Nuclear magnetic resonance spectroscopy to quantify major extracellular matrix components in fibro-calcific aortic valve disease

Figures

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  Figure 1. Detection of collagen, elastin, triacylglycerols and phospholipids by 13C MAS NMR spectroscopy in (a) control tissue and (b) fibro-calcific aortic valve disease (FCAVD) tissue. Top panel shows cross-polarized NMR spectra and the lower panel the respective directly excited NMR spectra for n = 5 specimen per group. Spectra are scaled per mg wet sample weight. Control spectra of the aliphatic region of collagen and different phospholipid membranes are shown in panel (c). Signals from internal standard (POM: polyoxymethylene) or the Teflon insert of the MAS rotor are indicated in grey, asterisks denote spinning sidebands (from CO signal). CO carbonyl group, E elastin, Hyp Cγ hydroxyproline Cγ, PL phospholipids, TAG triacylglycerols.

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  Figure 2. Quantitative analysis of the collagen content in aortic valve tissues based on integration of characteristic NMR signals. Quantification of hydroxyproline content (representative for the collagen amount) in control (CT) and fibro-calcific aortic valve disease (FCAVD) tissues. n = 5; *p < 0.01, error bars—standard error.

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  Figure 3. Detection of hydroxyapatite (HAP) in tissue samples using 31P MAS NMR spectra, upper panels show cross polarized, lower panels directly excited 31P NMR spectra of (a) control tissue (CT), (b) fibro-calcific aortic valve disease (FCAVD) tissue, and c HAP nanoparticles as control. Spectra are scaled per mg wet sample, except CT tissue in the lower panel, which is 30 times magnified. Spectra of HAP nanoparticles are scaled by a factor of 0.1. Chemical shift of HAP is indicated by dashed line; n = 5 per group.

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  Figure 4. Quantitative analysis of the phosphate content in aortic valve tissues based on integration of NMR signals. Quantification of the phosphate amount of control (CT) and fibro-calcific aortic valve disease (FCAVD) tissues. n = 5; *p < 0.01, error bars—standard error.

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  Figure 5. Quantification of mineralisation of valve interstitial cells treated with pro-calcifying medium to induce calcification. (a) Alizarin red staining for quantification of mineralization of valvular interstitial cells after treatment for 21 days with control medium (CM) or pro-calcifying medium (PM) and (b) quantification by 31P NMR. x-axis is scaled according to Fig. 4, to allow easy comparison between tissue and cell culture. n = 5; *p < 0,05.