Degradation effect of diepoxide fixation on porcine endogenous retrovirus DNA in heart valves: molecular aspects.

PURPOSE: Xenotransplantations of porcine cells, tissues, and organs involve a risk of zoonotic viral infections in recipients, including by porcine endogenous retroviruses (PERVs), which are embedded the genome of all pigs. An appropriate preparation of porcine heart valves for transplantation can prevent retroviral infection. Therefore, the present study focuses on the effect of epoxy compounds and glutaraldehyde on the PERV presence in porcine heart valves prepared for clinical use. METHODS: Porcine aortic heart valves were fixed with ethylene glycol diglycidyl ether (EDGE) at 5 °C and 25 °C as well as with glutaraldehyde (GA) for 4 weeks. Salting out was used to isolate genomic DNA from native as well as EDGE- and GA-fixed fragments of valves every week. Quantification of PERV-A, PERV-B, and PERV-C DNA was performed by real-time quantitative polymerase chain reaction (QPCR). RESULTS: All subtypes of PERVs were detected in native porcine aortic heart valves. The reduction of the PERV-A, PERV-B, and PERV-C DNA copy numbers was observed in the heart valves which were EDGE-fixed at both temperatures, and in GA-fixed ones in the following weeks. After 7 and 14 days of EDGE cross-linking, significant differences between the investigated temperatures were found for the number of PERV-A and PERV-B copies. PERV DNA was completely degraded within the first week of EDGE fixation at 25 °C. CONCLUSIONS: EDGE fixation induces complete PERV genetic material degradation in porcine aortic heart valves. This suggests that epoxy compounds may be alternatively used in the preparation of bioprosthetic heart valves in future.

Integrative measurements of calcifications in stented, antibiotic sterilized and cryopreserved sheep biological valves implanted for one year in tricuspid position.

UNLABELLED: The aim of the study was the characterization of calcification in the leaflets of a cryopreserved and alive heart valve depending on the diagnosed pathologic process. Sheep antibiotic sterilised and cryopreserved biological valves were implanted in tricuspid position in young sheeps for one year period. After this time the valves removed and studied morphologically. The control group consisted of 7 intact valves, the comparative group, so called group of valves after the processing antibiotic sterilization and cryopreservation consisted of 7 valves after mentioned procedures. Histological investigations were based on paraffin sections, calcium deposits were stained von Kossa technique. The measured values included integrative parameters as: 1. area fraction, 2. number of calcifications per area, 3. anisotropy. CONCLUSIONS: 1. A process of initial processing, sterilization and cryopreservation of biological valve increases a number of microcalcifications. 2. Cryopreserved biological valves explanted after one-year implantation into an animal in a tricuspid position possess fine calcifications and calcification foci. A number and size of fine calcifications decreases together with an intensification of degeneration and regressive processes of the connective tissue, especially in hyalinization. Hyalinization of the biological valve tissue seems to be favorable for a valve durability and as a pathological process decreasing calcification. 3. Mathematic analysis of morphometric features defining density and structure of calcifications indicate similarities among cryopreservation and initial processing groups, hyalinization, inflammation, whereas in a group of calcification foci, the similarity can be noticed between inflammation and hyalinization group.

Mathematical analysis of calcifications in stented, antibiotic sterilized and cryopreserved sheep biological valves implanted for one year in tricuspid position.

UNLABELLED: The aim of the study was morphometric and mathematic analysis of calcification profiles present in the leaflets of a cryopreserved and alive heart valve depending on the diagnosed pathologic process. Sheep antibiotic sterilised and cryopreserved biological valves were implanted in tricuspid position in young sheeps for one-year period. After this time the valves removed and studied morphologically. The control group consisted of 7 intact valves, the comparative group, so called group of valves after the processing antibiotic sterilization and cryopreservation consisted of 7 valves after mentioned procedures. Histologic investigations were based on paraffin sections of formalin-fixed valve cusps, stained with H&E and Masson trichrome, calcium deposits were stained von Kossa technique. The measured values included: 1. area and equivalent diameter, 2. length, 3. breadth, 4. perimeter, 5. elongation, 6. roundness, 7. fullness coefficient. CONCLUSIONS: 1. A process of initial processing and cryopreservation of biological valve increases a dimension and disturbs a shape of microcalcifications. 2. Cryopreserved biological valves explanted after one-year implantation into an animal in a tricuspid position possess microcalcifications and calcification foci. The size of microcalcifications decreases together with an intensification of degenerative processes of the connective tissue, especially in hyalinization. Hyalinization of the biological valve tissue seems to be favorable for a valve durability and as a pathological process decreasing calcification. 3. Mathematic analysis of morphometric features defining differences in size and shape of each calcification indicate morphologic and morphometric autonomy of calcifications, characteristic for the analyzed group of valve pathologic changes.

Cellular viability and ultrastructure of stented, antibiotic sterilized and cryopreserved sheep biological valves implanted for one year in tricuspid position.

UNLABELLED: The aim of the study was an evaluation of viability and damage of biological cells sterilized with antibiotics and cryopreserved a year after the implantation. Sheep antibiotic sterilised and cryopreserved biological valves were implanted in tricuspid position in young sheeps for one-year period. After this time the valves removed and studied morphologically. The control group consisted of 7 intact valves, the comparative group, so called group of valves after the processing antibiotic sterilization and cryopreservation consisted of 7 valves after mentioned procedures. Histologic investigations were based on paraffin sections of formalin-fixed valve cusps, stained with H&E and Masson trichrome. Valve viability was assessed using intravital staining with fluoresceine diacetate, whereas damaged cells were visualized by intravital staining with neutral red. Additionally, the ultrastructural studies were performed. The viability and ultrastructural results were compared with the pathologic process in the valve. CONCLUSIONS: 1. preliminary preparation with antibiotic sterilisation and cryopresrevation induces valve leaflet oedema and degenerative ultrastructural processes, colliquative cell necrosis and apoptotic morphology of a part of valve cells; 2. cryopreserved and antibiotic sterilised pulmonary valve after one-year implantation behaves lining cells, but ultrastructural changes indicates many degenerative phenomena in smaller degree than after preparation, sterilization and cryopreservation. Histopathologically the degenerative changes were prevalent.