Advertisement for orthosearch.org.uk
Results 1 - 1 of 1
Results per page:
Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XL | Pages 184 - 184
1 Sep 2012
Takahashi K Kambe Y Hayashi N Yamada K Yamamoto K Kojima K Tamada Y Tomita N
Full Access

INTRODUCTION

Several reports suggest that low-intensity pulsed ultrasound stimulation (LIPUS) facilitates chondrogenesis1). Recently it has been suggested that LIPUS may be transmitted via Integrin: a protein which mediates cellular attachment between cells and extracellular matrix2). In this study, the Arg-Gly-Asp (RGD) amino acid sequence, which is a ligand of Integrin, was induced to the fibroin substrates by either gene transfer or physical mixing, and the variation of chndrocyte response to LIPUS was evaluated.

EXPERIMENTAL METHODS

Three kinds of culture dishes coated with three diffrent fibroin aqueous solutions were prepared: 1 wild-type, 2 transgenic and 3 mixed. The wild-type aqueous solution was prepared from Bombyx mori silkworm cocoons. The transgenic aqueous solution was prepared from Bombyx mori silkworm cocoons in which RGD was interfused in the fibroin light chain3). The mixed aqueous solution was prepared simply by blending RGD peptides with the wild-type fibroin aqueous solution. Chondrocytes were asepically harvested from the joints of 4-week-old Japanese white rabbits and then subcultured on T-flasks and seeded at 2.0 × 105 cells/dish. LIPUS stimulation, with spatial and temporal average intensity of 30 mW/cm2 and a frequency of 1.71 MHz with a 200 ms tone burst repeated at 1.0 kHz, was applied to the chondrocytes at 12, 36, 60 hours and administered for 20 minutes each time. GAG production and the number of chondrocytes were measured by the Dimethylmethylene blue (DMMB) method4) and the LDH method5), respectively. Extracted mRNA from the chondrocytes was analyzed by using the Syber Green method, where the primers were designed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the house-keeping gene, aggrecan and Sox 9. This data was analyzed using the two-sided Student's t-test.