- Poster presentation
- Open Access
Abnormal collagen type I production in osteoarthritic subchondral bone is associated with a reduced capacity of osteoblasts to mineralize in vitro
Arthritis Res Thervolume 6, Article number: 87 (2004)
Osteoarthritis (OA) is characterized by cartilage loss, synovial inflammation, osteophytes, and abnormal subchondral bone remodeling including sclerosis. Bone sclerosis in OA is due to an abundant osteoid collagen matrix. Collagen type 1 synthesis is increased in in vivo OA bone tissue and there is an abnormal ratio of collagen type 1α1 chains (Coll1α1) to Coll1α2 chains in this tissue. The mechanisms responsible for this abnormal osteoid matrix remain unknown.
In this study using in vitro subchondral osteoblasts (Ob) from normal and OA individuals, we investigated the mechanisms responsible for abnormal collagen production.
We used primary human subchondral Ob from normal and OA individuals. Cells were stimulated or not with 100 ng/ml parathyroid hormone (PTH), 500 nM prostaglandin E2 (PGE2) or 50 nM 1,25(OH)2D3. RNA was extracted with TRIzol and used to perform RT-PCR and real-time PCR of Coll1A1 and Coll1A2. Coll1 synthesis was assessed as the release of the carboxy terminal peptide fragment (CICP), which reflects de novo collagen synthesis. Proteins were separated by SDS-PAGE and detected using selective antibodies against Coll1α1, Coll1α2, or membrane-type 1 or membrane-type 2 matrix metalloprotease (MT1-MMP and MT2-MMP). MMP-2 and MMP-9 activities were assessed by zymography. Mineralization was evaluated by the von Kossa staining of cells after 30 days of culture in the presence or not of 10 ng/ml bone morphogenic protein-2 (BMP-2).
Data showed that basal Coll1A1 mRNA levels were significantly increased in OA Ob compared with normal using real-time PCR, whereas Coll1A2 levels in OA Ob were similar to normal. This translated into an α1 to α2 collagen type I ratio of 2.5 in normal Ob whereas it increased to 7.2 in OA Ob. PTH and PGE2 both reduced Coll1A1 and Coll1A2 mRNA levels in normal Ob yet this was reduced for OA Ob. Indeed, PGE2 reduced Coll1A1 and Coll1A1 mRNA levels about half as in normal, and the effect of PTH was virtually absent in OA Ob. Basal collagen type I synthesis, determined by the release of the C-terminal propeptide and by western blot analysis, was also higher in OA Ob than normal. MMP-2 and MMP-9 were increased in OA Ob compared with normal as determined by zymography. Western blot analysis showed an increase in MT2-MMP but not in MT1-MMP in OA Ob. Finally, the mineralization of OA Ob was significantly reduced compared with normal as determined by Von Kossa staining under both basal conditions and following BMP-2 stimulation.
These results suggest that a cellular defect of OA Ob and an abnormal response to PTH and PGE2 challenge could explain abnormal production and ratio of α1 to α2 chains of mature collagen type 1 in these cells. Coupled to the increase in MMP activities, this could explain the abnormal collagen remodeling observed in OA bone tissue in vivo.