Feasibility Of Acellular Porcine Small Intestine Submucosa As A Scaffold For Corneal Epithelium Tissue Engineering

Background:Homeostasis of corneal epithelium plays an important role in keeping corneal transparency and visual function,limbal stem cells(LSCs)are responsible for renewal and restoration of the corneal epithelium.Ocular surface diseases such as Stevens-Johnson syndrome,chemical,thermal and radiation injuries,extensive microbial infection,and inherited disorders such as aniridia,can cause severe or total limbal stem cell deficiency(LSCD),which will result in corneal epithelial defects,conjunctivalization,neovascularization,chronic inflammation,corneal opacity,and eventually visual loss.In the context of corneal diseases and blindness resulting from LSCD,autologous limbal stem cell transplantation has been presented as effective therapeutic option for ocular surface regeneration.However,this approach suffers from two major disadvantages:limited availability of quality donor graft material,and occurrence of tissue rejection.As such ex-vivo construction and transplantation of tissue engineered corneal epithelium has become a growing topic of interest for ocular surface reconstruction on the treatment of LSCD,and the ideal scaffold material is the key elements.Objective:In this work,we decellularized porcine small intestine submucosa(SIS)by incubate in different concentrations of sodium dodecyl sulfate(SDS)and optimize a best concentration to produce an acellular SIS and investigate its histology,mechanical and biocompatibility properties for use in developing a suitable scaffold for cornea epithelium tissue engineering.Methods:Normal porcine SIS were decellularized with SDS(concentration of 0.1%,0.2%,0.3%,0.5%for 15min,30min,1h,2h)and obeserved the decellularization process at the same time.After decellularization,the scaffolds were examined through the HE,DAPI staining and the genomic DNA contect analysis(n=5)to optimize the best concentration to produce an acellular porcine SIS.Completely acellular SIS scaffolds were evaluated by uniaxial tensile testing.The resulting acellular matrices were then examined by scanning electron microscope(SEM)and transmission electron microscope(TEM);ELISA was performed to assessed the growth factors contents,The cytotoxicity of SIS extracts to rabbit corneal epithelial cells was determined by CCK-8 assay.Inflammatory reaction of SIS implanted subcutaneously in a rat was investigated.The biocompability was studied by rabbit interlamellar corneal transplantation and reseeding assay with cornea-derived cells.Immunofluorescent staining was used to detect the expression of CK3.Results:Acellular SIS was shown ivory,translucent and has certain transmittance.HE and DAPI staining with a light microscope revealed that when treatment with the concentration of 0.1%,0.2%SDS in the time of 30min the SIS were decellularized completely,the collagen fibres were appeared in an orderly fashion and preserved the overall tissue histoarchitecture.Genomic DNA contect analysis showed that in the time of 30min with the concentration of 0.1%,0.2%SDS no cell or DNA element were discovered.The uniaxial tensile testing indicated that decellularisation by 0.1%SDS for 30min did not significantly compromise the ultimate tensile strength of the tissue(P>0.05).TEM and SEM showed that collagen fibres were appeared in an orderly fashion and the collagen fibers porosity were increased;The content of growth factors within acellular SIS were well retained(P>0.05).CCK8 assay demonstrated that the acellular SIS extracts had no cytotoxicity to rabbit corneal epithelial cells.There was no sign that an immune reaction occurred with acellular SIS implanted subcutaneously in a rat.Moreover,in vivo implantation to rabbit interlamellar stromal pockets proved good biocompability.We also observed that clusters of rabbit corneal epithelial cells were growing well on the surface of the acellular SIS in vitro and the distinctive CK3 for corneal epithelial cells was detected.Conclusions:Our results suggest that the SIS developed by using 0.1%SDS for 30min can complete remove the heterogeneous cells and antigens composition of pigs,while preserving major natural acellular matrix and bioactive factor,the mechanical properties were preserved and biocompatible with cornea-derived cells,the acellular SIS might be a suitable biological scaffolds for cornea epithelium tissue engineering.