Neomycin enhances glutaraldehyde crosslinking and glycosaminoglycan stability in bioprosthetic heart valves

The native heart valve will open and close an astonishing 3 billion times in the average lifetime, implicating immense biomechanical ramifications that necessitate near-flawless structure and functional behavior [1,2]. Deviations from this idyllic function are a result of heart valve disease (HVD) which affects millions of individuals worldwide and results in over 300,000 heart valve replacements worldwide every year [3,4]. Glutaraldehyde (GLUT) cross-linked porcine aortic heart valves, a common type of bioprosthetic heart valve (BHV), are used frequently in these valve replacement surgeries. The native aortic valve leaflets entail a tri-composite design of type I collagen, elastin and glycosaminoglycans (GAGs); each of which are important structural and functional biomechanical components.;Our group has previously characterized the loss of GAGs from BHVs due to the inability of GLUT-crosslinking to stabilize these structures during in-vitro storage, fatigue, enzymatic degradation and in-vivo implantation [5-8]. Consequences of GAG loss include, but are not limited to, decreased hydration, loss of tissue compliancy, altered leaflet morphology and the potential compromise of collagen organization and mechanical integrity [5,9].;This study explicitly examines the ability of neomycin to enhance glutaraldehyde crosslinking (NG) and stabilize GAGs. Evidence for enhanced crosslinking using neomycin was supported by increased resistance to enzymatic collagen and elastin degradation compared to that of standard GLUT-crosslinking and by a small but significant increase in collagen denaturation temperatures as measured using differential scanning calorimetry. NG-crosslinked leaflets also exhibited a slightly diminished hydration capacity compared to GLUT leaflets, indicating potentially adverse biomechanical effects. However, biaxial tensile testing revealed no significant alterations in compliancy during NG versus GLUT-crosslinking. NG-crosslinked leaflets subjected to in-vitro storage, accelerated cyclic fatigue and enzyme-mediated GAG degradation revealed improved GAG stabilization versus standard GLUT-crosslinked leaflets, which sustained substantial decreases in GAG content. Lastly, ultrastructural analysis using transmission electron microscopy qualitatively assessed preservation of GAGs in NG-crosslinked leaflets and yielded insight into their morphological preservation utilizing NG crosslinking. Thus, we hypothesized that preservation of the GAG matrix using NG crosslinking may help maintain biomechanical function and ultimately improve tissue durability.