Light-Pipe FRESH 3D Bioprinting for Advanced Engineering of Functional Tissues

Introduction: Native tissues are highly complex with multiple cell types and spatial heterogeneity that is critical to structure and function. Tissue engineering aims to recapitulate this complexity in order to replace damaged tissues and organs, but to date there have been limited techniques to do so. Embedded 3D bioprinting methods, such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH), have demonstrated the biofabrication of architecturally-complex constructs with good fidelity by extruding bioinks within a support bath. Despite these advancements, spatial heterogeneity in terms of local biomechanics, ECM composition, growth factor gradients, etc. have proved challenging. We hypothesized that adding spatiotemporal control of photochemistry to the existing advantages of the FRESH technique would advance the complexity of engineered tissues. To do this, we have developed a new light-based bioprinting technique called light-pipe FRESH 3D bioprinting where the light delivered from the tip of a fiber-optic cannula acts as a point light source to achieve photocrosslinking directly in the support bath in a layer-by-layer manner.

Materials and

Methods: Light-pipe FRESH 3D bioprinting incorporates a photocrosslinkable support bath and a fiberoptic-coupled light-path (Fig 1A). The photocrosslinkable support bath is composed of gelatin methacryloyl (GelMA) microspheres surrounded by an aqueous mixture of GelMA, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator, and tartrazine as a photoabsorber. The viscoelastic behavior of the photocrosslinkable support bath was characterized through photorheology. The fiberoptic-coupled light-path was assembled as follows: the UV-visible light was collimated and then filtered to 365 nm, coupled via an adapter to a fiberoptic patch cord and then delivered from the tip of a fiberoptic cannula. The fiberoptic cannula was selected through measuring divergence angle and penetration depth of cannulas with different numerical apertures to minimize light scattering. Following the bioprinting process, the non-crosslinked GelMA support bath was melted and then constructs were imaged under optical coherence tomography (OCT). The effect of printing parameters on mechanical properties of the scaffolds was determined by microindentation analysis. Response of cells to the stiffness difference between scaffold regions was investigated by generating a skeletal muscle tissue model in which bipennate-shaped scaffolds were printed at four different printing speeds, corresponding to different elastic moduli, and then seeded with C2C12 mouse myoblast-laden collagen.

Results, Conclusions, and Discussions: Light-pipe FRESH 3D bioprinting was able to successfully integrate photochemistry into the extrusion printing process. Rheological characterization demonstrated yield-stress, shear-thinning and shear-recovering behavior of the GelMA support bath. The printability window was determined by investigating printing speed and photoabsorber concentration and then comparing the ratio of printed single fiber thickness to the designed fiber thickness (Fig 1B). Fibers with approximately 150 μm thickness were obtained with a support bath having 60 μM tartrazine and a printing speed of 25 to 40 mm/min at 8 µW power output. Microindentation results demonstrated elastic moduli in the kilopascal range with ~8 times difference between the scaffolds printed at 25 versus 40 mm/min (Fig 1C). Characterization of the cells by immunofluorescence and muscle contraction demonstrated that myoblasts favored less stiff scaffolds for tissue maturation, myotube formation and enhanced contractility (Fig 1D).

By combining FRESH with photochemistry, light-pipe FRESH 3D bioprinting technique is capable of locally engineering the structural, biological and mechanical properties of the biofabricated constructs in a way that cannot be achieved by other approaches.

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