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Substrate stiffness does affect the fate of human keratinocytes
P. Gupta, G. Hari Narayana S. N., U. Kasiviswanathan, T. Agarwal, K. Senthilguru, D. Mukhopadhyay, K. Pal, S. Giri, T.K. Maiti,
Published in Royal Society of Chemistry
Volume: 6
Issue: 5
Pages: 3539 - 3551
Recent reports on the paradoxical behaviour of epithelial cells in response to the varying stiffness of a polydimethyl siloxane (PDMS) substrate has made 'matrix stiffness dependent cellular mechanotransduction' an open issue for further investigation. In this context, we have tried to address the fundamental question, "Are human keratinocytes sensitive towards PDMS substrate stiffness?". To decipher the underlying relationship between PDMS substrate stiffness and keratinocyte mechanotransduction, we modulated the stiffness of the PDMS substrate across a physiological range (elastic modulus ranging from 1.6 MPa to 0.05 MPa) and characterized the behaviour of human skin keratinocytes (HaCaT cells). Preliminary analysis of the topographical features (contact angle, protein adsorption and distribution, and surface roughness) and mechanical properties (elastic modulus, stress relaxation time and ductility) ensured that all the PDMS substrates were topographically similar but differed in their mechanical properties. Matrix stiffness dependent variation in the cellular response was contoured, qualitatively and quantitatively, by mapping the cytoskeletal organization (FESEM and immunocytochemistry) and studying cell proliferation (live cell population assay, MTT assay in the presence and absence of mechanotransduction pathway blockers and flow cytometry analysis of proliferative cell population). The result showed that there was a significant increase in cell proliferation with increasing matrix stiffness whereas cell spreading was affected differentially. Mechanistic analysis revealed that stiffness induced cell proliferation was β-catenin independent but ERK1/2 dependent. Analysis at the nuclear level, showed that the soft surface caused nuclear mechanotransduction (evident from nuclear lamin A/C expression) and perturbed the transcription pattern of protein (VEGF as model protein) in HaCaT cells. This study confirmed that the human keratinocytes are mechanosensitive towards the stiffness of a PDMS substrate. © 2016 The Royal Society of Chemistry.
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JournalData powered by TypesetRSC Advances
PublisherData powered by TypesetRoyal Society of Chemistry