Chondro-protective effects of low intensity pulsed ultrasound

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Chondro-protective effects of low intensity pulsed ultrasound S.M.Z. Uddin, B. Richbourgh, Y. Ding, A. Hettinghouse, D.E. Komatsu, Y.-X. Qin, C.-J. Liu Osteoarthritis and Cartilage Volume 24, Issue 11, Pages 1989-1998 (November 2016) DOI: 10.1016/j.joca.2016.06.014 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 LIPUS stimulation setup. A) Human explant culture with and without IL-1β and LIPUS stimulation at 30 mW/cm2/per day for the duration of experiment. B) Micromass culture with and without IL-1β and LIPUS stimulation at 30 mW/cm2, 20 min per day for the duration of the experiment. Osteoarthritis and Cartilage 2016 24, 1989-1998DOI: (10.1016/j.joca.2016.06.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 LIPUS protects against IL-1β induced GAG depletion. A) IL-1β induced total depletion of GAG content in human cartilage explants (n = 3). LIPUS increased GAG content and rescued IL-1β induced depletion of GAG from cartilage matrix. B) IL-1β treated human explants show increased release of GAG contents into culture media. LIPUS reduced GAG breakdown in cartilage explants and significantly reduced IL-1β induced GAG depletion. C) Chondrocyte micromass cultures show disoriented and patchy GAG content in the presence of IL-1β, LIPUS protects GAG content in the presence of IL-1β and shows an apparent increase in GAG secretion in LIPUS treated samples (* represent statistical difference between Ctrl and IL-1β, P = 0.028, # statistical difference between IL-1β and LIPUS+IL-1β, P = 0.028, † represent statistical difference between Ctrl and LIPUS groups, P = 0.028. Error bars represent 95% confidence interval). Osteoarthritis and Cartilage 2016 24, 1989-1998DOI: (10.1016/j.joca.2016.06.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 LIPUS treated micromass cultures retain collagen. A) IL-1β treated cultures show reduced collagen staining with the lack of collagen fiber orientation. LIPUS stimulation intensified collagen staining and retained collagen fiber intensity and integrity in IL-β treated cultures. B) Matrix collagen levels significantly decreased in IL-1β treated micromass cultures. LIPUS stimulation increased matrix collagen content and inhibited IL-1β induced depletion of collagen. * represent statistical difference between Ctrl and IL-1β, P = 0.028, # statistical difference between IL-1β and LIPUS+IL-1β, P = 0.028, † represent statistical difference between Ctrl and LIPUS groups, P = 0.028. Error bars represent 95% confidence intervals. Osteoarthritis and Cartilage 2016 24, 1989-1998DOI: (10.1016/j.joca.2016.06.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 LIPUS regulates chondrocyte metabolism (n = 3). A) LIPUS significantly increase Col II, aggrecan, and COMP expression in chondrocytes and protects against IL-1β induced downregulation of anabolic markers. B) IL-1β significantly increases MMP3 and ADAMTS5 mRNA expression in C-28/I2 cells. LIPUS stimulation significantly reduces IL-1β induced MMP13 and ADAMTS5 mRNA expression. * represent statistical difference between Ctrl and IL-1β, P = 0.028, # statistical difference between IL-1β and LIPUS+IL-1β, P = 0.028, † represent statistical difference between Ctrl and LIPUS groups, P = 0.028. Error bars represent 95% confidence intervals. Osteoarthritis and Cartilage 2016 24, 1989-1998DOI: (10.1016/j.joca.2016.06.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 LIPUS enhances C-28/I8 cells migration and proliferation (n = 3). A) IL-1β inhibits chondrocyte migration, LIPUS stimulation enhances chondrocyte migration into the empty region and suppresses IL-1β anti-migratory effects. B) The scratch area remains mostly cell-free in IL-1β cultures, LIPUS reverses the IL-1β effects as more than 90% of the area is covered in LIPUS treated cultures. C) The ImageJ analysis shows the significantly higher number of cells (>500 cells) migrated into the scratch area in LIPUS treated cultures in comparison to <50 cells migrated in IL-1β treated culture. D) Proliferation study shows no significant differences at D1 and D3 but LIPUS stimulated cultures to show a significant increase at D5. IL-1β cultures show no or little effect on chondrocyte proliferation. * represent statistical difference between Ctrl and IL-1β, P = 0.028, # statistical difference between IL-1β and LIPUS+IL-1β, P = 0.028, † represent statistical difference between Ctrl and LIPUS groups, P = 0.028. Error bars represent 95% confidence intervals. Osteoarthritis and Cartilage 2016 24, 1989-1998DOI: (10.1016/j.joca.2016.06.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 LIPUS enhances chondrogenic differentiation of C3H10T1/2. LIPUS significantly increases the expression of Col II and Sox 9 in C3H10T1/2 cells and suppresses IL-1β mediated inhibition of A) Col II and B) Sox9. * represent the statistical difference between Ctrl and IL-1β, P = 0.028, # statistical difference between IL-1β and LIPUS+IL-1β, P = 0.028, † represent the statistical difference between Ctrl and LIPUS groups, P = 0.028. Error bars represent 95% confidence intervals. Osteoarthritis and Cartilage 2016 24, 1989-1998DOI: (10.1016/j.joca.2016.06.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 LIPUS downregulates IL-1β induced activation of NFκB pathway (n = 3): A) Phosphorylation of NFκB-p65 was significantly elevated in cartilage explants after IL- 1β exposure. LIPUS stimulation suppressed IL-1β induced phosphorylation of NFκB-p65. B) LIPUS suppressed IL-1β induced phosphorylation of IκBα in cartilage explants. C) IL-1β exposure increased the expression of IL-1R1. Stimulation with LIPUS significantly reduced the expression of IL-1R1. Expression levels were normalized to GAPDH expression levels. * represent the statistical difference between Ctrl and IL-1β, P < 0.001, # statistical difference between IL-1β and LIPUS+IL-1β. Error bars represent 95% confidence intervals. Osteoarthritis and Cartilage 2016 24, 1989-1998DOI: (10.1016/j.joca.2016.06.014) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions