Optimized Photoclick (Bio)Resins for Fast Volumetric Bioprinting

Abstract

Volumetric printing (VP) is a light-mediated technique enabling printing of complex, low-defect 3D objects within seconds, overcoming major drawbacks of layer-by-layer additive manufacturing. An optimized photoresin is presented for VP in the presence of cells (volumetric bioprinting) based on fast thiol-ene step-growth photoclick crosslinking. Gelatin-norbornene (Gel-NB) photoresin shows superior performance, both in physicochemical and biocompatibility aspects, compared to (meth-)acryloyl resins. The extremely efficient thiol-norbornene reaction produces the fastest VP reported to date (≈10 s), with significantly lower polymer content, degree of substitution (DS), and radical species, making it more suitable for cell encapsulation. This approach enables the generation of cellular free-form constructs with excellent cell viability (≈100%) and tissue maturation potential, demonstrated by development of contractile myotubes. Varying the DS, polymer content, thiol-ene ratio, and thiolated crosslinker allows fine-tuning of mechanical properties over a broad stiffness range (≈40 Pa to ≈15 kPa). These properties are achieved through fast and scalable methods for producing Gel-NB with inexpensive, off-the-shelf reagents that can help establish it as the gold standard for light-mediated biofabrication techniques. With potential applications from high-throughput bioprinting of tissue models to soft robotics and regenerative medicine, this work paves the way for exploitation of VPs unprecedented capabilities.

Keywords: bioprinting; gelatin; photoclick; thiol-ene; volumetric.

Figure 1

Overview of gelatin‐norbornene (Gel‐NB) synthesis. A) Schematic of the reaction: carbic anhydride (CA) grafting on nonprotonated gelatin‐free amines in pH 9 carbonate–bicarbonate (CB) buffer. B) Illustration of Gel‐NB synthesis methods investigated in this study with varying interval time between sequential addition of CA and pH adjustment. C) Comparison of degree of substitution (DS, left) and norbornene grafting yield (right) obtained with the three different methods in 2 g scale synthesis. The synthesis was performed in 100:1, 50:1, and 10:1 Gel:CA w/w ratio. For DS comparison the right y axis (%) refers only to average values, standard deviations refer to left y axis (mmol g⁻¹). D) Comparison of DS and grafting yield resulting from Gel‐NB synthesis at different reaction scales (2 g, 10 g, 50 g) using the fastest method (M3). The synthesis was performed with different Gel:CA ratio in order to target different DS. For DS comparison the right y axis (%) refers only to average values, standard deviations refer to left y axis (mmol g⁻¹). E) Comparison of Gel‐NB synthesis (≈50% DS) with previous reports using CA.^{[ 43} , ^{44 ]} Compared to the commonly used protocol developed by Muñoz et al.,^{[ 43 ]} no visible excess of unreacted reagent is obtained upon centrifugation step at pH 7.4 using M3‐based synthesis with 10:1 Gel:CA ratio (left). On the right, main improvements resulting from the use of M3 are highlighted.

Figure 2

Photorheology characterization of Gel‐NB‐based resin using 0.05% w/v LAP as photoinitiator. Unless otherwise specified, photoresins are composed of 5% Gel‐NB (DS ≈ 50%) and PEG4SH at 1:1 SH:NB molar ratio A) Thiol–ene crosslinking scheme illustration of photoresin composed of Gel‐NB and a thiolated crosslinker. Upon 405 nm excitation of LAP, the generation of radical initiation species leads to step‐growth crosslinking (top). Structures and MW of thiolated crosslinkers used in this study (bottom). B) Investigation of DS influence on final hydrogel mechanical properties. The wide DS range results in tunable hydrogel stiffness. DS of ≈3%, obtained with a Gel:CA ratio of 500:1, is also shown to be not enough to guarantee hydrogel formation. C) Investigation of Gel‐NB concentration influence on final hydrogel mechanical properties. Highest storage modulus is observed for Gel‐NB 10%. A reduction of polymer content is associated with a reduction of the final mechanical properties due to a less densely crosslinked network. D) Influence of SH:NB ratio on final hydrogel mechanical properties. The use of 5× norbornene or 5× thiols results in much weaker gels. E) Influence of different thiolated crosslinker on final hydrogel mechanical properties. The highest storage modulus is observed with PEG4SH, while a drastic reduction is shown with the use of bifunctional crosslinkers. A direct comparison between bifunctional crosslinker with different MW shows that also chain length plays an important role in determining hydrogel stiffness.

Figure 3

Volumetric printing with Gel‐NB. A) Illustration of volumetric printing principle of operation. A 405 nm laser beam (light purple) is directed toward a digital‐micromirror‐device (DMD), which generates dynamically evolving projection images (dark purple) in synchrony with the rotation of the glass vial containing the photosensitive resin. The desired object is solidified where the local light dose accumulation exceeds the gelation threshold. B) Printing parameter optimization using 5% and 2.5% Gel‐NB/PEG4SH resin (DS ≈ 50%). A branch model perfusable with a high‐MW blue‐dextran solution is obtained with a low light dose, corresponding to extremely fast (≈10–11 s) printing (scale bar: 2 mm). C) Print upscaling with 2.5% Gel‐NB/PEG4SH. The potential of fast printing is shown with the generation of replicas of twelve perfusable branch models (left, alternating perfusion with blue‐dextran and TRITC‐dextran) and eight pawn models (right). D) Printing of various 3D complex objects. i) VP with 2.5% Gel‐NB/PEG4SH. From left to right: pawn, rook (top‐left), knight (top‐right), bishop (bottom‐left), and queen (bottom‐right). Due to the hydrogel softness, tall structures such as the bishop and queen models tend to bend when not submerged in liquid. ii) Printing at higher concentration (5% Gel‐NB/PEG4SH) results in stiffer objects that can easily stand (scale bars: 2 mm). E) High cell viability (>90%) after bioprinting is shown for mouse myoblasts (C2C12) and normal human dermal fibroblasts (NHDF) over 1 week of culture in both 5% and 2.5% Gel‐NB/PEG4SH resin. F) Cellular constructs. i) Bioprinting of C2C12‐laden complex free‐form objects with 2.5% Gel‐NB/PEG4SH resin (left, scale bars: 2 mm). Bright‐field close up on cellular construct after 1 week of culture showing cell spreading and proliferation on the soft matrix (right, scale bars: 200 µm). Immunofluorescence evidence of myotubes differentiation after 3 weeks of culture (Myosin Heavy Chain: red, Nuclei: blue, scale bars: 200 µm). ii) Confocal imaging of branch model bioprinted with NHDF‐laden 2.5% Gel‐NB/PEG4SH photoresin and perfused with TRITC‐dextran (red) after 1 week of culture (scale bar: 500 µm).