The Study Of Mesenchymal Stem Cells In Synovial Fluid From Patients Of Knee Osteoarthritis

BackgroundOsteoarthritis (OA) is a joint disease that mostly occurs in the middle-aged and elderly population. The knees are most likely to be affected, resulting in progressive wear of surface cartilage. Mesenchymal stem cells (MSCs) are a kind of adult stem cells with self-replicating and multi-directional differentiation potential. It has been reported that MSCs are present in synovial fluid besides bone marrow, fat and other tissues. Further studies have found that the proportion of MSCs in patients with early osteoarthritis is higher than that in normal subjects, which is important for studying the pathogenesis and development of osteoarthritis. However, there are limitations in the methods of detection and identification of MSCs using the traditional flow cytometry.Therefore we apply FlowSight imaging multichannel flow cytometry and osteogenic differentiation of the cells to identify MSCs and calculate the proportion of MSCs between early and late osteoarthritis patients.Methods(1) Cells were cultured in the incubator with 5% CO2 at 37? after isolating from the synovial fluid. 6 fluorescent antibodies (CD44 PE, CD73 APC, CD90 FITC, CD 105 PerCP Cy5.5, CD34 eFluor 450, CD45 APC eFluor 780) were implanted to half of the cells which had been incubated for 4 weeks. FlowSight imaging multichannel flow cytometry were applied to detect antibodies and identify MSCs. The other half cells were cultured for 2 weeks and for another 2 weeks with osteogenic induction differentiation medium. Alizarin red staining was performed and calcium nodus were observed under microscope. (2)Patients were divided into early OA group (18 cases) and middle and late OA group (27 cases) according to the evaluation results. We apply FlowSight imaging multichannel flow cytometry to calculate the proportion of MSCs and evaluate the correlation between MSCs proportion and degrees of knee OA.ResultsLong spindle cells can be observed under microscope as well as cell colony formation.CD90, CD44, CD105, CD73 were expressed and CD34,CD45 were not expressed in the MSCs following the results of FlowSight imaging multichannel flow cytometry. Red calcium nodus were clearly seen under microscope. There was no significant difference in age, sex and BMI between the two groups.The mean proportion of MSCs without culture in the early OA group was lower than that in the middle and late OA group (2.12%±0.63<3.21% ± 0.84),the difference was statistically significant (P <0.05). The mean proportion of MSCs after culturing for 4 weeks in the early OA group was also lower than that in the middle and late OA group (48.63%±13.36 <56.35% ± 10.61), the difference was statistically significant (P <0.05).ConclusionsIn this study, FlowSight imaging multichannel flow cytometry was used to prove the presence of MSCs in synovial fluid of patients with knee OA and cell staining. The proportion of MSCs to total cells is related to the degree of development of knee OA. The proportion of MSCs in synovial fluid of patients with early osteoarthritis was lower than that of middle and late OA patients.