Panitch, Alyssa

Tissue autofluorescence provides fluorescence lifetime contrast between acellular tissue and that containing newly seeded cells. Fiber‐based fluorescence lifetime imaging (FLIm) can be used for tracking recellularization of engineered vascular grafts and potential matrix remodeling at large scale, without compromising sample integrity. FLIm cellular contrast was verified in a subset of samples seeded with eGFP‐labelled cells. Results suggests fiberbased FLIm is a suitable tool for monitoring recellularization of engineered tissue nondestructively.Further details can be found in the article by Alba Alfonso‐Garcia, Jeny Shklover, Benjamin E. Sherlock, et al. (e201700391). image

New techniques able to monitor the maturation of tissue engineered constructs over time are needed for a more efficient control of developmental parameters. Here, a label‐free fluorescence lifetime imaging (FLIm) approach implemented through a single fiber‐optic interface is reported for nondestructive in situ assessment of vascular biomaterials. Recellularization processes of antigen removed bovine pericardium scaffolds with endothelial cells and mesenchymal stem cells were evaluated on the serous and the fibrous sides of the scaffolds, 2 distinct extracellular matrix niches, over the course of a 7 day culture period. Results indicated that fluorescence lifetime successfully report cell presence resolved from extracellular matrix fluorescence. The recellularization process was more rapid on the serous side than on the fibrous side for both cell types, and endothelial cells expanded faster than mesenchymal stem cells on antigen‐removed bovine pericardium. Fiber‐based FLIm has the potential to become a nondestructive tool for the assessment of tissue maturation by allowing in situ imaging of intraluminal vascular biomaterials.

ABSTRACT Activation of cyclic nucleotide‐dependent signaling pathways leads to phosphorylation of the small heat shock‐related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose‐dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)‐PTD or FITC‐PTD‐HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD‐phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.