Development And Investigation Of The Prepareration Procedure The Amniotic Membrane

Objective To find a effective,efficient,safe and require no sepcial instrument procedures to prepare the amniotic membrane by comparing the common used prepareration procedures of the amniotic membrane.to investigate a new material to simplify the preparation and clinical steps.The study includes three parts:ExperimentⅠ:study on the influnce of different sterilisation on the properties of amniotic membraneExperimentⅡ: study on influnce of different perservation on the histological changes of amniotic membrane.ExperimentⅢ: Polypropylene Non-woven Substrates—a new substance used for attaching Amniotic Membrane,and compare with the traditional used material nitrocellulose paper.MethodExperimentⅠ: Four different procedures including Antibiotics-antimyotics,peracetic acid-ethanol-sterilisation(PES),γ-ray and 0.5%Iodophors were applicated for AM's sterilization.After the examination proved that they all met the clinic standard ,then Haematoxylin-eosin stain ( HE ) and light microscope observation,immunohistochemistry and RT-PCR were used to investigate the morphological changes of the amniontic epithelium and the protein and mRNA levevls of hepatocyte growth factor(HGF)and tissue inhibitor of metalloproteases-1(TIMP-1),and then compared influnce of different sterilisation procedures on the properties of amnitic membrane.ExperimentⅡ: Through proper procedure as normol, different reserving tempratures were used to reserve AM. Choose the AM reserved for 30 days , 90 days and 180 days to do macroscopic observation, histological section and HE stain, TEM examination. Then compared influnce of different reserving tempratures on the properties of amnitic membrane.ExperimentⅢ: A new substance-Polypropylene Non-woven Substrates-instead of the traditional used material nitrocellulose paper is selected to carry AM.After it is collceted and sterlized as normol process,AM is attached on the No Woven with the epithelium side up, dried under the room temperature (18~23℃)for 2~4h in the laminar flow cabinet and presvered with the No Woven above AM.To investigate the influnce of the No Woven to AM,the morphologic changes is tested by light microscopy and transmission electron miroscopy.Also the simplity of the new method provided for the preparation and clinical steps was tested.Result ExperimentⅠ: The resluts revealed that the amniotic epithelium was badly destoried by the irradiaton sterilisation.Procedures with PES and Antibiotics-antimyotics presented the tissue structure.The expression of HGF and TIMP-1 decreased by the sequence of PES group, Antibiotics-antimyotics group, 0.5%Iodophors group andγ-ray group (P<0.05).While the mRNA levels of HGF and TIMP-1 deceased in the sterilized group compared with the the unsterilized group,theγ-ray group expressed least (P<0.05)and the other three group showed no significant differences(P>0.05).ExperimentⅡ: The amniotic epithlium preserved under the 4℃were badly damaged ,they almost disappeared when it was reserved in the anhydro-glycerol at 4℃for 60days. Though the amniotic epithlium reserved in the anhydro-glycerol at -20℃and -80℃were lost with the preserved day passed by,the lost amount of the the amniotic epithlium were much more less than that of the 4℃group.In the -20℃group there were no significant differences about the amniotic epithlium between the 60days'and 180days'preservation.which as the same in the -80℃group.it can been found that -80℃group preserved the amniotic epithlium best,and -20℃group protceted them much better than the 4℃group. The sroma of the AM appeared normal and the ollagen fibers making up was well preserved except the AM reserved in the anhydro-glycerol at 4℃for 6 months. ExperimentⅢ: the hematoxylin and eosin (HE )staining and the TEM examinations showed that the majority of the amniotic epithlium in the group Non Woven showed little change comcering the morphology compared with the group nitrocellulose paper. the sroma of the AM also appeared normal and the ollagen fibers making up was well preserved. AM which attched on the No Woven could be easily seperated by the forcep just by diping into the 0.9% normal saline for a few seconds compared with the AM attched on the nitrocellulose paper. The use of this method not only resolves the problem companied with the use of nitrocellulose paper,but also simplify the preparationConclusion1. The PES was suggested as an alternative method for sterilisation because this method influnced the properties of the amniontic epithelium least.2. AM preserved under the -20℃was a alternative method for maintaining the properties of the amniontic epithelium.Considering this temperatura can be easily get by the normal freezer which can purchase in the market,it is concluded that this method could be widely adopted in the undeveloped countris.3. The use of this method not only resolves the problem compared with the use of nitrocellulose paper,but also simplify the preparation. Given examinations of the morphologic tests ,it is concluded that the method we chose is effective,efficient,safe and require no sepcial instrument.