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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

Attachment and proliferation of cells of HCE-T line on BMSF membranes. (a) Cellular attachment in serum-free medium; (b) Proliferation in serum-supplemented medium on non-treated fibroin membrane (black), genipin-crosslinked PEG-treated fibroin membrane (white) and TCP (grey). Numbers of cells were measured via quantification of DNA content (PicoGreen® assay). Bars represent mean ± standard error of the mean. The asterisk indicates that the difference is statistically significant (p < 0.05).
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jfb-06-00345-f006: Attachment and proliferation of cells of HCE-T line on BMSF membranes. (a) Cellular attachment in serum-free medium; (b) Proliferation in serum-supplemented medium on non-treated fibroin membrane (black), genipin-crosslinked PEG-treated fibroin membrane (white) and TCP (grey). Numbers of cells were measured via quantification of DNA content (PicoGreen® assay). Bars represent mean ± standard error of the mean. The asterisk indicates that the difference is statistically significant (p < 0.05).

Mentions: The attachment and proliferation of an SV40-immortalized cell line (HCE-T) was examined on membranes (ca. 6 µm in thickness) placed at the bottom of the culture-plate wells. Since these cells can be serially propagated in the absence of feeder cells, they provided a useful model of the human corneal epithelial cells’ growth in the absence of any accessory cells. The numbers of adherent cells was expressed as the total DNA content with the PicoGreen® assay (Figure 6). In a short-term attachment assay (over a period of 90 min), no quantitative difference between the numbers of cells attached to the genipin-crosslinked PEG-treated and those attached to non-treated membranes in serum-free conditions was noticed (Figure 6a), but they were significantly lower than the number of cells attached to the TCP control. In longer-term cultures (up to seven days), cell growth on the PEG-treated membrane was higher than that on non-treated membrane in serum-supplemented growth medium, albeit the differences were not statistically significant (Figure 6b). However, the level of cell growth on the non-treated membranes was found to be significantly lower than that on TCP substrata.


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)

Attachment and proliferation of cells of HCE-T line on BMSF membranes. (a) Cellular attachment in serum-free medium; (b) Proliferation in serum-supplemented medium on non-treated fibroin membrane (black), genipin-crosslinked PEG-treated fibroin membrane (white) and TCP (grey). Numbers of cells were measured via quantification of DNA content (PicoGreen® assay). Bars represent mean ± standard error of the mean. The asterisk indicates that the difference is statistically significant (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

jfb-06-00345-f006: Attachment and proliferation of cells of HCE-T line on BMSF membranes. (a) Cellular attachment in serum-free medium; (b) Proliferation in serum-supplemented medium on non-treated fibroin membrane (black), genipin-crosslinked PEG-treated fibroin membrane (white) and TCP (grey). Numbers of cells were measured via quantification of DNA content (PicoGreen® assay). Bars represent mean ± standard error of the mean. The asterisk indicates that the difference is statistically significant (p < 0.05).
Mentions: The attachment and proliferation of an SV40-immortalized cell line (HCE-T) was examined on membranes (ca. 6 µm in thickness) placed at the bottom of the culture-plate wells. Since these cells can be serially propagated in the absence of feeder cells, they provided a useful model of the human corneal epithelial cells’ growth in the absence of any accessory cells. The numbers of adherent cells was expressed as the total DNA content with the PicoGreen® assay (Figure 6). In a short-term attachment assay (over a period of 90 min), no quantitative difference between the numbers of cells attached to the genipin-crosslinked PEG-treated and those attached to non-treated membranes in serum-free conditions was noticed (Figure 6a), but they were significantly lower than the number of cells attached to the TCP control. In longer-term cultures (up to seven days), cell growth on the PEG-treated membrane was higher than that on non-treated membrane in serum-supplemented growth medium, albeit the differences were not statistically significant (Figure 6b). However, the level of cell growth on the non-treated membranes was found to be significantly lower than that on TCP substrata.

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