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High magnesium inhibits human osteoblast differentiation in vitro


Magnesium Research. Volume 24, Numéro 1, 1-6, March 2011, Original article

DOI : 10.1684/mrh.2011.0271

Summary  

Auteur(s) : Marzia Leidi, Federica Dellera, Massimo Mariotti, Jeanette A.M. Maier, Dipartimento di Scienze Cliniche Luigi Sacco, Università di Milano, Milano, Italy.

Illustrations

ARTICLE

mrh.2011.0271

Auteur(s) : Marzia Leidi, Federica Dellera, Massimo Mariotti, Jeanette AM Maier jeanette.maier@unimi.it

Dipartimento di Scienze Cliniche Luigi Sacco, Università di Milano, Milano, Italy

Correspondence. J.A.M. Maier, Dipartimento di Scienze Cliniche Luigi Sacco, Università di Milano, Via GB Grassi 74, Milano, Italy

Osteogenesis is a highly coordinated and tightly controlled process that involves the differentiation of mesenchymal cells into pre-osteoblasts and then osteoblasts that ultimately lead to the synthesis and deposition of bone matrix proteins [1]. Osteoblasts are also crucial in maintaining skeletal architecture, being involved not only in the deposition of bone matrix and in the mineralization of the osteoid, but also in regulating the differentiation and the activity of osteoclasts [2].

Magnesium (Mg), the second most abundant intracellular cation, plays an important role in a variety of cellular functions [3]. About 67% of Mg total body stores are found in bone [4]. Mg is important for bone homeostasis and health [5]. Several epidemiological studies have shown an association between low dietary Mg and osteoporosis [5]. Decrease in bone mass/strength during Mg deficiency seems to result from a decrease in bone formation. Several mechanisms are implicated. Mg is mitogenic for bone cells and also affects crystal formation [5]. In addition, Mg deficiency impairs PTH secretion and its effects on bone and kidney, and reduces serum levels of 1.25 (OH)2 -vitamin D [5]. In rodents, experimental Mg deficiency decreased osteoblastic activity [6] and histomorphometry demonstrated a reduced number of osteoblasts [7].

While hypomagnesemia is rather common in western countries, hypermagnesemia is more difficult to encounter due to the ability of the kidney to rapidly respond to elevated Mg serum levels [4]. Serum Mg gradually increases with the decrease of renal function and hypermagnesemia is rather frequent in patients subjected to hemodialysis and peritoneal dialysis [8]. Also, elevated Mg might have harmful effects on osseous metabolism and parathyroid gland function, leading to mineralization defects and contributing to osteomalacic renal osteodystrophy and adynamic bone disease [8]. It has also been demonstrated that prolonged maternal administration of MgSO4 for pre-term labor significantly increases neonatal Mg serum levels and these findings are associated with diffuse osteopenia [9,10]. These observations have been explained by the fact that maternal Mg, which crosses the placenta, can compete with the fetal calcium metabolism [10].

To gain insight into the regulation of osteoblast activity by Mg, we investigated the effects of different concentrations of Mg on the differentiation of human osteoblast-like SaOS-2 cells and normal human osteoblasts.

Materials and methods

Cell culture

SaOS-2, obtained by the American Type Culture Collection (Rockville, MD, USA), were cultured in DMEM containing 10% Fetal Bovine Serum (FBS). Normal Human Osteoblasts (NHOst) were obtained from Cambrex Bio Science (Milano, Italy) and maintained in Osteoblast Growth Media as indicated by the manufacturer (Lonza, Basel, Switzerland) at 37̊C in a humidified atmosphere containing 5% CO2. A Mg free medium (Invitrogen, San Giuliano M.se, Italy) was used to vary Mg concentration by adding MgSO4 (Sigma Aldrich, St Louis, MO).

Cell differentiation

SaOS-2 and NHOst cells at 80% confluence were cultured with a specific induction medium (osteogenic medium, OM) containing 10% FBS, 50 μM L-ascorbate-2-phosphate, 10 mM glycerophosphate, at 37̊C in a 5% CO2 for up to 14 and 19 days respectively, in a 6-well plate. The medium was changed every three days. The analyses were performed on day 14 on SaOS-2 cells and on day 19 on NHOst.

In SaOS-2 and NHOst cells, differentiation was evaluated by quantifying alkaline phosphatase (ALP) enzymatic activity. Briefly, the medium was removed and used for ALP activity, which was determined enzymatically by a colorimetric assay based on the hydrolysis of P-nitrophenyl phosphate by ALP. The absorbance was measured at 405 nm.

To analyze calcium deposition by SAOS-2 cells, the cells were rinsed with PBS followed by fixation (70% ethanol, 1h) and stained for 10 min with 2% Alizarin Red S (pH 4.2). Alizarin Red S staining was released from the cell matrix by incubation in 10% cetylpyridinium chloride in 10mM sodium phosphate (pH 7.0), for 15 min and the absorbance measured at 562 nm [11].

Nitric oxide release

Nitric oxide (NO) was measured as nitrite products in medium at the end of the experiment. Griess reagent was applied for spectrophotometric measurement at 550 nm [12]. The nitrite level was normalized to protein amounts measured by Bradford's method (Bio-Rad, Germany). The experiments were performed in triplicate and repeated 3 times with similar results. Corresponding controls in 0.1, 1.0 or 5.0 mM Mg served as 100%, and data from cells exposed to the osteogenic medium were expressed as percent of control.

Statistical analysis

All experiments were repeated at least three times in triplicate. Data are presented as means±SD. Statistical differences were determined using the unpaired two-tailed Student's t test. ** p<0.01.

Results

Effect of different concentrations of extracellular Mg on the deposition of calcium phosphate matrix by SaOS-2 cells

Physiologic circulating Mg levels are approximately 1.0 mM. 2 mM Mg is a concentration that falls within the physiological/pathophysiological range, whereas 5 mM is a very high concentration which has been previously utilized in in vitro studies [13, 14]. 0.1 mM Mg has been widely used as an experimental model to study the effects of low Mg on cultured mammalian cells [3, 13]. Based on these findings, we utilized the following concentrations of Mg in the culture media: 0.1, 1.0 and 5.0 mM.

Confluent osteoblast-like SaOS-2 cells were cultured in 0.1, 1.0 or 5.0 mM Mg and exposed to an osteogenic medium for 14 days. At the end of the experiment, the cells were stained with Alizarin Red (figure 1A) to evaluate their mineralizing activity by detecting the formation of calcium phosphate in culture [11]. After acid extraction the staining was quantified at 562 nm (figure 1B). Figure 1A and B shows that 5.0 mM Mg markedly inhibited the deposition of mineral matrix, while no significant difference was observed between SaOS-2 cells in 0.1 or 1.0 mM Mg. The inhibition of SaOS-2 differentiation was confirmed by measuring the activity of alkaline phosphatase (ALP), a marker of osteoblast differentiation, in the medium of cells cultured for 14 days in 0.1, 1.0 or 5.0 mM Mg with or without the osteogenic cocktail (figure 1C).

Nitric oxide (NO) is involved in coordinating some phases of osteoblast mineralizing activity [15, 16]. We therefore evaluated the release of NO after 14 days culture in 0.1, 1.0 and 5.0 mM Mg with or without osteogenic stimulus. We found that NO release was not modulated in SaOS-2 cells cultured in different concentrations of Mg and induced to differentiate when compared to the corresponding control (figure 2).

Effect of different concentrations of extracellular Mg on the differentiation of normal human osteoblasts

We then evaluated the behaviour of normal human osteoblasts (NHOst). The cells were cultured in different concentrations of Mg and exposed to an osteogenic medium for 19 days. We determined the activity of ALP at the end of the experiment. In NHOst cultured in 5.0 mM and exposed to the osteogenic medium, ALP did not increase, while it was induced in cells cultured 0.1 and 1.0 mM Mg (figure 3A). We then measured NO release at the end of the experiment and found a decrease of NO in differentiated NHOst in 0.1 mM Mg containing medium vs the corresponding control (figure 3B), while NO release remained unvaried in NHOst in 1.0 and 5.0 mM Mg cultured or not in osteogenic medium.

Discussion

The main function of the osteoblast throughout life is to produce bone extracellular matrix. This process is obviously important for longitudinal growth but also to maintain a constant bone mass through bone remodeling, the process by which bone constantly renews itself during adulthood. While it is widely accepted that Mg deficiency also contributes to osteopenia by inhibiting the growth and the activity of osteoblasts [5-7], less is known about the effects of high Mg on bones.

In our experimental model we show that excessive extracellular Mg inhibits the mineralizing activity of osteoblast-like SaOS-2 cells and blocks NHOst differentiation as detected by measuring ALP activity. Our data are in agreement with a report demonstrating that high extracellular Mg inhibited matrix mineralization in the prechondrogenic cell line ATDC5 [17]. In these cells, it has been suggested that high extracellular Mg might inhibit calcium-promoted mineralization mediated by matrix protein induction.

The inhibitory effect of high Mg on osteoblast differentiation and mineralizing activity explains, at least in part, some puzzling data from the literature. In patients with chronic renal failure or in individuals undergoing dialysis, serum Mg concentrations are frequently elevated and correlate with mineralization defects [8]. Another interesting set of studies was performed on premature infants presenting with osteopenia secondary to MgSO4 maternal administration for preterm labor [9,10]. Since Mg is a calcium antagonist, it is feasible to propose that high concentrations of Mg alter the calcium/Mg ratio, thus leading to dysregulated cell functions. In osteoblasts, calcium signalling is known to play a crucial role in proliferation and differentiation [18]. By competing for the same transporters, Mg might reduce intracellular calcium and, therefore, impair calcium-mediated events. In addition, the widely expressed ion channel TRPM7, which is essential for Mg homeostasis in mammals [19], is subjected to a strong negative feedback by intracellular Mg [20]. It is therefore possible that TRPM7, which is expressed in osteoblasts [20], is blocked by the higher concentrations of intracellular Mg in cells cultured in 5.0 mM Mg. Interestingly, apart from calcium and Mg, TRPM7 conducts other essential metals such as Mn, Zn and Co [21], which are cofactors for many essential cellular enzymes and/or transcription factors. We therefore propose that high extracellular Mg might alter the intracellular cation balance, thus interfering with various cellular functions.

Evidence from gene knock-out studies have shown that bone formation is, at least in part, mediated by NO, since mice deficient in endothelial NO synthase show bone abnormalities, and inducible NO synthase null mice show imbalances in bone osteogenesis and abnormalities in bone healing [15]. In our culture conditions, we ruled out the intervention of NO as a mediator of the action of different concentrations of Mg on SaOS-2 cells and NHOst. Indeed, while in SaOS-2 cells no differences of NO release were detected, a reduction of NO release was observed in NHOst cells cultured in 0.1 mM Mg after 19 days of culture in osteogenic medium. Since NO is required for osteoblast differentiation [15], we argue that this reduction is not sufficient to impair differentiation.

We are aware that the concentrations of extracellular Mg used in this study are extreme and unlikely to be reached in vivo. In in vitro experimental models, however, the use of very high or very low concentrations of different compounds, including magnesium, is dictated by the need to highlight significant differences. Obviously, caution is required when comparing in vitro and clinical data. However, on the basis of our results, we suggest that, in addition to low levels of Mg, high concentrations of Mg might also be detrimental to osteoblasts. A tight control of Mg homeostasis seems to be crucial to maintain bone integrity.

Disclosure

This work is supported by MIUR-PRIN 2007 grant number 2007ZT39FN.

None of the authors has any conflict of interest to disclose.

References

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