| Objectives:The amniotic membrane was air-dried in a constant temperature drying oven at 37℃,and the new amniotic membrane powder was prepared by grinding after pre-cooling with liquid nitrogen.The new amniotic membrane powder was combined with fibrin glue to make amniotic membrane-fibrin sealant(AM-FS)cement,using in vitro cell experiments with different concentration of the new amniotic membrane powder AM-FS cement influence on rabbit corneal epithelial cell proliferation,combined with animal studies in vivo AM-FS cement in the healing of severe ocular surface alkali burn in rabbits.Methods:Preparation of the new amniotic membrane powder:put the washed fresh amniotic membrane in a constant temperature drying oven at 37℃ to air dry,The liquid nitrogen was pre-cooled and then ground into a new type of amniotic membrane powder with a diameter of<260 feet,Pasteurization by γ rays.The expressions of bFGF,TGF β,NGF R,HGF and EGF in the new amniotic powder and fresh amniotic membrane were measured and compared using growth factor microchip.According to the preservation conditions,the new amniotic powder was divided into three groups(room temperature preservation group,4℃ preservation group and-20℃ preservation group).The expression of growth factors was detected and compared at intervals of 10d,20d and 30d to explore the best preservation method of the new amniotic powder.In vitro cell experiments:Preparation containing different concentration of the new amniotic membrane powder AM-FS cement(65 mg/ml,32 mg/ml,16 mg/ml,8 mg/ml,4 mg/ml,2 mg/ml,1 mg/ml,0.5 mg/ml,0.25 mg/ml,0.125mg/ml,0 mg/ml),to develop to the next generation of rabbit corneal epithelial cells were inoculated in covered by the above cement at the bottom of the cell culture plate,density of cells under a microscope observation of corneal epithelium,combined with CCK-8 testing AM-FS cement containing different concentrations of the new amniotic membrane powder effect on cell proliferation,The appropriate addition concentration of the new amniotic membrane powder in AM-FS was screened.Animal experiments:Immerse in 1 mol/ml sodium hydroxide solution of circular filter paper pieces(15mm diameter)in rabbit corneal surface 60 s,and then quickly rinse immediately with plenty of saline water damaged cornea and the surrounding tissue,production,severe ocular surface alkali burn in rabbit,according to the different treatment model animals were randomly divided into four groups(AM-FS cement group,the fresh amniotic membrane transplantation group,the FS group,experimental control group)for each group of eight,after 7d,14 d and 21d and 28d line slit lamp microscope examination,fluorescein sodium respectively the cornea staining observation to repair,The cornea was collected and fixed on the 28th day for HE staining,and the expression of MCP-1 and VEGF in the cornea was detected by immunohistochemistry.Results:Test and compare the new amniotic membrane powder and fresh amniotic membrane transplantation in bFGF,TGF β,NGF R,HGF,the expression of EGF,the results showed:the expression of NGF plays R in the new amniotic membrane powder amount is lower than fresh amnion,bFGF,HGF,expression of EGF in new amniotic membrane powder mass were higher than that of fresh amniotic membrane,differences were statistically significant(adj)P.V al<0.05 and logFC>0.263),TGF beta in the expression of the new amniotic membrane powder quantity there was no statistically significant difference compared with fresh amniotic membrane transplantation group(logFC<0.263).The new amniotic powder was stored at room temperature,4℃ and-20℃,and the changes in the expression levels of growth factors in each group were detected at different time points.The results showed that At 10d and 20d,the expressions of HGF,bFGF and EGF in each group were not lower than those in the fresh amniotic membrane group,and the expressions of HGF,NGF R,bFGF and EGF in the room temperature group were not lower than those in the 4℃ group and the-20℃ group.At 30 days,the expression of other factors except HGF and bFGF in each group decreased compared with that in the fresh amniotic membrane group,and the expression of HGF,bFGF and EGF in the room temperature group was not lower than that in the 4℃ group and the-20℃ group.It was speculated that the new amniotic membrane powder had stable properties and could be stored at room temperature.In vitro cell experiment results:no cell growth was observed in the new amniotic membrane powder with concentrations of 65mg/ml,32mg/ml,16mg/ml,8mg/ml,4mg/ml and 2mg/ml under the microscope after 72 hours of culture.The growth of corneal epithelial cells was observed from the 1mg/ml concentration group,and the most significant growth was observed from the 1mg/ml to 0.5mg/ml concentration group.CCK-8 detection results:absorbance(A)value increased with the decrease of the new amniotic powder concentration and reached the highest at 0.25mg/ml,but the difference between the results and 0.5mg/ml group was not statistically significant(P>0.05).The amniotic membrane powder concentration of 0.25mg/mL was screened for animal experiments.Animal experiment results:AM-FS cement group in the first 2 weeks to reduce corneal turbidity effect is not obvious(P>0.05)compared with control group,the lower the effect of corneal turbidity in the two weeks compared with control group(P<0.05),28 d AM-FS cement group corneal turbidity and there was no statistically significant difference of fresh amniotic membrane transplantation group(P<0.05),the FS group at various time points to reduce after corneal alkali burns of turbidity effect is not significant compared with control group(P>0.05);Each rabbit after alkali burn of cornea neovascularization area increases with time,but AM-FS cement group,the fresh amniotic membrane transplantation group,corneal neovascularization area at each point the FS group were smaller than that of control group(P<0.05),although the area of fresh amniotic membrane transplantation group of corneal neovascularization in 7 d,21 d,28 d were less than AM-FS cement group,the differences were statistically significant(P<0.05),but the new blood vessels area of each point in time between the two groups of difference are smaller(0.34,0.38,0.88).HE staining results:the corneal structure of the amniotic membrane transplantation group and the amniotic membrane transplantation group was intact,the epithelial arrangement tended to be normal,and the corneal healing was better than that of the FS group and the antibiotic control group.Immunohistochemical results:the positive expression of VEGF in the fresh amniotic membrane transplantation group was weaker than the other three groups,and the positive expression of mcp-1 in the AM-FS group and the fresh amniotic membrane transplantation group was similar.Conclusions:37℃ constant temperature air drying of fresh amniotic membrane and the liquid nitrogen precooling after grinding in the preparation of a new type of amniotic membrane powder active cytokines content high,stable properties can be kept at room temperature,its preparation combined with fibrin glue AM-FS cement in rabbit ocular surface alkali burn therapy can promote the cornea epithelial repair,inhibit inflammation and new angiogenesis,and easy to use,provide new way for the research and development of new amniotic membrane products. |
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