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Treatment of Silk Fibroin with Poly(ethylene glycol) for the Enhancement of Corneal Epithelial Cell Growth.

Suzuki S, Dawson RA, Chirila TV, Shadforth AM, Hogerheyde TA, Edwards GA, Harkin DG - J Funct Biomater (2015)

Bottom Line: The resulting membranes were thoroughly characterized and compared to the non-treated membranes.The crosslinking with genipin did not induce a significant improvement in mechanical properties.The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.

View Article: PubMed Central - PubMed

Affiliation: Queensland Eye Institute, South Brisbane, Queensland 4101, Australia. shuko.suzuki@qei.org.au.

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.

No MeSH data available.


Related in: MedlinePlus

Histology by confocal microscopy after cultivation of primary human CLECs for 12 days on non-treated (A,B) and genipin-crosslinked PEG-treated (C,D) BMSF membranes: without feeder cells (A,C); co-cultured with feeder cells (irradiated 3T3 murine fibroblasts) (B,D). The feeder cells have become dislodged during culture and subsequent preparation of samples for confocal microscopy. The genipin-crosslinked PEG-treated membranes were thicker than the non-treated membranes and displayed intense auto-fluorescence, as seen in (C) and (D).
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jfb-06-00345-f008: Histology by confocal microscopy after cultivation of primary human CLECs for 12 days on non-treated (A,B) and genipin-crosslinked PEG-treated (C,D) BMSF membranes: without feeder cells (A,C); co-cultured with feeder cells (irradiated 3T3 murine fibroblasts) (B,D). The feeder cells have become dislodged during culture and subsequent preparation of samples for confocal microscopy. The genipin-crosslinked PEG-treated membranes were thicker than the non-treated membranes and displayed intense auto-fluorescence, as seen in (C) and (D).

Mentions: After 12 days of growth, all cultures were fixed and subsequently stained with rhodamine phalloidin (to display F-actin filaments) and Hoechst nuclear dye (to display cell nuclei). Using confocal fluorescence microscopy, a high-resolution optical cross-section was obtained through each culture when folded and mounted in glycerol under a glass coverslip (Figure 8). This technique revealed that human CLEC cultures grown on fibroin membranes are consistently more stratified when an underlying layer of irradiated 3T3 cells is present, and the stratification was observed irrespectively of treatment with PEG. Nevertheless, the cells present within the cultures grown on PEG-treated membranes, in the presence of feeder cells, displayed a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. This observation tends to support the conclusions of Higa et al. [56] that superior growth is seen using PEG-treated membranes. Logically, this enhanced growth is due at least in part to the presence of feeder cells, but since cultures grown on non-treated membranes also displayed increased stratification, we cannot discount the potential role of changes in membrane topography created by PEG in conjunction with effects mediated by the feeder cells.


Treatment of Silk Fibroin with Poly(ethylene glycol) for the Enhancement of Corneal Epithelial Cell Growth.

Suzuki S, Dawson RA, Chirila TV, Shadforth AM, Hogerheyde TA, Edwards GA, Harkin DG - J Funct Biomater (2015)

Histology by confocal microscopy after cultivation of primary human CLECs for 12 days on non-treated (A,B) and genipin-crosslinked PEG-treated (C,D) BMSF membranes: without feeder cells (A,C); co-cultured with feeder cells (irradiated 3T3 murine fibroblasts) (B,D). The feeder cells have become dislodged during culture and subsequent preparation of samples for confocal microscopy. The genipin-crosslinked PEG-treated membranes were thicker than the non-treated membranes and displayed intense auto-fluorescence, as seen in (C) and (D).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493516&req=5

jfb-06-00345-f008: Histology by confocal microscopy after cultivation of primary human CLECs for 12 days on non-treated (A,B) and genipin-crosslinked PEG-treated (C,D) BMSF membranes: without feeder cells (A,C); co-cultured with feeder cells (irradiated 3T3 murine fibroblasts) (B,D). The feeder cells have become dislodged during culture and subsequent preparation of samples for confocal microscopy. The genipin-crosslinked PEG-treated membranes were thicker than the non-treated membranes and displayed intense auto-fluorescence, as seen in (C) and (D).
Mentions: After 12 days of growth, all cultures were fixed and subsequently stained with rhodamine phalloidin (to display F-actin filaments) and Hoechst nuclear dye (to display cell nuclei). Using confocal fluorescence microscopy, a high-resolution optical cross-section was obtained through each culture when folded and mounted in glycerol under a glass coverslip (Figure 8). This technique revealed that human CLEC cultures grown on fibroin membranes are consistently more stratified when an underlying layer of irradiated 3T3 cells is present, and the stratification was observed irrespectively of treatment with PEG. Nevertheless, the cells present within the cultures grown on PEG-treated membranes, in the presence of feeder cells, displayed a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. This observation tends to support the conclusions of Higa et al. [56] that superior growth is seen using PEG-treated membranes. Logically, this enhanced growth is due at least in part to the presence of feeder cells, but since cultures grown on non-treated membranes also displayed increased stratification, we cannot discount the potential role of changes in membrane topography created by PEG in conjunction with effects mediated by the feeder cells.

Bottom Line: The resulting membranes were thoroughly characterized and compared to the non-treated membranes.The crosslinking with genipin did not induce a significant improvement in mechanical properties.The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.

View Article: PubMed Central - PubMed

Affiliation: Queensland Eye Institute, South Brisbane, Queensland 4101, Australia. shuko.suzuki@qei.org.au.

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.

No MeSH data available.


Related in: MedlinePlus