Fabrication And Application Of Bioengineered Cornea By 3D Printing Of PCL Scaffold Combined With Rat Tail Collagen

Objectives: The main treatment of corneal injury is corneal transplantation.However,there is a severe shortage of donor corneas.This study aims to prepare a 3D scaffold as a replacement for corneal transplantation by tissue engineering technology,and to research its effect on inhibiting cornea stromal scarring after injury.Methods:(1)In this article,we fabricated a poly(ε-caprolactone)(PCL)microfibrous scaffold through the near field electrostatic spinning technology(NFES).The microstructure of the PCL microfibrous scaffold was observed by SEM;the light transmittance was determined using a spectrophotometer at wavelengths;the mechanical propert was measured by universal material testing machine.(2)The transmittance,water content and swelling ratio of rat tail collagen gels with different concentrations were measured by spectrophotometer to determine the optimal concentration,and the PCL/collagen scaffolds were prepared.(3)The biocompatibility of the composite scaffold was explored by CCK-8 test,the adhesion of cells on the scaffold were observed by immunofluorescence staining,the effects of PCL/collagen scaffold on cell proliferation and differentiation were detected by quantitative determination of labeled proteins.(4)A rat model of corneal stromal injury was established.In vivo experiments were conducted to explore the role of PCL/collagen scaffold in inducing the repair of corneal stromal injury.The expression and arrangement of extracellular matrix(ECM)influenced by PCL/collagen scaffold were observed by OCT、H&E staining and Sirius-red staining.The related proteins and ECM were detected in different periods of the corneal stroma repair by immunofluorescence and immunohistochemical staining and protein immunoblotting.RNA-sequencing on normal and injured corneas was carried out to find out the differential enriched pathways and gene expression,and real-time fluorescence quantitative polymerase chain reaction(q PCR)was used to detect the expression levels of related genes in different stages of corneal stromal repair.Results:(1)The PCL/collagen scaffold was designed to fabricate an optimal construct that simulates the stromal structure with properties that are most similar to the native cornea,which has excellent optical and mechanical properties.Its light transmittance(90.67±1.08%)was higher than that of natural cornea(87%),and its tensile strength(2.70±0.32 MPa)was higher than that of natural cornea(2.06±0.16 MPa);(2)The hydrophilicity of PCL/collagen scaffold prepared with 5 mg/ml rat tail collagen gel was similar to that of natural cornea.The rat tail collagen gel has good biocompatibility and no biotoxicity,and the differentiation of LSSCs was inhibited by the structure of PCL scaffold;(3)We established a corneal stromal injury model successfully,and in vivo experiments confirmed that the PCL/collagen scaffold(tissue engineering cornea)can inhibit the differentiation of limbal stromal stem cells and reduce fibrosis during the repair process of corneal stromal injury;(4)By RNA-sequencing,we observed that in injured group,ECM-related pathway was enriched and several ECM-related genes(MMP9、MMP13)were up-regulated,PCL/collagen composite scaffold could inhibit the up-regulation degree of corneal stromal injury.Conclusion: The PCL/collagen tissue-engineering cornea has good optical and mechanical properties,biocompatibility and inhibition of corneal stromal cell differentiation,which can effectively inhibit stromal scarring after corneal injury.