Clickable Dynamic Bioinks Enable Post‐Printing Modifications of Construct Composition and Mechanical Properties Controlled over Time and Space

Abstract Bioprinting is a booming technology, with numerous applications in tissue engineering and regenerative medicine. However, most biomaterials designed for bioprinting depend on the use of sacrificial baths and/or non‐physiological stimuli. Printable biomaterials also often lack tunability in terms of their composition and mechanical properties. To address these challenges, the authors introduce a new biomaterial concept that they have termed “clickable dynamic bioinks”. These bioinks use dynamic hydrogels that can be printed, as well as chemically modified via click reactions to fine‐tune the physical and biochemical properties of printed objects after printing. Specifically, using hyaluronic acid (HA) as a polymer of interest, the authors investigate the use of a boronate ester‐based crosslinking reaction to produce dynamic hydrogels that are printable and cytocompatible, allowing for bioprinting. The resulting dynamic bioinks are chemically modified with bioorthogonal click moieties to allow for a variety of post‐printing modifications with molecules carrying the complementary click function. As proofs of concept, the authors perform various post‐printing modifications, including adjusting polymer composition (e.g., HA, chondroitin sulfate, and gelatin) and stiffness, and promoting cell adhesion via adhesive peptide immobilization (i.e., RGD peptide). The results also demonstrate that these modifications can be controlled over time and space, paving the way for 4D bioprinting applications.


Fig. S9.
Stability of the soft (left) and stiff (right) dynamic hydrogels in culture medium.Clickable dynamic hydrogels were prepared in PBS then immersed in DMEM with or without HA-N3 (0.05% w/v for the soft and 0.125% w/v for the stiff formulations) for up to 30 days.Similarly to unmodified dynamic hydrogels, the clickable dynamic hydrogels showed minimal swelling/shrinking, confirming that the clickable moieties and the click reaction do not alter the hydrogel stability.Supplementary Table 1.Synthetic conditions of the modified polymers used for the design of clickable dynamic hydrogels.
Fig. S1.Swelling/stability study of the soft and stiff dynamic hydrogels in PBS at 37°C.

Fig. S4 .
Fig. S4.3D views of the distribution of primary human adipose-derived stromal cells (ASCs) encapsulated in the soft and stiff dynamic hydrogels or resuspended in an unmodified HA solution.Comparison of cell distribution 10 min vs 4 hours after encapsulation demonstrated the absence of cell sedimentation only for the dynamic bioinks.(Scale bar = 250 µm).

Fig
Fig. S5.Representative 1 H NMR (400 MHz, D 2 O) of unmodified HA, HA-glucamine, and BCNmodified HA-glucamine, confirming the successful chemical modification of HA.The 3 protons of the N-acetyl group of HA (2.1 ppm) served as a reference to calculate the degrees of BCN substitution of BCN-modified HA-glucamine (orange circles, including 2 protons at 1.1 ppm, 2 protons at 2.4 ppm, and 2 protons at 4.3 ppm).

Fig. S6 .
Fig. S6.Shear storage moduli (Gʹ, at 1 Hz) of the soft (left) and stiff (right) dynamic hydrogels either unmodified (unmod.)or modified with BCN for click reaction (BCN-mod.).Values were extracted from frequency sweep experiments performed at 37 °C with a dynamic shear rheometer.Data are shown as mean ± SD (n = 3) with statistical significance determined using Student's t-test (ns: not significant).

Fig. S8 .
Fig. S8.The incubation of an unmodified soft dynamic hydrogel (green fluorescence) with fluorescent HA-N3 (purple fluorescence) did not allow for subsequent polymer immobilization.After successful diffusion within the hydrogel, the fluorescence from HA-N3 disappears with washes, indicating the absence of HA-N3 immobilization within the non-clickable dynamic hydrogel (scale bar = 500 µm).

Fig. S11 .
Fig. S11.Time sweep experiments showing the stiffening of the soft (left) and stiff (right) clickable dynamic hydrogels after mixing with HA-N3 (N3:BCN molar ratio of 1:1), via dynamic shear rheometry.The red and black lines indicate the clickable dynamic hydrogel with and without HA-N3, respectively.The results show an important increase in shear moduli in the presence of HA-N3, suggesting successful covalent crosslinking.

Fig. S14 .
Fig. S14.Left: Top views of clickable dynamic hydrogels (CF488) incubated in CF647 HA (20 kDa) or CF647 HA-N3 (20 kDa) up to 3 days post-printing.Fluorescence imaging was performed after 24 hours of incubation, and before or after washes.The CF647 signal was measured along the white dashed lines.Right: CF647 fluorescence intensity along the dashed white arrows, confirming local modification of clickable dynamic hydrogels.

Fig. S15 .
Fig. S15.Temporal control over the composition adjustment of clickable dynamic hydrogels with N3-modified chondroitin sulfate (CS-N3).Clickable dynamic hydrogels were incubated for 24 hours with a solution containing fluorescent CS or CS-N3 (1 mg.mL -1 ) on day 0, 3, or 7 after hydrogel preparation.After washes, only CS-N3 remained in the hydrogel, suggesting specific immobilization of CS-N3 via click chemistry.CS-N3 immobilization was effective at the three investigated time points, confirming that the second click reaction can be performed at a chosen time (scale bar = 2.5 mm).

Fig. S16 .
Fig. S16.Representative images of encapsulated ASCs exposed or not to clickable RGD immobilization.These images reveal a decrease in cell sphericity only upon the addition of the clickable adhesive peptide.Cell adhesion could be triggered at different times (day 1 or day 2 after printing), demonstrating a temporal control over cell behavior after bioprinting.

Table 2 .
Composition of the dynamic and clickable dynamic hydrogels.