Measuring dimensionality of cell-scaffold contacts of primary human bone marrow stromal cells cultured on electrospun fiber scaffolds.

The properties and structure of the cellular microenvironment can influence cell behavior. Sites of cell adhesion to the extracellular matrix (ECM) initiate intracellular signaling that directs cell functions such as proliferation, differentiation, and apoptosis. Electrospun fibers mimic the fibrous nature of native ECM proteins and cell culture in fibers affects cell shape and dimensionality, which can drive specific functions, such as the osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs), by. In order to probe how scaffolds affect cell shape and behavior, cell-fiber contacts were imaged to assess their shape and dimensionality through a novel approach. Fluorescent polymeric fiber scaffolds were made so that they could be imaged by confocal fluorescence microscopy. Fluorescent polymer films were made as a planar control. hBSMCs were cultured on the fluorescent substrates and the cells and substrates were imaged. Two different image analysis approaches, one having geometrical assumptions and the other having statistical assumptions, were used to analyze the 3D structure of cell-scaffold contacts. The cells cultured in scaffolds contacted the fibers in multiple planes over the surface of the cell, while the cells cultured on films had contacts confined to the bottom surface of the cell. Shape metric analysis indicated that cell-fiber contacts had greater dimensionality and greater 3D character than the cell-film contacts. These results suggest that cell adhesion site-initiated signaling could emanate from multiple planes over the cell surface during culture in fibers, as opposed to emanating only from the cell's basal surface during culture on planar surfaces.

Optimization of Freeze-FRESH Methodology for 3D Printing of Microporous Collagen Constructs.

Freeform reversible embedding of suspended hydrogels (FRESH) is a layer-by-layer extrusion-based technique to enable three-dimensional (3D) printing of soft tissue constructs by using a thermo-reversible gelatin support bath. Suboptimal resolution of extrusion-based printing limits its use for the creation of microscopic features in the 3D construct. These microscopic features (e.g., pore size) are known to have a profound effect on cell migration, cell-cell interaction, proliferation, and differentiation. In a recent study, FRESH-based 3D printing was combined with freeze-casting in the Freeze-FRESH (FF) method, which yielded alginate constructs with hierarchical porosity. However, use of the FF approach allowed little control of micropore size in the printed alginate constructs. Herein, the FF methodology was optimized for 3D printing of collagen constructs with greater control of microporosity. Modifications to the FF method entailed melting of the FRESH bath before freezing to allow more efficient heat transport, achieve greater control on microporosity, and permit polymerization of collagen molecules to enable 3D printing of stable microporous collagen constructs. The effects of different freezing temperatures on microporosity and physical properties of the 3D-printed collagen constructs were assessed. In addition, finite element (FE) models were generated to predict the mechanical properties of the microporous constructs. Further, the impact of different micropore sizes on cellular response was evaluated. Results showed that the microporosity of 3D-printed collagen constructs can be tailored by customizing the FF approach. Compressive modulus of microporous constructs was significantly lower than the non-porous control, and the FE model verified these findings. Constructs with larger micropore size were more stable and showed significantly greater cell infiltration and metabolic activity. Together, these results suggest that the FF method can be customized to guide the design of 3D-printed microporous collagen constructs.

Evaluation of the effect of 3D porous Chitosan-alginate scaffold stiffness on breast cancer proliferation and migration.

Breast cancer (BCa) is one of the most common cancers for women and metastatic BCa causes the majority of deaths. The extracellular matrix (ECM) stiffens during cancer progression and provides biophysical signals to modulate proliferation, morphology, and metastasis. Cells utilize mechanotransduction and integrins to sense and respond to ECM stiffness. Chitosan-alginate (CA) scaffolds have been used for 3D culture, but lack integrin binding ligands, resulting in round cell morphology and limited cell-material interaction. In this study, 2, 4, and 6 wt% CA scaffolds were produced to mimic the stages of BCa progression and evaluate the BCa response to CA scaffold stiffness. All three CA scaffold compositions highly porous with interconnected pores and scaffold stiffness increased with increasing polymer concentration. MDA-MB-231 (231) cells were cultured in CA scaffolds and 2D cultures for 7 d. All CA scaffold cultures had similar cell numbers at 7 d and the 231 cells formed clusters that increased in size during the culture. The 2 wt% CA had the largest clusters throughout the 7 d culture compared with the 4 and 6 wt% CA. The 231 cell migration was evaluated on 2D surfaces after 7 d culture. The 6 wt% CA cultured cells had the greatest migration speed, followed by 4 wt% CA, 2D cultures, and 2 wt% CA. These results suggest that 231 cells sensed the stiffness of CA scaffolds without the presence of focal adhesions. This indicates that a non-integrin-based mechanism may explain the observed mechanotransduction response.