Lack of long-term durability remains the primary concern for BHVs with the majority failing within 12-15 years after implantation into adults. Although this may be adequate for many patients, BHV implantation can be contraindicated in younger individuals in order to avoid reoperation. Even in elder recipients, valve dysfunction can still cause death or reoperation that could be avoided with increased BHV durability. Therefore, investigation into methods for increased BHV durability is warranted in order to both widen the patient demographic as well as improve the quality of life for BHV recipients.
Improving BHV durability requires prevention of the two foremost causes of failure, calcification and non-calcific structural degradation. Glutaraldehyde crosslinking has been implicated as a contributor to both of these phenomena and has long been a target of improvement or replacement. Investigation into alternative fixation methods has yielded many options, many of which have demonstrated increased resistance to calcification. However, even with a fixation method that offers 100% reduction of mineralization, structural damage from mechanical sources would still lead to failure. This requires the development of fixation strategies that not only reduce calcification, but also resist the accumulation of structural damage.
The accumulation of non-calcific damage is highly dependent on cusp mechanics, and even minor biomechanical alterations can have profound implications on the fatigue life of BHVs. This stems from the lack of tissue repair mechanisms that leads to a cumulative effect for all incurred mechanical damage. Important biomechanical
molecules, GAGs, are not crosslinked by glutaraldehyde fixation and are consequently lost during implantation, storage, and cyclic mechanical fatigue. The increased flexural rigidity and tissue buckling that results from GAG loss may have direct repercussions on collagen integrity, which leads to cusp tears that cause valve insufficiency and failure.
Carbodiimide crosslinking using EDC and NHS is a particularly promising alternative fixation treatment. This method of crosslinking is capable of reacting with carboxyl groups as well as amines, enabling the crosslinking of both GAGs and collagen. This difference in crosslinking chemistry also causes changes to the ECM microarchitecture that may affect mechanical properties. As a result, improvements to tissue mechanics may occur through increased GAG preservation along with differing crosslink architecture. Additionally, tissues crosslinked using EDC and NHS have demonstrated improved resistance to calcification using in vivo models.
The crosslinking of GAGs alone may not be sufficient to preserve them, since they are still be prone to loss through enzymatic degradation. The use of neomycin, a hyaluronidase inhibitor, additionally protects GAGs by preventing enzymatic cleavage. Integration of neomycin into tissues is facilitated by the presence of multiple amine groups that permit integration into carbodiimide-initiated crosslinks.
We propose that the addition of neomycin into EDC/NHS fixation will demonstrate superior GAG stability while also exhibiting reduced calcification when compared to glutaraldehyde crosslinked tissue. Cusp tissue will be treated as such and assessed for general tissue stability as well as for the ability to resist GAG loss from all sources. Basic mechanical testing will also follow in order to obtain initial characterization on the effects of this crosslinking method on tissue mechanics.
2.2. Specific Research Aims
2.2.1. Aim I: What is the optimal concentration of neomycin needed to improve GAG preservation during in vitro degradation and storage?
Hypothesis: GAGs may be stabilized through EDC crosslinking due to bonding of available carboxyl groups to free amine groups on collagen. The presence of bound neomycin may further stabilize GAGs through the prevention of enzymatic degradation. Experimental Plan: Porcine aortic valve cusps will be treated using various concentrations of neomycin and then subjected to enzymatic degradation. Tissues crosslinked using the optimal treatment will be compared to EDC treated tissue with an inert amine functional molecule added in order to verify the specific contribution of neomycin to GAG preservation. GAG stability will be measured after enzymatic degradation and storage. Tissue GAG content will be quantified using hexosamine and DMMB assays, in addition to being visually verified via histology.
2.2.2. Aim II: Does neomycin further enhance EDC crosslinking of collagen and elastin in porcine aortic valve cusp tissue?
Hypothesis: The available amine functionalities on neomycin may enable interaction with EDC/NHS during crosslinking. The incorporation of neomycin into crosslinks may affect the stability of collagen and elastin.
Experimental Plan: NEN treated porcine aortic valve cusps will undergo collagenase and elastase treatment. Resistance to enzymatic degradation will be used as an indicator of
elastin and collagen stability. Differential scanning calorimetry will also be used to detect any changes in the collagen denaturation temperature of the tissue. Data will be compared to that of a GLUT control as an indicator for baseline performance.
2.2.3. Aim III: Does neomycin used with EDC crosslinking demonstrate improved
in vivo performance?
Hypothesis: The physical crosslinking along with neomycin protection in the NEN treatment may prevent the loss of GAGs due to in vivo degradation. Calcification may also be reduced for the NEN group due to the lack of glutaraldehyde crosslinks.
Experimental Plan: Rat subdermal implantation will be used to assess the performance of various crosslinking strategies. Hexosamine assay will be used to quantify the remaining GAGs after implantation, while atomic absorption spectroscopy will be used to quantify mineralization. Results will be also verified visually via histology.
2.2.4. Aim IV: Does neomycin used with EDC crosslinking affect the stiffness and extensibility of porcine aortic valve cusp tissue?
Hypothesis: The unique crosslinks formed within the NEN treated groups may cause the cusp tissue to exhibit different levels of stiffness and extensibility when compared to fresh tissue and GLUT fixed tissue.
Experimental Plan: Uniaxial tensile testing will be used to analyze stress-strain relationships for all groups in both the radial and circumferential directions.
3. MATERIALS AND METHODS