Y. Hagiwara, A. Ando, Y. Onoda, T. Takemura, T. Minowa, N. Hanagata, M

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Coexistence of fibrotic and chondrogenic process in the capsule of idiopathic frozen shoulders Y. Hagiwara, A. Ando, Y. Onoda, T. Takemura, T. Minowa, N. Hanagata, M. Tsuchiya, T. Watanabe, E. Chimoto, H. Suda, N. Takahashi, H. Sugaya, Y. Saijo, E. Itoi Osteoarthritis and Cartilage Volume 20, Issue 3, Pages 241-249 (March 2012) DOI: 10.1016/j.joca.2011.12.008 Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Histological features and elastic changes of capsules. A: H–E staining of IGHL in idiopathic frozen shoulders (IFS), B: H–E staining of IGHL in shoulders with rotator cuff tear (RCT), C: the number of cells in capsules, D: SAM gradation color image of IGHL in IFS, E: SAM gradation color image of IGHL in RCT, and F: average sound speed of capsules. Collagen bundles were dense in all the samples of IFS (A), however, there were interspaces between the bundles in all the samples of RCT (B). The number of cells was significantly higher in all the samples in IFS compared with those in RCT (C). High sound speed area (yellow to red) was observed in IFS (D), however, low sound speed area was in RCT (E). Average sound speed was significantly higher in all the samples in IFS compared with those in RCT (F). Scale bars: 100 μm (A and B). RI: the rotator interval capsule, MGHL: middle glenohumeral ligament, IGHL: inferior glenohumeral ligament. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Immunoreactivity of fibrotic factors in IGHL. A–D: IFS, E–H: RCT. Few immunoreactivity of Ki-67 was detected both in IFS and RCT (A and E). Stronger immunoreactivity of collagen type I was observed in IFS compared with those of RCT (B and F). There was no significant difference in immunoreactivity of collagen type III between IFS and RCT (C and G). Strong immunoreactivity of α-SMA was detected in blood vessels both in IFS and RCT. No immunoreactivity of α-SMA was observed in fibroblasts-like cells in the two groups. The number of blood vessels in the substance of the capsule increased in IFS (D and H). Scale bars: 100 μm. Arrow heads: immunoreactivity of Ki-67 (A and B). Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Chondrogenic differentiation of capsules. A: alcian blue staining of IGHL in IFS, B: alcian blue staining of IGHL in RCT, C: gene expressions, D and E: ISH of ACAN, F: ISH of collagen type II. The gene expressions are shown as a ratio of expression of EF1a1. Strong alcian blue staining was observed in IFS compared with that in RCT (A and B). The gene expressions of ACAN and COL2A1 were significantly higher in all the samples of IFS compared with those of RCT. The gene expression of COL10A1 was significantly higher in MGHL and IGHL of IFS compared with those of RCT (C). Stronger signals of ACAN were detected in IFS (D), however, there was no signal in RCT (E). There was no signal of collagen type II in IFS (F). Scale bars: 200 μm (A and B), 100 μm (D–F). Arrow heads: signals of ACAN (D). Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Table 1. Primary antibodies and their dilution. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Table 2. Polymerase chain reaction primer sequences. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Table 3. Polymerase chain reaction primer sequences. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Table 4. Inflammatory and fibrotic gene expressions of capsules. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Table 5. Gene expressions of MMPs and TIMPs of capsules. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Table 6. The gene expressions of chondrogenic factors of capsules. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Fig. 1: Histological features and elastic changes of RI and MGHL. A–D: H–E staining, E–H: SAM, I–L: alcian blue staining. Scale bars: 100 μm (A–D), 200 μm (I–L). RI: the rotator interval capsule, MGHL: middle glenohumeral ligament, IFS: idiopathic frozen shoulders, RCT: shoulders with rotator cuff tears. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Fig. 2. Image comparisons of SAM with IHC of collagen type I and alcian blue staining. A: Black and white image of SAM in IGHL, B-H: IHC of collagen type I, I-O: alcian blue staining. B and I: gray scale image, C and D: isolation of red, D and K: isolation of green, E and L: isolation of blue, F and M: isolation of the first component, G and N: isolation of the second component, H and O: isolation of the third component. SAM image (A) was not corresponded positively or negatively with all the images of IHC of collagen type I and alcian blue staining (B-O). Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions

Supplementary Fig. 3: Immunoreactivity of fibrotic factors in RI and MGHL. A-D: Ki 67, E-H: collagen type I, I-L: collagen type III, M-P: α-SMA. Arrow heads: immunoreactivity of Ki-67 (B). Scale bars: 100 μm. RI: the rotator interval capsule, MGHL: middle glenohumeral ligament, IFS: idiopathic frozen shoulders, RCT: shoulders with rotator cuff tears. Osteoarthritis and Cartilage 2012 20, 241-249DOI: (10.1016/j.joca.2011.12.008) Copyright © 2011 Osteoarthritis Research Society International Terms and Conditions