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Osteoblast Responses One Hour After Load-Induced Fluid Flow in a Three-Dimensional Porous Matrix

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Abstract

When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM. Strain-induced fluid flow was visualized with a microspheres ~3 μm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment.

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Acknowledgments

The authors appreciate Randall Duncan for cell cultures, Taffy Hooser for histology, Caroline Miller for SEM, and Razi Nalim and Kerem Pekkan for the flow analysis. This study was supported by The National Institutes of Health (R01EB001019) and Whitaker Foundation (H.Y.).

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Authors and Affiliations

  1. Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan

    Shigeo M. Tanaka

  2. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA

    Shigeo M. Tanaka, Hui B. Sun, David B. Burr & Hiroki Yokota

  3. Department of Orthopedic Surgery, Indiana University School of Medicine, Indianapolis, IN, USA

    David B. Burr & Charles H. Turner

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

    Ryan K. Roeder

  5. Department of Biomedical Engineering, Indiana University - Purdue University, Indianapolis, IN, USA

    Hui B. Sun, David B. Burr, Charles H. Turner & Hiroki Yokota

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  1. Shigeo M. Tanaka
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  2. Hui B. Sun
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  3. Ryan K. Roeder
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  4. David B. Burr
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  5. Charles H. Turner
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  6. Hiroki Yokota
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Correspondence to Hiroki Yokota.

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Tanaka, S.M., Sun, H.B., Roeder, R.K. et al. Osteoblast Responses One Hour After Load-Induced Fluid Flow in a Three-Dimensional Porous Matrix. Calcif Tissue Int 76, 261–271 (2005). https://doi.org/10.1007/s00223-004-0238-2

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