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Improved ultrastructural preservation of bioprosthetic tissue.

BACKGROUND AND AIMS OF THE STUDY: Poor ultrastructural tissue preservation of bioprosthetic heart valves is associated with a higher propensity for calcification. In spite of this realization, commercial valve fixation remains suboptimal. METHODS: In an attempt to maintain tissue integrity through improved cross-linking procedures, transmission electron microscopy and a 21-point damage score were applied to assess the ultrastructural preservation of aortic wall tissue-the main component of contemporary aortic valve bioprostheses. An ideal glutaraldehyde (GA) concentration was assessed by immediate tissue fixation at 4 degrees C comparing 0.2%, 0.5%, 0.65%, 1.0%, 2.0%, 3.0% and 4.0% GA in phosphate-buffered saline (PBS). Subsequently, an optimal concentration of 3.0% GA was used to determine the effect of fixation temperature (4 degrees, 22 degrees and 37 degrees C). Finally, the superior glutaraldehyde concentration (3.0%) and cross-linking temperature (4 degrees C) were used to assess tolerance towards delayed fixation. RESULTS: When different GA concentrations were used almost identical damage scores of 6.3 and 5.8 were found for 0.2% and 0.65% fixation. The first significant improvement was found at a concentration of 1.0% (score 3.3; p < 0.01) followed by a further improvement at 3.0% (score 2.6; p = 0.05). The optimal fixation temperature was 4 degrees C (3.7) with the worst results obtained at room temperature (score 9.2; p < 0.03). When fixation was delayed, the most significant damage occurred during the initial 30 min after slaughter (from 2.3 to 7.4; p < 0.02) followed by another significant deterioration between 4 and 16 h (from 5.6 to 9.7; p < 0.02). CONCLUSIONS: In summary, the prerequisites for an ideal ultrastructural preservation of bioprosthetic aortic wall tissue are immediate fixation (within 30 min), high GA concentrations (> 1.0%) and cold-temperature fixation (4 degrees C).

Animals↗

[Technical problems in microsurgical processing of preserved donor tissue].

The microsurgical processing of longterm preserved tissue is still a problem for lamellar and perforating keratoplasties. Advantages of donor tissue cut with rotortrephines versus manual trephines are presented. Optimal possibilities of examination and storage of donor tissue are explained. A lamellar microkeratome, an artificial anterior chamber for corneoscleral discs and a refractive workbench for non-freeze modification are shown.

Corneal Transplantation↗

Polymorphism of isoenzymes in preserved muscle tissues.

In muscles preserved in formalin enzymes were not found to be active. In muscles treated by ethanol the ESD, GLO, GPT and PGP enzymes were active. The best results were obtained in the case of acetone treatment. The phenotypes ESD, GLO, GPT and PGP in tissues corresponded with the ones in the comparative blood samples.

Acetone↗

Antifreeze solution improves DNA recovery by preserving the integrity of pathogen-infected blood and other tissues.

Preserving blood samples for shipping and later DNA extraction has been performed by cooling, freezing, drying, freeze-drying, and protease treatment, among other methods. Most methods to preserve field samples for further DNA extraction do not prevent cellular and DNA damage or are useful only in preserving them for short periods. This report introduces a novel method for blood and tissue that allows preservation in freezing temperatures for a prolonged period of time. The solution reported here (20% ethylene glycol-propylene glycol) preserves cells and tissues integrity, as judged by microscopic analysis, and improves DNA yield and quality.

Blood Preservation↗

Amelioration of rewarming ischemic injury of the pancreas graft during vascular anastomosis by increasing tissue ATP contents during preservation by the two-layer cold storage method.

Rewarming ischemic injury during vascular anastomosis severely compromises posttransplant pancreas graft survival because the graft has already been subjected to warm and cold ischemia before vascular anastomosis. We examined whether preservation of the pancreas graft by the two-layer method ameliorates rewarming ischemic injury of the graft during vascular anastomosis and also investigated the energy metabolism of the pancreas graft before, during and after rewarming ischemic period. After flushing with cold University of Wisconsin solution (UW), the pancreas grafts were preserved by the two-layer (UW/perfluorochemical [PFC]) method (group 1) or simple cold storage in UW (group 2) for 24-hr and then autotransplanted. In control, the pancreas grafts were flushed out with cold UW and immediately autotransplanted without preservation (group 3). After completion of vascular anastomosis, vascular clamp was not released until 90, 120, or 150 min of rewarming ischemia, including anastomosis time, has elapsed. After 90 min of rewarming ischemia, graft survival rates were 5/5, 100%, 5/5, 100%, and 5/5, 100% in groups 1, 2 and 3, respectively. After 120 min, all the grafts in groups 2 and 3 failed (0/5, 0%, and 0/5, 0%, respectively), however, all the grafts in group 1 survived (5/5, 100%). Even after 150 min, 1 of 3 grafts in group 1 survived (1/3, 33%). After 24 hr preservation, tissue adenosine triphosphate (ATP) and total adenine nucleotides (TAN) levels of the grafts in group 1 were about 2-fold the reference values before harvesting and significantly higher compared with group 2(p < 0.05; p < 0.05). After 120 min of rewarming ischemia, tissue ATP levels in group 1 were 84% of the reference values and significantly higher compared with group 2(p < 0.05). TAN levels of group 1 were also significantly higher compared with group 2(p < 0.05). Two hours after reperfusion, ATP and TAN levels in group 1 were significantly higher than group 2(p < 0.05). There were no remarkable difference between group 1 and group 2 concerning adenosine diphosphate (ADP), adenosine monophosphate (AMP) levels. We conclude that the two-layer (UW/PFC) method ameliorates rewarming ischemic injury of the pancreas graft during vascular anastomosis by increasing tissue ATP concentration and TAN levels during preservation and maintaining tissue ATP and TAN levels during vascular anastomosis. Consequently, ATP levels are rapidly recovered after reperfusion and the graft survives.

Adenosine Triphosphate↗

Periodontal and pulpal healing of monkey incisors preserved in tissue culture before replantation.

The effect of delayed replantation and tissue culture as a prevention against root resorption was examined in green Vervet monkeys (Cercopithecus aethiops). Extracted incisors were kept in tissue culture medium (Eagle's medium) for 5--14 days before replantation. The extra-alveolar dry period before tissue culture ranged from to 0 to 60 min. Incisors not subjected to tissue culture served as controls. The animals were sacrificed 8 weeks after replantation. The following histologic parameters were registered for each tooth: surface resorption, inflammatory resorption, replacement resorption (ankylosis), downgrowth of pocket epithelium, and periapical inflammatory changes. The evaluation of the pulp included the extent, recorded in mm, of pulp survival. Histometric evaluation showed that teeth immediately placed in tissue culture medium for 5--14 days showed improved periodontal healing, exhibiting significantly less inflammatory resorption than control teeth immediately replanted. Also the extent of pulp survival was significantly increased in teeth replanted after tissue culture. Teeth bench-dried for 60 min and thereafter placed in tissue culture medium also showed a significant reduction in the amount of inflammatory resorption and increased amount of pulp survival compared with control teeth replanted after the 60-min dry extra-alveolar period. Replacement resorption (ankylosis) was found in all teeth in both groups.

Animals↗