title: Functional impact of integrin α5β1 on the homeostasis of intervertebral discs: a study of mechanotransduction pathways using a novel dynamic loading organ culture system.
abstract: BACKGROUND
  Intervertebral disc (IVD) degeneration, a major cause of low back pain, is considered to be induced by daily mechanical loading. Mechanical stress is widely known to affect cell survival and extracellular matrix metabolism in many cell types. Although the involvement of integrin α5β1 transmembrane mechanoreceptor in IVD degeneration has been reported, the precise function of integrin α5β1 remains obscure.
  
  
  OBJECTIVE
  To reflect IVD tissue response to mechanical stress using a dynamic loading organ culture system and elucidate the functional impact of integrin α5β1 on the pathomechanism of IVD degeneration.
  
  
  METHODS
  An ex vivo study using a dynamic loading organ culture system.
  
  
  METHODS
  Ninety-six rat IVD explants were examined. Intervertebral discs were subjected to 1.3 MPa, 1.0 Hz dynamic compressive load in the presence or absence of an Arg-Gly-Asp (RGD) peptide with affinity to the fibronectin binding-site of integrin α5β1. Cell viability and histomorphology were assessed. The localization of integrin α5β1 in the IVD was assessed by immunohistochemistry. Gene expression levels of IVD cells were evaluated using real-time reverse transcription-polymerase chain reaction.
  
  
  RESULTS
  In the nucleus pulposus (NP), cell density and viability were reduced by dynamic compressive load. Histologic degenerative alterations, mainly seen in the NP, were the morphologic changes of NP cells. In both NP and annulus fibrosus (AF), immunohistochemistry revealed localization of integrin α5β1 and that the messenger-RNA expression of integrin α5β1 was increased by dynamic load. Dynamic load induced a catabolic effect, the stimulation of matrix metalloproteinase-3 and -13 gene expressions by NP and AF cells. The RGD peptide partially blocked the histologic alterations and the catabolic effect.
  
  
  CONCLUSIONS
  The dynamic loading organ culture system simulated cellular responses to mechanical loading of the IVD. Our results suggest that IVD cells recognize the mechanical stress through RGD integrins, particularly the α5β1 subtype that is highly expressed in NP and AF cells. Further experiments using this system will provide information about pathomechanisms of IVD degeneration through the mechanotransduction pathways.
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Takuto
authors.initials: T
authors.lastname: Kurakawa
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan. Electronic address: kenkakutani@gmail.com.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Kakutani
authors.affiliation: Department of Biomedical Engineering, Doshisha University, Karasuma-Higashi-iru, Kyoto, Kyoto Prefecture 602-8580, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Morita
authors.affiliation: Department of Biomedical Engineering, Doshisha University, Karasuma-Higashi-iru, Kyoto, Kyoto Prefecture 602-8580, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yurube
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Hirata
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Miyazaki
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Terashima
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Maeno
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takada
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Doita
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
authors.affiliation: Department of Biomedical Engineering, Doshisha University, Karasuma-Higashi-iru, Kyoto, Kyoto Prefecture 602-8580, Japan; Department of Orthopedic Surgery, Rush University Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612, USA.
authors.forename: Nozomu
authors.initials: N
authors.lastname: Inoue
authors.affiliation: Department of Orthopaedic Surgery, University of California San Diego School of Medicine, 9500 Gilman Drive, La Jolla, San Diego, CA 92093, USA.
authors.forename: Koichi
authors.initials: K
authors.lastname: Masuda
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyōgo Prefecture 657-8501, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Nishida
mesh_terms: D000818:Animals; D002452:Cell Count; D006706:Homeostasis; D007150:Immunohistochemistry; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D007403:Intervertebral Disc / Q000378:metabolism*; D055959:Intervertebral Disc Degeneration / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D040542:Mechanotransduction, Cellular; D009842:Oligopeptides; D009924:Organ Culture Techniques / Q000379:methods*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D013314:Stress, Mechanical
chemical_list: D039081:Integrin alpha5beta1; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D019278:Matrix Metalloproteinase 3
keywords: Compression; Integrin; Intervertebral disc degeneration; Mechanical load; Mechanoreceptor; Organ culture