet the criteria of the ANOVA method.
Results Effect of selected drugs on DNA synthesis. The addition of the different drugs at different concentrations (0.01 to 200 μg/ml) for 24 hours resulted in dose-dependent modulation of DNA synthesis in these human cells (Fig. 1). Each drug significantly stimulated DNA synthesis to a different extent. The most effective was rosuvastatin 5 μg/ml (by a mean of 225% (SD 20)) followed by metformin 10 μg/ml (by 185% (SD 10)), metoprolol 0.25 μg/ml (by 190% (SD 20)), citalopram 0.05 μg/ml (by 150% (SD 10), and finally omeprazole 0.001 μg/ml (by 145% (SD 5)). Effect of selected drugs on mineralisation. The addition of each drug at the optimal concentration for proliferation for 21 days resulted in no change in cell mineralisation (Fig. 2). Quantification of mineralisation demonstrated that to a small extent metformin and metoprolol, and to a much greater extent rosuvastatin, inhibited cell mineralisation by 85% (SD 5) for the two former drugs and by 43% (SD 5) for the latter (Fig. 2c).
Discussion The results of this study demonstrated that exposure of the bone cells to selected, commonly used medications caused a variety of both positive and negative modulations in the cell line. Statins are commonly used to treat hyperlipidaemia and to reduce the risk of coronary artery disease.11,12 Recent research has shown that some statins can increase bone formation both in vitro and in vivo when applied locally or systemically in high doses.13 Statins are capable of increasing the expression of bone morphogenic protein-2 (BMP2), which positively affects bone formation.14,15 In our study, even the short exposure of bone cells to rosuvastatin caused a significant increase in DNA synthesis. However, prolonged treatment of the cells with rosuvastatin markedly inhibited the mineralisation process, which is essential for bone strength. Statins have been reported to reduce osteoclast activity and induce osteoblast activity leading to bone formation, both in tissue culture and in animal models.16 Some clinical trials have shown that long-term administration of simvastatin is positively correlated with augmentation of bone mineral density at the hip and spine as well as a reduced risk of bone fracture.17 On the other hand, several other reports have suggested that statins have negative effects on fracture healing, and that they reduce bone mineral density.18-20 Increasing attention has recently been paid to the relationship between diabetes mellitus and the risk of osteoporotic fractures.21 Kanazawa22 reported that metformin is capable of reducing the rate of fracture occurrence, apparently mediated by a direct osteogenic effect on THE BONE & JOINT JOURNAL
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Agent concentration (µg/ml) Fig. 1c Graphs showing the effect of different concentrations of a) rosuvastatin (0.02 to 40 μg/ml) and metformin (0.1 to 200 μg/ml), b) citalopram (0.02 to 40 μg/ml) and metoprolol (0.002 to 4 μg/ml) and c) omeprazole (0.002 to 40 μg/ml) on DNA synthesis in the human bone cell line SaOS-2. Results are expressed as the mean percentage change in DNA synthesis (error bars show the standard error of the mean) for between five and eight repetitions. Significant differences from the control are shown by asterisks (* p < 0.05, ** p < 0.01). DNA synthesis of control cells was 20 712 dpm/well (SD 2171), where dpm is disintegrations per minute.
osteoblasts.23 We have been able to confirm that metformin induces osteoblastic proliferation (Fig. 1a), although mineralisation was slightly inhibited (Figs 2a and 2c). Antihypertensive drugs in general, and β-blockers in particular, are among the most commonly prescribed drugs for elderly people in developed countries.24 A wide variety of results have emerged from animal studies regarding the effects of β-blockers on bone healing.25 They are thought to exert positive effects by inhibiting the catabolic influence of the sympathetic nervous system on bone.26,27 Bonnet et al28 reported that low-dose propranolol prevents bone loss in rats following removal of the ovaries through the preservation of trabecular number and thickness and protection against increased cortical porosity. A meta-analysis of VOL. 95-B, No. 11, NOVEMBER 2013
fracture incidence in clinical trials on the use of β-blockers for heart failure found no significant difference between patients randomised to placebo or carvedilol (2.0% vs 2.3%).25 In our study, metoprolol stimulated DNA synthesis (Fig. 1b) and slightly inhibited mineralisation (Figs 2a and 2c). SSRIs represent a class of commonly used antidepressants. An accumulating body of evidence suggests that current SSRI use is associated with a reduction in bone marrow density (BMD) and bone loss.29,30 It has even been suggested that SSRIs may be as detrimental as glucocorticoids to bone loss.29 We demonstrated that citalopram enhanced proliferation (Fig. 1b) and did not affect mineralisation (Figs 2b and 2c) in an in vitro cell-line system. Future
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Fig. 2c Figures 2a and 2b – photographs showing the effect of a) metformin at 10 μg/ml, rosuvastatin at 2 μg/ml and metoprolol at 0.2 μg/ml and b) citalopram at 2 μg/ml and omeprazole at 0.002 μg/ml in the presence of dexamethasone on cell mineralisation in the human bone cell line SaOS-2 examined microscopically. Photographs were taken at 1 × 1 using a scanner. Figure 2c – graphs showing the mean quantified mineralisation as described in the experimental section, and given as mean optical density (OD; at 405 nm)/cells/well with standard error of the mean.
research is required to explore the effects of this class of drugs on bone. In the meantime, the currently available evidence suggests that SSRIs should be added to the list of medications that contribute to osteoporosis, and patients receiving SSRIs should be considered as candidates for BMD testing.30 By inhibiting the production and secretion of stomach acid, PPI therapy may cause a negative calcium balance through malabsorption, resulting in an increased rate of bone loss and a greater risk of fracture.31,32 Histing et al33 reported a delay in fracture repair in mice treated with pantoprazole. Our results demonstrated that low-dose omeprazole, available as an over-the-counter medication, resulted in a slight enhancement of cell proliferation, whereas a higher dose inhibited it (Fig. 1c). There was no noticeable
change in the mineralisation process. Well-designed in vivo studies and human clinical trials are required to address the important question of whether prolonged use of PPI therapy increases the prevalence of osteoporosis and hip fracture in the same population. We are aware of several limitations to this study. All of the experiments were conducted on SaOS-2 cells. The fact that this cell line originated from a child may somewhat affect the ability to apply the results to our patient group. Yet it should be noted that this cell line is used even in the research of age-related comorbidity states such as osteoporosis and atherosclerotic–cardiovascular diseases.34 Moreover, in vitro drug administration studies may not accurately predict in vivo interactions, as alternative metabolic or excretory routes may play a major role in the THE BONE & JOINT JOURNAL
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Fig. 3 Diagrammatic representation of the medications prescribed to patients, in relation to their effects on osteoblast proliferation and mineralisation (↑, enhancement; ↓, inhibition; NE, no effect; PU, peptic ulcer; HTN, hypertension; PPI, proton pump inhibitor; SSRI, selective serotonin re-uptake inhibitor; HTN, hypertension; PUD, peptic ulcer disease; NIDDM (non-insulin dependent diabetes mellitus).
clearance of a drug in vivo. Nevertheless, the importance of our in vitro approach should not be underrated, as our results indicate direct rather than systemic effects of these drugs on osteoblasts. In conclusion, osteoblasts demonstrated significant changes in proliferation and mineralisation following treatment with commonly used drugs (Fig. 3). The exact mechanism underlying those changes warrants investigation. Moreover, our results indicate the need to conduct epidemiological, laboratory and clinical studies to evaluate each old and new medication prescribed to patients with regard to its effects on osteoblasts, mineralisation (Fig. 3) and fracture healing. The potentially bone-preserving effects of the drugs could be particularly applicable in the clinical setting of fracture healing, and of osteoporosis treatment and prevention. VOL. 95-B, No. 11, NOVEMBER 2013
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. This article was primary edited by D. Rowley and first-proof edited by G. Scott.
References 1. Fogh J, Fogh JM, Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst 1977;59:221–226. 2. Rodan SB, Imai Y, Thiede MA, et al. Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res 1987;47:4961–4966. 3. Hausser HJ, Brenner RE. Phenotypic instability of Saos-2 cells in long-term culture. Biochem Biophys Res Commun 2005;333:216–222. 4. Lazzerini PE, Capperucci C, Spreafico A, et al. Rosuvastatin inhibits spontaneous and IL-1-induced interleukin-6 production from human cultured osteoblastic cells. Joint Bone Spine 2013;80:195–200. 5. Mai QG, Zhang ZM, Xu S, et al. Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats. J Cell Biochem 2011;112:2902–2909.
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6. Lai KB, Sanderson JE, Yu CM. High dose norepinephrine-induced apoptosis in cultured rat cardiac fibroblast. Int J Cardiol 2009;136:33–39. 7. Hodge JM, Wang Y, Berk M, et al. Selective serotonin reuptake inhibitors inhibit human osteoclast and osteoblast formation and function. Biol Psychiatry 2013;74:32– 39. 8. Sheraly AR, Lickorish D, Sarraf F, Davies JE. Use of gastrointestinal proton pump inhibitors to regulate osteoclast-mediated resorption of calcium phosphate cements in vivo. Curr Drug Deliv 2009;6:192–198. 9. Somjen D, Kohen F, Lieberherr M, et al. Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A. J Steroid Biochem Mol Biol 2005;93:293–303. 10. Katzburg S, Lieberherr M, Ornoy A, et al. Isolation and hormonal responsiveness of primary cultures of human bone-derived cells: gender and age differences. Bone 1999;25:667–673. 11. Davignon J. Beneficial cardiovascular pleiotropic effects of statins. Circulation 2004;109(Suppl):III39–III43. 12. Kwak B, Mulhaupt F, Myit S, Mach F. Statins as a newly recognized type of immunomodulator. Nat Med 2000;6:1399–1402. 13. Mundy G, Garrett R, Harris S, et al. Stimulation of bone formation in vitro and in rodents by statins. Science 1999;286:1946–1949. 14. Gutierrez GE, Zhang J, Jadhav S, Munoz SA, Mundy GR. Enhanced expression of BMP2/Smad pathway and SP7 by statins during fracture repair. Bone 2008;42:S17– 110. 15. Tang QO, Tran GT, Gamie Z, et al. Statins: under investigation for increasing bone mineral density and augmenting fracture healing. Expert Opin Investig Drugs 2008;17:1435–1463. 16. Maeda T, Matsunuma A, Kawane T, Horiuchi N. Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys Res Commun 2001;280:874–877. 17. Meier CR, Schlienger RG, Kraenzlin ME, Schlegel B, Jick H. HMG-CoA reductase inhibitors and the risk of fractures. JAMA 2000;283:3205–3210. 18. Cauley JA, Jackson R, Pettinger M, et al. Statin use and bone mineral density (BMD) in older women: The Women’s Health Initiative Observational Study. J Bone Min Res 2000;15(Suppl):155. 19. LaCroix AZ, Cauley JA, Jackson R. Does statin use reduce risk of fracture in postmenopausal women?: results from the Women’s Health Initiative Observational Study. J Bone Min Res 2000;15(Suppl):155.
20. van Staa TP, Wegman SLJ, de Vries F, Leufkens HGM, Cooper C. Use of statins and risk of fractures. J Bone Miner Res 2000;15(suppl):155. 21. Barrett-Connor E, Holbrook TL. Sex differences in osteoporosis in older adults with non-insulin-dependent diabetes mellitus. JAMA 1992;268:3333–3337. 22. Kanazawa I. Usefulness of metformin in diabetes-related bone disease. Clin Calcium 2009;19:1319–1325 (in Japanese). 23. Cortizo AM, Sedlinsky C, McCarthy AD, Blanco A, Schurman L. Osteogenic actions of the anti-diabetic drug metformin on osteoblasts in culture. Eur J Pharmacol 2006;536:38–46. 24. Straand J, Rokstad KS. Elderly patients in general practice: diagnoses, drugs and inappropriate prescriptions: a report from the Møre & Romsdal Prescription Study. Fam Pract 1999;16:380–388. 25. Reid IR. Effects of beta-blockers on fracture risk. J Musculoskelet Neuronal Interact 2008;8:105–110. 26. Cherruau M, Facchinetti P, Baroukh B, Saffar JL. Chemical sympathectomy impairs bone resorption in rats: a role for the sympathetic system on bone metabolism. Bone 1999;25:545–551. 27. Togari A. Adrenergic regulation of bone metabolism: possible involvement of sympathetic innervation of osteoblastic and osteoclastic cells. Microsc Res Tech 2002;58:77–84. 28. Bonnet N, Laroche N, Vico L, et al. Dose effects of propranolol on cancellous and cortical bone in ovariectomized adult rats. J Pharmacol Exp Ther 2006;318:1118– 1127. 29. Haney EM, Chan BK, Diem SJ, et al. Association of low bone mineral density with selective serotonin reuptake inhibitor use by older men. Arch Intern Med 2007;167:1246–1251. 30. Haney EM, Warden SJ, Bliziotes MM. Effects of selective serotonin reuptake inhibitors on bone health in adults: time for recommendations about screening, prevention and management? Bone 2010;46:13–17. 31. Graziani G, Como G, Badalamenti S, et al. Effect of gastric acid secretion on intestinal phosphate and calcium absorption in normal subjects. Nephrol Dial Transplant 1995;10:1376–1380. 32. O’Connell MB, Madden DM, Murray AM, Heaney RP, Kerzner LJ. Effects of proton pump inhibitors on calcium carbonate absorption in women: a randomized crossover trial. Am J Med 2005;118:778–781. 33. Histing T, Stenger D, Scheuer C, et al. Pantoprazole, a proton pump inhibitor, delays fracture healing in mice. Calcif Tissue Int 2012;90:507–514. 34. Klein BY, Rojansky N, Ben-Yehuda A, et al. Cell death in cultured human Saos2 osteoblasts exposed to low-density lipoprotein. J Cell Biochem 2003;90:42–58.
THE BONE & JOINT JOURNAL
Effects of commonly used medications on bone tissue mineralisation in SaOS-2 human bone cell line AN IN VITRO STUDY M. Salai, D. Somjen, R. Gigi, O. Yakobson, S. Katzburg, O. Dolkart From Tel-Aviv Sourasky Medical Center, Tel-Aviv University, Tel-Aviv, Israel
M. Salai, MD, Orthopaedic Surgeon, Professor, Chairman of the Orthopaedic Surgery Division R. Gigi, MD, Orthopaedic Surgeon O. Yakobson, PhD, Researcher O. Dolkart, PhD, Director of the Orthopaedic Research Unit Tel-Aviv Sourasky Medical Center, Division of Orthopedic Surgery, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. D. Somjen, PhD, Director of the Endocrinology Research Lab S. Katzburg, PhD, Researcher Tel-Aviv Sourasky Medical Center, Institute of Endocrinology, Metabolism and Hypertension, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. Correspondence should be sent to Dr O. Dolkart; e-mail: [email protected] ©2013 The British Editorial Society of Bone & Joint Surgery doi:10.1302/0301-620X.95B11. 31158 $2.00 Bone Joint J 2013;95-B:1575–80. Received 25 October 2012; Accepted after revision 9 July 2013
We analysed the effects of commonly used medications on human osteoblastic cell activity in vitro, specifically proliferation and tissue mineralisation. A list of medications was retrieved from the records of patients aged > 65 years filed in the database of the largest health maintenance organisation in our country (> two million members). Proliferation and mineralisation assays were performed on the following drugs: rosuvastatin (statin), metformin (antidiabetic), metoprolol (β-blocker), citalopram (selective serotonin reuptake inhibitor [SSRI]), and omeprazole (proton pump inhibitor (PPI)). All tested drugs significantly stimulated DNA synthesis to varying degrees, with rosuvastatin 5 μg/ml being the most effective among them (mean 225% (SD 20)), compared with metformin 10 μg/ml (185% (SD 10)), metoprolol 0.25 μg/ml (190% (SD 20)), citalopram 0.05 μg/ml (150% (SD 10)) and omeprazole 0.001 μg/ml (145% (SD 5)). Metformin and metoprolol (to a small extent) and rosuvastatin (to a much higher extent) inhibited cell mineralisation (85% (SD 5)). Our results indicate the need to evaluate the medications prescribed to patients in terms of their potential action on osteoblasts. Appropriate evaluation and prophylactic treatment (when necessary) might lower the incidence and costs associated with potential medicationinduced osteoporosis. Cite this article: Bone Joint J 2013;95-B:1575–80.
Our knowledge of the influence of medications used by patients who might also suffer from systemic osteoporosis is limited. We investigated the effects of five families of drugs that are widely prescribed to people aged > 65 years. The age-associated diseases that we addressed included hyperlipidaemia, diabetes mellitus, hypertension, mood disorders and peptic-ulcer disease. The effects of the drugs were tested in the SaOS-2 cell line. SaOS-2 (‘sarcoma osteogenic’) is a non-transformed cell line that was derived from the primary osteosarcoma of an 11-year-old Caucasian girl in 1973.1,2 One of the advantages of using the SaOS-2 line is that the cells become fully differentiated in a manner that osteoblastic cells do naturally, making them a valuable model for studying events associated with the late osteoblastic differentiation stage in human cells.3 In this study we evaluated the effects of some of the most commonly administered medications on bone biology using this cell culture model and analysing their influence on cellular activity.
Materials and Methods A list of medications was retrieved from the files of over two million patients aged
VOL. 95-B, No. 11, NOVEMBER 2013
> 65 years stored in the database of the largest Health Maintenance Organisation (HMO) in Israel. We identified the five most common diseases to be hyperlipidaemia, diabetes mellitus, cardiovascular diseases, mood disorders and peptic ulceration. The most common drug that was prescribed to treat each of these five conditions was chosen to be tested. We performed proliferation and mineralisation assays on rosuvastatin4 (statin), metformin5 (antidiabetic), metoprolol6 (β-blocker), citalopram7 (a selective serotonin re-uptake inhibitor (SSRI)) and omeprazole8 (proton pump inhibitor). The range of doses was chosen based on relevant clinical doses. We also performed dose–response analyses in order to identify the dose that induces the most robust osteoblasts proliferative response in vitro (i.e. DNA synthesis). An SaOS-2 human bone cell line was obtained from American Type Culture Collection (ATCC, Manassas, Virginia) and grown in Dulbecco’s Modified Eagle Medium (DMEM) (SigmaAldrich Israel Ltd, Rehovot, Israel) containing 10% fetal bovine serum (FBS), according to the manufacturer’s instructions. Cells were cultured without any stimulatory supplements or vitamins in Falcon flasks (BD Biosciences, San Jose, 1575
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California) in a humidified incubator at 37°C, using a standard mixture of 95% air and 5% CO2. We added 40 IU/ml of penicillin/streptomycin/ampicillin to each medium. As a control, sub-confluent cultured cells were treated with the culture medium alone. Different doses of rosuvastatin, metformin, metoprolol, citalopram and omeprazole were evaluated either for 24 hours for DNA synthesis as previously described,9 or the cells were grown for 21 days in the presence of dexamethasone 10-8M, 50 μg/ml sodium L-ascorbate (vitamin C) and 10 mM β-glycerophosphate. The growth medium was changed on day four of the month-long culture. All of the studied drugs were obtained from Enzo Life Sciences (Farmingdale, New York). All other chemicals were of analytical grade. Assessment of DNA synthesis. After the drugs had been treated for 22 hours, [H] thymidine was added for two hours and its incorporation into DNA was determined as previously described.9 Alizarin red S staining protocol for calcium detection. Alizarin Red S (Sigma Chemicals, St Louis, Missouri) is an anthraquinone derivative used to identify calcium in biological tissues, with which it forms a complex that stains red. After treatment, the cells were fixed with absolute methanol for ten minutes and stained with Alizarin Red S for one minute. The excess dye was washed with double-distilled water (DDW), and the cells were stained with Light Green (Sigma Chemicals) to detect mineralisation. Cultures were observed under a microscope.10 Quantification of SaOS-2 mineralisation. Cells were grown in six-well plates for 21 days, washed with DDW, fixed in 10% formaldehyde for 15 minutes at room temperature, washed again with DDW, and stained with Alizarin Red at 40 mM for 20 minutes at room temperature with gentle shaking. Extra dye was removed by DDW. The cells were observed microscopically and frozen at -20°C.9 In order to quantify the dye, 800 μl of 10% acetic acid was added to each well, incubated at room temperature for 30 minutes with shaking, scraped off the wells and transferred to Eppendorf tubes, vortexed and heated at 85°C for 30 seconds, cooled and centrifuged at 20 000 g for 15 minutes. The supernatant was transferred to new Eppendorf tubes (Axygen Scientific Inc., Union City, California), and 200 μl of 10% ammonium hydroxide was added to each tube to adjust the pH to 4.1 to 4.5, after which 150 μl of the liquid were transferred to 96-well plates and the optical density (OD) at 405 nm was determined.10 Statistical analysis. Statistical analyses were performed using SPSS v21.0 (SPSS Inc., Chicago, Illinois). The values are expressed as mean and standard deviation (SD) for a minimum of five independent repetitions, and a p-value ≤ 0.05 was considered statistically significant. One-way analyses of variance (ANOVA) were used. When significant differences were found they were followed by Tukey’s Honestly Significant Difference test for non-equal sample size for comparisons. When percentages were compared,
before the data were analysed by ANOVA they were subjected to an arcsine of square root-transformation for percentages and ratios to meet the criteria of the ANOVA method.
Results Effect of selected drugs on DNA synthesis. The addition of the different drugs at different concentrations (0.01 to 200 μg/ml) for 24 hours resulted in dose-dependent modulation of DNA synthesis in these human cells (Fig. 1). Each drug significantly stimulated DNA synthesis to a different extent. The most effective was rosuvastatin 5 μg/ml (by a mean of 225% (SD 20)) followed by metformin 10 μg/ml (by 185% (SD 10)), metoprolol 0.25 μg/ml (by 190% (SD 20)), citalopram 0.05 μg/ml (by 150% (SD 10), and finally omeprazole 0.001 μg/ml (by 145% (SD 5)). Effect of selected drugs on mineralisation. The addition of each drug at the optimal concentration for proliferation for 21 days resulted in no change in cell mineralisation (Fig. 2). Quantification of mineralisation demonstrated that to a small extent metformin and metoprolol, and to a much greater extent rosuvastatin, inhibited cell mineralisation by 85% (SD 5) for the two former drugs and by 43% (SD 5) for the latter (Fig. 2c).
Discussion The results of this study demonstrated that exposure of the bone cells to selected, commonly used medications caused a variety of both positive and negative modulations in the cell line. Statins are commonly used to treat hyperlipidaemia and to reduce the risk of coronary artery disease.11,12 Recent research has shown that some statins can increase bone formation both in vitro and in vivo when applied locally or systemically in high doses.13 Statins are capable of increasing the expression of bone morphogenic protein-2 (BMP2), which positively affects bone formation.14,15 In our study, even the short exposure of bone cells to rosuvastatin caused a significant increase in DNA synthesis. However, prolonged treatment of the cells with rosuvastatin markedly inhibited the mineralisation process, which is essential for bone strength. Statins have been reported to reduce osteoclast activity and induce osteoblast activity leading to bone formation, both in tissue culture and in animal models.16 Some clinical trials have shown that long-term administration of simvastatin is positively correlated with augmentation of bone mineral density at the hip and spine as well as a reduced risk of bone fracture.17 On the other hand, several other reports have suggested that statins have negative effects on fracture healing, and that they reduce bone mineral density.18-20 Increasing attention has recently been paid to the relationship between diabetes mellitus and the risk of osteoporotic fractures.21 Kanazawa22 reported that metformin is capable of reducing the rate of fracture occurrence, apparently mediated by a direct osteogenic effect on THE BONE & JOINT JOURNAL
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osteoblasts.23 We have been able to confirm that metformin induces osteoblastic proliferation (Fig. 1a), although mineralisation was slightly inhibited (Figs 2a and 2c). Antihypertensive drugs in general, and β-blockers in particular, are among the most commonly prescribed drugs for elderly people in developed countries.24 A wide variety of results have emerged from animal studies regarding the effects of β-blockers on bone healing.25 They are thought to exert positive effects by inhibiting the catabolic influence of the sympathetic nervous system on bone.26,27 Bonnet et al28 reported that low-dose propranolol prevents bone loss in rats following removal of the ovaries through the preservation of trabecular number and thickness and protection against increased cortical porosity. A meta-analysis of VOL. 95-B, No. 11, NOVEMBER 2013
fracture incidence in clinical trials on the use of β-blockers for heart failure found no significant difference between patients randomised to placebo or carvedilol (2.0% vs 2.3%).25 In our study, metoprolol stimulated DNA synthesis (Fig. 1b) and slightly inhibited mineralisation (Figs 2a and 2c). SSRIs represent a class of commonly used antidepressants. An accumulating body of evidence suggests that current SSRI use is associated with a reduction in bone marrow density (BMD) and bone loss.29,30 It has even been suggested that SSRIs may be as detrimental as glucocorticoids to bone loss.29 We demonstrated that citalopram enhanced proliferation (Fig. 1b) and did not affect mineralisation (Figs 2b and 2c) in an in vitro cell-line system. Future
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Fig. 2c Figures 2a and 2b – photographs showing the effect of a) metformin at 10 μg/ml, rosuvastatin at 2 μg/ml and metoprolol at 0.2 μg/ml and b) citalopram at 2 μg/ml and omeprazole at 0.002 μg/ml in the presence of dexamethasone on cell mineralisation in the human bone cell line SaOS-2 examined microscopically. Photographs were taken at 1 × 1 using a scanner. Figure 2c – graphs showing the mean quantified mineralisation as described in the experimental section, and given as mean optical density (OD; at 405 nm)/cells/well with standard error of the mean.
research is required to explore the effects of this class of drugs on bone. In the meantime, the currently available evidence suggests that SSRIs should be added to the list of medications that contribute to osteoporosis, and patients receiving SSRIs should be considered as candidates for BMD testing.30 By inhibiting the production and secretion of stomach acid, PPI therapy may cause a negative calcium balance through malabsorption, resulting in an increased rate of bone loss and a greater risk of fracture.31,32 Histing et al33 reported a delay in fracture repair in mice treated with pantoprazole. Our results demonstrated that low-dose omeprazole, available as an over-the-counter medication, resulted in a slight enhancement of cell proliferation, whereas a higher dose inhibited it (Fig. 1c). There was no noticeable
change in the mineralisation process. Well-designed in vivo studies and human clinical trials are required to address the important question of whether prolonged use of PPI therapy increases the prevalence of osteoporosis and hip fracture in the same population. We are aware of several limitations to this study. All of the experiments were conducted on SaOS-2 cells. The fact that this cell line originated from a child may somewhat affect the ability to apply the results to our patient group. Yet it should be noted that this cell line is used even in the research of age-related comorbidity states such as osteoporosis and atherosclerotic–cardiovascular diseases.34 Moreover, in vitro drug administration studies may not accurately predict in vivo interactions, as alternative metabolic or excretory routes may play a major role in the THE BONE & JOINT JOURNAL
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Fig. 3 Diagrammatic representation of the medications prescribed to patients, in relation to their effects on osteoblast proliferation and mineralisation (↑, enhancement; ↓, inhibition; NE, no effect; PU, peptic ulcer; HTN, hypertension; PPI, proton pump inhibitor; SSRI, selective serotonin re-uptake inhibitor; HTN, hypertension; PUD, peptic ulcer disease; NIDDM (non-insulin dependent diabetes mellitus).
clearance of a drug in vivo. Nevertheless, the importance of our in vitro approach should not be underrated, as our results indicate direct rather than systemic effects of these drugs on osteoblasts. In conclusion, osteoblasts demonstrated significant changes in proliferation and mineralisation following treatment with commonly used drugs (Fig. 3). The exact mechanism underlying those changes warrants investigation. Moreover, our results indicate the need to conduct epidemiological, laboratory and clinical studies to evaluate each old and new medication prescribed to patients with regard to its effects on osteoblasts, mineralisation (Fig. 3) and fracture healing. The potentially bone-preserving effects of the drugs could be particularly applicable in the clinical setting of fracture healing, and of osteoporosis treatment and prevention. VOL. 95-B, No. 11, NOVEMBER 2013
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. This article was primary edited by D. Rowley and first-proof edited by G. Scott.
References 1. Fogh J, Fogh JM, Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst 1977;59:221–226. 2. Rodan SB, Imai Y, Thiede MA, et al. Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res 1987;47:4961–4966. 3. Hausser HJ, Brenner RE. Phenotypic instability of Saos-2 cells in long-term culture. Biochem Biophys Res Commun 2005;333:216–222. 4. Lazzerini PE, Capperucci C, Spreafico A, et al. Rosuvastatin inhibits spontaneous and IL-1-induced interleukin-6 production from human cultured osteoblastic cells. Joint Bone Spine 2013;80:195–200. 5. Mai QG, Zhang ZM, Xu S, et al. Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats. J Cell Biochem 2011;112:2902–2909.
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6. Lai KB, Sanderson JE, Yu CM. High dose norepinephrine-induced apoptosis in cultured rat cardiac fibroblast. Int J Cardiol 2009;136:33–39. 7. Hodge JM, Wang Y, Berk M, et al. Selective serotonin reuptake inhibitors inhibit human osteoclast and osteoblast formation and function. Biol Psychiatry 2013;74:32– 39. 8. Sheraly AR, Lickorish D, Sarraf F, Davies JE. Use of gastrointestinal proton pump inhibitors to regulate osteoclast-mediated resorption of calcium phosphate cements in vivo. Curr Drug Deliv 2009;6:192–198. 9. Somjen D, Kohen F, Lieberherr M, et al. Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A. J Steroid Biochem Mol Biol 2005;93:293–303. 10. Katzburg S, Lieberherr M, Ornoy A, et al. Isolation and hormonal responsiveness of primary cultures of human bone-derived cells: gender and age differences. Bone 1999;25:667–673. 11. Davignon J. Beneficial cardiovascular pleiotropic effects of statins. Circulation 2004;109(Suppl):III39–III43. 12. Kwak B, Mulhaupt F, Myit S, Mach F. Statins as a newly recognized type of immunomodulator. Nat Med 2000;6:1399–1402. 13. Mundy G, Garrett R, Harris S, et al. Stimulation of bone formation in vitro and in rodents by statins. Science 1999;286:1946–1949. 14. Gutierrez GE, Zhang J, Jadhav S, Munoz SA, Mundy GR. Enhanced expression of BMP2/Smad pathway and SP7 by statins during fracture repair. Bone 2008;42:S17– 110. 15. Tang QO, Tran GT, Gamie Z, et al. Statins: under investigation for increasing bone mineral density and augmenting fracture healing. Expert Opin Investig Drugs 2008;17:1435–1463. 16. Maeda T, Matsunuma A, Kawane T, Horiuchi N. Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys Res Commun 2001;280:874–877. 17. Meier CR, Schlienger RG, Kraenzlin ME, Schlegel B, Jick H. HMG-CoA reductase inhibitors and the risk of fractures. JAMA 2000;283:3205–3210. 18. Cauley JA, Jackson R, Pettinger M, et al. Statin use and bone mineral density (BMD) in older women: The Women’s Health Initiative Observational Study. J Bone Min Res 2000;15(Suppl):155. 19. LaCroix AZ, Cauley JA, Jackson R. Does statin use reduce risk of fracture in postmenopausal women?: results from the Women’s Health Initiative Observational Study. J Bone Min Res 2000;15(Suppl):155.
20. van Staa TP, Wegman SLJ, de Vries F, Leufkens HGM, Cooper C. Use of statins and risk of fractures. J Bone Miner Res 2000;15(suppl):155. 21. Barrett-Connor E, Holbrook TL. Sex differences in osteoporosis in older adults with non-insulin-dependent diabetes mellitus. JAMA 1992;268:3333–3337. 22. Kanazawa I. Usefulness of metformin in diabetes-related bone disease. Clin Calcium 2009;19:1319–1325 (in Japanese). 23. Cortizo AM, Sedlinsky C, McCarthy AD, Blanco A, Schurman L. Osteogenic actions of the anti-diabetic drug metformin on osteoblasts in culture. Eur J Pharmacol 2006;536:38–46. 24. Straand J, Rokstad KS. Elderly patients in general practice: diagnoses, drugs and inappropriate prescriptions: a report from the Møre & Romsdal Prescription Study. Fam Pract 1999;16:380–388. 25. Reid IR. Effects of beta-blockers on fracture risk. J Musculoskelet Neuronal Interact 2008;8:105–110. 26. Cherruau M, Facchinetti P, Baroukh B, Saffar JL. Chemical sympathectomy impairs bone resorption in rats: a role for the sympathetic system on bone metabolism. Bone 1999;25:545–551. 27. Togari A. Adrenergic regulation of bone metabolism: possible involvement of sympathetic innervation of osteoblastic and osteoclastic cells. Microsc Res Tech 2002;58:77–84. 28. Bonnet N, Laroche N, Vico L, et al. Dose effects of propranolol on cancellous and cortical bone in ovariectomized adult rats. J Pharmacol Exp Ther 2006;318:1118– 1127. 29. Haney EM, Chan BK, Diem SJ, et al. Association of low bone mineral density with selective serotonin reuptake inhibitor use by older men. Arch Intern Med 2007;167:1246–1251. 30. Haney EM, Warden SJ, Bliziotes MM. Effects of selective serotonin reuptake inhibitors on bone health in adults: time for recommendations about screening, prevention and management? Bone 2010;46:13–17. 31. Graziani G, Como G, Badalamenti S, et al. Effect of gastric acid secretion on intestinal phosphate and calcium absorption in normal subjects. Nephrol Dial Transplant 1995;10:1376–1380. 32. O’Connell MB, Madden DM, Murray AM, Heaney RP, Kerzner LJ. Effects of proton pump inhibitors on calcium carbonate absorption in women: a randomized crossover trial. Am J Med 2005;118:778–781. 33. Histing T, Stenger D, Scheuer C, et al. Pantoprazole, a proton pump inhibitor, delays fracture healing in mice. Calcif Tissue Int 2012;90:507–514. 34. Klein BY, Rojansky N, Ben-Yehuda A, et al. Cell death in cultured human Saos2 osteoblasts exposed to low-density lipoprotein. J Cell Biochem 2003;90:42–58.
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