(L-463) DIFFERENTIAL DEVELOPMENT OF ECM IN THE FETAL MITRAL VALVE CHORDAE TENDINEAE AND LEAFLET: UNIQUE LESSONS FOR TISSUE ENGINEERING

Introduction:: When creating tissue-engineered scaffolds for replacements of load-bearing tissues, mechanical compatibility and viability are of great importance. It is sensible then to consider the main natural load-bearing protein in the body, collagen, for its robust properties that can be tailored to scaffold design. Tissue engineering can be further informed by fetal development since both involve the fabrication of collagenous scaffolds for future mechanical loading. This study examined the development of the bovine fetal mitral valve leaflet and chordae tendineae: collagenous tissues that experience unique mechanical loading conditions in the mature animal. Our goals were to (i) differentiate extracellular matrix development (ECM) between the leaflet and chordae and (ii) determine the mechanism by which chordae tendineae increase in number during gestation.


Materials and Methods:: Fetal bovine hearts were harvested from a local abattoir, and the crown-rump length of the fetus was used to determine gestational age. Once transported back to the lab, the valves were removed, then the anterior mitral valve leaflet and chordae were dissected. These were then imaged where chordae number was counted, and leaflet area measured using ImageJ. For histology, following dissection the anterior mitral valve leaflet and chordae were immediately fixed in 10% buffered neutral formalin before being embedded in paraffin wax. Sections of 4µm were used for all histological staining and images were taken using an AMScope MU1400 camera and a Nikon Eclipse E600 light microscope. Similar to histology, for samples undergoing transmission electron microscopy (TEM), fresh isolated chordae sections were fixed in a solution of 2.5% Glutaraldehyde diluted with 0.1M Sodium Cacodylate Buffer before transporting to the Dalhousie Electron Microscopy core for further processing.

Collagen contents were determined using Sircol™ Soluble (newly synthesized, immature) and Insoluble (crosslinked, mature) Collagen assays. Collagen crimp wavelength (distance between each peak) and percent crimp were visualized using picrosirius red staining combined with polarized light microscopy and quantified using ImageJ. In branched chordae, cross-sections of the common “trunk” and two “branches” were imaged using TEM and collagen fibril diameters and density were measured using ImageJ. The localization of extracellular matrix proteins over gestational time was visualized using Movat Pentachrome staining.


Results, Conclusions, and Discussions:: Leaflet area and chordae attachments increased during gestation however, only leaflet area continued increasing postnatally. Collagen percent crimp increased in both tissues during gestation, suggesting continuous collagen synthesis in response to increasing load. Yet, collagen deposition/maturation begins earlier in chordae than the leaflet. Soluble collagen accumulation began earlier in chordae with concentrations increasing during gestation while in the leaflet, the rate of accumulation was unchanged, and levels remained lower than in chordae. Insoluble collagen accumulation also began earlier in chordae but occurred at a faster rate than the leaflet. The low levels of soluble collagen in the leaflet suggest that rapid accumulation of insoluble collagen occurs via upregulation of crosslinking. Lengthening of collagen crimp also began earlier in chordae but increased at a similar rate over gestation in both tissues.

While collagen deposition and crosslinking begin earlier in chordae, functional, mature elastic fibers appear earlier in gestation in the leaflet. By mid-gestation, the leaflet contains prominent, dense elastic fibers while chordae show nearly none. This suggests differing elasticity requirements – where chordae may rely on microfibrillar elasticity until late gestation.

There were no differences in collagen fibril diameter or density between trunk and branch sections during gestation. This suggests that branches possess shared populations of collagen fibers running into them from the trunk. This supports our hypothesis that increases in chordae attachments occur through the longitudinal splitting of pre-existing chordae.

This is the first study to describe the anatomical development and ECM remodeling of the mitral valve during fetal development in large mammals. While leaflet area enlarges during gestation and postpartum, chordae multiplication occurs only during gestation, suggesting its governance by fetal mechanisms. Our results also suggest that while collagen and elastin synthesis is occurring in both tissues during gestation, it’s not at the same rates or intervals. These two tissues may possess unique and independent developmental processes—despite their anatomical proximity with continuous collagen fibers. This likely reflects cellular phenotypic differences between the leaflet and chordae. This deeper understanding of development in mechanically loaded fetal tissues may be harnessed to achieve more optimal mechanical viability in valve tissue engineering design.


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