Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity.

In order to ensure the long-term in vitro and in vivo functionality of cell-seeded 3D scaffolds, an effective and reliable method to control cell seeding efficiency and distribution is crucial. Static seeding on 3D additive manufactured scaffolds made of synthetic polymers still remains challenging, as it often results in poor cell attachment, high cell sedimentation and non-uniform cell distribution, due to gravity and to the intrinsic macroporosity and surface chemical properties of the scaffolds. In this study, the biocompatible macromolecules dextran and Ficoll (Ficoll-Paque) were used for the first time as temporary supplements to alter the viscosity and density of the seeding media, respectively, and improve the static seeding output. The addition of these macromolecules drastically reduced the cell sedimentation velocities, allowing for homogeneous cell attachment to the scaffold filaments. Both dextran and Ficoll-Paque -based seeding methods supported human mesenchymal stromal cells viability and osteogenic differentiation post-seeding. Interestingly, the improved cell distribution led to increased matrix production and mineralization compared to scaffolds seeded by conventional static method. These results suggest a simple and universal method for an efficient seeding of 3D additive manufactured scaffolds, independent of their material and geometrical properties, and applicable for bone and various other tissue regeneration. STATEMENT OF SIGNIFICANCE: Additive manufacturing has emerged as one of the desired technologies to fabricate complex and patient-specific 3D scaffolds for bone regeneration. Along with the technology, new synthetic polymeric materials have been developed to meet processability requirements, as well as the mechanical properties and biocompatibility necessary for the application. Yet, there is still lack of methodology for a universal cell seeding method applicable to all additive manufactured 3D scaffolds regardless of their characteristics. We believe that our simple and reliable method, which is based on adjusting the cell settling velocity to aid cell attachment, could potentially help to maximize the efficiency, and therefore, functionality of cell-seeded constructs. This is of great importance when aiming for both in vitro and future clinical applications.