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Dynamic Hydrogels: Switching of 3D Microenvironments Using Two‐Component Naturally Derived Extracellular Matrices

2010 WILEY-VCH Verlag Gm In native tissues, cells respond to changes in local threedimensional (3D) microenvironments during diverse processes such as tissue morphogenesis, wound healing and cancer progression. A number of methods have been developed to dynamically control cell spreading and migration on 2D surfaces. Few techniques, however, are available to dynamically modify the structural properties of natural 3D extracellular matrix (ECM), after the initial properties of the matrix are established. Recently, a technique has also been developed to spatially and temporally control cell spreading in 3D synthetic ECM materials after gelling, using ultraviolet radiation-induced degradation of synthetic polymers. In many instances, however, cellular behavior follows native processes more closely inside 3D naturally derived ECM, which contain native ligands for cell adhesion and signaling, than in synthetic polymers. This paper describes a method to dynamically and reversibly modify the structure of a 3D ECM, in order to permit temporal switching of properties such as rate of solute transport and cell spreading and mobility. Rather than using ECM consisting primarily of a single component that either permits or restricts cell spreading and migration, we hypothesized that the structural properties of a two-component ECM – in which one component acts as a stable structural element and another component gels or dissolves – could be dynamically modified (Fig. 1A). Such a concept could allow reversible switching of 3D microenvironments in a structurally solid gel, made of naturally derived ECM. Specifically, we explored the use of collagen I, a fibrous and cell-adhesive matrix that has a microporous structure (on the order of 1 to 20 micrometers), and alginate, a polysaccharide that lacks cell adhesion sites and has a nanoporous structure (on the orders of tens of nanometers). The state of crosslinking of the alginate component can be controlled by adding or removing divalent cations (Ca2þ, Zn2þ, Co2þ, Ba2þ, Ni2þ, Mn2þ, Pb2þ, Cu2þ, Cd2þ, Sr2þ), with Ca2þ being the most commonly used. In this manner, we sought to control the structure of the two-component 3D ECM by switching the state of alginate crosslinking. We pipetted a mixture of collagen and alginate into a chamber, and covered the solution with a membrane (either aluminum oxide or cellulose) that was permeable to small molecular-weight solutes but not alginate polymer chains (Fig. 1B). Compared to our previous work, this configuration robustly permitted multiple (rather than a single) rounds of reagent addition to permit reversible switching of the alginate crosslinking state. To form the composite gel, we first gelled the collagen component, and then crosslinked the alginate by placing a CaCl2 solution on top of the membrane. Subsequently, we could uncrosslink the alginate component by adding sodium citrate (a chelator of Ca2þ). To examine whether changes in the crosslinking state of alginate influence transport characteristics the gels, we first characterized diffusivity in two-component matrices (with single-component matrices as controls) using fluorescence recovery after photobleaching (FRAP) of fluorescein isothiocyanate (FITC)-tagged dextrans of 3, 70 and 500 kD. Lines were bleached through the center of a sample, and fluorescence recovery curves orthogonal to the bleach line were fit as described previously (see Supporting Information Fig. S1). Collagen alone did not appreciably restrict diffusion of any dextran (92 to 96% of measured diffusion coefficient in water), whereas pure alginate appreciably hindered the diffusion of 70 and 500 kD dextrans (Table 1). The diffusion coefficients of all three dextrans increased upon crosslinking the alginate, with significant (p< 0.05) differences in diffusion coefficients between the crosslinked and uncrosslinked states for 70 and 500 kD dextrans (see Supporting Methods). Importantly, after crosslinking and uncrosslinking, the diffusion coefficients returned to baseline values (i.e., the non-crosslinked state) for all three dextrans. These results suggested that the transport characteristics of the gels could indeed be reversibly switched, by adding calcium or citrate to the two-component ECM. In our model of composite ECM (Fig. 1), it is possible that changes to the state of the modulatory ECM component (alginate) could affect the structural component (collagen). To examine whether crosslinking of the alginate altered collagen fiber structure, we visualized the fine structure of the collagen fiber network using confocal reflectance microscopy on gels that were not fixed and without cells (since fixation and cell contraction alters fiber morphology). We then performed image segmentation to obtain estimates of collagen mesh size (Supporting Information Fig. S2). This analysis revealed that collagen fibers