Characteristics of solutions.

<p>Total initial concentration of [Ca²⁺]₀ = 1.8mM for DMEM and MEM and 1.05mM for DMEM:F12; activity coefficient from Davies equation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141751#pone.0141751.ref019" target="_blank">19</a>], γ = 0.32.</p><p>Ionic activity product of amorphous calcium phosphate, IAP(ACP) = [<i>a</i>(Ca²⁺)][<i>a</i>(PO₄^3-)]^0.74[<i>a</i>(H⁺)]^0.22 and K_sp(ACP) = 2.29 x 10^-11.</p><p>Ionic activity product of dicalcium phosphate dihydrate, DCPD, IAP(DCPD) = [<i>a</i>(Ca2+)][<i>a</i>(H₂PO₄^-)] and K_sp(DCPD) = 2.4 x 10^-7.</p><p>Ionic activity product of octacalcium phosphate, OCP, IAP(OCP) = [<i>a</i>(Ca²⁺)]⁸[<i>a</i>(H₂PO₄^-)]²[<i>a</i>(PO₄^-)]⁴ and K_sp(OCP) = 2.5 x 10^-99.</p><p>Ionic activity product of hydroxyapatite, HAP, IAP(HAP) = [<i>a</i>(Ca²⁺)]¹⁰[<i>a</i>(PO₄^3-)]⁶[<i>a</i>(OH^-)]² and</p><p>K_sp(HAP) = 5.5 x 10^-118.</p><p>The supersaturation ratios were calculated for the pH values at time 0 (pH₀) and in equilibrium with 5% CO₂ (pH_∞) in air, and they are defined as <math><mi>S</mi><mo>=</mo><mrow><mo>(</mo><mrow><mrow><mi>I</mi><mi>A</mi><mi>P</mi></mrow><mrow><msub><mrow><mi>K</mi></mrow><mrow><mi>s</mi><mi>P</mi></mrow></msub></mrow></mrow><mo>)</mo></mrow><mrow><mrow><mn>1</mn></mrow><mrow><mi>υ</mi></mrow></mrow></math>, where ν is the sum of the exponent numbers in IAP expression.</p><p>Characteristics of solutions.</p

DLS analysis of the nanocrystal formation rate.

<p>(A) Graphs showing the formation of nuclei in MEM or DMEM without cells, in the CO₂ incubator, at the indicated times, and in the presence of 2 or 3mM Pi. DLS determinations were made outside the CO₂ incubator. (B) The same experiment as in A, in the presence of VSMC, with the indicated conditions of Pi concentration and incubation times.</p

Mechanisms of cell death.

<p>(A) Prevention of calcification for 12 days with 12μM PPi in MEM. ANOVA, p < 0.0001 for calcification without PPi; *Significantly different from 1mM Pi with Tukey’s multiple comparison test. B. Prevention of Pi-induced cell death with PPi in MEM; statistics are as in A. (C) Alkaline phosphatase activity relative to the 1 mM Pi condition in cell lysates incubated for 4 or 8 days with 1, 2, or 3mM Pi in MEM. ANOVA at 4 days, p = 0.0065; ANOVA at 8 days, p = 0.0195. *Significantly different from 1mM Pi with Tukey’s multiple comparison test. (D) Treatment of VSMC with MEM containing 1, 2, or 3mM Pi and 0.2–10% FCS, for 14 days. Asterisks indicate significant differences of the means with a t-test, and p values are shown. (E) Treatment with DMEM as in A, for 8 days. Statistics as in C. (F) Variation of the calcium concentration in MEM after 4 and 8 days of incubation using the indicated Pi concentrations. *p < 0.0001 with t-tests. (G) Cell mortality after incubating VSMC with the indicated concentrations of calcium in lab-made MEM.</p

Critical Parameters of the <i>In Vitro</i> Method of Vascular Smooth Muscle Cell Calcification

<div><p>Background</p><p>Vascular calcification (VC) is primarily studied using cultures of vascular smooth muscle cells. However, the use of very different protocols and extreme conditions can provide findings unrelated to VC. In this work we aimed to determine the critical experimental parameters that affect calcification <i>in vitro</i> and to determine the relevance to calcification <i>in vivo</i>.</p><p>Experimental Procedures and Results</p><p>Rat VSMC calcification <i>in vitro</i> was studied using different concentrations of fetal calf serum, calcium, and phosphate, in different types of culture media, and using various volumes and rates of change. The bicarbonate content of the media critically affected pH and resulted in supersaturation, depending on the concentration of Ca²⁺ and Pi. Such supersaturation is a consequence of the high dependence of bicarbonate buffers on CO₂ vapor pressure and bicarbonate concentration at pHs above 7.40. Such buffer systems cause considerable pH variations as a result of minor experimental changes. The variations are more critical for DMEM and are negligible when the bicarbonate concentration is reduced to ¼. Particle nucleation and growth were observed by dynamic light scattering and electron microscopy. Using 2mM Pi, particles of ~200nm were observed at 24 hours in MEM and at 1 hour in DMEM. These nuclei grew over time, were deposited in the cells, and caused osteogene expression or cell death, depending on the precipitation rate. TEM observations showed that the initial precipitate was amorphous calcium phosphate (ACP), which converts into hydroxyapatite over time. In blood, the scenario is different, because supersaturation is avoided by a tightly controlled pH of 7.4, which prevents the formation of PO₄^3--containing ACP.</p><p>Conclusions</p><p>The precipitation of ACP <i>in vitro</i> is unrelated to VC <i>in vivo</i>. The model needs to be refined through controlled pH and the use of additional procalcifying agents other than Pi in order to reproduce calcium phosphate deposition <i>in vivo</i>.</p></div

Cell death during in vitro calcification.

<p>(A) Microphotographs of rat VSMC at passage 3, immunodecorated with smooth muscle markers. Bar, 20μm. (B) Osteogene RNA expression in VSMC cultured in MEM containing 1 or 2mM Pi for 1, 8, or 10 days. ANOVA p < 0.0001 for all three genes. *Significantly different from the corresponding 1mM Pi with Tukey’s and a t-test. (C) LDH activity, as a percentage of day 0, in VSMC incubated using Pi concentrations for up to 12 days in MEM. ANOVA p < 0.0001, *Different from the corresponding 1mM Pi with Tukey’s test. At 12 days, the difference at 2 and 1mM Pi is also significant with a t-test (p < 0.0001). (D) Calcium deposition at days 8, 10, and 12. ANOVA, p < 0.0001; *Different from the corresponding 1mM Pi with Tukey’s test. (E) Calcium deposition and LDH activity correlation after 12 days of calcification <i>in vitro</i>. (F) Phase contrast and EB/AO staining microphotographs after 12 days. Bar, 50μm. (G) VSMC, total protein (ANOVA, p < 0.005), and DNA content (ANOVA p < 0.005) after 12 days of Pi treatments; *Different from 1mM with Tukey’s test.</p

Ultrastructural analysis of deposits.

<p>(A) TEM images of deposits at different magnifications, from cells incubated as indicated at the left of each row. Images in the first column, at low magnification, are indicative of the density of the deposits, which is clearly increasing with the Pi concentration; images in the second column, with medium magnification, show the microstructures of the deposits, which gradually adopt a spherulite arrangement of needle-like particles as the concentration increases; images in the third column, at high magnification, show the shape and size of crystalline nanoparticles. (B) Electron diffraction patterns of cell culture deposits from A, showing an increasing crystallinity from MEM to DMEM, and from 2mM Pi to 3mM Pi. Using MEM at 2mM Pi for 10 days, ED revealed no reflections from crystal planes, however, when DMEM was used instead, crystallization rings at 2.81 and 3.52Å were observed, compatible with those of hydroxyapatite (2.82 and 3.44Å). MEM at 2.5 and 3mM Pi for 16 days also revealed a hydroxyapatite crystallization pattern, with clear reflections at 3mM.</p

Effect of culture parameters on Pi cytotoxicity in VSMC.

<p>(A) Effect of the indicated MEM volume and Pi concentrations on cell viability. ANOVA, p < 0.0001; *Different from the corresponding 1mM with Tukey’s test. For simplicity, asterisks show the comparisons of single means after combining the values from the different media volumes at the same Pi concentration. <i>n</i>.<i>s</i>.: non-significant differences caused by media volumes. (B) Calcium deposition in the same experiment as A. Statistical analysis as in A. (C) Effect of culture media type on Pi cytotoxicity after 8 days. MEM and DMEM ANOVA are p < 0.0001; *Different from the corresponding 1mM with Tukey’s test. (D) Quantification of calcium in deposits after 8 days of Pi treatments, using either MEM or DMEM, or MEM plus a bicarbonate concentration as for DMEM. All three ANOVAs (one per culture media) are p < 0.0001; *Different from the corresponding 1mM with Tukey’s test.</p