Characterization of a polyepoxy compound fixed porcine heart valve bioprosthesis.

Concerns with the currently available bioprostheses are calcification, long-term durability, and suboptimal hemodynamic performance. It is well known that these concerns are all more or less related to the cross-linking reagent, glutaraldehyde or formaldehyde, used in fixing bioprostheses. To address these concerns, we undertook the development of a porcine bioprosthesis fixed with a polyepoxy compound. In the development of this polyepoxy compound valve, it was found that the porcine leaflets fixed with polyepoxy compound were softer and more pliable than those fixed with glutaraldehyde. In this study, a special microtoming technique was developed to section the biological tissue so that the fixation uniformity in distinct layers of porcine aortic wall could be characterized. The fixation index and the denaturation temperature measurements in distinct layers of aortic walls showed that the cross-linking density was uniform throughout the entire aortic wall for the polyepoxy compound fixed porcine valve. It was also noted that the fixation index of the polyepoxy compound fixed aortic wall (91.5 +/- 0.5, n = 3) was not significantly different from that of its valvular leaflet (90.6 +/- 0.8, n = 3). Similarly, the denaturation temperature of the polyepoxy compound fixed aortic wall (80.4 +/- 0.9 degrees C, n = 5) was statistically comparable to that of its valvular leaflet (79.0 +/- 0.5 degrees C, n = 5). The results of this study indicated that polyepoxy compound can adequately fix the entire porcine aortic wall as well as its valvular leaflets.(ABSTRACT TRUNCATED AT 250 WORDS)

Fixation of bioprosthetic tissues with monofunctional and multifunctional polyepoxy compounds.

Collagen from a native tissue is fixed with a polyepoxy compound (PC) for use as a new biologic prosthetic material. Prior studies have shown that this biomaterial has comparable properties with collagen fixed with glutaraldehyde (GA), and thus has great promise for biomedical applications. A prior kinetic study indicated that the reaction between the functional groups of collagen and the multifunctional epoxy EX-313 is a 2.5th-order reaction. The purpose of this study was to understand the mechanism of the amino acid-PC reactions in a fixation process. Bovine arteries were fixed with a monofunctional PC (EX-131) and a multifunctional PC (EX-313) as a function of fixation time. A sequential fixation with a second fixative was used to identify the available remaining reactive sites from a prior fixation. The denaturation temperature (Td) was measured on each sample. Because the denaturation temperature is a direct indication of crosslinking of individual amino acids with the fixative, the increase in Td of a subsequent fixation may be indicative of the available remaining amino acids. The fixation index was measured on each sample to reflect the increase of fixation completion in a sequential fixation process. The fixation index and crosslink data also revealed that the reactive amino acids for EX-131 and EX-313 may not be exactly the same. The data in this study suggest that a monofunctional fixative can pre-react with the amino acids of collagen to effectively block further fixation of collagen with a second fixative. This amino acid masking may be associated with collagen branching. Collagen branching and its effect on denaturation temperature are described.

A newly developed porcine heart valve bioprosthesis fixed with an epoxy compound. An experimental evaluation.

Concerns with currently available bioprostheses are calcification, long-term durability, and functional and hemodynamic performance. It has been well known that these concerns are all more or less related to the fixatives, glutaraldehyde or formaldehyde, used in preserving bioprostheses. To address these concerns, we undertook the development of a porcine bioprosthesis fixed with an epoxy compound. It was discovered that the porcine leaflets fixed with the epoxy compound appeared more natural than those preserved with glutaraldehyde. The performance of this newly developed epoxy compound bioprosthesis (three samples) was evaluated in a juvenile sheep model. The results were compared to those of its glutaraldehyde counterpart (three samples). Two-dimensional echocardiographic inspection of the valvular leaflet motion indicated that the epoxy compound leaflets were more pliable than their glutaraldehyde counterparts. In addition, the epoxy compound valve appeared to open more widely than the glutaraldehyde valve. Color Doppler flow mapping demonstrated that the blood flow distal to the epoxy compound valve was slightly broader than that observed distal to the glutaraldehyde valve. Moreover, at retrieval, less calcium and pannus ingrowth were observed in the epoxy compound valve than its glutaraldehyde counterpart. The results of this preliminary evaluation indicated that the performance of this newly developed epoxy compound valve was at least equivalent to its glutaraldehyde counterpart, if not better.

Evaluation of collagen modification and surface properties of a bovine artery via polyepoxy compound fixation.

Collagen of bovine internal thoracic artery (BITA) was treated with glutaraldehyde (GA) or polyepoxy compounds (PC). This study was to evaluate the surface properties as a result of tissue tanning reaction with PC. The fixation resulted in a significant reduction of available lysine, histidine, and other amino acid residues in PC fixed grafts as compared to fresh pre-fixed arteries. Among them, the lysine (Lys) content was reduced by about 80%, indicating that PC reactions mainly involve with Lys residues. Both PC and GA treatment led to crosslinking as evidenced by the increase in the denaturation temperature. The critical surface tension and the Fourier Transform Infrared Spectrum (FTIR) on a pre-implant and its 96 days explant were evaluated and found to be similar. The FTIR analysis of a pre-implant and the 96 day explant indicated that there was no lipid deposition.

A preliminary study of the fixation mechanism of collagen reaction with a polyepoxy fixative.

A new biomaterial has been developed by fixing native collagens with a polyepoxy compound (PC) fixative. Prior studies have shown that this biomaterial has comparable properties as compared to collagen fixed with glutaraldehyde (GA) and thus has a great promise for use as an implantable bioprosthesis. The purpose of this study was to understand the mechanism of the amino acids-PC reactions in the fixation process. Bovine arteries were fixed with PC under various pH, concentration and temperature conditions as a function of fixation time. Individual amino acid components in the fresh and the fixed arteries were assayed using a Beckman amino acid analyzer to determine the degree of tanning. The denaturation temperature (Td) was also measured on each sample. Since the denaturation temperature is a direct indication of cross-linking of individual amino acids with the fixative, the difference in the degree of tanning for the same increase in Td may be indicative of the quantity of the masked, non-cross-linked amino acids. The fixation reaction data indicated that not all amino acids were cross-linked upon contacting the PC fixative. Masking appeared to be more substantial with a fixation at higher pH values.

Comparison of the cross-linking characteristics of porcine heart valves fixed with glutaraldehyde or epoxy compounds.

The concerns about currently available bioprosthetic heart valves are calcification, long-term durability, and functional and hemodynamic performance. These concerns are all more or less related to the cross-linking reagents, glutaraldehyde or formaldehyde, used in fixing bioprostheses. To address these concerns, the authors undertook the development of a porcine heart valve cross-linked with an epoxy compound. This study compared the cross-linking characteristics, shrink temperature, and moisture content of porcine heart valves fixed with epoxy compounds or glutaraldehyde. Two types of epoxy compounds, Denacol EX-313 and EX-810, or a 0.625% glutaraldehyde were used to fix the porcine aortic valves procured from a slaughter house. Samples of each group were removed at various elapsed fixation times. The shrink temperature and moisture content of the valvular leaflet and distinct layers of aortic wall of each sample were measured. Fresh porcine aortic valve was used as a control. It was found that the shrink temperature of the glutaraldehyde leaflet was the highest, whereas the moisture content of the EX-313 leaflet was the greatest among the three test groups. No significant difference in shrink temperature was observed among the epoxy compound fixed inner, middle, outer, and entire aortic walls. This implied that the cross-linking density of the epoxy compound valve was uniform throughout the entire aortic wall. The same also was observed for the glutaraldehyde fixed aortic wall.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study of complement activation by Denaflex, Bioflow, and BioPolyMeric vascular grafts.

This study was performed to evaluate the degree of complement activation by three bovine arterial graft materials: Bioflow (Bio-Vascular Inc., a bovine artery fixed with dialdehyde starch), BioPolyMeric (St. Jude Medical Inc., a collagen conduit of bovine arterial origin, tanned with glutaraldehyde and covered with a Dacron mesh), and Denaflex (Baxter Edwards CVS Division, a bovine artery fixed with polyepoxy compounds). The grafts were rinsed by following the manufacturer's recommended procedures and thereafter incubated with normal human serum. CH50 assays were performed on the serum after incubation, and the percentage of complement activation for each sample was calculated relative to its control serum. The results indicated that the BioPolyMeric grafts activated the most complement, with about a 48% decrease in the CH50. The BioPolyMeric graft is composed of an outer polyester mesh and an inner collagenous tubing, exhibiting a nonreversible negative surface charge. After the polyester mesh was removed, the BioPolyMeric graft showed the highest complement activation in this study, suggesting that the glutaraldehyde fixed graft is more prone to complement activation than either the polyepoxy compound or dialdehyde starch fixed grafts. The complement fragment, C5a, generated during complement activation is strongly chemotactic for polymorphonuclear leukocytes and monocytes, which likely play early and long-lasting roles in regulating tissue reaction to the implanted graft.

Development and evaluation of a pliable biological valved conduit. Part I: Preparation, biochemical properties, and histological findings.

Different types of external valved conduits have been used for the repair of complex congenital cardiac anomalies that may have otherwise been inoperable. However, an ideal conduit has yet to be found due to complications such as stenosis, thrombosis, calcification of the valve and graft wall, and "peeling" of the neointima. To address those problems, a new extracardiac valved conduit made of bovine jugular vein was developed and evaluated in a preliminary animal study. Harvested bovine vein containing a naturally existing valve was initially incorporated with protamine on the inner surface and then was cross-linked in diglycidyl ether (DE). Fixation with DE allowed the vein and its leaflets to retain a tissue-like elasticity. To provide antithrombogenicity to the graft, heparin was introduced into the lumen to bind ionically to the pre-entrapped protamine. The biological valved conduit of approximately 14 mm diameter was implanted from the right ventricle to pulmonary artery as bypass graft in three dogs. After implantation, the native main pulmonary artery was ligated between the anastomotic sites of the bypass conduit. No anticoagulant or antiplatelet drugs were administered after surgery. One DE-fixed valved conduit was retrieved at 3 months, and the others were removed at 5 months. Only small thrombus areas were found on the white luminal surfaces. The valves and the conduits maintained softness and pliability, similar to before implantation. Additionally, the collagen content, shrink temperature, and tanning index of this newly developed biological valved conduit before and after fixation were measured in the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Development and evaluation of a pliable biological valved conduit. Part II: Functional and hemodynamic evaluation.

Many congenital cardiac malformations may require a valved conduit for the reconstruction of the right ventricular outflow tract. In spite of many endeavors made in the last 25 years, the clinical results of right ventricular outflow tract reconstruction with currently available valved conduits are still not satisfactory. Specific problems encountered clinically include suboptimal hemodynamic performance, conduit kinking or compression, and fibrous peeling from the luminal surface. To address these deficiencies, we undertook the development of a biological valved conduit: a bovine external jugular vein graft with a retained native valve cross-linked with a diglycidyl ether (DE). This study, using a canine model, was to evaluate the functional and hemodynamic performance of this newly developed valved conduit. Three 14 mm conduits, implanted as bypass grafts, right ventricle to pulmonary artery, were evaluated. The evaluation was conducted with a noninvasive color Doppler flow mapping system at pre-implantation, immediately post implantation, one- and three-months post implantation, and prior to retrieval (five-months post implantation). The two-dimensional tomographic inspection of the leaflet motion at various periods post implantation showed that the valvular leaflets in the DE treated conduit was quite pliable. No cardiac failure or valvular dysfunction was observed in any of the studied cases. The color Doppler flow mapping study demonstrated that the valve in the DE treated conduit was competent, with no conduit kinking or compression observed in any of the three cases. The spectral Doppler velocity study evidenced that the transvalvular pressure gradients of the DE treated conduit were minimal as compared to those of the currently available conduits. In conclusion, from the functional and hemodynamic performance points of view, this newly developed valved conduit is superior to those currently available.

A compliant biological vascular prosthesis.

One requirement of the mechanical parameters for an acceptable vascular prosthesis is compliance. The compliance of a vascular prosthesis is defined as the fractional change in luminal volume per unit change in applied pressure. A compliant prosthesis has been correlated to prosthesis patency and long-term efficacy in an animal study. However, there have been very few reports on how to manufacture a compliant prosthesis. It is the objective of this study to research the processing methods to manufacture a reasonably compliant vascular prosthesis. A new fixative, polyepoxy compound, was used to fix an artery. The arteries were fixed under different degrees of longitudinal retraction. By locking in the collagen micro-structure at an overly relaxed state and then crosslinking said collagen, the resulting biological prosthesis exhibited extreme compliance and pliability. A prosthesis matching its arterial origin in tensile modulus was achieved by crosslinking an artery at its 45% retraction longitudinally. This flexible prosthesis showed a volumetric compliance index of 18.4 +/- 0.9 % delta/100 mmHg and a longitudinal tensile modulus of 942 grams/cm2. Our current study indicated that a prosthesis fixed with polyepoxy compounds has shown more pliability than that with glutaraldehyde. Further animal study to correlate prostheses patency to different degrees of compliance is needed to confirm this proposed manufacturing approach.

Kinetic study of collagen fixation with polyepoxy fixatives.

A new biomaterial has been developed by fixing native collagen with a polyepoxy compound (PC) fixative. In this study, bovine internal thoracic arteries were fixed with PC under various conditions to help understand the kinetics of the collagen-PC reactions and optimize the fixation process. At predetermined time intervals, small samples were cut from the arteries to determine the quantities of the remaining unreacted amino acids in the collagen. Temperature, concentration, and solution pH were among the key parameters studied. The overall fixation rate was found to be reaction-rate controlled, as the rate of fixation was relatively slow compared with the rate of diffusion of PC. As might be expected, the reaction rate was favored by a higher temperature, concentration, and solution pH. A kinetic model, with a 2.5th reaction order with respect to the reactive functional groups of collagen and a first order with respect to PC, was developed that gave a good fit to the experimental data. Based on this model, the degree of fixation, X, as a function of time, t, is given by (1 - X)-1.5 = 1 + Kt, where K is a constant related to the initial concentrations and the reaction rate constant.

Noninvasive color Doppler inspection of small-diameter vascular grafts implanted in canine carotid and femoral arteries.

To noninvasively evaluate a small-diameter vascular graft (approx. 4 mm in diameter) developed for coronary artery bypass application, a state-of-the-art color Doppler flow mapping system was applied to inspect various grafts implanted in 5 canines. The grafts, including Denaflex, Gore-Tex ePTFE, and Bioflow, were implanted interpositionally in the carotid and femoral arteries. Inspections were conducted with a 5 MHz linear vascular transducer at 6 weeks postimplantation and 12 weeks postimplantation, immediately prior to retrieval. In the carotid artery position, all 5 Denaflex grafts were patent throughout the implantation period while 2 of the 5 Gore-Tex grafts were occluded at 6 weeks, and 1 more was occluded at 12 weeks. In the femoral artery position, all 5 Denaflex grafts were patent at 6 weeks; however 2 were occluded at 12 weeks. For the Bioflow grafts at 6 weeks, 3 were patent, and 2 were occluded. The same results were observed at 12 weeks. The color Doppler inspection results indicated a higher patency rate in the carotid artery position than in the femoral artery position. Furthermore, the volumetric flow rate and the wall shear stress measured with the pulsed Doppler in the carotid artery were greater than in the femoral artery. These findings suggest that the hemodynamic "environment" in which the graft was implanted may affect the graft patency rate. The Doppler inspection results obtained at 12 weeks, identical with those observed after retrieval, demonstrated that color Doppler flow mapping is a reliable method to noninvasively inspect blood flow through small-diameter vascular grafts.

Evaluation of a polyepoxy compound fixed biological vascular prosthesis and an expanded polytetrafluoroethylene vascular graft.

Antithrombogenicity is one essential requirement for the successful use of small caliber vascular prostheses. In this study, a polyepoxy compound fixed, heparinized 4 mm diameter Baxter Denaflex vascular graft was evaluated against a 4 mm diameter Gore-Tex expanded polytetrafluoroethylene (ePTFE) vascular graft in the canine model. In addition to the thromboresistant characteristic conferred by heparinization, the crosslinking agent allowed the Denaflex graft to retain the original color of the native artery. Six centimeter long graft segments were implanted into the carotid arteries bilaterally in 5 dogs. The patency rate at 3 months for the Denaflex graft was 100% (five out of five) whereas in the control ePTFE graft, it was 40% (two out of five). The explanted Denaflex grafts exhibited softness and flexibility, and their luminal surfaces maintained a white color like that before implantation. To the contrary, the patent ePTFE grafts felt hard, and red thrombi covered large portions on their inner surfaces. Under microscopic observation, neointima formation was limited to regions near the anastomotic sites for both types of grafts. This experiment showed that the Denaflex vascular graft has an excellent antithrombogenic property and has a compliance similar to native arteries.

Dynamic internal compliance of a vascular prosthesis.

A new technique was used to measure dynamic internal compliance of a blood vessel or vascular graft subjected to dynamic internal pressure. The internal compliance can be broken into three categories: the volumetric compliance (CV), defined as (dV/V)/dP; the longitudinal compliance (CL), defined as (dL/L)/dP; and radial compliance (CR), defined as (dR/R)/dP. It can be shown mathematically that CV = 2 CR + CL. Thus, measuring any two of the three entities will also give the value for the third. A Dynatek DCT1 dynamic compliance tester was used for measuring the compliance of DenaflexTM biologic grafts and fresh bovine internal thoracic arteries, from which the Denaflex grafts were obtained by fixation. Volumetric compliance was obtained with the test sample mounted in a loose loop that allowed the sample to move both radially and longitudinally. By mounting the sample in a straight fashion that limited longitudinal movement, the radial compliance was determined. The longitudinal compliance was then calculated from the above relationship. Test results show that the fresh bovine artery had an average volumetric compliance of 26.1%/100 mmHg, radial compliance of 9.5%/100 mmHg, and longitudinal compliance of 7.2%/100 mmHg. The Denaflex vascular graft showed a reduction in longitudinal and radial compliance, compared to the fresh raw artery, as a result of extensive fixation.