Background/Purpose: Spondyloarthritis (SpA) is common, with a prevalence of ~1% in the United States. Patients with SpA suffer from pain and disability due to inflammation and ossification at enthesial sites, the mechanism of which remains unclear. BMP2 has been reported to be upregulated in mesenchymal stem cells derived from AS patients and has been implicated as an essential factor in bone formation and fracture repair in other settings, but the specific role of BMP2 in entheseal bone formation in SpA is not known.

Methods: Bone forms at enthesial sites in SpA through the process of endochondral bone formation, in which cartilage is laid down and subsequently remodeled into bone. We used a murine model of inflammatory arthritis, the antigen-induced arthritis (AIA) model, in which enthesial bone formation occurs at predictable sites around the knee, to determine the essential role of BMP2 in enthesial bone formation. By crossing the BMP2 ^fl/fl mouse with limb mesenchymal cell-specific (Prx1) Cre transgenic mice, we generated conditional knock out mice (KO) in which BMP2 expression is deleted in limbs, and littermate control mice (WT). We induced AIA in knee joints at 8 and 14 weeks of age.

Results: At 8 and 14 weeks, both KO and WT mice showed similar degrees of inflammation in arthritic knees. Histologic analysis of 8 week-old WT mice revealed endochondral bone formation with cartilage development by day 9, angiogenesis (CD-31 expressiong cells), TRAP-positive osteoclasts and bone at entheses by day 15. In contrast, KO mice developed only cartilage, but not bone, at entheses. Furthermore, angiogenesis and osteoclasts were absent at entheses in KO mice. To determine whether entheseal bone formation was simply delayed in KO mice, we prolonged AIA until day 36. Bone formation, angiogenesis and osteoclasts were still not observed at entheses of KO mice. AIA induced in 14 week-old WT mice showed entheseal bone formation by day 15. In contrast, KO mice did not develop cartilage or bone at the enthesis. Entheseal cells and bone marrow stromal cells (BMSCs) were then harvested from 14 week-old WT and KO mice. After 3 weeks in osteoblast differentiation media, entheseal cells and BMSCs from WT mice differentiated and mineralized, but cells from KO mice did not. To determine the molecular mechanism, we dissected entheses from 14 week-old WT and KO mice with AIA (day 9) using laser capture microscopy. Gene expression was analyzed using Affymetrix whole transcriptome arrays. The Wnt antagonist, Sfrp4 (known to inhibit Wnt signaling and osteoblast proliferation), was upregulated but chondroadherin (known to mediate chondrocyte adhesion) and Cxcl5 (known to promote bone metastasis) were down regulated in KO entheses. qPCR analysis revealed that Sfrp4 mRNA was also upregulated in BMSCs from KO mice.

Conclusion: These results demonstrate that in this model, mesenchymal cell-derived BMP2 is essential for entheseal bone formation in the setting of inflammation. A role for Wnt signaling is likely in this process. These results have implications for the inhibition of enthesial bone formation in SpA.