Mechanical properties of cellularly responsive hydrogels and their experimental determination

Abstract

Hydrogels are increasingly employed as multidimensional cell culture platforms often with a necessity that they respond to or control the cellular environment. Specifically, synthetic hydrogels, such as poly(ethylene glycol) (PEG)-based gels, are frequently utilized for probing the microenvironment's influence on cell function, as the gel properties can be precisely controlled in space and time. Synthetically tunable parameters, such as monomer structure and concentration, facilitate initial gel property control, while incorporation of responsive degradable units enables cell- and/or user-directed degradation. Such responsive gel systems are complex with dynamic changes occurring over multiple time-scales, and cells encapsulated in these synthetic hydrogels often experience and dictate local property changes profoundly different from those in the bulk material. Consequently, advances in bulk and local measurement techniques are needed to monitor property evolution quantatively and understand its effect on cell function. Here, recent progress in cell-responsive PEG hydrogel synthesis and mechanical property characterization is reviewed.

Figure 1. PEG hydrogels

Two common methods of forming PEG-based hydrogels are chain growth and step growth polymerization of multifunctional PEG monomers. (A) PEG di(meth)acrylate monomers (left, gray PEG chain with red (meth)acrylate end groups; PEGDMA) are polymerized by free radical chain polymerization, forming coiled poly(methacrylate) chains (right, red coils) connected by PEG (gray linkers). Here, the molecular mesh size of the network (ξ) is dictated by the length of the PEG chain and the concentration of PEGDMA in the gel-forming solution. In addition, the crosslinking density of the network (ρ_x) is controlled by the concentration of PEGDMA, dictating the resultant gel modulus, equilibrium water content, and solute diffusivity. Degradation is easily introduced within these gels by incorporation of a degradable moiety (purple and orange hexagon; water, enzyme, or light degradable group) within the PEGDMA crosslinking monomer. (B) Multifunctional PEG monomers (gray 4-arm PEG with red box reactive end group) are reacted stoichiometrically with degradable linkers (orange linker with open red box reactive end groups and purple hexagon degradable group) to form nearly perfect networks by step growth polymerization. The reactive end groups may include acrylates and thiols polymerized by base-catalyzed Michael-type reaction, norbornenes and thiols by free radical initiation, and azide and alkynes by copper-based or copper-free click reaction. Here, the mesh size is again dictated by the length of the PEG chain, whereas the crosslinking density is dictated by the length and concentration of the PEG crosslinker.

Figure 2. Complexities of the cell environment and cellular response to substrate mechanics

(A) Cells respond to multiple signals within their microenvironment. Studies often focus on the effect of soluble factors (red circles) and genetics (green DNA strands) on cell function while less attention is given to the structure and mechanics of the gel with which the cell is interacting, where the cell binds to ligands tethered to its microenvironment (purple coils) and pulls on the surrounding structure. To address this issue, 2D studies of cell-gel interactions have been undertaken. (B) Cells cultured on low modulus substrates exhibit a diffuse cytoskeleton (orange actin fibers), whereas cells cultured on high modulus substrates exhibit a dense actin cytoskeleton, leading to different differentiation pathways for hMSCs, neurogenic (~ 1 kPa), myogenic (~ 10 kPa), and osteogenic (> 25 kPa), through mechanotransduction.^[41] (C) Similarly, cell shape has been shown to influence cytoskeletal organization and cell fate, where cells cultured on small islands of fibronectin (green) undergo apoptosis (left) while cells on larger islands (> 10 µm) undergo proliferation (right).^[43]

Figure 3. Cell response to mechanical stimulation in 3D

Cells encapsulated within a gel respond to applied mechanical strain non-uniformly (top). Specifically, individual chondrocytes show varying degrees of deformation in response to increasing strain, as observed with confocal microscopy, where cells in gels under 20% strain deform between 1 and 32%. While the gel structure is heterogeneous on the nanoscale, the cellular response to gel structure and mechanics is heterogeneous on the micro and macroscale.^[47] Reproduced with permission from ^[47] [permission pending]. Copyright 2004 Wiley.

Figure 4. Dynamic modification of the microenvironment

Cells encapsulated within PEG gels initially exhibit a rounded morphology (left). Within an enzymatically degradable gel, cell-secreted enzymes (purple coils) begin to breakdown the local matrix surrounding the cell, enabling cell spreading and movement (middle) and subsequently cell division (right). Monitoring these dynamic and local changes in gel mechanics in response to cell degradation is challenging.

Figure 5. Detection of local gel degradation

FRET-based linkers within gels provide a means to locally detect cell-directed gel degradation. As the cell degrades crosslinks within the gel (left, orange hexagon degradable blocks cleaved by purple cell secreted enzymes), increased fluorescence is observed (middle, green hexagon fluorescent groups). Cells can subsequently extend processes and move within the degraded gel, leaving tracks of increased fluorescence (right, scale bar 20 µm).^[60] Reproduced with permission from ^[60] [permission pending]. Copyright 2007 El Sevier.

Figure 6. Bulk property measurement with rheometry

(A) As the modulus is a function of crosslinking density, (B) the polymerization progression can be monitored utilizing oscillatory bulk rheometry. Initially monomeric and oligomeric species yield a viscous response to oscillatory deformation, where the viscous modulus (G″) dominates the viscoelastic spectrum. With polymerization, the formation of the incipient gel occurs (shown in the second inset of Panel B), defining the gel point of the material. The gel point is typically in the vicinity of, but rarely at, the elastic and viscous moduli crossover. At the end of the polymerization, the hydrogel network becomes fully formed with the elastic modulus (G′) dominating the viscoelastic spectrum.

Figure 7. AFM for local surface measurement of gel modulus and crosslinking density

(A) Cells on substrates of different crosslinking densities exhibit different cytoskeletal organization and moduli. A cell on a highly crosslinked gel (right) has a cytoskeleton dense with actin fibers (orange) and focal adhesions (green), pulling on the network through integrin-ligand binding (brown and purple) and responding to the elasticity of the substrate, whereas a cell on a less crosslinked substrate has a diffuse cytoskeleton (left). With AFM, the modulus, and thus crosslinking density, of the underlying gel as well as the modulus of the cell’s cytoskeleton can be measured by controlled indentation of the sample surface during 2D cell culture (top) to explore this interplay between gel structure and cell function. (B) A correlation between substrate stiffness and cell stiffness was observed with AFM measurements of fibroblasts cultured on polyacrylamide gels of varying crosslinking density.^[91] Reproduced with permission from ^[91] [permission pending]. Copyright 2007 Cell Press.

Figure 8. Illustration of passive tracer microrheology in a hydrogel with a crosslinking density gradient

The embedded probe particles (red spheres) undergo Brownian motion, whose path is characteristic of the surrounding hydrogel (yellow chains). The insets show typical paths for probe particles in a hydrogel having a gradient crosslinking density. With increasing crosslinking density, there is a decrease in the mean squared displacement of individual particles. After multiple particles have been observed, the ensemble averaged viscoelastic properties are obtained.