Effects of Epidermal Growth Factor and Keratinocyte Growth Factor on the Growth of Oropharyngeal Keratinocytes in Coculture with Autologous Fibroblasts in a Three-Dimensional Matrix

Abstract

Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of β₁-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15–1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigel™. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct.

Effects of epidermal growth factor on the proliferation and cell cycle regulation of cultured human amnion epithelial cells

“…This is similar with previous studies on other epithelial cells such as epidermal keratinocytes (11,12,16) and oropharyngeal keratinocytes (14). In some other studies, different concentrations of EGF were added to HAECs culture medium (2,3,7,8) to support the cells' proliferation.…”

“…EGF plays an important role in cell proliferation, migration and locomotion (11e13). It induces the proliferation of different types of cells, for instance, the growth of keratinocytes of the skin and oropharynx, and retinal pigment epithelial cells, without inducing differentiation (11,12,14,15). EGF and its related peptides have been implicated in the promotion of cell proliferation in wound healing such as in re-epithelialization (12).…”

Adaptive informatics for multifactorial and high-content biological data

“…5a), we observed maximum variability at physiologically relevant doses of drug and ligand (estimated to be 0.1–1.5 ng/mL for EGF and 0.4–50 µM for gefitinib 21,22 ). Mean value and variance in response changed over time, such that 20 hours post-EGF/gefitinib treatment, IC 50 was less dependent on [EGF] but the variance increased.…”

A New System for Cultivation of Human Keratinocytes on Acellular Dermal Matrix Substitute with the Use of Human Fibroblast Feeder Layer

“…This may be related to the increased amount of fibroblasts. KGF (KGF/FGF7) acts specifically on epithelial cells, promoting proliferation and inhibiting apoptosis of keratinocytes and preventing them from differentiation [Blaimauer et al, 2006]. IL-6 is an epithelial cell mitogen, able to speed up epithelialization of the wound surface, while IL-8 is a proinflammatory cytokine, able to promote growth and proliferation of epithelial cells.…”