Morphological transformation of hBMSC from 2D monolayer to 3D microtissue on low-crystallinity SF-PCL patch with promotion of cardiomyogenesis.

The effects of the stiffness of substrates on the cell behaviours of human bone marrow-derived mesenchymal stem cells (hBMSC) have been investigated, but the effects of the secondary structures of proteins in the substrates on the morphological transformation and differentiation of hBMSC have yet been elucidated. To investigate these issues, silk fibroin-poly(ε-caprolactone) SP cardiac patches of poly(ε-caprolactone; P), on which is grafted by silk fibroin (SF) with various ß-sheet contents (or crystallinity) to provide various degrees of stiffness, were produced to examine the in vitro behaviours of hBMSC during proliferation, and cardiomyogenesis on the SP patches. ß-sheet contents of SF from 20% to 44% (SP20 to SP44, respectively) were induced on patches, which were examined by attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, and analysed using the Fourier self-deconvolution method. The stiffness of the SP patches, quantified by their Young's moduli and elasticities, increased with the crystallinity of the SF. During 3 days of proliferation, hBMSC migrated and morphologically transformed into 3D microtissues with diameters of approximately 150-200 µm on low-stiffness SP20 and SP30 patches, whereas 2D monolayers were observed on the SP37 and SP44 patches. The 3D microtissues/patch yielded more extensive in vitro cardiomyogenesis of hBMSC than the 2D cell monolayer with significantly higher expressions of all examined cardiac-specific proteins after induction by 5-aza. Notably, in vivo subcutaneously growing 3D microtissues on SP20 patches and a 2D monolayer on SP44 patches were preliminarily demonstrated in a rat model. Morphological transformations of hBMSC from a 2D monolayer to a 3D microtissue by low-stiffness SP cardiac patches, promoting cardiomyogenesis, provide a new opportunity for cardiac tissue engineering.

Macromolecular transport in heart valves. I. Studies of rat valves with horseradish peroxidase.

The present study aims to experimentally elucidate subtle structural features of the rat valve leaflet and the related nature of macromolecular transport across its endothelium and in its subendothelial space, information necessary to construct a rational theoretical model that can explain observation. After intravenous injection of horseradish peroxidase (HRP), we perfusion-fixed the aortic valve of normal Sprague-Dawley rats and found under light microscopy that HRP leaked through the leaflet's endothelium at very few localized brown spots, rather than uniformly. These spots grew nearly as rapidly with HRP circulation time before euthanasia as aortic spots, particularly when the time axis only included the time the valve was closed. These results suggest that macromolecular transport in heart valves depends not only on the direction normal to, but also parallel to, the endothelial surface and that convection, as well as molecular diffusion, plays an important role in macromolecular transport in heart valves. Transmission electron microscopy of traverse leaflet sections after 4-min HRP circulation showed a very thin ( approximately 150 nm), sparse layer immediately beneath the endothelium where the HRP concentration was much higher than that in the matrix below it. Nievelstein-Post et al.'s (Nievelstein-Post P, Mottino G, Fogelman A, Frank J. Arterioscler Thromb 14: 1151-1161, 1994) ultrarapid freezing/rotary shadow etching of the normal rabbit valve's subendothelial space supports the existence of this very thin, very sparse "valvular subendothelial intima," in analogy to the vascular subendothelial intima.