Resistance to tearing of calf and ostrich pericardium: Influence of the type of suture material and the direction of the suture line

The tearing of the valve leaflet of a cardiac bioprosthesis can cause early failure of this device, which is employed to replace a diseased native valve. This report involves the study of the behavior of 312 tissue samples (152 of calf pericardium and 160 of ostrich pericardium) treated with glutaraldehyde and subsequently subjected to tear testing. The samples were cut in the two principal directions: longitudinally, or root to apex, and transversely. They included a series of control samples that were left unsutured, and the remaining samples were repaired with the use of two different suture techniques: a running suture in the direction of the load and a telescoping suture perpendicular to the load. Four commercially available suture materials were employed: Pronova, nylon, Gore-Tex, or silk. The unsutured control samples of both types of pericardium exhibited a similar anisotropic behavior in the tear test. The mean resistance to tearing of the calf pericardium was 24.29 kN m in samples cut longitudinally and 34.78 kN m in those cut transversely (p =.03); the values were 28.08 kN m and 37.12 kN m (p =.002), respectively, in ostrich pericardium. The series repaired with the telescoping suture always exhibited greater resistance to tearing, with values that ranged between 44.34 and 64.27 kN for the samples of calf pericardium and from 41.65 to 47.65 kN for those obtained from ostrich. These assays confirm the anisotropic behavior of calf and ostrich pericardium treated with glutaraldehyde when subjected to tear testing, as well as the loss of this behavior in ostrich pericardium after suturing. Suturing techniques, such as the telescoping model, that provide a greater resistance to tearing should be studied for use in the design of the valve leaflets of cardiac bioprostheses made of biological materials.     

Comparison of the mechanical behaviors of biological tissues subjected to uniaxial tensile testing: pig,calf and ostrich pericardium sutured with Gore-Tex

The purpose of this study was to compare the mechanical behavior of calf pericardium, pig pericardium and ostrich pericardium when subjected to tensile testing. Tensile stress was applied to 108 tissue samples, 36 of each type of tissue, until rupture. Groups of three adjacent strips measuring 12 x 2 cm(2) were cut longitudinally. Each group consisted of an unsutured center sample, or control, and the two contiguous samples, that on the right sutured with Gore-Tex at a 90 degrees angle with respect to the longitudinal axis and that on the left sewn with the same suture material at 45 degrees angle. The sutured samples showed a statistically significant loss of resistance (p<0.001) when compared with the corresponding unsutured tissue. The mean stresses at rupture for sutured ostrich pericardium were 21.81 and 20.81 MPa in the samples sewn at 45 degrees and 90 degrees, respectively, higher than those corresponding to unsutured calf and pig pericardium, 14.0 and 11.49 MPa, respectively, at rupture.The analysis of the stress/strain curve shows a smaller difference between sutured and unsutured ostrich pericardium than those observed in the other two biomaterials. These results demonstrate that, in addition to its greater resistance, ostrich pericardium also presents a less pronounced interaction with the suture material. Its capacity to absorb the shearing stress produced by the suture is greater. This report also confirms that the method of selection using paired samples ensures their homogeneity and makes it possible to predict the behavior of a sample by determining that of the other half of the pair.     

Biochemical and mechanical behavior of ostrich pericardium as a new biomaterial

We have performed a comparative analysis of glutaraldehyde-preserved ostrich pericardium, as a novel biomaterial, with bovine pericardium. The biochemical characteristics (histology, water content, amino acid composition, and collagen and elastin contents), mechanical properties, and in vivo calcification in a subcutaneous rat model were examined. Ostrich pericardium is slightly thinner and shows a higher water content (70+/-2% vs. 62+/-2%) than bovine pericardium. Additionally, ostrich pericardium presents 1.6-fold lower elastin content and a lower percentage of collagen in reference to the total protein content (68+/-2% vs. 76+/-2%). However, ostrich pericardium shows better mechanical properties, with higher tensile stress at rupture (32.4+/-7.5 vs. 11.5+/-4.6) than calf pericardium. In vivo calcification studies in a rat subcutaneous model show that ostrich pericardium is significantly less calcified than bovine pericardium (23.95+/-13.30 vs. 100.10+/-37.36 mg/g tissue) after 60 days of implantation. In conclusion, glutaraldehyde-stabilized ostrich pericardium tissue shows better mechanical properties than calf tissue. However, calcium accumulation in implanted ostrich tissue is still too high to consider it a much better alternative to bovine pericardium, and anticalcification treatments should be considered.     

The relationship between stress and relaxation in calf pericardium used in the construction of cardiac bioprostheses

Assessment of relaxation (loss of load within a given time) without apparent deformation is a necessary step before durability assay of biomaterials. From results obtained using calf pericardium, the following conclusions were drawn: (a) there is no limit to relaxation for this biomaterials; (b) the lesser the load applied, the greater the relaxation; and (c) the relaxation curve follows the logarithmic function y = K1 - K2 - Int. These findings demonstrate the poor behaviour of the biomaterial at supposedly low loads and suggest that rupture-point load is not a reliable reference to determine the safety coefficient of calf pericardium used in cardiac bioprostheses.     

Mechanical behavior of chemically treated ostrich pericardium subjected to uniaxial tensile testing: influence of the suture

The mechanical behavior of sutured ostrich pericardium was studied by uniaxial tensile testing. One hundred forty-four tissue specimens were assessed: 96 sutured samples (48 in which a centrally located suture was placed at an angle of 90 degrees with respect to the longitudinal axis, whereas in the remaining 48, a centrally located suture was placed at a 45 degrees angle to the longitudinal axis, in sets of 12 samples each, sewn with sutures made of Gore-Tex, nylon, Prolene, or silk), and 48 unsutured controls. Each group of 24 samples sewn at one angle or the other with the different suture materials was assayed together with a corresponding control group of 12 unsutured samples. The mean tensile strengths in the unsutured controls ranged between 30.16 MPa and 43.42 MPa, whereas those of the sutured sets ranged from 14.68 MPa to 21.91 MPa. The latter presented a statistically significant loss of resistance (p < 0.01) when compared with the unsutured tissue samples. The angle of the suture with respect to the longitudinal axis influenced the degree of shear stress produced by the suture, as well as the behavior of the different suture materials used. The set of samples sewn with Prolene appeared to be that most sensitive to changes in the angle of the suture, whereas tissue sewn at a 45 degrees angle with Gore-Tex presented lower shear stress values in comparison with samples in which the other three materials were used. A method of tissue selection based on morphological and mechanical criteria was used to ensure the homogeneity of the results in such a way that the coefficients of determination (R2) for the stress/strain curve fitting equation ranged between 0.888 and 0.995. This excellent fit made it possible, applying regression analysis, to predict the mechanical behavior of a specimen by determining that of a contiguous tissue sample. Thus, it should be possible, at least theoretically, to characterize the behavior of a specific region or zone of the biomaterial. In conclusion, ostrich pericardium exhibits strong resistance to rupture, even when sutured. The selection method used ensures the homogeneity of the samples and, thus, of the results. The angle of the suture with respect to the longitudinal axis, where the load is centered, determines the shear stress produced by the suture and the mechanical behavior of each suture material.     

Uniaxial and biaxial tensile strength of calf pericardium used in the construction of bioprostheses: biomaterial selection criteria

Using morphological and mechanical criteria and applying a method involving paired samples that is widely employed in epidemiology, we obtained an excellent prediction of the mechanical behavior of the calf pericardium used in the construction of cardiac bioprostheses. The method of selection employed in this study may be a highly useful tool for guaranteeing the mechanical resistance of calf pericardium, with a very low level of error.     

Durability of a cardiac valve leaflet made of calf pericardium: fatigue and energy consumption

We studied the mechanical behavior of membranes of calf pericardium, similar to those employed in prosthetic valve leaflets, when subjected to tensile fatigue. The objective was to assess its durability, as a fundamental property of cardiac bioprosthesis, and analyze the energy consumption. For this purpose, the authors built a hydraulic simulator to subject a spherical valve leaflet made of calf pericardium to cyclic stress mimicking cardiac function. A total of 522 assays were performed in 40 samples, subjected to cyclic pressures greater than 6 atm, and 482 subjected to pressures ranging between 2 and 6 atm. The mathematical expression that establishes the relationship between the pressure exerted and the frequency was obtained. If we assume that the function is continuous, this equation provides the range of fatigue tolerated for a given number of cycles. Using the optimal values (the five highest values per series), the expression was found to be y = 9.95x(-0 1214) (R(2) = 0.955), where x represents the frequency in cycles per second and y the pressure in atmospheres. In addition, we established the mathematical relationship between the energy consumed and the frequency, which was a function of the pressure exerted, regardless of the region or zone from which the samples had been obtained. The methods of manual and morphology-based selection employed produced widely dispersed results. When a mechanical criterion was included, the similarity in the energy consumed during fatigue testing markedly improved the correlation, with a coefficient of determination between paired samples of R(2) = 0.7477. A mechanical criterion, such as energy consumption, can help to improve sample selection and produce more consistent results. Finally, we obtained the mathematical expression that relates the energy consumed to the pressure exerted and the number of cycles per second to which the valve leaflet was subjected. This procedure enables us to establish the limit to the energy that a biomaterial can consume over a period of time during which it is subjected to a working pressure and, thus, calculate more precisely its durability.     

Effects of periodate and chondroitin 4-sulfate on proteoglycan stabilization of ostrich pericardium. Inhibition of calcification in subcutaneous implants in rats

Chemical modification of biological materials used in the manufacture of cardiac valves tends to reduce the relatively high degree of biodegradation and calcification of the implanted bioprostheses. The most widely used treatment to reduce biodegradability of the valves is glutaraldehyde fixation. However, this treatment is potentially toxic and induces tissue calcification. In order to minimize these undesirable effects, we have analyzed the effect of a pre-fixation of endogenous proteoglycans and exogenous glycosaminoglycans, as well as the borohydride reduction influence on the different modified ostrich pericardium implants after subcutaneous implantation in rats. The presence of calcific deposits was detected in all implanted GA-fixed samples; however, calcification was highly reduced in both groups of periodate-prefixed materials, which showed also a very low Ca/P molar ratio. Borohydride post-treatment of these biomaterials resulted in a significant increase in calcium phosphate precipitation, with the appearance of calcium deposits mainly in an amorphous form even though X-ray diffraction allowed the detection of brushite- and apatite-like crystals. Regarding tissue stability, no significant differences were found among the borohydride-untreated implants but higher levels of matrix metalloproteinases were observed by gelatin zymography in the periodate pre-fixed materials. This increase was partially reduced by pre-fixation of exogenous chondroitin 4-sulfate. On the other hand, borohydride post-treatment not only increased calcification, but also reduced tissue stability and increased the presence of matrix-degrading activities.     

The telescoping suture--Part II: A novel method to improve the mechanical behavior of a new biomaterial: ostrich pericardium

Ostrich pericardium, sutured using a telescoping or overlapping technique, was studied to determine its mechanical behavior. From each of 12 pericardial sacs, four contiguous strips were cut longitudinally, from root to apex, and another four contiguous strips were cut in transverse direction. One of the strips in each set of four was used as an unsutured control and the remaining three were sutured by overlapping 0.5 cm of the tissue and sewing with Gore-tex, Prolene or Pronova. These 96 samples were then subjected to tensile testing along their major axes until rupture. The tensile stresses recorded in the suture materials at the moment tears appeared in the pericardium ranged between 55.99 MPa and 70.23 MPa for Gore-tex in samples cut in the two directions. Shear stress became ostensible at 56 MPa, with clearly evident tears. However, microfracture of the collagen fibers must be produced at much lower stress levels. The comparison of the resistance in kilograms (machine-imposed), without taking into account the sections in which the load was applied, demonstrated only a slight loss of load when the telescoping suture was employed in ostrich pericardium samples. Ostrich pericardium may continue to be an alternative biological material for the construction of heart valve leaflets.     

Comparison of fortified calf serum, serum substitutes and fetal calf serum with or without extenders for propagation of cell cultures for virus plaque assays

Two studies comparing sewage-isolated and laboratory stock viruses were conducted to determine if alternative forms of serum or serum extenders could be used in place of fetal bovine serum without a significant loss of viral titer. In the first study, BGM cells were grown in standard MEM-L15 medium which was supplemented with Nuserum, Sigma serum replacement (CPSR-1), HyClone defined iron supplemented calf bovine serum, fetal bovine serum (FBS) or FBS supplemented with either SerXtend or Mito serum extenders. Comparison of virus titers showed that CPSR-1 gave the best overall results and was comparable to FBS. Of the serum extenders, only SerXtend improved virus recovery from sewage samples. In the second study, all sera were tested with and without SerXtend. In these experiments, SerXtend enhanced virus sensitivity of the BGM cell line grown in the HyClone serum but reduced the sensitivity of those cultured in Sigma serum. In both series, the growth of BGM cells was monitored for 12 weeks and all test products were shown to support long-term cell growth.     

Mechanical resistance of a new biomaterial, ostrich pericardium, and a new method of joining tissues combining suturing and a biological adhesive

We studied the mechanical behavior in response to tensile stress of samples of ostrich pericardium bonded with a cyanoacrylate glue or sewn with a rectangular, overlapping suture that was subsequently sealed with the same bioadhesive. Seventy-two trials were performed in three series of 24 samples each: series AG, glued with an overlap of 1 cm2; series ASG, sewn with a rectangular, overlapping suture and sealed; and series AC, control samples that were left intact. The mean stress at rupture in series AG (glued) was 0.1 MPa, much lower than the working stress of a human valve leaflet, which is approximately 0.25 MPa. In the control series, this stress was 26.28 MPa. At rupture in series ASG (sutured/glued), the suture material was being subjected to a stress of 64.91 MPa, thus confirming the existence of an interaction between the suture and the shear stress exerted by the suture on the samples of pericardium. In series ASG, the mean value for the resistance to rupture when measured in machine kg was 8.83 kg, lower than but similar to that recorded in the control series AC (10.26 kg). The percentages of reversible deformation, or elongation, once the samples were torn were similar in series AC (19.15%) and ASG (21.93%). This phenomenon can only be explained by the damage to the collagen fibers in the area around the rupture, while other more distant regions work at a lower load within the elastic limit. We conclude that cyanocrylate adhesives alone are not suitable as bonding materials in cardiac bioprostheses. The results with the rectangular, overlapping suture, when subsequently sealed with an adhesive, can be considered good because, although this approach does not impede shear stress, it does maintain an excellent degree of resistance to rupture of the samples thus joined. We stress the need to take into account the concentration of the load in the design of bioprostheses.     

Pre-hatch lung development in the ostrich

We studied development of the ostrich lung using light microscopy as well as electron microscopy techniques. At E24, the lung comprised a few epithelial tubes, interspersed with abundant mesenchyme with scattered profiles of incipient blood vessels. Between E24 and E39, the epithelial thickness was reduced by 90% from 13.5 ± 0.41 μm to 1.33 ± 0.014 μm (mean ± SD, respectively). Atria were evident at E32, and by E35, the first portions of the blood-gas barrier (BGB) measuring 3.41 ± 1.12 μm were encountered. Gas exchange tissue was well formed by E39 with atria, infundibulae, air capillaries and a mature blood-gas barrier (BGB). BGB formation proceeded through the complex processes of secarecytosis and peremerecytosis, which entailed decapitation of epithelial cells by cutting or pinching off respectively and by E39, the BGB was thin at 2.21 ± 1.21 μm. Vascular remodeling by intussusceptive angiogenesis was a late stage process mediated by intraluminal pillars in the pulmonary vasculature.     

The adverse effect of some calf sera on the isolation and propagation of bluetongue virus in tissue culture

Summary Isolation and propagation of bluetongue virus was attempted in 4 tissue culture systems. Good results were demonstrated in all 4 systems using high titre virus well adapted to tissue culture. Using material containing early passage virus of low titre no isolations were achieved in LK cells maintained in medium containing calf serum; the results were poor in LK cells with horse serum in the medium and in BHK cells with calf serum in the medium and only in BHK cells with horse serum incorporated in the medium were satisfactory results obtained. Substances inhibitory to BTV were demonstrated in varying amounts in 5 different batches of calf sera.     

Tannic acid-stabilized pericardium tissue: IR spectroscopy, atomic force microscopy, and dielectric spectroscopy investigations

Infrared (IR) spectroscopy, atomic force microscopy (AFM), and dielectric spectroscopy methods were employed to study structural and dynamic changes in the tannic acid (TA)-stabilized pericardium tissue. Chemically stabilized pericardium tissue is widely used in construction of the tissue derived bioprostheses. IR spectra recorded in the range 400-4000 cm-1 allowed us to recognize different types of TA-collagen interactions. Formation of hydrogen bonds between amine as well as amide NH groups from collagen and hydroxyl groups of TA was analyzed. The AFM imaging showed that the stabilization procedure with TA introduces considerable changes in both surface topography and thickness of collagen fibrils as well as in fibril arrangement on the tissue surface. It was found, that these structural changes have an impact on the dielectric behavior of the TA-stabilized tissue. The dielectric spectra for the native and TA-stabilized tissues were measured in the frequency and temperature ranges of 10(-1) -10(7) Hz and 120-270 K, respectively. The dielectric spectra revealed the relaxation process due to orientation of bound water supplemented by the fluctuation of collagen polar side groups. At the temperatures above approximately 210 K, the relaxation due to ion migration process was observed. It was found that both relaxation processes were influenced by the TA-collagen interaction.     

Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes.

Listeria monocytogenes is able to escape from the phagolysosome and grow within the host cell cytoplasm. By 3 h after initiation of infection, actin filaments begin to concentrate at one end of the bacterium. Polarization of F-actin is associated with intracellular bacterial movement, long projections of actin filaments forming directly behind the moving bacteria. New actin monomers are added to the region of the projection in proximity to the bacterium. The rate of new actin filament growth correlates closely with the speed of bacterial migration. This actin structure is anchored within the cytoplasm, serving as a fixed platform for directional expansion of the actin filament network. The actin projection progressively lengthens as the bacterium migrates. Cytochalasin blocks both elongation of the projection and bacterial movement but does not result in complete depolymerization of the bacterially induced actin structure, residual actin and alpha-actinin persisting in proximity to one end of the bacterium. Bacteria initially migrate within the cortical cytoplasm but later move to the peripheral membrane, where they form filopodiumlike structures which pivot and undulate in the extracellular medium. In the filopodia, bacteria are occasionally seen to abruptly change direction, turn 180 degrees, and move back into the medullary region of the host cell. All filopodium movement ceases once the bacterium containing the F-actin projection returns to the cortical cytoplasm. These results indicate that host cell actin polymerization is necessary for intracellular migration of listeriae and suggest that directional actin assembly may in fact generate the propulsive force for bacterial and filopodial movement.     

Cholinesterase-positive and catecholamine-containing nerves in the guinea-pig pericardium

The functions of the pericardium are traditionally defined in terms of lubrication and support. However, its complement of autonomic nerve fibers, shown by physiologc experimnts to be sensory, suggests that the pericardium may also serve as a mechanoreceptor site. In this study acetycholinesterase-positive, and catecholamine-containing elements of the pericardial plexus of the guinea-pig were visualized, using thiocholine and fluorescence techniques, respectively. A cholinesterase-positive nerve net, containing the preterminal segments of unmyelinated fibers and simple nerve endings, extended over the entire parietal pericardium. Some of the cholinesterase-positive endings, apparently separate from effector structures were assumed to be sensory. A few heavily myelinated fibers were contributed by the phrenic nerve. The parietal pericardium covering the atria contained many adrenergic nerves and endings. Not all pericardial adrenergic endings were linked to effector structures, such as blood vessels. Some fibers ended freely. It is suggested that liberation of catecholamines from “unattached terminals” may serve to lower the threshold, and prolong the adaptation of adjacent cholinesterase-positive mechanoreceptor terminals.