Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium.

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167±0.14 mmol/g of liberated amine groups vs. 598±60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.

Decellularization of pericardial tissue and its impact on tensile viscoelasticity and glycosaminoglycan content.

Bovine pericardium is a collagenous tissue commonly used as a natural biomaterial in the fabrication of cardiovascular devices. For tissue engineering purposes, this xenogeneic biomaterial must be decellularized to remove cellular antigens. With this in mind, three decellularization protocols were compared in terms of their effectiveness to extract cellular materials, their effect on glycosaminoglycan (GAG) content and, finally, their effect on tensile biomechanical behavior. The tissue decellularization was achieved by treatment with t-octyl phenoxy polyethoxy ethanol (Triton X-100), tridecyl polyethoxy ethanol (ATE) and alkaline treatment and subsequent treatment with nucleases (DNase/RNase). The quantified residual DNA content (3.0±0.4%, 4.4±0.6% and 5.6±0.7% for Triton X-100, ATE and alkaline treatment, respectively) and the absence of nuclear structures (hematoxylin and eosin staining) were indicators of effective cell removal. In the same way, it was found that the native tissue GAG content decreased to 61.6±0.6%, 62.7±1.1% and 88.6±0.2% for Triton X-100, ATE and alkaline treatment, respectively. In addition, an alteration in the tissue stress relaxation characteristics was observed after alkaline treatment. We can conclude that the three decellularization agents preserved the collagen structural network, anisotropy and the tensile modulus, tensile strength and maximum strain at failure of native tissue.

Optimal selection of biological tissue using the energy dissipated in the first loading cycle.

Calf pericardium, similar to that used in the manufacturing of prosthetic valve cusps, was fatigue tested. After six batches of 100 cycles of 1 MPa of loading pressure, half of the samples broke. The mean energy dissipated in the first cycle by the surviving samples was 0.16 J, which is lower than the 0.28 J dissipated by the specimens that broke (p = 0.005). The hysteresis of the first cycle was characteristic and different from the following ones and correlated superbly with fatigue resistance. Setting a threshold value for the energy of the first cycle of 0.20 J, the performance index (the percentage of true predictions) was almost 80%, and the area under the ROC curve was 0.823 (maximum value is 1). When including the mean thickness in the selection parameters, as an indirect measure of the specimen mass, the performance index grew over 95%, meaning that the error of the predictions was less than 5%. Combining both parameters in one, a high performance index is maintained at 87.5% and the area under the ROC curve increases to 0.917. This non-destructive method should help optical methods in the process of selecting the most appropriate and homogenous biological material.

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. Homsy's 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.

Propagation of tears in pericardium from young bulls: influence of the suture.

The tearing of the collagen fibers of biological materials utilized in implants or bioprostheses is an important, and sometimes early cause of the failure of these devices. We studied the force necessary to propagate a tear in a biomaterial, pericardium from young bulls, and the influence of the suture. An Elmendorf pendulum capable of measuring the force necessary to tear a given length of tissue was employed. We analyzed 112 trials (70%) that proved valid after achieving the homogeneity of the samples according to their thickness, thus making the results comparable. Mean forces ranging between 19.87 and 150 N were required to propagate tears measuring from 0.25 to 2.0 cm. In the samples with a 1-cm-long suture, sewn using an edge-to-edge technique, the propagation of the tear required a mean force of 15.75 N when the suture was made of nylon and 28.73 N when Prolene was utilized. When these results were compared with the mean recorded in an unsutured control series (56.76 N), the loss of resistance was significant in both sutured series (P = 0.000 and P = 0.011, respectively). Finally, the equation that relates the force (y) with the length of the tear made in unsutured tissue (x) was also obtained: y = 58.14 + 9.62x(2) (R(2) = 0.924). The force necessary to produce a microtear, thus estimated, can be utilized as a parameter for comparison.

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.

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.