Novel in vitro microfluidic platform for osteocyte mechanotransduction studies

2020 ◽  
Vol 12 (12) ◽  
pp. 303-310
Author(s):  
Liangcheng Xu ◽  
Xin Song ◽  
Gwennyth Carroll ◽  
Lidan You
Keyword(s):  
Shear Stress ◽  
Drug Targets ◽  
Fluid Shear Stress ◽  
Bone Cells ◽  
Osteoclast Differentiation ◽  
Bone Cell ◽  
Interstitial Fluid Flow ◽  
Molecule Transport ◽  
Flow Shear Stress

Abstract Osteocytes are the major mechanosensing cells in bone remodeling. Current in vitro bone mechanotransduction research use macroscale devices such as flow chambers; however, in vitro microfluidic devices provide an optimal tool to better understand this biological process with its flexible design, physiologically relevant dimensions and high-throughput capabilities. This project aims to design and fabricate a multi-shear stress, co-culture platform to study the interaction between osteocytes and other bone cells under varying flow conditions. Standard microfluidic design utilizing changing geometric parameters is used to induce different flow rates that are directly proportional to the levels of shear stress, with devices fabricated from standard polydimethylsiloxane (PDMS)-based softlithography processes. Each osteocyte channel (OCY) is connected to an adjacent osteoclast channel (OC) by 20-μm perfusion channels for cellular signaling molecule transport. Significant differences in RANKL levels are observed between channels with different shear stress levels, and we observed that pre-osteoclast differentiation was directly affected by adjacent flow-stimulated osteocytes. Significant decrease in the number of differentiating osteoclasts is observed in the OC channel adjacent to the 2-Pa shear stress OCY channel, while differentiation adjacent to the 0.5-Pa shear stress OCY channel is unaffected compared with no-flow controls. Addition of zoledronic acid showed a significant decrease in osteoclast differentiation, compounding to effect instigated by increasing fluid shear stress. Using this platform, we are able to mimic the interaction between osteocytes and osteoclasts in vitro under physiologically relevant bone interstitial fluid flow shear stress. Our novel microfluidic co-culture platform provides an optimal tool for bone cell mechanistic studies and provides a platform for the discovery of potential drug targets for clinical treatments of bone-related diseases.

The Effects of the Inhibition of Connexin 43 on Pre-Osteoblasts and Their Response to Mechanical Stimulation

2008 ◽  
Author(s):  
Danese M. Joiner ◽  
Ethan L. H. Daley ◽  
Steven A. Goldstein
Keyword(s):  
Shear Stress ◽  
Gap Junctions ◽  
Cell Signaling ◽  
Mechanical Stimulation ◽  
Connexin 43 ◽  
Fluid Shear Stress ◽  
Bed Rest ◽  
Bone Cell ◽  
Fluid Shear

It is well established that bone can adapt to the demands of daily mechanical usage. Mechanical loading can result in bone formation depending on the magnitude, duration, and frequency. Unloading, which can occur during bed rest, micro-gravity exposure and a variety of clinical conditions, can result in bone resorption. In vitro studies have demonstrated that osteoblasts and osteocytes respond to mechanical stimulation, especially oscillatory fluid shear stress. Mechano-responses have included increases in inter- and intra-cellular communication through gap junctions and soluble factors such as nitric oxide and prostaglandin E2 [1]. Bone cell gap junctions are primarily comprised of connexin 43 (Cx43). Mice lacking Cx43 have an osteopenic phenotype and when subjected to cyclic 4 pt. bending loads have an increased tibia bone marrow area [2, 3]. These observations may represent altered cell signaling. To investigate the role of Cx43 in cell signaling and bone mechanotransduction the Cx43 gene was silenced in MC3T3-E1 pre-osteoblast cells subjected to oscillatory fluid shear stress.

A Novel Mechanical Bioreactor System Allowing Simultaneous Strain and Fluid Shear Stress on Cell Monolayers

2011 ◽  
Author(s):  
W. Scott Van Dyke ◽  
Eric Nauman ◽  
Ozan Akkus
Keyword(s):  
Shear Stress ◽  
Fluid Flow ◽  
Fluid Shear Stress ◽  
Compressive Strain ◽  
Bone Adaptation ◽  
Bone Architecture ◽  
Mechanical Stimuli ◽  
Interstitial Fluid Flow ◽  
Loading Mode

The causes, mechanisms, and biology of bone adaptation have been under intense investigation ever since Julius Wolff proposed that bone architecture is determined by mathematical laws as a result of mechanical loading. How bone responds to mechanical loads by converting the mechanical signals into chemical signals is known as mechanotransduction. The in vivo environment of bone is complex, and most studies of cell-level phenomena have relied on the use of in vitro experiments using mechanical bioreactors. The main types of bioreactors are fluid flow shear stress, tensile and/or compressive strain, and hydrostatic pressure [1–2]. Of these bioreactors, the most intuitive mechanical stimulus for bone would be the tensile and compressive strain bioreactors. However, many researchers now claim that shear stress via interstitial fluid flow in the lacunar-canalicular porosity is the primary mechanosensory stimulus [3]. A handful of studies have attempted to compare the effects of both of these mechanical stimuli on osteoblasts, but these studies are lacking in two respects [4–6]. First, if both fluid flow and strain are performed in the same bioreactor, the magnitude of one loading mode is explicitly determined through constitutive equations, while the other is only estimated. Second, if the magnitudes of the loading modes are able to be explicitly determined they are performed in different bioreactors, providing the cells different extracellular environments. Therefore, a highly controllable dual-loading mode mechanical bioreactor, as described and characterized in this study, is a necessary tool to further understand the mechanotransduction of bone.

scholarly journals Distinct Concentration-Dependent Molecular Pathways Regulate Bone Cell Responses to Cobalt and Chromium Exposure from Joint Replacement Prostheses

2021 ◽  
Vol 22 (10) ◽  
pp. 5225
Author(s):  
Karan Shah ◽  
Mark Dunning ◽  
Alison Gartland ◽  
J Wilkinson
Keyword(s):  
Gene Expression ◽  
Joint Replacement ◽  
Bone Cells ◽  
Osteoclast Differentiation ◽  
Gene Clusters ◽  
Bone Cell ◽  
Metal Exposure ◽  
Metal Debris ◽  
Gene Expression Response

Systemic cobalt (Co) and chromium (Cr) concentrations may be elevated in patients with metal joint replacement prostheses. Several studies have highlighted the detrimental effects of this exposure on bone cells in vitro, but the underlying mechanisms remain unclear. In this study, we use whole-genome microarrays to comprehensively assess gene expression in primary human osteoblasts, osteoclast precursors and mature resorbing osteoclasts following exposure to clinically relevant circulating versus local periprosthetic tissue concentrations of Co2+ and Cr3+ ions and CoCr nanoparticles. We also describe the gene expression response in osteoblasts on routinely used prosthesis surfaces in the presence of metal exposure. Our results suggest that systemic levels of metal exposure have no effect on osteoblasts, and primarily inhibit osteoclast differentiation and function via altering the focal adhesion and extracellular matrix interaction pathways. In contrast, periprosthetic levels of metal exposure inhibit both osteoblast and osteoclast activity by altering HIF-1α signaling and endocytic/cytoskeletal genes respectively, as well as increasing inflammatory signaling with mechanistic implications for adverse reactions to metal debris. Furthermore, we identify gene clusters and KEGG pathways for which the expression correlates with increasing Co2+:Cr3+ concentrations, and has the potential to serve as early markers of metal toxicity. Finally, our study provides a molecular basis for the improved clinical outcomes for hydroxyapatite-coated prostheses that elicit a pro-survival osteogenic gene signature compared to grit-blasted and plasma-sprayed titanium-coated surfaces in the presence of metal exposure.

Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE2 but no change in mineralization

2001 ◽  
Vol 90 (5) ◽  
pp. 1849-1854 ◽  
Author(s):  
E. A. Nauman ◽  
R. L. Satcher ◽  
T. M. Keaveny ◽  
B. P. Halloran ◽  
D. D. Bikle
Keyword(s):  
Shear Stress ◽  
Fluid Flow ◽  
Fluid Shear Stress ◽  
Bone Cells ◽  
Shear Stresses ◽  
Long Term Effects ◽  
Fluid Shear ◽  
Short Term

Although there is no consensus as to the precise nature of the mechanostimulatory signals imparted to the bone cells during remodeling, it has been postulated that deformation-induced fluid flow plays a role in the mechanotransduction pathway. In vitro, osteoblasts respond to fluid shear stress with an increase in PGE2production; however, the long-term effects of fluid shear stress on cell proliferation and differentiation have not been examined. The goal of this study was to apply continuous pulsatile fluid shear stresses to osteoblasts and determine whether the initial production of PGE2 is associated with long-term biochemical changes. The acute response of bone cells to a pulsatile fluid shear stress (0.6 ± 0.5 Pa, 3.0 Hz) was characterized by a transient fourfold increase in PGE2 production. After 7 days of static culture (0 dyn/cm2) or low (0.06 ± 0.05 Pa, 0.3 Hz) or high (0.6 ± 0.5 Pa, 3.0 Hz) levels of pulsatile fluid shear stress, the bone cells responded with an 83% average increase in cell number, but no statistical difference ( P > 0.53) between the groups was observed. Alkaline phosphatase activity per cell decreased in the static cultures but not in the low- or high-flow groups. Mineralization was also unaffected by the different levels of applied shear stress. Our results indicate that short-term changes in PGE2 levels caused by pulsatile fluid flow are not associated with long-term changes in proliferation or mineralization of bone cells.

scholarly journals In Vitro Bone Cell Models: Impact of Fluid Shear Stress on Bone Formation

2016 ◽  
Vol 4 ◽  
Author(s):  
Claudia Wittkowske ◽  
Gwendolen C. Reilly ◽  
Damien Lacroix ◽  
Cecile M. Perrault
Keyword(s):  
Shear Stress ◽  
Bone Formation ◽  
Fluid Shear Stress ◽  
Bone Cell ◽  
Cell Models ◽  
Fluid Shear

scholarly journals The collective influence of 1, 25-dihydroxyvitamin D3with physiological fluid shear stress on osteoblasts

2017 ◽  
Author(s):  
Yan Li ◽  
Jiafeng Yuan ◽  
Qianwen Wang ◽  
Lijie Sun ◽  
Yunying Sha ◽  
...  
Keyword(s):  
Shear Stress ◽  
Fluid Shear Stress ◽  
Mechanical Stimuli ◽  
Fluid Shear ◽  
Dihydroxyvitamin D ◽  
Physiological Fluid ◽  
Flow Shear Stress ◽  
System 1 ◽  
Collective Influence

Abstract1, 25-dihydroxyvitamin D3(1, 25 (OH)2D3) and mechanical stimuli in physiological environment play an important role in the pathogenesis of osteoporosis. The effects of 1, 25-dihydroxyvitamin D3alone and mechanical stimuli alone on osteoblasts have been widely investigated. This study reports the collective influences of 1, 25-dihydroxyvitamin D3and flow shear stress (FSS) on biological functions of osteoblasts. 1, 25 (OH)2D3were constructed in various kinds of concentration (0, 1, 10, 100 nmmol/L), while physiological fluid shear stress (12 dynes/cm2) were produced by using a parallel-plate fluid flow system. 1, 25 (OH)2D3affects the responses of ROBs to FSS, including the inhibition of NO releases and cell proliferation as well as the promotion of PGE2releases and cell differentiation. These findings provide a possible mechanism by which 1, 25(OH)2D3influences osteoblasts responses to FSS and may provide guidance for the selection of 1, 25(OH)2D3concentration and mechanical loading in order toin vitroproduce functional bone tissues.

scholarly journals Rapid Fabrication of Microfluidic Devices for Biological Mimicking: A Survey of Materials and Biocompatibility

Micromachines ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 346
Author(s):  
Hui Ling Ma ◽  
Ana Carolina Urbaczek ◽  
Fayene Zeferino Ribeiro de Souza ◽  
Paulo Augusto Gomes Garrido Carneiro Leão ◽  
Janice Rodrigues Perussi ◽  
...  
Keyword(s):  
Shear Stress ◽  
Cell Viability ◽  
Cellular Response ◽  
Fluid Shear Stress ◽  
Umbilical Vein ◽  
Microfluidic Devices ◽  
In Vitro Assays ◽  
Stress Effect

Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept.

scholarly journals The Effect of Fluid Flow Shear Stress and Substrate Stiffness on Yes-Associated Protein (YAP) Activity and Osteogenesis in Murine Osteosarcoma Cells

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3128
Author(s):  
Thomas R. Coughlin ◽  
Ali Sana ◽  
Kevin Voss ◽  
Abhilash Gadi ◽  
Upal Basu-Roy ◽  
...  
Keyword(s):  
Shear Stress ◽  
Fluid Flow ◽  
Bone Cells ◽  
Bone Cancer ◽  
Substrate Stiffness ◽  
Flow Shear ◽  
Fluid Movement ◽  
Flow Shear Stress ◽  
Fluid Flow Shear Stress ◽  
New Treatments

Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness—two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity—which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression.

scholarly journals Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro.

1986 ◽  
Vol 83 (7) ◽  
pp. 2114-2117 ◽  
Author(s):  
P. F. Davies ◽  
A. Remuzzi ◽  
E. J. Gordon ◽  
C. F. Dewey ◽  
M. A. Gimbrone
Keyword(s):  
Shear Stress ◽  
Endothelial Cell ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cell ◽  
Vascular Endothelial ◽  
Cell Turnover ◽  
Fluid Shear ◽  
Turbulent Fluid

scholarly journals ShearFAST: a user-friendly in vitro toolset for high throughput, inexpensive fluid shear stress experiments.

2020 ◽  
Author(s):  
Thomas Brendan Smith ◽  
Alessandro Marco De Nunzio ◽  
Kamlesh Patel ◽  
Haydn Munford ◽  
Tabeer Alam ◽  
...  
Keyword(s):  
Shear Stress ◽  
High Throughput ◽  
Fluid Shear Stress ◽  
Tracking System ◽  
Frequency Measurement ◽  
Camera Motion ◽  
Fluid Shear ◽  
Mean Frequency

Fluid shear stress is a key modulator of cellular physiology in vitro and in vivo, but its effects are under-investigated due to requirements for complicated induction methods. Herein we report the validation of ShearFAST; a smartphone application that measures the rocking profile on a standard laboratory cell rocker and calculates the resulting shear stress arising in tissue culture plates. The accuracy with which this novel approach measured rocking profiles was validated against a graphical analysis, and also against measures reported by an 8-camera motion tracking system. ShearFASTs angle assessments correlated well with both analyses (r ≥0.99, p ≤0.001) with no significant differences in pitch detected across the range of rocking angles tested. Rocking frequency assessment by ShearFAST also correlated well when compared to the two independent validatory techniques (r ≥0.99, p ≤0.0001), with excellent reproducibility between ShearFAST and video analysis (mean frequency measurement difference of 0.006 ± 0.005Hz) and motion capture analysis (mean frequency measurement difference of 0.008 ± 0.012Hz). These data make the ShearFAST assisted cell rocker model make it an attractive approach for economical, high throughput fluid shear stress experiments. Proof of concept data presented reveals a protective effect of low-level shear stress on renal proximal tubule cells submitted to simulations of pretransplant storage.

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