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1. High-Fidelity Simulation of Flows in Bone-Like Environment to Investigate the Growth of Cancer Cells

   https://doi.org/10.1115/DMD2021-1081  

   Akerkouch, Lahcen; Le, Trung; Jasuja, Haneesh; Katti, Kalpana; Katti, Dinesh (April 2021, 2021 Design of Medical Devices Conference)

   Abstract Metastatic cancer in bones is incurable, which causes significant mobility and mortality to the patients. In this work, we investigate the role of interstitial fluid flow on cancer cells' growth within the interconnected pores of human bone. In-vitro experiments were carried out in a bio-reactor which includes bone-like scaffold specimens. A pump is used to maintain a laminar flow condition inside the bioreactor to resemble fluid flow in bones. The scaffold specimens are harvested after 23 days in the bioreactor. The scaffold specimen is scanned with Micro-CT under the resolution of 70 micrometers. We created a full-scale 3D computational model of the scaffold based on the micro-CT data using the open-source software Seg3D and Meshmixer. Based on the geometrical models, we generated the computational grids using the commercial software Gridgen. We performed Computational Fluid Dynamics (CFD) simulations with the immersed boundary method (Gilmanov, Le, Sotiropoulos, JCP 300, 1, 2015) to investigate the flow patterns inside the pores of the scaffolds. The results reveal a non-uniform flow distribution in the vicinity of the scaffold. The flow velocity and the shear stress distributions inside the scaffold are shown to be convoluted and very sensitive to the pore sizes. Our future work will further quantify these distributions and correlate them to cancer cells' growth observed in the experiments. 

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2. A Computational Fluid Dynamics Model for Investigation of Material Flow in a Perfusion Bioreactor Toward Optimal Biomedical Design of Cell-Laden Scaffolds for Bone Regeneration

   https://doi.org/10.1115/IMECE2024-139666  

   Rollins, Hannah; Salary, Roozbeh “Ross” (November 2024, American Society of Mechanical Engineers (ASME) - International Mechanical Engineering Congress and Exposition (IMECE 2024))

   Abstract In tissue engineering, once a scaffold has completed mechanical property testing, it must then undergo biological characterization which determines if the scaffold is capable of supporting cell viability. To perform biological tests, cells must be seeded onto a scaffold with the help of bioreactors, the four main types being: (i) rotating wall, (ii) spinner flask, (iii) compression, and (iv) perfusion bioreactor. In perfusion bioreactors, a consistent flow of material is introduced (using a pump) into the inlet of the bioreactor chamber where multiple scaffolds of a disc geometry are located. However, the intrinsic, complex interaction between the scaffolds and material flow as it goes through the bioreactor chamber affects the viability of the seeded stem cells. Therefore, there is a need to identify consequential fluid dynamics phenomena governing the material flow in a perfusion bioreactor. In this study, using a CFD model, the effects of critical scaffold parameters (such as the number of scaffolds, scaffold diameter, scaffold thickness, and number of pores) on the main flow properties (i.e., flow pressure, wall shear stress, and streamline velocity) influential in cell proliferation and bone development will be investigated. It was observed that increasing the number of pores, in addition to decreasing the pore diameter had an adverse effect on the maximum forces occurring on the scaffold. In addition, changing the overall scaffold diameter did not appear to have as much as an effect as the other parameters. Furthermore, it was observed that a decrease in porosity would lead to an increase in wall shear stress and consequently in cell death. Overall, the outcomes of this study pave the way for optimal design, fabrication, and preparation of cell-laden bone scaffolds for treatment of bone fractures in clinical settings. 

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3. Fluid Flow Analysis for Suitable 3D Bio-Printed Scaffold Architectures to Incubate in a Perfusion Bioreactor: A Simulation Approach

   https://doi.org/10.1115/msec2023-104324  

   Clark, Scott; Quigley, Connor; Mankowsky, Jack; Habib, MD Ahasan (June 2023, Proceedings of the ASME 2023 18th International Manufacturing Science and Engineering Conference (MSEC2023))

   Abstract Due to the three-dimensional nature of the 3D bio-printed scaffolds, typical stagnant cell culturing methods don’t ensure entering medium inside areas or passing through the scaffolds. The bioreactor has frequently provided the required growth medium to encapsulated- and seeded-cells in 3D bio-printed scaffolds. To address this issue, we developed a customized perfusion bioreactor to supply the growth medium dynamically to the cells encapsulated or seeded in the scaffolds. The dynamic supply of fresh growth medium may help improve cell viability and proliferation. Because of its uniform nutrition distribution and flow-induced shear stress within the tissue-engineering scaffold, perfusion bioreactors have been used in a variety of tissue engineering applications. Including a modified setup of our designed bioreactor may improve the in vivo stimuli and conditions, eventually enhancing the overall performance of tissue regeneration. In this paper, we explored the response of fluid flow to certain types of scaffold pore geometries and porosities. We used a simulation technique to determine fluid flow turbulence through various pore geometries such as uniform triangular, square, diamond, circular, and honeycomb. We used variable pore sizes of the scaffold maintaining constant porosity to analyze the fluid flow. Based on the results, optimum designs for scaffolds were determined. 

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4. A Simulation Approach to Determine Internal Architecture of 3D Bio-Printed Scaffold Suitable for A Perfusion Bioreactor

   Clark, S.; Quigley, C.; Mankowsky, J.; & Habib, M. A. (May 2023, Proceedings of the IISE Annual Conference & Expo 2023)

   Babski-Reeves, K; Eksioglu, B; Hampton, D. (Ed.)

   Traditional static cell culture methods don't guarantee access to medium inside areas or through the scaffolds because of the complex three-dimensional nature of the 3D bio-printed scaffolds. The bioreactor provides the necessary growth medium encapsulated and seeded cells in 3D bioprinted scaffolds. The constant flow of new growing medium could promote more viable and multiplying cells. Therefore, we created a specialized perfusion bioreactor that dynamically supplies the growth medium to the cells implanted or encapsulated in the scaffolds. A redesigned configuration of our developed bioreactor may enhance the in vivo stimuli and circumstances, ultimately improving the effectiveness of tissue regeneration. This study investigated how different scaffold pore shapes and porosities affect the flow. We employed a simulation technique to calculate fluid flow turbulence across several pore geometries, including uniform triangular, square, circular, and honeycomb. We constructed a scaffold with changing pore diameters to examine the fluid movement while maintaining constant porosity. The impact of fluid flow was then determined by simulating and mimicking the architecture of bone tissue. The best scaffold designs were chosen based on the findings. 

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5. The Differential Effects of Fluid Shear Stress on Mesenchymal Stem Cell Differentiation

   Robertson, T; Van_Wie, B; Driskell, R; Gozen, A; Bonassar, L; Thiessen, D; Dong, W; Schuler, A (October 2024, Biomedical Engineering Society)

   Introduction: Directing mesenchymal stem cell (MSC) chondrogenesis by bioreactor cultivation provides fundamental insight towards engineering healthy, robust articular cartilage (AC). The mechanical environment is represented by compression, fluid shear stress, hydrostatic pressure, and tension which collectively contribute to the distinct spatial organization of AC. Mimicking this cell niche is necessary for dictating cell growth, fate, and role. Researchers have shown that different mechanical stimulus types improve MSC chondrogenic commitment demonstrated by increases in key chondrogenic gene and protein markers. However, challenges remain in manufacturing spatially, anisotropic AC consisting of defined regions such as native tissue. Our strategy towards furthering this effort involves exposing MSC-laden alginate scaffolds in a multi-chambered, perfusion bioreactor with controlled fluid shear stress magnitudes to better mimic the native AC microenvironment leading to defined regions throughout the scaffold marked by varied cellular phenotypes. Validations made from assessing biochemical content, mRNA expression, western blot analysis, and cell viability will provide meaningful insight towards regulating MSC chondrogenesis. Methods: MSCs grown up to passage 4 were expanded to confluency in a T-175 flask then released from the surface using trypsin. Cells were stored in -80 ℃ freezer until experimentation. Our bioreactor system was sterilized by UV radiation for 4 hours then perfused with 70% ethanol overnight. Cell-laden scaffolds were prepared by first dissolving 1.5% alginate into deionized water. The polymeric solution was sterilely filtered and stored until usage. Cryopreserved MSCs were thawed and suspended in α-MEM medium containing essential supplements. Cells were counted and resuspended in alginate at a density of 106 cells/mL. The mixture was transferred to our multi-chambered bioreactor where they were allowed to crosslink in CaCl2 solution for 45 min. Separate scaffolds (N = 3) were molded within an identical reactor system and removed to serve as a control to compare effects of fluid shear stress on MSC differentiation. All, structures were washed with PBS then supplied with DMEM/F-12 medium containing 10% FBS, 1% penicillin/streptomycin , 1% L-glutamine, 100 nM dexamethasone, 50 µg/mL L-ascorbic acid, and10 ng/mL TGF-β3. The flowrate for the bioreactor was adjusted to 20 mL/min which provided desired fluid shear ranges of 2-87 mPa to stimulate the cells . Cell cultures were grown for 7 days, and medium changed every 3 days. Sectioned samples were analyzed for biochemical content, mRNA expression, and western blot to understand the impact of fluid shear stress magnitudes on MSC differentiation. Results: Directed fluid shear stress across a cell-laden alginate scaffold contained within an individual chamber in our bioreactor indicates varied cellular behavior within the superficial and deep regions of the construct marked by spatially secreted biochemical content as well as mRNA expression. This observation is supported by superficial MSCs stimulated by high and medium mechanical stimulation which indicates a 1.3 and 1.2-fold increase in total collagen production, respectively, when directly compared to cells deep in the construct. A similar effect is supported by total GAG secretion where high and medium shear stress across the fluid hydrogel interface yielded 1.2 and 1.3-fold upregulation of protein secretion, respectively, when observed under similar conditions. Perfused MSCs show upregulation to 3 and 20-fold for Sox9 and aggrecan, respectively, compared to a static culture. Shear ranges distributed throughout our cell-laden alginate scaffold correlates to differential chondrogenic commitment shown by variance of Sox9 expression when assessed by location and depth. Additional information on COL10A1 expression demonstrates mechanical stimulation that reduces hypertrophic cell differentiation contrary to a static culture. Discussion: In this investigation we emphasize that cells respond differently to mechanical stimulation when located in either the superficial or deep region of an alginate scaffold. This observation is supported by enhanced matrix production of chondrogenic protein for cells near the perfused fluid and hydrogel interface compared to deeper areas when stimulated by high and medium fluid shear loading regimes. Most importantly, maintenance of a healthy fluid shear gradient in our TBR provides evidence of promoting MSC chondrogenesis by spatially upregulating anabolic cartilage-like markers in addition to diminishing the onset of cell hypertrophy. Our efforts in monitoring mRNA expression of our samples reveals enhancement of chondrogenic cell differentiation for a perfused sample marked by increases in Sox9 and aggrecan genes; whereas a static sample stimulated only by TGF-β3 leads to undesirable expression of COL10A1. Key takeaways from our study support the contributions from previous researchers in recreating the native AC mechanical environment to encourage MSC differentiation. The development of our TBR system for controlled delivery of fluid shear stresses to MSCs furthers efforts in spatially guiding MSC chondrogenesis which is critical for engineering zonally differentiated AC. 

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