Research article

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

Ryohei Takeuchi¹ , Akihide Ryo^2,3 , Noriko Komitsu¹ , Yuko Mikuni-Takagaki⁴ , Atsuko Fukui¹ , Yuta Takagi⁵ , Toshihiko Shiraishi⁵ , Shin Morishita⁵ , Yoshiyuki Yamazaki¹ , Ken Kumagai¹ , Ichiro Aoki³ and Tomoyuki Saito¹

¹Department of Orthopaedic Surgery, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama City, Kanagawa 236-0004, Japan

²First Research Group, AIDS Research Center, National Institute of Infectious Diseases, 4-7-1 Gagkuen, Musashimurayama, Tokyo 208-0011, Japan

³Department of Pathology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama City, Kanagawa 236-0004, Japan

⁴Department of Functional Biology, Kanagawa Dental College, 82 Inaokachyo, Yokosuka City, Kanagawa 238-8580, Japan

⁵Department of Environment and Information Sciences, Yokohama National University Graduate School, 79-5 Tokiwadai, Hodogaya-ku, Yokohama City, Kanagawa 240-851, Japan

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Arthritis Research & Therapy 2008, 10:R77doi:10.1186/ar2451

Published:	11 July 2008

Abstract

Introduction

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.

Conclusion

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.