Abstract
Hydrogel substrates with a stiffness gradient have been used as a surrogate of the extracellular matrix (ECM) to investigate how cells respond to the stiffness of their surrounding matrix. Various fabrication methods have been proposed to create a stiffness gradient in the hydrogel substrate, and some of them rely on generating a concentration gradient in a prepolymer solution before photo-polymerization. One easy way to do so is to coalesce two prepolymer solution drops of different stiffness values in a narrow confinement formed by two glass surfaces and then to induce polymerization using ultraviolet (UV) light irradiation, as proposed by Lo et al. [Biophys. J. 2000, 79:144–152]. We have improved their method to enable modulating the obtained stiffness gradient and characterized fabricated polyacrylamide (PAAM) gels. We controlled the coalescence and mixing duration of two prepolymer drops using the lab-built Hele-Shaw cell device and glass surfaces with a superhydrophobic barrier. Limited mixing between the drops created a concentration gradient of the gel ingredient, which was converted to a stiffness gradient by UV-based photo-polymerization. Atomic force microscopy (AFM) indentation showed that the fabricated gels had the stiffness gradient zone at the center and that the width of the zone increased with the mixing duration.
Similar content being viewed by others
References
Discher DE, Janmey P, Wang Y-L (2005) Tissue cells feel and respond to the stiffness of their substrate. Science 310:1139–1143
Watt FM, Huck WTS (2013) Role of the extracellular matrix in regulating stem cell fate. Nat Rev Mol Cell Biol 14:467–473
Charras G, Sahai E (2014) Physical influences of the extracellular environment on cell migration. Nat Rev Mol Cell Biol 15:813–824
Janson IA, Putnam AJ (2014) Extracellular matrix elasticity and topography: material-based cues that affect cell function via conserved mechanisms. J Biomed Mater Res A 103:1248–1258
Levental I, Georges PC, Janmey PA (2007) Soft biological materials and their impact on cell function. Soft Matt 3:299–306
Trappmann B, Chen CS (2013) How cells sense extracellular matrix stiffness: a material's perspective. Curr Op Biotechnol 24:948–953
Wen JH, Vincent LG, Fuhrmann A, Choi YS, Hribar KC, Taylor-Weiner H, Chen S, Engler AJ (2014) Interplay of matrix stiffness and protein tethering in stem cell differentiation. Nat Mater 13:979–987
Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689
Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, Zahir N, Ming W, Weaver V, Janmey PA (2005) Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskel 60:24–34
Chen L, Zhang Z, Qiu J, Zhang L, Luo X, Jang J (2014) Chaperonin CCT-mediated AIB1 folding promotes the growth of ERα-positive breast cancer cells on hard substrates. PLoS One 9:e96085
Whang M, Kim J (2016) Synthetic hydrogels with stiffness gradients for durotaxis study and tissue engineering scaffolds. Tissue Eng Regen Med 13:126–139
Burdick JA, Khademhosseini A, Langer R (2004) Fabrication of gradient hydrogels using a microfluidics/photopolymerization process. Langmuir 20:5153–5156
Zaari N, Rajagopalan P, Kim SK, Engler AJ, Wong JY (2004) Photopolymerization in microfluidic gradient generators: microscale control of substrate compliance to manipulate cell response. Adv Mater 16:2133–2137
Sundararaghavan HG, Monteiro GA, Firestein BL, Shreiber DI (2009) Neurite growth in 3D collagen gels with gradients of mechanical properties. Biotechnol Bioeng 102:632–643
Byfield FJ, Wen Q, Levental I, Nordstrom K, Arratia PE, Miller RT, Janmey PA (2009) Absence of filamin a prevents cells from responding to stiffness gradients on gels coated with collagen but not fibronectin. Biophys J 96:5095–5102
Isenberg BC, DiMilla PA, Walker M, Kim S, Wong JY (2009) Vascular smooth muscle cell durotaxis depends on substrate stiffness graident strength. Biophys J 97:1313–1322
Orsi G, Fagnano M, De Maria C, Montemurro F, Vozzi G (2017) A new 3D concentration gradient maker and its application in building hydrogels with a 3D stiffness gradient. J Tissue Eng Regen Med 11:256–264
Lo C-M, Wang H-B, Dembo M, Wang Y-L (2000) Cell movement is guided by the rigidity of the substrate. Biophys J 79:144–152
Wang H-B, Dembo M, Hanks SK, Wang Y-I (2001) Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc Natl Acad Sci U S A 98:11295–11300
Raab M, Swift J, Dingal PCDP, Shah P, Shin J-W, Discher DE (2012) Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain. J Cell Biol 199:669–683
Nemir S, Hayenga HN, West JL (2010) PEGDA hydrogels with patterned elasticity: novel tools for the study of cell response to substrate rigidity. Biotechnol Bioeng 105:636–644
Diederich VEG, Studer P, Kern A, Lattuada M, Storti G, Sharma RI, Snedeker JG, Morbidelli M (2013) Bioactive polyacrylamide hydrogels with gradients in mechanical stiffness. Biotechnol Bioeng 110:1508–1519
Du Y, Hancock MJ, He J, Villa-Uribe JL, Wang B, Cropek DM, Khademhosseini A (2010) Convection-driven generation of long-range material gradients. Biomaterials 31:2686–2694
Wong JY, Velasco A, Rajagopalan P, Pham Q (2003) Directed movement of vascular smooth muscle cells on gradient-compliant hydrogels. Langmuir 19:1908–1913
Kidoaki S, Matsuda T (2008) Microelastic gradient gelatinous gels to induce cellular mechanotaxis. J Biotechnol 133:225–230
Tse JR, Engler AJ (2011) Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate. PLoS One 6:e15978
Marklein RA, Burdick JA (2010) Spatially controlled hydrogel mechanics to modulate stem cell interactions. Soft Matt 6:136–143
Khetan S, Burdick JA (2010) Patterning network structure to spatially control cellular remodeling and stem cell fate within 3-dimensional hydrogels. Biomaterials 31:8228–8234
Stowers RS, Allen SC, Suggs LJ (2015) Dynamic phototuing of 3D hydrogel stiffness. Proc Natl Acad Sci U S A 112:1953–1958
Kloxin AM, Benton JA, Anseth KS (2010) In situ elasticity modulation with dynamic substrates to direct cell phenotype. Biomaterials 31:1–8
Johnson PM, Reynolds TB, Stansbury JW, Bowman CN (2005) High throughput kinetic analysis of photopolymer conversion using composition and exposure time gradients. Polymer 46:3300–3306
Sunyer R, Jin AJ, Nossal R, Sackett DL (2012) Fabrication of hydrogels with steep stiffness gradients for studying cell mechanical response. PLoS One 7:e46107
García S, Sunyer R, Olivares A, Noailly J, Atencia J, Trepat X (2015) Generation of stable orthogonal graidents of chemical concentration and substrate stiffness in a microfluidic device. Lab Chip 15:2606–2614
Kloxin AM, Tibbitt MW, Kasko AM, Fairbairn JA, Anseth KS (2010) Tunable hydrogels for external manipulation of cellular microenvironments through controlled photodegradation. Adv Mater 22:61–66
Tong X, Jiang J, Zhu D, Yang F (2016) Hydrogels with dual gradients of mechanical and biochemical cues for deciphering cell-niche interactions. ACS Biomater Sci Eng 2:845–852
Yanagawa F, Mizutani T, Sugiura S, Takagi T, Sumaru K, Kanamori T (2015) Partially photodegradable hybrid hydrogels with elasticity tunable by light irradiation. Colloid Surf B 126:575–579
Kawano T, Kidoaki S (2011) Elasticity boundary conditions required for cell mechanotaxis on microelastically-patterned gels. Biomaterials 32:2725–2733
Mosiewicz KA, Kolb L, van der Vlies AJ, Lutolf MP (2014) Microscale patterning of hydrogel stiffness through light-triggered uncaging of thiols. Biomater Sci 2:1640–1651
Frey MT, Wang Y-L (2009) A photo-modulatable material for probing cellular responses to substrate rigidity. Soft Matt 5:1918–1924
Wang P-Y, Tsai W-B, Voelcker NH (2012) Screening of rat mesenchymal stem cell behaviour on polydimethylsiloxane stiffness gradients. Acta Biomater 8:519–530
Gray DS, Tien J, Chen CS (2003) Repositioning of cells by mechanotaxis on surfaces with micropatterned Young's modulus. J Biomed Mater Res A 66:605–614
Choi YS, Vincent LG, Lee AR, Kretchmer KC, Chirasatitsin S, Dobke MK, Engler AJ (2012) The alignment and fusion assembly of adipose-derived stem cells on mechanically patterned matrices. Biomaterials 33:6943–6951
Cheung YK, Azeloglu EU, Shiovitz DA, Costa KD, Seliktar D, Sia SK (2009) Microscale control of stiffness in a cell-adhesive substrate using microfluidics-based lithography. Angew Chem Int Ed 48:7188–7192
Shu Y, Chan HN, Guan D, Wu H, Ma L (2017) A simple fabricated thickness-based stiffness gradient for cell studies. Sci Bull 62:222–228
Kuo C-HR, Xian J, Brenton JD, Franze K, Sivaniah E (2012) Complex stiffness gradient substrates for studying mechanotatic cell migration. Adv Mater 24:6059–6064
Chao PG, Sheng S-C, Chang W-R (2014) Micro-composite substrates for the study of cell-matrix mechanical interactions. J Mech Behav Biomed Mater 38:232–241
Kim TH, An DB, Oh SH, Kang MK, Song HH, Lee JH (2015) Creating stiffness gradient polyvinyl alcohol hydrogel using a simple gradual freezing-thawing method to investigate stem cell differentiation behaviors. Biomaterials 40:51–60
Hopp I, Michelmore A, Smith LE, Robinson DE, Bachhuka A, Mierczynska A, Vasilev K (2013) The influence of substrate stiffness gradients on primary human dermal fibroblasts. Biomaterials 34:5070–5077
Tse JR, Engler AJ (2010) Preparation of hydrogel substrates with tunable mechanical properties. In: Current protocols in cell biology. John Wiley & Sons, Inc: 1–16
Lakins JN, Chin AR, Weaver VM (2012) Exploring the link between human embryonic stem cell organization and fate using tension-calibrated extracellular matrix functionalized polyacrylamide gels. In: Mace KA, Braun KM (eds) Progenitor cells, vol 916. Methods in molecular biology. Humana Press, Totawa, pp 317–350
Lee D, Rahman MM, Zhou Y, Ryu S (2015) Three-dimensional confocal microscopy indentation method for hydrogel elasticity measurement. Langmuir 31:9684–9693
Lee D, Ryu S (2017) A validation study of the repeatability and accuracy of atomic force microscopy indentation using polyacrylamide gels and colloidal probes. J Biomech Eng 139:044502
Graham PJ, Farhangi MM, Dolatabadi A (2012) Dynamics of droplet coalescence in response to increasing hydrophobicity. Phys Fluids 24:112105
Hancock MJ, Yanagawa F, Jang Y-H, He J, Kachouie NN, Kaji H, Khademhosseini A (2012) Designer hydrophilic regions regulate droplet shape for controlled surface patterning and 3D microgel synthesis. Small 8:393–403
Hermanowicz P, Sarna M, Burda K, Gabryś H (2014) AtomicJ: an open source software for analysis of force curves. Rev Sci Instrum 85:063703
Crank J (1975) The mathematics of diffusion. 2nd edn. Oxford University Press, Oxford, UK
Carey AE, Wheatcraft SW, Glass RJ, O'Rourke JP (1995) Non-Fickian ionic diffusion across high-concentration gradient. Water Resour Res 31:2213–2218
Küntz M, Lavallée P (2004) Anomalous diffusion is the rule in concentration-dependent diffusion processes. J Phys D Appl Phys 37:L5–L8
Wu Z, Nguyen N-T, Huang X (2004) Nonlinear diffusive mixing in microchannels: theory and experiments. J Micromech Microeng 14:604–611
Chen J, Kim HD, Kim KC (2013) Measurement of dissolved oxygen diffusion coefficient in a microchannel using UV-LED induced fluorescent method. Microfluid Nanofluid 14:541–550
Jimenez M, Dietrich N, Cockx A, Hébrard G (2013) Experimental study of O2 diffusion coefficient measurement at a planar gas-liquid interface by planar laser-induced fluorescence with inhibition. AICHE J 59:325–333
Jimenez M, Dietrich N, Grace JR, Hébrard G (2014) Oxgen mass transfer and hydrodynamic behaviour in wastewater: determination of local impact of surfactants by visualization techniques. Water Res 58:111–121
Roht YL, Auradou H, Hulin J-P, Salin D, Chertcoff R, Ippolito I (2015) Time dependence and local structure of tracer dispersion in oscillating liquid hele-Shaw flows. Phys Fluids 27:103602
Xu F, Jimenez M, Dietrich N, Hébrard G (2017) Fast determination of gas-liquid diffusion coefficient by an innovative double approach. Chem Eng Sci 170:68–76
Long R, Hall MS, Wu M, Hui C-Y (2011) Effects of gel thickness on microscopic indentation measurements of gel modulus. Biophys J 101:643–650
Calvet D, Wong JY, Giasson S (2004) Rheological monitoring of polyacrylamide gelation: importance of cross-link density and temperature. Macromolecules 37:7762–7771
Buxboim A, Rajagopalan K, Brown AEX, Discher DE (2010) How deeply cells feel: methods for thin gels. J Phys Condens Matter 22:194116
Markert CD, Guo X, Skardal A, Wang Z, haradwaj S, Zhang Y, Bonin K, Guthold M (2013) Characterizaing the micro-scale elastic modulus of hydrogels for use in regenerative medicine. J Mech Behav Biomed Mater 27:115–127
Abidine Y, Laurent VM, Michel R, Duperray A, Palade LI, Verdier C (2015) Physical properties of polyacrylamide gels probed by AFM and rheology. Eur Phys Lett 109:38003
Boudou T, Ohayon J, Picart C, Tracqui P (2006) An extended relationship for the characterization of Young's modulus and Poisson's ratio of tunable polyacrylamide gels. Biorheology 43:721–728
Damljanovic V, Lagerholm BC, Jacobson K (2005) Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays. Biotechniques 39:847–851
Takigawa T, Morino Y, Urayama K, Masuda T (1996) Poisson's ratio of polyacrylamide (PAAm) gels. Polym Gel Netw 4:1–5
Geissler E, Hecht AM (1980) The Poisson ratio in polymer gels. Macromolecules 13:1276–1280
Kalcioglu ZI, Mahmoodian R, Hu Y, Suo Z, Van Vliet KJ (2012) From macro- to microscale poroelastic characterization of polymeric hydrogels via indentation. Soft Matt 8:3393–3398
Geissler E, Hecht AM (1981) The Poisson ratio in polymer gels. 2. Macromolecules 14:185–188
Acknowledgements
This study was supported by Bioengineering for Human Health grant from the University of Nebraska-Lincoln (UNL) and the University of Nebraska Medical Centre (UNMC). KG appreciates UNL Undergraduate Creative Activities and Research Experiences (UCARE) program, and AM and BG appreciate UNL Summer Research Program and NSF Research Experiences for Undergraduates (REU) grant. AFM measurements were performed at the NanoEngineering Research Core Facility of UNL (part of the Nebraska Nanoscale Facility), which is partially funded from Nebraska Research Initiative Funds.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lee, D., Golden, K., Rahman, M.M. et al. Fabrication of Hydrogels with a Stiffness Gradient Using Limited Mixing in the Hele-Shaw Geometry. Exp Mech 59, 1249–1259 (2019). https://doi.org/10.1007/s11340-018-0416-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11340-018-0416-1