Speaker
Description
Introduction
The development of smart materials for bioprinting unlocks flexibility and control over the final construct characteristics and composition.1 Dynamic crosslinking allows the development of stimuli-responsive inks with intrinsic reversibility triggered by tunable physical and chemical conditions.2 Typically made of densely packed or jammed microgels, granular hydrogels offer unique properties, compared to bulk hydrogels, making them attractive for biofabrication. In the jammed state, the physical interactions between particles result in rheological behaviors (e.g., yield stress, shear thinning behavior) that contribute to their extrudability and shape fidelity post-printing. While microgel jamming is normally done through centrifugation, sedimentation, and vacuum filtration, in this study, we propose a chemical approach through reversible covalent bonds (Fig 1A). We focus on introducing stimuli responsiveness (volume transitions) into individual microgels, by the selective cleavage of disulfide bonds (Fig 1B) and consequently improve the rheological properties and printability of the granular hydrogel-based inks.
Methods
Specifically, we synthesized poly(N-isopropylacrylamide) (pNiPAM) based microgels by surfactant-free radical polymerization and introduced the cleavable dynamic covalent disulfide bonds within the microgels’ network, with the crosslinker N,N′-bis(acryloyl)cystamine (BAC). The granular hydrogel inks were prepared by directly dispersing the microgels in 1x-PBS. The reductant agent tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) was used to cleave the disulfide bonds, swell the microgels and consequently jam them (Fig 1C). The inks, before and after swelling/jamming, were characterized by DLS, Wet-AFM, Cryo-TEM, and rheology. A BioScaffolder 3.3 (Gesim) was used to print square-mesh scaffolds up to 32 layers, and shape fidelity was investigated. The obtained 3D printed scaffolds were immersed in a sodium periodate (NaIO4) solution to render back the disulfide bonds and crosslink the 3D printed constructs (Fig 1C). The obtained dimensionally stable scaffolds were used in biocompatibility tests with primary fibroblasts.
Results
pNiPAM microgels were prepared with an average diameter of 818 ± 25 nm at 20°C, and the successful incorporation of the disulfide units in the microgels network was proved by proton NMR and UV-Vis spectroscopy. The addition of TCEP effectively led to microgel swelling by cleavage of the disulfide bonds, which efficiently increases the yield-stress values of the microgel dispersions (Fig 1D). Moreover, inks that initially exhibited poor shape fidelity after 3D printing, were transformed into stable, multi-layered 3D structures capable of supporting their own weight, up to 32 layers, after reduction (Fig 1E). Post-printing annealing was done by immersion in a NaIO4 solution, to form intra and inter-particle disulfide bonds and obtain a dimensionally stable scaffold in physiological conditions. Its lack of toxicity prospers its use for tissue engineering purposes.
Conclusions
We successfully jammed disulfide crosslinked pNiPAM granular hydrogel systems which printability was tuned, by the weight fraction of particles and by the extent of swelling of the particles. Reversible covalent bonds allowed particle swelling triggered by the addition of reductant, and maintenance of the construct's stability by the immersion in an oxidant solution.
References
1.S. Vanaei, M.S. Parizi, S. Vanaei, F. Salemizadehpriz, H.R. Vanaei, Eng. Regeneration. Volume 2, 2021,1-18.
2.Muir VG, Burdick JA. Chem Rev. 2021, 121, 18.
3.A. C. Daly, Adv. HealthcareMater. 2023,2301388.
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