| BackgroundClinically, preservation of arterial allografts is usually by freezing. The mechanism of cryopreservation is their metabolism decreased under the condition of low temperature. But the manipulation of freezing is complicated and time consuming, and the viability of the arteries preserved by freezing is limited. Recently, a new method of cryopreservation called vitrification, of which manipulation is simple and less damage to cells and tissues on account of ice-free in the process of cryopreservation. Up to present, many kinds of cells have been vitrified successfully, and vitrification of tissues is also started. However, vitrification of arterial allografts has not been published in the literature.ObjectiveTo find the appropriate conditions for vitrifying preservation of arterial allografts. And comparing with freezing grafts in vitro and in vivo, in order to evaluate the feasiability of its clinical applicat ion.MethodsThere are five steps as follow: 1) By using the orthogonal design and rapid detection technique of vascular viability (TTC testing), to find out the appropriate conditions of vitrification and freezing of femoral arterial grafts in rabbits. 2) The vitreous arterial grafts were done under the cell culture, to observe its cellular viability by comparing with fresh arteries and frozen arteries. 3) The morphologic changes of vitreous arterial grafts were under the gross, histological and ultrastructural observations. We could observe vasomotor function by using KC1, NE, Ach and SNP, and we could also observe vascular mechanical capability through longitudinal pulling test of single axis. By means of comparing with fresh arteries and frozen arteries in vitro, thus we could know the benefit and shortcoming of vitrification method. 4) After transplanted femoral arterial allografts, the rabbits were sacrificed in two weeks, one month, two months or four months, and grafts were harvested to observe the morphological changes, thrombosis and immunologic rejection. By means of comparing with fresh arterial autografts and frozen arterial allografts, to evaluate the feasibility of vitrified arteries being used as allografts. 5) Use the one year vitrifying arterial grafts to observe the morphologic changes, vasomotor function and vascular mechanical capability in vitro, followed by bacteria culture of arteries and preservation solution, finally transplant vitrified arterial allografts to rabbits, harvest grafts after four months and observe vascular structural changes. By comparing with the vitrified arterial allografts that were preserved for two weeks, to evaluate the long-term stability of vitrification.ResultsThe results are as follow: 1) The factors that affect vitrification were cooling, balancing, thawing, vitrification solution and washing in turn, and affect freezing were maintaining time, cooling, cryopreservation solution, thawing, balancing and washing. The vascular viability of optimized set of vitrification maintained 82.67%, which was significantly better than 73.29% of freezing. 2) The cells that cultivated from vitrified arteries were smooth muscle cells, which growing out from tissue in the seventh day, cells passaged in the sixteenth day, and counting of cells was 2.50 X 106. The cells that cultivated from frozen arteries were also smooth muscle cells, which growing out from tissue in the tenth day, cells passaged in the twenty-fourth day, and counting of eel Is was 2.00 X 106. 3) Both vascular smooth muscle cells and extracellular matrix could be preserved perfectly by vitrification and freezing method. Even the vitri fication does fewer traumas to endothelial cell than freezing in morphology. Vitrified arteries could reserve 96. 55% of maximal contraction of fresh vessels to KC1, 99. 00% of maximal contraction of fresh vessels to NE, which were significantly better than 68.97% and 82.42% of frozen arteries. Endothelium~dependent relaxant responses of vitrified arteries to Ach was markedly reduced to 76. 37% of fresh controls, which was still significantly bett... |
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