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Shear Stress in Bone Marrow has a Dose Dependent Effect on cFos Gene Expression in In Situ Culture

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Abstract

Introduction

Mechanical stimulation of bone is necessary to maintain its mass and architecture. Osteocytes within the mineralized matrix are sensors of mechanical deformation of the hard tissue, and communicate with cells in the marrow to regulate bone remodeling. However, marrow cells are also subjected to mechanical stress during whole bone loading, and may contribute to mechanically regulated bone physiology. Previous results from our laboratory suggest that mechanotransduction in marrow cells is sufficient to cause bone formation in the absence of osteocyte signaling. In this study, we investigated whether bone formation and altered marrow cell gene expression response to stimulation was dependent on the shear stress imparted on the marrow by our loading regime.

Methods

Porcine trabecular bone explants were cultured in an in situ bioreactor for 5 or 28 days with stimulation twice daily. Gene expression and bone formation were quantified and compared to unstimulated controls. Correlation was used to assess the dependence on shear stress imparted by the loading regime calculated using computational fluid dynamics models.

Results

Vibratory stimulation resulted in a higher trabecular bone formation rate (p = 0.01) and a greater increase in bone volume fraction (p = 0.02) in comparison to control explants. Marrow cell expression of cFos increased with the calculated marrow shear stress in a dose-dependent manner (p = 0.002).

Conclusions

The results suggest that the shear stress due to interactions between marrow cells induces a mechanobiological response. Identification of marrow cell mechanotransduction pathways is essential to understand healthy and pathological bone adaptation and remodeling.

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Acknowledgments

This material is based on work supported by the National Science Foundation under Grant No. CMMI-1453467 and CMMI-1100207. K.J.C. was supported by the Walther Cancer Foundation through a Notre Dame Harper Cancer Research Institute Interdisciplinary Interface Training Program fellowship and the Notre Dame Advanced Diagnostics and Therapeutics Institute’s Leiva Graduate Fellowship in Precision Medicine.

Conflict of interest

Kimberly J. Curtis, Thomas R. Coughlin, Mary A. Varsanik, and Glen L. Niebur declare no conflicts of interest.

Ethical Standards

No human subjects research was carried out by the authors for the research in this article, and no animal experiments were carried out by the authors for the research in this article.

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Authors and Affiliations

  1. Tissue Mechanics Laboratory, University of Notre Dame, Notre Dame, IN, 46556, USA

    Kimberly J. Curtis, Thomas R. Coughlin, Mary A. Varsanik & Glen L. Niebur

  2. Bioengineering Graduate Program, University of Notre Dame, 147 Multidisciplinary Engineering Research, Notre Dame, IN, 46556, USA

    Kimberly J. Curtis, Thomas R. Coughlin & Glen L. Niebur

  3. Department of Aerospace and Mechanical Engineering, University of Notre Dame, Notre Dame, IN, 46556, USA

    Glen L. Niebur

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  1. Kimberly J. Curtis
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Supplementary Figure 1

Cell metabolism and proliferation assays indicated that the cultured trabecular bone explants maintain their cell populations and that cells proliferate, although we have not ascertained whether the relative numbers of cell types are maintained. (A) Four trabecular bone explants were harvested and cultured in the bioreactor for 22 days. Three explants were exposed to LMMS at 50 Hz, 0.3 xg in 30 min bouts, twice per day, 5 days per week throughout culture. The remaining three explants were cultured with fluid flow, but no mechanical stimulation. A metabolic assay (CellTiter-Blue, Promega, Madison, WI) was carried out following manufacturer’s instructions. Briefly, on day 4 and day 21, 20% CellTiter-Blue reagent was added to the media and incubated for 24hr at 37 °C. After incubation, fluorescence of the supernatant from each explant was measured in triplicate in a 96-well plate at 570 nm excitation and 600 nm emission. Cell viability was not different between any conditions or time points, indicating that cells in the marrow remained viable during culture (ANOVA; p = 0.08). (B) Eight different trabecular bone explants excised from two pigs were cultured in the bioreactor for 5 days. Four explants were subjected to LMMS at 50 Hz, 0.3 xg in 30 min bouts, twice per day, on days 2 through 5 of culture. The remaining four explants were cultured statically. Twenty-four hours after explants were placed in the bioreactor, 10 µM BrdU (ThermoFisher) was added to the media for the remaining culture period. After culture, explants were fixed in 10% formalin, demineralized in 10% EDTA, processed, and embedded in paraffin. Explants were sectioned at 6 μm and mounted on microscope slides. Sections were dried at 60 °C, dewaxed in xylene, and hydrated through descending concentrations of ethanol. Incubation in HCl was used to break the DNA structure of the labeled cells followed by neutralization in borate buffer. Sections were blocked before being stained with rat anti-BrdU (ab6326) at a 1:40 dilution overnight at 4 °C. A goat anti-rat secondary antibody conjugated to Alexafluor 488 (ab150165) was applied at a dilution of 1:200 for one hour at room temperature. The nuclei were stained using propidium iodide (Sigma) before being mounted with a coverslip. Two sections per explant were imaged in five regions at 100X and BrdU positive nuclei were counted using ImageJ (NIH) and normalized to total area imaged. A representative image of BrdU-positive cells (white arrows) in the marrow is shown, indicating that marrow cells are proliferating during culture. We did not attempt to identify specific cell lineages. Scale bar is 50 μm. (C) There was no difference in cell proliferation between loaded and control samples after 5 days of bioreactor culture (Student’s t-test; p = 0.12). (EPS 3550 kb)

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Curtis, K.J., Coughlin, T.R., Varsanik, M.A. et al. Shear Stress in Bone Marrow has a Dose Dependent Effect on cFos Gene Expression in In Situ Culture. Cel. Mol. Bioeng. 12, 559–568 (2019). https://doi.org/10.1007/s12195-019-00594-z

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