Hierarchical patterning via dynamic sacrificial printing of stimuli-responsive hydrogels

Abstract

Inspired by stimuli-tailored dynamic processes that spatiotemporally create structural and functional diversity in biology, a new hierarchical patterning strategy is proposed to induce the emergence of complex multidimensional structures via dynamic sacrificial printing of stimuli-responsive hydrogels. Using thermally responsive gelatin (Gel) and pH-responsive chitosan (Chit) as proof-of-concept materials, we demonstrate that the initially printed sacrificial material (Gel/Chit-H+ hydrogel with a single gelatin network) can be converted dynamically into non-sacrificial material (Gel/Chit-H+–Citr hydrogel with gelatin and an electrostatic citrate–chitosan dual network) under stimulus cues (citrate ions). Complex hierarchical structures and functions can be created by controlling either the printing patterns of citrate ink or the diffusion time of citrate ions into the Gel/Chit-H+ hydrogel. Specifically, mechanically anisotropic hydrogel film and cell patterning can be achieved via two-dimensional (2D) patterning; complex external and internal 3D structures can be fabricated in stimuli-responsive hydrogel and other hydrogels that are not stimuli-responsive under experimental conditions (also owing to the erasable properties of Gel/Chit-H+–Citr hydrogel) via 3D patterning; and an interconnected or segregated fluidic network can be constructed from the same initial 3D grid structure via 4D patterning. Our method is very simple, safe and generally reagentless, and the products/structures are often erasable, compatible and digestible, enabling advanced fabrication technologies (e.g. additive manufacturing) to be applied to a sustainable materials platform.

Natural Origin Biomaterials for 4D Bioprinting Tissue‐Like Constructs

“…[16] This strategy can also be applied to achieve other desirable properties, [16] such as solubility in water, [54] control over the degradation rate, [55] and enhanced mechanical properties. [56] Gathering on this, some of the most explored natural polymers for 4D bioprinting including alginate, [33,57] collagen, [58] gelatin, [34,59,60] hyaluronic acid, [18,33] and chitosan [61] are depicted in Figure 1. Within 3D bioprinting applications, alginate hydrogel bioinks are undoubtedly one of the most broadly researched natural biomaterials.…”

“…Collagen and gelatin possess an exceptional trait for ECM-biomimetic hydrogel bioinks' formulation, since, in essence, they are natural-ECMderived biomaterials, [70] i.e., collagen proteins make up a significant portion of the natural ECM in mammalian tissues, and gelatin corresponds to the denatured form of collagen, which significantly improves cell attachment, proliferation, and activity, making them particularly suitable for bioprinting applications and TERM. [7] This feature, allied with the inherent temperature [61] and pH [32] responsiveness of gelatin, makes it a particularly suitable precursor for 4D biomaterial inks' formulation and dynamic constructs' manufacture. Moreover, gelatin modified with methacrylic groups (methacrylated gelatin, GelMA) has been frequently employed as a bioink.…”

“…[35] Mammalian-derived gelatin is another example of a thermoresponsive naturally derived polymer that can experience reversible sol-gel transitions upon temperature variations (at temperatures above 30 °C, gelatin is in a soluble state, while at temperatures below 25 °C gelatin is in a gel state). [61] Importantly, these temperature ranges are highly dependent on gelatin origin, with fish skin gelatin presenting an entirely different temperature profile (for example, 4-8 °C gelling and 16-18 °C melting [90] ), which is also dependent on the type of fish living areas (i.e., cold or warm water).…”

Biomaterials for protein delivery for complex tissue healing responses

“…Hydrogels have been extensively investigated for tissue regeneration applications due to their innate hydrophilic characteristic, which makes them suitable delivery vehicles for cells and proteins. 16,17 Hydrogels are 3D networks consisting of various natural and/or synthetic polymers, including alginate, 18 gelatin, 19 hyaluronic acid, 20 polyethylene glycol (PEG), 21 and devitalized or decellularized ECM. 22 Synthetic polymer hydrogels are highly reproducible and can be extensively functionalized for modular addition of proteins, cells, and other bioactive ligands; however, since synthetic polymers are typically biologically inert, multiple functional groups may be required to achieve a desired biological response.…”

Development of an electrically responsive hydrogel for programmable in situ immobilization within a microfluidic device

“…22,23 In addition, the repeatable/cyclic sol-gel transformation of hydrogels enables the design of smart engineering materials as stimulable catalytic supports with immobilized active sites. 24 Among others, the design of naturally accessible polysaccharides, such as alginic acid and chitosan, is of particular interest due to their sustainable disposition. 5,13,[25][26][27] Sodium or potassium salts of alginic acid form viscous, homogeneous aqueous solutions that are converted to ionotropic hydrogels by crosslinking with divalent or multivalent metal cations, typically Ca 2+ .…”