PEG-phosphorylcholine hydrogels as tunable and versatile platforms for mechanobiology.

We report here the synthesis of a new class of hydrogels with an extremely wide range of mechanical properties suitable for cell studies. Mechanobiology has emerged as an important field in bioengineering, in part due to the development of synthetic polymer gels and fibrous protein biomaterials to control and quantify how cells sense and respond to mechanical forces in their microenvironment. To address the problem of limited availability of biomaterials, in terms of both mechanical range and optical clarity, we have prepared hydrogels that combine poly(ethylene glycol) (PEG) and phosphorylcholine (PC) zwitterions. Our goal was to create a hydrogel platform that exceeds the range of Young's moduli reported for similar hydrogels, while being simple to synthesize and manipulate. The Young's modulus of these "PEG-PC" hydrogels can be tuned over 4 orders of magnitude, much greater than commonly used hydrogels such as PEG-diacrylate, PEG-dimethacrylate, and polyacrylamide, with smaller average mesh sizes and optical clarity. We prepared PEG-PC hydrogels to study how substrate mechanical properties influence cell morphology, focal adhesion structure, and proliferation across multiple mammalian cell lines, as a proof of concept. These novel PEG-PC biomaterials represent a new and useful class of mechanically tunable hydrogels for mechanobiology.

[1]  Kazuhiko Ishihara,et al.  Synthesis of hydrophilic cross-linker having phosphorylcholine-like linkage for improvement of hydrogel properties , 2004 .

[2]  Kazuhiko Ishihara,et al.  Preparation of cross-linked biocompatible poly(2-methacryloyloxyethyl phosphorylcholine) gel and its strange swelling behavior in water/ethanol mixture , 2002, Journal of biomaterials science. Polymer edition.

[3]  Joyce Y. Wong,et al.  Surface probe measurements of the elasticity of sectioned tissue, thin gels and polyelectrolyte multilayer films : correlations between substrate stiffness and cell adhesion , 2004 .

[4]  T. Jones,et al.  Swelling behaviour of crosslinked hydrogels based on (2-hydroxyethyl methacrylate) with a zwitterionic comonomer (1-3-sulfopropyl-2-vinyl-pyridinium-betaine) , 2007 .

[5]  Mikala Egeblad,et al.  Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling , 2009, Cell.

[6]  P. Parker,et al.  Integrin-specific signaling pathways controlling focal adhesion formation and cell migration , 2003, The Journal of cell biology.

[7]  M. Schwartz,et al.  Mechanotransduction in vascular physiology and atherogenesis , 2009, Nature Reviews Molecular Cell Biology.

[8]  D. Seliktar,et al.  Protein-polymer conjugates for forming photopolymerizable biomimetic hydrogels for tissue engineering. , 2007, Biomaterials.

[9]  J. Hubbell,et al.  Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing. , 1998, Journal of biomedical materials research.

[10]  Milan Makale,et al.  Cellular mechanobiology and cancer metastasis. , 2007, Birth defects research. Part C, Embryo today : reviews.

[11]  Jianping Fu,et al.  Synergistic regulation of cell function by matrix rigidity and adhesive pattern. , 2011, Biomaterials.

[12]  W. G. van der Wiel,et al.  Josephson supercurrent through a topological insulator surface state. , 2011, Nature materials.

[13]  Dorian Liepmann,et al.  Cell-shape regulation of smooth muscle cell proliferation. , 2009, Biophysical journal.

[14]  L. Addadi,et al.  Hierarchical assembly of cell-matrix adhesion complexes. , 2004, Biochemical Society transactions.

[15]  Sanjay Kumar,et al.  Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform. , 2011, Biomaterials.

[16]  S. Chien,et al.  Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity , 2011, Proceedings of the National Academy of Sciences.

[17]  Antonios G Mikos,et al.  Effect of poly(ethylene glycol) molecular weight on tensile and swelling properties of oligo(poly(ethylene glycol) fumarate) hydrogels for cartilage tissue engineering. , 2002, Journal of biomedical materials research.

[18]  Takehiko Kitamori,et al.  The biological performance of cell-containing phospholipid polymer hydrogels in bulk and microscale form. , 2010, Biomaterials.

[19]  Giacomo Belli,et al.  Liver stiffness measurement predicts severe portal hypertension in patients with HCV‐related cirrhosis , 2007, Hepatology.

[20]  Stéphane Laurent,et al.  Structural and Genetic Bases of Arterial Stiffness , 2005, Hypertension.

[21]  N. Peppas,et al.  Correlation between mesh size and equilibrium degree of swelling of polymeric networks. , 1989, Journal of biomedical materials research.

[22]  S. Omata,et al.  Variations in local elastic modulus along the length of the aorta as observed by use of a scanning haptic microscope (SHM) , 2011, Journal of Artificial Organs.

[23]  J. Hubbell,et al.  Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels. , 1998, Biomaterials.

[24]  W. Xue,et al.  Observations on the swelling characteristics of the zwitterionic hydrogel of poly(1-3-sulfopropyl)-2-vinyl-pyridinium-betaine hydrogel , 2006 .

[25]  P. Janmey,et al.  Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli. , 2009, Tissue engineering. Part A.

[26]  Shelly R Peyton,et al.  The effects of matrix stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D biosynthetic hydrogel system. , 2008, Biomaterials.

[27]  E. Cosgriff-Hernandez,et al.  Compositional control of poly(ethylene glycol) hydrogel modulus independent of mesh size. , 2011, Journal of biomedical materials research. Part A.

[28]  Albert J. Keung,et al.  Substrate modulus directs neural stem cell behavior. , 2008, Biophysical journal.

[29]  J. Hubbell,et al.  Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration. , 2005, Biophysical journal.

[30]  P. Janmey,et al.  Biomechanics and Mechanotransduction in Cells and Tissues Cell type-specific response to growth on soft materials , 2005 .

[31]  Chu Zhang,et al.  Microfabricated electrospun collagen membranes for 3-D cancer models and drug screening applications. , 2009, Biomacromolecules.

[32]  Cynthia A. Reinhart-King,et al.  Indentation measurements of the subendothelial matrix in bovine carotid arteries. , 2011, Journal of biomechanics.

[33]  Stephanie J Bryant,et al.  Controlling the spatial distribution of ECM components in degradable PEG hydrogels for tissue engineering cartilage. , 2003, Journal of biomedical materials research. Part A.

[34]  Paul A. Janmey,et al.  Soft biological materials and their impact on cell function. , 2007, Soft matter.

[35]  C M Lapiere,et al.  In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton. , 2001, Cardiovascular research.

[36]  P Zioupos,et al.  Mechanical properties and the hierarchical structure of bone. , 1998, Medical engineering & physics.

[37]  Lewis,et al.  Phosphorylcholine-based polymers and their use in the prevention of biofouling. , 2000, Colloids and surfaces. B, Biointerfaces.

[38]  D J Mooney,et al.  Alginate hydrogels as synthetic extracellular matrix materials. , 1999, Biomaterials.

[39]  Shaoyi Jiang,et al.  Physical, chemical, and chemical-physical double network of zwitterionic hydrogels. , 2008, The journal of physical chemistry. B.

[40]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by substrate flexibility. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Joyce Y. Wong,et al.  Directed Movement of Vascular Smooth Muscle Cells on Gradient-Compliant Hydrogels† , 2003 .

[42]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by the mechanical properties of the substrate. , 1998, The Biological bulletin.

[43]  B. Geiger,et al.  Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Adam J. Engler,et al.  Myotubes differentiate optimally on substrates with tissue-like stiffness , 2004, The Journal of cell biology.

[45]  Jennifer S. Park,et al.  The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β. , 2011, Biomaterials.

[46]  Radhika Desai,et al.  ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix , 2003, The Journal of cell biology.

[47]  Brian P Helmke,et al.  Mechanisms of mechanotransduction. , 2006, Developmental cell.

[48]  H. Yoshida,et al.  Assessing liver tumor stiffness by transient elastography , 2007, Hepatology international.

[49]  Daniel Gioeli,et al.  Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype , 2010, PloS one.

[50]  K. Anseth,et al.  Attachment of fibronectin to poly(vinyl alcohol) hydrogels promotes NIH3T3 cell adhesion, proliferation, and migration. , 2001, Journal of biomedical materials research.

[51]  D. Irvine,et al.  Inverse opal hydrogel-collagen composite scaffolds as a supportive microenvironment for immune cell migration. , 2008, Journal of biomedical materials research. Part A.

[52]  S. Bhatia,et al.  Three-Dimensional Photopatterning of Hydrogels Containing Living Cells , 2002 .

[53]  Shelly R. Peyton,et al.  Extracellular matrix rigidity governs smooth muscle cell motility in a biphasic fashion , 2005, Journal of cellular physiology.

[54]  Yung Chang,et al.  Biofouling-resistance expanded poly(tetrafluoroethylene) membrane with a hydrogel-like layer of surface-immobilized poly(ethylene glycol) methacrylate for human plasma protein repulsions , 2008 .

[55]  Rachelle N. Palchesko,et al.  Development of Polydimethylsiloxane Substrates with Tunable Elastic Modulus to Study Cell Mechanobiology in Muscle and Nerve , 2012, PloS one.

[56]  Douglas A Lauffenburger,et al.  Marrow‐Derived stem cell motility in 3D synthetic scaffold is governed by geometry along with adhesivity and stiffness , 2010, Biotechnology and bioengineering.

[57]  Jacques Ohayon,et al.  Mapping elasticity moduli of atherosclerotic plaque in situ via atomic force microscopy. , 2011, Journal of structural biology.

[58]  T. Emrick,et al.  Polymeric phosphorylcholine-camptothecin conjugates prepared by controlled free radical polymerization and click chemistry. , 2009, Bioconjugate chemistry.

[59]  Sanjay Kumar,et al.  Mechanics, malignancy, and metastasis: The force journey of a tumor cell , 2009, Cancer and Metastasis Reviews.

[60]  M. Dembo,et al.  Cell movement is guided by the rigidity of the substrate. , 2000, Biophysical journal.

[61]  N. Tirelli,et al.  Network connectivity, mechanical properties and cell adhesion for hyaluronic acid/PEG hydrogels. , 2011, Biomaterials.

[62]  Shelly R. Peyton,et al.  The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function , 2007, Cell Biochemistry and Biophysics.

[63]  Nic D. Leipzig,et al.  The effect of substrate stiffness on adult neural stem cell behavior. , 2009, Biomaterials.

[64]  Jason A. Burdick,et al.  Spatially controlled hydrogel mechanics to modulate stem cell interactions , 2010 .

[65]  S. Sen,et al.  Matrix Elasticity Directs Stem Cell Lineage Specification , 2006, Cell.

[66]  Dennis Discher,et al.  Substrate compliance versus ligand density in cell on gel responses. , 2004, Biophysical journal.

[67]  Florian Rehfeldt,et al.  Hyaluronic acid matrices show matrix stiffness in 2D and 3D dictates cytoskeletal order and myosin-II phosphorylation within stem cells. , 2012, Integrative biology : quantitative biosciences from nano to macro.

[68]  N. Jeon,et al.  The effect of matrix density on the regulation of 3-D capillary morphogenesis. , 2008, Biophysical journal.

[69]  Jennifer L West,et al.  Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fluorogenic substrate to visualize collagenase activity during three-dimensional cell migration. , 2007, Biomaterials.

[70]  Shelly R. Peyton,et al.  The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells. , 2006, Biomaterials.

[71]  Joyce Y Wong,et al.  Evaluation of polydimethylsiloxane scaffolds with physiologically-relevant elastic moduli: interplay of substrate mechanics and surface chemistry effects on vascular smooth muscle cell response. , 2005, Biomaterials.

[72]  Paul A. Janmey,et al.  Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation , 2009, PloS one.

[73]  B. Nebe,et al.  Control of focal adhesion dynamics by material surface characteristics. , 2005, Biomaterials.

[74]  Kazuhiko Ishihara,et al.  Super-hydrophilic silicone hydrogels with interpenetrating poly(2-methacryloyloxyethyl phosphorylcholine) networks. , 2010, Biomaterials.

[75]  Dany J. Munoz-Pinto,et al.  Uncoupled investigation of scaffold modulus and mesh size on smooth muscle cell behavior. , 2009, Journal of biomedical materials research. Part A.

[76]  Laurent Castera,et al.  Accuracy of liver stiffness measurement for the diagnosis of cirrhosis in patients with chronic liver diseases , 2006, Hepatology.

[77]  Tatiana Segura,et al.  The effect of enzymatically degradable poly(ethylene glycol) hydrogels on smooth muscle cell phenotype. , 2008, Biomaterials.

[78]  Wesley R. Legant,et al.  Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels , 2013, Nature materials.

[79]  D. Lauffenburger,et al.  Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[80]  Adam J Engler,et al.  Hydrogels with time-dependent material properties enhance cardiomyocyte differentiation in vitro. , 2011, Biomaterials.

[81]  V. Weaver,et al.  Effect of substrate stiffness and PDGF on the behavior of vascular smooth muscle cells: Implications for atherosclerosis , 2010, Journal of cellular physiology.