Histological Study In Cryopreserv-ing Composite Tissue With Three Dif-ferent Freezing Methods

Objective Cryopreservation can maintain composite tissue over a long period because shortage of energy may weaken biochemical reaction and metabolism. However, tissue was susceptible to damage when it underwent freezing and thawing. Up to mow, the problems in cryopreserving composite tissue remain poorly understood. In the present study, the histological study was applied in the pedicle artery of cryopreserved composite tissue with three different freezing methods in order to find an optimum freezing method and lay the theoretical and experimental foundation for clinical application.Method Thirty-two Wistar rats were randomly divided into three experimental groups and control group. One hinder claw was harvested using microsurgical technique and perfused by cold cryoprotective agent. The control group specimens didn't undergo freezing. The group A specimens were frozen to -196℃ in vitrification manner. In groupB, freezing was carried out to -80℃ using ultra-low refrigerator, then frozen to -196℃. In group C, freezing was carried out in a stepwise manner to -80℃ using a computer-programmed freezer, then frozen to -196℃. After thawing, the pedicle artery was dissected and its endothelium and wall structure were examined by optical microscope, scanning electron microscope and transmission electron microscope. The results were analyzed statistically.Result In group A and group B, examination of optical microscope showed that sloughed endothelial cells were separated from the intima in the cryopreserved artery and the integrity of the arterial wall was damaged, and there was significant difference statistically between the control group and group A (P<0. 01) or group B (P<0. 01). In groupC, the integrity of cryopreserved arterial wall was kept intact in six specimens, and there was no significant difference statistically between the control group and group C (P> 0. 05). The ultrastructure of endothelial cells and intima in cryopreserved artery in group C were well preserved.Conclusion (1)It is imperative to apply two-step freezing method in cryopreservation of composite tissue. Especially, the stepwise computer-programmed freezing and liquid nitrogen preservation method can completely keep arterial tissue structure of the pedicle of composite tissue. But the vitrification cryopreservation method may severely damage the arterial tissue structure.(2) The composite tissue perfused by cryoprotective agent should be frozen with optimum freezing velocity in two-step freezing method. (3) The optimum velocity of stepwise computer-programmed freezing should be selected and affirmed according to the size and type of composite tissue, this is helpful to cryopreservation and transplantation of cryopreserved allograft composite tissue.