title: Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in three-dimensional cultures: a basic science study.
abstract: BACKGROUND
  The effect of low-intensity pulsed ultrasound (LIPUS) on cell growth was examined in three-dimensional-cultured chondrocytes with a collagen sponge. To elucidate the mechanisms underlying the mechanical activation of chondrocytes, intracellular signaling pathways through the Ras/mitogen-activated protein kinase (MAPK) and the integrin/phosphatidylinositol 3 kinase (PI3K)/Akt pathways as well as proteins involved in proliferation of chondrocytes were examined in LIPUS-treated chondrocytes.
  
  
  METHODS
  Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly sacrificed pigs. Isolated chondrocytes mixed with collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. Experimental cells were cultured with daily 20-minute exposures to LIPUS. The chondrocytes proliferated and a collagenous matrix was formed on the surface of the sponge. Cell counting, histological examinations, immunohistochemical analyses and western blotting analysis were performed.
  
  
  RESULTS
  The rate of chondrocyte proliferation was slightly but significantly higher in the LIPUS group in comparison with the control group during the 2-week culture period. Western blot analysis showed intense staining of type-IX collagen, cyclin B1 and cyclin D1, phosphorylated focal adhesion kinase, and phosphorylated Akt in the LIPUS group in comparison with the control group. No differences were detected, however, in the MAPK, phosphorylated MAPK and type-II collagen levels.
  
  
  CONCLUSIONS
  LIPUS promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a three-dimensional culture using a collagen sponge. In addition, the anabolic LIPUS signal transduction to the nucleus via the integrin/phosphatidylinositol 3-OH kinase/Akt pathway rather than the integrin/MAPK pathway was generally associated with cell proliferation.
authors.affiliation: Department of Orthopaedic Surgery, Yokohama City University School of Medicine, Yokohama City, Kanagawa, Japan. take0822@hotmail.com
authors.forename: Ryohei
authors.initials: R
authors.lastname: Takeuchi
authors.forename: Akihide
authors.initials: A
authors.lastname: Ryo
authors.forename: Noriko
authors.initials: N
authors.lastname: Komitsu
authors.forename: Yuko
authors.initials: Y
authors.lastname: Mikuni-Takagaki
authors.forename: Atsuko
authors.initials: A
authors.lastname: Fukui
authors.forename: Yuta
authors.initials: Y
authors.lastname: Takagi
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Shiraishi
authors.forename: Shin
authors.initials: S
authors.lastname: Morishita
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Ken
authors.initials: K
authors.lastname: Kumagai
authors.forename: Ichiro
authors.initials: I
authors.lastname: Aoki
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Saito
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology; D049109:Cell Proliferation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000000981:diagnostic imaging / Q000378:metabolism*; D024081:Collagen Type IX / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013552:Swine; D018617:Ultrasonography, Doppler, Pulsed; D051176:beta Catenin / Q000378:metabolism
chemical_list: D024081:Collagen Type IX; D019926:Cyclin B; D051176:beta Catenin; D019938:Cyclin D1; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt