Bioinspired and Photo-Clickable Thiol-Ene Bioinks for the Extrusion Bioprinting of Mechanically Tunable 3D Skin Models

Abstract

Bioinks play a fundamental role in skin bioprinting, dictating the printing fidelity, cell response, and function of bioprinted 3D constructs. However, the range of bioinks that support skin cells' function and aid in the bioprinting of 3D skin equivalents with tailorable properties and customized shapes is still limited. In this study, we describe a bioinspired design strategy for bioengineering double crosslinked pectin-based bioinks that recapitulate the mechanical properties and the presentation of cell-adhesive ligands and protease-sensitive domains of the dermal extracellular matrix, supporting the bioprinting of bilayer 3D skin models. Methacrylate-modified pectin was used as a base biomaterial enabling hydrogel formation via either chain-growth or step-growth photopolymerization and providing independent control over bioink rheology, as well as the mechanical and biochemical cues of cell environment. By tuning the concentrations of crosslinker and polymer in bioink formulation, dermal constructs were bioprinted with a physiologically relevant range of stiffnesses that resulted in strikingly site-specific differences in the morphology and spreading of dermal fibroblasts. We also demonstrated that the developed thiol-ene photo-clickable bioinks allow for the bioprinting of skin models of varying shapes that support dermis and epidermis reconstruction. Overall, the engineered bioinks expand the range of printable biomaterials for the extrusion bioprinting of 3D cell-laden hydrogels and provide a versatile platform to study the impact of material cues on cell fate, offering potential for in vitro skin modeling.

Keywords: bioink; click chemistry; dermis; epidermis; extrusion bioprinting; hydrogels; in vitro model; mechanical cues; pectin; skin bioprinting.

Figure 1

Schematic illustration of bioink design and extrusion bioprinting of 3D skin equivalents. (a) Chemical modification of pectin using methacrylic anhydride, yielding pectin methacrylate (PECMA), bearing methacrylates for photocrosslinking and carboxylic groups for binding with calcium. (b) Cell-adhesive and MMP-sensitive peptide sequences used for hydrogel biofunctionalization and photocrosslinking, respectively, via a thiol-ene reaction between methacrylates in the polymer backbone and cysteines (highlighted as green) in the peptide sequences. (c) A photocrosslinkable bioink is prepared from PECMA polymer and custom-made peptide sequences and its rheology is tuned via the addition of calcium chloride for ionic crosslinking, followed by mixing and homogenization to obtain a physically crosslinked bioink. Then, the bioink is loaded with dermal fibroblasts and bioprinted for dermal reconstruction, followed by in vitro dermis maturation. Afterwards, HaCaT cells are seeded onto the dermis, cultured under submerged conditions, and subsequently subjected to ALI culture to generate the bilayer skin equivalent.

Figure 2

Rheological properties of pre-crosslinked PECMA inks. Effect of CaCl₂ concentration (0 mM and 6 mM) on the shear viscosity (a) and yield stress (b) of inks prepared at varying polymer concentrations. (c) Viscosity of pre-crosslinked (6 mM CaCl₂) PECMA inks at the yield point.

Figure 3

Hydrogel crosslinking and tunable mechanical properties. (a) Illustration of dual-crosslinked hydrogel network formed via either chain-growth or step-growth mechanisms, showing the establishment of ionic bonds via calcium crosslinking in both networks and the formation of chemical carbon–carbon crosslinks or thioether crosslinks, depending on the absence or presence of the peptide crosslinker, respectively. (b) Impact of chain-growth (no crosslinker) or step-growth (0.5 mM peptide crosslinker) mechanisms on the formation and mechanical properties of 1.5% PECMA hydrogels. (c) Mechanical properties of chain-growth and step-growth hydrogels (1.5% PECMA) prepared with varying photocrosslinking times. (d) Influence of peptide crosslinker content and (e) PECMA concentration on the mechanical properties of step-growth hydrogels (**** p < 0.0001).

Figure 4

Bioprinting and characterization of dermal equivalents. (a) Bioprinting strategy to generate tissue-engineered dermis using dermal fibroblast-loaded thiol-ene bioink (cysteines highlighted as green). (b) Macroscopic images of bioprinted 3D dermal equivalents using bioinks with varying composition after 14 days of culture (scale bar: 2 mm). (c) Representative confocal images of fibroblasts stained for F-actin (F-ACT, green) and nuclei (DNA, blue), showing the effect of hydrogel elastic moduli on cell morphology at the center of the hydrogel (scale bar: 100 μm). (d) Cross-section confocal images of cells within bioprinted hydrogels stained for F-actin (green) and nuclei (blue) at day 14 (scale bar: 100 μm), showing the morphology of cells located at the hydrogel center and periphery. (e) Confocal images depicting the deposition of fibronectin (FN, red) within bioprinted dermis at day 14 (F-actin: green; nuclei: blue; left image scale bar: 100 μm; right image scale bar: 50 μm).

Figure 5

Bioprinted 3D skin models. (a) Illustration of bilayered models with circular and square shapes, as well as their structural integrity after 28 days of culture (scale bar top: 2.5 mm; scale bar down: 5 mm). (b) Immunostaining of paraffin-embedded samples using antibodies directed against cytokeratin (keratinocytes) in epidermis and vimentin (fibroblasts) in the dermis (epidermis: cytokeratin (green) and nuclei (blue); dermis: vimentin (red) and nuclei (blue); scale bar: 100 μm).