| Corneal disease as a major cause of blindness in the world affects more than 10 million people worldwide,and remains second only to cataracts.Keratoplasty is currently the only treatment that has been proven to be effective for corneal blindness.However,there is a significant lack of donor corneas availabile to meet the clinical demand,which causes many patients with corneal blindness can not get timely and effective treatment.There were about 300,000 patients that need corneal transplantation for the treatment of corneal diseases,but the annual patients treated with corneal transplantation were less than 4000 cases due to insufficient donor cornea.Under these circumstances,people focused on the development of artificial cornea replacement in recent years.Keratoprosthesis had been approved for clinical use,but the poor biocompatibility and postoperative complications limited the generalization of this kind of corneal substitute.Tissue-engineered cornea as a replacement of donated cornea is constructed by culturing seed cells on degradable scaffold,which shares similar structure and function with native cornea.Due to good biocompatibility,tissue-engineered cornea becomes the focus of public attention.Tissue-engineered corneal equivalents have experienced major progress in recent years.Acellular porcine corneas and recombinant human collagen as graft replacement have been used for lamellar keratoplasty in clinical or animal experiment.Penetrating keratoplasty remains widely used for the treatment of various corneal diseases,such as keratoconus and corneal perforation.Due to the influence of traditional ideas,there are few corneal donations in China.Moreover,with the increasing popularity of refractive surgery,the supply of donor cornea suitable for penetrating keratoplasty is expected to diminish even further.Construction of tissue-engineered full-thickness corneal substitute(TECS)holds a great promise for solving the shortage of donor graft suitable for penetrating keratoplasty.Furthermore,compared to other organs,cornea contains fewer cell types mainly including corneal epithelial cell,corneal endothelial cell,and keratocytes.Therefore,the construction of tissue-engineered cornea may be simpler and easier than that of other organs.Scaffolds and seed cells are two key elements in the construction of tissue-engineered cornea.Acellular porcine cornea matrix(APCM)possesses the critical properties of cornea,such as optical transparency,good biocompatibility and biomechanical properties,low immunogenicity,the toughness to withstand surgical procedures,and so on.Again,there is a wide source of porcine cornea in China.Therefore,APCMs were used as scaffold for the construction of tissue-engineered cornea in this study.It was reported that APCM remained stable and retained the thickness for a year after implantation of cornea,and the stromal cells started to invade into it on postoperative 3 weeks.Based on this,corneal stromal cells were not seeded in TECS during the construction.Moreover,porcine cornea is thicker than human cornea,and its thickness may further increase after decellularized process.The thickness mismatch between graft and host may affect corneal wound healing.Therefore,how to fabricate the tissue-engineered cornea with similar thickness to natural cornea still needs further study.Seed cells as the other key element of tissue engineering should have a wide source and strong proliferative ability.Whereas,primary cultured corneal epithelial and endothelial cells showed limited proliferative potential in vitro,and gene-transfected corneal cell lines were potentially tumorigenic,which limited their clinical application.HESCs possess the features of strong proliferation and the ability to differentiate into the cells from all three embryonic germ layers,and became a potential source of corneal cells in the past few years.In our previous studies,we had developed the protocols to differentiate hESCs into LEC-like and CEC-like cells.LEC-like cells and CEC-like cells showed similar morphology and function to primary cultured limbal epithelial cells and corneal endothelial cells,respectively,which might serve as the seed cells in corneal tissue engineering.Corneal epithelial cells and endothelial cells located on the different sides of cornea.Therefore,the three-dimensional culture protocol is needed for coculturing LEC-like cells and CEC-like cells on the scaffold.Hence,first,we developed a cutting tool that could be used to prepare APCM lamellae with similar thickness to native cornea.Next,we developed a three-dimensional culture protocol suitable for culturing cells on both sides of the scaffold,and investigated the possibility of constructing TECS by coculturing LEC-like cells and CEC-like cells on APCM in this three-dimensional culture system.We found that the prepared scaffold and the construct showed similar thickness and biomechanical properties with native cornea,and the three-dimensional culture method we developed was suitable for the construction of TECS.Similar to native cornea,the construct with stratified corneal epithelium and a corneal endothelium monolayer expressed corneal epithelial cell and corneal endothelial cell markers in its epithelium and endothelium,respectively.Furthermore,the construct possessed similar endothelial cell density and biomechanical properties with native cornea,and showed some function of native rabbit cornea in vivo.But the transparency of the construct is slightly lower than native cornea,which still needs further improvement in future.Part I Preparation of APCM scaffold[Purpose]This study aimed to investigate the feasibility of APCM lamellae prepared using an APCM cutting tool designed by ourselves as the scaffold of TECS.[Methods]1.Preparation of APCM.Fresh pig eyes were washed with phosphate buffer saline(PBS)containing 1%penicillin-streptomycin in the clean bench.The central corneas were excised from the eyes using a trephine(11 mm diameter),and then treated with 1.5 M sterile sodium chloride solution and 5 U/mL DNase/RNase to remove the cell composition of porcine corneas.2.Preparation of APCM lamellae.APCM was placed in our self-designed cutting tool,and then was pressurized to reduce moisture.The tool was adjusted to 400μm-scale,and APCM was cut through the gap between two columns with a thin blade(Fig.1 in Part Ⅰ).The prepared APCM lamellae was stored at-20℃ and used as the scaffold of TECS.3.Biological characteristics of APCM scaffolds.The scaffold was analyzed using HE and DAPI staining,immunofluorescence for type IV collagen and laminin in corneal epithelial basement membrane,a standard micrormeter,UV-VIS spectrophotometer,and Instron electromechanical universal tester.Its water content were also examined as previous reports.[Results]HE and DAPI staining showed that cell composition was completely removed from APCM lamellae,and the collagen was well preserved.Type IV collagen and laminin were detected and continuous arrangement on the front surface of APCM scaffold.The scaffold showed similar thickness and biomechanical properties but slightly higher water content and lower transmittance compared with native rabbit cornea.After being soaked in sterile glycerol to remove the moisture,the transmittance of the scaffold increased,even higher than that of native rabbit cornea.[Conclusion]The cellular material could be completely removed with high osmotic sodium chloride(NaCl)plus nucleases,and epithelial basement membrane was completely intact.The prepared APCM scaffold showed similar thickness and biomechanical properties to native rabbit cornea,and may be used as the scaffold of TECS.Part Ⅱ Differentiation of hESCs into LEC-like and CEC-like cells[Purpose]The aim of this study was to isolate ABCG2-positive LEC-like and N-cadherin-positive CEC-like cells from the induced hESCs in vitro.[Methods]1.Culture and identification of hESCs:hESC line H1 was cultured on hES-qualified Matrigel coated plates with mTeSRl medium,and passaged every five days.The markers of hESCs,OCT4 and SSEA-3,were detected by immunofluorescence.2.Culture and identification of limbal epithelial cells and corneal stromal fibroblasts:(1)Culture of limbal epithelial cells:The remaining corneal ring after transplantation was washed with PBS containing 1%penicillin-streptomycin,and Descemet’s membrane was mechanically stripped.Then,it was immersed in dispase Ⅱ(2.4U/ml)for 1.5 hours,and trypsinized for 10 minutes at 37℃.The digested LECs were seeded on the plates coated with 2%growth factor reduced Matrigel.The markers of LECs-ABCG2,p63,and CK3,were detected by immunofluorescent staining.(2)Culture of corneal stromal fibroblasts.The remaining tissue was trimmed to 1 X 1 mm,and digested in 0.02%collagenase A for 2 hours.After centrifugation,the cells were seeded on the plates in the medium containing 10%fetal bovine serum.Fibroblast markers,vimentin and alpha-SMA,were examined by immunofluorescence.3.Preparation of LEC-conditioned medium and CEC differentiation medium.(1)For LEC-conditioned medium,it was composed of hLEC medium and the collected medium from LECs at 70%-90%confluence in a ratio of 1:3,similar to our previous reports.(2)Preparation of CEC differentiation medium.According to our previously described method,lens epithelial cell-conditioned medium was obtained by collecting the medium from these cultured cells at 70%-90%confluence,and mixed with corneal stromal fibroblast(CSFs)medium at a ratio of 3:1 as CEC differentiation medium.4.Differentiation of LEC-like and CEC-like cells from hESCs.HESCs were detached with 2 mg/ml Dispase and grown in EB medium in the low adhesion plates to form embryoid bodies(EBs).The differentiation of LEC-like and CEC-like cells was performed as the protocols that we previously developed.For the differentiation of LEC-like cells,according to our previously reports,approximately 10-15 EBs were plated into each well of 24-well plates coated with Type IV collagen and fed with LEC-conditioned medium for 9 days.To induce CEC-like cell differentiation,approximately 80 EBs per well were plated in 6-well tissue culture plate coated with the coating buffer that composed of fibronectin,laminin and chondroitin sulfate,and cocultured with coreal stromal fibroblasts that were plated at 1,800/cm2 on 0.4-mm-pore transwell inserts.The culture lasted 5 days.Then,the medium was changed into CEC differentiation medium,and culturing was continued to 2 weeks.5.Identification and sorting of LEC-like cells and CEC-like cells.For LEC-like cells,LEC markers ABCG2,p63,and CK3 were detected using immunofluorescent staining,and the cells that expressed ABCG2 were sorted by fluorescence-activated cell sorting(FACS)in the differentiated cells as LEC-like cells.For CEC-like cells,the CEC markers N-cadherin,ZO-1,and Na+/K+ ATPase were examined using immunofluorescent staining,and the N-cadherin-positive cells were sorted by FACS as CEC-like cells.[Results]Similar to primary cultured LECs,LEC-like cells showed cobble stone-like morphology and high expression of ABCG-2,p63 and CK3 as demonstrated by immunofluorescent staining.The differentiated CEC-like cells displayed polygonal shapes,and formed an endothelial monolayer at the periphery of plated EBs with expression of CEC markers N-cadherin,ZO-1,and Na+/K+ ATPase.FACS analysis showed that averaged 34.94%of the cells were ABCG2-positive in differentiated LEC-like cells,and averaged 10.91%of the cells were N-cadherin-positive in the differentiated CEC-like cells.The sorted LEC-like cells co-expressed ABCG2 and p63,and co-expression of N-cadherin,ZO-1 and Na+/K+ ATPase was also found in the sorted CEC-like cells.[Conclusion]The differentiated LEC-like cells and CEC-like cells with our protocols were similar to primary cultured LECs and CECs in cell morphology and expression of the markers.Moreover,LEC-like and CEC-like cells with high purity can be obtained in the differentiated cells by cell sorting.Part Ⅲ Constructing tissue-engineered full-thickness cornea in vitro and evaluating its performance in vivo[Purpose]This study aimed to investigate the feasibility of constructing TECS using the three-dimensional culture protocol that developed by ourselves,and explored the characteristics and in vivo performance of TECS to evaluate whether it could be used in penetrating keratoplasty.[Methods]1.Seeding LEC-like cells and CEC-like cells on APCM scaffold.APCM scaffold was soaked in CSFs medium for 24 hours,and plated on the inner convex mesa of the hollow insert of 24-well plates(Fig.1 in Part Ⅲ).The sorted CEC-like cells were seeded at 3000 mm2 on stromal side of the scaffold and cultured in CSFs medium for 2 weeks.Then,the sorted LEC-like cells were seeded at 1,5×104 mm2 on Bowman’s membrane of the scaffold.The tissue-engineered grafts were cultured in submersion condition for 7 days,and then changed to airlift culture for another 7 days to form the stratified epithelium.2.Morphological and biological characteristics of TECS.The morphology of TECS was analyzed by HE staining,and the markers of corneal epithelium and endothelium were detected by immunofluorescence.CEC-like cells on TECS were counted under optical microscopy after double-staining with alizarin red S and trypan blue.The thickness of TECS was examined using a standard micrometer,and the light transmittance percentage was measured using the UV-VIS spectrophotometer.Furthermore,the tensile strength,elastic modulus and elongation at break of the construct were analyzed with an Instron electromechanical universal tester.3.Penetrating keratoplasty.The rabbits from experimental group were treated with TECS as grafts for penetrating keratoplasty,and the other rabbits were transplanted with APCM scaffolds as controls.After surgery,the eyes were followed using slit lamp microscopy,optical coherent tomography(OCT)in anterior segment,and Tonopen tonometer.The transparency and thickness of the graft,neo-vascularization,and immune rejection were assessed.On postoperative 8 weeks,confocal laser microscopy was used to evaluate endothelial cells.4.Histological analysis.One rabbit cornea was taken on postoperative 1 week,and 2,4,8 weeks,respectively.The epithelium and endothelium of the graft,stromal cell ingrowth,and inflammatory cell infiltration were analyzed by H&E staining.Anti-human nuclei antibody was used to detected human-derived cells by immunofluorescence.The markers of LECs and CECs were examined using immunofluorescent staining.[Results]1.Detecting the morphological and biological characteristics of TECS in vitro.HE staining showed tight cell coverage with three or four layers of epithelium-like cells on the Bowman’s membrane side and a uniform monolayer of the endothelium on the other side.CK3 but not p63 could be detected in the epithelium of the construct,and ABCG2 was only found in part of the basal cells in the epithelial cell layers.N-cadherin,ZO-1 and Na+/K+ ATPase could be detected in the endothelium of the construct.The construct showed similar endothelial cell density,thickness,and biomechanical properties with native rabbit cornea,but the transmittance was slightly lower than that of native rabbit cornea.2.Evaluating the in vivo performance of TECS.After transplantation,the thickness of the graft increased in both experimental and control groups.After one week,the thickness of TECS decreased gradually,and the transparency increased,but the APCM scaffold became thicker and more opaque.At 2 to 4 weeks,the neovascularization began to grow into the cornea in both groups.However,it grew faster in the control group than that in experimental groups.On postoperative 8 weeks,the grafts in control group were covered with new blood vessels,which was porcelain white and opaque with an average thickness of 780μm.Neovascularization appeared in the periphery of the TECS grafts at 8 weeks after surgery,and the iris could be seen through the TECS with an average thickness of 460μm.HE staining showed that TECS maintained stratified corneal epithelium and corneal endothelial cell monolayer during follow-up period.In the control group,epithelium could be found on the graft after postoperative 4 weeks,but endothelium was not observed on the graft during the follow-up time.Inflammatory cell infiltration was observed at 2 weeks postoperatively,but it had no significant increase during the follow-up period.Anti-human nuclear antibody staining positive cells were observed in endothelium and part of epithelium of TECS at 4 weeks,but it was only observed in the endothelium on postoperative 8 weeks.In control group,there was not cells stained by anti-human nuclear antibody at all time-points of observation.CK3-positive but not ABCG2-positive and p63-positive human cells could be found in part of the epithelial cells of TECS at 4 weeks postoperatively.On postoperative 8 weeks,there were not CK3-positive human cells that could be detected in graft epithelium.However,the cells expressed the markers of human endothelial cell could be detected in the endothelium of TECS within 8 weeks after surgery.[Conclusion]The three-dimensional culture protocol in this study is suitable for the construction of tissue-engineered full-thickness cornea,and the construct showed similar endothelial cell density,thickness,and biomechanical properties with native rabbit cornea.In vivo,TECS showed some functions of donated corneal graft,and the rejection was not severe after surgery,but its transparency was lower than that of native cornea.Therefore,how to improve in vivo transparency of the construct still need further investigation. |
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