Effects of vibration and hyaluronic acid on activation of three‐dimensional cultured chondrocytes

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Abstract
Objective To investigate the effects of vibration (Vib) and hyaluronic acid (HA) on 3-dimensional cultured cartilage. Methods Chondrocytes were obtained from metatarsophalangeal joints of freshly killed 6-month-old pigs. Twenty-four–well plates containing type I collagen sponge disks were used to culture samples. The frequency and the amplitude of the vibration of the well plate were 100 Hz and 0.5 nm, respectively. We produced 3-dimensional cartilage tissue using HA and vibration with collagen sponge as a carrier. Four different culture conditions were examined: a control HA−Vib− group, an HA−Vib+ group, an HA+Vib− group, and an HA+Vib+ group. Each group was cultured for 2 weeks. After culture days 3, 7, 10, and 14 (every 3.5 days), the levels of chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S) isomers synthesized in each culture medium were measured. Histologic analysis, immunohistochemical analysis, and electron microscopic examination were performed. Results Mean C4S and C6S synthesis had increased rapidly after 7 days of culture and continued to increase thereafter. There were significant differences among the 4 groups (P < 0.01). Synthesis of both C4S and C6S was most abundant in the HA+Vib+ group and the lowest in the HA−Vib− group. After 1 and 2 weeks of culture, the chondrocytes had formed stratified structures on the collagen sponges in all groups, although the thickest structure was observed in the HA+Vib+ group and the thinnest in the HA−Vib− group. Under immunofluorescence, the HA+Vib+ group exhibited the strongest chromatic features. Under electron microscopy, the chondrocytes in the HA+Vib+ group exhibited many long and slender prominences on their surface, and extracellular substance could be observed associated with the cells. Conclusion Our results indicate that the combination of vibration and HA activates the production of proteoglycan in 3-dimensional cultured chondrocytes and stimulates MAPK and β-catenin. This suggests that some mechanoreceptors for vibration exist on the plasma membrane of chondrocytes and activate the intracellular signal transduction system.

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