A new in vitro test method for calcification of bioprosthetic heart valves

To investigate the calcification behavior of different bioprosthetic heart valves and verify possible hypotheses of the etiology of valve calcification, an accelerated pulse tester for bioprostheses was developed, whereby up to ten valves can be tested under identical test conditions. Each valve was mounted in a separate compartment on a piston and cyclically moved through a calcifying solution at frequencies of up to 800/min at 37 degrees C: An appropriate calcifying solution was evaluated by incubation tests of bovine and porcine tissue. Calcification was confirmed by measuring Ca and phosphate depletion by atomic absorption spectroscopy, von Kossa staining, EDAX, and microradiography. The first tests were successfully carried out on porcine valves that had been nondestructively assessed for tissue/stress anomalies by holographic interferometry prior to the calcification test. The tests showed that 75% of irregular fringe pattern areas corresponded to the calcification areas.     

Symptomatic spinal calcinosis in systemic sclerosis (scleroderma)

Two patients with diffuse cutaneous systemic sclerosis and spinal calcification, involving the lumbar spine in one and the cervical spine in the other, are described. Computed tomography-guided aspiration of the calcific masses was performed, and material aspirated from one patient was shown to be apatite, Ca5(PO4)3OH. One patient showed improvement following lumbar laminotomy, hemilaminectomy, and diskectomy.     

X-ray diffraction study of in vitro calcification of tendon collagen

Decalcified samples of turkey leg tendon were submitted to in vitro calcification in the presence of metastable solutions of calcium phosphate at different concentrations. The structural relationship between apatitic deposits and collagen fibrils was examined by high- and small-angle X-ray diffraction using conventional and synchrotron radiation sources. At high supersaturation the apatitic crystallites were deposited on the collagen fibrils with their crystallographic c-axis preferentially oriented parallel to the fibril axis. At lower supersaturation, a fraction of the apatitic crystallites also grew with the c-axis preferentially oriented parallel to the collagen fibril axis, whereas other exhibited a preferential orientation perpendicular to the fibril axis. The analysis of the small-angle X-ray diffraction data indicates that the deposition of the apatitic phase in the sample stored in solution at lower supersaturation induced modifications of the collagen electron density distribution in the axial direction, which can be attributed to the deposition of the inorganic crystallites inside the gap region of the collagen structure.     

An in vitro diffusion model for the study of calcification of bovine pericardium tissue

Bovine pericardium (BP) is extensively used for the production of heart valve bioprostheses. BP has excellent mechanical properties but a limited lifespan because of intrinsic subsurface calcification in vivo. In this study, the in vitro mineralization of BP was investigated by a novel diffusion cell model. In two sets of experiments, glutaraldehyde-treated BP membranes were placed between two compartments, both of which contained calcium phosphate solutions made by equilibration of octacalcium phosphate (Exp I) or dicalcium phosphate dihydrate (Exp II) in phosphoric acid. The movement of calcium (Ca), phosphate (P), and protons through the BP membrane was followed throughout the diffusion process. Histology, scanning electron microscopy, wet chemical analysis, and energy dispersive X-ray analyses provided good evidence of subsurface mineralization of BP that resembled in vivo mineral deposition. Energy dispersive x-ray microanalyses found a Ca/P heterogeneity of the early subsurface mineral that formed in the membrane. The use of a diffusion cell to model BP calcification under well-characterized conditions has led to in vitro mineralization that more closely matches that observed in vivo. The results suggest that this in vitro diffusion model can be used to study the mechanism of pathological mineralization. This model has the potential to provide rapid, inexpensive, basic information about the mineralization process.     

Complement activation is involved in the structural deterioration of bovine pericardial bioprosthetic heart valves

Disintegrated collagen fibers surrounded with protein deposits are a morphologic feature in torn, folded, and disrupted cusps of pericardial prostheses explanted for clinical dysfunction. New technologies for valve bioprostheses with improved durability require further investigation of molecular mechanisms initiating the deterioration of bioprosthetic valves. The authors' aim was to obtain experimental evidence of biologic factors contributing to the degradation of the bioprosthetic matrix. Clinically failed Mitroflow (22), Hancock (3), Ionescu-Shiley (2), and Sorin (1) valves were explanted after 69-170 months. Non calcific deterioration of the prosthetic matrix was studied with labeled antibodies to plasma proteins and cells. IgG, and complement proteins C1q, C3, and C4 were accumulated close to dissociated collagen bundles (26/28) throughout the prostheses. Fibrin was identified on the cuspal surface and in the deep disrupted areas. The fibrin peptides and proteolytic breakdown products of the complement components, the latter consistent with complement activation and chemotaxis for monocytes, were shown by immunoenzymic assay on Western blots from the valve extracts. The complement activation triggered by the IgG aggregates generates bioactive peptide signals that can activate macrophages (22/28) and neutrophil granulocyte elastase (22/24) able to cooperate with the mechanical stress in the breakdown of the chemically processed, non hemocompatible, and non-self macromolecular matrix.     

In vitro and in vivo calcification of vascular bioprostheses

Efficacy of different chemical treatments on calcification of vascular graft in vitro and in vivo was studied. Culture medium-filled rat aortas were separately treated in 0.2% glutaraldehyde and epoxy compound, and photooxidized in 0.01% methylene blue for a shorter period (group 1). Another group of rat aortas were separately treated in the same chemicals for a longer period (group 2). All fresh and treated aortas of both groups were cultured for 21 days in an organ culture medium and implanted (except for group 1) in weanling rats for five months. Histology and immunohistochemistry revealed that differently treated aortas of group 1 grow and calcify, and the smooth muscle cells between elastin fibers are the primary site of calcium deposition. In contrast, differently treated aortas of group 2 neither grew, nor did calcify in the medium except the epoxy compound cross-linked aorta of group 2 which did not grow but did calcify. Untreated aorta did not calcify. All fresh and differently treated aortic homografts calcified severely in rats. Our whole arterial segment-calcification system would be useful for analyzing the molecular and cellular mechanisms of both bioprosthetic and atherosclerotic calcification of vascular graft. New anticalcification technique is the only hope for better outcome of future vascular bioprostheses.     

No-react detoxification process: a superior anticalcification method for bioprostheses

BACKGROUND: Glutaraldehyde pretreatment of bioprosthetic heart valves is the major pathogenic factor in their calcific degeneration. This comparative study investigates the merit of the No-React aldehyde detoxification process as an alternative modifier of xenograft tissues. METHODS: Glutaraldehyde- and No-React-pretreated porcine aortic valve cusps were implanted subcutaneously in 6-week-old rats (n = 20). At 3, 6, and 14 weeks, randomly selected animals were sacrificed and the explants underwent mineral and morphologic analyses. Glutaraldehyde- and No-React-treated bovine pericardium and porcine aortic valve cusp were incubated in fibroblast cell culture plates. Cell viability was observed under reversed microscope at 6, 24, 48, and 96 hours. Erythrosin B dye exclusion test was used to validate percent cell death. RESULTS: Pretreatment with No-React significantly inhibited calcification of aortic cusp subcutaneous implants throughout the 14-week period (mean tissue Ca2+ content = 1.3 +/- 0.7 micrograms/mg at 14 weeks.) Glutaraldehyde-treated cusps underwent protracted calcification (Ca2+ content = 190.6 +/- 89.5 micrograms/mg; p < 0.01). Morphologic findings correlated with mineral analyses. One-hundred percent of fibroblast cells survived in the presence of No-React-treated tissue, with a growth pattern indistinguishable from control cell culture (ie, in the presence of no tissue). The cells incubated with glutaraldehyde-treated tissue showed signs of nonviability by 6 hours, with 100% cell death by 48 hours. Dye exclusion tests validated these findings. CONCLUSIONS: The No-React detoxification process completely abolishes the cytotoxicity of the xenograft tissue and inhibits calcific degeneration.     

Long-term Doppler echocardiographic results of aortic or mitral valve replacement with Biocor porcine bioprosthesis

OBJECTIVES: Our objectives were to evaluate the long-term bioprosthetic and cardiac functional outcome after insertion (over a 10-year period) of a new-generation porcine zero pressure-fixed Biocor bioprosthesis, as well as to determine the echocardiographic accuracy for selection of patients requiring reoperation. The long-term systematic Doppler echocardiographic assessment after valve replacement with this bioprosthesis is lacking. METHODS: Between January 1983 and January 1993, we inserted 756 Biocor prostheses in the aortic (619) or mitral (137) positions. All 51 patients who had a reoperation during the follow-up time were evaluated echocardiographically before reoperation. Additionally, 263 of 446 patients (59%) with aortic bioprostheses and 42 of 74 patients (57%) with mitral bioprostheses who were alive in January 1993 had long-term echocardiographic follow-up. RESULTS: Group A: Normally functioning bioprostheses were found in the aortic position in 242 of 263 patients and in the mitral position in 33 of 42 patients. Group B: Thirty patients had abnormal bioprosthetic function. Eleven patients had regurgitation, 3 had a combined lesion, and signs of calcification appeared in 16 patients with aortic valves, all with a peak gradient of above 60 mm Hg. Group C: Patients who had a reoperation (41 aortic and 10 mitral) within the follow-up period were followed up echocardiographically from the detection of a possible valve dysfunction until reoperation, and the findings accorded well with those at operation in 49 of 51 patients. CONCLUSIONS: These findings suggest that, during a long-term follow-up, most bioprostheses function normally, facilitating improved heart function. Abnormalities in a bioprosthesis usually develop gradually, enabling their detection by Doppler echocardiographic evaluations performed regularly or in case of any symptomatic deterioration.     

Atherosclerotic plaque components in human aortas contrasted by ex vivo imaging using fast spin-echo magnetic resonance imaging and spiral computed tomography

RATIONALE AND OBJECTIVES: Imaging techniques that distinguish atherosclerotic plaque components may be useful in identifying the nature of the atherosclerotic lesion and determining the best method of treatment for obstructive vascular mining the best method of treatment for obstructive vascular disease. This study compares fast spin-echo (FSE) magnetic resonance (MR) and spiral computed tomography (CT) images of excised human atherosclerotic aortas to determine which imaging technique provides the best contrast between plaque components ex vivo. METHODS: Aortas were imaged using four FSE sequences in MR with and without frequency-selective fat saturation, and using spiral CT without contrast. The average signal intensity of a region of calcification, thrombosis, fatty plaque, and normal vessel wall was measured on all images and compared. RESULTS: The use of fat saturation pulses in MR did not significantly alter the signal from atherosclerotic plaque for the sequences used. Proton density-weighted FSE sequences that collected early echoes were better than other FSE sequences and CT at differentiating calcification from all soft tissues. T2-weighted FSE sequences that collected later echoes were best at soft-tissue discrimination. CONCLUSIONS: The FSE techniques used were superior to nonenhanced spiral CT in discriminating plaque components ex vivo, including calcification.     

Impact of glutaraldehyde on calcification of pericardial bioprosthetic heart valve material

BACKGROUND: This study was conducted to investigate the impact of the preservation method of bioprosthetic heart valve materials on calcification rates and biocompatibility of the biologic tissue. METHODS: In subcutaneous rat implants, conventionally preserved bioprosthetic heart valve material was compared with bovine pericardium that was treated with L-glutamic acid to reduce residual glutaraldehyde released from the fixed tissue. Both these methods were compared with bovine pericardium that was stabilized by a dye-mediated photooxidation reaction without glutaraldehyde. Biocompatibility of these biomaterials was tested in vitro using human endothelial cell cultures. RESULTS: Conventionally preserved bovine pericardium with a high amount of glutaraldehyde incorporated into the tissue resulted in severe calcification 63 days after subcutaneous implantation in rats (165.4 +/- 20 mg Ca2+/g dry weight). Postfixation treatment with L-glutamic acid, which reduces free, unbound aldehyde groups, showed a significant decrease in calcification (89.6 +/- 14 mg Ca2+/g dry weight). Glutaraldehyde-free preservation by dye-mediated photooxidation showed no calcification after 63 days of subcutaneous implantation (1.0 +/- 0.4 mg Ca2+/g dry weight). Regular endothelial cell proliferation was observed on photooxidized and L-glutamic acid-treated tissue, whereas conventionally treated tissue caused endothelial cell death. CONCLUSIONS: This study underlines the detrimental role of glutaraldehyde in the calcification process of bioprosthetic heart valve materials and emphasizes alternative preservation methods that reduce or avoid the use of glutaraldehyde.     

Acidic lipids associated with the local mechanism of calcificaiton: a review

Two current areas of research on hard tissues have focused upon acidic lipids associated with the local mechanism of calcification and the presence of matrix vesicles as the loci for initial mineralization. Data which show that acidic phospholipids are a component of the vesicle membrane provide a common denominator between these two areas. Furthermore, studies on vertebrate, microbial and synthetic lipoprotein calcification indicate that acidic phospholipids play a pivotal role in the local mechanism. Through a sequence of initial Ca2++ binding followed by desolvation and ion concentration, the acidic phospholipid rich membrane provides an environment in which calcification can be facilitated. A proposed mechanism includes in additon to Ca2++ binding, the formation of (CaHPO4)2 dimers and their condensation into a Ca9 (PO4)6 unit bound to the membrane. The bound unit functions as a nucleus for the formation of additonal units to form an amorphous calcium phosphate cluster. Conversion to crystalline apatite would require dehydration of the environment, either by mineral build up exceeding the hydrophobic domain or by breakdown of the membrane.     

Importance of intrinsic calf vasodilator capacity in determining distribution of skeletal muscle perfusion during supine bicycle exercise in patients with left ventricular dysfunction

Although there is interest in forming synthetic analogs of hard tissues at physiologic temperature, significant gaps in knowledge exist with respect to the mechanisms by which precursor solids convert to apatites and also with respect to the apatite compositions that may be formed. In this study calcium-deficient HAp [Ca9(HPO4)(PO4)5OH] was prepared by hydrolysis of tricalcium phosphate (TCP), alpha-Ca3(PO4)2. The kinetics of HAp formation were studied as a function of temperature by isothermal calorimetry. TCP hydrolyzed completely within about 12 h, and the hydrolysis reaction evolved 133 kJ/mol of HAp formed. Although the kinetics of hydrolysis exhibited a strong temperature dependence, the mechanistic path taken appeared independent of temperature. The fluoridation of hydroxyapatite compositions having Ca/P ratios higher than 1.59 previously has been investigated. However, little work has been done on the fluoridation of more calcium-deficient hydroxyapatite. Ca9(HPO4)(PO4)5OH was formed at temperatures between 37.4 degrees and 55 degrees C to vary its morphology. These preparations then were reacted in NaF solution and the kinetics of fluoride incorporation studied. Solution chemical analyses were used to determine the amounts of fluoride incorporated. The extent of hydroxyl replacement by fluoride ranged from 17 to 72% and correlated with the surface area of the parent HAp.     

Development of an avian model for restenosis

Recurrence of atherosclerotic plaque growth after interventional therapy, restenosis, is a significant clinical problem occurring in 20%-50% of cases. We have developed a new avian model for the investigation of restenosis after arterial injury in cholesterol fed White Leghorn roosters. Atherosclerotic plaque growth 1-30 weeks after angioplasty balloon mediated endothelial injury in the abdominal aorta was studied in 37 roosters. Roosters were maintained on either normal poultry diet or high cholesterol diet. Twelve cholesterol fed roosters were also fed a hormone supplemented diet in order to modify plaque morphology. The procedural success rate was high. Angiographic stenoses (mean 36% with maximum of 74%) were detectable in cholesterol fed roosters after balloon angioplasty with associated histological evidence of plaque growth (P < 0.017). Cholesterol feeding enhanced fatty plaque growth; hormone manipulation increased calcific and ulcerated plaque but with high associated morbidity. Three interventional devices were subsequently examined in 32 roosters (16 laser angioplasty, 7 atherectomy, and 9 stent implant). Plaque development was again assessed by contrast angiography and histological analysis. We conclude that balloon mediated arterial injury in cholesterol fed roosters produces early proliferative and late, complex atherosclerotic lesions providing an inexpensive model for plaque development after intimal injury.     

Clinical, echocardiographic, and pathologic features of aortic wall dehiscence of porcine bioprosthetic valves: a cause of rapidly progressive mitral regurgitation and heart failure after bioprosthetic mitral valve replacement

The aim of this study was to define the clinical, echocardiographic, and pathologic correlates of commissural dehiscence of aortic wall from the stent post of the porcine bioprostheses in the mitral position. This form of valve degeneration was found in 5 of 23 explanted mitral bioprostheses. A thickened, separated aortic wall at multiple commissural sites along with other evidence of valve degeneration was identified in the three patients who had chronic congestive heart failure. A large dehiscence at a single commissural site with otherwise normal valve morphology was present in the two patients who had acute heart failure. Two dimensional/Doppler echocardiography showed a prolapsing or a flail anteriorly positioned leaflet and an eccentric posteriorly directed mitral regurgitation jet in all patients. These echocardiographic findings in patients with a porcine bioprosthetic mitral valve should suggest commissural dehiscence from the aortic wall as a possible mechanism of valve failure. Exclusive involvement of the porcine aortic bioprosthesis placed in the mitral position along with involvement of strut of the bioprosthesis facing the aortic root in all cases suggests excessive hemodynamic stress on the valve in the mitral position and in particular on the anteriorly placed strut as the potential cause of this form of valve degeneration.     

Iterative selection from a Salmonella typhimurium cosmid library can lead to the isolation of an atypically small plasmid

OBJECTIVE: To determine whether ocular cicatricial pemphigoid (OCP) may represent a distinct immunopathologic disease when it is pure ocular cicatricial pemphigoid (POCP) (e.g., only confined to the conjunctiva) or when it is associated with skin or extraocular mucous membrane lesions or both (OCP+). DESIGN: Prospective, immunologic, and immunopathologic study with special emphasis on direct immunoelectron microscopy. PARTICIPANTS: Six patients with POCP and seven patients with OCP+. INTERVENTION: After informed consent was obtained, a conjunctival biopsy was performed in all patients. Skin and extraocular mucosa biopsy specimens were harvested in selected cases only. MAIN OUTCOME MEASURES: Results of direct immunofluorescence and direct immunoelectron microscopy without freezing on conjunctival and skin biopsy specimens, indirect immunofluorescence, and Western immunoblotting analysis were analyzed. RESULTS: Results of direct immunoelectron microscopic examination of the conjunctiva showed the presence of immune deposits in the upper lamina lucida of the basement membrane zone in the six patients with POCP, whereas the immune reactants were located in the lower part of the lamina lucida and in the lamina densa of the basement membrane zone (conjunctiva, buccal mucosa, and skin) in the seven patients with OCP+. Direct immunofluorescence was positive in the biopsy specimens of three patients with POCP (50%) and the seven patients with OCP+ (100%). Results of indirect immunofluorescence study showed circulating autoantibody levels only in two patients with OCP+, and results of Western immunoblot analysis were negative. CONCLUSIONS: Results of direct immunoelectron microscopic examination of the conjunctiva support the hypothesis that POCP may be a disease entity distinct from mucocutaneous cicatricial pemphigoid.     

A comparison of outcomes in men 11 years after heart-valve replacement with a mechanical valve or bioprosthesis. Veterans Affairs Cooperative Study on Valvular Heart Disease

BACKGROUND: Mechanical heart valves are durable but thrombogenic, and their use requires that the patient receive anticoagulants. In contrast, bioprosthetic valves are less thrombogenic, but they have limited durability because of tissue deterioration. METHODS: To compare the outcomes of patients who receive these two types of valves, we randomly assigned 575 men scheduled to undergo aortic-valve or mitral-valve replacement to receive either a mechanical or a bioprosthetic valve. The primary end points were death from any cause and any valve-related complication. RESULTS: During an average follow-up of 11 years, there was no difference between the two groups in the probability of death from any cause (11-year probability for mechanical valves, 0.57; for bioprostheses, 0.62; P = 0.57) or in the probability of any valve-related complication (0.65 and 0.69, respectively; P = 0.39). There was a much higher rate of structural valve failure among patients who received bioprosthetic valves (11-year probability, 0.15 for the aortic valves and 0.36 for the mitral valves) than among those who received mechanical valves (no valve failures; P < 0.001). However, this difference was offset by a higher rate of bleeding complications among patients with mechanical valves than among those with bioprosthetic valves (11-year probability, 0.42 and 0.26, respectively; P < 0.001) and by a greater frequency of peri-prosthetic valvular regurgitation among patients with mechanical mitral valves than among those with mitral bioprostheses (11-year probability, 0.17 and 0.09, respectively; P = 0.05). CONCLUSIONS: After 11 years, the rates of survival and freedom from all valve-related complications were similar for patients who received mechanical heart valves and those who received bioprosthetic heart valves. However, structural failure was observed only with the bioprosthetic valves, whereas bleeding complications were more frequent among patients who received mechanical valves.     

Prevention of calcification of tissue valves

In this study an attempt was made to find an optimum method of chemical treatment to prevent the calcification of bioprosthetic heart valves. Bovine pericardium was washed in a 5% sodium chloride solution followed by trypsin (Tr) treatment and was kept in 0.1% glutaraldehyde (GA) with a gradual increase in concentration up to 0.25% GA and finally posttreated with a 4% chitosan (Ch) solution. Fresh, 0.2% GA, 0.625% GA, and sodium chloride-Tr-GA treated pericardial samples were taken for comparative study. Tensile testing showed comparable strength and elongation at the breaking point for all groups. The thermal shrinkage studies indicated merit of the proposed treatment (5% sodium chloride-trypsin-glutaraldehyde treated pericardia with chitosan and without chitosan posttreatment). Collagenase assay showed that all differently treated (GA) materials were equally resistant to collagenase. All samples were implanted subcutaneously in rats for 2, 4, 8, or 12 weeks for calcification study. Morphological and mineral analyses showed complete prevention of calcification in sodium chloride-trypsin-GA-chitosan treated pericardium (Ca was 1.1 +/- 0.27 mg/g, von Kossa 0) at the 12th week of implantation.     

Nanobacteria: an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation

Calcium phosphate is deposited in many diseases, but formation mechanisms remain speculative. Nanobacteria are the smallest cell-walled bacteria, only recently discovered in human and cow blood and commercial cell culture serum. In this study, we identified with energy-dispersive x-ray microanalysis and chemical analysis that all growth phases of nanobacteria produce biogenic apatite on their cell envelope. Fourier transform IR spectroscopy revealed the mineral as carbonate apatite. The biomineralization in cell culture media resulted in biofilms and mineral aggregates closely resembling those found in tissue calcification and kidney stones. In nanobacteria-infected fibroblasts, electron microscopy revealed intra- and extracellular acicular crystal deposits, stainable with von Kossa staining and resembling calcospherules found in pathological calcification. Previous models for stone formation have led to an hypothesis that elevated pH due to urease and/or alkaline phosphatase activity is a lithogenic factor. Our results indicate that carbonate apatite can be formed without these factors at pH 7.4, at physiological phosphate and calcium concentrations. Nanobacteria can produce apatite in media mimicking tissue fluids and glomerular filtrate and provide a unique model for in vitro studies on calcification.     

An evaluation of nasal patency with acoustic rhinometry--preop. and postop. comparisons

PURPOSE: This prospective study was designed to assess the effect of primary hyperparathyroidism on heart muscle, valves, and myocardial function. Echocardiography was used to evaluate changes in mechanical performance, the thickness of the left ventricular wall, myocardial calcific deposits, and valvular calcifications in patients with primary hyperparathyroidism. METHODS: Echocardiography was performed in 54 patients with hyperparathyroidism prior to surgery and 12 +/- 2 months after successful parathyroidectomy. A matched control group was followed for comparison. RESULTS: In a blinded fashion, aortic and mitral valve calcifications were detected in 63% and 49% of patients with primary hyperparathyroidism (controls: 12% and 15%, respectively). Calcific deposits in the myocardium were found in 69% of patients with hyperparathyroidism and 17% of the control subjects. After parathyroidectomy and 12 months of normocalcemia, a significant regression of left ventricular hypertrophy (p < 0.001) was observed. CONCLUSIONS: The present data show a high incidence of left ventricular hypertrophy, calcific deposits in the myocardium, and/or aortic and mitral valve calcification in patients with primary hyperparathyroidism. A 1-year follow-up after parathyroidectomy (and restoration of normocalcemia) discloses regression of hypertrophy, while calcifications persist without evidence of progression.     

含银无机抗菌剂的研制和抗菌性能初探

采用不同工艺制备了载银磷酸钙和载银碳酸钙粉末 ,通过大肠杆菌培养实验 (抑菌环法 )比较了银离子存在形式对抑菌性能的影响 ,证实了银离子释放是灭菌的关键。