Speaker
Description
Introduction:
Deep vat printing (DVP) techniques, such as tomographic (Figure 1A) and filamented light (FLight) (Figure 1B) printing, have advanced biofabrication by enabling high-resolution, layer-free fabrications at unprecedented speeds[1]. Despite, collagen being the most widely used matrix in tissue engineering, its compatibility with DVP remained unexplored. Within this work, we introduce photoclickable collagen-based bioresins for DVP, achieving high printing fidelity (~50 µm), fast throughput (<20 s/cm³), and excellent biocompatibility as demonstrated by increased integrin β1 (ITGB1) expression in encapsulated C2C12 myoblasts compared to pure gelatin-based resin systems.
Methods:
We synthesized norbornene-functionalized collagen (ColNB), enabling rapid photocrosslinking with thiolated crosslinkers under 405 nm light. As a potential application, we demonstrated multi-material and multicellular DVP using ColNB resins in both tomographic and FLight printing to fabricate facile myotendinous tissue interfaces.
Figure 1. A. Schematic of tomographic printing, enabling the fabrication of entire 3D objects within seconds through volumetric light-exposure. B. Illustration of the FLight biofabrication technique, producing highly aligned filamented constructs. C. Tomographically printed perfusable 3D construct (Scale bar: 1 mm). D. 3D reconstruction of a FLight- construct with microarchitectural analysis (Scale bar: 40 µm). E. Tomographically fabricated muscle–connective tissue constructs after maturation. Immunofluorescence staining for sarcomeric α-actinin (SAA) indicates increased myotube formation in the transition and muscle regions (Scale bar: 500 µm). F. FLight-muscle–connective tissue construct. Myoblasts show highly aligned sarcomere structures with consistent SAA striation and spacing (Scale bars: map: 250 µm; inset: 100 µm; sarcomere detail: 50 µm). G. Quantification of sarcomere spacing in FLight contructs and comparison of myotube diameters.
Results:
Tomographic printing enabled the fabrication of complex, perfusable constructs (Figure 1C) with high spatial control. In contrast, FLight printing yielded high-aspect-ratio constructs with precisely aligned microfilaments throughout the porous gel (Figure 1D). These filament networks acted as structural guidance cues significantly impacting cellular behavior.
While cells aligned in tomographically printed tissues, along stress-induced cues (Figure 1 E), encapsulated myoblasts and fibroblasts within FLight constructs exhibited highly anisotropic cytoskeletal organization (Figure 1F), with ~90% of intracellular f-actin filaments aligned within ±10° of the predominant orientation angle.
Both DVP techniques supported C2C12 myoblast fusion and myotube formation, FLight constructs yielded thicker myotubes (~28 µm) compared to tomographically printed constructs (~16 µm) (Figure 1G). Immunostaining further revealed well-organized sarcomeric α-actinin structures in FLight samples with regular 2.6 µm spacing, a hallmark of contractile function. In contrast, such sarcomere structures were absent in tomographic constructs at identical timepoints.
Additionally, myotendinous tissue interfaces could be fabricated using multi-material printing, showing gradual transitions in MyHC expression and f-actin alignment across zones.
Discussion:
We demonstrate that photoclickable collagen-based resins can be effectively used in DVP for the fabrication of complex, multicellular tissues. Compared to widely used gelatin-based resins (e.g., GelMA, GelNB-GelSH), ColNB offers enhanced bioactivity and ECM-like composition. Our results highlight the potential of collagen-derived materials in advancing DVP-based tissue engineering, especially in applications requiring aligned, functional muscle-like tissues.
[1] H. Liu, P. Chansoria, P. Delrot, E. Angelidakis, R. Rizzo, D. Rütsche, L. A. Applegate, D. Loterie, M. Zenobi-Wong, Advanced Materials 2022, 34, 2204301.
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