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Haptotaxis Is Cell Type Specific and Limited by Substrate Adhesiveness

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Haptotaxis Is Cell Type Specific and Limited by Substrate Adhesiveness Cellular and Molecular Bioengineering (Ó 2015) DOI: 10.1007/s12195-015-0398-3 Haptotaxis is Cell Type Specific and Limited by Substrate Adhesiveness 1 2 1 1 3 JESSICA H. WEN, ONKIU CHOI, HERMES TAYLOR-WEINER, ALEXANDER FUHRMANN, JEROME V. KARPIAK, 3,4 1,5 ADAH ALMUTAIRI, and ADAM J. ENGLER 1Department of Bioengineering, UC San Diego, La Jolla, CA 92093, USA; 2Department of Biomedical Engineering, National Yang-Ming University, Beitou District, Taipei City 112, Taiwan; 3Department of Biomedical Sciences, UC San Diego, La Jolla, CA 92093, USA; 4Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093, USA; and 5Sanford Consortium for Regenerative Medicine, La Jolla, CA 92037, USA (Received 23 April 2015; accepted 22 May 2015) Associate Editor Michael R. King oversaw the review of this article. Abstract—Motile cells navigate through tissue by relying on these cues are often found together in vivo via normal tactile cues from gradients provided by extracellular matrix tissue variation or pathological conditions, e.g., the such as ligand density or stiffness. Mesenchymal stem cells post-infarct myocardial scar that is stiffer than, (MSCs) and fibroblasts encounter adhesive or ‘haptotactic’ and compositionally different from, healthy cardiac gradients at the interface between healthy and fibrotic tissue 4,36,38 as they migrate towards an injury site. Mimicking this muscle. These gradients are thought to aid in phenomenon, we developed tunable RGD and collagen directional migration through tissue; for example, gradients in polyacrylamide hydrogels of physiologically mesenchymal stem cells (MSCs) whose fate can be relevant stiffness using density gradient multilayer polymer- regulated by ECM stiffness14,42,45 also durotax or mi- ization to better understand how such ligand gradients grate in the direction of an increasing stiffness gradi- regulate migratory behaviors. Independent of ligand compo- 43 sition and fiber deformation, haptotaxis was observed in ent. Dermal fibroblast migration into cutaneous mouse 3T3 fibroblasts. Human MSCs however, haptotaxed wound spaces is also well known and critical for only when cell-substrate adhesion was indirectly reduced via assembly of new ECM.27,29 However, spatial variation addition of free soluble matrix ligand mimetic peptides. in tissue stiffness in vivo is often accompanied by var- Under basal conditions, MSCs were more contractile than fibroblasts. However, the presence of soluble adhesive iation in adhesive ligand density. During fibrosis, ECM peptides reduced MSC-induced substrate deformations; in- accumulates and may present several ligand gradients creased contractility may contribute to limited migration, but at the interface of healthy and pathological tissue.18 modulating cytoskeletal assembly was ineffective at promot- Because in vivo spatial distributions of ECM proteins18 ing MSC haptotaxis. When introduced to gradients of and stiffness4 have been shown to change with fibrosis increased absolute ligand concentrations, 3T3s displayed and animal models have documented directed migra- increased contractility and no longer haptotaxed. These data 27,29 suggest that haptotactic behaviors are limited by adhesion tion, it is of interest to develop systems to inves- and that although both cell types may home to tissue to aid tigate cell migratory behaviors in response to spatial in repair, fibroblasts may be more responsive to ligand gradients in vitro. Although we and others have gradients than MSCs. developed simplified systems to probe durotaxis,30,31,43,46 few systems with the ability to separately modulate Keywords—Migration, Mesenchymal stem cell, Fibroblast, directed migration due to adhesive ligand gradients,6 i.e., Ligand gradient, Surface modification, Elasticity. haptotaxis, and migration due to substrate stiffness have been developed. Adhesive ligand gradients have been shown to in- duce directed cell migration in human dermal fibrob- INTRODUCTION lasts in vitro.10 However, cell type-specific differences in the degree of haptotaxis could be simultaneously Anchorage dependent cells sense and respond to influenced by physiologically-relevant substrate stiff- properties of the extracellular matrix (ECM), e.g., ness, which is a major regulator of stem cell fate.14,45 stiffness30,43,46 and ligand density.13,17,34 Gradients of Moreover, haptotactic mechanisms remain unclear; this is in part due to the fact that systems commonly Address correspondence to Adam J. Engler, Department of used to study haptotaxis are often limited in Bioengineering, UC San Diego, La Jolla, CA 92093, USA. Electronic gradient resolution, and typically utilize substrates of mail: [email protected] Ó 2015 Biomedical Engineering Society WEN et al. non-physiologically relevant stiffness. Furthermore, 3 min, washed with ethanol, and dried. All chemicals haptotaxis studies often lack direct comparisons were obtained from Sigma-Aldrich and other products between cell and adhesive ligand types. For example, from ThermoFisher unless otherwise indicated. human melanoma cells undergo haptotaxis when exposed to gradients of collagen type IV, fibronectin, DGMP Hydrogel Fabrication and laminin using Boyden chambers; these chambers however, can create only step gradients.2 Myoblast DGMP involves layering pre-polymer hydrogel haptotaxis has been observed with more gradual nan- solutions into rubber molds cut to fit between two glass odot adhesive gradients; however, these gradients were slides, followed by UV polymerization. 5 9 5mm printed on rigid glass.35 Literature suggests that cell molds cut from super-soft 35A silicone rubber sheets forces exerted on adhesive matrix proteins contribute (McMaster-Carr) were clamped between a glass slide, a to cellular mechanical signaling41,45 and that in vivo functionalized glass coverslip to facilitate gel attach- gradients are gradual.4,42 Reductionist systems ment, and a dimethylchlorosilane- (DCDMS-) treated employing compliant hydrogels deformable by cells, glass slide to facilitate gel detachment. Pre-polymer e.g., polyacrylamide20,22 and polyethylene glycol,10 solutions composed of acrylamide, bis-acrylamide, with gradual haptotactic gradients are thus needed to acrylated poly(ethylene) glycol conjugated to RGD determine which signaling networks within a variety of (aPEG-RGD) or acrylic acid, 2,2¢-Azobis(2-methyl- cell types regulate haptotaxis. Although not yet studied propion0amidine) dihydrochloride photoinitiator in detail, mechanisms regulating haptotaxis likely (Sigma), and serial dilutions of iodixanol (Sigma), an involve adhesive and/or cytoskeletal proteins given inert density modifier, were layered into the silicone their roles in other migratory control mechanisms in molds in order of decreasing density. Hydrogels with processes such as chemotaxis,1 and the establishment gradients of adhesive ligands were fabricated by of cell polarity and shape.21,44 allowing two 12 lL layers of pre-polymer solution of Gradients of protein attachment to compliant distinct composition (Fig. 1a) to diffuse and settle for hydrogels have been achieved via microcontact protein 10 s creating a gradient at the mixing interface printing, self-assembled monolayers, and microfluidic (Fig. 1b). Layered hydrogels were subsequently poly- mixing devices.10,26,33 Despite the use of compliant merized via exposure to 350 nm UV light for 4.5 min. hydrogels, these methods are often difficult to scale 10/0.15% acrylamide/bis-acrylamide solutions down, tune, and require significant quantities of pro- composed of 0.1 or 10 mM aPEG-RGD with 1 or 4% tein or peptide, much of which does not bind. Here, we iodixanol respectively, were used to obtain PA-PEG- used density gradient multilayer polymerization RGD hydrogels with adhesive RGDS ligand gradients (DGMP), to construct compliant culture substrates of approximately 10 mM/mm that span an 800 lm with spatial gradients of composition.22,23 DGMP, a region across the center of the hydrogel (Fig. 1c). A simple method that does not require specialized hydroxycoumarin dye (excitation 350 nm) pre-conju- equipment, ingredients, or expertise, relies on density- ated to aPEG-RGD allowed us to visualize serial mediated phase separation to construct layers of dis- dilutions of adhesive peptide; differences in cell tinct composition. By using this method to fabricate spreading and fluorescent intensity confirm that sub- gradients of adhesive peptide RGD or collagen type I strate adhesiveness scales with aPEG-RGD concentra- on a polyacrylamide (PA) hydrogel platform, we tion (Fig. 1d). aPEG-RGD, or acryl-PEG3400-GRGD- demonstrate that while fibroblasts undergo haptotaxis amide and acryl-PEG3400-[K-7-hydroxycoumarin-3- in response to both gradient types under basal condi- OH]GGGRGD-amide were custom ordered (21st tions, MSC haptotaxis appears to be limited by cell- Century Biochemicals). substrate adhesiveness, and is observed only when cell To investigate the migration behavior of cells in affinity for the substrate is reduced. response to solely a ligand gradient on a substrate of physiologically relevant stiffness, AFM was used to characterize the hydrogel stiffness in order to ensure MATERIALS AND METHODS that no simultaneous stiffness gradients were created as a consequence of the haptotactic gradient fabrication Functionalized Coverslips process (Fig. 1e). Iodixanol and aPEG-RGD did not To covalently attach hydrogel substrates to glass, significantly alter hydrogel stiffness at the concentra- 25 mm square glass coverslips
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