A simple layer-stacking technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels

Abstract

Physicochemical and biological gradients are desirable features for hydrogels to enhance their relevance to biological environments for three-dimensional (3D) cell culture. Therefore, simple and efficient techniques to generate chemical, physical and biological gradients within hydrogels are highly desirable. This work demonstrates a technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels by stacking and crosslinking prehydrogel solution in a layer by layer manner. Partial crosslinking of the hydrogel allows mixing of prehydrogel solution with the previous hydrogel layer, which makes a smooth gradient profile, rather than discrete layers. This technique enables the generation of concentration gradients of bovine serum albumin in both gelatin methacryloyl (GelMA) and poly(ethylene glycol) diacrylate hydrogels, as well as mechanical gradients across a hydrogel containing varying gel concentrations. Fluorescence microscopy, mechanical testing, and scanning electron microscopy show that the gradient profiles can be controlled by changing both the volume and concentration of each layer as well as intensity of UV exposure. GelMA hydrogel gradients with different Young's moduli were successfully used to culture human fibroblasts. The fibroblasts migrated along the gradient axis and showed different morphologies. In general, the proposed technique provides a rapid and simple approach to design and fabricate 3D hydrogel gradients for in vitro biological studies and potentially for in vivo tissue engineering applications. RECEIVED

Gradient Hydrogels—The State of the Art in Preparation Methods

“…Ko and co-workers formed three-dimensional concentrations and mechanical gradients by means of a layer-stacking technique in order to generate 3D mechanical and biomolecular gradients [1]. The gradients were created using two different types of photocrosslinkable hydrogel-poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA).…”

“…The prepared hydrogel, when used as scaffolds, offered good biocompatibility, good adsorption properties, and supported cell growth and proliferation. The technique is similar to that used by Ko and co-workers [1] but requires a freeze-dryer and thus can also be more time-consuming. Otherwise, the technique is quite general and not limited only to the studied system and application.…”

“…The proposed gels could be used in tissue engineering for connecting implants with underlying tissue. This method is similar to that used by Ko and co-workers [1] and is based on changing the composition of an identical gel basis, thereby giving different crosslinking densities and allowing controlled over-laying, which should prevent the mixing of different compositions but still enable the creation of chemical junctions between them.…”

Functionally Graded Biomaterials for Use as Model Systems and Replacement Tissues

“…[29] Fibroblasts migrated and exhibited distinct morphologies on these materials possessing a stiffness gradient, with cells on more compliant areas remaining round while cells on stiffer regions becoming more spindle-like. [29] Light-based gradient fabrication utilizes methods with high resolution and facilitates high throughput formation. The lightresponsive photoinitiator functions through the generation of free radicals that crosslink macromers, yet this approach may impair cell or DNA damage due to UV light.…”

Magnetic Fields Enable Precise Spatial Control of Electrospun Fiber Alignment for Fabricating Complex Gradient Materials

“…Recently, increasing focus is on the development of spatially organized, heterogenous scaffolds in order to spatially control cell behavior and facilitate interfacial tissue regeneration. [13][14][15][16] A variety of fabrication techniques have been used to spatially control material properties, including: freeze drying to create structural gradients, [17][18][19] controlling polymer crosslinking density to create stiffness gradients, 20,21 photopatterning to create biochemical signaling gradients, 22,23 solvent casting and particulate leaching to create porosity gradients, 24 and others. 25,26 Of particular interest, electrospinning is a commonly used fabrication method for producing scaffolds that mimic the fibrous structure of the ECM.…”