Abstract: Objective To investigate the effect of stiffness-controlled thermoresponsive PEG-NIPAM hydrogel system with adjustable stiffness on EnMT of rabbit corneal endothelium cultured in vitro. Methods Six 4-5 weeks New Zealand rabbit were selected.After anesthesia,the rabbits were sacrificed and bilateral bulbus oculi were taken.The corneas were cut and descemet's membrane with corneal endothelial cells were peeled after soaking with tobramycin solution.The P0 generation primary corneal endothelium cells (CECs) were extracted by two-steps digestion method and cultured in six-well cell culture plates.When the cell density>90%,passaging CECs were divided and cultured in 10 groups of mediums containing PEG with concentrations of 1%,10% and 20% stiffness-controlled thermoresponsive hydrogels,TCP coated by different pre-coating (chondroitin sulfate + laminin,FNC,Gelatin) and TCP alone.After the three generations cultivation,the expression of EnMT marker α-SMA was observed by immunofluorescence. Results The hardness of the hydrogel examined by the AFM method was (346.4±26.5 Pa) at the concentration of 1% PEG,(8 782.0±444.4 Pa) at the concentration of 10%,and (11 518.0±791.1 Pa) at the concentration of 20%,with significent difference between each two groups (P<0.001).Morphological result and the expression of α-SMA indicated the occurrence of EnMT in cultured rabbit CECs in vitro.The α-SMA positive CECs cells increased gradually from P1 to P3,and the cytoskeleton changed into fibrotic morphology.The α-SMA fluorescence intensity and positive cells number in the 20% PEG-NIPAM group were significantly lower compared with the TCP control group;and they were also significantly lower in the laminin and chondroitin sulfate mixed coating compared with FNC coating. Conclusion The process of EnMT can be alleviated when rabbit CECs cultured on a PEG-NIPAM hydrogel bionic materials close to the natural rabbit descemet's membrane.