Engineered Cell-Secreted Extracellular Matrix Modulates Cell Spheroid Mechanosensing and Amplifies Their Response to Inductive Cues for the Formation of Mineralized Tissues.

The clinical translation of mesenchymal stromal cell (MSC)-based therapies remains challenging due to rapid cell death and poor control over cell behavior. Compared to monodisperse cells, the aggregation of MSCs into spheroids increases their tissue-forming potential by promoting cell-cell interactions. However, MSCs initially lack engagement with an endogenous extracellular matrix (ECM) when formed into spheroids. Previously the instructive nature of an engineered, cell-secreted ECM is demonstrated to promote survival and differentiation of adherent MSCs. Herein, it is hypothesized that the incorporation of this cell-secreted ECM during spheroid aggregation would enhance MSC osteogenic potential by promoting cell-matrix and cell-cell interactions. ECM-loaded spheroids contained higher collagen and glycosaminoglycan content, and MSCs exhibited increased mechanosensitivity to ECM through Yes-associated protein (YAP) activation via integrin α2ß1 binding. ECM-loaded spheroids sustained greater MSC viability and proliferation and are more responsive to soluble cues for lineage-specific differentiation than spheroids without ECM or loaded with collagen. The encapsulation of ECM-loaded spheroids in instructive alginate gels resulted in spheroid fusion and enhanced osteogenic differentiation. These results highlight the clinical potential of ECM-loaded spheroids as building blocks for the repair of musculoskeletal tissues.

Alginate-Based Bioinks for 3D Bioprinting and Fabrication of Anatomically Accurate Bone Grafts.

To realize the promise of three-dimensional (3D) bioprinting, it is imperative to develop bioinks that possess the necessary biological and rheological characteristics for printing cell-laden tissue grafts. Alginate is widely used as a bioink because its rheological properties can be modified through precrosslinking or the addition of thickening agents to increase printing resolution. However, modification of alginate's physiochemical characteristics using common crosslinking agents can affect its cytocompatibility. Therefore, we evaluated the printability, physicochemical properties, and osteogenic potential of four common alginate bioinks: alginate-CaCl2 (alg-CaCl2), alginate-CaSO4 (alg-CaSO4), alginate-gelatin (alg-gel), and alginate-nanocellulose (alg-ncel) for the 3D bioprinting of anatomically accurate osteogenic grafts. While all bioinks possessed similar viscosity, printing fidelity was lower in the precrosslinked bioinks. When used to print geometrically defined constructs, alg-CaSO4 and alg-ncel exhibited higher mechanical properties and lower mesh size than those printed with alg-CaCl2 or alg-gel. The physical properties of these constructs affected the biological performance of encapsulated bone marrow-derived mesenchymal stromal cells (MSCs). Cell-laden constructs printed using alg-CaSO4 and alg-ncel exhibited greater cell apoptosis and contained fewer living cells 7 days postprinting. In addition, effective cell-matrix interactions were only observed in alg-CaCl2 printed constructs. When cultured in osteogenic media, MSCs in alg-CaCl2 constructs exhibited increased osteogenic differentiation compared to the other three bioinks. This bioink was then used to 3D print anatomically accurate cell-laden scaphoid bones that were capable of partial mineralization after 14 days of in vitro culture. These results highlight the importance of bioink properties to modulate cell behavior and the biofabrication of clinically relevant bone tissues. Impact statement Alginate-based bioinks are widely used for three-dimensional (3D) bioprinting of bone tissues. However, a direct systematic comparison between alginate-based bioinks is needed to assess the optimal bioink properties for mesenchymal stromal cell survival and osteogenesis. This study evaluates the printability, physical properties, biocompatibility, and osteogenic potential of four commonly used alginate-based bioinks and establishes the importance of bioink properties for advancing toward the clinical translation of 3D bioprinted bone grafts.

Three-Dimensional Printed Stamps for the Fabrication of Patterned Microwells and High-Throughput Production of Homogeneous Cell Spheroids.

Aggregation of cells into spheroids and organoids is a promising tool for regenerative medicine, cancer and cell biology, and drug discovery due to their recapitulation of the cell-cell and cell-matrix interactions found in vivo. Traditional approaches for the production of spheroids, such as the hanging drop method, are limited by the lack of reproducibility and the use of labor-intensive and time-consuming techniques. The need for high-throughput approaches allowing for the quick and reproducible formation of cell aggregates has driven the development of soft lithography techniques based on the patterning of microwells into nonadherent hydrogels. However, these methods are also limited by costly, labor-intensive, and multistep protocols that could impact the sterility of the process and efficiency of spheroid formation. In this study, we describe a one-step method for the fabrication of patterned nonadherent microwells into tissue culture plates using three-dimensional (3D) printed stamps and evaluate the production of cell spheroids of different sizes and cell sources. The generation of bone marrow-derived mesenchymal stromal cell and endothelial cell spheroids by the use of 3D printed stamps was superior in comparison with a widely used multistep mold technique, yielding spheroids of larger sizes and higher DNA content. The 3D stamps produced spheroids of more consistent diameter and DNA content when compared with other commercially available methods. These 3D printed stamps offer a tunable, simple, fast, and cost-effective approach for the production of reproducible spheroids and organoids for a wide range of applications.