Spatial and temporal analysis of extracellular matrix proteins in the developing murine heart: a blueprint for regeneration.

The extracellular matrix (ECM) of the embryonic heart guides assembly and maturation of cardiac cell types and, thus, may serve as a useful template, or blueprint, for fabrication of scaffolds for cardiac tissue engineering. Surprisingly, characterization of the ECM with cardiac development is scattered and fails to comprehensively reflect the spatiotemporal dynamics making it difficult to apply to tissue engineering efforts. The objective of this work was to define a blueprint of the spatiotemporal organization, localization, and relative amount of the four essential ECM proteins, collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN) in the left ventricle of the murine heart at embryonic stages E12.5, E14.5, and E16.5 and 2 days postnatal (P2). Second harmonic generation (SHG) imaging identified fibrillar collagens at E14.5, with an increasing density over time. Subsequently, immunohistochemistry (IHC) was used to compare the spatial distribution, organization, and relative amounts of each ECM protein. COLIV was found throughout the developing heart, progressing in amount and organization from E12.5 to P2. The amount of COLI was greatest at E12.5 particularly within the epicardium. For all stages, FN was present in the epicardium, with highest levels at E12.5 and present in the myocardium and the endocardium at relatively constant levels at all time points. ELN remained relatively constant in appearance and amount throughout the developmental stages except for a transient increase at E16.5. Expression of ECM mRNA was determined using quantitative polymerase chain reaction and allowed for comparison of amounts of ECM molecules at each time point. Generally, COLI and COLIII mRNA expression levels were comparatively high, while COLIV, laminin, and FN were expressed at intermediate levels throughout the time period studied. Interestingly, levels of ELN mRNA were relatively low at early time points (E12.5), but increased significantly by P2. Thus, we identified changes in the spatial and temporal localization of the primary ECM of the developing ventricle. This characterization can serve as a blueprint for fabrication techniques, which we illustrate by using multiphoton excitation photochemistry to create a synthetic scaffold based on COLIV organization at P2. Similarly, fabricated scaffolds generated using ECM components, could be utilized for ventricular repair.

Ex vivo canine vocal fold lamina propria rehydration after varying dehydration levels.

OBJECTIVES: To examine the recoverability of canine vocal fold (VF) lamina propria (LP) on rehydration from varying dehydration levels. STUDY DESIGN: Open, controlled experimental trial. METHODS: The VF LP was excised en bloc using a scalpel from 10 canine larynges, providing 20 tissue samples. The initial volume of each sample was measured. Ten samples were dehydrated to 30% by mass and the other 10 samples to 70%. Each sample was rehydrated in 0.9% saline until the mass stabilized. The liquid mass and volume fractions, liquid:solid mass and volume ratios, and the fractions of the original tissue masses and volumes were calculated. RESULTS: All calculated parameters were significantly different between 30% and 70% dehydration recovery, with all parameters lesser in the 70% dehydration treatment group. Half of the tissue samples subjected to 30% dehydration fully recovered to their original volumes, whereas only one of the 10 samples subjected to 70% dehydration fully recovered its volume. CONCLUSIONS: The level of attainable rehydration recovery of VF LP tissue in an ex vivo setting depends on the level of dehydration. The results correspond to the biphasic theory and may be used to help model the biomechanical and physiological properties of VF LP tissue during rehydration.

Parameters quantifying dehydration in canine vocal fold lamina propria.

OBJECTIVES/HYPOTHESIS: The goal of this study was to measure the solid and liquid volume and mass of canine vocal fold lamina propria tissue at varying dehydration levels, and to calculate parameters to test the biphasic theory of vocal fold physiology and biomechanics. STUDY DESIGN: Open, controlled, experimental trial. METHODS: The vocal fold lamina propria was dissected from 15 canine larynges, yielding 30 tissue samples. The initial volumes and masses of the tissue samples were measured. The masses of the tissue samples were then measured every 2 minutes during 30%, 50%, and 70% dehydration, with 10 samples subjected to each of the three treatments, followed by complete dehydration to yield the solid component of the tissue. The liquid mass and volume fractions and liquid:solid mass and volume ratios of the vocal fold lamina propria samples were calculated. RESULTS: The liquid mass and volume fractions and liquid:solid mass and volume ratios were significantly different at each dehydration level, except for the liquid:solid volume ratios at 30% versus 50% dehydration. Linear regression analysis suggested that all of the solid and liquid parameters measured could be predicted by dehydration level based on inverse, linear relationships. CONCLUSIONS: These results provide further experimental evidence supporting the biphasic theory and suggest that the extent of vocal fold lamina propria tissue dehydration may be quantified based on the biphasic model parameters.