Neomycin prevents enzyme-mediated glycosaminoglycan degradation in bioprosthetic heart valves

Abstract

Bioprosthetic heart valves (BHVs) derived from glutaraldehyde crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, BHVs have limited durability and fail either due to degeneration or calcification. Glycosaminoglycans (GAGs), one of the integral components of heart valve cuspal tissue, are not stabilized by conventional glutaraldehyde crosslinking. Previously we have shown that valvular GAGs could be chemically fixed with GAG-targeted chemistry. However, chemically stabilized GAGs were only partially stable to enzymatic degradation. In the present study an enzyme inhibitor was incorporated in the cusps to effectively prevent enzymatic degradation. Thus, neomycin trisulfate, a known hyaluronidase inhibitor, was incorporated in cusps via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry followed by glutaraldehyde crosslinking (NEG). Controls included cusps crosslinked with either EDC/NHS followed by glutaraldehyde (ENG) or only with glutaraldehyde (GLUT). NEG group showed improved resistance to in vitro enzymatic degradation as compared to GLUT and ENG groups. All groups showed similar collagen stability, measured as a thermal denaturation temperature by differential scanning calorimetry (DSC). The cusps were implanted subdermally in rats to study in vivo degradation of GAGs. NEG group preserved significantly more GAGs than ENG and GLUT. NEG and ENG groups showed reduced calcification than GLUT.

Figure 1

Immunoperoxidase staining for Neomycin sulfate; A. Cusps in the NEG group showed uniform brown stain for neomycin, B. Cusps in the ENG group where neomycin was not added showed no staining.

Figure 2

Alcian blue staining of cusps for GAGs after one week of fixation and four weeks of storage in 0.2% GLUT. (A) GLUT (B) ENG and (C) NEG. GLUT group showed least staining while NEG group showed intense staining for GAGs. Most of the GAGs were in the middle spongiosa layer.

Figure 3

A. Cuspal tissue hexosamine content after one week of fixation and four weeks of storage in 0.2% GLUT showed higher GAG content in the NEG group than the ENG and GLUT groups (p<0.05). NEG group also retained more GAGs after hyaluronidase and chondroitinase treatment (p<0.05); B. GAGs released from the cusps into the enzyme solution was measured by DMMB assay. Significantly fewer amounts of GAGs were released in NEG group showing better stabilization efficiency (p<0.05). C. Hexosamine assay performed on the cusps after papain digestion showed highest GAG retention in NEG group. Some retention was also seen in ENG group (p<0.05); D. DMMB assay on papain solution showed least amount of GAGs released (p<0.05) into papain solution in the NEG group confirming that GAGs were stabilized efficiently in this group.

Figure 4

A. Hexosamine assay on cusps explanted after three weeks. The NEG group has significantly greater GAGs retained as compared to the other two groups (p<0.05). B. Calcium assay on three weeks explanted cusp samples showed lower calcification in NEG and ENG groups as compared to GLUT (p<0.05).

Figure 5

Alcian blue staining of cusps after three weeks explantation showing more GAGs retained in the NEG group. (A) GLUT (B) ENG (C) NEG

Figure 6

HA gel zymography on soluble proteins extracted from the capsule surrounding the three weeks explanted cusps revealed equal amounts of hyaluronidase activity (migrating at ~ 55 kD) in all the groups. Addition of 1mM neomycin trisulfate to the developing buffer inhibited enzyme activity (lanes DN1 and DN2).