Effects of Shear Stress Gradients on Ewing Sarcoma Cells Using 3D Printed Scaffolds and Flow Perfusion.

In this work, we combined three-dimensional (3D) scaffolds with flow perfusion bioreactors to evaluate the gradient effects of scaffold architecture and mechanical stimulation, respectively, on tumor cell phenotype. As cancer biologists elucidate the relevance of 3D in vitro tumor models within the drug discovery pipeline, it has become more compelling to model the tumor microenvironment and its impact on tumor cells. In particular, permeability gradients within solid tumors are inherently complex and difficult to accurately model in vitro. However, 3D printing can be used to design scaffolds with complex architecture, and flow perfusion can simulate mechanical stimulation within the tumor microenvironment. By modeling these gradients in vitro with 3D printed scaffolds and flow perfusion, we can identify potential diffusional limitations of drug delivery within a tumor. Ewing sarcoma (ES), a pediatric bone tumor, is a suitable candidate to study heterogeneous tumor response due to its demonstrated shear stress-dependent secretion of ligands important for ES tumor progression. We cultured ES cells under flow perfusion conditions on poly(propylene fumarate) scaffolds, which were fabricated with a distinct pore size gradient via extrusion-based 3D printing. Computational fluid modeling confirmed the presence of a shear stress gradient within the scaffolds and estimated the average shear stress that ES cells experience within each layer. Subsequently, we observed enhanced cell proliferation under flow perfusion within layers supporting lower permeability and increased surface area. Additionally, the effects of shear stress gradients on ES cell signaling transduction of the insulin-like growth factor-1 pathway elicited a response dependent upon the scaffold gradient orientation and the presence of flow-derived shear stress. Our results highlight how 3D printed scaffolds, in combination with flow perfusion in vitro, can effectively model aspects of solid tumor heterogeneity for future drug testing and customized patient therapies.

Extrusion-Based 3D Printing of Poly(propylene fumarate) in a Full-Factorial Design.

3D printing has emerged as an important technique for fabricating tissue engineered scaffolds. However, systematic evaluations of biomaterials for 3D printing have not been widely investigated. We evaluated poly(propylene fumarate) (PPF) as a model material for extrusion-based printing applications. A full-factorial design evaluating the effects of four factors (PPF concentration, printing pressure, printing speed, and programmed fiber spacing) on viscosity, fiber diameter, and pore size was performed layer-by-layer on 3D scaffolds. We developed a linear model of printing solution viscosity, where concentration of PPF had the greatest effect on viscosity, and the polymer exhibited shear thinning behavior. Additionally, linear models of pore size and fiber diameter revealed that fiber spacing and pressure had the greatest effect on pore size and fiber diameter, respectively, but interplay among the factors also influenced scaffold architecture. This study serves as a platform to determine if novel biomaterials are suitable for extrusion-based 3D printing applications.

Evaluating changes in structure and cytotoxicity during in vitro degradation of three-dimensional printed scaffolds.

This study evaluated the structural, mechanical, and cytocompatibility changes of three-dimensional (3D) printed porous polymer scaffolds during degradation. Three porous scaffold designs were fabricated from a poly(propylene fumarate) (PPF) resin. PPF is a hydrolytically degradable polymer that has been well characterized for applications in bone tissue engineering. Over a 224 day period, scaffolds were hydrolytically degraded and changes in scaffold parameters, such as porosity and pore size, were measured nondestructively using micro-computed tomography. In addition, changes in scaffold mechanical properties were also measured during degradation. Scaffold degradation was verified through decreasing pH and increasing mass loss as well as the formation of micropores and surface channels. Current methods to evaluate polymer cytotoxicity have been well established; however, the ability to evaluate toxicity of an absorbable polymer as it degrades has not been well explored. This study, therefore, also proposes a novel method to evaluate the cytotoxicity of the absorbable scaffolds using a combination of degradation extract, phosphate-buffered saline, and cell culture media. Fibroblasts were incubated with this combination media, and cytotoxicity was evaluated using XTT assay and fluorescence imaging. Cell culture testing demonstrated that the 3D-printed scaffold extracts did not induce significant cell death. In addition, results showed that over a 224 day time period, porous PPF scaffolds provided mechanical stability while degrading. Overall, these results show that degradable, 3D-printed PPF scaffolds are suitable for bone tissue engineering through the use of a novel toxicity during degradation assay.