Direct Freeform Fabrication of Spatially Heterogeneous Living Cell-Impregnated 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 [5,9]. This technology has the potential to overcome challenges to scaffolding and could enable the efficient fabrication of spatially heterogeneous, patient-specific, living implants.Mechanical Engineerin

A Study of Variable Stiffness Alginate Printing for Medical Applications

Technologies for multi-material 3D-printing of anatomical shapes are useful both for fabrication of heterogeneous cell-seeded implants as well as for fabrication of synthetic models for surgical planning and training. For both these applications, it would be desirable to print directly with biological materials to best emulate the target’s properties. Using a novel material platform, we describe a series of experiments attempting to print variable-stiffness hydrogels. We vary compliances by alternating 2% alginate hydrogel and a Dextran-infused calcium chloride post-crosslinker. Stiffness throughout the construct ranged from 4 kPa to 20 kPa as a function of post-crosslinker concentration, which was spatially specified by the user.Mechanical Engineerin

Improved Quality of 3D-Printed Tissue Constructs Through Enhanced Mixing of Alginate Hydrogels

While alginate hydrogel is a desirable material platform for Solid Freeform Fabrication (SFF) of cell-seeded tissue engineering scaffolds, achieving consistently high-quality results can be challenging. Local variations in the material properties cause inconsistent material deposition behavior and consequently decrease the resultant geometric fidelity of the construct. The effects of gel mixing on material property consistency, geometric fidelity, and cell viability were characterized in an attempt to improve the formulation’s compatibility with SFF processing. Material homogeneity was quantified through a novel experimental setup composed of an EnduraTEC mechanical test-frame and custom syringe-extrusion jig. Cell viability and geometric fidelity were assessed using standard protocol. The baseline mechanical stiffness of the printed samples was 16±3 kPa (n=6). We found that increasing mixing reduced material inconsistency and improved geometric fidelity, without adversely affecting cell viability: the printed construct quality was drastically improved by increasing mixing well beyond previously established limits.Mechanical Engineerin

Cell and protein compatible 3D bioprinting of mechanically strong constructs for bone repair

Rapid prototyping of bone tissue engineering constructs often utilizes elevated temperatures, organic solvents and/or UV light for materials processing. These harsh conditions may prevent the incorporation of cells and therapeutic proteins in the fabrication processes. Here we developed a method for using bioprinting to produce constructs from a thermoresponsive microparticulate material based on poly(lactic-co-glycolic acid) at ambient conditions. These constructs could be engineered with yield stresses of up to 1.22 MPa and Young's moduli of up to 57.3 MPa which are within the range of properties of human cancellous bone. Further study showed that protein-releasing microspheres could be incorporated into the bioprinted constructs. The release of the model protein lysozyme from bioprinted constructs was sustainted for a period of 15 days and a high degree of protein activity could be measured up to day 9. This work suggests that bioprinting is a viable route to the production of mechanically strong constructs for bone repair under mild conditions which allow the inclusion of viable cells and active proteins

Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes

Background and purpose Three-dimensionally (3D-) embedded chondrocytes have been suggested to maintain the chondrocytic phenotype. Furthermore, mechanical stress and growth factors have been found to be capable of enhancing cell proliferation and ECM synthesis. We investigated the effect of mechanical loading and growth factors on reactivation of the 3D-embedded chondrocytes

Articular cartilage and changes in Arthritis: Cell biology of osteoarthritis

The reaction patterns of chondrocytes in osteoarthritis can be summarized in five categories: (1) proliferation and cell death (apoptosis); changes in (2) synthetic activity and (3) degradation; (4) phenotypic modulation of the articular chondrocytes; and (5) formation of osteophytes. In osteoarthritis, the primary responses are reinitiation of synthesis of cartilage macromolecules, the initiation of synthesis of types IIA and III procollagens as markers of a more primitive phenotype, and synthesis of active proteolytic enzymes. Reversion to a fibroblast-like phenotype, known as 'dedifferentiation', does not appear to be an important component. Proliferation plays a role in forming characteristic chondrocyte clusters near the surface, while apoptosis probably occurs primarily in the calcified cartilage