Three-dimensional micropatterning of bioactive hydrogels via two-photon laser scanning photolithography for guided 3D cell migration

Abstract

Micropatterning techniques that control three-dimensional (3D) arrangement of biomolecules and cells at the microscale will allow development of clinically relevant tissues composed of multiple cell types in complex architecture. Although there have been significant developments to regulate spatial and temporal distribution of biomolecules in various materials, most micropatterning techniques are applicable only to two-dimensional patterning. We report here the use of two-photon laser scanning (TPLS) photolithographic technique to micropattern cell adhesive ligand (RGDS) in hydrogels to guide cell migration along pre-defined 3D pathways. The TPLS photolithographic technique regulates photo-reactive processes in microscale focal volumes to generate complex, free from microscale patterns with control over spatial presentation and concentration of biomolecules within hydrogel scaffolds. The TPLS photolithographic technique was used to dictate the precise location of RGDS in collagenase-sensitive poly(ethylene glycol-co-peptide) diacrylate hydrogels, and the amount of immobilized RGDS was evaluated using fluorescein-tagged RGDS. When human dermal fibroblasts cultured in fibrin clusters were encapsulated within the micropatterned collagenase-sensitive hydrogels, the cells underwent guided 3D migration only into the RGDS-patterned regions of the hydrogels. These results demonstrate the prospect of guiding tissue regeneration at the microscale in 3D scaffolds by providing appropriate bioactive cues in highly defined geometries.

Figure 1

Degradation profiles of GGGLGPAGGK-derivative PEG hydrogels in protease solutions. Each hydrogel sample was swollen in HBS with 1 mM CaCl₂ and 0.2 mg/mL sodium azide at 37 °C for 24 hr, and its weight was monitored in 0.2 mg/mL protease solution at 37 °C: (□) HBS, (◇) collagenase, (△) plasmin, (○) proteinase K. Hydrogels incubated with either collagenase or proteinase K underwent protease-mediated degradation.

Figure 2

The overall methodology for three-dimensional RGDS patterning by two-photon laser scanning (TPLS) photolithography. First, HDFs encapsulated in fibrin clusters were photopolymerized into collagenase-sensitive PEG hydrogels by exposure to long wavelength UV light. The hydrogels were soaked in PEG-RGDS solution, allowing its diffusion into the bulk of materials. The TPLS photolithographic technique was used to irradiate the hydrogels according to the pre-designed virtual patterns, conjugating PEG-RGDS in 3D network of hydrogels in pre-determined patterns. After washing steps, cell migration was subsequently monitored over time.

Figure 3

The characterization of RGDS-patterned region by confocal microscopy (A) XZ cross-sectional image of hydrogel (green fluorescence represents RGDS channel) was used to obtain (B) horizontal and (C) longitudinal fluorescence intensity profile across the RGDS channel. The horizontal and longitudinal cross-sections show sharp contrast between irradiated and non-irradiated regions, demonstrating high fidelity of the TPLS photolithographic technique.

Figure 4

Quantification of RGDS concentration conjugated in the hydrogels based on a standard calibration curve generated with known concentrations of FITC-RGDSK-PEG-acrylate. The patterned regions in the hydrogels used for cell migration studies were calculated to contain 0.97 μmol/mL of RGDS.

Figure 5

(A-C) DIC and confocal fluorescence images of HDFs undergoing 3D migration within degradable PEG hydrogel with homogenously distributed RGDS; (A) DIC image after 10 days of cell culture, (B) higher magnification DIC image of panel (A), and (C) DAPI and rhodamine-phallodin staining of cells shown in panel (B). (D) A cluster of HDFs encapsulated in a non-degradable hydrogel with RGDS did not show any signs of cell migration after 5 days of cell culture as cells could not penetrate the hydrogel network lacking collagenase-sensitive peptide linker. Scale bars = 100 μm.

Figure 6

Confocal microscope image of HDFs undergoing 3D migration within a RGDS patterned region inside a collagenase-sensitive PEG hydrogel; an overlaid image shows FITC-RGDS-PEG-acrylate, rhodamine-phalloidin, and DAPI. Scale bar = 100 μm.