M. Ishikawa, K. Yoshioka, K. Urano, Y. Tanaka, T. Hatanaka, A. Nii

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Biocompatibility of cross-linked hyaluronate (Gel-200) for the treatment of knee osteoarthritis M. Ishikawa, K. Yoshioka, K. Urano, Y. Tanaka, T. Hatanaka, A. Nii Osteoarthritis and Cartilage Volume 22, Issue 11, Pages 1902-1909 (November 2014) DOI: 10.1016/j.joca.2014.08.002 Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Histological evaluation of the air pouch in rats after injection of four different samples. Values represent the means ± 95% CI (n = 3–6). (A) Thickness of fibrous belt was increased in the hylan G-F 20 (**P = 0.002) and carrageenan group (**P = 0.002) compared to the saline group at day 14. On days 28 and 56, only the carrageenan group showed significant increase (***P < 0.001 for both). (B) The score for granulomatous inflammation with multinucleated giant cells was higher in all three time points in the hylan G-F 20 (*P = 0.02, 0.01, and 0.02 at days 14, 28, and 56, respectively) compared to the saline group. N.D.: not determined. (C) The foamy macrophage infiltration score was increased in the Gel-200 group at days 14 (*P = 0.02) and 28 (*P = 0.02) and in the carrageenan group at days 14 (**P = 0.008), 28 (*P = 0.02), and 56 (*P = 0.02) compared to the saline group. (D) The eosinophil infiltration score was increased in the hylan G-F 20 group at days 28 (*P = 0.01) and 56 (**P = 0.005) and in the carrageenan group at days 14 (*P = 0.01) and 28 (*P = 0.02) compared to the saline group. (E–J) Histological appearance of the rat air pouches at day 14. (E) In the saline group, fibrous belt composed of fibroblasts and collagenous matrices is seen (HE × 40). (F) In the carrageenan group, accumulation of large-sized macrophages and granulomatous inflammation are seen in the upper-half of the figure. A mature fibrous belt with inflammatory cells is seen in the lower-half of the figure (HE × 40). (G and H) In the Gel-200 group, foamy macrophages (arrowheads) containing the test article (asterisks, stained blue in (H)) are seen in and on the surface of the fibrous belt (G; HE stain × 40, H; MP stain × 40). (I and J) In the hylan G-F 20 group, granulomatous nodules (arrows) primarily composed of macrophages, multinucleated giant cells, and eosinophils (arrowheads) accompanied with the test article (asterisks, stained blue in (J)) in the center of the nodules are seen in the fibrous belt (I; HE stain × 40, J; MP stain × 40). Scale bars: 50 μm (E–J). Osteoarthritis and Cartilage 2014 22, 1902-1909DOI: (10.1016/j.joca.2014.08.002) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Histological evaluation of rabbit knee joints after injection of three different samples. Values represent the means ± 95% CI (n = 6). (A) The average synovial thickening score was increased in the Gel-200 and hylan G-F 20 group, but not significantly so compared to the saline group. (B) Granulomatous inflammation with multinucleated giant cells was observed only in the hylan G-F 20 group but change in the score was not significant. (C) Foamy macrophage infiltration score was increased significantly in the Gel-200 group compared to the saline group in three-injection regimen (**P = 0.0098). (D) Eosinophil infiltration was observed in the hylan G-F 20 group and significantly higher than the saline group in three-injection regimen (**P = 0.004). (E–G) Histological appearance of characteristic tissue reaction around test samples 15 days after single administration (HE × 40). (E) In the saline group, no abnormalities were observed. (F) In the Gel-200 group, fragments of the test article (asterisks) with infiltration with macrophages in the adipose tissue are seen. (G) In the hylan G-F 20 group, granulomatous nodules with multinucleated giant cells (arrow) and eosinophils (arrowheads, distinguished from neutrophils histologically), which were accompanied by the test article (asterisk) in the center of the nodules, were seen in the adipose tissue. Scale bars: 50 μm (E–G). Osteoarthritis and Cartilage 2014 22, 1902-1909DOI: (10.1016/j.joca.2014.08.002) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Cell recruitment in the synovial fluid in rabbits after single (A) or three weekly (B) injections of three different samples. Values represent the means ± 95% CI (n = 6). (A) Marginal increases compared to the saline group were observed for total cells (P = 0.06), monocytes (P = 0.06), and lymphocytes (P = 0.09) in the hylan G-F 20 group. (B) Total cells (*P = 0.01), monocytes (**P = 0.008), and heterophils (*P = 0.03) were significantly increased in the hylan G-F 20 group compared to the saline group. Gel-200 did not showed significant increases in cell numbers in either treatment regimen. Osteoarthritis and Cartilage 2014 22, 1902-1909DOI: (10.1016/j.joca.2014.08.002) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions