3D Printed Hydrogel Corneal Implants While Mediating Cell Behavior For Corneal Regeneration

Corneal blindness is one of the major causes of blindness worldwide,and the severe shortage of corneal transplant donors makes the development of corneal repair materials the key to restoring vision for corneal blindness patients.Traditional corneal repair materials had problems of poor tissue adaptability,difficult epithelialization and corneal fibrosis,which were due to the fact that they focused on the biomimetic of corneal components,and ignored the curvature,an important factor affecting the corneal repair process,thus limiting their clinical application.The curvature structure of the cornea plays a decisive role in maintaining the normal physiological function of the cornea,and 3D printing technology has advantages in preparing high-precision complex structure tissues,and can be customized according to the patient’s defect situation,which is a good way to prepare curvature corneas.However,there are still some technical difficulties in 3D printing corneas that have not been overcome,such as “step effect”,which greatly limits the development of 3D printing technology in corneal materials.Therefore,how to overcome these problems and prepare a corneal repair material with smooth surface and excellent performance is a key for obtaining ideal corneal repair effect.Therefore,this paper intended to fabricate a convex corneal regeneration material through extruding-temperature printing technique with suppressing step effect,then we clarified the regulation rules of curvature structure on corneal cells from the perspectives of material mechanics and cell mechanics,and explored the intrinsic regulation mechanism of3 D printed curvature cornea on corneal cells and its impact on corneal repair by combining in vitro gene level,protein level and other functional verification with in vivo rabbit corneal repair process,which provided new ideas for guiding the design and preparation of corneal regeneration materials.Based on this,this paper carried out the following research:Firstly,we used type I collagen and Gel MA to construct biomimetic curved structures of the cornea based on 3D printing,the proposed “temperature controlling” strategy can develop high-precision and good-performance curvature cornea materials,then we studied the influence of inks compositions and printing process on 3D printed cornea.The addition of collagen can improve the printability of ink and the mechanical properties,enzymatic resistance and cell compatibility of curvature cornea,which was beneficial for preparing printed curvature cornea.When the concentration of Gel MA and collagen were 10% and 0.6%,respectively,and the nozzle printing temperature was 27.5 ℃,the obtained curvature cornea had the smoothest surface and the best comprehensive performance,meeting the needs of corneal repair.Secondly,we systematic studied of the growth behavior and pattern of RCECs on 3D printed convex corneas.In order to verify that convex cornea also had anisotropy of natural cornea,according to ophthalmology division,curvature cornea was divided into four regions according to different slopes,and flat cornea was used as a control to illustrate the advantages of curvature.Unlike traditional flat corneas,cells growing on curvature corneas showed obvious polarization and orientation alignment behaviors,and their growth trajectories tended to grow along the direction of curvature meridians.Through observation of cell skeleton and orientation changes,it was found that as slope increased,cells gradually became slenderer and orientation degree gradually increased.RCECs adhering tightly to the cornea was the key to corneal regeneration.Through screening and analysis of signal pathways,key integrin-related genes and pathways affecting adhesion were found out.Through adhesion force measurement,immunofluorescence staining,molecular biology and FEM simulation,it was found that curvature and slope can improve cell adhesion ability.The regulation rules and internal mechanism of RCECs by convex cornea were clarified.Thirdly,we designed animal experiments to verify whether convex corneas can complete corneal repair.Through rabbit experiment,it can be seen that curvature cornea had excellent biocompatibility.At 180 days in vivo,material basically remained intact.In lamellar keratoplasty,compared with flat cornea,convex cornea could fit better to the corneal tissue with the same suture pattern,it can complete epithelialization process within 3 days,and RCECs growing on curvature cornea adhered firmly while epithelial function behaved well.From 180 days continuous observation,the cornea remained transparent,and corneal stroma and nerve regeneration were observed,which proved the regeneration was excellent.Fourthly,we constructed biological convex cornea carrying RCSCs by 3D printing to cope with corneal fibrosis during regeneration.Inspired by native cornea structures,we tried to maintain the phenotype and functions of RCSCs through curvature design.The primary problem was to solve solubility of high molecular weight type I collagen under neutral conditions.We added EPTAC that formed hydrogen bond interaction with collagen,weakening interaction between collagen molecular chains and improving its solubility.Then we used the EPTAC-Col bioinks to load RCSCs and printed a biological convex cornea,the printed biological cornea had good physical and chemical properties with excellent cell survival rate(95%).Through q-PCR and protein analysis,it can be shown that RCSCs in biomimetic convex cornea had characteristic expression of stroma cells,while cells in flat biological cornea had higher α-SMA expression,proving that the printed biological convex cornea can inhibit conversion of RCSCs to fibroblasts,and it had potential as a corneal regeneration material that can inhibit corneal fibroblast.