Effect Of Alendronate On Proliferation And Osteogenic Differentiation In MG-63 Cells

BackgroundBisphosphonates are the clinically most important class of antiresorptive agents available to treat diseases characterized by osteoclast-mediated bone resorption such as post-menopausal osteoporosis, Paget disease, and tumor-associated bone diseases. They are synthetic, metabolically stable analogues of inorganic pyrophosphate in which the P-O-P bond has been replaced with a non-hydrolyzable P-C-P bond. The diphosphate configuration of both P-O-P and P-C-P contributes to a three-dimensional structure capable of binding divalent ions such as Ca2+ and is the basis for the bone-targeting property of these compounds.Bisphosphonates are traditionally divided into nitrogen-containing (N-) and non-nitrogen-containing (non-N-) categories. N-BPs impede the mevalonate pathway of cholesterol synthesis by competitively inhibitingthe binding of isopentenyl pyrophosphate to farnesyl diphosphate synthase, ultimately resulting in decreased amounts of farnesyl pyrophosphate, an essential donor of prenyl moieties. This decrease results ultimately in lack of prenylation of small GTPases that are essential for osteoclast functions and survival, leading to cell death. Non-N-BPs are incorporated into inert intracellular ATP analogues that have no releasable energy content, thus leading to cell death.It is a common misconception that the mechanism of action of the bisphosphonates is specific to the osteoclast. However, more and more studies have indicated that bisphosphonates may also have a direct action on osteoblasts. For example, Giuliani et al. reported alendronate and etidronate enhanced IL-6 production in osteoblasts. Shanbhag et al. observed that in addition to inhibiting osteolysis, bisphosphonate treatment resulted in a 115% increase in mineralized bone in acetabular implant porosities. Plotkin et al. reported bisphosphonates inhibited apoptosis of osteocyte cell lines and primary murine osteoblasts, as well as human osteoblasts. Pan et al. demonstrated that zolendronate decreased transmembrane RANKL expression on osteoblasts by increasing the activity of a RANKL sheddase, TACE (TNF-a converting enzyme). Thus based on these studies,we have hypothesized that bisphosphonates may have anabolic effects on osteoblasts as well.However, there are some discrepancy about the action ofbisphosphonates on osteoblasts. Im et al. showed that alendronate could increase osteoblast and osteoblast progenitor numbers. While Fumiaki et al. considered alendronate had no effect. Garcia-Moreno et al. reported that high concentrations of alendronate inhibited osteoblast proliferation. And Renholz et al. showed high concentrations of bisphosphonates could lead to impaired mineralization. But D' Aoustp et al. showed that moderately high doses of bisphosphonates (particularly HEBP) affect cell proliferation and increase the production of type I collagen and expression of alkaline phosphatase, osteopontin and bone sialoprotein in osteoblastic cells.ObjectiveTo confirm whether bisphosphonates have direct effects on proliferation and osteogenic differentiation, furthermore, to determine in which concentration bisphosphonates would exert the best efficacy in MG-63 osteoblast-like cell line.Materials and methodsMG-63 osteoblast-like cells were cultured at a density of 400,000 cells/cm² in H-DMEM medium supplemented with 10% fetal bovine serum, L-glutamine (2 mM), 10mM β-glycero-phosphate and 50ng/ml L-ascorbic at 37℃ with 95% humidity and 5% CO₂. Every three or fourdays, when cells grew to 80% confluence, cells were isolated from culture dishes by trypsinization and washed with phosphate-buffered saline(PBS).After passage, MG-63 cells were divided into five groups. Group A: negative control (medium alone); Group B: positive control, treated with 10^-8 Mdexamethasone; Group C, D, E were experimental groups, treated with 10^-6M, 10^-8M, 10^-10M alendronate respectively. After some days, we carried out MTT, alkaline phosphatase bioassay, RT-PCR, von kossa's staining.All statistical analyses were performed with commercially available software (SPSS 11.0). The results are presented as a percentage over controls. The mean and standard errors were obtained from sums of each culture. The data was then analyzed by one-way ANOVA, and the post-hoc Student's twotailed t-test. Statistical significance was tested at an avalue of 0.05.ResultsAlendronate significantly increased the cell number over controls, attaining peak levels at a concentration of 10^-8M. Alkaline phosphatase activity was increased; gene expression of BMP-2, Type I collagen and osteocalcin was enhanced. And alendronate stimulated the formation of mineralization.ConclusionAlendronate aside from its role as inhibitor of osteoclastic bone resorption, is promoter of osteoblast proliferation and maturation.