Hierarchically Structured and Tunable Hydrogel Patches: Design, Characterization, and Application

Abstract

Recent studies show the importance of hydrogel geometry for various applications, such as encoding, micromachines, or tissue engineering. However, fabricating hydrogel structures with micrometer-sized features, advanced geometry, and precise control of porosity remains challenging. This work presents hierarchically structured hydrogels, so-called hydrogel patches, with internally deviating regions on a micron-scale. These regions are defined in a one-step, high-throughput fabrication process via stop-flow lithography. Between the specified projection pattern during fabrication, an interconnecting lower crosslinked and more porous hydrogel network forms, resulting in at least two degrees of crosslinking within the patches. A detailed investigation of patch formation is performed for two material systems and pattern variations, revealing basic principles for reliable patch formation. In addition to the two defined crosslinked regions, further regions are implemented in the patches by adapting the pattern accordingly. The variations in pattern geometry impact the mechanical characteristics of the hydrogel patches, which display pattern-dependent compression behavior due to predefined compression points. Cell culture on patches, as one possible application, reveals that the patch pattern determines the cell area of L929 mouse fibroblasts. These results introduce hierarchically structured hydrogel patches as a promising and versatile platform system with high customizability.

Keywords: biomaterial; hierarchical cellular material; hydrogel; porosity; projection lithography.

Figure 1

Hydrogel formation using mask projection lithography. a) Schematics of the hydrogel formation events in the side and top view for a so‐called hexagonal spot arrangement, showing the network connections. b,c) Occurrence of the formation events depending on the applied exposure parameters during fabrication, displayed for three radiant exposures (38 mJ/mm²  (square), 50 mJ/mm²  (triangle), and 63 mJ/mm²  (circle)) with four parameter combinations (radiant exposure | exposure time) each. The occurring formation events for a (b) PEGDA‐based and a (c) PNIPAM‐based material system are displayed at these exposure parameters equal for both systems: no patch (unfilled symbols), patch formation (filled symbols and a gray area), and sticking (strikethrough symbols). A transparency mask with circle‐shaped spots of 40 µm diameter was used with the circles having an edge‐to‐edge distance of 100 µm to all six neighboring circle shapes on the mask (hexagonal arrangement).

Figure 2

Influence of the dimensions of a transparency mask with a hexagonal arrangement of circle‐shaped spots on the hydrogel formation events: no patch (unfilled symbols), patch formation (filled symbols and a gray area), and sticking (strikethrough symbols). PNIPAM hydrogels fabricated at a constant radiant exposure (38 mJ/mm² ), investigated for a) a varying spot distance at a constant spot diameter of 40 µm and b) a varying spot diameter at a constant spot distance of 100 µm for four exposure combinations each.

Figure 3

Varying patch geometries by different spot shapes. a) Mask schemes and corresponding brightfield images of patches with different shapes of the HC regions induced by the irradiated mask spots. From left: circle‐shaped, square‐shaped, snowflake‐shaped, star‐shaped. All other mask parameters are held constant for all patch types: irradiation spot area, center‐to‐center spot distance, and hexagonal spot arrangement. Scale bar (100 µm) applies to all micrographs. b,c) Field emission scanning electron microscopy (FESEM) images of patches with (b) circle‐shaped or (c) star‐shaped irradiation spots. (b) Black lines and arrows indicate regions with different directionalities within the porous structure of the patch, most likely resulting from freeze‐drying.

Figure 4

Patches series with induced third level of porosity by missing out irradiation spots within a regular hexagonal spot arrangement of circle‐shaped spots with 7 µm diameter on the patches. From left to right: without additional induced porosity, induced 44 µm porous regions by single missing irradiation spots, induced 95 µm pores by missing an arrangement of seven spots. Visualization of the patches by a) mask schemes and corresponding brightfield images of the patches and b) FESEM images with levels of porosity being indicated. Scale bars (a: 100 µm, b: 50 µm) apply to all micrographs each.

Figure 5

Mechanical properties of selected patches. a) Investigated patches (top view) grouped according to geometrical variations: structure, directionality, and porosity. Scale bar (200 µm) applies to all micrographs. b,c) Schematic microfluidic channel geometry and experimental process of (b) lateral compression with the compressive force being applied along with the flow direction and (c) squeezing experiments, exemplarily shown for a patch with circle‐shaped HC regions with 52 µm diameter in a hexagonal arrangement (circle | 52 µm). Scale bar (500 µm) applies to all micrographs. d) Compression behavior of the patches, displayed by the change in their surface area over increasing applied pressure. e) Squeezing behavior of the patches, characterized by the pressure needed for the patches to break through the channel constriction.

Figure 6

Brightfield images showing the squeezing states of the porosity patch types w/o pore, 44 µm pore, and 95 µm pore. Scale bar (500 µm) applies to all images. The shape of the through‐pores indicates local deformation states during the translocation through the constriction.

Figure 7

Cell cultivation (L929 mouse fibroblasts) on patches with varying structures and two different materials: a) PEGDA patches and b) PNIPAM patches with fibronectin coating. Patch patterns include circle‐shaped HC areas with diameters of 7 µm (Circle 7 µm), with 52 µm diameters (Circle 52 µm), and structureless (Reference) patches. Comparison of (a,b) visual cell appearance after two days of cultivation, c) cell area of isolated cells after this time, and d) Young's modulus of the patches. Scale bar (50 µm) applies to all micrographs.

Figure 8

Cell cultivation (L929 mouse fibroblasts) on a circle 95 µm pore patch with HC regions of 7 µm diameter in a hexagonal arrangement, regularly interrupted by through‐pores with 95 µm diameter after four days. The patch material is based on the PEGDA‐material system with the addition of GMA to enable fibronectin coating.