Most studies examining the influence of growth factor on keratocytes in vitro have used serum supplemented media which promotes fibroblastic differentiation. The purpose of this study was to evaluate the effect of growth factors on the regulation of the keratocyte phenotype and extracellular matrix (ECM) production in vitro using a serum free media that has been shown to promote a keratocyte phenotype.Methods
Human keratocytes were cultured in keratocyte media (KM) consisting of Advanced DMEM, supplemented with GlutaMax, FGF-2 and L-ascorbic-2-phosphate. Cells were cultures for 14 days and supplemented with IGF-1, TGFβ-1 or TGFβ-3. DNA, sGAG and collagen synthesis were determined via biochemical assay. Expression of corneal, fibrotic and ECM related genes were examined by real-time PCR. ECM production was also examined by histological and immunofluorescent staining.Results
TGFβ-3 stimulated the highest level of proliferation and sGAG synthesis. In contrast, IGF-1 and TGFβ-1 showed significantly lower levels of DNA synthesis and sGAG production, comparable to KM alone. KM and TGFβ-3 showed the highest level of expression for keratocan. ECM and collagen production was evident for both the KM and TGFβ-3 group. The expression of αSMA was greatest for TGFβ-3, while KDM demonstrated the highest expression levels for both collagen type I and III.Conclusion
These results demonstrate the influence different growth factors have on the phenotypic behaviour of keratocytes in serum free media. Further study in 3D culture will provide a useful model to further understand the effect growth factors have on keratocyte behaviour.