Biocompatibility Of Collagen Membranes Based On Genipin-cross-linked Chitosan Bends With Rabbit Limbal Stem Cells

Objective: This study evaluated the adhesion and proliferation of rabbit limbal stem cells on genipin-crosslinked chitosan-collagen scaffolds and the cytotoxicity of scaffold materials to cells,and observed the adhesion and growth of rabbit limbal stem cells on scaffold materials.,and changes in cell morphology.It was initially confirmed that chloronidyl cross-linked Ch-Col and rabbit limbal stem cells have good biocompatibility.Methods: In this study,collagen and chitosan were mixed in a ratio of 3:1,genipin was cross-linked,and freeze-dried to make scaffold materials for the experimental group;the other group was directly treated with genipin cross-linked collagen and chitosan by 3: 1 mix,freeze-dried to make a control stent material.The rabbit limbal stem cells were isolated and cultured.The expression of p63 in the passaged LSCs was detected by immunofluorescence assay.The cells were inoculated on chlorpheniramine cross-linked Ch-Col and Ch-Col scaffolds and blank well plates and divided into experimental groups.,Control group,negative control group.Observed with an optical microscope.MTT assay was used to detect the adhesion and proliferation of cells on experimental materials.Results: The scaffold material was generally viewed with no significant difference between the two groups of stents.Under the electron microscope,the Ch-Col scaffold material crosslinked by genipin has many voids,and the pores are connected to each other.However,the Ch-Col scaffold material not crosslinked by genipin is vague and has few structural features.After inoculation,the cells were inspected under a microscope.The limbal stem cells in the experimental group and the control group were denser and more crowded.The distance between limbal stem cells in the negative control group was looser,and the number of cells gradually increased with the increase of the observation days.No growth was observed.Obvious necrosis.The MTT assay of proliferation of limbal stem cells on scaffolds and cytotoxicity experiments showed that the absorbance values of the early experimental group and the control group were lower than the negative control group(P<0.05),and the differences between the groups were significant;and with observation days The increase of the absorbance value of the experimental group and the control group gradually increased,and the experimental group and the control group were significantly better than the negative control group(P <0.05),and the difference was greater with time.According to the increase of the observation days,the absorbance values of the experimental group and the control group were more and more superior to the negative control group(P<0.05).There was no significant difference in the absorbance value between the experimental group and the control group(P>0.05).The cytotoxicity rating of all three scaffolds was 0.Conclusion: It shows that the cross-linked Ch-Col tissue engineering scaffold of genipin has good affinity with rabbit limbal stem cells,and the cells can adhere,migrate and proliferate well on the scaffold;the cross-linked scaffold material has an excellent three-dimensional space structure and can Provides a broader space for cell growth and proliferation.The cross-linking of genipin compensates for the shortcomings of the Ch-Col complex and does not produce significant cytotoxicity.