Glutaraldehyde affects biocompatibility of bioprosthetic heart valves.

A marked release of glutaraldehyde from commercially available pericardial bioprosthetic heart valve (BHV) material in washing solutions was found by high performance liquid chromatography (up to 1.8 ppm of glutaraldehyde per gram of dry tissue). In vitro endothelial cell proliferation rate was impaired dose-dependently in the presence of increasing glutaraldehyde concentrations of the cultivation medium (r = 0.9; p less than 0.05). Cultivation of endothelial cells was impossible on the surface of commercially available BHV material, but successful and uninhibited when toxic glutaraldehyde ligands of the BHV material were antagonized by treatment with L-glutamic acid.     

Relation of calcification to torn leaflets of spontaneously degenerated porcine bioprosthetic valves

The gross appearance of 54 spontaneously degenerated porcine bioprosthetic valves was evaluated to determine the relation of calcium deposition to cusp disruption. Tears or perforations were shown in 89% (48) of the degenerated valves. The most common site of tears or perforations was near the commissural attachment (60% of all tears). Grossly visible deposits of calcium salts that ruptured to the surface of the cusps or caused changes in the topography were observed in 70% (38) of the 54 valves. Calcification was adjacent to tears or perforations in 56% (27) of the 48 valves with torn cusps. Among the valves that showed calcification, the deposits of calcium salts were adjacent to tears or perforations in 71% (27 of 38). The location of deposits of calcium did not relate to the age or sex of the patient or to the position of the valve, but valves with calcium were inserted longer than valves with no calcium (87 +/- 4 versus 58 +/- 7 months; p less than 0.001). The outflow surfaces showed more calcification than the inflow surfaces, irrespective of whether the valves were in the aortic or mitral position. Among the 38 valves with calcification, 92% (35) showed calcification at the commissural attachments, 53% (20) showed calcification in the body of 1 or more cusps, 11% (4) near the base, and 8% (3) near the free edge. In conclusion, most patients with spontaneous porcine valve degeneration showed calcification. The calcification was associated with tears or perforations of the cusps in 50% of all degenerated valves, in 56% of valves with torn cusps, and in 71% of valves that showed gross calcification.     

Mechanisms of bioprosthetic heart valve calcification

BACKGROUND: Cryopreserved human heart valves are used in approximately 20% of the tissue heart valve procedures performed annually. The pathophysiology of allograft failure is not fully understood. The authors proposed the hypothesis that the rapid deterioration observed in some allograft heart valve recipients is caused by disruptive interstitial ice damage that occurs during cryopreservation and subsequently leads to accelerated valve degeneration on implantation. METHODS: This hypothesis was tested by comparison of the standard commercial heart valve freezing method of cryopreservation and an ice-free, vitrification method of cryopreservation with fresh controls in a subcutaneous, juvenile rat implant model of calcification. Calcium concentration in explants was determined by atomic absorption spectroscopy. RESULTS: Statistically significant calcification (P<0.05) was observed in both syngeneic and allogeneic cryopreserved valves relative to fresh valves. The ice-free cryopreservation method demonstrated significant reduction of allogeneic heart valve calcification (P<0.01). Comparison of fresh syngeneic and allogeneic grafts at the 3-week time point demonstrated significantly higher calcium content in allograft valve explants (P<0.005). CONCLUSIONS: These findings demonstrate that allogeneic valve calcification is influenced by two factors, the cryopreservation method used and immunogenicity. Alternative cryopreservation methods that avoid ice formation may improve the in vivo performance of cryopreserved allogeneic heart valves.     

Effect of warfarin on calcification of spontaneously degenerated porcine bioprosthetic valves

Synthesis of a calcium-binding amino acid, gamma-carboxyglutamic acid, is a vitamin K-dependent enzymatic process. Warfarin inhibits gamma-carboxyglutamic acid synthesis and, therefore, might diminish the calcification of porcine bioprosthetic valves. To evaluate this, we studied 40 porcine bioprosthetic valves removed because of spontaneous degeneration; 17 patients were treated with warfarin (prothrombin time greater than or equal to 1.5 control) and 23 were untreated. Gross visualization of calcification corresponded closely to x-ray visualization of calcification in explanted valves. No grossly visible calcification or only a single localized nodule was shown in 11 of 17 valves (65%) in treated patients and in only five of 23 valves (22%) in untreated patients (p less than 0.02). Histologic examination showed no calcium or only fine specks of calcium in nine of 13 valves (69%) among warfarin-treated patients and three of 19 valves (16%) from untreated patients. Warfarin, therefore, administered in usual clinical doses, appeared to diminish calcification in spontaneously degenerated porcine bioprosthetic valves.     

New aspects of the degeneration of bioprosthetic heart valves after long-term implantation.

Bioprosthetic heart valves removed 76 to 150 months after implantation were morphologically investigated to correlate structural alterations with clinical failure modes. Traditional morphologic methods of evaluating valvular heterografts, such as microradiography and electron microscopy, were complemented by undecalcified ground sections, a new technique for analyzing the distribution of mineral deposits. Apart from well-investigated mechanisms that accelerate tissue degeneration, our observations point to additional facts: (1) phagocytosis of collagen fibrils and elastic material by macrophages and foreign body giant cells in areas near tears and perforations and (2) initial calcification indicated by delicate crystals in the intercellular space arranged in close relation to the periodicity of the cross-striation pattern of collagen fibrils. The present report not only calls attention to degenerative changes that are enhanced by mechanical stress but also underlines phagocytosis as an important mechanism in the destruction of bioprosthetic heart valves.     

Prevention of leaflet calcification of bioprosthetic heart valves with diphosphonate injection therapy. Experimental studies of optimal dosages and therapeutic durations

Ethanehydroxydiphosphonate therapy was studied for prevention of calcification of bioprosthetic heart valve cusps (from glutaraldehyde-preserved porcine aortic valves) implanted subcutaneously in 3-week-old male rats. Animals received daily subcutaneous injections of the drug (1, 5, 10, 15, or 25 mg/kg/24 hr) for 21 days with maximal inhibition of bioprosthetic heart valve calcification at a dosage of 15 mg/kg/24 hr (calcium level of diphosphonate-treated bioprostheses 3.5 +/- 0.5 micrograms/ml; calcium level of control bioprostheses, 161.2 +/- 5.0 micrograms/mg), but with irreversibly diminished bone and somatic growth. A dosage optimum was observed at 10 mg/kg/24 hr with significant inhibition of bioprosthetic heart valve calcification (at 21 days, the calcium level was 16.4 +/- 3.6 micrograms/mg) and an absence of adverse effects on epiphyseal development and overall growth. Bioprosthetic heart valves retrieved from animal receiving ethanehydroxydiphosphonate (15 mg/kg/24 hr) for only the first week after implantation had significantly more calcification after 21 days than did bioprostheses from animals treated for 2 or 3 weeks. Bioprostheses explanted after 110 days from animals receiving the drug (15 mg/kg/24 hr) for the first 3 weeks had calcification equivalent to that of untreated control rats. Diphosphonate (15 mg/kg/24 hr) was most efficacious when initiated within 48 hours of bioprosthesis implantation, but was totally ineffective if administered after 1 week. It is concluded that ethanehydroxydiphosphonate optimally prevents bioprosthesis calcification without significant adverse effects on epiphyseal development and overall somatic growth at a dosage of 10 mg/kg/24 hr in rat subdermal implants, but it must be administered by continuous daily injections beginning within 48 hours of the implantation; this approach should be pursued in further long-term circulatory experimental studies because of its possible clinical relevance.     

Antimineralization treatments for bioprosthetic heart valves. Assessment of efficacy and safety.

Since calcification limits the durability of contemporary bioprosthetic heart valves, antimineralization treatments are being widely investigated. Potential antimineralization treatments must have sustained prevention of mineralization without adverse effects. The preclinical investigation of efficacy and safety of antimineralization treatments comprises four essential steps: (1) subcutaneous implantation in small animals, (2) in vitro biomechanical studies of hemodynamics and durability, (3) morphology of unimplanted valves, and (4) circulatory implants in large animals.     

Dynamic in vitro calcification of bioprosthetic porcine valves: evidence of apatite crystallization

OBJECTIVE: Calcification is the most important cause of structural deterioration of glutaraldehyde-fixed bioprosthetic valves. Devitalization of tissue favors calcium deposits in the shape of apatite crystals. Host factors influence the extent and progression of calcification, but the phenomenon can also occur in vitro in the absence of a viable milieu. Whether calcific deposits obtained in vitro are similar to those found in vivo is unknown. METHODS: Four porcine frame-mounted bioprostheses (St Jude Medical Bioimplant; St Jude Medical, Inc, St Paul, Minn) were tested in vitro by using a pulsatile accelerated calcification testing device at a frequency of 300 cycles per minute at 37 degrees C for 19 x 10(6) cycles with a rapid synthetic calcification solution (final product [calcium x phosphate], 130 mg/dL(2)). Three of the same type of xenografts explanted from human subjects because of calcific failure (time in place, 108 +/- 25.63 mo) served as control grafts. Each sample underwent gross and x-ray examination, histology, transmission and scanning electron microscopy, atomic absorption spectroscopy, electron microprobe analysis, and x-ray powder diffraction methods. RESULTS: All in vitro bioprostheses were heavily calcific, with intrinsic Von Kossa stain-positive deposits and a mean calcium content of 205.285 +/- 64.87 mg/g dry weight. At transmission electron microscopy, nuclei of calcification involved mostly collagen fibers and interfibrillar spaces and, more rarely, cell debris and nuclei. Electron microprobe analysis showed a Ca/P atoms ratio of 4.5:3, a value intermediate between hydroxyapatite and its precursor, octacalciumphosphate. X-ray powder diffraction showed a well-separated and sharp peak, which is typical of hydroxyapatite. Aggregates of plate-like crystals up to 8 microm in size were observed at scanning electron microscopy, with a typical tabular hexagonal shape consistent with apatite. The morphologic and chemical findings in human explants were similar. CONCLUSIONS: Intrinsic calcification of glutaraldehyde-fixed porcine valves was induced in vitro. Electron microprobe analysis and x-ray powder diffraction findings were in keeping with apatite crystallization, such as that occurring in valve xenografts implanted in vivo. The model may be of value to accelerate the screening of anticalcific agents and may reduce the need for animal experiments.     

Incidence of primary tissue valve failure in porcine bioprosthetic heart valves

This report provides retrospective follow-up data on 324 consecutive patients who received a Hancock-I porcine valve in the aortic or the mitral position, or in both positions, between June, 1974, and December, 1976. This analysis included 319 valves (193 mitral, 126 aortic) available for study of the incidence of primary tissue valve failure after 10 to 12.5 years of follow-up. Of the 319 prostheses at risk, 114 instances of primary tissue valve failure occurred. Seventy-three of the failed valves were in the mitral position, and 41 were in the aortic position. The calculated actuarial probability of freedom from primary tissue valve failure was 52 +/- 5% for the mitral and 58 +/- 6% for the aortic prostheses at 12.5 years of follow-up. For patients older than 40 years at the time of operation, the rate of freedom from primary failure was 68 +/- 8% and 55 +/- 6% for aortic and mitral prostheses, respectively, at 12.5 years. Comparison of both actuarial curves disclosed no meaningful difference. However, a tendency toward greater failure rate was observed in the mitral prosthesis group.     

Anticoagulation or antiplatelet therapy of bioprosthetic heart valves recipients: an unresolved issue.

Improvements in the performance and longevity of biological valve prostheses have steadily increased their rates of implantation in recent years. Aortic bioprostheses, which are commonly used in the elderly or when the risks of anticoagulating are high, have generally been associated with low rates of long-term complications. Freedom from anticoagulation, therefore, represents the main theoretical advantage of biological, compared with mechanical, aortic prostheses. While a variety of anticoagulant and antiplatelet drug regimens have been described, a precise antithrombotic protocol for the early postoperative period after bioprosthetic aortic valve replacement has not been developed. There are also important differences between the international guidelines published. This review examines the clinical evidence concerning the use of vitamin K antagonist and antiplatelet therapy in the early management of the antithrombotic complications after bioprosthetic aortic valve replacement.     

Binding of preformed xenoantibodies to porcine bioprosthetic valves

We have investigated whether preformed antibodies against xenoantigens bind to cellular elements remaining on porcine bioprosthetic valves after various methods of preservation. Fresh porcine valves treated with either acetone, 4% formaldehyde, or 0.625% glutaraldehyde, as well as an unfixed valve, were incubated with antiserum against porcine xenoantigens. This serum was prepared using the affinity purification method with porcine lymphocytes as the target. The valves were stained with secondary fluorescein-conjugated antibody against immunoglobulin M or immunoglobulin G and examined under fluorescent microscopy. Intense binding of immunoglobulin M to the endocardium was observed in the unfixed valve as well as in valves fixed in acetone and formaldehyde. Glutaraldehyde fixation eliminated binding of antibody. Binding was not noted within the connective tissue. No binding of antiimmunoglobulin G was noted on the endocardium of any of the sections. Examination of three glutaraldehyde-treated porcine valves explanted from the aortic position after 10 years in situ showed no immunoglobulin deposition. These results demonstrate the elimination of antigenicity to preformed antibodies in the endocardium and connective tissue of glutaraldehyde-preserved porcine valves. The findings may, in part, explain the poor performance of formaldehyde-preserved bioprosthetic xenograft valves in the past and support the use of glutaraldehyde as a preferred agent for preservation of bioprosthetic endovascular materials.     

High glutaraldehyde concentrations mitigate bioprosthetic root calcification in the sheep model

Background. Fixation at high glutaraldehyde (GA) concentrations mitigated bioprosthetic calcification in the rat model. The present study intended to verify this observation in the circulatory sheep model.

Methods. Porcine aortic roots were either fixed in 0.2%, 1.0%, or 3.0% GA. Eight roots per group were implanted in the distal aortic arch of sheep. After six weeks and six months calcification and inflammation were quantitatively and qualitatively assessed.

Results. By increasing the GA concentration from 0.2% to 3.0%, aortic wall calcification could be reduced by 38% after 6 weeks and 34% after 6 months of implantation (p < 0.01). Mineralization coincided with the presence of elastin although calcium was predominantly found in cell nuclei and membranes. Leaflet calcification was absent in all groups after 6 weeks but in a few leaflets presented as heterogeneous, nodular spongiosa deposits after 6 months. Overall, differences between 0.2%-, 1.0%-, and 3.0%-fixed tissue were quantitative but not qualitative regarding distribution patterns. There was no significant difference in inflammatory host reaction between all groups.

Conclusions. We have shown in the circulatory sheep model that the anticalcific effect of better cross-linking seems to outweigh the intrinsic pro-calcific effect of GA accumulation in bioprosthetic aortic wall tissue.     

Mechanical stresses on closed cusps of porcine bioprosthetic valves: correlation with sites of calcification

The purpose of this study was to (1) evaluate the magnitude and distribution of mechanical stresses on closed leaflets of porcine bioprosthetic valves (PBVs) by using a nonlinear, finite-element model, and (2) determine if a relationship exists between the magnitude of stresses at various sites on the leaflet and the incidence of calcification at those sites. Mechanical stresses were found to be highest near the commissures and lowest at the base of the leaflet. At a pressure of 100 mm Hg applied to the closed valve, the maximal principal normal stress was 160 kPa near the commissures, 140 kPa in the body of the leaflet, 70 kPa at the free margin, and 60 kPa near the base. Similarly, the maximal shear stress, at an applied pressure of 100 mm Hg, was 80 kPa near the commissures, 70 kPa in the body of the leaflet, 50 kPa at the free margin, and 40 kPa near the base. This distribution of mechanical stresses on the PBV leaflet coincided with the incidence of calcification of the various regions of the leaflet. Calcification was found most frequently near the commissures, less frequently in the body of the leaflet and free margin, and least frequently at the base. These observations suggest a possible causative relation between the magnitude of mechanical stresses and the site of calcification of PBV leaflets.     

Inhibition of bioprosthetic heart valve calcification with aminodiphosphonate covalently bound to residual aldehyde groups

Calcification is the principal mode of failure of bioprosthetic heart valves (BPHV) fabricated from glutaraldehyde-pretreated porcine aortic valves or bovine pericardium. Covalent binding of aminopropanehydroxy-diphosphonate (APDP) to residual glutaraldehyde in pericardial BPHV tissue was studied as an approach for the inhibition of calcification. BPHV tissue was preincubated in 0.14 M APDP at pH 7.4, 9.0, and 11.0 for various durations (1 hour to 8 days). The need for NaBH4 stabilization of the tissue-bound APDP was also examined in vitro. The bound APDP was determined using 14C-labeled APDP. APDP uptake was dependent on incubation duration and pH. Calcification of APDP-pretreated BPHV was studied using 21-day rat subdermal implants. Calcification inhibition was directly related to the amount of tissue APDP incorporation. Inhibition of calcification to less than 15% of control was achieved with a concentration of bound APDP of greater than or equal to 30 nM/mg dry tissue with more than 1 hour of incubation at pH 11.0 (bound APDP, 33.55 nM/mg; BPHV calcium content = 3.1 +/- 0.9 micrograms/mg). No adverse effects such as rat growth inhibition or disruption of bone architecture were observed after any treatment. Additionally, in vitro, NaBH4 stabilized tissue-bound APDP. In conclusion, APDP covalently bound to residual aldehyde functions markedly inhibited calcification of BPHV tissue. This inhibition was dependent on the amount of APDP incorporated. NaBH4 stabilized APDP-glutaraldehyde covalent bonds.     

Degenerative pathologic findings after long-term implantation of bovine pericardial bioprosthetic heart valves

Degeneration of bioprosthetic heart valves constitutes the most important limitation to their long-term durability and the factor that avoids a wider clinical use of these devices. We studied 26 degenerated bovine pericardial valves that belong to a series of 55 prostheses explanted for various reasons. Age of the patients at implantation of the valve and other factors predisposing to primary tissue failure did not seem to significantly influence the results obtained. Mean implantation time was longer for aortic than for mitral valves (p less than 0.05). Also, the mode of failure was different for mitral and aortic prostheses. Tearing of one or more leaflets without mineralization was more frequent (p less than 0.0025) among mitral than among aortic specimens. Coverage of the valve cusps by a macroscopically visible host sheath was more extensive on the outflow than on the inflow aspect (p less than 0.0015 aortic valves; p less than 0.015 mitral valves). On radiological examination the majority of valves had diffuse and severe mineralized lesions. Collagen degeneration was the most frequent histologic lesion to be found in both mineralized and calcium-free valves. Calcification was also frequent and appeared as mineral deposits that extended between different collagen planes. Scanning electron microscopy revealed the almost complete lack of "endothelium-like" cover on any of the valves and exposure of the underlying fibrous components of the pericardial tissue in areas subjected to abrasion. Transmission electron microscopy confirmed the collagen degeneration and disclosed electron-dense microparticles (probably mineralized) both in the extracellular space and within degenerated host connective tissue cells.