Bovine pericardium versus porcine aortic valve: comparison of tissue biological properties as prosthetic valves

The choice of a bioprosthetic valve substitute remains controversial with the major concern being primary tissue failure after implantation. We compared biological properties of the two most frequently used bioprosthetic valve materials, bovine pericardium and porcine aortic valve, before and 90 days after subcutaneous implantation in rats. Before implantation, tissue collagen and water content were measured in nine pieces of bovine pericardium and porcine valves, each fixed in 0.625% glutaraldehyde; calcium, tissue collagen, and water content were measured in another nine pieces of the same tissues after 90 days' implantation. Bovine pericardium had higher collagen content than that of porcine valve (hydroxyproline, 7.98 +/- 0.05* vs. 4.56 +/- 0.02 micrograms/mg, dry weight) but lesser water content (72.16 +/- 3.22%* vs. 87.36 +/- 1.62%) before implantation (*p < 0.001, mean +/- SD, t test); after implantation, bovine pericardium still maintained higher collagen content (hydroxyproline, 4.89 +/- 0.04* vs. 2.61 +/- 0.06 micrograms/mg, dry weight) but contained the same amount of water (60.24 +/- 5.08% vs. 61.43 +/- 9.00%) and calcium (214.43 +/- 34.34 vs. 199.33 +/- 53.44 micrograms/mg, dry weight) (*p < 0.001, mean +/- SD, t test). We conclude that bovine pericardium has superior intrinsic biological properties for prosthetic valve manufacture. With proper integration of properties and design it will in some applications be superior to the porcine aortic valve.     

Improved postfixation treatment of glutaraldehyde fixed porcine aortic valves by monosodium glutamate

The use of bioprosthetic valves remains limited due to poor long-term durability primarily because of tissue calcification-associated degeneration. Release of locally cytotoxic residual aldehyde after glutaraldehyde fixation is one of the major causes of this degeneration. In this study, monosodium glutamate was used as postfixation treatment to bind residual aldehyde in order to block its toxic effects. Thirty-six pieces of fresh porcine aortic valves were fixed by 0.625% glutaraldehyde for 14 days, and then 18 of them were treated with 1% monosodium glutamate for another 3 days before they were implanted subcutaneously into the backs of two groups of rats (n = 9 in each group) for 45 and 90 days, respectively. Retrieved specimens were examined grossly, and calcium analysis and measurements of tissue collagen and water content were carried out. The results showed that, compared with glutaraldehyde fixed specimens, monosodium glutamate postfixation treated specimens had less calcification (calcium 104.93 + 50.94 versus 141.58 +/- 58.10 at 45 days and 103.07 +/- 76.48 versus 199.33 +/- 53.44 at 90 days, micrograms/mg dry weight, p < 0.01), higher collagen content (hydroxyproline 5.50 +/- 1.29 versus 3.58 +/- 1.48 at 45 days and 5.64 +/- 0.87 versus 4.25 +/- 0.65 at 90 days, micrograms/mg wet weight, p < 0.01), and higher water content (68.00 +/- 6.95% versus 61.33 +/- 8.83% at 90 days, p < 0.05) (mean +/- SD, paired t test). We conclude that monosodium glutamate couples with residual aldehyde, which significantly reduces calcification of glutaraldehyde fixed porcine aortic valves while preserving a higher tissue collagen and water content after implantation. The preserved tissue collagen and water content of the implants is closer to that of unimplanted native valves.     

Octanediol treatment of glutaraldehyde fixed bovine pericardium: evidence of anticalcification efficacy in the subcutaneous rat model

OBJECTIVE: Anticalcification strategies of glutaraldehyde-fixed xenograft tissue aim to extract lipids or to neutralize toxic aldehyde residuals. The purpose of this study was to evaluate the efficacy of octanediol compared to standard treatments of glutaraldehyde-fixed bovine pericardium in the subdermal rat model. Octanediol treatment is an ethanolic solution (40%) containing a long chain aliphatic alcohol (5% 1,2-octanediol) that removes lipids without diminishing the stability of collagen. METHODS: Octanediol and standard glutaraldehyde fixed bovine pericardium were both implanted in 24 Sprague-Dawley rats, explanted after 30-75 days (12 animals each) and submitted to X-ray (score 0-4), histology, electron microscopy and elemental analysis by spectroscopy (Ca and P content). Unimplanted octanediol and standard glutaraldehyde fixed pericardium served as control. RESULTS: At 30 days octanediol-treated pericardium showed calcium content of 0.20+/-0.1 vs 20.07+/-36.79 mg/g dry weight for standard pericardium. The difference was also evident at 75 days: calcium content of 2.36+/-7.38 mg/g dry weight for octanediol vs 165.61+/-23.35 mg/g dry weight for standard (p<0.0001). Differences were also detected at X-ray (mean score 0.7+/-0.6 octanediol vs 3.8+/-0.4 standard at 75 days). Equally, mean P content was 11.69+/-21.33 mg/g dry weight for standard vs 0.60+/-1.45 mg/g dry weight for octanediol samples at 30 days, and 90.90+/-12.61 mg/g dry weight for standard vs 1.42+/-4.34 mg/g dry weight for octanediol at 75 days (p<0.0001). At electron microscopy collagen appeared well preserved regardless of the type of treatment; in octanediol treated pericardium cell membranes almost disappeared and only few profiles of endoplasmic reticulum and rare mitochondria were visible. CONCLUSIONS: Treatment with octanediol strongly prevents calcification of glutaraldehyde fixed bovine pericardium in rat subdermal model, even in the long-term. Evidence of octanediol efficacy may entail important implications for new generation bioprosthetic valves.     

A multi-step approach in anti-calcification of glutaraldehyde-preserved bovine pericardium

AIM: Bioprosthetic cardiovascular substitutes, manufactured from glutaraldehyde-preserved bovine or porcine tissues, are prone to calcification after implantation. The aim of the study was to evaluate the ultrastructure, material stability and calcification behaviour of glutaraldehyde-preserved bovine pericardium, treated with a multi-step anti-calcification process which addresses each of the major causes of calcification and tissue degeneration. METHODS: Bovine pericardium samples were divided into 2 groups. Group I (control) consisted of tissue fixed with 0.625% glutaraldehyde and Group II (study group) consisted of tissue fixed with 0.625% glutaraldehyde and exposed to a multi-step anti-calcification process. Ultrastructure was examined by scanning electron microscopy and material stability was assessed by mechanical testing, shrinkage temperature and enzymatic degradation. Calcification was assessed by histology (Von Kossa stain) and by atomic absorption spectrophotometry in the subcutaneous rat model. RESULTS: Bovine pericardium in the study group revealed less visible changes in the ultrastructure of the collagen matrix, improved material stability (P<0.05) and significantly (P<0.001) reduced calcification compared to control tissues (4.5+/-1.2 versus 136.03+/-11.39 ug/mg tissue). CONCLUSIONS: In conclusion, results demonstrate that the multi-step anticalcification process improved the material stability and reduced the calcification potential of bovine pericardial tissue. These improvements in the quality of the bovine pericardium should enhance the long-term durability of the tissue as a bioprosthetic substitute for cardiovascular application.     

Biocompatibility of aldehyde-fixed bovine pericardium. An in vitro and in vivo approach toward improvement of bioprosthetic heart valves.

Aldehyde-induced side effects limit the clinical usefulness of bioprosthetic heart valves. Treatment of aldehyde-fixed pericardium with L-glutamic acid at pH 3.5 and storage in a nontoxic, bacteriostatic solution resulted in a lower degree of calcification in 63-day subcutaneous implants in rats (13.3 +/- 2 mg calcium per gram dry weight of tissue), as compared with commercially available tissue (169 +/- 24 mg/gm, p less than 0.05). Endothelial cells died within 1 day after seeding on the commercial tissue; however, considerable endothelial cell proliferation was measured, even 14 days after seeding on L-glutamic acid-treated pericardium. Improved biocompatibility of this alternative treatment may be due to stable chemical binding of free, reactive aldehyde groups.     

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.     

Evaluation of kangaroo pericardium as an alternative substitute for reconstructive cardiac surgery

BACKGROUND: Bioprosthetic materials (human, bovine and porcine) are used in various cardio-thoracic repair and replacement procedures because of excellent performance and low thrombogenicity. These bioprosthetic substitutes fail due to degeneration and calcification. This study examines the morphology, tensile properties and calcification potential of kangaroo pericardium in vitro and in vivo. METHODS: Bovine (control tissue) and kangaroo pericardium, fixed in 0.625% buffered glutaraldehyde, were examined by light and scanning electron microscopy. A standard method was used for biaxial testing. Pericardial strips (10 x 5 mm) were implanted subcutaneously into male Wistar rats and retrieved after 4, 6 and 8 weeks and examined by Von Kossa's stain technique and atomic absorption spectrophotometry. RESULTS: Histology revealed serosa and fibrosa cell layers in both tissues. Electron microscopy showed a densely arranged collagen matrix in kangaroo pericardium. Kangaroo pericardium calcified significantly less than bovine pericardium at 4 weeks (0.80+/-0.28 versus 21.60+/-4.80 microg/mg) at 6 weeks (0.48+/-0.08 versus 32.80+/-14.4 microg/mg) and at 8 weeks (2.40+/-1.20 versus 30.40+/-17.20 microg/mg), respectively. CONCLUSIONS: Kangaroo pericardium has a densely arranged collagen matrix with a higher extensibility and significantly lower calcification potential. Therefore, kangaroo pericardium could be used as an alternative substitute in cardiac surgery because of its low calcification potential.     

Is the Dog a Useful Model for Accelerated Calcification Study of Cardiovascular Bioprostheses?

Abstract: Chitosan posttreatment has been shown to be effective in prevention of calcification of the glutaral-dehyde treated bovine pericardium when implanted sub-dermally in rats for 12 weeks. The efficacy of chitosan posttreatment in complete calcium mitigation of the glutaraldehyde treated porcine aortic valves implanted in the right side of the heart in dogs was well-documented in our previous study. In this study, an attempt has been made to evaluate the merit of the chitosan posttreatment in prevention of calcification of the glutaraldehyde (GA) treated porcine aortic valved conduits in the systemic circulation in dogs for a period of 5 months. Eleven mongrel dogs underwent left thoracotomy. Porcine aortic valved conduits treated with 0.625% GA (n = 5) and GA-chitosan (n = 6) were implanted in the descending thoracic aortas of the dogs for 5 months. Gross histological observations showed no calcification in either the 0.625% GA treated or in the GA-chitosan treated valved conduits at 5 months. This was confirmed by results of quantitative analyses for calcium in each explant. There was no significant difference in calcium content between the GA only (Ca, 0.43 ± 0.26 mg/g) and GA-chitosan treated (Ca, 0.51 ± 0.19 mg/g; p = 0.5959) valved conduits. This study suggests that the dog is not a suitable model for evaluating the efficacy of a calcium mitigating agent in bioprostheses implanted in systemic circulation.     

Diphenylhydantoin inhibits calcification of bovine pericardial implants and myocardium: a preliminary study.

Calcification is a major cause of glutaraldehyde-fixed bioprosthetic valve failure. Recent studies have shown that dystrophic calcification shares basic features with normal bone mineralization, including crystal initiation through the mediation of cell membranes, usually in the form of extracellular vesicles. In this study, we observed that calcification of the myocardium of DBA/2J mice was inhibited or reversed by diets supplemented with 100 mg/kg diet diphenylhydantoin (dilantin) for 70 days, with a calcification incidence of 25% in the dilantin group versus 58% in control. We further studied the effects of dilantin on bioprosthetic valve calcification. Three groups of young male Sprague-Dawley rats (100 g, 9/group) were implanted subcutaneously with 1-cm2 pieces of glutaraldehyde-fixed bovine pericardium. Controls were fed a ground chow for 45 or 90 days postimplantation; experimentals received the same chow for the first 45 days postimplantation and then were fed the same diet supplemented with 1000 mg dilantin/kg for the succeeding 45 days. Calcium content (microgram/mg dry weight) of the implants in the dilantin group was 137 +/- 18.6 versus 214 +/- 34.3 in 90 days control and 79.9 +/- 41.5 in 45 days control (mean +/- SD, P < 0.01 and P < 0.05 respectively, t test). The tibia calcium content of the dilantin group was not significantly different from 90 days control. We conclude that orally administered dilantin inhibits calcification of glutaraldehyde-fixed bovine pericardial implants preferentially. It does not cause decalcification either of implants that have already calcified or of the bones. The anti-calcification effect of dilantin may be associated with its anti-vitamin D effect.     

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

Abstract: 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 glutaralde-hyde-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 ex-planted 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.     

Calcification resistance with aluminum-ethanol treated porcine aortic valve bioprostheses in juvenile sheep

BACKGROUND: Calcification of glutaraldehyde fixed bioprosthetic heart valve replacements frequently leads to the clinical failure of these devices. Previous research by our group has demonstrated that ethanol pretreatment prevents bioprosthetic cusp calcification, but not aortic wall calcification. We have also shown that aluminum chloride pretreatment prevents bioprosthetic aortic wall calcification. This study evaluated the combined use of aluminum and ethanol to prevent both bioprosthetic porcine aortic valve cusp and aortic wall calcification in rat subcutaneous implants, and the juvenile sheep mitral valve replacement model. METHODS: Glutaraldehyde fixed cusps and aortic wall samples were pretreated sequentially first with aluminum chloride (AlCl3) followed by ethanol pretreatment. These samples were then implanted subdermally in rats with explants at 21 and 63 days. Stent mounted bioprostheses were prepared either sequentially as previously described or differentially with AlCl3 exposure restricted to the aortic wall followed by ethanol pretreatment. Mitral valve replacements were carried out in juvenile sheep with elective retrievals at 90 days. RESULTS: Rat subdermal explants demonstrated that sequential exposure to AlCl3 and ethanol completely inhibited bioprosthetic cusp and aortic wall calcification compared with controls. However the sheep results were markedly different. The differential sheep explant group exhibited very low levels of cusp and wall calcium. The glutaraldehyde group exhibited little cusp calcification, but prominent aortic wall calcification. All sheep in the two groups previously described lived to term without evidence of valvular dysfunction. In contrast, animals in the sequential group exhibited increased levels of cusp calcification. None of the animals in this group survived to term. Pathologic analysis of the valves in the sequential group determined that valve failure was caused by calcification and stenosis of the aortic cusps. CONCLUSIONS: The results clearly demonstrate that a combination of aluminum and ethanol reduced aortic wall calcification and prevented cuspal calcification. Furthermore, this study demonstrates that exclusion of aluminum from the cusp eliminated the cuspal calcification seen when aluminum and ethanol treatments were administered in a sequential manner.     

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.     

Biocompatibility and Calcification of Bovine Pericardium Employed for the Construction of Cardiac Bioprostheses Treated With Different Chemical Crosslink Methods

The use of biological materials in the construction of bioprostheses requires the application of different chemical procedures to improve the durability of the material without producing any undesirable effects. A number of crosslinking methods have been tested in biological tissues composed mainly of collagen. The aim of this study was to evaluate the in vitro biocompatibility, the mechanical properties, and in vivo calcification of chemically modified bovine pericardium using glutaraldehyde acetals (GAAs) in comparison with glutaraldehyde (GA) treatment. Homsy's tests showed that the most cytotoxic treatment is GA whereas GAA treatments showed lower cytotoxicity. Regarding the mechanical properties of the modified materials, no significant differences in stress at rupture were detected among the different treatments. Zeta‐Potential showed higher negative values for GA treatment (?4.9 ± 0.6 mV) compared with GAA‐0.625% (?2.2 ± 0.5 mV) and GAA‐1% (?2.2 ± 0.4 mV), which presented values similar to native tissue. Similar results were obtained for calcium permeability coefficients which showed the highest values for GA treatment (0.12 ± 0.02 mm²/min), being significantly lower for GAA treatments or non‐crosslinked pericardium. These results confirmed the higher propensity of the GA‐treated tissues for attraction of calcium cations and were in good agreement with the calcification degree obtained after 60 days implantation into young rats, which was significantly higher for the GA group (22.70 ± 20.80 mg/g dry tissue) compared with GAA‐0.625% and GAA‐1% groups (0.49 ± 0.28 mg/g dry tissue and 3.51 ± 3.27 mg/g dry tissue, respectively; P < 0.001). In conclusion, GAA treatments can be considered a promising alternative to GA treatment.     

Synergistic effect of released aspirin/heparin for preventing bovine pericardial calcification

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde pretreated bovine pericardium (GATBP). Aspirin, a potent antiplatelet drug, and heparin, an anticoagulant, are commonly used for postimplant complications such as thrombosis and thromboembolism. Aspirin and heparin were embedded in chitosan/polyethylene vinylacetate co-matrix to develop a prolonged release form. The effect of these drugs towards the bioprosthetic calcification was investigated by in vitro and in vivo models. In vitro and in vivo evaluation suggest that the released aspirin/heparin from the co-matrix had a synergistic effect in inhibiting GATBP calcification. In vivo subcutaneous co-implantation was performed with PEG-20,000 grafted bovine pericardium (PEG-GABP), aspirin, and heparin. Biochemical, histological, and scanning electron microscopic evaluation of retrieved samples demonstrated a significant reduction in calcium deposition and alkaline phosphatase activity on PEG-GABP compared to GATBP. It seems that the aspirin/heparin combination synergistically inhibits the pericardial calcification in addition to their antithrombotic function.     

Improved Biocompatibility of Bioprosthetic Heart Valves by L-Glutamic Acid Treatment

Treatment of glutaraldehyde-fixed pericardium with L-glutamic acid and storage in bacteriostatic preservatives (paraben) stably antagonizes free, reactive aldehyde groups within the fixed bioprosthetic heart valve tissue. In 63-day subcutaneous implants in rats, the calcification rate of this treatment (13.3 +/- 2 mg calcium/g wt tissue) was markedly reduced as compared to conventionally treated tissue (169 +/- 24 mg/g; p less than 0.05). To test the influence of tissue-released toxic aldehydes on spontaneous endothelial cell ingrowth in vivo, vascular grafts (8-cm long, 6-mm diameter) from fixed pericardium treated with L-glutamic acid were interposed into the carotid arteries in ten sheep. They were compared to grafts from conventionally treated pericardium implanted at the contralateral side. Following 3 months of implantation, planimetry revealed 49% +/- 20% of the surface of conventionally preserved pericardium to be covered with red thrombus, but only 12% +/- 5% in L-glutamic acid treated pericardium (p less than 0.05). The ultrastructural findings of a closed endothelial cell layer on the graft surface reveals the new technique to be a promising approach towards increased biocompatibility of aldehyde-fixed bioprosthetic heart valves.     

Kangaroo vs. porcine aortic valves: calcification potential after glutaraldehyde fixation

The aim of this study was to evaluate and compare the calcification potential of kangaroo and porcine aortic valves after glutaraldehyde fixation at both low (0.6%) and high (2.0%) concentrations of glutaraldehyde in the rat subcutaneous model. To our knowledge this is the first report comparing the time-related, progressive calcification of these two species in the rat subcutaneous model. Twenty-two Sprague-Dawley rats were each implanted with two aortic valve leaflets (porcine and kangaroo) after fixation in 0.6% glutaraldehyde and two aortic valve leaflets (porcine and kangaroo) after fixation in 2% glutaraldehyde respectively. Animals were sacrificed after 24 h and thereafter weekly for up to 10 weeks after implantation. Calcium content was determined using inductively coupled plasma-mass spectrometry and confirmed histologically. Mean calcium content per milligram of tissue (dry weight) treated with 0.6 and 2% glutaraldehyde was 116.2 and 110.4 microg/mg tissue for kangaroo and 95.0 and 106.8 microg/mg tissue for porcine valves. Calcium content increased significantly over time (8.8 microg/mg tissue per week) and was not significantly different between groups. Regression analysis of calcification over time showed no significant difference in calcification of valves treated with 0.6 or 2% glutaraldehyde within and between the two species. Using the subcutaneous model, we did not detect a difference in calcification potential between kangaroo and porcine aortic valves treated with either high or low concentrations of glutaraldehyde.     

A new model to test the calcification characteristics of bioprosthetic heart valves.

BACKGROUND: Tissue degeneration and calcification are the two chief obstacles to the successful application of bioprosthetic heart valves. To enable the study of the durability of bioprosthetic heart valves and the efficacy of anti-calcification treatment, it has become necessary to develop animal models. The aim of this study is to validate a new model for implantation in the pulmonary position. METHODS: Three juvenile sheep underwent implantation of Carpentier-Edwards pericardial valves in the pulmonary position (experimental group). These three valves were compared with three Carpentier-Edwards pericardial valves in the aortic position in patients which had been explanted due to primary tissue failure (clinical group). The valves were analyzed. RESULTS: The findings of macroscopic, X-ray and light microscopic examination were very similar between the two groups. Scattered irregular calcification was seen near the commissures and at the base of the cusps in both groups. Quantitative calcium content analysis showed that calcification of the cusps had progressed to almost the same degree in both groups (experimental group, 3.7+/-0.2 micro g/mg dry tissue; clinical group, 4.3+/-0.3; p>0.05). In the experimental group, calcification in the commissural area of the cusp was pronounced (6.5+/-1.0). In the clinical group, calcification had also progressed in the commissural area of the cusp (6.0+/-1.5), and extended to the base area of the cusp (6.6+/-1.2). CONCLUSIONS: This model is promising for preclinical evaluation of bioprosthetic heart valves. The degree of calcification is not significantly different between our experimental results after three months of implantation in sheep and clinical results after 10 years of implantation in elderly patients. However, the pattern of calcification is somewhat different between the two groups.     

A new storage solution for porcine aortic valves.

The aim of this study was to assess the calcification tendency of two biovalves manufactured by different fixation techniques and compare their biocompatibility when implanted subcutaneously in rats. Two biological valve types (Intact) and Mosaic, stored in either glutaraldehyde or in a solution recently developed in our department, were investigated ultrastructurally and their calcium content was measured following 12 weeks subcutaneous implantation in rats. All valves tested in this study showed a considerable loss of the endothelial cover, as judged by scanning electron microscopy. Independent of fixation conditions, the bioprostheses demonstrated a partial destruction of collagen fibers and a rearrangement of the extracellular matrix. The calcium content of Intact valves was significantly higher than that of Mosaic valves (66+/-2.6 versus 3.6+/-0.6 mg/g dry tissue, p<0.0001). Low calcium content of the bioprostheses is considered to result from effective anti-calcification treatment. Ultrastructural changes of prosthetic tissue seem to promote degenerative calcification. The valves stored in the new storage solution exhibited a calcium content which was reduced by approximately 50% compared to those stored in glutaraldehyde. The percentage of reduction in calcification of the valves stored in our newly developed solution is independent of the fixation conditions (p=0.886). The advantage of the new storage solution is based on the fact that rinsing is unnecessary before implantation and, most importantly, a clear reduction in the calcification tendency is achieved.     

Anticalcification Treatments of Bioprosthetic Heart Valves: In Vivo Studies in Sheep

Studies performed by other investigators have shown that a number of preimplantation processes inhibit the calcification of pieces of porcine aortic valves and of bovine parietal pericardium subcutaneously implanted in rats. To evaluate biological reactivity with these biomaterials functioning in an intracardiac position, mitral and tricuspid valve replacements were performed in young sheep to assess the effects of the following preimplantation processes: (1) surfactants, including sodium dodecyl sulfate, polysorbate-80, Triton X-100 and N-lauryl sarcosine; (2) covalently bound aminohydroxypropane diphosphonic acid; (3) toluidine blue; and (4) incorporation of polyacrylamide into valvular tissues. Quantitative calcium analyses showed that only the surfactants substantially reduced calcification, and only in porcine aortic valvular bioprostheses. However, morphological studies showed that some of these agents also induced alterations that decreased the durability of the valves. Toluidine blue decreased calcification to a degree that was statistically significant, but not biologically important. Polyacrylamide incorporation and diphosphonate binding increased calcification. Thus, data regarding anticalcification treatments obtained from subcutaneous implantation studies in small animal models should be cautiously interpreted and validated by studies with intracardiac valvular implantation in large animals.     

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.