PEG-based hydrogels with collagen mimetic peptide-mediated and tunable physical cross-links

Abstract

Mechanical properties of tissue scaffolds have major effects on the morphology and differentiation of cells. In contrast to two-dimensional substrates, local biochemical and mechanical properties of three-dimensional hydrogels are difficult to control due to the geometrical confinement. We designed synthetic 3D hydrogels featuring complexes of four-arm poly(ethylene glycol) (PEG) and collagen mimetic peptides (CMPs) that form hydrogels via physical cross-links mediated by thermally reversible triple helical assembly of CMPs. Here we present the fabrication of various PEG-CMP 3D hydrogels and their local mechanical properties determined by particle tracking microrheology. Results show that CMP mediated physical cross-links can be disrupted by altering the temperature of the gel or by adding free CMPs that compete for triple helix formation. This allowed modulation of both bulk and local stiffness as well as the creation of stiffness gradients within the PEG-CMP hydrogel, which demonstrates its potential as a novel scaffold for encoding physicochemical signals for tissue formation.

Figure 1

Schematics of PEG-based CMP hydrogel synthesis. (A) Reacting PEG-NHS with CMP9 yielded the target product with average conjugations of 3.04 CMP9 per PEG. PEG-CMP9 forms physical crosslinks mediated by CMP triple helices (arrowheads). (B) PEG-CMP9-K hydrogel contains covalent crosslinks due to the bifunctionality of CMP9-K. In addition to chemical crosslinks, PEG-CMP9-K can form triple helix mediated physical crosslinks (arrowhead).

Figure 2

Circular dichroism melting curves of CMP9, CMP9-K, RCMP-K, and their PEG conjugates. CMP9 and CMP9-K peptides exhibited sigmoidal melting transitions indicative of a triple helical structure both as free peptides in solution (left) and after PEG conjugation (right). RCMP-K peptides with a scrambled CMP sequence, G₃-PGOGPGPOPOGOGPOPGOOPGGOOPPG-K, did not show triple helical melting transition upon heating.

Figure 3

Particle tracking data for PEG-CMP9-K and PEG-RCMP-K hydrogels (10 wt%) at room temperature. (A) Sample particle trajectories. (B) and (C) Over 50 representative particle MSDs and the ensemble-average values. (D) Comparison of ensemble-average MSD between PEG-CMP9-K and PEG-RCMP-K hydrogels with data reported as mean ± SEM (n ≥ 4). (E) α exponents of PEG-CMP9-K and PEG-RCMP-K hydrogel calculated by fitting ensemble-average MSDs to <Δr²(τ)> ~ kτ^α.

Figure 4

Comparison of viscoelastic properties for PEG-CMP9-K and PEG-RCMP-K hydrogels (10 wt%). (A) and (B) Storage (G′) and loss (G″) moduli of the two hydrogels as a function of frequency with phase angle shown in the inset. (C) Storage (G′) and loss (G″) moduli at ω = 7.5Hz (PEG-RCMP-K crossover frequency) with data reported as mean ± SEM error bars (n ≥ 4). (D) Ensemble-average MSDs and storage moduli G′ (at ω = 30 Hz) of PEG-CMP9-K, PEG-RCMP-K, and PEG-CMP9-K after free CF-CMP9 addition.

Figure 5

Particle tracking data for PEG-CMP9 hydrogels (10 wt%). (A) Distribution of particle MSDs in PEG-CMP9 showing viscoelastic behavior, elastic behavior, and the total combined ensemble-average values. (B) Sample particle trajectories in viscoelastic and elastic gel regions. (C) Storage (G′) and loss (G″) moduli (ω = 30 Hz) corresponding to viscoelastic regions, elastic regions, and combined average values.

Figure 6

Effect of temperature on the elasticity of physically crosslinked PEG-CMP9 hydrogels (7 wt%). (A) Emsembles-average MSDs of PEG-CMP9 at 25°C and 45°C with SEM error bars (n > 100). (B) α exponents determined by fitting MSDs to <Δr²(τ)> ~ kτ^α for PEG-CMP9 and PEG-RMCP-K.

Figure 7

Modulation of mechanical properties of PEG-CMP9 hydrogels by addition of free CMP9 that can compete for triple helix formation. (A) Ensemble-average MSDs with SEM error bars (n > 100) shown for PEG-CMP9 hydrogels (10 wt%) before and after addition of 40 μL of 27.2 mM free CMP9 solution or H₂O (control experiment). (B) Corresponding storage (G′) and loss (G″) moduli (ω = 30 Hz) for ensemble-average MSDs. (C) Corresponding α exponents determined by fitting the ensemble-average MSDs to <Δr²(τ)> ~ kτ^α.

Figure 8

Spatial modification of PEG-CMP9 hydrogel with CF-CMP9. (A) Reconstituted fluorescence micrograph of 10% PEG-CMP hydrogel in the well of 96-well plate after injecting the CF-CMP9 solution to bottom right corner (marked ×) of the gel. (B) Ensemble-average MSD data with SEM error bars (n > 100) from the particle tracking measurements at the injection area and the opposite area of the gel. (C) Corresponding storage (G′) and loss (G″) moduli (ω = 30 Hz) for ensemble-average MSDs at injection and opposite sites of the gel.