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Unique Shrinkage Method Improves 3D Printed Hydrogel Constructs

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A group of international authors delve further into bioprinting and the structures that make it possible to keep cells alive during tissue engineering, releasing their findings in the recently published ‘Complexation-induced resolution enhancement of 3D-printed hydrogel constructs.’ In this study, the research team focuses on post-printing treatment of hydrogel constructs, immersing them in a solvent meant to transform dimensions via a unique shrinking method.

3D printing is impacting the medical field in many ways, and bioprinting, tissue engineering, and fabrication of a variety of bioinks and scaffolding structures are a source of fascination around the globe as we wait for the ultimate—true 3D printed human organs that may eventually change the face of medicine, and do away with waiting lists one day for patients in dire need. The authors of this latest research point out that while 3D printing and bioprinting have allowed for huge strides, limitations remain:

“ … existing printing strategies all have their minimally producible resolutions, which are factors of multiplexed parameters, such as the printer hardware and ink properties. For example, in extrusion printing using hydrogels as inks, the resolutions are typically sub-millimeter for the dispensed microfibers,” explain the researchers.

“The same holds true for microfluidic coaxial printing, where the diameters of the created hollow microfibers usually fall in the range of a couple hundred micrometers or larger. Although some other printing strategies, such as those based on light (e.g., two-photon lithography) can achieve varying degrees of higher resolutions, their instrumentation is usually complicated limiting the broader adoption for general use.”

Much research has been devoted to refining inks and hardware for better performance; however, the authors point out that much of the progress so far has been ‘impractical for some applications.’ Other proposed methods for improving hydrogels via shrinkage technology have fallen somewhat flat too.

Shrinking printing, also known as complexation-induced resolution enhancement in 3D printing, offers improved resolution—without the need to transform printers or ink. The research team used anionic inks like used hyaluronic acid methacrylate (HAMA), gelatin methacryloyl (GelMA), and alginate.

“Following standard printing procedures, we subject the HAMA-, GelMA-, or alginate-based hydrogel constructs to immersion in a polycationic chitosan solution,” explained the researchers. “Through charge complexation and subsequent expulsion of water from the gels, these printed constructs are found to reduce in their linear dimensions in various degrees.”

Proof-of-concept was performed via:

  • Direct extrusion printing
  • Sacrificial printing
  • Microfluidic hollow fiber printing

The researchers also demonstrated versatility of this technique in using polyanionic alginate to shrink polycationic chitosan-based hydrogel constructs, along with showing success in keeping cells alive within the bioprinted hydrogels.

a) Schematics showing the shrinking effect based on charge compensation. b) Photograph showing size change of fabricated HAMA hydrogel (1.0 w/v%) before (lower) and after (upper) shrinking in 2.0 w/v% MMw chitosan dissolved in 1.0 v/v% acetic acid aqueous solution. c) Schematic representation of a HAMA hydrogel disc together with the dimensions and corresponding quantitative analyses showing the dimensions and volume changes before and after shrinking in 2.0 w/v% MMw chitosan dissolved in 1.0 v/v% acetic acid aqueous solution. d) Corresponding quantitative analyses showing the shrinking of HAMA hydrogels in perchloric acid solution (pH = 1.0) or in 1.0 v/v% acetic acid aqueous solutions (pH = 4.7) with 2.0 w/v% of chitosan of different molecular weights and types. e) Confocal images showing the diffusion of FITC-Q. chitosan solution in PBS into a 2.0 w/v% HAMA hydrogel at 3 and 24 h of shrinking. The bright-field image at 0 h serves as the size reference of the initial hydrogel. f) Photograph showing size change of HMw chitosan hydrogels (2.0 w/v%), where the lower one was swollen in 1.0 v/v% acetic acid aqueous solution and the upper one was shrunken in 2.0 w/v% alginate in 1.0 v/v% acetic acid aqueous solution. **P < 0.01; one-way ANOVA (c, d, compared with the values of corresponding as-prepared samples); mean ± s.d, (n = 3).

Sample cylindrical HAMA hydrogels were fabricated for the study, with comparisons noted before and after chitosan shrinkage.

“Although the concentrations of HAMA in the shrunken hydrogels were much higher than those of the initial constructs, water was still the main constituent maintaining their hydrogel nature for various relevant applications,” explained the authors.

a) Schematics showing the concept of shrinking printing, where a printed hydrogel structure is post-treated to reduce its size and achieve higher resolution. b, c) Printability mapping of HAMA inks at different concentrations and extrusion pressures. d) Photographs (upper) and micrographs (lower) showing size changes of printed HAMA hexagons (2.0 w/v%) immersed in 2.0 w/v% HMw chitosan dissolved in 1.0 v/v% acetic acid aqueous solution during the 24h shrinking process. e, f) Corresponding quantitative analyses of size changes of the printed HAMA hexagons (2.0 w/v%), include e side-to-side distance and f thickness, during the 24h shrinking process. g) Photographs (upper) and micrographs (lower) showing size changes of printed HAMA hexagons (2.0 w/v%) at 2 h and 24 h of shrinking in different concentrations of HMw chitosan (0.5–5.0 w/v%) dissolved in 1.0 v/v% acetic acid aqueous solution, h, i) Corresponding quantitative analyses of size changes in h side-to-side distance and i thickness. j) Vector-field maps comparing the 24-h shrinking images (magenta) to the corresponding 2-h shrinking images (green) by a B-spline-based non-rigid registration algorithm, where the overlaps appear in white and the grids show local distortions. Note that the 2-h shrinking images have been rescaled to match the sizes of the 24-h shrinking images to enable comparisons. **P < 0.01; one-way ANOVA (e, f, compared with the values of corresponding as-prepared samples); mean ± s.d. (e, f, n = 40; h, i, n = 10).

“Notably, our data showed that these printed constructs could reduce in their sizes by different degrees, comparing to their original dimensions. In addition, results indicated that this method is broadly applicable, i.e., a printed anionic hydrogel structure might be shrunken by a cationic polymer, or vice versa,” concluded the researchers. “We finally demonstrated that successive shrinking could preserve, in a cell type-dependent manner, the viability of cells embedded in the printed hydrogel matrices compared to a single, longer shrinking procedure, revealing the potential applications of our shrinking printing method towards tissue biofabrication.

“We therefore anticipate widespread adoption of our unique technology in future printing of hydrogel constructs for various application areas with further optimizations.”

What do you think of this news? Let us know your thoughts! Join the discussion of this and other 3D printing topics at 3DPrintBoard.com.

a) Schematic illustrations of the core-sheath coaxial nozzle used as the printhead, where the ink is delivered through the sheath flow and the CaCl2 solution is co-delivered through the core flow. b) Printing of the cannular construct and its subsequent shrinkage. c, d) Photographs and micrographs showing the size changes of the tubes, coaxial-printed with inks containing different concentrations of HAMA (0.5–2.5 w/v%), before and after 24 h of shrinkage in 2.0 w/v% HMw chitosan dissolved in 1.0 v/v% acetic acid aqueous solution. e, f, g) Corresponding quantitative analyses of diameter (e, inner diameter; f, outer diameter; g, wall thickness) changes before and after shrinkage. **P < 0.01; one-way ANOVA (e, f, g, compared with the corresponding as-printed structures); mean ± s.d. (n = 40).

[Source / Images: ‘Complexation-induced resolution enhancement of 3D-printed hydrogel constructs’]

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