An Experimental Study On The Morphology And Biological Vitality Of Preserved Amniotic Membranes

Objective:To investigate the effects on the amniotic membrane(AM) morphology and vitality of different preservation methods and periods, pick out a method which can keep AM structure and vitality well, and provide the experimental evidence for clinical use. Methods:Fresh AM was preserved by 4 ways,DMEM/glycerol at -80 ℃ , glycerol at 4℃,ethanol and vacuum dry storage, each way was run for 1,3 and 12 months(DMEM/glycerol and glycerol). The histological properties (light and transmission electron microscopic) were analyzed. Immunohistochemical method with image analysis system was used to investigate the changes in the expression of hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), collagen type Ⅳ(colⅣ) and laminin(LN). Enzyme linked immunosorbent assay technique was used to measure the contents of HGF and bFGF. All compared with fresh AM . Results: 1 Light microscopic study: AM structure was kept well since preserved in DMEM/glycerol at -80℃ for 1 and 3 months, but a few epithelial cells were lost 12 months later; Most of the epithelial cells were destroyed in AM preserved in glycerol at 4℃ for 1,3 and 12 months, and stromal structure was destroyed 12 months later; AM structure preserved in ethanol was kept well 1 and 3 months later; Most of the epithelial cells and several stromal structure were destroyed in vacuum dry storage AM 1 and 3 months later. 2 Transmission electron microscopic study: The epithelium in AM preserved in DMEM/glycerol at -80℃ for 1,3 and 12 months was observed as intact structure, nucleus dissolved partly and mitochondria showed tumefaction, while basement membrane still normal; The epithelial cells were destroyed and basement membrane was loose in AM preserved in glycerol at 4℃ for 1,3 and 12 months; The epithelial cell membrane was observed destroyed, cytoplasm and nucleus dissolved, basement membrane thinner in AM preserved in ethanol for 1 and 3 months; The epithelial cell membrane was kept intact in vacuum dry storage AM for 1 and 3 months, but nucleus dissolved, chromation homogenization, and basement membranes thinner. 3 HGF and bFGF were distributed in AM epithelium mostly. The express of HGF in the epithelial cells of AM preserved in four groups at 1 and 3 months decreased significantly compared with fresh AM (p<0.05). The express of bFGF in vacuum dry storage group at 1 and 3 months and the other three groups at 3 months also decreased significantly(p<0.05).The contents of HGF in four preserved AMs (except in DMEM/glycerol at 1 month) and bFGF in vacuum dry storage and glycerol AM at 3 months,ethanol AM at 1 and 3 months decreased significantly compared with fresh AM( p<0.05). 4 ColⅣ and LN were distributed in AM basement membrane mostly. The express of ColⅣ in the basement membrane of AM preserved in DMEM/glycerol at 1 and 3 months, glycerol and ethanol at 1 month were as the same as that in the fresh control group (p>0.05). The express of LN in DMEM/glycerol and glycerol group at 1,3 and 12 months, ethanol and dry storage group at 1 months were as the same as that in the fresh control group (p>0.05). Conclusions: 1 Four AM preservation methods all have influences on AM structure and vitality. 2 AM preserved in DMEM/glycerol can keep structures well, better than in glycerol , ethanol and dry storage are the worst. 3 The growth factors decreased significantly in four groups, and the vality of AM epithelial cells decreased gradually with time. But the basement membrane components of DMEM/glycerol and glycerol group remained nearly the same as that in the fresh AM.