Long-term observation after transplantation of cultured human corneal endothelial cells for corneal endothelial dysfunction.

BACKGROUND: Corneal transplantation is the only way to treat serious corneal diseases caused by corneal endothelial dysfunction. However, the shortage of donor corneal tissues and human corneal endothelial cells (HCECs) remains a worldwide challenge. We cultivated HCECs by the use of a conditioned medium from orbital adipose-derived stem cells (OASC-CM) in vitro. Then the HCECs were used to treat animal corneal endothelial dysfunction models via cell transplantation. The purpose of this study was to conduct a long-term observation and evaluation after cell transplantation. METHODS: Orbital adipose-derived stem cells (OASCs) were isolated to prepare the conditioned medium (CM). HCECs were cultivated and expanded by the usage of the CM (CM-HCECs). Then, related corneal endothelial cell (CEC) markers were analyzed by immunofluorescence. The cell proliferation ability was also tested. CM-HCECs were then transplanted into monkey corneal endothelial dysfunction models by injection. We carried out a 24-month postoperative preclinical observation and verified the long-term effect by histological examination and transcriptome sequencing. RESULTS: CM-HCECs strongly expressed CEC-related markers and maintained polygonal cell morphology even after 10 passages. At 24 months after cell transplantation, there was a CEC density of more than 2400 cells per square millimeter (range, 2408-2685) in the experimental group. A corneal thickness (CT) of less than 550 µm (range, 490-510) was attained. Gene sequencing showed that the gene expression pattern of CM-HCECs was similar to that of transplanted cells and HCECs. CONCLUSIONS: Transplantation of CM-HCECs into monkey corneal endothelial dysfunction models resulted in a transparent cornea after 24 months. This research provided a promising prospect of cell-based therapy for corneal endothelial diseases.

The Effect of SPARC on the Proliferation and Migration of Limbal Epithelial Stem Cells During the Corneal Epithelial Wound Healing.

Secreted protein acidic and rich in cysteine (SPARC) shows a specific colocalization with limbal epithelial stem cells (LESCs) in vivo; however, the inherent relationship between SPARC and LESCs is still unclear. This study investigated the effects of SPARC on the maintenance of LESC stemness and corneal wound healing. To test the influence of different concentration of exogenous SPARC on the proliferation of LESCs, cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine staining were performed and the results indicated that 1 µg/mL SPARC was the optimum concentration for enhanced LESC proliferation. Compared with a control group, SPARC-treated group showed a higher expression of LESC-positive markers p63α, ABCG-2, and Bmi-1, and a lower level of differentiation marker cytokeratin-3 (CK3), thereby suggesting that SPARC could maintain LESC characteristic phenotype and suppress spontaneous epithelial differentiation in vitro. In vivo, exogenous SPARC accelerated the wound-healing process by both the enhancement of LESC proliferation and promoting the migration of the proliferating cells. However, the intact epithelium impaired this function of SPARC by contact inhibition.

Spontaneous resolution of a traumatic cataract in a patient with an open-globe ocular injury: a case report.

BACKGROUND: We report a case of spontaneous resolution of a traumatic cataract in a patient with an open-globe ocular injury. This case highlights the importance of conservative management in these types of cases, as excellent visual outcome is possible without invasive surgical intervention. CASE PRESENTATION: A 13-year-old boy presented with a corneal laceration in the left eye caused by a neuter pen. He underwent emergency repair of the corneal laceration under general anesthesia, and at 3 days post-op, a dense posterior cortical cataract was observed. Based on the patient's age and normal visual development, in addition to preserving accommodative potential, the patient received conservative management and follow-up. Interestingly, the cataract spontaneously resolved over the following 9 months and the corrected distance visual acuity in the injured eye was restored from finger counting at 50 cm, to 20/25 + 3. CONCLUSIONS: To optimize treatment in pediatric traumatic cataract, several critical factors such as age, visual development and the preservation of accommodative potential, need to be comprehensively considered. Conservative management with lens preservation is important to consider in young, traumatic cataract patients where invasive surgical intervention may not be required.

SPARC promotes self-renewal of limbal epithelial stem cells and ocular surface restoration through JNK and p38-MAPK signaling pathways.

The purpose of this study was to investigate the effects of secreted protein acidic and rich in cysteine (SPARC) on the maintenance of limbal epithelial stem cell (LESC) stemness and restoration of ocular surface. To determine the suitable concentration of SPARC for LESC culture, the marker expression, mitogenic effect, and holoclone-forming capacity of LESCs treated with different concentrations of SPARC were analyzed. To investigate the mechanism of SPARC's action on the preservation of LESCs stemness, the phosphorylation of related signaling pathways was evaluated by Western blotting. A corneal wound model was established to verify the function of SPARC in ocular surface repair. Consecutive subculturing, colony-forming efficiency, immunofluorescence, and 5-ethynyl-2-deoxyuridine incorporation assays indicated that 1 µg/mL SPARC was a suitable concentration to stimulate LESC proliferation and preserve their proliferative potential. Compared with a control group, 1 µg/mL SPARC effectively increased the expression of ABCG-2, Bmi-1, and Ki67, while decreasing that of CK3/12. The mitogenic effect of SPARC on LESCs was found to be mediated by the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-MAPK signaling pathways, whereas the inhibitors of JNK and p38 MAPK reduced the marker expression and mitogenic capacity of LESCs. In a corneal injury model, SPARC facilitated corneal epithelial wound healing and promoted the proliferation of p63α-positive cells both in the limbus and in the epithelial healing front. SPARC promotes proliferation while suppressing spontaneous differentiation of LESCs through JNK and p38-MAPK signaling pathways, suggesting that SPARC is a promising factor for the improvement of LESCs culture in vitro and in vivo.

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro.

AIM: To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS: The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4', 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS: Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION: Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.

Effect of poly(DL-lactide-co-glycolide) on scar formation after glaucoma filtration surgery.

BACKGROUND: Glaucoma filtering surgery (GFS) is the most common procedure performed in the treatment of glaucoma. Although antiscarring agents help prevent postsurgical scarring and improve glaucoma surgical outcomes, they may be associated with an increased incidence of severe and potentially blinding complications. Poly(DL-lactide-co-glycolide) (PDLLA/GA) is a bioresorbable polymer, which can be prepared with a large range of physical, mechanical, and biological properties and has been widely used in medicine, including as an absorbable suture and a drug carrier and especially as a scaffold in tissue engineering. This study aimed to evaluate the effect of PDLLA/GA on scar formation after glaucoma filtration surgery (GFS). METHODS: Forty-eight New Zealand white rabbits were divided into two groups randomly and GFS was performed on the right eye of each. PDLLA/GA membranes were put under the sclera flap for evaluation. GFS with no membrane inserted served as control. Clinical evaluations of intraocular pressure (IOP) and the presence of a filtration bleb were performed at intervals (3 days, 1, 2, 4, 8, 12, 20, and 24 weeks) postoperatively. At each time point, three eyes per group were excised to observe histological changes such as inflammation and scar formation and the expression of collagen type IV, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1). The expression of connective tissue growth factor (CTGF) mRNA was determined by reverse transcription-polymerase chain reaction. RESULTS: The lower IOP level and an effective bleb were maintained for a long time after GFS in the PDLLA/GA group. The histological analysis showed less inflammation and scar formation, weaker expression of collagen type IV and PCNA, more intense MMP-9 and TIMP-1, slightly elevated ratio of MMP-9 and TIMP-1, and a smaller increase in CTGF mRNA postoperatively in the PDLLA/GA group but less than the control group (P < 0.05). CONCLUSION: PDLLA/GA membranes may be promising for preventing fibrosis after GFS.

In vivo biological stability of chemically pretreated silicone gel inserts intended for use in keratoprostheses.

BACKGROUND: Pretreatment with chemical agents could alter the surface chemistry of the silicone gel, which makes it suitable for epithelial migration onto its surface and thus enhances the cytobiocompatibility. This study aimed to evaluate the biological response of the corneal stroma to porous silicone gel pretreated with different chemical agents in vivo. METHODS: The porous silicone gels were treated with a mixed acid solution containing 23.2% H2SO4 and 0.8% K2Cr2O7 for 10 or 15 minutes or with 30% H2O2 for 15 minutes. Discs (4 mm in diameter) were inserted into interlamellar stromal pockets of New Zealand white rabbits and followed up for a period of 3 months. Clinical evaluations such as corneal infiltration, edema and neovascularization were performed daily. At 3 months, the fibroplasias and collagen deposition were examined under light and scanning electron microscopy (SEM) and by immunohistochemical analysis. RESULTS: Pretreatment of the discs obviously decreased conjunctival congestion, discharge, cornea edema, and the extent of neovascularization. More fibroblasts migrated into the pretreated discs than into the control, and collagen was deposited, indicating that the biocompatibility of the corneal replacements was enhanced by the chemical pretreatments. From immunohistochemical analysis, Type I collagen deposition in the pretreated silicone discs was greater than in the control. CONCLUSIONS: Chemical treatment of silicone gel is effective in decreasing rabbit corneal inflammation, encouraging fibroblast in-growth, and enhancing tissue compatibility. Pretreated gels show good biological stability when used as a skirt material in Keratoprosthesis (Kpros).

Effect of different biomedical membranes on alkali-burned cornea.

OBJECTIVE: To evaluate the effects of different biomedical membranes on alkali-burned cornea in vivo. METHODS: 12 New Zealand rabbits were chosen and randomly divided into four groups. The right cornea of each rabbit was made into an alkali-burned model with 1 mmol/l NaOH. Poly-D,L-lactic acid (PDLLA), PDLLA modified with collagen (PDLLA/collagen) and PDLLA modified with chitosan (PDLLA/chitosan) membranes were transplanted onto the alkali-burned corneas for evaluation. Clinical evaluations were performed daily with a slit lamp. On the 12th day after surgery, the progress in wound healing was compared by clinical and histological examination. The reepithelialization of each cornea was evaluated with fluorescein staining and 3 corneas of each group were excised to observe histological changes such as corneal wound healing, inflammation and collagen synthesis. RESULTS: The wound healing rate of the PDLLA/chitosan group was higher than in the other groups. A more orderly arrangement of collagen and mild inflammation was observed. The control group had the next best performance, while the PDLLA/collagen and PDLLA alone treatment groups showed the worst results. CONCLUSION: PDLLA/chitosan promoted wound healing of alkali-burned corneas in vivo and decreased scar tissue formation, while the effect of the PDLLA/collagen and PDLLA membranes was to promote corneal ulcers, which suggests that PDLLA/chitosan membrane transplantation is a potential effective strategy for treatment of alkali-burned cornea.

[The role of connective tissue growth factor, transforming growth factor and Smad signaling pathway during corneal wound healing].

OBJECTIVE: To study the expression and location of connective tissue growth factor (CTGF) and transforming growth factor-beta(1) (TGF-beta(1)) protein and mRNA in rabbit cornea during the wound healing process. To assess the interaction between CTGF and TGF-beta(1), as well as the Smad signaling pathway involved. METHODS: Twenty-six Albino white rabbits were used as experimental animals and randomly divided into 4 groups: (1) CONTROL GROUP: two rabbits. (2) Simple corneal injury group: a 3 mm diameter and 0.05 mm depth corneal tissue was excised by a trephine at the anterior central cornea as a corneal wound model in 12 rabbits. Two rabbits were randomly sacrificed at 2 h, 6 h, 1 d, 3 d, 7 d and 21 d after the trauma. (3) TGF-beta(1) antibodies treated group: 6 rabbits were injected with TGF-beta(1) antibodies (15.5 microg) subconjunctivally after corneal trephine. Two rabbits were randomly sacrificed at 3 d, 7 d and 21 d after the injection. (4) Smad4 antibodies treated group: 6 rabbits were injected with Smad4 antibodies (20 microg) subconjunctivally after corneal trephine. Two rabbits were randomly sacrificed at 3 d, 7 d and 21 d after the injection. Protein of CTGF, TGF-beta(1), and FN was assessed with immunohistochemistry. CTGF and type one collagen mRNA were measured in by in situ hybridization. RESULTS: (1) CTGF protein or mRNA did not exist in normal rabbit corneas, but TGF-beta(1) protein was expressed in normal rabbit cornea epithelium. (2) Cornea fibroblasts activated 6 h after the operation. Expression of CTGF, TGF-beta(1), FN protein and mRNA of CTGF and type one collagen were upregulated after cornea injury, and reached the highest level in 3 days. The expression was reduced to the basal level 21 days later. (3) Injection of TGF-beta(1) antibodies reduced the expression of CTGF, TGF-beta(1) and FN in the cornea stroma and down-regulated the expression of CTGF in corneal epithelial cells. (4) Injection of Smad4 antibodies inhibited the expression of TGF in the stroma but did not change the expression of CTGF. CONCLUSIONS: Injury can upregulate the expression of CTGF, TGF-beta(1), FN and type one collagen proteins and mRNA of CTGF and TGF-beta(1). TGF-beta(1) and CTGF affect the wound healing process. TGF can induce the activation of corneal fibroblasts and upregulate the expression of CTGF. CTGF modulates the production of FN and Type one collagen. These data support the hypothesis that TGF-beta(1) and CTGF promote cornea scar formation and imply that the regulation of CTGF and TGF-beta(1) synthesis may be a therapy target for reducing corneal scarring.

Effect of chemical treatment of silicon gel on tissue compatibility.

BACKGROUND: Silicon gel is unfavourable for cell attachment and growth. This study was to study if pretreating the surface of silicon gel with chemical agents affects the proliferation of epithelial cells. METHODS: Silicon gel was made and treated with either mixed acid solution (containing 232 g/dm(3) of H(2)SO(4) and 8 g/dm(3) of K(2)Cr(2)O(7)) or 300 cm(3)/dm(3) peroxide for 5, 10, and 15 minutes or 10, 15, and 20 minutes, respectively. The cultured corneal epithelial cells were seeded onto those silicon gels and kept for 13 days. Immunohistochemical investigations were then carried out for integrin (alpha 6 or beta 4) and actin. RESULTS: Growth of the epithelial cells in silicon gels treated with mixed acid solution for 10 minutes and 15 minutes was much significant than that in the untreated gels. After a 12-hour culture, a small number of corneal epithelial cells were proliferated on the surface of the silicon gels that had been treated with peroxide for 15 minutes. After a 3-day culture, those cells were further proliferated and fused together. The corneal epithelial cells did not grow well in the silicon gels treated with peroxide for 10 or 20 minutes. Immunostaining revealed the expression of actin and integrin alpha 6 or beta 4 on the silicon gels that were treated with mixed acid solution for 10 minutes or peroxide for 15 minutes. CONCLUSION: Silicon gels treated either with mixed acid solution for 10 or 15 minutes or with peroxide for 15 minutes improves cell proliferation.