Direct Freeform Fabrication of Spatially Heterogeneous Living Pre-cell-seeded Implants

The objectives of this work are the development of the processes, materials, and tooling to directly “3-D print” living, pre-seeded, patient-specific implants of spatially heterogeneous compositions. The research presented herein attempts to overcome some of the challenges to scaffolding, such as the difficulty of producing spatially heterogeneous implants that require varied seeding densities and/or cell-type distributions. In the proposed approach, living implants are fabricated by the layer-wise deposition of pre-cell-seeded alginate hydrogel. Although alginate hydrogels have been previously used to mold living implants, the properties of the alginate formulations used for molding were not suitable for 3-D printing. In addition to changing the formulation to make the alginate hydrogels “printable,” we developed a robotic hydrogel deposition system and supporting CAD software to deposit the gel in arbitrary geometries. We demonstrated this technology’s capabilities by printing alginate gel implants of multiple materials with various spatial heterogeneities, including, implants with completely embedded material clusters. The process was determined to be both viable (94±5% n=15) and sterile (less than one bacterium per 0.9 μL after 8 days of incubation). Additionally, we demonstrated the printing of a meniscus cartilage-shaped gel generated directly from a CT Scan. The proposed approach may hold advantages over other tissue printing efforts [4,5]. This technology has the potential to overcome challenges to scaffolding and could enable the efficient fabrication of spatially heterogeneous, patient-specific, living implants. INTRODUCTION Alginate gels have been previously used for molding of living implants. One of the aims of this research was to change the formulation in order to make it suitable for printing. Alginate is a seaweed derivate which, when combined with certain calcium cross-linking compounds, forms a polymer hydrogel [1]. Cells can be evenly mixed into this gel and then injection molded in order to create a desired geometry [2]. When the cell-seeded gel is placed in a growth medium and incubated, the cells consume the medium and replace the alginate gel, resulting in living tissue. The material properties of the gel are governed by the type of alginate, the type of cross-linker, the concentrations of each, as well as other factors such as temperature and age. The material properties that are optimal for molding the gels are not the same as those required for printing the gels. In fact, it turns out that the desired material properties for both cases are essentially opposite of each other. The discrepancies between the material properties required for molding and printing pose a major design challenge that this project addresses. The type of cell that is seeded into the gel can also dictate the required material properties. In this research, bovine cartilage chondrocyte cells are used because they do not require a blood supply for nutrition, but the challenge of growing cells that do require a blood supply is a separate open research field. Another consideration when dealing with alginate gels is the need for sterility. Much in the same way that the alginate gel supports growth of seeded cells, it will also support the growth of bacteria and fungi; hence, sterile print conditions are essential to prevent infection.