| ObjectiveTo investigate the characteristics and cytocomptibility ofhuman-amniotic membrane(HAM) fixed with genipin in vitro and toprovide a proper biomaterials for ocular surface reconstruction,futhermore,we also studied the effect of genipin-crosslinked amnioticmembrane for corneal alkali burn. Our aim is to provide a properbiomaterials for ocular surface reconstructionThe study includes three parts:PartⅠ: the preparation of genipin-crosslinked HAM.PartⅡ: the characterization in vitro of the genipin-crosslinked HAM.Prat Ⅲ: the effect of genipin-crosslinked HAM for corneal alkaliburn.MethodsPart Ⅰ: the deepithelialized HAM were treated by genipin for48h,thenexamined with HE and SEM. Part Ⅱ:1、 Biomechanical MeasurementsThe tensile strengths of non-crosslinked and crosslinked HAM weredetermined using a microcomputer-controlled universal testing machine.Toprepare specimens for tensile strength testing,each HAM was cut into3.5cm×1cm strips.8tissue strips in each group were tested at a10mm/minextension rate.After measurement,the maximum tensile strength and theultimate elastic modulus was calculated directly by the testing machine.2、HAM pachymetryManual pachymetry was performed in0.5%genipin-corsslinked HAMapplied topically for30min(n=8) and in non-corsslinked HAM(n=8)3、Stability against enzymatic degradationThe stability of the crosslinked HAM against enzymatic degradationwas carried out by bacterial collagenase A digestion at a final concentrationof0.25%dissolved in DMEM at25℃.After weighting the initialweight,specimens were completely immersed in the0.25%collagenaseAsolution(PH=7.4) and incubated at37℃.Every2days weighted theresidual specimens and replaced the solution with fresh media.All residualspecimens were washed with distilled water,air dried and weighed.Thedegradation rate (△W%) was calculated according to the formula:△W%=(Wo-Wt)/Wo×100%4、The cytotoxicity of the genipin-crosslinked HAMMouse embryonic fibroblasts(BALB/C3T3)were cultured in a basal medium(a2:1mixture of DMEM/F12and PBS) with10%(V/V)fetalbovine serum,100U/ml penicillin,100Ug/ml streptomycin and2mML-glutamine. The cytotoxicity of the genipin-crosslinked HAM wasassessed in vitro by its ability to stimulate the proliferation of BALB/3T3cells. The genipin-crosslinked HAM samples were put in5ml microifugetubes with a certain amount of basal medium at the ratio of1mlmedia/1cm2G-HAM.after3days,media were collected from thetubes.BALB/c3T3cells were then seeded in96-well plate at a density of5000cells/well. After24h, replace the medium with the collected medium.After a time span of2d、4d、7d, cell numbers were determined by a MTTassay.PartⅢ:60Rabbits without eye diseases were randomly divided into3groups,20each group,groupA:genipin-crosslinked HAM,groupB:HAM,group C:control group.A round filter paper soaked in NaOHsolution was put on the center of the cornea to make a burn. Then AMTwas done. check the eyes of the cornea edema and conjuctive hyperaemiaeveryday,observing the dissolve time of AM,and scoring theneovascularization and transparency of the cornea.The day7、14、28aftersurgery,corneas of each group were examed for HE.ResultsPart Ⅰ: The genipin-crosslinked HAM is a light blue opalescencemembrane，with much stronger tension strength、elastic modulus、 elongation at break than AM.PartⅡ:1、HAM pachymetryThe mean pachymetry was (0.040±0.009)mm in HAMs treated withgenipin0.5%and(0.038±0.010)mm in untreated HAMs. There was nostatistically significant difference between the two groups(P>0.05)2、G-HAM:tension strength:（1.489±0.405）Mpa,elongation atbreak:（25.337±5.137）%,elastic modulus:（0.0047±0.0021）Gpa.Thetensile strength、 the elongation at break and the elastic modulus ofG-HAM are more than those of the AM(P<0.05).3、In vitro enzymatic degradation of crosslinked HAMAfter in vitro enzymatic degradation using collangenase,the relativeweight loss of all crosslinked samples was significantly lower than that ofnon-crosslinked samples.The untreated HAM was completely hydrolyzedafter24hours’ digestion. The treated HAM was hydrolyzed much moreslowly than untreated HAM. And only（4.48±0.86）%was hydrolyzed after24h digestion. the digestion rates of7,14,28days were （15.25±4.08）%（34.93±6.07）%、（50.49±4.77）%respectively.4、Effect of genipin crosslinked HAM on proliferation of mouse embryoniccellsAfter7days of culture, no significant difference there was in cellgrowth between the genipin treated HAM and the negative group,theresults indicated that there was no toxicity of genipin Part Ⅲ:1、The average time of genipin crosslinked HAM dissolved is(20.00±10.418)d, AM is (9.20±10.618)d.there is significantdifference between the dissolved time of G-HAM and AM(P<0.01).2、day14、28、42,the score of CNV showed signnificent differencebetween G-HAM and control group(P<0.01). For AM group,though,thescore of CNV is less than the control group,there is no significantdifference(P>0.05).3、day14,the score of corneal transparency in both G-HAM group andAM group showed no significant difference compared with control group(P>0.05).Day28and42the score showed significant difference betweenG-HAM、AM and comparative group(P<0.01).But,there is no significantdifference between G-HAM and AM group(P>0,05).4、For the whole1.5month,there was1eye in AM group,3eyes incontrol group had perforatio cornea.Conclusion1. Young modulus and stiffness in treated HAM increasedsignificantly than the fresh HAM. Genipin-crosslinked HAM increasedresistance to collagenase significantiy in comparison with the fresh HAM.The RGR of genipin-fixed HAM was high which presumed a lowcytotoxicity of this material. the Genipin-fixed HAM should be a promisingmaterial for ocular surface reconstruction.2. Compared with ordinary membrane,genipin-crosslinked HAM showed better effect for corneal alkali burn.The genipin-crosslinked HAMcan keep on the surface of corneal for a longer time,it can suppress thePMNS and the formation of neovascularization,accelerate theepithelializtion, and improve corneal transparency then reconstruct thesurface of the cornea.3、Genipin-crosslinked HAM maybe a promising material forocular reconstruction... |
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