Self-assembly of collagen building blocks guided by electric fields

Show me the way: protein building blocks are programmed to assemble hierarchically and yield a defined fiber morphology of micrometric length and precise nanometric diameter. The key step of this method is to align the building blocks with an AC field prior to assembly. The resulting protein nanofibers are straightforwardly integrated with the circuitry for potential applications in bionanotechnology

Self-assembly of collagen building blocks guided by electric fields

Show me the way: protein building blocks are programmed to assemble hierarchically and yield a defined fiber morphology of micrometric length and precise nanometric diameter. The key step of this method is to align the building blocks with an AC field prior to assembly. The resulting protein nanofibers are straightforwardly integrated with the circuitry for potential applications in bionanotechnology. (Figure Presented)

Characterization of Porcine Aortic Valvular Interstitial Cell 'Calcified' Nodules

Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-beta 1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-beta 1 (OST+TGF-beta 1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-beta 1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-beta 1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that 'calcified' nodules formed from PAVICs grown in OST+TGF-beta 1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.</p

Raman bands contributing to the distinction between PAVICs grown in OST+TGF-β1 media, CTL media and MOBs mineralised nodules.

<p>Inorganic peaks are italicized. * indicates collagen assignments.</p

Transmission Electron Micrographs of cultured PAVICs.

<p>PAVICs were grown in <b>A -</b> CTL medium and <b>B -</b> OST+TGF-β1 medium (VIC – valvular interstitial cell, ECM – extracellular matrix) (scale = 2 µm).</p

Micrographs showing the morphology and staining of PAVICs and MOBs in culture.

<p><b>A,D,G -</b> Phase contrast images of cultured PAVICs grown in CTL medium for 21 days, PAVICs grown in OST+TGF-β1 medium for 21 days, and MOBs grown in OST medium for 21 days respectively (scale = 100 µm). <b>B,E,H -</b> Alizarin Red S staining negative for PAVICs grown in CTL medium for 21 days, positive for PAVICs grown in OST+TGF-β1 medium for 21 days, and positive for MOBs grown in OST medium respectively (scale = 100 µm). <b>C,F,I</b> - SEM images of cultured PAVICs grown in CTL medium for 21 days, PAVICs grown in OST+TGF-β1 medium for 21 days, and MOBs grown in OST medium for 21 days respectively (scale = 50 µm).</p

Gene expression and cross sectional staining of cultured PAVICs.

<p><b>A -</b> Gene expression of collagen I (* = p<0.05, ** = p<0.0001, *** = p<0.05;**** = p<0.001) and <b>B -</b> Osteocalcin (bone gamma-carboxyglutamate protein (BGLAP)) (* = p<0.05; ** = p<0.001; *** = p<0.0001) comparing PAVICs expression at day 7 (D7), day 14 (D14), and day 21 (D21) in both OST+TGF-β1 and CTL media. <b>C –</b> Cross sectional plane of cultured nodules used for the D,E,F,G,H, and I. <b>D,E -</b> Modified Verhoeff van Gieson stain [purple – cell nuclei, pink – collagen, black-elastin] of a cross section of PAVICs cultured in CTL media for 21 days and PAVICs cultured in OST+TGF-β1 media for 21 days respectively (scale = 50 µm). <b>F,G -</b> Peroxidase stain with Sirius red staining [brown - αSMA, red – collagen] for PAVICs grown in CTL medium for 21 days and PAVICs grown in OST+TGF-β1 medium for 21 days respectively (scale = 50 µm) <b>H,I -</b> Fluorescence staining [red - αSMA, blue - cell nuclei] of a PAVICs nodule cultured for 21 days in CTL medium and a PAVICs nodule cultured in OST+TGF-β1 medium for 21 days respectively (scale = 50 µm).</p