Activated osteoclasts were seen on day 5 after arthritis induction and peaked on day 10. 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 AURKA 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. was calculated by subtracting swelling measured on day 0 from swelling measured on day test, and parametric data were analyzed by using a two-sided unpaired test or one-way analysis of variance. For multiple group comparisons, the KruskalCWallis test and Dunns multiple comparisons test as a posttest were used. A value less than 0.05 was considered significant, and levels of significance were assigned as *periarticular pannus, inflammation, subchondral granulation tissue. c Histopathological scoring of inflammation, extraarticular pannus, and subchondral granulation tissue at indicated time points after arthritis induction by evaluation of H&E-stained tissue sections. Sequential development of joint destruction and new bone formation Destruction of the joint structures was assessed by looking at tissue sections stained with Safranin O/Fast Green, which stains cartilage red and bone blue. Most of the cartilage damage was seen as cartilage irregularity and loss of cartilage integrity (Fig.?2a). Bone resorption facilitated by the periarticular pannus and the subchondral granulation tissue occurred early during arthritis, as shown in the scoring of cartilage damage and bone resorption (Fig.?2b). This suggests that inflammation directly induces a strong osteoclastic and proteolytic response, which drives tissue destruction. Open in a separate window Fig. 2 Inflammation in delayed-type hypersensitivity arthritis (DTHA) is accompanied by early cartilage damage and bone resorption that precedes new bone formation. a Representative examples of Safranin O/Fast Green and collagen type X (COLX)- and osteocalcin (OC)-stained tissue sections of CW-069 DTHA or control [phosphate-buffered saline (PBS)] paws at indicated time points. cartilage, bone, inflammation, bone marrow, new cartilage, new bone. Safranin O/Fast Green original magnification??100, COLX and OC original magnification??200. b Histological scores of cartilage damage and bone resorption performed on Safranin O/Fast GreenCstained tissue sections and of new bone formation performed on COLX- and OC-stained sections (indicate osteoclasts, and indicates vascular channels. Tissue response in DTHA involves enhanced proteolytic and osteoclastic activity and increased transcription of osteoclast- and osteoblast-specific genes To identify mediators involved in tissue and bone destruction, we assessed inflammatory tissue destruction CW-069 by measurement of the activity of cathepsin B, a protease expressed in fibroblasts and other cells. For this purpose, we used in vivo FMT and also determined serum levels of MMP3. The peak of both local cathepsin B activity CW-069 and systemic MMP3 levels was synchronized with the peak of paw swelling (Fig.?4a and b). RANKL messenger RNA (mRNA) expression in the CW-069 arthritic paw followed a similar time course. In contrast, mRNA expression of TRAP and cathepsin K increased from day 3 and peaked on day 8 after arthritis induction, reflecting increased osteoclastogenesis (Fig.?4c). Open in a separate window Fig. 4 Delayed-type hypersensitivity arthritis (DTHA) is accompanied by enhanced proteolytic osteoclast activity and increased transcription of genes associated with bone erosion and new.