Granulation Tissue Eroding the Subchondral Bone Also Promotes New Bone Formation in Ankylosing Spondylitis.

OBJECTIVE: We previously suggested that fibroblast-rich granulation tissue eroding the subchondral bone is instrumental in the joint remodeling that occurs in ankylosing spondylitis (AS). The purpose of this study was to determine if this granulation tissue also carries bone-forming capabilities, which we approached by searching for bone-forming cells (hypertrophic chondrocytes, osteoblasts) in its vicinity. We also assessed adipogenic tissue transformation, which has been suggested to be an intermediate feature in AS bone formation based on imaging studies. METHODS: The facet joints of AS patients, osteoarthritis (OA) patients, and autopsy subjects (controls) were screened for subchondral granulation tissue. We searched for hypertrophic chondrocytes by assessing RUNX-2, type X collagen, and matrix metalloproteinase 13 (MMP-13) expression, for osteoblasts by analyzing RUNX-2, CD56, and type I collagen expression, as well as for signs of new bone formation. Adipocytes and lipid accumulation were assessed in Safranin O-stained sections. RESULTS: In the joints of AS and OA patients, RUNX-2-positive cells were found to be lining the granulation tissue. These cells coexpressed type I collagen but lacked type X collagen and MMP-13 expression, confirming their osteoblastic nature. In 91% of AS joints and in 20% of OA joints (P < 0.05), we observed foci of new bone formation at contact zones between the granulation tissue and the cartilage. Joints containing bony spots showed greater replacement of the adjacent bone marrow by granulation tissue than did joints without bone formation (P < 0.05). The granulation tissue often contained adipocytes and lipid accumulations. Replacement of the subchondral bone marrow by fat tissue was also frequently found but was not associated with new bone formation. CONCLUSION: The subchondral granulation tissue carries osteoblasts, which promote new bone formation, leading to intraarticular ankylosis of the facet joints in AS.

Anti-RANKL treatment inhibits erosive joint destruction and lowers inflammation but has no effect on bone formation in the delayed-type hypersensitivity arthritis (DTHA) model.

BACKGROUND: The aims of the present study were to determine the relationship between bone destruction and bone formation in the delayed-type hypersensitivity arthritis (DTHA) model and to evaluate the effect of receptor activator of nuclear factor κB ligand (RANKL) blockade on severity of arthritis, bone destruction, and bone formation. METHODS: DTHA was induced in C57BL/6 mice. Inflammation, erosive joint damage, and new bone formation were semiquantitatively scored by histology. Osteoclast activity was assessed in vivo, and messenger RNA (mRNA) expression of mediators of bone destruction and bone formation were analyzed by mRNA deep sequencing. Serum concentrations of tartrate-resistant acid phosphatase 5b, carboxy-terminal telopeptide I (CTX-I), matrix metalloproteinase 3 (MMP3), and serum amyloid P component (SAP) were determined by enzyme-linked immunosorbent assay. Anti-RANKL monoclonal antibody treatment was initiated at the time of immunization. RESULTS: Bone destruction (MMP3 serum levels, cathepsin B activity, and RANKL mRNA) peaked at day 3 after arthritis induction, followed by a peak in cartilage destruction and bone erosion on day 5 after arthritis induction. Periarticular bone formation was observed from day 10. Induction of new bone formation indicated by enhanced Runx2, collagen X, osteocalcin, MMP2, MMP9, and MMP13 mRNA expression was observed only between days 8 and 11. Anti-RANKL treatment resulted in a modest reduction in paw and ankle swelling and a reduction of serum levels of SAP, MMP3, and CTX-I. Destruction of the subchondral bone was significantly reduced, while no effect on bone formation was seen. CONCLUSIONS: Anti-RANKL treatment prevents joint destruction but does not prevent new bone formation in the DTHA model. Thus, although occurring sequentially during the course of DTHA, bone destruction and bone formation are apparently not linked in this model.

Cartilage in facet joints of patients with ankylosing spondylitis (AS) shows signs of cartilage degeneration rather than chondrocyte hypertrophy: implications for joint remodeling in AS.

INTRODUCTION: In ankylosing spondylitis (AS), joint remodeling leading to joint ankylosis involves cartilage fusion. Here, we analyzed whether chondrocyte hypertrophy is involved in cartilage fusion and subsequent joint remodeling in AS. METHODS: We assessed the expression of chondrocyte hypertrophy markers runt-related transcription factor 2 (Runx2), type X collagen (COL10), matrix metalloproteinase 13 (MMP13), osteocalcin and beta-catenin and the expression of positive bone morphogenic proteins (BMPs) and negative regulators (dickkopf-1 (DKK-1)), sclerostin, (wingless inhibitory factor 1 (wif-1)) of chondrocyte hypertrophy in the cartilage of facet joints from patients with AS or osteoarthritis (OA) and from autopsy controls (CO) by immunohistochemistry. Sex determining region Y (SRY)-box 9 (Sox9) and type II collagen (COL2) expression was assessed as indicators of chondrocyte integrity and function. RESULTS: The percentage of hypertrophic chondrocytes expressing Runx2, COL10, MMP13, osteocalcin or beta-catenin was significantly increased in OA but not in AS joints compared to CO joints. Frequencies of sclerostin-positive and DKK-1-positive chondrocytes were similar in AS and CO. In contrast, wif-1- but also BMP-2- and BMP-7-expressing and Sox9-expressing chondrocytes were drastically reduced in AS joints compared to CO as well as OA joints whereas the percentage of COL2-expressing chondrocytes was significantly higher in AS joints compared to CO joints. CONCLUSIONS: We found no evidence for chondrocyte hypertrophy within hyaline cartilage of AS joints even in the presence of reduced expression of the wnt inhibitor wif-1 suggesting that chondrocyte hypertrophy is not a predominant pathway involved in joint fusion and remodeling in AS. In contrast, the reduced expression of Sox9, BMP-2 and BMP-7 concomitantly with induced COL2 expression rather point to disturbed cartilage homeostasis promoting cartilage degeneration in AS.

Histomorphologic and histomorphometric characteristics of zygapophyseal joint remodeling in ankylosing spondylitis.

OBJECTIVE: To unravel the mechanisms that control bony ankylosis in ankylosing spondylitis (AS). METHODS: Histomorphologic and histomorphometric analyses were performed on zygapophyseal joints obtained from 18 patients with AS, 9 patients with osteoarthritis (OA), and 10 cadaver donors without a rheumatic disease (controls). The proteoglycan content of the cartilage was determined by Safranin O staining and the chondrocyte apoptosis according to caspase 3 expression. RESULTS: AS joints were categorized into 3 groups according to the morphology of the joint surfaces and joint space: group 1 were joints with an open joint space, group 2 were joints with cartilaginous fusion, and group 3 were joints with bony fusion of the joint surfaces. Progressive loss of the joint space from group 1 joints to group 3 joints suggests that this grouping corresponds to sequential stages of joint remodeling. Cartilage thickness and subchondral bone plate thickness declined from group 1 to group 3 (P < 0.01). Increased chondrocyte apoptosis rates were found in groups 1 and 2 (P < 0.05), while in group 3, a reduction in the proteoglycan content was found (P < 0.001). Bone marrow replacement and invasion of the subchondral bone plate by fibrous tissue was found predominantly in AS joints in group 2. CONCLUSION: Cartilage degeneration, indicated by cartilage thinning, enhanced chondrocyte apoptosis, and proteoglycan loss, and subchondral bone thinning, promoted by invasion of the subchondral bone plate by a fibrous tissue originating from the bone marrow, are hallmarks of joint remodeling in AS.

In situ analysis of interleukin-23- and interleukin-12-positive cells in the spine of patients with ankylosing spondylitis.

OBJECTIVE: The interleukin-12 (IL-12) family of cytokines has been suggested to play a critical role in inflammatory autoimmune diseases, and recent studies analyzing peripheral blood and synovial fluid from patients with spondyloarthritides suggest that IL-23 might be a proinflammatory factor in these disorders. This study was undertaken to investigate the presence and source of IL-23 in the spines of patients with ankylosing spondylitis (AS). METHODS: The frequency of IL-23-positive and IL-12-positive cells within the subchondral bone marrow and within fibrous tissue replacing normal bone marrow in facet joints of patients with AS was analyzed by immunohistochemistry. The origin of IL-23-positive cells was determined by double staining of CD163+ macrophages, CD68+ macrophages, CD1a+ dendritic cells, tryptase-positive mast cells, myeloperoxidase-positive cells, CD20+ B cells, and CD3+ T cells. Findings in 28 facet joints from 22 AS patients were compared with those in 20 facet joints from 13 patients with osteoarthritis (OA) and 10 normal control specimens. RESULTS: The frequency of IL-23-positive cells in subchondral bone marrow from the joints of AS patients (mean ± SD 42.50 ± 32.81/high-power field [hpf]) was significantly increased compared to that in subchondral bone marrow from OA patients (OA 15.63 ± 29.90/hpf) (P = 0.0017) or controls (19.36 ± 16.8/hpf) (P = 0.03). Myeloperoxidase-positive cells and, to a lesser extent, macrophages and dendritic cells were found to be the origin of IL-23 in the bone marrow. In AS and OA patients, the frequency of IL-23-positive cells was significantly higher than that of IL-12-positive cells (P < 0.001 in both patient groups). Within fibrous tissue from AS and OA facet joints, IL-23 was predominantly produced by CD163+ macrophages (mean ± SD 0.64 ± 0.59/hpf and 4.36 ± 3.4/hpf, respectively) and CD68+ macrophages (2.3 ± 0.65/hpf and 6.54 ± 4.1/hpf, respectively). CONCLUSION: IL-23 is expressed in the subchondral bone marrow and in fibrous tissue replacing bone marrow in facet joints of patients with AS. It might have a role in inflammatory processes and in chronic changes in AS joints, which makes it an interesting potential therapeutic target in this disease.