Effects of antibiotics on cellular viability in porcine heart valve tissue.

The purpose of this study was to find an antibiotic disinfecting solution for cardiovascular tissues which would allow maximal cellular viability while maintaining antibacterial and antifungal activity. Cellular viability of porcine heat valves sterilised using antibiotics was assessed by radiolabelled proline transport assays. The results show that storage of valve tissues in RPMI 1640 tissue culture medium (4 degrees C) alone decreases the cellular viability by 50%, 60% and 90% within 12, 24, and 72 h, respectively. Amphotericin B was shown to be the toxic component of the antibiotic sterilising solution recommended by Strickett and coworkers, accounting for all the observed antibiotic toxicity. It reduced the cellular viability by 100% within 12 h of storage at 4 degrees C. Our study also showed that streptomycin is responsible for the loss of cellular viability in the antibiotic medium utilised by O'Brien's group. The results show that after storage of valve tissues for 12 h in O'Brien's antibiotic solution, the cellular viability was reduced by 60%, and after 24 h by 90%. Cefoxitin, lincomycin, polymyxin B, vancomycin and penicillin had no apparent effects on viability of cells in the cardiovascular tissues utilised in this study. In conclusion, heart valve tissue may be sterilised effectively using selective antibiotics without causing significant damage to cellular viability.

Isolated valvular amyloid.

Two cases are described of grossly evident amyloid infiltration of the cardiac valves, while only minor deposits were found in other locations. The right-sided valves were more heavily involved than the left. No pre-existing disease of the valves was found, and the lesions appeared to have no adverse effect upon the subjects. Only one other similar case was found in the literature. The only consistently associated condition among the recognized cases was advancing age. The name proposed for the condition is isolated valvular amyloid.

Autofluorescence of bovine ligamentum nuchae, cartilage, heart valve and lung measured by microscopy and fibre optics.

The fluorescence of bovine tissues was measured post mortem by microscopy of frozen sections and by using optical fibres to excite fluorescence and to measure fluorescence emission spectra. Mechanical disruption of the tissue (by comminution or sectioning) did not appreciably change tissue fluorescence spectra. Ligamentum nuchae had the strongest fluorescence and lung tissue had the weakest. In samples measured with a minimum prior exposure to ultraviolet light, the peak fluorescence emission was at 410 or 420 nm (with excitation at 365 nm). Exposure to ultraviolet light for about 1 minute shifted the fluorescence peak to 450 to 470 nm. Further exposure (about 30 minutes) caused a loss of the 450 to 470 nm fluorescence peak, while emissions above 530 nm were maintained or strengthened. Microscopy showed that the fluorescence that was measured by fibre optics from intact connective tissues originated mostly from collagen and elastin fibres.

Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.

To study the roles played by cardiac valvular endothelium in normal and pathologic conditions, we have established and characterized a system of bovine valvular endothelial cells (VEC) in culture. Viable VEC from calf atrioventricular valves were obtained by a non-enzymatic procedure using 3 mM ethylenediamine-tetraacetic acid (EDTA) as dissociating agent. The cells grown in Dulbecco's modified Eagle's medium supplemented with non-essential amino acids, vitamins and 20% fetal calf serum, developed as monolayers of closely apposed polygonal cells which were subcultured for up to seven passages. VEC maintained in culture the general ultrastructure displayed in vivo, expressed von Willebrand factor, presented angiotensin converting enzyme activity and synthesized a rich extracellular matrix. VEC preserved the cell surface anionic sites (detected with cationized ferritin, pI 8.4) and cationic sites (visualized with haemeundecapeptide pI 4.85), and took up, especially by adsorptive endocytosis, albumin-gold conjugate. The cells were coupled by functional communicating (gap) junctions, as demonstrated by microinjection of 6-carboxyfluorescein. VEC in culture produced fibronectin, prostacyclin, hyaluronic acid and heparin-like glycosaminoglycans (identified by electrophoresis, enzyme digestion, and deaminative cleavage of molecules). These properties render cultured VEC a suitable model for investigating their functions and involvement in normal and pathologic heart valves.

Heart valve involvement in familial amyloidosis with polyneuropathy.

Involvement of the heart is common in the various types of amyloidosis. Little attention has, however, been paid to the presence and significance of amyloid in cardiac valves. We examined the heart valves of twelve autopsy cases with familial amyloidosis with polyneuropathy. All leaflets showed more or less abundant amyloid infiltration with significant valvular aortic stenosis due to degenerative calcification in two cases. In familial amyloidosis with polyneuropathy, amyloid deposits are thus invariably present in the valves, but the valvular function is often preserved. Amyloid may, however, produce hemodynamically significant valvular lesions, and the importance of valvular involvement in the various types of amyloidosis may still be underestimated.

Penetration of pefloxacin in human heart valves.

Pefloxacin concentrations were measured by HPLC in serum, subcutaneous tissue, heart muscle and heart valves of 41 patients receiving a one hour infusion of 800 mg iv 4, 8, 12, 18, or 24 h before open heart surgery for prosthetic cardiac valve insertion. In all but three of the 46 valvular samples, pefloxacin concentrations were higher than the mode MIC of Staphylococcus, Enterobacteriaceae and Pseudomonas microorganisms causing infections following open heart surgery. Pefloxacin therefore has good penetration of heart valves and is present for up to 24 h. It could be considered as a useful drug in the prophylaxis of cardiac surgery and in therapy of bacterial endocarditis due to susceptible micro-organisms.

The identification of the vitamin K-dependent bone protein osteocalcin as one of the gamma-carboxyglutamic acid containing proteins present in calcified atherosclerotic plaque and mineralized heart valves.

Calcium binding proteins containing gamma-carboxyglutamic acid (Gla) have previously been demonstrated to occur in calcified atherosclerotic plaque and calcified cardiac valves. Experiments were carried out to determine if one of the Gla-containing proteins in human cardiovascular calcifications is the vitamin K-dependent bone protein, osteocalcin. A radio-immunoassay for human osteocalcin was employed, and EDTA extractions of calcified atheromata, and aortic valves as well as relevant noncalcified material were analyzed. Tissue calcium levels were also determined, as were Gla levels as a measure of total vitamin K-dependent protein content. Osteocalcin was present at low levels in all calcified cardiovascular tissues (4.5-175.7 ng osteocalcin/mg protein) with trace levels or nondetectable amounts present in noncalcified tissue. Osteocalcin accounted for a small proportion of the total protein-bound Gla (0.01-0.05%). The relationship of osteocalcin to the other Gla-containing proteins of atherosclerotic plaque including atherocalcin, the principal extractable Gla-containing protein of calcified plaque, is discussed.

Penetration of gentamicin into heart valves, subcutaneous and muscular tissue of patients undergoing open heart surgery.

Concentrations of gentamicin in plasma, heart valves, subcutaneous tissue and muscle were determined in 38 patients undergoing open heart surgery. Gentamicin reached peak levels in plasma and tissue within 60 min after a 5 min intravenous bolus injection of 1.5 mg/kg body weight. Subcutaneous and muscle concentrations varied between 0.51 microgram/g and 2.1 microgram/g. Gentamicin peak concentrations in cardiac valvar tissue wre 3.6 mug/g between 2 and 5 hours after administration; gentamicin heart valve concentrations varied between 1.2 microgram/g and 1.59 microgram/g. Gentamicin tissue concentrations during open heart surgery are high enough to inhibit most Klebsiella/Enterobacter and Staphylococcus aureus and epidermidis strains. However Gentamicin heart valve concentrations do not exceed 1.5 microgram/g for more than 1 h, which may explain treatment failures of patients with endocarditis.

[Use of mezlocillin in open heart surgery (author's transl)]. / Mezlocillin-Konzentrationen in Gewebe und Serum während Herzoperationen.

Concentrations of mezlocillin in serum and heart tissue were studied in 29 patients undergoing open heart surgery. The antibiotic agent was administered three times as a bolus injection of 2 grams each: after induction of anaesthesia, after initiation of extracorporeal bypass (ECC) and shortly after discontinuation of ECC. Serum levels were measured in 25 patients from samples drawn 30 minutes after the bolus injection and subsequently biologic assays were carried out by the agar diffusion method. The mean initial values during the three phases of surgery were 119.2, 170.6 and 236.0 micrograms/ml, respectively; at 60 minutes the values were 61.3, 100.8 and 101.9 micrograms/ml. Calculation of the exponential curve enabled a mathematical comparison of the half-life of the substance during the pre-ECC, ECC and post-ECC periods. The most rapid elimination was found to occur prior to initiation of ECC, the slowest during ECC, while in the post-ECC period the elimination was similar to, but somewhat slower than that of, the pre-ECC period. The concentration, measured in the tissue of eleven aortic valves, averaged 35.96 (range 8.4 to 63.4) micrograms/ml. The mean concentration found in papillary muscles of the left ventricle, resected at the time of mitral valve replacement in six patients, was 31.54% (range 17.7 to 58.33) micrograms/ml. Mean tissue concentration found in five resected mitral valves was 43.77 (range 27.33 to 71.5) micrograms/ml. The findings indicate that mezlocillin, administered as described, will reach serum and tissue concentrations at all periods of open heart surgery well above those of the minimum inhibitory concentration of most clinically relevant bacteria.

Characterization of pepsin-solubilized bovine heart-valve collagen.

Collagens extracted from heart valves by using limited pepsin digestion were fractionated by differential salt precipitation. Collagen types were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, amino acid analysis and cleavage with CNBr. Heart-valve collagen was heterogeneous in nature, consisting of a mixture of type-I and type-III collagens. The identity of type-III collagen was established on the basis of (a) insolubility in 1.7 M-NaC1 at neutral pH, (b) behaviour of this collagen fraction on gel electrophoresis under reducing and non-reducing conditions, (c) amino acid analysis showing a hydroxyproline/proline ratio greater than 1, and (d) profile of CNBr peptides on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showing a peak characteristic for type-III collagen containing peptides alpha1(III)CB8 and alpha1(III)CB3. In addition to types-I and -III collagen, a collagen polypeptide not previously described in heart valves was identified. This polypeptide represented approx. 30% of the collagen fraction precipitated at 4.0 M-NaCl, it migrated between beta- and alpha1-collagen chains on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its electrophoretic behaviour was not affected by disulphide-bond reduction. All collagen fractions from the heart valves contained increased amounts of hydroxylysine when compared with type-I and -III collagens from other tissues. The presence of beta- and gamma-chains and higher aggregates in pepsin-solubilized collagen indicated that these collagens were highly cross-linked and suggested that some of these cross-links involved the triple-helical regions of the molecule. It is likely that the higher hydroxylysine content of heart-valve collagen is responsible for the high degree of intermolecular cross-linking and may be the result of an adaptive mechanism for the specialized function of these tissues.

Solubilization of bovine heart-value collagen.

Complete solubilization of bovine heart-valve gollagen was obtained by using sequential pepsin digestion and extraction with dithiothreitol under non-denaturing conditions. Use of the reducing agent was the crucial step resulting in solubilization. These findings suggest that disulphide-bonded proteins may be in part responsible for the insolubility of bovine heart-valve collagen.

Simultaneous morphological and stress-strain studies of the fibrous components in wet heart valve leaflet tissue.

Simultaneous morphological and stress-strain studies have been carried out on heart valve leaflet tissue maintained in its unaltered functional condition. A microtensile tissue testing device is described which inserts directly into the stage of an optical microscope fitted with a Nomarski interference contrast facility. Glutaraldehyde fixation of tissue subjected to different levels of loading reveals that the current stressed state of the collagen fibers is "frozen" in by crosslinking process, and as a consequence marked alterations in the preserved tissue properties are produced. This is shown to be relevant to the preparation of the glutaraldehyde treated heterograft heart valve. The mechanical roles of the collagen and elastin components are investigated and a "complementation" mechanism is postulated whereby the widely different mechanical properties of the two types of fibers are combined in a non-competing manner to produce a diversity of composite tissue characteristics.

The collagen of heart valve.

A hydroxylysine-rich type I collagen has been isolated from pepsin-digested porcine heart valve. The ratio of alpha1 to alpha2 in the isolated molecule was 2:1. The component alpha chains exhibited unusual chromatographic behavior in comparison to corresponding chains from human dermis and lathyritic rat skin collagen. The composition of component cyanogen bromide peptides identified the alpha chains as authentic type I chains and demonstrated hydroxylysine enrichment throughout the length of the chain. delta6-Dehydro-5,5'dihydroxylysinonorleucine, a collagen cross-link derived from two hydroxylysyl residues and ordinarily found in hard tissue collagens was found to be the predominant cross-link in heart valve.