Abstract
A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.
Highlights
The silk produced by the larvae of domesticated silkmoth (Bombyx mori) or some wild silkmoths have been known in the textile manufacturing for millennia [1,2,3,4]
Considering the rather ambiguous results reported with a poly(ethylene glycol) (PEG) of high molecular weight (MW) [44,47,55], and the promising results reported [56] using a PEG of low MW, we developed freestanding B. mori silk fibroin (BMSF) membranes that were modified with PEG of MW 300 Da, with the expectation of increasing permeability and of generating topographic features on the surface of the membranes
Since an underlying culture of irradiated 3T3 murine fibroblasts can influence positively the growth of human corneal epithelial cells (HCECs) when cultivated on non-treated membranes, it would appear that even without PEG-treatment the BMSF substrata are perhaps sufficiently permeable to support the manufacture of clinically suitable cultures
Summary
The silk produced by the larvae of domesticated silkmoth (Bombyx mori) or some wild silkmoths have been known in the textile manufacturing for millennia [1,2,3,4]. The enhancement of substratum’s transport properties and of the adhesion and growth of cells would be important for the development of better tissue-engineered constructs, and strategies to achieve it have been actively pursued by some dedicated research groups To this aim, methods for creating surface topographic features and/or rendering the substratum porous were investigated in order to improve colonization by corneal cells of the BMSF templates. The cultures of primary rabbit corneal limbal epithelial cells on the PEG-treated substrata resulted in stratified epithelial layers, while only monolayers were noticed on the original BMSF substrata [56] This finding could be a consequence of favorable combined effects of higher permeability and rougher surface topography. The aim of this study was to investigate whether the treatment of BMSF substrata with a PEG of low MW is of benefit to corneal epithelial cellular growth due to the potential synergism of higher permeability and irregular patterning of the surface
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