J. L. Castillo-Olivares

Study of the calcification of bovine pericardium: analysis of the implication of lipids and proteoglycans

A major problem with cardiac bioprosthesis is the calcification of the tissue used in their manufacture, an event which may be promoted by multiple factors. A subcutaneously implanted model was used to determine, by selective extractions of proteoglycans and lipids, the role played by these compounds in the calcification of the bovine pericardial tissue used in the construction of some biological valves. The selective extraction of proteoglycans resulted in a great accumulation of calcium salts in the tissue, which, moreover, had a reduced hydrothermal stability. On the other hand, lipid extraction produced no modification in the stability of the tissue and resulted in a lesser calcium accumulation than in the control group. Proteoglycans and lipids may, therefore, be implicated in mineralization of the pericardial tissue.

Calcification of pericardial tissue pretreated with different amino acids.

Since the development of cardiac prostheses, numerous chemical treatments have been assayed to prevent the process of their mineralization. The effect of chemical treatment with amino acids is assessed in a subcutaneous implantation model in rats. Pericardial tissue from young calves was treated with L-lysine, L-glutamine, L-arginine or L-glutamic acid, each at a concentration of 0.5 M, following treatment with 0.625% glutaraldehyde. Then, the tissue was implanted into young rats for periods of 21 and 60 d, after which the calcium accumulated was quantified by atomic absorption spectroscopy. Values similar to or higher than those found in control samples indicated a lack of effectiveness of these treatments. Only in the 21-d implantation samples treated with L-lysine and L-arginine was less calcium accumulated than in the control tissue. After 60 d of implantation, all groups showed high levels of calcium deposition. The values obtained after 60 d of subcutaneous implantation were 87.5 +/- 52.4 mg Ca2+ per g dry weight of tissue for L-lysine, 108.7 +/- 43.5 mg Ca2+ per g dry weight of tissue for L-glutamine, 130.4 +/- 22.4 mg Ca2+ per g dry weight of tissue for L-glutamic acid, 119.3 +/- 27.6 mg Ca2+ per g dry weight of tissue for L-arginine and 100.0 +/- 38.3 mg Ca2+ per g dry weight of tissue for the control group. Treatment with amino acids does not appear to prevent the calcification of cardiac bioprostheses or of collagen-based biomaterials when assayed in a model of subcutaneous implantation.

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.

Selection and interaction of biomaterials used in the construction of cardiac bioprostheses

The mechanical behavior of calf pericardium employed in the manufacture of cardiac bioprostheses was assessed according to the region from which it was selected. For this purpose, selected samples of the tissue were sewn with different types of commercially available sutures and subjected to tensile testing, the results of which were compared with the findings in selected, but not sutured, tissue used as a control. The results confirm a loss of resistance--that is, a reduction of the capacity of sutured samples of the biomaterial to withstand breakage stress compared with control samples. Taking into account the marked resistance to breakage of the suture thread, this phenomenon can only be explained as a consequence of the deleterious mechanical interaction between the suture and chemically treated pericardium. This interaction is illustrated by the shearing force which is responsible for the loss of resistance in the tested samples. These trials demonstrate that the results can be improved and the deleterious interaction diminished, although not eliminated, when the pericardium is selected from a given region.

Chemical treatment and tissue selection: factors that influence the mechanical behaviour of porcine pericardium

Calcification and mechanical failure are the major causes of the loss of cardiac bioprostheses. The chemical treatments used to stabilize the tissue employed are considered to play a fundamental role in the development of these two phenomena, although the problem is multifactorial and the underlying causes are yet to be fully identified. Currently, there is an ongoing search for chemical treatments capable of reducing or eliminating the process of calcification while preserving the mechanoelastic characteristics of the tissue. One of the approaches to this effort is the elimination of the phospholipid component from the biological tissue employed in prosthesis construction. There is evidence that this component may be responsible for the precipitation of calcium salts. The present study compares two delipidating chemical treatments involving chloroform/methanol and sodium dodecyl sulfate (SDS) with the use of glutaraldehyde (GA) alone. For this purpose, porcine pericardial tissue was subjected to tensile strength testing employing a hydraulic simulator. A total of 234 samples were studied 90 treated with GA, 72 treated with chloroform/methanol and 72 treated with SDS. The mean breaking strength was significantly higher in the samples treated with GA (between 43.29 and 63.01 MPa) when compared with those of tissue treated with chloroform/methanol (29.92-42.30 MPa) or with SDS (13.49-19.06 MPa). In a second phase of the study, selection criteria based on morphological and mechanical factors were applied to the pericardial membranes employing a system of paired samples. The mathematical analysis of the findings in one fragment will aid in determining the mechanical behavior of its adjacent twin sample. In conclusion, the anticalcification chemical treatments tested in the experimental model conferred a lesser mechanical resistance than that obtained with GA. On the other hand, the utilization of paired samples was found to be useful in the prediction of the mechanical behavior of porcine pericardial tissue. Nevertheless, in order for our method of selection to be considered the most adequate approach, it will be necessary to validate these findings in dynamic studies involving a real, functional model.

Resistance and elasticity of the suture threads employed in cardiac bioprostheses

The mechanoelastic features of five types of sutures were studied. The breaking stress for each was determined by means of tensile tests in which a constant strain rate was applied, and a tensile test with graduated stress and relaxation defined the elastic limit, i.e. the point beyond which deformation becomes irreversible. The study of the stress-strain curve during this elastic period enabled us to obtain the mathematical function that governs these reversible deformations, which shows excellence of fit (R2 > 0.98). The prime derivative at each point of the resulting functions is the elastic modulus, the best parameter for comparing the elasticities of the suture threads. Since breaking stress alone does not suitably define the mechanical quality of a suture, we propose the use of other parameters during the elastic period, such as percentage of elongation at a point 10 times lower than the elastic limit (safety coefficient of 10), and tensile stress and elastic modulus at the said point, which are more reliable in the assessment of the resistance and elasticity of these threads.

Ostrich pericardium, a biomaterial for the construction of valve leaflets for cardiac bioprostheses: mechanical behaviour, selection and interaction with suture materials

The mechanical behavior of ostrich pericardium was studied for the purpose of assessing its utility in the construction of bioprosthetic cardiac valve leaflets. The tissue was tested biaxially using a hydraulic simulator that subjected it to increasing stress until rupture. One hundred eighty trials were performed, 36 with unsutured pericardium and four series of 36 trials each with pericardium sutured with silk, Prolene, nylon or Gore-Tex. The samples were tested in pairs from three different pericardial regions. One sample from each pair (the predictive specimen) was assessed according to morphological and mechanical criteria, while the other (the predicted or selectable specimen) was subjected only to morphological analysis. The findings show that ostrich pericardium treated with glutaraldehyde according to standard methods has an excellent resistance to rupture in biaxial testing, withstanding stresses of up to 100 MPa, and never lower than 30 MPa. Its resistance to rupture is lowered by suturing, a loss that is less pronounced when silk sutures are used. The results with Gore-Tex are very homogeneous and the elastic behavior of the pericardium/suture unit appears to be similar to that of unsutured tissue, suggesting that the interaction between the two biomaterials is minor. Similar results were observed in the series sutured with Prolene and nylon. The use of paired samples makes it possible to closely estimate the mechanical behavior of the tissue in a given zone by determining that of its mate. The statistical study shows that this estimation is not conditioned by the suture employed, thus validating this approach and providing more precise criteria for tissue selection.

Comparison of elasticities of components of a cardiac bioprosthesis leaflet

The mechanoelastic behavior of calf pericardium employed in cardiac bioprostheses was compared with that of three types of thread (Nylon, Prolene, and silk) used to suture this biological tissue. The elastic limit (EL) of each material was determined by means of tensile tests and the mathematical functions that govern the stress/strain curves within the EL have been described. The first derivative of these functions for each point to the curves allowed the immediate calculation of the elastic modulus (EM), which was considered the best parameter for comparing the elasticities of the materials being assessed. It was observed that the deformation of the pericardium produced by the working stress of a pericardial leaflet was approximately 1000 times greater than that produced in the surgical threads. When the elasticities were compared on the basis of the EM, that of pericardium was 749.06, 626.95, and 1253.17 times greater than that of the Nylon, Prolene, and silk suture threads, respectively. These results demonstrate that the interaction between these materials (pericardium and the threads) could be generating detrimental forces that can diminish the durability of the leaflets of the bioprostheses constructed of calf pericardium.

Elastic behaviour of sutured calf pericardium: influence of the suture threads

The purpose of this study was to assess the elastic behaviour of calf pericardium used in the construction of cardiac bioprosthesis valve leaflets, sutured with different types of commercially available sutures: silk, Gore-Tex, Surgilene and nylon. Thirty-two samples (four series of eight samples each) were subjected to tensile strength testing to breakage. The breaking stress (MPa) ranged between 4.89 MPa for samples sutured with Gore-Tex and 5.22 MPa for those sewn with nylon. Three samples from each series were subjected to a stepwise stress test, involving increasing levels of stress followed by return to zero, to define the elastic limit (the cut-off point beyond which strain is no longer reversible). Analysis of the results provided the mathematical functions that govern the elastic behaviour (stress/strain) within the elastic range for each type of sutured sample. The series sutured with Surgilene presented the highest mean value (1.649 MPa). Finally, a statistical study was carried out to determine which series showed the greatest probability of having the least interaction between the thread and the pericardium. Allowing an interval of +/- 10%, Gore-Tex showed the best probability in this respect. However, real fatigue testing is necessary to definitively determine which is the best suture to use.

ANALYSIS OF SHEARING STRESS IN THE LIMITED DURABILITY OF BOVINE PERICARDIUM USED AS A BIOMATERIAL

The objective of the study was to determine the shearing stress exerted by the suture thread under conditions of normal working stress. Thirty-six samples of calf pericardium, similar to that employed in the manufacture of bioprosthetic cardiac valve leaflets, were subjected to tensile testing. Prior to the trial, a continuous suture was sewn in the central zone of each sample, at a 45° angle to the longest axis of the sample, using commercially-available threads (silk, Gore-Tex, Surgilene and nylon). Application of the Mohr circle for combined wear revealed that the shearing stress ranged between 2.68-fold greater (for samples sewn with silk) and 5.48-fold greater (for samples sewn with nylon) than the working tensile stress in the region of the suture. It is concluded that the shearing stress is responsible for the limited durability of sutured samples of calf pericardium prepared to simulate bioprosthetic cardiac valve leaflets.