Effects Of The Supernatant From Ankylosing Spondylitis Synovial Cell Primary Culture On The Osteogenic Differentiation Of Ligament Fibroblasts

BackgroundAnkylosing spondylitis is a common inflammatory rheumatic disease that affects the axial skelton,causing characteristic inflammatory back pain,which can lead to structural and functional impairments and a decrease in quality of life. New imaging techniques and therapies have substantially changed the managements of this disease in the past decade. Whether inhibition of radiographic progression and structural damage canbe reached with available drugs is as yet unclear. Furthermore, treatment with non-steroidal anti-inflammatory agents and physiotherapy remains an important approach to long-term management of patients with ankylosing spondylitis. The new treatment options with tumour necrosis factor blokers seems a breakthrough for patients refractory to conventional treatment. However, current radiographic follow-up data suggest that these drugs do not affect the process of new bone formation.The pathological study of ankylosing spondylitis found that both enthesitis and subchondral granulation tissue, are accompanied by a large number of abnormal proliferation of fibroblasts. Furthermore, fibroblast cells have the potential osteogenic differentiation capacity in vivo and in vitro. Therefore, we speculated that fibroblast cells may through the differentation of osteoblasts and then contribute to the new bone formation in AS. In addition, studies have shown that a variety of cytokines presented in the synovial tissue of AS, such as transforming growth factor-β, bone morphogenetic protein-2(BMP-2), fibroblast growth factor and tumor necrosis factor-a. These factors have different levels of the promotion of osteogenic differentiation. Therefore, we further speculated that synovial cells of AS may can secrete these growth factors and promote fibroblasts differentiate into osteoblasts.ObjectivesTo determine the osteogenic capacity of ligament fibroblasts that derived from hip joints of ankylosing spondylitis and the effects of synovial fluid on the differention of ligament fibroblasts into osteoblasts.MethodsSpecimens of ligament and synovium were derived from the hip joints of ankylosing spondylitis and aseptic necrosis of femoral head patients, respectively. The primary cell culture of ligament fibroblasts and synovial cells were processed and then the supernatant of synovial cells in the first generation was collected. Four different conditioned media were used to induce ligament cells of the third-generation into osteoblastic differentiation, that is:A:osteogenic induction medium(OIM)(DMEM/F12 containing 2×10-8 mmol/L dexamethasone, 0.1mg/ml of vitamin C,20mmol/Lβ-glycerophosphate disodium and 10% fetal bovine serum)+DMEM/F12 containing 10% fetal bovine serum; B:OIM+ DMEM/F12 containing 10% fetal bovine serum with 0.3ug/ml BMP-2; C:OIM+the supernatant of ANFH synovial cells; D:OIM+ the supernatant of AS synovial cells. Osteogenic phenotype tests were processed at the days of 0,5,10,15 and 20, respectively. Quantitative and qualitative changes in alkaline phosphatase(ALP) activity was determined. The expression of human osteocalcin(OC) was determined by ELISA. Calcium deposits were detected by Von Kossa staining.ResultsThe expression levels of alkaline phosphatase and osteocalcin were increased with the extension of time, in which alkaline phosphatase activity was significantly higher at day 10 and reached a peak, osteocalcin was significantly higher at day 15 and reached a peak. Calcium deposits can be detected at day 20 in each group. Comparing four different conditioned media' effects of osteoginic differentation on the ligament fibroblasts find that the combination of dexamethasone and BMP-2 have a intense effect on increasing the expression levels of ALP and OC, that significantly higher than the other three groups. In addition, the expression levels of ALP and OC were significantly higher in D groups than C groups, P<0.05. Furthermore, ligament fibroblast cells from AS expressed higher levels of ALP and OC than that from ANFH.ConclusionsLigament fibroblasts that derived from AS have higher osteogenic potentials than that from ANFH in vitro. In AS, synovial cells may play a certain role in promoting ligament fibroblasts osteogenic differentiation.This study confirmed that liagment fibroblasts that from AS have the capacity of osteogenic differentiation in vitro, and also found that synovial cells of AS may have a certain role in promoting ligament fibroblast cells differente into osteoblast and then in the new bone formation.