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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

Relative permeability of BMSF membranes to VEGF. (a) Schematic representation of the permeability experimental setup. (b) Comparison of VEGF concentrations after 24 h in the apical and basal compartments delimiting the membranes.
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jfb-06-00345-f005: Relative permeability of BMSF membranes to VEGF. (a) Schematic representation of the permeability experimental setup. (b) Comparison of VEGF concentrations after 24 h in the apical and basal compartments delimiting the membranes.

Mentions: To estimate the permeability of the BMSF membranes to biomolecules, the growth factor VEGF (vascular endothelial growth factor) was chosen as the permeant molecule, and a method was designed for the purpose (Figure 5a). VEGF has a MW of 26–28 kDa, and plays an important role in certain pathophysiological processes in the eye. In this study, we determined the relative permeability of the non-treated and of the crosslinked PEG-treated membranes. As shown in Figure 5b, the PEG-treated membranes were relatively more permeable to VEGF molecule as compared to the non-treated membranes. This clearly indicates that by blending BMSF with PEG (MW 300 Da), the permeability is enhanced, thus supporting the observations of Higa et al. [56]. Interestingly, approximately 50% and 70% of VEGF (i.e., 7.5 and 10.5 ng) for the non-treated and PEG-treated membranes, respectively, were lost as shown by comparing the total amounts of protein in the apical and basal compartments after 24 h to the initial amounts. This could be due to the trapping of VEGF within BMSF due to electrostatic interactions of positively charged VEGF and negatively charged fibroin molecules.


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)

Relative permeability of BMSF membranes to VEGF. (a) Schematic representation of the permeability experimental setup. (b) Comparison of VEGF concentrations after 24 h in the apical and basal compartments delimiting the membranes.
© Copyright Policy
Related In: Results  -  Collection

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

jfb-06-00345-f005: Relative permeability of BMSF membranes to VEGF. (a) Schematic representation of the permeability experimental setup. (b) Comparison of VEGF concentrations after 24 h in the apical and basal compartments delimiting the membranes.
Mentions: To estimate the permeability of the BMSF membranes to biomolecules, the growth factor VEGF (vascular endothelial growth factor) was chosen as the permeant molecule, and a method was designed for the purpose (Figure 5a). VEGF has a MW of 26–28 kDa, and plays an important role in certain pathophysiological processes in the eye. In this study, we determined the relative permeability of the non-treated and of the crosslinked PEG-treated membranes. As shown in Figure 5b, the PEG-treated membranes were relatively more permeable to VEGF molecule as compared to the non-treated membranes. This clearly indicates that by blending BMSF with PEG (MW 300 Da), the permeability is enhanced, thus supporting the observations of Higa et al. [56]. Interestingly, approximately 50% and 70% of VEGF (i.e., 7.5 and 10.5 ng) for the non-treated and PEG-treated membranes, respectively, were lost as shown by comparing the total amounts of protein in the apical and basal compartments after 24 h to the initial amounts. This could be due to the trapping of VEGF within BMSF due to electrostatic interactions of positively charged VEGF and negatively charged fibroin molecules.

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