Abstract
Purpose: :
Biomechanics of the extracellular microenvironment is crucial for cell functions including growth and differentiation. Therefore, we created an experimental microenvironment for corneal epithelial cells to analyze these functions at defined biomechanics.
Methods: :
Corneal keratinocytes were cultured on micropillar interfaces for up to 72 hours. For defined biomechanics, interfaces comprised a micropattern range from 3 - 11µm. Cell specimen were proceeded for scanning electron microscopy (SEM) and indirect immunofluorescence (IIF) for keratins K12, K19, involucrin, fillagrin and ABCG2 indicating progressive differentiation.
Results: :
In SEM, regular morphogenesis concomitant with proceeding cell growth was denoted at small pillar micropatterns, i.e. 4, 5 and 5µm, while progressively deranged morphology was observed at rising interpillar distances, suggesting higher adhesion efficiency at small micropatterns. IIF revealed that terminal differentiation marker expression K12, fillagrin and involucrin increased with rising interpillar distances, while early marker K19 showed inverse expression. ABCG2 showed increasing expression with larger interpillar distances, which points to the increased expression of the marker during cell stress. This demonstrates that smaller micropattern favour early, and large-scale pillar patterns terminal differentiation of corneal keratinocytes.
Conclusions: :
We show that morphogenesis, adhesion and growth of corneal keratinocytes in conjunction with expression of biomarkers indicating early and late stages of differentiation are clearly directed by the pillar micropattern, indicating that environmental biomechanics governs cell behaviour. This points to a special microenvironment needed for each cell type and differentiation status, in order to have cells fulfil their function. Determination of the biomechanics of this microenvironment might be crucial for the construction of an artificial niche for corneal stem cells.
Keywords: cornea: epithelium