| Objective Corneal alkali burn can cause ulcer,perforation and even corneal blindness in severe cases due to corneal epithelial defect and massive cell necrosis.Previous studies have found that Limbal stem cells(LSCs)transferred by chitosan-based in situ hydrogel(ACH)can repair corneal alkali burn to some extent,but due to different pore sizes of the carrier,the cell entrapment rate is low and the repair effect is not ideal.In this study,a new type of cell carrier was prepared to improve the cell entrapment rate and repair effect.Methods Chitosan acetic acid solution,carboxymethyl chitosan(CMCTS)solution and sodium β-glycerophosphate solution with a certain concentration were prepared respectively,and the three solutions were mixed according to a certain proportion to prepare chitosan-based thermosensitive hydrogel(CTH).The 4D-CTH with uniform pore size was prepared by 4D bioprinting technology at low temperature of-20℃.The microstructure of the carrier surface was watched by scanning electron microscope,and its swelling ratio,viscoelasticity and chemical structure were detected.LSCs of SD rats were extracted and cultured in vitro,and the positive rate of P63 cells was identified by immunofluorescence experiment.Cell counting kit-8(CCK-8)assay was applied to examine the cytotoxicity of 4D-CTH,and scratch test was applied to examine the influence of 4D-CTH on the migration of LSCs,and Calcein-AM/PI cell staining test was used to detect whether 4D-CTH has influence on the proliferation and morphology of LSCs,thus verifying the cytocompatibility of 4D-CTH.Wounds of moderate and severe alkali burn of cornea were made in SD rats,and LSCs were transferred to injured cornea by 4D-CTH for repair.Observe corneal opacification and neovascularization through slit lamp,and evaluate the healing of injury site through sodium fluorescein staining.Paraffin sections and frozen sections of corneal tissue were made for HE and immunofluorescence staining respectively,which were applied to evaluate the therapeutic effect of 4D-CTH transferring LSCs on corneal alkali burn.Results The prepared 4D-CTH carrier had uniform pore size and was gel-like at 37℃.Scanning electron microscope showed that the carrier had regular pore size,swelling rate was about 1100%,and viscoelasticity test results showed that the storage modulus of the prepared hydrogel was always greater than the loss modulus,showing typical hydrogel-like characteristics.In vitro cultured rat LSCs grew adherent and fused with each other,mainly polygonal,with a large ratio of nucleus to cytoplasm,and arranged in a "paving stone" shape.Immunofluorescence experiments showed that the positive rate of P63 was over90%(P>0.05),while the positive rate of K3+K12 was less than 20%(P<0.001),which confirmed that the extracted LSCs had high purity.CCK-8 assay indicated that 4D-CTH cell carriers could not markedly hinder LSCs proliferation(P>0.05).Scratch test showed that the wound healing rates of 4D-CTH group at 24 h and 48 h were 53.49% and 93.28%respectively,which were significantly higher than those of the control group at the same period(39.39% and 72.08%,P<0.01),which indicated that 4D-CTH cell carrier could promote the migration and growth of LSCs.Calcein-AM/PI staining assay indicated that4D-CTH cell carriers would not damage the activity of LSCs,nor would they change the morphology of LSCs(P>0.05).4D-CTH treatment group could significantly reduce corneal neovascularization rate,reduce corneal turbidity and accelerate corneal epithelial reconstruction when compared with the other two groups(P<0.05).HE staining results of paraffin sections of corneal tissue showed that after 15 days of 4D-CTH treatment,the corneal structure was complete,the boundary between epithelial layer and stroma layer was clear,and the epithelial layer was continuous and smooth.Immunofluorescence staining of frozen sections of corneal tissue showed that after 15 days,the epithelium of4D-CTH treatment group had continuous expression of K3+K12 protein,and the cornea basically returned to normal.Conclusion The 4D-CTH cell vector had good cell compatibility and uniform pore size,which significantly improved the cell transfer rate.Transferring LSCs with 4D-CTH vector could significantly improve the local cell growth microenvironment,promote corneal repair,accelerate the reconstruction of injured epithelial tissue and promote the repair of injured tissue. |
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