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A new method to investigate how mechanical loading of osteocytes controls osteoblasts

Periodical: Front Endocrinol (Lausanne) ISBN: 1664-2392  Date: 2014/12/30  Language: eng  Pages: 208

Authors:Vazquez, M., Evans, B. A., Riccardi, D., Evans, S. L., Ralphs, J. R., Dillingham, C. M., Mason, D. J.
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Abstract
Mechanical loading, a potent stimulator of bone formation, is governed by osteocyte regulation of osteoblasts. We developed a three-dimensional (3D) in vitro co-culture system to investigate the effect of loading on osteocyte-osteoblast interactions. MLO-Y4 cells were embedded in type I collagen gels and MC3T3-E1(14) or MG63 cells layered on top. Ethidium homodimer staining of 3D co-cultures showed 100% osteoblasts and 86% osteocytes were viable after 7 days. Microscopy revealed osteoblasts and osteocytes maintain their respective ovoid/pyriform and dendritic morphologies in 3D co-cultures. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) of messenger ribonucleic acid (mRNA) extracted separately from osteoblasts and osteocytes, showed that podoplanin (E11), osteocalcin, and runt-related transcription factor 2 mRNAs were expressed in both cell types. Type I collagen (Col1a1) mRNA expression was higher in osteoblasts (P < 0.001), whereas, alkaline phosphatase mRNA was higher in osteocytes (P = 0.001). Immunohistochemistry revealed osteoblasts and osteocytes express E11, type I pro-collagen, and connexin 43 proteins. In preliminary experiments to assess osteogenic responses, co-cultures were treated with human recombinant bone morphogenetic protein 2 (BMP-2) or mechanical loading using a custom built loading device. BMP-2 treatment significantly increased osteoblast Col1a1 mRNA synthesis (P = 0.031) in MLO-Y4/MG63 co-cultures after 5 days treatment. A 16-well silicone plate, loaded (5 min, 10 Hz, 2.5 N) to induce 4000-4500 muepsilon cyclic compression within gels increased prostaglandin E2 (PGE2) release 0.5 h post-load in MLO-Y4 cells pre-cultured in 3D collagen gels for 48, 72 h, or 7 days. Mechanical loading of 3D co-cultures increased type I pro-collagen release 1 and 5 days later. These methods reveal a new osteocyte-osteoblast co-culture model that may be useful for investigating mechanically induced osteocyte control of osteoblast bone f
Keywords
Esophageal squamous cell carcinoma, Hypoxia-inducible factor-1a, RNA interference, Vasculogenic mimicry, VE-cadherin

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CellLine: MC3T3-E1
  Morphology: Osteoblast-like
  Origin: Bone
  Species: Mouse
CellLine: MG63
  Morphology: Cancerous-Osteosarcoma
  Origin: Bone
  Species: Human
CellLine: MLO-Y4
  Morphology: Osteoclast precursor
  Origin: Bone
  Species: Mouse
Scaffold Form: gel/hydrogel
Scaffold Material: Collagen
Scaffold Origin: natural