Tissue-engineered Corneal Epithelium Research Articles

Downregulation of p38 MAPK Signaling Pathway Ameliorates Tissue-Engineered Corneal Epithelium.

Tissue-engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation, and insufficient stem cell during construction affect the quality of tissue-engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the p38 mitogen-activated protein kinase (MAPK) signaling pathway during construction of tissue-engineered corneal epithelium. With application of SB203580, tissue-engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ATP-binding cassette sub-family G member 2, tumor protein p63, keratin 14, and Wnt family member 7A was increased, differentiation markers keratin 12, paired box 6, keratin 10, and keratin 13 and trans-differentiation markers actin alpha 2, smooth muscle and snail family transcriptional repressor 1 was decreased, while cell junction markers claudin 1 and cadherin 1 was increased in the tissue-engineered corneal epithelium. The Wnt/catenin beta 1 signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue-engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness and prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, and thus can improve the quality of tissue-engineered corneal epithelium. Impact statement Downregulation of p38 MAPK signaling pathway helps maintain the self-renewal of limbal stem cells and prevents terminal differentiation and abnormal differentiation of corneal epithelial cells. Small molecules modulating p38 MAPK signaling pathway ameliorate tissue-engineered corneal epithelium.

Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells

PurposeTo construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation. MethodsA stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, real-time quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks. ResultsThe hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch, Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype. ConclusionThe tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.

An Ultra-thin Amniotic Membrane as Carrier in Corneal Epithelium Tissue-Engineering.

Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.

Construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells in vitro

Background Corneal transplantation is an effective treatment to severe corneal diseases, but the shortage of cornea donor limits its application.Tissue-engineered cornea is being a new approach to corneal diseases. Objective This study was to investigate the possibility of construction of tissue-engineered corneal epithelium by culturing human amniotic mesenchymal stem cells (hAMSCs) in vitro. Methods Fresh human amniotic membranes were obtained under the approval of Ethic Committee of Affiliated First Hospital of Jinan University and informed consent of maternal women.The 6 cm×6 cm amniotic membrane tissue explant was digested using trypsin+ EDTA, and then the amniotic epithelial cells (AECs) were scraped before putting into collagenase Ⅱ digestion medium to isolate hAMSCs.hAMSCs of passage 3 were cultured to achive 80%-90% confluence, and then the cells were incubated on rabbit deepithelial corneal stroma at a 1×105/ml density.The corneal stroma was co-cultured with hAMSCs at an air-liquid interface till 14 days.Rabbit deepithelial corneal stroma with and without hAMSCs (experimental group and control group) were fixed in 4% para formaldehyde, and sections were prepared for histopathological examination.Immunochemistry and immunofluorescence were empoyed to detect the expressions of cytokeratin3 (CK3) and CK12 in hAMSCs. Results hAMSCs grew well and formed a stratified epidermal structure resembling native corneal epithelium on rabbit corneal stroma in cultured 14 days in the experimental group, with the oval nucleus at basement and fusiform nucleus on the surface of corneal stroma.There was no cell structure in the control group.Immunochemistry revealed brown staining for CK3, CK12 in cytoplasm of hAMSCs on the rabbits corneal stroma, and the green fluorescence for CK3 and CK12 was also seen in the hAMSCs.However, the response for CK3 and CK12 was absent in the control sections either immunochemistry or immunofluorescence test. Conclusions hAMSCs can be induced to differentiate into corneal epithelioid cells at an air-liquid interface on the rabbit corneal stroma. Key words: Amnion; Mesenchymal stem cells; Cell culture; Tissue engineering; Epithelium, corneal; Differentiation; Humans

Xenogeneic acellular conjunctiva matrix as a scaffold of tissue-engineered corneal epithelium.

Amniotic membrane-based tissue-engineered corneal epithelium has been widely used in the reconstruction of the ocular surface. However, it often degrades too early to ensure the success of the transplanted corneal epithelium when treating patients with severe ocular surface disorders. In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium. Native porcine conjunctiva was decellularized with 0.1% sodium dodecyl sulfate (SDS) for 12 h at 37°C and sterilized via γ-irradiation. Compared with native conjunctiva, more than 92% of the DNA was removed, and more than 90% of the extracellular matrix components (glycosaminoglycan and collagen) remained after the decellularization treatment. Compared with denuded amniotic membrane (dAM), the aCM possessed favorable optical transmittance, tensile strength, stability and biocompatibility as well as stronger resistance to degradation both in vitro and in vivo. The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM. The aCM-based tissue-engineered corneal epithelium was more effective in the reconstruction of the ocular surface in rabbits with limbal stem cell deficiency. These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface.

Transfection efficiency and cytotoxicity of lentivirus-GFP on rabbit adipose-derived stem cells

Objective To evaluate the transfection efficiency and cytotoxicity of lentivirus containing green fluorescence protein(GFP)(lentivirus-GFP) on rabbit adipose-derived stem cells(rADSCs),and establish a method of cell tracer marker in the study of reconstruction of tissue engineered corneal epithelium.Methods rADSCs were transfected with lentivirus-GFP at different multiplicity of infection(MOI).Cell transfection was observed under fluorescent microscope,and influence of lentivirus transfection on proliferation of rADSCs was detected by MTT.Results rADSCs were successfully transfected at MOI of 50,100 and 500,and the transfection efficiency could reach more than 50% 120 h later.Lentivirus-GFP had no significant effect on proliferation of rADSCs at MOI of 50 and 100,while the proliferation of rADSCs was significantly inhibited at MOI of 500.Conclusion rADSCs are successfully transfected with lentivirus-GFP,which can be used in the study of reconstruction of tissue engineered corneal epithelium.

Biocompatibility of acellular corneal stroma and transplantation of tissue-engineered corneal epithelium

To evaluate the biocompatibility of xenogeneic acellular corneal stroma, the feasibility of tissue engineered corneal epithelial transplantation, and verify the long term survival of epithelial allograft. It was a experimental study. Porcine corneal stroma was treated by dispase followed by Triton-X-100 detergent. Treated porcine corneal stroma (group A) or fresh corneal stroma (group B) were put into the sac of rabbit cornea. Rabbit cornea without implantation of porcine corneal stroma was used as the control group (group C). Immunological rejection was evaluated in morphology, histopathology and immunohistochemistry in month 1, 3, 6. Female rabbit underwent lamellar keratoplasty (LK) using male tissue engineered corneal epithelium as donors, and immunological rejection after LK was analyzed. The corneas were collected at day 1, 3, 7, 15 and 30 after LK and evaluated by histopathology, immunohistochemistry and sex-determining region of Y-chromosome (SRY)-PCR analysis. All corneas became transparent gradually after the transplantation of treated porcine corneal stroma and were not rejected. The epithelium, stroma, endothelium, Bowman's and Descemet's membrane were preserved in all corneas of group A and B in histological observations, collagen fibers were parallel, a few keratocytes presented in the acellular and fresh corneal stromas. The corneas of group C were normal in histological sections. No significant immune rejection was noted in any of the corneas. The corneas in the study of transplantation of tissue engineered cornea epithelium recovered smoothly in 3 or 4 days, turned transparent in 15 or 20 days after surgery and were not stained by fluorescein. Well-differentiated corneal epithelium were recognized at 15 and 30 days after LK. Many keratocytes infiltrated into the scaffold. SRY-PCR analysis showed that allogenic donor corneal epithelium cells could survive in recipients after a long-term observation. Acellular porcine corneal stroma shows a satisfying biocompatibility. Tissue engineered corneal epithelium using acellular porcine corneal stroma as carrier could be used as donors for LK with satisfactory results. Donor cells have the potential to survive in recipients after long-term observation.

Reconstruction tissue-engineered corneal epithelium using xenogeneic acellular corneal stroma as scaffold

To compare two methods for preparing acellular corneal stroma and evaluate the possibility of culturing corneal epithelium on xenogeneic acellular corneal stroma. Experimental study, applying completely randomized design method. Dispase followed by Triton-X-100 detergent and sodium chloride SDS detergent followed by trypsinase were applied respectively to treat the rabbit cornea. The characteristics of corneal stroma and acellular status after treatment were examined with slit lamp, optical microscope and transmission electron microscope. The rabbit limbal cells were then cultured on the acellular porcine Bowman's membrane/stroma. Rabbit corneal epithelium lamella reconstructed in vitro and evaluated in morphology, histopathology and immunohisto-chemistry. Acellular corneal stroma prepared by two different methods is quite similar in morphology, being gray and opaque with visible edema and soft texture. Collagen fibers of the stroma were regularly in histopathology and ultrastructure. But the one prepared by the NaCl-SDS-Trypsinase method retained a amounts of cell debris, while there was none in the other did by Dispase-Triton-X-100 method. The limbal cells began to shift out at 24 hours after being inoculated on xenogeneic acellular corneal stroma, then attached on it, formed a confluent monolayer containing normal-appearing in 7 days. The tissue engineering corneal epithelium was cryosectioned and characterized immunohistochemically at 14 days after inoculation. Meanwhile, epithelium associated antigen CK3 in endochylema was stained. Dispase-Triton-X-100 was proved better in obtaining acellular corneal stroma. It is possible to reconstruction tissue-engineered rabbit's corneal epithelium on acellular porcine corneal Bowman's membrane/stroma.

Tissue engineering of the cornea.

Tissue engineering of the cornea represents a paradigm shift in medical treatment to overcome the present disadvantages of corneal transplantation, primarily immune rejection and the shortage of donor corneas. Transplantation of cultivated corneal epithelial cells expanded ex vivo from corneal epithelial stem cells has been developed and has already entered the clinical realm. However, there remain many hurdles to be overcome. The author and colleagues are developing a method to transplant cultivated cell sheets that uses a temperature-responsive culture dish. This article reviews the present situation regarding tissue-engineered corneal epithelium and introduces the results of this program of research.