Thiol-Ene Alginate Hydrogels as Versatile Bioinks for Bioprinting

Abstract

Bioprinting is a powerful technique that allows precise and controlled 3D deposition of biomaterials in a predesigned, customizable, and reproducible manner. Cell-laden hydrogel ("bioink") bioprinting is especially advantageous for tissue engineering applications as multiple cells and biomaterial compositions can be selectively dispensed to create spatially well-defined architectures. Despite this promise, few hydrogel systems are easily available and suitable as bioinks, with even fewer systems allowing for molecular design of mechanical and biological properties. In this study, we report the development of a norbornene functionalized alginate system as a cell-laden bioink for extrusion-based bioprinting, with a rapid UV-induced thiol-ene cross-linking mechanism that avoids acrylate kinetic chain formation. The mechanical and swelling properties of the hydrogels are tunable by varying the concentration, length, and structure of dithiol PEG cross-linkers and can be further modified by postprinting secondary cross-linking with divalent ions such as calcium. The low concentrations of alginate needed (<2 wt %), coupled with their rapid in situ gelation, allow both the maintenance of high cell viability and the ability to fabricate large multilayer or multibioink constructs with identical bioprinting conditions. The modularity of this bioink platform design enables not only the rational design of materials properties but also the gel's biofunctionality (as shown via RGD attachment) for the expected tissue-engineering application. This modularity enables the creation of multizonal and multicellular constructs utilizing a chemically similar bioink platform. Such tailorable bioink platforms will enable increased complexity in 3D bioprinted constructs.

Figure 1

Schematic overview of the strategy employed to develop photoactive alginate bioink (Alg-norb) for bioprinting of hydrogels reported in the current work.

Scheme 1. Reaction Schemes of a) the Carbodiimide Reaction between Alginate and Norbornene Methylamine and Photoinitiated Thiol–Ene Reactions of Alg-norb with b) RGD Peptide Sequence (CGGGRGDS)

Figure 2

¹H NMR spectra (in D₂O) of a) Alg-norb, b) CGGGRGDS, and c) Alg-norb reacted with 1 mM CGGGRGDS. The observed decrease in intensity of the double bonds of the norbornene groups after the reaction (highlighted in the blue box at 6.2 ppm) and the appearance of the peaks corresponding to the peptide sequence are highlighted in the red box at 1.7 ppm.

Figure 3

Alg-norb hydrogel (2 w/v%) cross-linked with 10 mol % of 1500 Da PEG dithiol (Alg-norb10-1) swelled in a) water at 23 °C, b) PBS at 23 °C, c) PBS at 37 °C, and d) DMEM at 37 °C.

Figure 4

Shear storage (G′) and loss (G″) moduli, G′, measured as a function of a) UV illumination time, b) frequency, and c) strain for Alg-norb with different PEG cross-linkers (1 = Alg-norb10-1; 2 = Alg-norb10-2; 3 = Alg-norb10-3; 4 = Alg-norb10-4). d) Strain sweep of Alg-norb10-1 (blue lines) and after addition of 100 mM CaCl₂ for 3 min (red lines). UV intensity is 1 W/cm² for all rheological measurements.

Figure 5

Images of 3D bioprinted hydrogels loaded with cells at a) day 0 and b) day 7. Green and red cell tracker labeled L929 as two different bioinks printed as alternating fibers c) in the X-Y plane and d) in the Z direction.

Figure 6

Scaffolds bioprinted in a) the geometry of a pyramid. b) and c) the geometry of a cube. Porous-like structures can be seen in the cube scaffold shown in d) X-Y and e) Z planes when imaged between two glass coverslips. Of note, the bioprinting conditions used to produce these scaffolds match those optimized for high-cell viability. These scaffolds have shown stability in PBS for over two months. Theoretical side length = 6.9 mm (13 strands, 0.53 mm between strands), total height = 5.2 mm (200 μm/layer, 26 layers).

Figure 7

Viability of ATDC5 and L929 cells in bioprinted scaffolds over day 1 and 7. Green stain represents live cells and red stain represents dead cells. ATDC5 scaffolds were bioprinted at 5 mm/s with 30 kPa pressure, and L929 scaffolds were bioprinted at 10 mm/s with 30 kPa pressure.