Glutaraldehyde-treated Bovine Pericardium Research Articles

The effects of collagen fiber orientation on the flexural properties of pericardial heterograft biomaterials

Improving cardiac valve bioprostheses (BHV) utilizing heterograft biomaterials requires a better understanding of their mechanical behavior. Flexure is a major mode of deformation for BHV leaflets during valve operation, inducing more complex deformation patterns within the tissue compared to tensile loads. In this study, we investigated the relation between collagen fiber preferred direction and the resulting flexural properties of native and glutaraldehyde-treated bovine pericardium. 20 mm×4 mm strips were cut from the presorted sheets of bovine pericardium and divided into four groups: two directions of collagen fiber orientation in two groups of native and chemically treated specimens. Specimens were flexed in two different directions using a three-point bending technique (ASAIO J. 45(1999)59) and their flexural mechanical response compared. Results indicated that: (1) the relationship between the applied flexing moment and change of curvature of specimens was non-linear in both native and chemically fixed groups, (2) there were no directional differences in flexural properties when the bovine pericardium is flexed towards either the epi-pericardial or visceral surfaces in both native and chemically fixed specimens, (3) native and chemically fixed bovine pericardium were stiffer when flexed perpendicular to local preferred collagen fiber direction, and (4) chemical fixation increased the flexural rigidity of bovine pericardium. Results of this study indicate that the flexural properties of bovine pericardium are dominated by inter-fiber cross-links as opposed to the stiffness of the collagen fibers themselves. These findings can be used to guide the development of novel chemical treatment methods that seek to optimize biomechanical properties of heterograft biomaterials.

Response of heterograft heart valve biomaterials to moderate cyclic loading.

We have recently demonstrated that noncalcific tissue damage can lead to significant collagen degradation in clinically explanted bioprosthetic heart valves (BHVs). In the present study we quantified the early response of glutaraldehyde treated bovine pericardium (GLBP) to cyclic tensile loading to begin to elucidate the mechanisms of noncalcific tissue degeneration in BHV biomaterials. GLBP specimens were cycled at 30 Hz to a maximum uniaxial strain of 16% (corresponding to approximately 1-MPa peak stress), with the loading direction parallel to the preferred collagen fiber (PD) direction. After 30 x 10(6) cycles, specimens were subjected to biaxial mechanical testing, then cycled until 65 x 10(6) cycles. The results indicated a permanent change in the unloaded tissue dimensions of +7.1% strain in the PD direction and -7.7% strain in the cross fiber direction (XD) after 65 x 10(6) cycles and an increase of the collagen crimp period from 40.6 to 45.2 microm by 65 x 10(6) cycles (p = 0.05). Fourier transform IR spectroscopy analysis indicated that cyclic fatigue of GLBP leads to both collagen conformational changes and early denaturation. Furthermore, no significant changes in areal strain were found under 1-MPa equibiaxial stress, indicating that cyclic loading changed the collagen fiber orientation but not the overall tissue compliance. These observations suggest that while deterioration of collagen begins immediately, fiber straightening and reorientation dominates the changes in the mechanical behavior up to 65 x 10(6) cycles. The present study underscores the complexity of the response of biologically derived biomaterials to cyclic mechanical loading. Improved understanding of these phenomena can potentially guide the development of novel chemical treatment methods that seek to improve BHV durability by minimizing these degenerative processes.

Glutaraldehyde-treated bovine pericardium: effects of lyophilization on cytotoxicity and residual aldehydes.

This work assesses the effect of lyophilization on the cytotoxicity and residual aldehyde concentration of glutaraldehyde-treated and lyophilized bovine pericardium (group A), comparing it to conventional glutaraldehyde-treated bovine pericardium (group B). Cytotoxicity was measured by incubating a pericardium sample from each group in saline and assessing the eluant's influence on cellular growth. Residual aldehydes were measured by HPLC. Although both groups' eluants exhibited some cytotoxicity, the eluant from group A was less cytotoxic, with a cytotoxicity index (IC50(%)) of 41%. Group B eluants all had marked cytotoxic effects; cell growth was 24.15% of the negative control at the most dilute eluant concentration (6.25%). The mean residual glutaraldehyde level was less in group A than in group B (2.36 +/- 0.11 and 9.90 +/- 3.70 g/l, respectively; n=3, P < 0.05), but residual formaldehyde levels did not differ. These results demonstrate that compared with conventional glutaraldehyde-treated bovine pericardium, lyophilized pericardium is less cytotoxic, with fewer glutaraldehyde residues.

Partial mitral valve replacement for acute endocarditis

We present a case of acute endocarditis involving the posteromedial commissure and both leaflets of the mitral valve, including a vegetation on and perforation of the anterior leaflet, in a young man with active Crohn’s disease. Repair was performed using glutaraldehyde-treated bovine pericardium. Competence of the valve was achieved with no recurrence of endocarditis. This case demonstrates that extensive destruction of both leaflets of the mitral valve does not prohibit repair.

Implant Technique for the Sorin Stentless Pericardial Valve

Bovine pericardium has a long and checkered lfistory as a leaflet material for bioprosthetic heart valves 1 (Fig 1). Early mechanical failures in first-generation pericardial valves emanated from deficiencies of design and manufacture rather titan from the intrinsic properties of bovine pericardium itself. 2 With appropriate changes in tissue preservation and stent design, pericardial valves currently manifest durability equivalent to and perhaps exceeding that of porcine xcnografts. 3 Although preservation methods account for some differences in tile durability of bioprostheses, accelerated fatigue tests show the valve stent to be the major factor determining stress on the leaflet nmteriaP (Fig 2). The stentless pericardial valve (Sorin, Italy) has been available for more than 10 years and provides arguably the best hemodynamic performance of any valve replacement device, including the aortic homograft 5 (Fig 3). This is because the stentless pericardial valve is very thin-walled and malleable. However, the high tissue flexibility increases the risk of problems stemming from imprecise surgical teclmiqne. Imperfect commisural ali~mlent or size discrelmncy between the native annulus and sinotubular junction can cause aortic regurgitation. Nonetheless, an accurate implant is functionally equivalent to a nornml human aortic valve. We have previously analyzed our learning curve for this prosthesis in an attempt to refine tile implant procedure and provide the anticipated result even in a disadvantageous aortic root. 6 Tlle Sorin stentless pericardial valve is constructed from two separate sheets of glutaraldehyde-treated bovine pericardium (Fig. 4). A new anticalcification process is currently used in the preparation. The external cylinder is trimmed at the inflow to provide a three-ram rim of pericardimn (<one mm thick) beneath the pericardial cusps, to accommodate the inflow suture line. Fabric reinforcement is not required, because the pericardium provides appropriate mechanical suture-holding strength. The external cylinder extends above the pcricardial cusps on the outflow side, where it is scalloped to match the shape of the commisure, The external cylinder is trimmed at Surgery so as not to encroach on the native sinuses of Valsalva. The resuhing prosthesis is thin and pliable, allowing inversion at the annulus as was at one time undertaken (inadvisably) ~ith an aortic homograft. The versatility of the construction process allows a range of implant sizes from 15 to 29 ram. We have used this prosthesis by subcoronary implantation in 150 cases anti also for puhnonary valve replacement anti aortic root replacement (by suturing the valve into a Dacron graft). Besides providing superlative hemodynamics, the valve is also user friendly.

Implant technique for the sorin stentless pericardial valve

Implant technique for the sorin stentless pericardial valve

Mechanical evaluation and design of a multilayered collagenous repair biomaterial.

One method of fabricating implantable biomaterials is to utilize biologically derived, chemically modified tissues to form constructs that are both biocompatible and remodelable. Rigorous mechanical characterization is a necessary component in material evaluation to ensure that the constructs will withstand in vivo loading. In this study we performed an in-depth biaxial mechanical and quantitative structural analysis of GraftPatch (GP), a biomaterial constructed by assembling chemically treated layers of porcine small intestinal submucosa (SIS). The mechanical behavior of GP was compared to both native SIS and to glutaraldehyde-treated bovine pericardium (GLBP) as a reference biomaterial. Under biaxial loading, GP was found to be stiffer than native SIS and mechanically anisotropic, with the preferred fiber direction demonstrating greater stiffness. Quantitative structural analysis using small-angle light scattering indicated a uniform fiber structure similar to GLBP and SIS. To enable test-protocol-independent quantitative comparisons, the biaxial mechanical data were fit to an orthotropic constitutive model, which indicated a similar degree of mechanical anisotropy between the three groups. We also demonstrate how the constitutive model can be used to design layered biocomposite materials that can undergo large deformations.

Quantification of the fiber architecture and biaxial mechanical behavior of porcine intestinal submucosa.

Porcine small intestinal submucosa (SIS) has been shown to serve as a remodelable tissue scaffold in a wide range of applications. Despite the large number of experimental studies, there is a lack of fundamental information on SIS anisotropic mechanical behavior and how this behavior changes postimplantation. As a first step in our study of remodeling biomaterials, we performed biaxial mechanical testing to quantify the anisotropic mechanical behavior and used small-angle light scattering (SALS) to quantify the gross fiber structure of fresh, unimplanted SIS. Structural results indicate that SIS displays primarily a single, continuous preferred fiber direction oriented parallel to the long axis of the intestine. Occasionally, two distinct fiber populations oriented at approximately +/-28 degrees with respect to the longitudinal axis could be distinguished. Consistent with this structure, SIS exhibited a nonlinear, anisotropic mechanical response with higher stresses along the longitudinal axis. Further, the circumferential stress-strain response was strongly affected by the maximum longitudinal strain level, but the maximum circumferential strain level only weakly affected the longitudinal stress-strain response. This asymmetric mechanical coupling suggests strong mechanical interactions on a fiber level. SIS stress-strain response also was similar to glutaraldehyde-treated bovine pericardium, attesting to the substantial strength of SIS in the fresh, untreated state. The results of this study will provide a basis for a future analysis of the structural and mechanical changes during the remodeling process.

Changes in pericardial calcification due to antiplatelet agents: in vitro studies.

To develop tissue valves for prolonged use in the cardiovascular system, the complicated process of surface induced calcification must be better understood. Calcification was examined for 60 days on glutaraldehyde treated bovine pericardium (GABP) and enzyme extracted tissues fixed in glutaraldehyde (GATBP) incubated in metastable solutions of calcium phosphate, and the roles of aspirin and persantine in conjunction with vitamins C, B, or E, gentamycin (antibiotic), or pentothal sodium (anesthetic) in the medium were examined. Further, the diffusion of calcium across the GATBP was evaluated using a diffusion cell with 2 compartments. Pericardial calcification was also observed using scanning electron microscopy (SEM) techniques. It seems that the examined antiplatelet agents can modify the pericardial surfaces and subsequently their mineralization processes (GATBP, 31.7 micrograms/mg tissue; in the presence of 5 mg% vitamin C, 13.1 micrograms/mg tissue; in 1.5 mg% aspirin, 17.2 micrograms/mg tissue; and 1 mg% gentamycin, 14.8 micrograms/mg tissue) on exposure with the metastable calcium phosphate solution for 60 days. In addition, these agents may modify calcium transport and interfere with the adsorption at the surface, hence reducing calcium nodulation on GATBP. Scanning electron micrographs also revealed a reduction in calcium deposition on the pericardium due to these antiplatelet agents. It may be hypothesized that the influx of calcium on GATBP may be due to the cellular components or the involvement of plasma proteins like the fibrinogen molecule. The exact mechanism of these changes in the calcification of the pericardium are still unknown. From these in vitro findings, it appears that a combined vitamin therapy with low doses of aspirin may be beneficial for platelet suppression and thereby for prevention of thrombosis and calcification. However, more in vivo studies are needed to develop applications.

Glutaraldehyde treated bovine pericardium: changes in calcification due to vitamins and platelet inhibitors.

Cardiovascular calcification, the formation of calcium phosphate deposits in cardiovascular tissue, is a common endstage phenomenon affecting a wide variety of bioprostheses. The purpose of the present paper is to study the possibility that some antiplatelet drugs (aspirin and persantine) and certain vitamins (vitamin C, vitamin B6, and vitamin E) and their combinations might prevent the mineralization of glutaraldehyde treated bovine pericardium (GABP) by modifying the pericardial surface. In this experimental protocol, we used Golomb and Wagner's (1991) in vitro model for studying GABP calcification and a diffusion cell with 2 compartments for evaluating the diffusion of calcium across the GABP. The results showed that a combination of aspirin and vitamins (0.5 mg% aspirin, 1.5 mg% vitamin C, 4 mg% vitamin B6, and 2 mg% vitamin E) in a metastable calcium phosphate solution not only reduced the transport of calcium ions through GABP, but along with the combinations of 0.5 mg% aspirin and 5 mg% persantine also produced significant reductions in GABP calcification. The exact mechanism of these changes in the calcification of GABP are still unknown. From these in vitro findings, it appears that a combined vitamin therapy with low doses of aspirin may be beneficial for platelet suppression and thereby prevent thrombosis. In addition, the vitamins may modify calcium transport and interfere with the adsorption at the surface, thus reducing GABP calcification. However, an important question that remains unanswered is whether this inhibitory effect would continue if the antiplatelet drugs and vitamins were discontinued. For the answer, more in vivo studies are needed to develop applications.

Efficacy of the chitosan posttreatment in calcification prevention of the glutaraldehyde-treated porcine aortic noncoronary cusp implanted in the right ventricular outflow tract in dogs.

Calcific tissue failure results in poor performance of the bioprosthetic heart valve. Chitosan post-treatment has been shown to be effective in calcification prevention of the glutaraldehyde-treated bovine pericardium when implanted subdermally in rats for 12 weeks. The present study investigated the effectiveness of the chitosan posttreatment in prevention of calcification of the glutaraldehyde-treated porcine aortic noncoronary cusp 5 months after implantation in the right ventricular outflow tract (RVOT) in mongrel dogs. Either 0.625% glutaraldehyde-treated (Group 1, n = 6) or glutaraldehyde-chitosan-treated (Group 2, n = 6) porcine aortic noncoronary cusp with the aortic wall was sewn to the RVOT. Gross histological observations showed moderate calcification of the glutaraldehyde-treated cusps, but no calcification was noticed in the glutaraldehyde-chitosan-treated grafts at 5 months. This was confirmed by results of quantitative analyses for calcium in half of each explanted cusp with aortic wall. The calcium content of the 0.625% glutaraldehyde-treated cusps (Ca, 40.6 +/- 24.9 mg/g dry wt) was significantly (p < 0.01) higher than that of glutaraldehyde-chitosan-treated cusps (Ca, 1.3 +/- 0.29 mg/g dry wt). These findings suggest that chitosan post-treatment is effective in complete calcium mitigation of the glutaraldehyde-treated porcine aortic noncoronary cusps implanted in the RVOT in dogs.

Structural studies on bovine bioprosthetic tissues and their in vivo calcification: prevention via drug delivery

Cardiovascular calcification, the formation of calcium phosphate deposits in cardiovascular tissue, is a common end-stage phenomenon affecting a wide variety of bioprostheses. To study the process of calcification in tissue prosthetics, glutaraldehyde-treated bovine pericardium, dura mater and fascialata were implanted subcutaneously in rats and retrieved 21 days later and thereby morphological findings were correlated with biochemically determined levels of calcium. Transmission electron microscopy showed that calcification primarily involved the surface of collagen fibrils and the interfibrillar spaces. The deposition of calcium was higher with dura and fascia prostheses compared to pericardium. However, the release of Fe 3+ ions from chitosan matrix had substantially inhibited the deposits of calcium in all implanted tissues. It seems that the structural and anatomical features of the tissue is one of the important factors for tissue-associated calcification. It is also confirmed that glutaraldehyde-preserved pericardium is the most suitable material for the development of cardiac prosthesis, with an appropriate drug combination therapy for prevention of pathological calcification.

Anesthetic and ferric-magnesium ion combinations as calcium antagonists for glutaraldehyde-treated pericardial tissues

The principal cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated bovine pericardial valves is calcification. The present investigation describes the mineralization of glutaraldehyde-treated bovine pericardium (GBP), in an extra-circulatory environment and the possible methods of prevention via metal ions. Calcification was examined on GBP incubated in metastable solutions of calcium phosphate and the role of certain anesthetic drugs, ferric ions and magnesium ions in the media was evaluated. It seems that the addition of ethyl alcohol, pentothal and xylocaine in the calcium phosphate solutions, variably inhibited the GBP calcification. The metals like Fe 3+ ions and Mg 2+ ions and their combinations also substantially reduced the GBP mineralization. It is assumed that ferric ions may slow down or retard the calcification process by delaying the proper formation of hydroxyapatite while magnesium ions disrupt the growth of these crystals by replacing Ca 2+. Hence, it is conceivable that a combination therapy-via local delivery of low levels of ferric ions and magnesium ions-may prevent the GBP-associated calcification. Further, a very low daily intake of alcohol appears to be beneficial to reduce the profile of calcium deposition at tissue interfaces.

Inhibition of extracellular-matrix degrading proteases in pericardial-derived cardiovascular bioprostheses

The presence of intrinsic remnant extracellular-matrix degrading proteases in glutaraldehyde-treated bovine pericardium per se and in explanted bioprosthetic heart valves has been demonstrated recently by us. These enzymes were shown to degrade type I collagen and thus could contribute to a possible in vivo slow tissue degeneration in time. We have investigated the potential use of protease inhibitors for effective control of this degenerative process. Collagenase, trypsin and chondroitinase were inhibited in vitro by 0.5% glutaraldehyde to 10–20% of initial activity; these remnant activities could be further inhibited by their specific inhibitors suggesting that a combination of glutaraldehyde and a mixture of enzyme inhibitors could be applied to bovine pericardium. Analysis of glutaraldehyde-treated tissue extracts by gelatin zymography revealed the presence of at least four distinct enzyme species which were active towards a collagen substrate and were inhibited most effectively by ethylenediaminetetraacetic acid and thus could be included in the matrix-metallo-proteinase family. Bovine pericardial fragments that were treated with a combination of protease inhibitors and glutaraldehyde showed a reduced colagenolytic activity compared to glutaraldehyde alone; furthermore, upon implantation in the rat subcutaneous model the proteolytic activities found in these preparations were further reduced.

Detection of remnant proteolytic activities in unimplanted glutaraldehyde-treated bovine pericardium and explanted cardiac bioprostheses.

The presence and activity of proteolytic enzymes has been investigated in vitro on soluble and insoluble preparations obtained from both unimplanted and implanted glutaraldehyde-treated bovine parietal pericardium. Using detection by colorimetric techniques, soluble preparations were shown to hydrolyze enzyme substrates that are characteristic for trypsin-like proteases, cathepsin-like proteases, and collagenase. As detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gradient gels and gel filtration on Sepharose CL-6B, insoluble (pellet) preparations degraded denatured type I collagen in a time-dependent pattern, producing low-molecular-weight fragments. These activities were partially inhibited by phenylmethylsulfonyl fluoride, N-ethyl maleimide, soybean trypsin inhibitor, para-chloromercuribenzoic acid, or ethylenediaminetetraacetic acid, suggesting the presence of a heterogeneous enzymatic mixture. Insoluble preparations incubated with pure pericardial dermatan sulfate proteoglycan detached the glycosaminoglycan chains from their core protein carrier, producing a digestion pattern similar to Cathepsin C. These findings demonstrate the presence of active proteases in glutaraldehyde-fixed bovine pericardium per se and in explanted pericardial bioprosthetic cardiac valves, an additional factor that might contribute to intrinsic extracellular matrix degeneration in pericardial bioprosthetic devices.

Mapping of glutaraldehyde‐treated bovine pericardium and tissue selection for bioprosthetic heart valves

Glutaraldehyde-crosslinked bovine pericardium is widely used in bioprosthetic heart valve fabrication. In an attempt to set a scientific basis for more reproducible tissue selection, we produced and analyzed topographical maps of glutaraldehyde-treated bovine pericardium. Whole pericardia were divided into specific anatomical areas and their thickness was measured and mapped on templates. In each area, the suture holding power was determined in both parallel and perpendicular (to the base-apex line) directions; analyses of the tearing patterns in each fragment were used to evaluate predominant fiber orientation, and observations were confirmed by polarized light microscopy. Complete maps were superimposed graphically to aid in the selection of certain areas that would have known fiber orientation, high suture holding power, and suitable thickness. Our results describe regional heterogeneity of bovine pericardial structure and mechanical properties, specifically demonstrating variations in thickness, suture holding power, and collagen fiber orientation. Two areas of choice (representing about 35% of the total) were described as suitable for use in bioprosthetic heart valve fabrication.

Alkaline phosphatase activity of glutaraldehyde-treated bovine pericardium used in bioprosthetic cardiac valves.

Bioprosthetic valves fail frequently because of pathological mineralization, a process that begins in cell remnants of the glutaraldehyde (GLUT) fixed tissue. Other pathological cardiovascular calcification and physiological mineralization in skeletal/dental tissues are both largely initiated in cell-derived membranous structures (often called "matrix vesicles"), and the enzyme alkaline phosphatase (AP) likely has an important function in the pathogenesis of mineral nucleation. This study tested the hypothesis that AP might also be present in and contribute to calcification of bioprosthetic valves. AP activity of fresh and GLUT-treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. Following 24 hours in 0.6% HEPES-buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained in GLUT-treated tissue relative to that of fresh tissue (Vmax, 24 vs. 45 mumol reaction product/min/mg tissue protein, respectively), although binding was somewhat reduced (Km, 1.9 X 10(3) vs. 1.4 X 10(3) microM substrate, respectively). Enzyme reaction product was demonstrated in both fixed and fresh tissue by light microscopic histochemical studies, confirming the biochemical results. Reaction product was noted along membranes of vascular endothelial cells and interstitial fibroblasts, the sites of early calcific deposits in bioprosthetic valves, by ultrastructural examination of GLUT-treated tissue. We conclude that GLUT-treated bovine pericardium retains much of the hydrolytic activity of AP, an enzyme associated with normal skeletal and pathological cardiovascular and noncardiovascular mineralization, and suggest that further examination of the mechanistic role of this enzyme may stimulate new approaches for slowing or preventing calcification of bioprosthetic tissue.

Clinical experience with stentless pericardial aortic monopatch for aortic valve replacement

A stentless pericardial aortic monopatch was used in 60 consecutive patients undergoing aortic valve replacement. The monopatch is constructed of a sheet of glutaraldehyde-treated bovine pericardium, tailored and shaped to fit the aortic anulus, and is sutured in place without a stent or sewing ring. The valve area is effectively preserved by this method. Results indicate that this technique is simple, inexpensive, and applicable to all cases of aortic valve disease. It does not require anticoagulation and may allow for annular growth when used in children. This technique is particularly suitable for patients with infective endocarditis because the amount of foreign material is minimized.

Biomaterials-Associated Pathology of Cardiac Valve Prostheses: Clinical Explant Analysis and Studies of Tissue Valve Calcification

ABSTRACTStudies of the pathology of explanted clinical and experimental valve specimens were done to determine the role of biomaterials in the failure of heart valve prostheses. From 7/80 through 12/84, 129 patients had valve prostheses removed at our hospital. Thrombosis/thromboembolism was the most frequent cause of the 39 mechanical valve failures (23%). Structural failure (poor durability) caused 12%. In contrast, with tissue-derived bioprostheses (90 patients), primary tissue degenerative failure was frequent (70%), and was usually associated with calcification (CALC). Infection caused 19% of all valve failures with a frequency not differing among valve types. Other causes of failure were not biomaterials-related. Thus, although frank materials degradation necessitates only a minority of mechanical valve removals, the suboptimal biologic properties of contemporary heart valve biomaterials contribute to some frequent complications (such as thrombosis/thromboembolism). Furthermore, degeneration due to mineralization is the overwhelming mode of failure of bioprosthetic valves.To understand and potentially eliminate bioprosthetic valve CALC, the kinetics, morphology and host and implant determinants of this process were studied using subcutaneous implants in rats. In this model, in which the pathologic features were similar to those of actual experimental and clinical valve replacements, CALC of glutaraldehyde-treated porcine aortic valve and bovine pericardium began within 48 hours following implantation; mineral levels were comparable to those in failed clinical valves after 84–126 days. Calcific deposits were initially associated with connective tissue cells, but later involved collagen. Systemically-administered ethane-hydroxy-diphosphonate (EHDP), a drug used to treat some forms of metabolic bone disease, inhibited CALC by 97%, but caused significant bone growth retardation. However, EHDP given by site-specific controlled-release largely mitigated CALC at only 1% of the total effective systemic dose, but without adverse effects.