Objective To review research progress of corneal tissueengineering.Methods The recent articles on corneal tissue engineering focus on source and selection of corneal cells, the effects of growth factors on culture of corneal cells in vitro. The preparation and selection of three-dimensional biomaterial scaffolds and their b and weak points were discussed. Results The corneal tissue engineering cells come from normal human corneal cells. The embryo corneal cell was excellent. Several kinds of growth factors play important roles in culture, growth and proliferation of corneal cell, and incroporated into matrix.Growth factors including basic fibroblast growth factor, keratinocyte growth factor, transforming growth factor β1 and epidermal growth factor was favor to corneal cell. Collagen, chitosan and glycosaninoglycans were chosen as biomaterial scaffolds. Conclusion Human tissue engineering cornea can be reconstructed and transplanted. It has good tissue compatibility and can be used as human corneal equivalents.
In the evaluation of tear film stability based on corneal topography, a pretreatment algorithm for tear film video was proposed for eye movement, eyelash reflection and background interference. First, Sobel operator was used to detect the blur image. Next, the target image with highlighted ring pattern was obtained by the morphological open operation performed on the grayscale image. Then the ring pattern frequency of the target image was extracted through the Hough circle detection and fast Fourier transform, and a band-pass filter was applied to the target image according to the ring pattern frequency. Finally, binarization and morphological closed operation were used for the localization of the ring pattern. Ten tear film videos were randomly selected from the database and processed frame by frame through the above algorithm. The experimental results showed that the proposed algorithm was effective in removing the invalid images in the video sequence and positioning the ring pattern, which laid a foundation for the subsequent evaluation of tear film stability.
Objective To observe the biocompatibility of the acellular corneal stroma materials prepared by three different methods. Methods Three different serial digestion methods were used to produce the acellular corneal stroma materials. The biocompatibility of the materials was investigated by the cell seeding and the materials were implanted into the rabbit corneal stroma layer. Results The cells in the materials 1 and 2 were not decellularized completely. The rabbit corneal fibroblasts died on the materials 1 and 2 after the cell seeding for 3-4 days. An obvious rejection could be observed after the implantation. The cells in material 3 were decellularized completely and the collagen fibers or elastic fibers were reserved integrally，showing a typical three-dimensional net work. The rabbit corneal fibroblasts could expand on the materials in vitro. No obvious rejection could be observed and the materials were gradually absorbed. Conclusion The acellular porcine cornea stroma materials prepared by trypsin-Dnase-Rnase are suitable for reconstruction of the tissue engineered cornea.
ObjectiveTo investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene.
MethodsThe adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs.The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A),pcDNA3.1 empty vector transfection (group B),and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C),respectively.At 48 hours after transfection,the cells in groups B and C were selected with G418.The cell morphology changes were observed under the inverted microscope.Pax6 protein and level of corneal epithelial cells specific molecular-cytokeratin 12 (CK-12) were measured by Western blot.Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12.
ResultsNo morphology change was observed in groups A and B.Two different cell clones were found in group C.No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells;No.2 selected clone showed a net-like appearance,with 3-7 cell processes.The Western blot results showed the Pax6 protein expression in 2 clones of group C,but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C,and no expression in the others.The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64±0.73,which was significantly higher than that of No.2 selected clone of group C (0.55±0.42),group B (1.36±0.40),and group A (1.00±0.00) (P<0.05),and there was no significant difference among groups A,B and No.2 selected clone of group C (P>0.05).
ConclusionPax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels.This result provides a promising strategy of generating corneal epithelilcm-like cells for construction of tissue engineered cornea.
Objective
To analyze the risk factors for persistent corneal epithelial defects (PCED) after pars plana vitrectomy (PPV) in patients with proliferative diabetic retinopathy (PDR).
Methods
A total of 201 PDR patients (201 eyes) who received PPV were enrolled in this retrospective study. There were 86 males (86 eyes) and 115 females (115 eyes). The patients aged from 30 to 81 years, with the mean age of (57.94±9.65) years. Among them, 159 patients were ≥50 years of age, and 42 patients were ＜50 years of age. There were 36 patients with HbA1c ＜7.0%, 165 patients with HbA1c ≥7.0%. There were 93 right eyes and 108 left eyes. There were 93 right eyes and 108 left eyes. The diabetic retinopathy stages were as follows: stage Ⅳ in 24 eyes, stage Ⅴ in 78 eyes and stage Ⅵ in 99 eyes. The operation time was ranged from 1 to 4 hours, with an average of 2 hours. Among the 201 eyes, corneal epidermis were scraped on 25 eyes; 70 eyes were combined with cataract surgery; a laser photocoagulation count ＜1000 points was performed in 78 eyes, and ＞1000 points were performed in 123 eyes. Sixty-one eyes involved intravitreal silicone oil tamponade, 18 eyes involved intravitreal tamponade with C3F8, and 122 eyes were not involved with intraocular tamponade. Postoperative persistent intraocular hypertension was defined as an intraocular pressure (IOP) ≥21 mmHg (1 mmHg=0.133 kPa) after PPV with necessary treatment using IOP-lowering medications for ≥2 weeks. The diagnostic criteria for corneal epithelial defects were taken from the Expert Consensus on Clinical Diagnosis and Treatment of Corneal Epithelial Defect in China (2016). The corneal epithelial defect was diagnosed as PCED if it was treated with common methods such as a lacrimal substitute or corneal contact lens, but showed no improvement and no signs of healing for ≥2 weeks. The incidence of PCED after eye surgery was recorded and its related risk factors were analyzed. A multivariate logistic regression was used to analyze the risk factors for PCED, which were expressed as a odds ratio (OR) and a 95% confidence interval (CI).
Results
Of 201 eyes, 16 eyes (7.96%) presented with PCED after surgery and 185 eyes (92.04%) with no PCED. There was no significant difference in the age, sex, and eyes between the patients with or without PCED (χ2=6.548, 0.927, 0.044; P=0.011, 0.336, 0.833). A multivariate logistic regression showed that intraoperative epithelial debridement (OR=13.239, 95%CI 2.999?58.442, P=0.001), intraoperative treatment in combination with cataract surgery (OR=7.448, 95%CI 1.975?28.091, P=0.003), intravitreal tamponade with C3F8 (OR=11.344, 95%CI 2.169?59.324, P=0.004), and postoperative persistent intraocular hypertension (OR=10.462, 95%CI 2.464?44.414, P=0.001) were risk factors for PCED after PPV.
Conclusion
Intraoperative epithelial debridement, intraoperative treatment in combination with cataract surgery, intravitreal tamponade with C3F8, and postoperative persistent intraocular hypertension are risk factors for PCED in patients with PDR after PPV.
The aim of this article is to study the effect of tumor necrosis factor alpha (TNF-α) on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in keratoconus fibroblasts in vitro. Normal cornea and keratoconus fibroblasts were extracted using enzyme digestion method and were cultured in the medium containing TNF-α (0, 10 and 100 ng/mL). The expression of MMPs proteins in the supernatant of corneal fibroblasts and the expression of TIMPs in the normal cornea and keratoconus fibroblasts were detected by Western blot and real-time quantitative polymerase chain reaction respectively. The active form of MMP1 could be detected in the supernatant of keratoconus fibroblasts and upregulated by TNF-α. TNF-α could increase the protein expression of MMP2, MMP3, MMP9 in the supernatant of keratoconus fibroblasts and decrease the gene expression of TIMP1, TIMP2 in keratoconus fibroblasts. The increased MMPs and the decreased TIMPs can increase the degradation of the extracellular matrix. TNF-α may play an important role in the occurrence and development of keratoconus by regulating the expression of MMPs/TIMPs.
The decrease of corneal stiffness is the key factor leading to keratoconus, and the corneal collagen fiber stiffness and fiber dispersion are closely related to the corneal biomechanical properties. In this paper, a finite element model of human cornea based on corneal microstructure, namely collagen fiber, was established before and after laser assisted in situ keratomileusis (LASIK). By simulating the Corvis ST process and comparing with the actual clinical results, the hyperelastic constitutive parameters and corneal collagen fiber stiffness modulus of the corneal material were determined before and after refractive surgery. After LASIK, the corneal collagen fiber stiffness modulus increased significantly, and was highly correlated with central corneal thickness (CCT). The predictive relationship between the corneal collagen fiber stiffness modulus and the corresponding CCT before and after surgery was: k1 before = exp(9.14 ? 0.009CCTbefore), k1 after = exp(8.82 ? 0.008CCTafter). According to the results of this study, the central corneal thickness of the patient can be used to estimate the preoperative and postoperative collagen fiber stiffness modulus, and then a personalized corneal model that is more consistent with the actual situation of the patient can be established, providing a theoretical reference for more accurately predicting the safe surgical cutting amount of the cornea.
Study of the mechanical properties of in vivo corneal materials is an important basis for further study of corneal physiological and pathological phenomena by means of finite element method. In this paper, the elastic coefficient (E) and viscous coefficient (η) of normal cornea and keratoconus under pulse pressure are calculated by using standard linear solid model with the data provided by corneal visualization scheimpflug technology. The results showed that there was a significant difference of E and η between normal cornea and keratoconus cornea (P < 0.05). Receiver operating characteristic curve analysis showed that the area under curve (AUC) for E, η and their combined indicators were 0.776, 0.895 and 0.948, respectively, which indicated that keratoconus could be predicted by E and η. The results of this study may provide a reference for the early diagnosis of keratoconus and avoid the occurrence of keratoconus after operation, so it has a certain clinical value.
Objective To evaluate the relative factors of effect of vitrectomy on corneal endothelial cells. Methods Retrospective analysis of the results of corneal endothelium microscopy performed on 213 eyes of 213 patients undergone vetrectomy operations including single vitrectomy (78 eyes), vitrectomy combined with cataract extraction (135 eyes), silicone oil injection (34 eyes), and C3F8 injection (53 eyes) before and after 1 week, 1 and 3 moths of these surgical procedures. Results There was no significant difference between pre- and postoperative corneal endothelium density in single vitrectomy group and vitrectomy combined with cataract extraction with posterior capsule integrity group (Pgt;0.05). The corneal endothelium density significantly decreased postoperatively in C3F8or silicone oil injection group with broken posterior capsule (Plt;0.05). Conclusion C3F8 and silicone oil may damnify corneal endothelium in patients undergo vitrectom y combined with cataract extraction with broken posterior capsule. (Chin J Ocul Fundus Dis，2004，20：101-103)
It is important to design a long-period transparent bioactive material for corneal repair in the process of corneal tissue renovation. This article discusses the silk fibroin and formamide blend membranes as a corneal stroma repair material. Silk fibroin solution was mixed with formamide in different proportions to obtain insoluble transparent silk fibroin film by casting method. The blending membranes had excellent mechanical properties, cell compatibility and long-term transparent properties. Rabbit corneal stromal cells were seeded on the sterilized composite films. The rate of cell surface adhesion was over 90% after cells were placed on it for 5 hours. When cells were seeded on blend membranes from one day to seven days, Alma Blue was added to complete medium. Compared with the cell culture plate, there was no significant difference in cell proliferation on formamide/silk films. The results indicated that formamide/silk films might be used as a corneal stroma repair material and worth of further investigation.
