Macroporous Aligned Hydrogel Microstrands for 3D Cell Guidance

Abstract

Tissue engineering strongly relies on the use of hydrogels as highly hydrated 3D matrices to support the maturation of laden cells. However, because of the lack of microarchitecture and sufficient porosity, common hydrogel systems do not provide physical cell-instructive guidance cues and efficient transport of nutrients and oxygen to the inner part of the construct. A controlled, organized cellular alignment and resulting alignment of secreted ECM are hallmarks of muscle, tendons, and nerves and play an important role in determining their functional properties. Although several strategies to induce cellular alignment have been investigated in 2D systems, the generation of cell-instructive 3D hydrogels remains a challenge. Here, we report on the development of a simple and scalable method to efficiently generate highly macroporous constructs featuring aligned guidance cues. A precross-linked bulk hydrogel is pressed through a grid with variable opening sizes, thus deconstructing it into an array of aligned, high aspect ratio microgels (microstrands) with tunable diameter that are eventually stabilized by a second photoclick cross-linking step. This method has been investigated and optimized both in silico and in vitro, thereby leading to conditions with excellent viability and organized cellular alignment. Finally, as proof of concept, the method has been shown to direct aligned muscle tissue maturation. These findings demonstrate the 3D physical guidance potential of our system, which can be used for a variety of anisotropic tissues and applications.

Multiscale Hybrid Fabrication: Volumetric Printing Meets Two-Photon Ablation

“…Every 10 min, 0.5 g of CA was added for a total amount of 2 g. Twenty minutes after the last addition, the solution was diluted 2-fold in prewarmed mQ H2O, and the pH was adjusted to 7 with the addition of HCl 1 M. Then, 2.5 g of NaCl was added and the solution was filter-sterilized (0.2 μm) and dialyzed (12.4 kDa cutoff cellulose tubing) for 4-5 days against mQ H2O at 30 °C with frequent water changes before freeze-drying. The degree of substitution (DS) was calculated using 1 H NMR (Bruker Ultrashield 400 MHz, 1024 scans), as previously reported, 8, 28 and found to be ~ 0.091 mmol g -1 .…”

“…Gel-SH was stored under inert atmosphere at −20 °C prior to use. The degree of substitution (DS) was determined by 1 H NMR (Bruker Ultrashield 400 MHz, 1024 scans) as previously reported, 28 and found to be ~ 0.276 mmol g -1 .…”

“…Gelatin-Norbornene (Gel-NB) synthesis: The synthesis protocol was adapted from Rizzo et al. 8,28 In short, 25 g of gelatin type A from porcine skin was first dissolved in 0.5 M carbonatebicarbonate pH 9 buffer at 10% at 40 °C. When completely dissolved, 0.5 g of cis-5norbornene-endo-2,3-dicarboxylic anhydride (CA) was added to the reaction mixture under vigorous stirring.…”

“…Gelatin-Thiol (Gel-SH) synthesis: The synthesis protocol was adapted from Rizzo et al. 28 In short, 25 g of gelatin type A from porcine skin was first dissolved in 1.25 L of 150 mM MES pH 4 buffer warmed up to 40 °C for a final concentration of 2%. When completely dissolved, 2.38 g (10 mmol) of 3,3′-dithiobis(proprionohydrazide) (DTPHY) was added to the reaction solution under stirring.…”

Synergizing algorithmic design, photoclick chemistry and multi-material volumetric printing for accelerating complex shape engineering

“…Matrix synthesis. The norbornene or thiol-modified gelatin were synthesized using procedures previously established in our lab (17,41). For formulation of GelNB, porcinederived (Type A) gelatin was dissolved in 0.5 M carbonate-bicarbonate buffer (pH~9, obtained by adding 38.2 g/l of sodium bicarbonate and 4.7d/l of sodium carbonate in deionized (DI) water) at 50°C to get a 10% w/v solution.…”