Flow perfusion bioreactors have been extensively investigated as a promising culture method for bone tissue engineering, due to improved nutrient delivery and shear force-mediated osteoblastic differentiation. However, a major drawback impeding the transition to clinically-relevant tissue regeneration is the inability to non-destructively monitor constructs during culture. To alleviate this shortcoming, we investigated the distribution of fluid shear forces in scaffolds cultured in flow perfusion bioreactors using computational fluid dynamic techniques, analyzed the effects of scaffold architecture on the shear forces and monitored tissue mineralization throughout the culture period using microcomputed tomography. For this study, we dynamically seeded one million adult rat mesenchymal stem cells (MSCs) on 85% porous poly(L-lactic acid) (PLLA) polymeric spunbonded scaffolds. After taking intermittent samples over 16 days, the constructs were imaged and reconstructed using microcomputed tomography. Fluid dynamic simulations were performed using a custom in-house lattice Boltzmann program. By taking samples at different time points during culture, we are able to monitor the mineralization and resulting changes in flow-induced shear distributions in the porous scaffolds as the constructs mature into bone tissue engineered constructs, which has not been investigated previously in the literature. From the work conducted in this study, we proved that the average shear stress per construct consistently increases as a function of culture time, resulting in an increase at Day 16 of 113%.

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.

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Introduction Flow‐induced shear stresses have been found to be a stimulatory factor in pre‐osteoblastic cells seeded in 3D porous scaffolds and cultured under continuous flow perfusion. However, due to the complex internal structure of the scaffolds, whole scaffold calculations of the local shear forces are computationally intensive. Instead, representative volume elements (RVEs), which are obtained by extracting smaller portions of the scaffold, are commonly used in literature without a numerical accuracy standard. Objective Hence, the goal of this study is to examine how closely the whole scaffold simulations are approximated by the two types of boundary conditions used to enable the RVEs: “wall boundary condition” (WBC) and “periodic boundary condition” (PBC). Method To that end, lattice Boltzmann method fluid dynamics simulations were used to model the surface shear stresses in 3D scaffold reconstructions, obtained from high‐resolution microcomputed tomography images. Results It was found that despite the RVEs being sufficiently larger than 6 times the scaffold pore size (which is the only accuracy guideline found in literature), the stresses were still significantly under‐predicted by both types of boundary conditions: between 20% and 80% average error, depending on the scaffold's porosity. Moreover, it was found that the error grew with higher porosity. This is likely due to the small pores dominating the flow field, and thereby negating the effects of the unrealistic boundary conditions, when the scaffold porosity is small. Finally, it was found that the PBC was always more accurate and computationally efficient than the WBC. Therefore, it is the recommended type of RVE.

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