The Potential of Pulsed Low Intensity Ultrasound to stimulate Chondrocytes in a 3D Model System
Pulsed low intensity ultrasound (PLIUS) is used clinically to accelerate fracture healing. although the mode of action is unclear. However studies suggest that PLIUS may stimulate endochondral ossification and consequently PLIUS may also be beneficial for cartilage regeneration, either in vivo or as part of a tissue engineered approach. Preyious studies using chondrocytes cultured in monolayer have suggested that PLIUS may stimulate glycosaminoglycan (GAG) synthesis, and that calcium signalling is implicated in this process. Therefore the present studies set out to investigate the influence of PLIUS on bovine articular chondrocytes in monolayer and agarose culture. This required the design of a bioreactor system which enabled cell-agarose constructs to be subjected to PLIUS, as well as a microscope-mounted test rig enabling confocal visualisation of intracellular calcium dynamics. A PLIUS system and signalling characteristics were provided by Smith and Nephew, Inc. (York, UK). Chondrocytes in agarose demonstrated a reduction in cell viability associated with PLIUS above a spatial averaged time averaged (SAT A) intensity of 200m W /cm2 , presumably associated with transducer heating. In subsequent studies, 30 and 100mW/cm2 were applied to monolayer and agarose cultures for up to 20 days, and biosynthesis was examined by assessment of GAG synthesis and cell proliferation using biochemical and radio-labelling protocols. Intracellular calcium signalling was investigated as a possible mechanotransduction pathway, using confocal mIcroscopy and the calcium indicator Fluo-4. In monolayer culture PLIUS did not stimulate total GAG content or cell proliferation at either 30 or 100 mW/cm2 • In agarose cultures, PLIUS had no effect on total GAG content at 30 mW/cm2 • At 100 mW/cm2 PLIUS induced a very small increase in total GAG content but there was no detectable effect on the rate of GAG synthesis in either model system at either 30 or 100 mW/cm2 . There were no PLIUS associated changes in the levels of intracellular calcium signaling in either monolayer or agarose cultures. Preliminary studies using Fluorescent Recovery after Photobleaching (FRAP) showed that PLIUS at 30mW/cm2 increased diffusion of 70kDa FITC-dextrans, although this clearly had no effect on GAG synthesis or cell proliferation. These studies indicate that PLIUS-induced fracture healing, or any potential use of PLIUS for cartilage repair. is unlikely to involve direct stimulation of proteoglycan synthesis or cell proliferation. Indeed the proposed use of PLIUS in cartilage tissue engineering is more limited than previously suggested.
AuthorsVaughan, Natalie Marlene
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