Poster O19 - Novel 4D Printing Approach to Generate Aligned Collagen Scaffolds for Ligamentous Applications

Introduction: Introducing anisotropy in collagen scaffolds is a promising strategy to generate biomimetic constructs with enhanced mechanical and topographical properties to control cell function. While biofabrication techniques such as 3D printing allows for generation of application-specific tailormade collagenous scaffolds with high geometric precision, the scaffolds produced from this method lack collagen anisotropy. In this work, 3D printing was combined with the magnetic alignment approach in an innovative 4D printing approach to introduce high degree of collagen fiber alignment over a larger-scale custom scaffold. The goal of this study was to optimize the 4D printing process parameters including magnetic field strength, streptavidin-coated magnetic particle (SMP) concentration, and ink composition to yield magnetically aligned collagen scaffolds with high print fidelity and anisotropy. In addition, the impact of aligned collagen topography on ligamentous differentiation of mesenchymal stem cells (MSCs) was investigated.

Materials and

Methods: Col (6 mg/ml) and xanthum gum (XG; 10 mg/ml) were combined in varying ratios (Col:XG - 1:1, 4:1, or 9:1) to which a photoinitiator (0.1% w/v), and SMP (0, 0.2, 0.4 mg/ml) were added. An extrusion-based printer was used to 4D print collagenous constructs in the presence of a magnetic field (0 or 0.2 T) and the prints were incubated at 37 °C for 30 min to induce gelation followed by crosslinking using UV light. Rheological analyses were performed to compare the yield stress and viscosity of different ink compositions. Print fidelity was quantified by measuring the areas of the printed constructs using ImageJ. Qualitative analysis of collagen fiber alignment in 4D printed constructs was performed using polarized light microscopy (PLM) and scanning electron microscopy (SEM). MSCs were seeded on aligned and unaligned scaffolds and the effects of collagen alignment on cell metabolic activity (AlamarBlue assay), cell morphology (AlexaFluor 488 cytoskeleton staining), and ligamentous differentiation (scleraxis (SCX) expression) was assessed. Data were analyzed using one-way ANOVA with Tukey post hoc test. Statistical significance was set at p < 0.05.

Results, Conclusions, and Discussions: Results showed that increasing the XG composition (Col:XG 1:1) improved collagen ink viscosity (Fig. 1) and yielded scaffolds with good print fidelity (Fig. 2) but poor collagen alignment as seen in the results from SEM and PLM imaging (Fig 3). On the other hand, use of inks with lower XG composition (Col:XG 4:1 and 9:1) together with 0.4 mg/ml SMP concentration yielded scaffolds with high degree of collagen alignment albeit with suboptimal print fidelity (Fig 2, Fig. 3). Optimization of printing paramters by reducing the print flow speed from 3 mm/s to 2 mm/s with 4:1 Col:XG inks and 0.4 mg/ml SMP resulted in an improved print fidelity of the collagen scaffolds while maintaining the high level of collagen fiber alignment. Results from the cell studies revealed that MSCs orient uniformly on aligned collagen scaffolds as observed from cytoskeleton staining (Fig. 4A). Cells seeded on aligned scaffolds exhibited higher cell metabolic activity compared to unaligned scaffolds (Fig. 4D). SCX which is an early marker of ligamentous differentiation, was expressed very early in culture on day 3 and increased with time on aligned collagen scaffolds (Fig. 5) indicating that MSCs can sense and respond to the topographical cues provided by collagen anisotropy and differentiate specifically to the ligamentous lineage.
This work presents a novel 4D printing strategy that combines extrusion-based 3D printing and the magnetic alignment approach to generate reproducible multilayered scaffolds with a high degree of collagen fiber alignment. The relevance lies in incorporating a fourth dimension to leverage real-time matrix remodeling of fiber-forming collagen molecules, to generate highly anisotropic scaffolds that recapitulate the native microarchitecture of ligamentous tissue. In conclusion, optimization of ink composition and SMP concentration can deliver 4D printed scaffolds with a high degree of collagen fiber alignment. Overall, this study presents a novel 4D printing strategy to deliver biomimetic scaffolds with a high degree of collagen anisotropy and tailored printability for use in tendon tissue engineering applications.

Acknowledgements (Optional): The authors would like to acknowledge the financial support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R15AR071102.