Chitosan-based Hydrogel With Limbal Stem Cells For Corneal Alkali Burn Wounds Repair

Objective Evaluate the physicochemical properties and biological properties of in situ alginate-chitosan hydrogel(ACH)crosslinked by carboxymethyl chitosan(CMCTS)and sodium alginate(SA).Study the application of in situ chitosan-based hydrogel with limbal stem cells(LSCs)for corneal alkali burn wounds repair.Methods Sodium alginate was oxidized with sodium periodate according to a certain degree of oxidation to obtain the modified alginate dialdehyde,and then its free aldehyde groups were uniformly mixed with the free amino groups of CMCTS at room temperature for preparation of in situ alginate-chitosan hydrogel.After freeze-drying,the surface micro-structure was observed by scanning electron microscopy,and figure out the swelling rate.Transmission of in situ alginate-chitosan hydrogel was measured by Multiskan Spectrum Spectrophotometer in the visible wavelength range from 400 nm to 800 nm.MTT assay analyze the cytotoxicity of the ACH by observing the proliferation activity of L929 mice fibroblasts cells at different time periods and different concentrations of the extract liquid of ACH.The ACH was injected intramuscularly in mice and in vivo degradability and histocompatibility were evaluated by hematoxylin-eosin(HE)staining of tissue section.The degradation toxicity of the ACH in vivo was evaluated by weighting of mouse liver and kidney.LSCs were identified by Western blot and immunofluorescence analysis.And the compatibility was evaluated by 5,6-carboxyfluorescein diacetate succinimidyl ester(CFSE)staining and MTT assay.The LSCs were loaded into the hydrogels as a transplant carrier to transform in the surface of the corneal alkali burns wounds of New Zealand white rabbits.The effect of the wounds repair was evaluated by the general observation,slit lamp,HE staining of tissue section and immunofluorescence staining of frozen section,respectively.Results The luminousness of the ACH was almost more than 70% under the tested visible wavelengths.The morphology of the ACH showed an irregular porous structure,which was suit for the cell growth.The result of MTT assay showed that relative proliferation rate(RGR)of L929 mouse fibroblast were reached more than 80%,which proved that the ACH had a low cytotoxicity,and it conformed to the requirements of the international biomaterial cytotoxicity.The ACH showed a steady degradation rate and relatively low toxicity in liver and kidney.After four weeks,the ACH was completely degraded in skeletal muscles.By Western blot and immunofluorescence analysis,it showed that about p63(LSCs corneal limbus stem cells marker protein)positive rate was as high as 90%,and K3+12(corneal epithelium cells marker protein)positive rate below 20%.In short,the cells cultured in vitro were certainly identified as LSCs.The result of CFSE staining showed that the ACH possessed good cytocompatibility to LSCs,which was in line with the result of the MTT assay.As the hydrogel-loaded with LSCs were injected into rabbit ocular surface of cornea alkali burn,the burn wound was repaired within 28 days,the cornea was almost transparent and no secretion,the repairing effects of the experimental group were significantly improved compared with the model group.Conclusion The ACH hydrogel-prepared by CMCTS and SA showed good physical and biological properties.The repairing effects of cornea alkali burn were significantly improved.Chitosan-based hydrogel with limbal stem cells could be a promising prospect for corneal alkali burn wounds repair in the field of cornea tissue engineering and provide the valid data and solid foundation for treatment of corneal alkali burn.