Our main finding was that the bovine pericardium preserved in glutaraldehyde presented no changes in its structure when deployed with different faces to the vessel lumen. Its production followed a rough standardization of manufacturing and quality control. In order to reduce the variables that could influence the results, the pericardium was obtained from only one source and the patch was made with the aid of the same mold.
It is important to note the presence of an expansion of the retail in the aortic wall. There was dilation of the material with the formation of an aneurysm wall at the implant site, which was significantly higher when the smooth face was turned to the light of the aorta. Furthermore, we observed that the bovine pericardium patch was well integrated into the wall of the aorta, confirming what Pires et al [9
] observed. The structure of the patch provided by surgical retail, when implanted in the aorta of dogs, promotes clear and precise limits in preserved shape, and it is already well defined at the end of the first month after surgery [9
]. Also, this type of procedure is vulnerable to infections [16
In the group where the rough face was turned into the aorta, we observed the formation of a covering layer on the inner surface with more plasticity, modeling elastic tendon and bone formation, which provided greater tension resistance to the surgical patch, preventing its expansion. Pires et al [9
] suggested that the internal apposition fibrosis originates from the fibrous organization of blood components deposited on the surface of the retail, because this plan was not observed in any case in retail implanted in the pericardium. In the group in which the flat edge of the retail was turned into the aorta this phenomenon was more intense on the suture line and was related to the roughness of the area and local release of thromboplastin by damaged tissues. Our results suggest that the wrinkled face of the retail would facilitate the capture and adhesion of blood components across its surface, with subsequent assemblage by the release of platelet factors and secondarily by thromboplastin released from damaged tissues [19
]. We also verified that in this group there was the formation of a thin inner cover layer, resulting in dilatation of the patch area of surgery. These results may suggest that fibrosis internal apposition would support retail and strengthen the structure of the surgical patch. Its histological structure reveals a dense fibrous connective tissue shaped pattern tendon with great tensile resistance.
Researchers have offered different hypotheses to explain why glutaraldehyde helped preserve bovine pericardium from changes in its structure when deployed with different faces to the vessel lumen. Ishihara et al [11
] reported that bovine pericardium treated with glutaraldehyde loses the mesothelial cells of serosal surface, exposing the submesotelial layer of connective smooth tissue, while Pires et al [9
] observed that the surface of the pericardium was covered by a layer of pavement cells and fibrous connective tissue, which they called internal apposition tissue. On the other hand Schoen et al [8
] reported that platelet aggregation, on the pericardial membrane, would be a factor for accelerating the calcification process. We observed that in both sides of the retail implanted in animals, an inner surface composed of endothelium was formed. However, when the retail was implanted with the rough side facing the lumen surface, it was isolated from bovine pericardium through blood, thus reducing the intensity and incidence of aortic dilatation in relation to the adjacent aorta. Without this coverage, we would expect an increase in calcification. Additionally, we observed that this layer of tissue was differentiated from the other sides and better trained when it had the wrinkled face toward the lumen. Furthermore, modeling elastic tendon and bone formation, formed a surgical patch level with the retail bovine pericardium which had good resistance and was well integrated into the aortic wall, probably because of the uniformity resulting from the molding process.
The presence of osteo-cartilaginous metaplasia in the fibrous layer of internal apposition was observed in only one case as an isolated focus, occurring in the group in which the rough face was turned into the aorta. These data are not in agreement with the findings of Pires et al [9
] who described a large amount of calcium present in the inner layer of apposition. The authors hypothesized that this calcification represented an osteo-cartilaginous metaplasia and ossification of fibrous tissue covering the graft, indicating the transformation of fibroblast into osteoblasts. Nonetheless, we feel confident that our findings are valid. Our methodology was validated by Sucu et al [20
] who demonstrated that the use of microscopic sections merged for evaluation of calcification was more precise than the use of the chemical method of extraction of calcium from the patch.
The calcification of biological tissue is the main cause of bioprostheses dysfunction. According to Schoen et al [7
], Chanda et al [21
], Vasudev et al [22
] and Pires et al [9
] this calcification is a multifactorial phenomenon, but it is not well defined and varies in different animal models. The authors pointed out that calcification in bovine pericardium used as a vascular patch presented a different connotation of calcification than valvular prostheses, because it implied a reduction in leaflet mobility, resulting in dysfunction of the prosthesis, which was not observed in vascular grafts.
We observed calcification of the retail irrespective of the side facing the lumen; the same was true regarding the retail implanted in the peritoneum of the animal. However, these finding are not consistent with findings of Schoen et al [6
] and Rossi et al [23
] who found calcification of retail implanted in rats. The authors described calcification of bovine pericardium from 24 hours of implantation and progressively increased with time, but did not report the presence of internal tissue apposition. We did not find this tissue in the patches implanted in the peritoneum (without direct contact with the bloodstream). These findings are consistent with findings by Gabbay et al [24
], Bortolotti et al [25
] and Pires et al [10
], who indicated the major influences of the implant site of bovine pericardium treated with glutaraldehyde and its direct contact with the bloodstream.
We used a bovine pericardium produced by Braile Biomedica. However, several other industries also produce the same patch. Some industries use special procedures to reduce the prevalence of calcification. For example, Synovis, who produces Vascu-Guard [26
], follows Apex-Processing. Briefly, in this process, the levels of residual glutaraldehyde are below the limits of detection by the sophisticated analytical methods now available (< 0.5 ppm) and products undergoing Apex-Processing have levels of cellularity that are four times lower than a variety of competitive materials including products conventionally treated.
According to our data, there was no calcification in the retail implanted in the peritoneum in almost half of the animals. Peritoneal calcification is a rare condition developed in uremic patients on continuous ambulatory peritoneal dialysis. Once peritoneal calcification is detected, it is essential to assess whether encapsulating peritoneal sclerosis develops [27
Bovine pericardium is also implanted for the vascular reconstruction in the femoral or carotid artery as patchplasty and venous patch angioplasty [28
]. Moreover, the bovine pericardium is not only used in cardiovascular surgery but also in hernia [29
] or thoracic surgery [30
]. Based on our data, we confirm the use of the rough face in those types of surgery. We propose future studies to investigate this possibility.
Our investigation presents some points that should be addressed: we did not perform blood analysis, i.e., cholesterol, triglycerides and blood glucose levels. However, we aimed to focus only on calcification. Hematoxylin and eosin is not the best method for investigating tissue calcification and it does not quantify proteins related to fibrosis. On the other hand, this is the first study to investigate the utilization of different faces of bovine pericardium conserved in glutaraldehyde as a vascular patch. The glutaraldehyde treatment, the resulting increased stiffness of the treated tissue and the presence of fixed cellular material in the tissue are all factors contributing to the calcification of cardiovascular implants. We did not perform microbiological investigation of the patch. We suggest this procedure for future studies. The time the bovine patch stayed in the circulation was not constant, possibly this factor may be a bias for our findings.