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.

Glutaraldehyde-fixed kangaroo aortic wall tissue: histology, crosslink stability and calcification potential.

Stentless aortic heart valve substitutes, manufactured from biological tissues, are fixed with glutaraldehyde to cross-link collagen, reduce antigenicity, and sterilize the tissue. Despite improved cross linking, reduced antigenicity, and various anticalcification measures, the aortic wall tissue present in these prostheses tends to calcify. The aim of this study was to assess the morphology, collagen cross-link stability, and calcification potential of glutaraldehyde-preserved kangaroo aortic wall tissue as opposed to porcine aortic wall tissue. Porcine and kangaroo aortic wall tissues were fixed in 0.625% buffered glutaraldehyde. Histology and cross-link stability were examined. Calcification potential was determined in the subcutaneous rat model. Kangaroo aortic wall tissue was significantly (p < 0.01) less calcified than porcine aortic wall tissue (26.67 +/- 6.53 versus 41.959 +/- 2.75 microg/mg tissue) at 8 weeks. In conclusion, the histological differences between kangaroo and porcine aortic wall tissue correlate well with the reduced calcification potential of kangaroo aortic wall tissue. The reduced calcification potential could result in improved long-term durability of stentless kangaroo heart valves as bioprostheses.

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.

Evaluation of kangaroo aortic valved conduits in a juvenile sheep model: preliminary findings.

BACKGROUND: Biological heart valve substitutes, manufactured from either porcine or bovine tissue, have been in use for more than 30 years. Despite low thrombogenicity and excellent performance, bioprosthetic heart valves tend to degenerate and calcify early in young patients because of patient and valve related factors. The aim of this study was to examine the calcification behavior of glutaraldehyde-preserved kangaroo heart valves in a juvenile sheep model. METHODS: Porcine (n = 10) and kangaroo (n = 10) valved conduits were implanted in the descending aortic position of juvenile sheep and retrieved after 6, 8, and 12 months. Retrieved valved conduits were examined for morphological changes and calcification of the valve tissue, using Von Kossa's stain technique and atomic absorption spectrophotometry. RESULTS: Structural valve deterioration, characterized by increased stiffness and severe calcification, occurred in 100% of the porcine conduits within 4 months. Kangaroo valve leaflets were significantly (p < 0.001) less calcified at 6 months (3.39+/-1.80 microg/mg), 8 months (5.86+/-4.57 microg/mg), and at 12 months (14.38+/-6.72 microg/mg), compared to porcine valves at 3 months (176.45+/-42.88 microg/mg ) and at 4 months (154.67+/-52.67 microg/mg ). Porcine aortic wall tissue was more calcified (118.24+/-42.86 microg/mg) than kangaroo aortic wall tissue (79.55+/-26.40 microg/mg). CONCLUSIONS: Kangaroo heart valves calcify less than porcine heart valves. These findings suggest that a different donor valve tissue has a lower calcification potential probably due to a difference in the morphological ultrastructure. This could result in improved long-term durability of kangaroo heart valves.

Kangaroo versus porcine aortic valve tissue--valve geometry morphology, tensile strength and calcification potential.

OBJECTIVE: Valve related factors and patient related factors are responsible for calcification of valvular bioprostheses. Recent studies showed different donor and recipient species have different influences on the total calcification rate of bioprostheses. This study was performed to evaluate and compare Kangaroo aortic valve leaflets with porcine aortic valve leaflets. Experimental design. Prospective study. Setting. Cardio-thoracic experimental research of a university department. MATERIALS AND METHODS: Glutaraldehyde-fixed Kangaroo and porcine valve leaflets were evaluated in vitro according to valve geometry (internal diameter and leaflet thickness), morphology (light and electron microscopy) and tensile strength. In vivo evaluation consisted of implantation in a rat model for 8 weeks, Von Kossa stain for calcium and atomic absorption spectrophotometry for total extractable calcium content. RESULTS: Kangaroo valves indicated a smaller internal valve diameter as well as a thinner valve leaflet (p<0.01, ANOVA) at corresponding body weight, less proteoglycan spicules in the fibrosa, increased elasticity (p<0.05) and low calcification potential (p<0.01, confidence interval 95%). CONCLUSIONS: Kangaroo aortic valve leaflets have different valvular qualities compared to porcine valve tissue. Kangaroo valve leaflets are significantly superior to porcine valve leaflets as far as calcification is concerned. These results are encouraging and suggest further in vivo evaluation in a larger animal model before clinical application can be considered.

Osteogenic sarcoma of the left atrium.

A primary cardiac osteogenic sarcoma is described in a patient presenting with a left atrial obstructive lesion. Wide surgical excision was possible with left atrial reconstruction and mitral valve replacement. The patient survived the operation and was symptom free for 1 year, finally dying at 18 months of cerebral metastases.

Ultrasonic coronary angioplasty during coronary artery bypass grafting.

This preliminary study in 20 patients demonstrated that ultrasonic coronary angioplasty in the setting of bypass grafting is feasible, safe, and able to recanalize atherosclerotic vessels. Shorter monorail probes were superior to longer probes without guidewires in terms of success of vessel recanalization; >95% of particle debris was <25 microm in size.

Antibiotic prophylaxis in cardiac operations.

This clinical trial, which was composed of 1,031 adults undergoing cardiac operations, compared the efficacy of a single dose of 1 g of ceftriaxone with a 48-our regimen consisting of flucloxacillin and gentamicin. There was no significant difference (p = 0.89) in the overall incidence of major infections: 30 of 515 patients (5.8%; 95% confidence interval, 5.4% to 6.2%) taking ceftriaxone and 29 of 516 patients (5.6%; 95% confidence interval, 5.2% to 6.0%) taking flucloxacillin and gentamicin. Subgroup analyses, with a lower statistical power, failed to show a significant difference between patients who received ceftriaxone and those who received flucloxacillin/gentamicin: major sternal wound infections arose in 2.7% of the patients taking ceftriaxone versus 1.6% in those on the 48-hour regimen (p = 0.20) and major limb wound infections arose in 4.2% and 5.4%, respectively (p = 0.44). Single-dose prophylaxis was associated with fewer intravenous administrations (864 doses versus 9,570 doses) and cost less (A$17,248 versus A$78,510). Although the regimen that included gentamicin was associated with the greatest biochemical impairment of renal function, the overall toxicity for both groups was low. We conclude that a single dose of ceftriaxone provided cost-efficient prophylaxis for adults undergoing cardiac operations when compared with a 48-hour regimen of gentamicin and flucloxacillin. The general principle revealed by our data is that the short-term administration of an appropriate antibiotic regimen represents optimal prophylaxis for patients undergoing cardiac procedures.

Esophageal malignancies: is preoperative radiotherapy the way to go?

During the period 1969 to 1988, 68 patients with esophageal malignancy were subjected to preoperative radiotherapy consisting of 30 Gy over 3 weeks followed by esophagectomy after a wait of 2 weeks. Sixty-two of 68 patients (91%) were followed up retrospectively (5-year survival rate, 17.2%; 10-year survival rate, 4.8%). Radiosensitivity of the tumor was graded by a radiation effect scoring method devised by us, and this was found to correlate with long-term prognosis (p = 0.05). Radiotherapy "sterilized" malignant involvement of adventitia and draining lymph nodes in 8 patients and thereby converted "palliative" into potentially "curative" resections with a mean survival of 57.2 months in this group of patients. Preoperative radiotherapy was found to increase the survival, decrease the incidence of residual tumor at the resected stump, reduce the incidence of local recurrence, and increase the resectability rate.

Molecular models illustrating the possible distributions of 'holes' in simple systematically staggered arrays of type I collagen molecules in native-type fibrils.

Clear 3/8'' Lucite rod models at a scale of 1'' = D (the native-type period), and using four different colored ribbon markers to indicate one set of 'equivalent bands' in the SLS band pattern, clearly show the possible distribution of 'holes' and staining loci in native-type fibrils. Close-packed ordered arrays of the 4.4'' long rods (based on the Hodge-Petruska model) in which nearest neighbors are systematically staggered by D must have ordered distributions of the 0.6D long 'holes'. In one of the two simplest cases, holes occur singly, and are separated laterally by two molecular diameters. In the other case, contiguous holes form transversely continuous channels or slots within the fibril. Electron micrographs of both fish bone and embryonic chick bone clearly show that at least the initial mineralization is intrafibrillar in these systems.

AVEC-DIC and electron microscopic analyses of axonally transported particles in cold-blocked squid giant axons.

Anterogradely and retrogradely transported membranous organelles were analysed separately by focally cooling axons (cold-blocking) for 2-4 h. Video-enhanced differential interference contrast light microscopy (AVEC-DIC) and dark field light microscopy showed that particles accumulated in large numbers on both the anterograde and the retrograde sides of the cold-block and that the accumulated particles resumed their transport when the preparation was rewarmed to 18 degrees C. The particles accumulated in files on both sides of the cold-block suggesting that particles move along linear pathways in the axoplasm. Comparisons of the results obtained by AVEC-DIC light microscopy with those obtained by electron microscopy indicate that the AVEC-DIC method is capable of detecting all of the different types of rapidly transported membranous organelles, including the smallest (35-80 nm) vesicles that move anterogradely. Electron microscopic analyses of the transported particles demonstrate that the anterogradely transported organelles are structurally distinct from those that are transported retrogradely. The anterogradely transported particles consisted of normal mitochondria and small (35-80 nm) tubulovesicular profiles. By contrast, the retrogradely transported particles were 150 nm or larger and they often contained complex membranous inclusions. The largest retrogradely transported particles appeared to be degenerating mitochondria. The results are consistent with the hypothesis that the direction of organelle movement is related to the physiological state of the organelle. That is, organelles containing newly synthesized membrane components move primarily anterogradely and organelles that contain transformed and degraded membrane components move retrogradely.

Direct visualization of particle velocity distribution by pseudostereoscopic viewing of time-lapsed sequential images: application to fast axonal transport.

We describe a simple method for direct visualization of the velocity distribution of particles moving against an immobile background. The technique involves pseudostereoscopic viewing of image pairs separated by an appropriate time interval in a sequential recording of the subject. Under these conditions, the positive or negative parallax arising from particle motion results in the binocular image of a particle being perceived as raised or lowered relative to an immobile background plane depending on its direction of movement, and with the degree of perceived elevation being proportional to its speed. In effect, the binocular optic axis becomes a velocity (speed) axis under these conditions. The technique is illustrated with examples of image pair sequences showing fast axonal transport in lobster and squid axons using video-enhanced differential interference contrast microscopy. However, the pseudostereoscopic method is quite generally applicable to both microscopic and macroscopic time-dependent phenomena. Particle speeds can be quantitated using standard procedures for measuring frame-to-frame particle displacements, or alternatively, by determination of parallax using stereogrammatic methods. It should be also readily adaptable for on-line monitoring of particle velocity distribution, particularly in video systems where frame buffers can be utilized to extract and present serial image pairs having any desired time separation from video-taped sequences.

Neurofilamentous network and filamentous matrix preserved and isolated by different techniques from squid giant axon.

The contribution of the neurofilamentous network to the structure of the squid giant axon was analyzed electron-microscopically. Axial 10-nm filaments cross-linked by radial 5-nm bridges form a network that is present in preparations prepared by a variety of techniques. The axoplasm is differentiated into dense and less dense regions. In the presence of Co(II) ions, the neurofilamentous network was remarkably well preserved and appeared to be associated with a dense web of fine filament matrix, which also was identified in extracted axoplasm and in fractions enriched with neurofilament protein complex. In the presence of La(III) ions, the neurofilamentous network had a coarse and open appearance. The stereo images of extracted and critical-point dried axoplasm suggested that the neurofilamentous network contains ordered lattice-like regions. Extracted preparations of extruded axoplasm and fractions enriched with neurofilament protein complex suggested that the properties of the network are determined by the neurofilament protein complex. It is proposed that the neurofilamentous network is the essential determinant of the form of the axon, and that the order within the network is determined by the radial components of the network. The structures observed in the different preparations are not artifacts, but rather are related closely to their native state in the axon.

A case of fatal fat embolism syndrome following cardiopulmonary bypass.

A case of fat embolism syndrome following median sternotomy and cardiopulmonary bypass, with fatal outcome, is described. This syndrome is present subclinically in the majority of these surgical procedures. The etiology and possible mechanisms of production are discussed; management is empirical and aimed at maintaining the function of embolized microvascular endothelium.

The effect of chronic cardiac volume overload on regional myocardial blood flow in the dog.

1. Regional myocardial blood flow was studied in the anaesthetized, open-chest dog with a large chronic aorto-caval fistula, using carbonized microspheres of 7-10 mum diameter. The results from fourteen dogs with fistulae of 4-84 days duration were compared to those from nine normal animals. 2. Myocardial blood flow to all areas of both ventricles was increased to between 180 and 250% of the normal despite lower aortic diastolic (coronary diastolic) pressure. Myocardial blood flow in the right ventricle was lower than in the left in both groups of dogs although the increase above normal in dogs with fistulae was relatively greater in the right ventricle. Increased myocardial blood flow is attributed to coronary autoregulation resulting from increased myocardial oxygen consumption due to increase in myocardial tension development. 3. Although absolute blood flow to the inner left ventricular wall was markedly increased in all dogs with fistulae, in those with aortic diastolic pressure below 55 mmHg the ratio of flow in the inner to that in the outer free left ventricular wall was significantly less than in those with aortic diastolic pressure above 55 mmHg (P less than 0 with 02). Low aortic diastolic pressurere and diastolic coronary perfusion pressure probably resulted in relative ischaemia of the inner left ventricular wall.

The role of the sympathetic nervous system in hypoglycaemia-stimulated gastric secretion.

The gastric secretory response to insulin is mediated predominantly by the vagus. The associated hypoglycaemia stress response is mediated by the sympathetic nervous system. Inhibition of the sympathetic response by simultaneous alpha and beta receptor blockade was studied in five healthy young adults. No appreciable modification of gastric secretory output resulted.