Chemical contribution
Glut crosslinking make tissues biocompatible and nontrombogenic, while maintaining anatomic integrity, leaflet strength and flexibility (45). Its preference among aldehydes is related to its availability, low price, quick action and capacity to react with a large number of amino acids (45).
Glut is responsible for the effective crosslinking of collagen, the most common structural component of the valves. It forms covalent bonds by the formation of Schiff bases (reaction of an aldehyde group with an amino group of lysine or hydroxylysine) and/ or by aldol condensation between two adjacent aldehydes (46). Cross-linking increases tissues durability, reducing resistance to proteolysis of the cross-linked proteins. However, after Glut fixation residual aldehydes remain expressed on the tissue surface and may act as calcification locations.
Although its action is essential for valve preservation and to eliminate cellular components to reduce tissue immunogenicity, the induced chemical reactions are probably the most important part of bioprosthetic valve degeneration. After Glut fixation, valvular interstitial cells lose their viability (42). However, some studies have showed that Glut retains many of the viscoelastic proprieties of the collagen (45), with haemodynamic proprieties of the prostheses similar to those of living tissues (45).
Additionally, as part of the fixation and fabrication process, the cellular content of the tissues is modified, with loss of endothelial cells, loss of interstitial cell viability and interstitial cell degeneration (41). Indeed, bioprosthetic heart valves show several histological differences from native heart valves, with flattening of the cuspal corrugations, loss of the endothelium or mesothelium surfaces, disruption of interstitial cells and loss of GAG (42). With the loss of interstitial cells viability, the mechanical proprieties and durability of the valve depends primarily on the quality of the collagen and the remaining viscoelastic proprieties are not enough to avoid valve tissue degeneration.
Schoen et al described the calcification process as having two phases: nucleation (or initiation) and propagation (41). One important change to initiate calcification is the abnormal extrusion of calcium ions from the nonviable cells. Cross-linking to proteins of the cellular membrane alters their proprieties, resulting in a different permeability in the non-viable cells. Additionally, there is a the reduction of the functional transmembrane ion pumps and an increased permeability to calcium ions that contribute to the onset of calcifications (47). Usually calcium concentration is 1000 to 10.000 times lower in the cytoplasm due to the healthy ion pumps that carry calcium out of the cells. With deregulated calcium levels inside the cells, cellular membranes and other intracellular structures bind calcium and serve as nucleators for calcifications. Indeed, calcification seems to start predominantly at the cell membranes and other intracellular structures rich in phospholipids, while the loss of proteoglycans may enhance this phenomenon by removing calcification inhibitors (48). Glut also reacts directly with intracellular structures, predisposing to calcification in the presence of high intracellular calcium levels (49). The debris of interstitial cells that remain in valve tissue also serves as initiation sites for calcification.
Collagen and elastic fibers can also serve as nucleation sites, independent of cellular components. One important difference is that calcification of collagen requires cross-linking alterations, while calcification of elastin occur independently of cross-linking (50).
After the nucleation, calcification is influenced by all the metabolic and pathologic changes in calcium and phosphorus metabolism, with calcium-enriched crystals growing to eventually culminate in prosthesis malfunction (propagation phase).
Another important characteristic of native heart valves is their remodeling and reparation capacity. In biological prosthesis the fixed and nonviable tissue is incapable to maintain the ongoing repair, and every damage do the extracellular matrix is cumulative. Moreover, endothelial is denuded or absent and adjacent smooth muscle cells might proliferate and migrate freely to the non-endothelized valve surface (51), also contributing to valve dysfunction.
The dynamic role of native valve cells’ and their importance for the durability of bioprosthesis is now recognized, and new strategies of repopulation and regeneration have been proposed to minimize the problem. Repopulation defines the process of using a clean connective tissue matrix valve, repopulated with the recipient cells’, before or after prosthesis implantation (52). A completely “self-populated” prosthesis would maintain tissues invisible, avoiding host reaction to the graft. Although this technology reached clinical practice, the results were not as good as expected. Regeneration involves the implantation of a remodeling matrix with the proteins and cells of the recipient that can resemble the dynamic changes of native heart valves (52), and remains in the pre-clinical development.
Storage is another important issue regarding bioprosthetic valve dysfunction. Actually, several bioprosthesis are stored in liquids containing aldehydes, which are toxic and a source for calcification, as previously described in this chapter. Regardless fixation and production procedures with a reduced aldehyde content, tissues are exposed once again to deleterious free aldehydes when they are stored in aldehyde enriched solutions. Even pre-implant rinsing does not guarantee complete removal of toxic aldehydes with such storage solutions, and new technologies regarding storage are also an active field of research.