The goal of this study was to evaluate the potential suitability of an artificial membrane composed of silk fibroin (SF) functionalized by different ratios of chitosan (CS) as a substrate for the stroma of the cornea. Keratocytes were cultured on translucent membranes made of SF and CS with different ratios. The biophysical properties of the silk fibroin and chitosan (SF/CS) membrane were examined. The SF/CS showed tensile strengths that increased as the CS concentration increased, but the physical and mechanical properties of chitosan-functionalized silk fibroin scaffolds weakened significantly compared with those of native corneas. The resulting cell scaffolds were evaluated using western blot in addition to light and electron microscopy. The cell attachment and proliferation on the scaffold were similar to those on a plastic plate. Keratocytes cultured in serum on SF/CS exhibited stellate morphology along with a marked increase in the expression of keratocan compared with identical cultures on tissue culture plastics. The biocompatibility was tested by transplanting the acellular membrane into rabbit corneal stromal pockets. There was no inflammatory complication detected at any time point on the macroscopic level. Taken together, these results indicate that SF/CS holds promise as a substrate for corneal reconstruction.

Purpose: To construct a scaffold using silk fibroin (SF) and chitosan (CS) that could replace the corneal stroma with the biological characteristics of the scaffold materials still intact. Methods: To develop an organotypic corneal stroma, SF and CS were chosen to synthesise the tissue-engineered bioscaffold. We cultured primary rabbit corneal epithelial cells and corneal stromal cells in vitro. Keratocytes were used to assess cytotoxicity on SF-CS (SFCS) blends, which was determined by a Cell Counting Kit-8 assay. The corneal lamellar scaffolds were developed with sequential culture techniques to form cell-scaffold constructs. Implantation was tested in 15 New Zealand White rabbits. The corneal substitutes were analysed by light and electron microscopy. Results: The reconstructed lamellar cornea was comparable to native tissue, with high levels of K3/12 expression in the corneal epithelial cells and vimentin in the stromal cells; moreover, the morphology and the position of the cells could be distinguished by histological methods. There was no sign of any immune reaction in or around the transplanted discs 12 weeks after implantation. Conclusion: A SFCS scaffold might be a suitable blend for corneal tissue engineering.

Cataracts are the most common eye disease to cause blindness in patients. The abnormal deposition of laminins (LMs) in the lens capsule and the disruption of capsular epithelium contribute to cataract development, although the mechanism by which this occurs is currently unclear. The present study aimed to reproduce HLE B-3 basement membranes (BMs) using HLE B-3 cells and to analyze the similarities of LM expression between HLE B-3 BMs and human anterior lens capsule (ALC). Immunohistochemistry (IHC), ELISA, western blot analysis and immunoprecipitation (IP)-western blot analysis were used to detect total LMs, LM trimers and 11 LM subunits in HLE B-3 cells, HLE B-3 BMs and human ALCs. In IHC staining, HLE B-3 cells and human ALCs were positive for LMs. In LM ELISA, all samples analyzed were positive for LMs. Western blot analysis detected all LM subunits except for LMγ3 in HLE B-3 cell lysate, 4 subunits (LMα4, LMα2, LMα1 and LMγ1) in HLE B-3 cell culture supernatant, 5 subunits (LMα4, LMα2, LMα1, LMβ3 and LMγ1) in HLE B-3 BMs, and 3 subunits (LMα4, LMγ2 and LMγ1) in human ALCs. The results of IP-western blot analysis revealed that the LM411 trimer was detected in HLE B-3 cell culture supernatant. These results indicated that HLE B-3 BMs were similar to human ALCs in terms of LM expression. Therefore, HLE B-3 BMs could be used as an in vitro ALC model to determine the role of LMs in ALC in the pathogenesis of cataracts and to select potential anti-cataract drugs.

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Senescence is a leading cause of age-related cataract (ARC). The current study indicated that the senescence-associated protein, p53, total laminin (LM), LMα4, and transforming growth factor-beta1 (TGF-β1) in the cataractous anterior lens capsules (ALCs) increase with the grades of ARC. In cataractous ALCs, patient age, total LM, LMα4, TGF-β1, were all positively correlated with p53. In lens epithelial cell (HLE B-3) senescence models, matrix metalloproteinase-9 (MMP-9) alleviated senescence by decreasing the expression of total LM and LMα4; TGF-β1 induced senescence by increasing the expression of total LM and LMα4. Furthermore, MMP-9 silencing increased p-p38 and LMα4 expression; anti-LMα4 globular domain antibody alleviated senescence by decreasing the expression of p-p38 and LMα4; pharmacological inhibition of p38 MAPK signaling alleviated senescence by decreasing the expression of LMα4. Finally, in cataractous ALCs, positive correlations were found between LMα4 and total LM, as well as between LMα4 and TGF-β1. Taken together, our results implied that the elevated LMα4, which was possibly caused by the decreased MMP-9, increased TGF-β1 and activated p38 MAPK signaling during senescence, leading to the development of ARC. LMα4 and its regulatory factors show potential as targets for drug development for prevention and treatment of ARC.