Preparation And Preclinical Study Of Fibrin-Binding Amniotic Membrane

Objective To research the production technique, preservation condition, histomorphology, biomechanical characters and cellular biocompatibility in vitro of a new medical biomaterial—fibrin-binding amniotic membrane(FBAM). To approach its feasibility and superiority using in ocular surface graft and tissue repair. The study includes four parts:ExperimentⅠ: Study on the production technique and morphological observation of fibrin-binding amniotic membrane.ExperimentⅡ: Study on biomechanic assessment of fibrin-binding amniotic membrane.ExperimentⅢ: Study on cellular biocompatibility of fibrin-binding amniotic membrane in vitro.Experiment IV: Primary studies on reservation condition of fibrin-binding amniotic membrane.MethodsExperimentⅠ:Through proper procedure, we bind fibrin to amniotic membrane to prepare the FBAM. Choose fresh FBAM, do macroscopic observation and examination with a operating microscope, do histological section, HE stain and light microscope observation, take pictures when it is possible.ExperimentⅡ: Choose twelve films of FBAM at random,test their tensile strength using electronic universal testing machine, the same quantity of the bilayer amniotic membrane(BAM) and the monolayer amniotic membrane(MAM) as control. The results include tensile strength, elongation at break, elastic modulus, the stress-strain curve and so on.ExperimentⅢ: Get fresh rabbit eyeball under sterile conditions, peel corneal epithelial tissues, do corneal epithelial cell primary culture, the monolayer fibrin binding amniotic membrane as the carrier. Observe cell morphous in different stages under inverted microscope. Observe cell growth overlay area in different time under grid eyepiece, cell culture on the denuded human amniotic membrane as control.Experiment IV: According to the amniotic membrane routine reserving methods, we use the anhydro-glycerol at 4℃to reserve FBAM. Choose the FBAM reserved for 20 days and 40 days to do macroscopic observation, histological section and HE stain, the fresh FBAM as control. Choose the monolayer fibrin binding amniotic membrane reserved for 6 months to observe its surface conditions using the SEM, fresh monolayer fibrin binding amniotic membrane as control. Assay protein concentration in preservation solution of the fibrin reserved for one month. ResultsExperimentⅠ: The FBAM is obviously thicker than the BAM and the MAM,about 0.1-0.3㎜ thick as a whole.It is hardly separable under an operating microscope.The FBAM tissue section shows that fibrin and the amniotic membrane are structurally-complete, the fibrin binds to the amniotic membrane tightly. Basal surface of amniotic membrane binds to basal surface of another amniotic membrane (the bilayer fibrin binding amniotic membrane ). Basal surface of amniotic membrane binds to fibrin (the monolayer fibrin binding amniotic membrane). Simple columnar amniotic membrane epithelium cells can be seen on the liber amniotic membrane epithelium. There is no gap between amniotic membrane and fibrin. There is no obvious amniotic membrane shrinkage. The fibrin in the middle is uniform. There is no obvious delamination and degeneration of fiber.ExperimentⅡ: FBAM: tensile strength: (0.727±0.142)Mpa, elongation at break: 24.130±4.523%, elastic modulus: (1.283±0.482)Mpa. The shape of the stress-strain curve of FBAM is regular, the curve can be repeated well. The tensile strength and the elongation at break of FBAM are more than those of the MAM and the BAM.The elastic modulus of the FBAM is smaller than that of the MAM and the BAM(P<0.01).ExperimentⅢ: Corneal limbal epithelial cells adhere to the FBAM, grow and proliferate on it, form simple epithelial cells in vitro culture on the 7th day. Disparity of cell growth overlay area possess the significance(P<0.05)from the 2nd to the 7th day between the experimental group and the control group (the denuded human amniotic membrane as control).Experiment IV: The FBAMs reserved in the anhydro-glycerol at 4℃for 20 days and 40days are brittler than fresh FBAMs. The histological section reveals that the AM shrinks, but the amniotic membrane still binds to the fibrin tightly. The fiber reticular formation is fairly fuzzy on the surface of the FBAM reserved for 6 months while the fiber reticular formation on the surface of fresh FBAM is distinct. The fibrin reserved in the anhydro-glycerol at 4℃for one month is degraded partially.Conclusion1. The amniotic membrane and the fibrin can be combined into a new material—FBAM. The amniotic membrane binds to the fibrin tightly, its morphous is regular under a microscope.2. The FBAM composites has more ideal biomechanical properties than the MAM and the BAM.3. Corneal limbal epithelial cells adhere to the FBAM, grow and proliferate on it. The FBAM can be a carrier raising epithelial cells, this aspect still needs further study.4. The anhydro-glycerol at 4℃can be a reserving method of FBAM, but under which temperature or condition the reserving result can be the best still needs further study. 5. The FBAM can be a material to reconstruct ocular surface, it has superiority over common AM.