ObjectiveTo investigate the role of sphingosine-1-phosphate （S1P） in abnormal ossification in ankylosing spondylitis （AS）， clarify the relationship between S1P and “angiogenesis-osteogenesis” coupling， and provide new strategies for AS treatment. MethodsFemoral heads from AS patients and patients undergoing routine hip replacement were collected for immunohistochemical （IHC） staining to evaluate osteogenesis and H-type vessel formation. In vitro， ELISA was used to quantify the synthesis of S1P and analyze the expression changes of S1P signaling pathway-related molecules during the osteogenic differentiation of mesenchymal stem cells derived from patients with ankylosing spondylitis （ASMSCs） and those from healthy donors （HDMSCs）， to evaluate the activation status of S1P pathway during osteogenesis. Sphingosine kinase 1 （SK1） expression was knocked down in MSCs， and the S1P receptor inhibitor FTY720 was applied to block S1P signaling. Alkaline phosphatase （ALP） activity and Alizarin Red S （ARS） quantification were used to assess the effect of S1P on ASMSCs osteogenesis. Conditioned medium from osteogenically induced MSCs was used to treat human umbilical vein endothelial cells （HUVECs） to evaluate the effect of S1P on angiogenesis. An AS mouse model （SKG mice） was treated with FTY720 or the SK1 inhibitor PF-543 citrate. IHC staining and micro-CT scanning were used to assess abnormal ossification and spinal fusion， and immunofluorescence was used to evaluate H-type vessel formation. ResultsCompared with Osteonecrosis of the Femoral Head（ONFH） patients， AS patients exhibited excessive osteogenesis and H-type vessel formation （OCN P＜0.001， CD31 P＜0.001， EMCN P＜0.001）. During osteogenic differentiation， S1P expression and secretion were significantly higher in ASMSCs than in HDMSCs （P=0.0179）. Inhibition of S1P signaling with FTY720 or SK1 knockdown significantly suppressed osteogenic differentiation （compared with ASMSC， ARS： HDMSC P=0.001 8， FTY720 P＜0.001， si-SK1 P＜0.001； ALP： HDMSC P=0.032 8， FTY720 P=0.001 6， si-SK1 P＜0.001） of ASMSCs and the angiogenesis of HUVEC（compared with ASMSC， cell-covered area， total loops， total tube length and total branch points P＜0.001）. Treatment with FTY720 or PF-543 markedly inhibited abnormal ossification and spinal fusion（compared with Curdlan， arthritis index score， P＜0.001； OCN：control P=0.002， PF-543 P=0.010 7， FTY720 P=0.015 9 ） in AS mice and reduced H-type vessel formation （CD31⁺EMCN⁺： compared with curdlan， control P＜0.001， PF-543 P=0.001 7， FTY720 P=0.002 1）. ConclusionIncreased S1P synthesis in ASMSCs promotes osteogenic differentiation via autocrine mechanisms and further enhances ossification by facilitating H-type angiogenesis. Inhibiting S1P secretion in ASMSCs significantly suppresses abnormal ossification in AS.

A method for the determination of three volatile elements (mercury, arsenic and selenium) in soils by one-time digestion was established.The digestion of samples was carried out in an automatic program temperature controlled graphite digestion instrument by aqua regia + hydrofluoric acid+boric acid.Hydride generation atomic fluorescence spectrometry (HG-AFS) was used to determine the contents of mercury, arsenic and selenium in the same digestion solution.The accuracy of the method was verified by the results of the soil environment samples of certified reference materials GSS-1-GSS-8 from the Center of the National Standard.The contents of soil mercury, arsenic and selenium obtained by this method were consistent with the standard values of these elements provided by the Center of the National Standard.In comparison with the current standard methods, the one-time digestion method was simplified, the pre-processing time was saved, and the reagent consumption was reduced.The method had wide range of application, high sensitivity, low detection limit, which was especially suitable for trace analysis of bulk samples, and also it could be used as a rapid digestion method for the measurement and governance of heavy metals in polluted soils.

BACKGROUND:Cartilage tissue engineering has been widely used to achieve cartilage regeneration in vitro and repair cartilage defects. Tissue-engineered cartilage mainly consists of chondrocytes, cartilage scaffold and in vitro environment. OBJECTIVE:To mimic the environment of articular cartilage development in vivo, in order to increase the bionic features of tissue-engineered cartilage scaffold and effectiveness of cartilage repair. METHODS: Knee joint chondrocytes were isolated from New Zealand white rabbits, 2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracelular matrix in the concentration of 1×106/L to prepare cel-scaffold composites. Cel-scaffold composites were cultivated in an Instron bioreactor with mechanical compression (1 Hz, 3 hours per day, 10% compression) as experimental group for 7, 14, 24, 28 days or cultured staticaly for 1 day as control group. RESULTS AND CONCLUSION:Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time (P < 0.05). Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradualy with mechanical stimulation time. These results were consistent with the findings of proteoglycan kit. Real-time quantitative PCR revealed that mRNA expressions of colagen type I and colagen type II were significantly higher in the experimental group than the control group (P < 0.05). The experimental group showed the highest mRNA expression of colagen type I and colagen type II at 21 and 28 days of mechanical stimulation, respectively (P < 0.05). With the mechanical stimulation of bioreactor, the cel-scaffold composite can produce more extracelular matrix, such as colagen and proteoglycan, strengthen the mechanical properties to be more coincident with thein vivo environment of cartilage development, and increase the bionic features. With the progress of tissue engineering, the clinical bioregeneration of damaged cartilage wil be achieved.