E. Jorge-Herrero

Influence of different chemical cross-linking treatments on the properties of bovine pericardium and collagen

The use of biological materials in the construction of bioprostheses requires the application of different chemical or physical procedures to improve the mechanical performance of the material without producing any undesirable effects. A number of cross-linking methods have been tested in biological tissues composed mainly of collagen. The basis for most of them is the use of glutaraldehyde (GA), which acts on the Lys or Hyl residues. We have studied the effects of alternative chemical treatments: diphenylphosphorylazide (DPPA) and ethyldimethylaminopropyl carbodiimide (EDAC). Their mechanism of action is based on the activation of the carboxyl groups, which then permits their cross-linking to amino groups. As a control, we employed conventional treatment with GA, applying it to bovine pericardium and collagen membranes removed from bovine pericardium. The analysis of the Lys and Hyl residues showed that DPPA and EDAC produced 50% of the chemical change provoked by GA. This value was even lower in the trials with collagen. In terms of the resistance to collagenase degradation, chemical cross-linking with GA provided much greater protection in both materials (3.81 +/- 3.47 nmol of amino acid/mg dry tissue for pericardium and 4.41 +/- 1.13 nmol of amino acid/mg dry tissue for collagen). Treatment with DPPA also protected pericardium (13.11 +/- 6.57 nmol amino acid/mg dry tissue) although the values for collagen was lower (50.0 +/- 32.4 nmol amino acid/mg dry tissue). Treatment with EDAC was much less protective than the other two chemical reagents (43.28 +/- 17.4 and 55.85 +/- 14.57 nmol amino acid/mg dry tissue for pericardium and collagen, respectively). The degree of tissue calcification after implantation of the chemically treated materials into young rats was considerably greater for GA and DPPA (32.9 +/- 18.8 and 36.3 +/- 13.3 mg g(-1) dry tissue, respectively) than with EDAC (18.0 +/- 7.2 mg g(-1) dry tissue; P < 0.001). After 60 days of implantation, the values for GA and EDAC were higher(124.1 +/- 31.3 and 124.6 +/- 21.0 mg g(-1) dry tissue, respectively) versus 34.6 +/- 19.2 mg g(-1) dry tissue for DPPA. There were no significant differences in collagen levels in samples treated with GA or EDAC after 30 days of implantation, although both groups showed significant differences when compared with DPPA-treated samples (P < 0.001). After 60 days of implantation, there were no significant differences among these three treatments in terms of the calcium accumulated on samples.

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.

Biocompatibility and Calcification of Bovine Pericardium Employed for the Construction of Cardiac Bioprostheses Treated With Different Chemical Crosslink Methods

The use of biological materials in the construction of bioprostheses requires the application of different chemical procedures to improve the durability of the material without producing any undesirable effects. A number of crosslinking methods have been tested in biological tissues composed mainly of collagen. The aim of this study was to evaluate the in vitro biocompatibility, the mechanical properties, and in vivo calcification of chemically modified bovine pericardium using glutaraldehyde acetals (GAAs) in comparison with glutaraldehyde (GA) treatment. Homsys tests showed that the most cytotoxic treatment is GA whereas GAA treatments showed lower cytotoxicity. Regarding the mechanical properties of the modified materials, no significant differences in stress at rupture were detected among the different treatments. Zeta-Potential showed higher negative values for GA treatment (-4.9 +/- 0.6 mV) compared with GAA-0.625% (-2.2 +/- 0.5 mV) and GAA-1% (-2.2 +/- 0.4 mV), which presented values similar to native tissue. Similar results were obtained for calcium permeability coefficients which showed the highest values for GA treatment (0.12 +/- 0.02 mm(2)/min), being significantly lower for GAA treatments or non-crosslinked pericardium. These results confirmed the higher propensity of the GA-treated tissues for attraction of calcium cations and were in good agreement with the calcification degree obtained after 60 days implantation into young rats, which was significantly higher for the GA group (22.70 +/- 20.80 mg/g dry tissue) compared with GAA-0.625% and GAA-1% groups (0.49 +/- 0.28 mg/g dry tissue and 3.51 +/- 3.27 mg/g dry tissue, respectively; P < 0.001). In conclusion, GAA treatments can be considered a promising alternative to GA treatment.

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.

Tissue heart valve mineralization: Review of calcification mechanisms and strategies for prevention

Attempts to replace diseased human valves with prostheses began more than 30 yrs ago. Heart valve prostheses can be broadly classified into mechanical prostheses (made out of non-biological materials) and bioprostheses made out of biological tissue. Biological valves are made from animal tissue bovine pericardium and porcine valves. The use of these tissues became commercially available after the introduction of the glutaraldehyde (GA) fixation technique. GA reacts with tissue proteins to form inter- and intramolecular crosslinks, resulting in improved durability. The advantage of bioprostheses compared with mechanical valves is the freedom from thromboembolism; and therefore, the avoidance of long-term anticoagulation therapy. These prostheses are preferable in elderly people and in patients who do not tolerate anticoagulants. However, tissular calcification and primary tissue failure (caused by the mechanical stress) are the main unresolved problems. The causes of calcification are numerous and, to date, a satisfactory solution to this question has not been found, although chemical treatments with metal cations, diphosphonates and treatments eliminating phospholipids have proved to mitigate calcification. In addition, alterna-tive approaches to GA chemical treatment fixation are being proposed to provide the tissue with greater resistance to this process. Studies are under way using polyepoxy compounds, derivates of amino oleic acid (AOA), agents such as diphenylphosphorylazide, carbodiimide, amino acids etc. Further improvements in fixation techniques, as well as in bioprosthesis design (stentless valves) are being made to improve the durability and functional characteristics of bioprosthetic heart valves. The development of a biomaterial capable of withstanding calcification and mechanical stress, while being as durable as mechanical prostheses, would convert the bioprostheses into the replacement of choice by eliminating the need for anticoagulation therapy.

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.

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.

Behaviour of the bovine pericardium used in cardiac bioprostheses when subjected to a real fatigue assay.

The behaviour of bovine pericardium was studied using a fatigue assay. Twenty-three samples were assayed, maintaining the preset initial stress and measuring the time it took for the onset of load loss due to permanent deformation. The results indicated a mathematical relationship defined by the expression: log y = 1.3 - 0.211 log t, where y is the fatigue stress (MPa) and t the duration of the assay. The correlation coefficient was 0.948 (P < 0.001). The safety coefficient of the material diminished significantly as the period of time during which it was subjected to fatigue increased. The theoretical durability of the tissue was much greater than the real durability of the prostheses, which is determined by unsolved problems such as calcification and those derived from suture-related cutting.

Assessment of Pericardium in Cardiac Bioprostheses: A Review

Cardiac valve bioprostheses are assessed in terms of their present and future clinical utility. The problems concerning durability basically involve early failure due to tears in the valve leaflets and late failure mainly associated with calcification of the biological tissue. New strategies for selection and chemical treatment of the biomaterials employed are analyzed, and the available knowledge regarding their mechanical behavior is reviewed. It is concluded that the durability of these devices, and thus their successful use in the future, depends on the knowledge of the interactions among the different biomaterials of which they are composed, the development of new materials, and the engineering design applied in their construction.

Influence of stress on calcification of delipidated bovine pericardial tissue employed in construction of cardiac valves

Since the development of cardiac bioprostheses, numerous chemical treatments have been assayed to prevent mineralization. The effectiveness of chemical treatments that eliminate lipids from the tissue was tested by combining two models. First, handmade bovine pericardial bioprostheses, subjected to chemical treatment with chloroform/ methanol and glutaraldehyde or treated with glutaraldehyde alone for use as controls, were subjected to mechanical stress in a heart valve, accelerated wear tester (100 x 10(6) consecutive cycles). Then, the bioprostheses were unstitched and tissue samples were taken from the portion subjected to maximal stress (P1) and from that surrounding the sewing ring, which had not been subjected to mechanical stress (P2), for subcutaneous implantation. After 21 and 60 days of implantation, we observed calcification of the samples subjected to mechanical stress, even after delipidating treatment, with no significant differences with respect to the control group. However, the treated samples from the portion not subjected to mechanical stress presented a slighter accumulation of calcium after 60-day implantation (5.60 +/- 3.09 mg Ca2 +/g dry weight of tissue) versus the control group (47.17 +/- 20.4 mg Ca2+/g dry weight of tissue), the difference of which was statistically significant (p < 0.01). At the time of these medium-term studies, marked calcification was observed in tissue subjected to delipidating treatment in the zones that underwent mechanical stress.