Hybrid Elastin-like Polypeptide–Polyethylene Glycol (ELP-PEG) Hydrogels with Improved Transparency and Independent Control of Matrix Mechanics and Cell Ligand Density

Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP’s lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly cross-links with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent anti-Stokes Raman scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 °C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell–matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (>98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics.

[1]  K. Anseth,et al.  Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells. , 2008, Nature materials.

[2]  Murat Guvendiren,et al.  Engineering synthetic hydrogel microenvironments to instruct stem cells. , 2013, Current opinion in biotechnology.

[3]  Mitsuhiro Shibayama,et al.  Spatial inhomogeneity and dynamic fluctuations of polymer gels , 1998 .

[4]  Jason A. Burdick,et al.  Sequential crosslinking to control cellular spreading in 3-dimensional hydrogels , 2009 .

[5]  Ashutosh Chilkoti,et al.  Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair. , 2002, Biomacromolecules.

[6]  P. Ma,et al.  Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties. , 2001, Biomaterials.

[7]  E Ruoslahti,et al.  RGD and other recognition sequences for integrins. , 1996, Annual review of cell and developmental biology.

[8]  Jayanta Debnath,et al.  Modelling glandular epithelial cancers in three-dimensional cultures , 2005, Nature Reviews Cancer.

[9]  Kevin M. Cherry,et al.  Characterization of resilin-based materials for tissue engineering applications. , 2012, Biomacromolecules.

[10]  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.

[11]  Annika Enejder,et al.  Coherent Anti-Stokes Raman Scattering Microscopy of Cellular Lipid Storage , 2010, IEEE Journal of Selected Topics in Quantum Electronics.

[12]  Ravi A. Desai,et al.  Mechanical regulation of cell function with geometrically modulated elastomeric substrates , 2010, Nature Methods.

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

[14]  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.

[15]  K. Lampe,et al.  Tetrakis(hydroxymethyl) phosphonium chloride as a covalent cross-linking agent for cell encapsulation within protein-based hydrogels. , 2012, Biomacromolecules.

[16]  W. Oppermann,et al.  Polyacrylamide-clay nanocomposite hydrogels : Rheological and light scattering characterization , 2007 .

[17]  S. Heilshorn,et al.  Protein-engineered biomaterials: highly tunable tissue engineering scaffolds. , 2010, Tissue engineering. Part B, Reviews.

[18]  Jane A Dickerson,et al.  Current Applications of Liquid Chromatography / Mass Spectrometry in Pharmaceutical Discovery After a Decade of Innovation , 2008 .

[19]  Julie C. Liu,et al.  Cell response to RGD density in cross-linked artificial extracellular matrix protein films. , 2008, Biomacromolecules.

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

[21]  Sarah C. Heilshorn,et al.  Independent tuning of multiple biomaterial properties using protein engineering , 2009 .

[22]  Junmin Lee,et al.  Directing stem cell fate on hydrogel substrates by controlling cell geometry, matrix mechanics and adhesion ligand composition. , 2013, Biomaterials.

[23]  A. Metters,et al.  Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Jonathan R. McDaniel,et al.  Self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation. , 2013, Angewandte Chemie.

[25]  Hamidreza Ghandehari,et al.  Recombinant protein-based polymers for advanced drug delivery. , 2012, Chemical Society reviews.

[26]  David J Mooney,et al.  Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: a combined computational and experimental approach. , 2007, Biomaterials.

[27]  G. Mattson,et al.  A practical approach to crosslinking , 1993, Molecular Biology Reports.

[28]  D. Urry.  Physical Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers† , 1997 .

[29]  D. G. T. Strange,et al.  Extracellular-matrix tethering regulates stem-cell fate. , 2012, Nature materials.

[30]  R. Misra,et al.  Biomaterials , 2008 .

[31]  Ashutosh Chilkoti,et al.  Evaluation of an elastin-like polypeptide-doxorubicin conjugate for cancer therapy. , 2003, Journal of controlled release : official journal of the Controlled Release Society.

[32]  R. Marchant,et al.  Design and synthesis of biomimetic hydrogel scaffolds with controlled organization of cyclic RGD peptides. , 2009, Bioconjugate chemistry.

[33]  F Javier Arias,et al.  Synthesis and characterization of macroporous thermosensitive hydrogels from recombinant elastin-like polymers. , 2009, Biomacromolecules.

[34]  Song Li,et al.  Control of cell adhesion on poly(methyl methacrylate). , 2006, Biomaterials.

[35]  Annika Enejder,et al.  Sequence‐Specific Crosslinking of Electrospun, Elastin‐Like Protein Preserves Bioactivity and Native‐Like Mechanics , 2013, Advanced healthcare materials.

[36]  Kevin E Healy,et al.  Hydrogels as artificial matrices for human embryonic stem cell self-renewal. , 2006, Journal of biomedical materials research. Part A.

[37]  D. Schaffer,et al.  Engineering biomaterials for synthetic neural stem cell microenvironments. , 2008, Chemical reviews.

[38]  Douglas A Lauffenburger,et al.  Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus. , 2002, Journal of cell science.

[39]  J. Elisseeff,et al.  The independent roles of mechanical, structural and adhesion characteristics of 3D hydrogels on the regulation of cancer invasion and dissemination. , 2013, Biomaterials.

[40]  Xinqiao Jia,et al.  Resilin-Like Polypeptide Hydrogels Engineered for Versatile Biological Functions. , 2013, Soft matter.

[41]  Amit Jain,et al.  Probing cellular mechanobiology in three-dimensional culture with collagen-agarose matrices. , 2010, Biomaterials.

[42]  K. Neoh,et al.  Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion. , 2008, Biomaterials.

[43]  A. Chilkoti,et al.  Protein Purification by Fusion with an Environmentally Responsive Elastin‐Like Polypeptide: Effect of Polypeptide Length on the Purification of Thioredoxin , 2001, Biotechnology progress.

[44]  Junmin Zhu,et al.  Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering. , 2010, Biomaterials.

[45]  Mark W. Tibbitt,et al.  Hydrogels as extracellular matrix mimics for 3D cell culture. , 2009, Biotechnology and bioengineering.

[46]  A. Sinz,et al.  Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters , 2008, Analytical and bioanalytical chemistry.

[47]  B. Pruitt,et al.  Hydrogel crosslinking density regulates temporal contractility of human embryonic stem cell-derived cardiomyocytes in 3D cultures. , 2012, Soft matter.

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

[49]  Adrian Ranga,et al.  Engineering 3D cell instructive microenvironments by rational assembly of artificial extracellular matrices and cell patterning. , 2011, Integrative biology : quantitative biosciences from nano to macro.

[50]  S. Heilshorn,et al.  Multifunctional Materials through Modular Protein Engineering , 2012, Advanced materials.

[51]  David J. Mooney,et al.  Harnessing Traction-Mediated Manipulation of the Cell-Matrix Interface to Control Stem Cell Fate , 2010, Nature materials.

[52]  S. Heilshorn,et al.  Dynamic, 3D‐Pattern Formation Within Enzyme‐Responsive Hydrogels , 2009 .

[53]  Brendon M. Baker,et al.  Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues , 2012, Journal of Cell Science.

[54]  Yunxiao Liu,et al.  Hydrogel based on interpenetrating polymer networks of dextran and gelatin for vascular tissue engineering. , 2009, Biomaterials.

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

[56]  T. M. Parker,et al.  Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity , 1991 .

[57]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[58]  F. Pampaloni,et al.  The third dimension bridges the gap between cell culture and live tissue , 2007, Nature Reviews Molecular Cell Biology.

[59]  Kristi S Anseth,et al.  Three-dimensional hMSC motility within peptide-functionalized PEG-based hydrogels of varying adhesivity and crosslinking density. , 2013, Acta biomaterialia.

[60]  K. Lampe,et al.  Design of three-dimensional engineered protein hydrogels for tailored control of neurite growth. , 2013, Acta biomaterialia.

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

[62]  N. Kotov,et al.  Three-dimensional cell culture matrices: state of the art. , 2008, Tissue engineering. Part B, Reviews.

[63]  Kristi L. Kiick,et al.  Designing degradable hydrogels for orthogonal control of cell microenvironments , 2013, Chemical Society reviews.

[64]  Joe Tien,et al.  Mechanotransduction at cell-matrix and cell-cell contacts. , 2004, Annual review of biomedical engineering.

[65]  Glenn D Prestwich,et al.  Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel. , 2004, Journal of biomedical materials research. Part A.

[66]  Jason A. Burdick,et al.  Bioengineering: Cellular control in two clicks , 2009, Nature.

[67]  Kristi S Anseth,et al.  Three-dimensional growth and function of neural tissue in degradable polyethylene glycol hydrogels. , 2006, Biomaterials.

[68]  S. Bryant,et al.  Cell encapsulation in biodegradable hydrogels for tissue engineering applications. , 2008, Tissue engineering. Part B, Reviews.

[69]  Sanjay Kumar,et al.  The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells. , 2009, Cancer research.