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Hydrogel Viscoelasticity Influences Mechanotransduction and Chondrogenesis of Mesenchymal Stem Cells

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Hydrogel Viscoelasticity Influences Mechanotransduction and Chondrogenesis of Mesenchymal Stem Cells HYDROGEL VISCOELASTICITY INFLUENCES MECHANOTRANSDUCTION AND CHONDROGENESIS OF MESENCHYMAL STEM CELLS Matthew Walker (1), Marco Cantini (1) 1. Centre for the Cellular Microenvironment, University of Glasgow, UK Introduction increased early chondrogenesis (SOX9 upregulation Hydrogel stiffness has been long recognised to and RUNX2 downregulation) and late determine human mesenchymal stem cell chondrogenesis (COL2A1 and aggrecan (hMSC) differentiation, whereas viscous upregulation), while preventing an hypertrophic interactions are only recently emerging as powerful phenotype. hMSC behaviour and fate was also regulators of stem cell fate [1, 2]. For example, while influenced by ratios of RGD/HAVDI. Importantly, viscoelastic hydrogels can promote hMSC spreading 3D culture within viscous PEG gels also promoted and proliferation, elastic hydrogels can inhibit cell enhanced chondrogenesis via similar proliferation and, in the case of chondrocytes, limit mechanotransductive mechanisms. cartilage matrix formation [3]. Moreover, hydrogels Discussion can be functionalised with peptide motifs to These novel findings demonstrate that viscous improve hMSC attachment and influence cell fate: interactions both in 2D and 3D can be modulated to for example, chondrogenesis can be controlled by target key mechanotransductive events, leading to peptide gradients using integrin receptor RGD and the activation of chondrogenic pathways in hMSCs cadherin ligand HAVDI [4]. However, as a consequence of substrate viscosity. investigations into how the viscoelastic properties of hydrogels influence the chondrogenic differentiation of hMSCs are lacking. In this work, we investigate whether viscoelastic hydrogels with variable viscous properties and consistent elasticity can provide a cellular micro- environment conducive to the chondrogenic differentiation of hMSCs. Methods Polyacrylamide (PAAM) and polyethylene glycol (PEG) hydrogels were prepared using acrylamide and cross-linker N, N'-Methylene bisacrylamide, or PEG macromers and protease degradable VPM peptide cross-linkers, respectively. Elastic and viscous properties of the hydrogels were measured using AFM and rheology. hMSCs were cultured on RGD/HAVDI-functionalised PAAM hydrogels and within RGD-functionalised PEG hydrogels. Cell cultures were analysed by immunoflourescence Figure 1. A) Sketch and AFM mechanical microscopy and qPCR for adhesion behaviour, as characterization by microrheology (left) and well as markers of mechanotransduction, early nanoindentation (right) of viscoelastic PAAM gels. chondrogenesis, cartilage matrix, and chondrocyte B) Mechanotransduction (quantification of IF of hypertrophy. YAP and nuclear lamins). C) Early chondrogenesis (SOX9) and neocartilage formation (Col II). D) Results Representative IF images of matrix secretion. We prepared viscoelastic PAAM and PEG gels with References different polymer:crosslinker ratios that maintain similar Young’s moduli (~13 kPa), whilst exhibiting 1. Chaudhuri O et al. Nat Mater 15:326, 2016. 2. Cantini M et al. Adv Healthc Mater 1901259, 2019. differences in viscosity (~two-fold loss modulus 3. Lee HP et al. Nat Mater 16:1243, 2017. change), as measured by AFM nanoindentation and 4. Vega S et al. Nat Comm 9, 2018. microrheology, and confirmed by bulk rheology. Acknowledgements 2D culture on PAAM gels functionalised with RGD showed that more viscous substrates prompted The authors acknowledge funding from MRC and decreased hMSC spreading, modulation of adhesion UKRMP (MR/S005412/1). Help and guidance by proteins expression, different mechanotransduction members of the Centre for the Cellular Micro- (decreased nuclear YAP and lamin A/C:B1 ratio), environment is also acknowledged. 26th Congress of the European Society of Biomechanics, July 12-15, 2020, Milan, Italy .
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