Experimental Study On Cryopreservation Of Human Small-Diameter Artery With Four Cryoprotective Agents

Objective: To evaluate the protective effect of dimethyl sulfoxide,glycerol,propylene glycol and ethylene glycol in cryopreservation of human small-diameter artery,and to select appropriate cryopreservation agents and proportions,so as to provide theoretical basis for cryopreservation of blood vessels.Methods: This study collected 45 fragments of small-diameter artery after amputation in The Fifth Affiliated Hospital of Zunyi Medical University(Zhu Hai)from September 2016 to September 2018,each of which was about 0.5 cm.According to the different kinds and proportions of cryoprotective agents,they were randomly divided into 9 groups(8 experimental groups,1 control group):30% dimethyl sulfoxide group,40% dimethyl sulfoxide group,30% glycerol group,40% glycerol group,30% propylene glycol group,40% propylene glycol group,30% ethylene glycol group,40% ethylene glycol group and the control group.The experimental groups were cryopreserved for 30 days with different kinds and proportions of cryoprotective agents,and the control group was cryopreserved for 30 days without cryoprotective agents.Making glass slide after rapid rewarming.The tissue structure of human small-diameter artery was observed by HE staining.The expression of vascular endothelial growth factor in human small-diameter artery was observed by immunohistochemical.Statistical methods were used to compare the tissue structure changes and VEGF expression levels,and the differences between the groups were analyzed.Results: Results of HE staining of human small-diameter artery: In the control group,lots of endothelial cells were lost and the intima was disorganized,and large areas of internal elastic lamina were broken and missed,and the structure of smooth muscle was damaged.In the group of 30% ethylene glycol,lots of endothelial cells were lost,the inner elastic lamina were partially broken and missed,and the structure of smooth muscle was partially damaged.In the group of 40% ethylene glycol,lots of endothelial cells were exfoliated,the internal elastic lamina was partially damaged,and the smooth muscle cell structure was invaded.In the group of 30% propylene glycol,the endothelial cells were lost in small pieces,and there were some gaps between the endothelial cells and the internal elastic lamina,and the internal elastic lamina was rarely ruptured.In the group of 40% propylene glycol,a partial loss of endothelial cells were observed,and there were some gaps between the endothelial cells and the internal elastic lamina,and the internal elastic lamina was not ruptured.In the group of 30% glycerol,the endothelial cells were slightly lost,and there were a few of gaps between the endothelial cells and the internal elastic lamina.The structure of smooth muscle were not damaged.In the group of 40% glycerol,the endothelial cells were continuous,and the subendothelial layer,internal elastic lamina and smooth muscle cells were well organized.In the group of 30% dimethyl sulfoxide,the endothelial cells were rarely lost,the internal elastic lamina and the smooth muscle cells were organized.In the group of 40% dimethyl sulfoxide,the endothelial cells were continuous and aligned orderly,the subendothelial layer,internal elastic lamina and smooth muscle cells were well organized.Expression of VEGF of human arterioles in each group: When the proportions of the cryoprotective agents were 30% and 40%,compared with the control group,the IOD of VEGF in the small-diameter arteries of the glycerol group,the dimethyl sulfoxide group and the propylene glycol group were significantly increased(P<0.05);Compared with the control group,the IOD of VEGF increased in the ethylene glycol group(P>0.05).Compared with the ethylene glycol group,the IOD of the dimethyl sulfoxide group,the glycerol group and the propylene glycol group were significantly increased(P<0.05).Compared with propylene glycol group,the IOD of VEGF in small-diameter artery of glycerol group and dimethyl sulfoxide group were significantly increased(P<0.05).Compared with the glycerol group,the IOD of VEGF in the small-diameter artery of dimethyl sulfoxide group was significantly increased(P<0.05).With the increased of proportion,the IOD of VEGF were significantly increased in the dimethyl sulfoxide group,glycerol group and propylene glycol group(P<0.05),while significantly decreased in the glycol group(P<0.05).Conclusion: 1.Dimethyl sulfoxide,glycerol and propylene glycol have protective effects on the cryopreservation of human small-diameter artery,while the cryoprotective effect of ethylene glycol is not obvious.2.The cryoprotective effect of dimethyl sulfoxide on small-diameter artery is better than glycerol,propylene glycol and ethylene glycol.3.The 40% dimethyl sulfoxide may be more suitable for cryopreservation of small-diameter artery.