The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells.

Hydrogels based on poly(ethylene glycol) (PEG) are of increasing interest for regenerative medicine applications and are ideal materials to direct cell function due to the ability to confer key functionalities of native extracellular matrix (ECM) on PEG's otherwise inert backbone. Given extensive recent evidence that ECM compliance influences a variety of cell functions, PEG-based hydrogels are also attractive due to the ease with which their mechanical properties can be controlled. In these studies, we exploited the chemical and mechanical tunability of PEG-based gels to study the impact of ECM chemistry and mechanics on smooth muscle cells (SMCs) in both 2-D and 3-D model systems. First, by controlling the extent of crosslinking and therefore the mechanical properties of PEG-based hydrogels (tensile moduli from 13.7 to 423.9kPa), we report here that the assembly of F-actin stress fibers and focal adhesions, indicative of the state of actin contractility, were influenced by the compliance of 2-D PEG gels functionalized with either short adhesive peptides or full-length ECM proteins. Varying ECM ligand density and identity independent of gel compliance affected the physical properties of the focal adhesions, and also influenced SMC spreading in 2-D. Furthermore, SMCs proliferated to a greater extent as gel stiffness was increased. In contrast, the degree of SMC differentiation, which was qualitatively assessed by the extent of smooth muscle alpha-actin bundling and the association of calponin and caldesmon with the alpha-actin fibrils, was found to decrease with substrate stiffness in 2-D cultures. In 3-D, despite the fact that their viability and degree of spreading were greatly reduced, SMCs did express some contractile markers indicative of their differentiated phenotype when cultured within PEG-RGDS constructs. Combined, these data suggest that the mechanical and chemical properties of PEG hydrogels can be tuned to influence SMC phenotype in both 2-D and 3-D.

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

[2]  D. Mooney,et al.  Cyclic strain inhibits switching of smooth muscle cells to an osteoblast‐like phenotype , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[3]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

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

[5]  Joyce Y Wong,et al.  Direct comparison of the spread area, contractility, and migration of balb/c 3T3 fibroblasts adhered to fibronectin- and RGD-modified substrata. , 2004, Biophysical journal.

[6]  A. Tedgui,et al.  Signal transduction of mechanical stresses in the vascular wall. , 1998, Hypertension.

[7]  Martin Bastmeyer,et al.  Cell behaviour on micropatterned substrata: limits of extracellular matrix geometry for spreading and adhesion , 2004, Journal of Cell Science.

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

[9]  J. Thyberg,et al.  Regulation of differentiated properties and proliferation of arterial smooth muscle cells. , 1990, Arteriosclerosis.

[10]  S. Bryant,et al.  Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. , 2002, Journal of biomedical materials research.

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

[12]  J. West,et al.  Cell migration through defined, synthetic extracellular matrix analogues , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  A. Tedgui,et al.  Cellular mechanics and gene expression in blood vessels. , 2003, Journal of biomechanics.

[14]  Shelly R. Peyton,et al.  Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells. , 2006, American journal of physiology. Cell physiology.

[15]  Qingbo Xu,et al.  Mechanical stress-initiated signal transductions in vascular smooth muscle cells. , 2000, Cellular signalling.

[16]  G. Osol.  Mechanotransduction by vascular smooth muscle. , 1995, Journal of vascular research.

[17]  Joe Tien,et al.  Repositioning of cells by mechanotaxis on surfaces with micropatterned Young's modulus. , 2003, Journal of biomedical materials research. Part A.

[18]  Kytai Truong Nguyen,et al.  Photopolymerizable hydrogels for tissue engineering applications. , 2002, Biomaterials.

[19]  Eben Alsberg,et al.  FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Jon A. Rowley,et al.  Controlling Mechanical and Swelling Properties of Alginate Hydrogels Independently by Cross-Linker Type and Cross-Linking Density , 2000 .

[21]  Christopher S. Chen,et al.  Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. , 2004, Developmental cell.

[22]  Jason A Burdick,et al.  Photoinitiated crosslinked degradable copolymer networks for tissue engineering applications. , 2003, Biomaterials.

[23]  J. Hubbell,et al.  Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering , 2005, Nature Biotechnology.

[24]  Jennifer L West,et al.  Cell adhesion peptides alter smooth muscle cell adhesion, proliferation, migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds. , 2002, Journal of biomedical materials research.

[25]  B. Williams.  Mechanical influences on vascular smooth muscle cell function , 1998, Journal of hypertension.

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

[27]  L. Griffith,et al.  Tissue Engineering--Current Challenges and Expanding Opportunities , 2002, Science.

[28]  A. Mikos,et al.  Growing new organs. , 1999, Scientific American.

[29]  Kenneth M. Yamada,et al.  Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions. , 2000, Molecular biology of the cell.

[30]  D. Mooney,et al.  Independent Control of Rigidity and Toughness of Polymeric Hydrogels , 2003 .

[31]  E. Ruoslahti,et al.  Effects of modifications of the RGD sequence and its context on recognition by the fibronectin receptor. , 1989, The Journal of biological chemistry.

[32]  David J. Mooney,et al.  Cyclic mechanical strain regulates the development of engineered smooth muscle tissue , 1999, Nature Biotechnology.

[33]  R M Nerem,et al.  Vascular tissue engineering. , 2001, Annual review of biomedical engineering.

[34]  P. Martens,et al.  Tailoring the degradation of hydrogels formed from multivinyl poly(ethylene glycol) and poly(vinyl alcohol) macromers for cartilage tissue engineering. , 2003, Biomacromolecules.

[35]  K. Anseth,et al.  Synthetic hydrogel niches that promote hMSC viability. , 2005, Matrix biology : journal of the International Society for Matrix Biology.

[36]  H. S. Kim,et al.  Phenotypic Modulation of Smooth Muscle Cells during Progression of Human Atherosclerosis as Determined by Altered Expression of Myosin Heavy Chain Isoforms , 1994, Annals of the New York Academy of Sciences.

[37]  Y. Yazaki,et al.  Redifferentiation of smooth muscle cells after coronary angioplasty determined via myosin heavy chain expression. , 1997, Circulation.

[38]  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.

[39]  Jennifer L. West,et al.  Tethered-TGF-β increases extracellular matrix production of vascular smooth muscle cells , 2001 .

[40]  C. S. Chen,et al.  Geometric control of cell life and death. , 1997, Science.

[41]  Donald E Ingber,et al.  Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress. , 2004, American journal of physiology. Cell physiology.

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

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

[44]  Stephanie J Bryant,et al.  Encapsulating chondrocytes in degrading PEG hydrogels with high modulus: Engineering gel structural changes to facilitate cartilaginous tissue production , 2004, Biotechnology and bioengineering.

[45]  K. Beningo,et al.  Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions. , 2002, Methods in cell biology.

[46]  P. Wolf,et al.  Heart disease and stroke statistics--2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. , 2006, Circulation.

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

[48]  J. Gunn,et al.  Adhesive and mechanical properties of hydrogels influence neurite extension. , 2005, Journal of biomedical materials research. Part A.

[49]  S. Aota,et al.  Molecular diversity of cell-matrix adhesions. , 1999, Journal of cell science.

[50]  D. Seliktar,et al.  Biosynthetic hydrogel scaffolds made from fibrinogen and polyethylene glycol for 3D cell cultures. , 2005, Biomaterials.

[51]  J. West,et al.  Val-ala-pro-gly, an elastin-derived non-integrin ligand: smooth muscle cell adhesion and specificity. , 2003, Journal of Biomedical Materials Research. Part A.

[52]  W McIntosh,et al.  Transdermal photopolymerization for minimally invasive implantation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[53]  J L West,et al.  Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering. , 2001, Biomaterials.

[54]  R Langer,et al.  Functional arteries grown in vitro. , 1999, Science.

[55]  David J. Mooney,et al.  Tissue engineering using synthetic extracellular matrices , 1996, Nature Medicine.

[56]  Jason A Burdick,et al.  Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. , 2002, Biomaterials.

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