Neomycin prevents enzyme-mediated glycosaminoglycan degradation in bioprosthetic heart valves.

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.

Physical characteristics of small intestinal submucosa scaffolds are location-dependent.

Using biodegradable scaffolds as an alternative to engineer new tissues has become an attractive candidate in various transplantation protocols. In particular, small intestinal submucosa (SIS), a dense connective matrix harvested from the small intestine, has gained attention due to a number of favorable properties. However, use of SIS is constrained by obtaining reliable, reproducible products in large-scale preparations that affect the regenerative process. To better understand the heterogeneous nature of SIS, this study focused on evaluating the location-dependent alterations in the physical characteristics of the matrices harvested from distal and proximal ends and processed in-house (referred as hand-made). Additionally, results were compared with a commercially available machine-made Cook SIS. Tensile properties during monotonic loading and cyclical loading were compared in wet conditions. Furthermore, permeability of these membranes to urea was analyzed using a custom-built chamber, and the microarchitecture was analyzed via scanning electron microscopy. These results showed that distal samples were more elastic and less permeable to urea relative to other samples. However, permeability in each sample was direction-dependent, that is, mucosal to serosal direction was less permeable compared to sorasal to mucosal direction in all the samples. Cook SIS was more susceptible to cyclical loading and had a shorter range of load carrying capacity. In summary, results show that physical characteristics of SIS are location-dependent.

Bioengineered self-seeding heart valves.

OBJECTIVE: Mechanical and biological prostheses are used to replace damaged heart valves but are associated with significant morbidities. Although there is increased interest in bioengineering cell-seeded heart valve scaffolds, it is a time-consuming and technically difficult process. The goal of this project was to engineer self-seeding heart valves that mature quickly in vivo and have a shorter preparation time. METHODS: Porcine pulmonary valves were decellularized using detergent methods and then either (1) left untreated (unconjugated, n = 6), (2) reseeded with autologous endothelial progenitor cell-derived endothelial cells (cell-seeded, n = 4), or (3) conjugated with CD133 antibodies (conjugated, n = 8). The valve constructs were transplanted into the pulmonary position of sheep using standard surgical techniques. After 1 or 3 months, the implants were removed and assessed for cell and matrix content as well as biomechanical properties. RESULTS: Endothelial cells expressing von Willebrand factor lined the entire length of both ventricular and arterial surfaces of conjugated valves by 1 month after implantation. Interstitial cell and structural protein content of conjugated valves increased from 1 month to 3 months with interstitial expression of metalloproteinase-9 and new collagen formation. In contrast, there were few endothelial or interstitial cells associated with unconjugated, or cell-seeded valves at any time point. No calcification or thrombi were noted on any of the valves. Young's modulus and tensile strength was greater in the conjugated valves versus unconjugated or cell-seeded valves. CONCLUSIONS: Results indicate that tissue-engineered heart valve replacement constructs can be made quickly and therefore may be a clinically relevant option for patients needing heart valve surgery in a timely fashion.

Neomycin fixation followed by ethanol pretreatment leads to reduced buckling and inhibition of calcification in bioprosthetic valves.

Glutaraldehyde crosslinked bioprosthetic heart valves (BHVs) have two modalities of failure: degeneration (cuspal tear due to matrix failure) and calcification. They can occur independently as well as one can lead to the other causing co-existence. Calcific failure has been extensively studied before and several anti-calcification treatments have been developed; however, little research is directed to understand mechanisms of valvular degeneration. One of the shortcomings of glutaraldehyde fixation is its inability to stabilize all extracellular matrix components in the tissue. Previous studies from our lab have demonstrated that neomycin could be used as a fixative to stabilize glycosaminoglycans (GAGs) present in the valve to improve matrix properties. But neomycin fixation did not prevent cuspal calcification. In the present study, we wanted to enhance the anti-calcification potential of neomycin fixed valves by pre-treating with ethanol or removing the free aldehydes by sodium borohydride treatment. Ethanol treatment has been previously used and found to have excellent anti-calcification properties for valve cusps. Results demonstrated in this study suggest that neomycin followed by ethanol treatment effectively preserves GAGs both in vitro as well as in vivo after subdermal implantation in rats. In vivo calcification was inhibited in neomycin fixed cusps pretreated with ethanol compared to glutaraldehyde (GLUT) control. Sodium borohydride treatment by itself did not inhibit calcification nor stabilized GAGs against enzymatic degradation. Neomycin fixation followed by ethanol treatment of BHVs could prevent both modalities of failure, thereby increasing the effective durability and lifetime of these bioprostheses several fold.

Neomycin binding preserves extracellular matrix in bioprosthetic heart valves during in vitro cyclic fatigue and storage.

Bioprosthetic heart valve (BHV) cusps have a complex architecture consisting of an anisotropic arrangement of collagen, glycosaminoglycans (GAGs) and elastin. Glutaraldehyde (GLUT) is used as a fixative for all clinical BHV implants; however, it only stabilizes the collagen component of the tissue, and other components such as GAGs and elastin are lost from the tissue during processing, storage or after implantation. We have shown previously that the effectiveness of the chemical crosslinking can be increased by incorporating neomycin trisulfate, a hyaluronidase inhibitor, to prevent the enzyme-mediated GAG degradation. In the present study, we optimized carbodiimide-based GAG-targeted chemistry to incorporate neomycin into BHV cusps prior to conventional GLUT crosslinking. This crosslinking leads to enhanced preservation of GAGs during in vitro cyclic fatigue and storage. The neomycin group showed greater GAG retention after both 10 and 50 million accelerated fatigue cycles and after 1 year of storage in GLUT solution. Thus, additional binding of neomycin to the cusps prior to standard GLUT crosslinking could enhance tissue stability and thus heart valve durability.