Different doses of strontium ranelate and mechanical vibration modulate distinct responses in the articular cartilage of ovariectomized rats A.G.H. Mierzwa,

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Presentation transcript:

Different doses of strontium ranelate and mechanical vibration modulate distinct responses in the articular cartilage of ovariectomized rats A.G.H. Mierzwa, J.F. Campos, M.F. Jesus, H.B. Nader, M. Lazaretti-Castro, R.D. Reginato Osteoarthritis and Cartilage Volume 25, Issue 7, Pages 1179-1188 (July 2017) DOI: 10.1016/j.joca.2017.02.793 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Histomorphometric analysis to quantify the number of chondrocytes and clusters in the WB and NWB areas from the articular cartilage of OVX rats. Representative light micrographs of the WB area, showing the number of chondrocytes and clusters. H&E stain. Scale bars represent 20 μm. (A) Control, (B) SrR300, (C) SrR625, (D) MV, and (E) SrR625 + MV. (F) Quantification of chondrocyte number in the WB areas. P values and 95% CIs of difference between groups: MV vs. Control: P = 0.0014, CI 4.36−21.49; SrR625 + MV vs. Control: P < 0.0001, CI 13.99−30.51; MV vs. SrR300: P = 0.0047, CI 2.93−20.07; SrR625 + MV vs. SrR300: P < 0.0001, CI 12.57−29.08; SrR625 + MV vs. SrR625: P < 0.0001, CI 8.79−25.30; SrR625 + MV vs. MV: P = 0.0212, CI 1.07−17.58. (G) Quantification of chondrocyte number in the NWB area. P values and 95% CIs of difference between groups: SrR625 + MV vs. Control: P = 0.0027, CI 2.71−15.76; SrR625 + MV vs. SrR300: P = 0.0050, CI 2.16−15.21; SrR625 + MV vs. SrR625: P = 0.0062, CI 1.98−15.03. (H) Quantification of chondrocyte clusters in the WB areas. P values and 95% CIs of difference between groups: MV vs. Control: P < 0.0001, CI −19.60 to −8.49; SrR625 + MV vs. Control: P < 0.0001, CI −18.84 to −8.13; MV vs. SrR300: P < 0.0001, CI −17.80 to −6.69; SrR625 + MV vs. SrR300: P < 0.0001, CI −17.04 to −6.33; MV vs. SrR625: P < 0.0001, CI −16.40 to −5.29; SrR625 + MV vs. SrR625: P < 0.0001, CI −15.64 to −4.93. (I) Quantification of chondrocyte clusters in the NWB area. P values and 95% CIs of difference between groups: MV vs. Control: P < 0.0001, CI −8.36 to −2.57; SrR625 + MV vs. Control: P < 0.0001, CI −8.59 to −3.02; MV vs. SrR300: P < 0.0001, CI −8.53 to −2.74; SrR625 + MV vs. SrR300: P < 0.0001, CI −8.76 to −3.18; MV vs. SrR625: P = 0.0002, CI −8.06 to −2.27; SrR625 + MV vs. SrR625: P < 0.0001, CI −8.29 to −2.72. Data are presented as the mean ± 95% CI (n = 6−7 per group); ∗ indicates statistically significant differences between groups. Osteoarthritis and Cartilage 2017 25, 1179-1188DOI: (10.1016/j.joca.2017.02.793) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Analysis of CS and HA in the articular cartilage of OVX rats. The values are expressed as μg per mg of dry tissue. (A) Quantification of CS. P values and 95% CIs of difference between groups: MV vs. Control: P = 0.0019, CI 0.09−0.51; MV vs. SrR300: P < 0.0001, CI 0.27−0.67; MV vs. SrR625: P < 0.0001, CI 0.16−0.56; SrR625 + MV vs. Control: P = 0.0011, CI 0.10−0.48; SrR625 + MV vs. SrR300: P < 0.0001, CI 0.28−0.64; SrR625 + MV vs. SrR625: P < 0.0001, CI 0.17−0.53. Data are presented as the mean ± 95% CI (n = 6−9 per group); ∗ indicates statistically significant differences between groups. (B) Quantification of HA. P values and 95% CIs of difference between groups: SrR300 vs. Control: P < 0.0001, CI −0.01 to −0.01; SrR300 vs. SrR625: P < 0.0001, CI 0.01−0.01; SrR300 vs. MV: P < 0.0001, CI 0.01−0.02; SrR300 vs. SrR625 + MV: P < 0.0001, CI 0.01−0.01. Data are presented as the mean ± 95% CI (n = 3−8 per group); ∗ indicates statistically significant differences between groups. Osteoarthritis and Cartilage 2017 25, 1179-1188DOI: (10.1016/j.joca.2017.02.793) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Representative light micrographs of the NWB area from the articular cartilage of OVX rats, subjected to type II collagen immunohistochemistry (diffuse brown color) and counterstained with hematoxylin. Scale bars represent 100 μm. (A) Control, (B) SrR300, (C) SrR625, (D) MV, and (E) SrR625 + MV (n = 3 per group). Osteoarthritis and Cartilage 2017 25, 1179-1188DOI: (10.1016/j.joca.2017.02.793) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Immunohistochemistry analysis of caspase-3 expression in WB and NWB areas from the articular cartilage of OVX rats. Representative light micrographs of the WB area, showing caspase-3 positive chondrocytes (arrow). Scale bars represent 20 μm. (A) Control, (B) SrR300, (C) SrR625, (D) MV, and (E) SrR625 + MV. (F) Quantification of caspase-3 expression in the WB areas. P values and 95% CIs of difference between groups: SrR300 vs. Control: P = 0.0427, CI 0.43−36.41; SrR625 + MV vs. Control: P = 0.0036, CI −39.16 to −5.99; SrR625 + MV vs. SrR300: P < 0.0001, CI −59.43 to −22.56; SrR625 + MV vs. SrR625: P = 0.0002, CI −49.99 to −13.12; SrR625 + MV vs. MV: P < 0.0001, CI −49.58 to −15.44. (G) Quantification of caspase-3 expression in the NWB area. P values and 95% CIs of difference between groups: SrR625 + MV vs. Control: P < 0.0001, CI −76.31 to −37.39; SrR625 + MV vs. SrR300: P < 0.0001, CI −70.94 to −26.62; SrR625 + MV vs. SrR625: P < 0.0001, CI −69.72 to −25.40; SrR625 + MV vs. MV: P = 0.0005, CI −57.29 to −16.25; MV vs. Control: P = 0.0418, CI −39.54 to −0.62. Data are presented as the mean ± 95% CI (n = 3−5 per group); ∗ indicates statistically significant differences between groups. Osteoarthritis and Cartilage 2017 25, 1179-1188DOI: (10.1016/j.joca.2017.02.793) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunohistochemistry analysis of TNF-α expression in the WB and NWB areas from the articular cartilage of OVX rats. Representative light micrographs of the WB area, showing TNF-α positive chondrocytes (arrow). Scale bars represent 20 μm. (A) Control, (B) SrR300, (C) SrR625, (D) MV, and (E) SrR625 + MV. (F) Quantification of TNF-α expression in the WB areas. P values and 95% CIs of difference between groups: MV vs. SrR300: P = 0.0005, CI 8.75−35.36; MV vs. SrR625: P = 0.0001, CI 10.98−37.58; MV vs. SrR625 + MV: P = 0.0002, CI −38.72 to −10.81. (G) Quantification of TNF-α expression in the NWB area. Data are presented as the mean ± 95% CI (n = 3 per group); ∗ indicates statistically significant differences between groups. Osteoarthritis and Cartilage 2017 25, 1179-1188DOI: (10.1016/j.joca.2017.02.793) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Immunohistochemistry analysis of MMP-9 expression in WB and NWB areas from the articular cartilage of OVX rats. Representative light micrographs of the WB area, showing MMP-9 positive chondrocytes (arrow). Scale bars represent 20 μm. (A) Control, (B) SrR300, (C) SrR625, (D) MV, and (E) SrR625 + MV. (F) Quantification of MMP-9 expression in the WB areas. P values and 95% CIs of difference between groups: SrR625 vs. Control P < 0.0001, CI 20.10−63.62; MV vs. Control: P = 0.0029, CI 8.66−52.19; SrR625 vs. SrR625 + MV: P = 0.0003, CI −58.21 to −14.68; MV vs. SrR625 + MV: P = 0.0181, CI −46.77 to −3.25. (G) Quantification of MMP-9 expression in the NWB area. P values and 95% CIs of difference between groups: MV vs. Control: P = 0.0036, CI 21.01−116.20; MV vs. SrR625 + MV: P = 0.0026, CI −118.90 to −23.72. Data are presented as the mean ± 95% CI (n = 4 per group); ∗ indicates statistically significant differences between groups. Osteoarthritis and Cartilage 2017 25, 1179-1188DOI: (10.1016/j.joca.2017.02.793) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Representative light micrographs of the WB area from the articular cartilage of OVX rats. Safranin O and Fast Green stain. Scale bars represent 100 μm. (A) Control, (B) SrR300, (C) SrR625, (D) MV, and (E) SrR625 + MV. (F) OARSI histological score for articular cartilage degeneration. P values: SrR300 vs. Control: P = 0.0002, SrR300 vs. MV: P = 0.0170, SrR300 vs. SrR625 + MV: P < 0.0001; SrR625 vs. Control: P = 0.0309 and SrR625 vs. SrR625 + MV: P = 0.0040. Data are presented as the mean ± 95% CI (n = 8 per group); ∗ indicates statistically significant differences between groups. Osteoarthritis and Cartilage 2017 25, 1179-1188DOI: (10.1016/j.joca.2017.02.793) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions