The purpose of this study was to determine if non-mulberry varieties of silk are suitable for the culture of corneal endothelium (CE). Methods: Aqueous silk fibroin derived from Philosamia ricini (PR), Antheraea assamensis (AA), and Bombyx mori (BM) were cast as approximately 15 μm films with and without pores on which human CE cells were cultured. Tensile strength, elasticity, transmittance in visible range, and degradation properties of the films were characterised. Adhesion of CE to the silk films was quantified using MTT assay in addition to quantifying the number and area of focal adhesions using paxillin. Expression of CE markers was determined at the gene and protein levels using PCR and immunostaining, respectively. Barrier integrity of the cultured cells was measured as permeability to FITC dextran (10 kDa) in the presence or absence of thrombin. Results: The films exhibited robust tensile strength, >95% transmittance and a refractive index comparable to the native cornea. BM degraded significantly faster when compared to PR and AA. A comparison between the three varieties of silk showed that significantly more cells were adhered to PR and AA than to BM. This was also reflected in the expression of stable focal adhesions on PR and AA, thus enabling the formation of intact monolayers of cells on these varieties unlike on BM. Treatment with thrombin significantly increased cellular permeability to dextran. Conclusions: Our data shows that PR and AA varieties sufficiently support the growth and function of CE cells. This could be attributed to the presence of natural cell binding motifs (RGD) in these varieties.
Developing a scaffold for culturing human corneal endothelial
(HCE)
cells is crucial as an alternative cell therapeutic approach to bridge
the growing gap between the demand and availability of healthy donor
corneas for transplantation. Silk films are promising substrates for
the culture of these cells; however, their tensile strength is several-fold
greater than the native basement membrane which can possibly influence
the dynamics of cell–matrix interaction and the extracellular
matrix (ECM) secreted by the cells in long-term culture. In our current
study, we assessed the secretion of ECM and the expression of integrins
by the HCE cells on Philosamia ricini (PR) and Antheraea assamensis (AA)
silk films and fibronectin-collagen (FNC)-coated plastic dishes to
understand the cell–ECM interaction in long-term culture. The
expression of ECM proteins (collagens 1, 4, 8, and 12, laminin, and
fibronectin) on silk was comparable to that on the native tissue.
The thicknesses of collagen 8 and laminin at 30 days on both PR (4.78
± 0.55 and 5.53 ± 0.51 μm, respectively) and AA (4.66
± 0.72 and 5.71 ± 0.61 μm, respectively) were comparable
with those of the native tissue (4.4 ± 0.63 and 5.28 ± 0.72
μm, respectively). The integrin expression by the cells on the
silk films was also comparable to that on the native tissue, except
for α3 whose fluorescence intensity was significantly higher
on PR (p ≤ 0.01) and AA (p ≤ 0.001), compared to that on the native tissue. This study
shows that the higher tensile strength of the silk films does not
alter the ECM secretion or cell phenotype in long-term culture, confirming
the suitability of using this material for engineering the HCE cells
for transplantation.
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