Keloid scar formation is associated with aberrant inflammatory signalling however the underlying causes remain poorly understood.  Additionally, keloid scars have been shown to be 10-50 times stiffer than normal skin and often form in areas of the body where skin tension is high. As a result, it is thought that the formation of keloid scars may be influenced by mechanical forces. The aim of this study was to investigate the direct effects of tissue stiffness on the behaviour of normal (NF) and keloid fibroblasts (KF) and explore the potential cross-talk with inflammatory signalling pathways. First, we characterised the stiffness dependent responses within an immortalised fibroblast cell line, HCA2, when cultured onto collagen-coated polyacrylamide hydrogels mimicking the stiffness of normal skin (8kPa) and keloid skin (70-214kPa). HCA2 cells showed stiffness dependent changes in cell spreading, F-actin intensity, and focal adhesion (FA) assembly.  In addition, phospho-SMAD2 activation following stimulation with TGF-β was higher on stiff gels. Secondly, we extended this technique to study primary NFs and KFs from three different donors. KFs were more spread, exhibited more FAs and more intense F-actin stress fibres than NFs, particularly on the stiff hydrogels. Additionally, both cell types demonstrated a stiffness dependent response in phospho-SMAD2 when stimulated with TGF-. Finally, after culturing both KFs and NFs on the hydrogels for 10 days we studied the cell derived matrix of each cell type. This analysis revealed that substrate stiffness regulates extracellular matrix (ECM) assembly with increased total fibronectin deposited on the stiff substrates. The ECM on the stiff substrates showed smaller but more densely packed fibronectin fibres, and the ECM assembled by the KFs showed thicker and more aligned fibronectin fibres compared to NFs. Collectively, these results provide further insight into the mechanical regulation of dermal fibroblast behaviour showing a distinctive difference between KFs and NFs. The increased stress fibre and FA formation suggest that KFs are more contractile and mechano-sensitive than NFs.

Mechano-sensing and inflammatory signalling in normal and keloid dermal fibroblasts.