Poster M18 - Shear-induced collagen fibril alignment using FRESH 3D bioprinting

Introduction: Collagen type I is the most abundant fibrillar protein in the human body, making up load bearing tissues such as tendons, ligaments, and muscles. The anisotropy of the extracellular matrix (ECM) in tissue impacts its mechanical properties and depends in large part on the multiscale organization of collagen molecules into fibrils and fibers through a process called fibrillogenesis. In recent years, several methods have been developed to engineer collagen scaffolds with anisotropy through electrospinning and microfluidic systems. However, they require complex and expensive equipment and are limited by the 2D area that can be covered with aligned fibers.1,2 Many 3D printing approaches have been developed to direct collagen fibril alignment in situ, however, these methods use low concentration inks or decellularized ECM proteins that limits the density and control on morphology of aligned fibrils formed.3 There is a critical need to develop improved methods to engineer collagen fibrillar scaffolds with high density collagen fibrils from high concentration collagen bioinks as this is known to mimic native tissues and enhances cell attachment.4 Here we propose to use Freeform Reversible Embedding Suspended Hydrogels (FRESH) 3D bioprinting5 to align collagen fibrils in situ as the collagen crosslinks during the printing process. FRESH 3D bioprinting provides a novel solution, as the printing method allows for shear-induced fiber alignment as the fibrils are being formed in situ, which can be further controlled by tuning various print parameters.

Materials and

Methods: FRESH 3D bioprinting was performed on a Maker Gear M2 modified with a custom designed syringe pump extruder. A gelatin microparticulate support bath was prepared via complex coacervation based on previously published methods.5 Lifeink® 240 collagen type I bioink acidified at 35 mg/ml and Lifeink® 260 acidified at 70 mg/ml (Advanced Biomatrix) were used. Post printing, the collagen constructs were released by melting the gelatin support bath at 37 ºC. The constructs were then washed 3x times in 200 mM HEPES (Corning®) followed by 3x times washes in DI water to remove all the gelatin and salts. The printed collagen constructs were then dried on a glass slide overnight. Various parameters such as collagen ink concentration, HEPES concentration, needle diameter, print speed and volumetric flowrate were modified to increase collagen fibrillar alignment with print direction.

Results, Conclusions, and Discussions: For assessing collagen fibril alignment, a window calibration model was designed with overall dimensions of 8x8x1 mm and the lines in between were designed to be 100 um in width and 100 um in height (Figure 1 (A)). This construct was printed with high fidelity using the 70 mg/ml high concentration collagen bioink (Figure 1 (B)). In-situ collagen fibrils formed using the 70 mg/ml collagen ink during FRESH printing was confirmed by observing D-banding (Figure 1 (C) (i), (ii)) from the collagen fibrils when the individual line segments of the window calibration were imaged post drying of the printed constructs. To further quantify the alignment of Collagen fibrils with various print parameter, Orientation J plugin in Image J will be used to obtain an orientation index based on the orientation distribution. These results demonstrate that collagen fibrils can be formed in situ during FRESH 3D printing. With change in various parameters such as HEPES concentration, nozzle diameter, print speed and volumetric flow rate we expect to see an increase in shear induced collagen fibril alignment within FRESH printed constructs. Future studies will also focus on in situ collagen fibril formation and alignment in 3D directions by printing structures in non-planar 3D space that will mimic native tissue architectures.

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