The role of subchondral bone resorption pits in osteoarthritis: MMP production by cells derived from bone marrow A. Shibakawa, M.D., Ph.D., K. Yudoh,

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The role of subchondral bone resorption pits in osteoarthritis: MMP production by cells derived from bone marrow A. Shibakawa, M.D., Ph.D., K. Yudoh, M.D., Ph.D., K. Masuko-Hongo, M.D., Ph.D., T. Kato, M.D., Ph.D., K. Nishioka, M.D., Ph.D., H. Nakamura, M.D., Ph.D. Osteoarthritis and Cartilage Volume 13, Issue 8, Pages 679-687 (August 2005) DOI: 10.1016/j.joca.2005.04.010 Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Outline of the knee joint (A) and topographic location of the osteochondral blocks from representative osteoarthritic (B) and non-arthritic joint (C). Blocks were resected along the dotted line and each section was prepared from each block. MFC: medial femoral condyle, LFC: lateral femoral condyle, MTC: medial tibial condyle, LTC: lateral tibial condyle, PFJ: patellar femoral joint, P: patella. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Histological findings of subchondral plate with HE staining. (A) Grade 0: subchondral plate with no invasion of bone marrow. (B) Grade I: subchondral bone resorption pit formation is limited within the calcified cartilage, (C) Grade II: bone marrow tissue infiltrate into the articular cartilage beyond the tidemark. Arrow: tidemark, AC: articular cartilage, CC: calcified cartilage, SB: subchondral bone, bar=100μm. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 The density of grade I and grade II subchondral bone resorption pit in OA and control. The density of the pits is expressed as the number of pits per 10mm of the subchondral bone. Values are the mean±SD. *P<0.05. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 The density of subchondral bone resorption pits in the medial and the lateral condyles. Values are the mean±SD. Med: medial femoral condyle and medial tibial condyle, Lat: lateral femoral condyle and lateral tibial condyle, *P<0.05. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Correlation of subchondral bone resorption pit and the degradation of articular cartilage. (A) The mean mMankin score in articular cartilage overlying subchondral bone with (+) or without (−) grade II subchondral bone resorption pits. **P<0.01. (B) Correlation of the number of pits and mMankin score overlying the pits (r=0.459, n=112, P<0.001). (C–E) Representative view stained by safranin-O, (C) no pit formation with mMankin score=2, (D) two-pit formation with mMankin score=4, (E) multiple pit formation with mMankin score=7. Arrowhead: subchondral bone resorption pit, bar=1000μm. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 Depletion of proteoglycan by subchondral bone resorption pit in OA. Safranin-O staining is positive in articular cartilage in the grade 0 (A) and the grade I (B) pits. (C) Depletion of safranin-O staining in articular cartilage around the tip of grade II pit. Arrow: tidemark, bar=100μm. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 7 Osteoclast-like cells attached to the inner surface of the pit and seemed to absorb calcified cartilage as well as articular cartilage. Serial sections showed the presence of multinucleated cells positive for TRAP (A) and VNR-α chain (B) in the grade II pit. AC: articular cartilage, CC: calcified cartilage, BM: bone marrow, arrow: tidemark, bar=100μm. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 8 Immunohistochemistry of the subchondral bone resorption pit for MMP-1 (A,D), MMP-3 (B,E) and MMP-13 (C,F) in the grade II (A–C) and the grade I (D–F) pit. Polynuclear giant cells or mononuclear cells expressed MMPs in their cytoplasm. AC: articular cartilage, CC: calcified cartilage, SB: subchondral bone, arrow: tidemark, bar=100μm. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 9 Co-expression of MMP-13 (A) and VNR-α chain (B) in polynuclear cells in the grade II subchondral bone resorption pit. AC: articular cartilage, arrowhead: positive cells, bar=50μm. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 10 Some pits permitted vimentin positive cells to invade (A) and other pits were lined with BMP positive cells (B). There were some pits that were surrounded by mature lamellar bone (HE staining) (C). Bony indentation surrounded by mature lamellar bone (C) was found, which made the subchondral plate irregular (D). (A,B) Immunohistochemistry, (C,D) HE staining. Arrow: tidemark, bar=100μm. Osteoarthritis and Cartilage 2005 13, 679-687DOI: (10.1016/j.joca.2005.04.010) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions