| Objective:Observe the synergistic effect of Clematis chinensis Osbeck and low-intensity pulsed ultrasound on proliferation、collagen type Ⅱ secretion and TGF-β1expression of rabbit knee articular chondrocytes cultured in vitro, and then explore the effect and possible mechanism of Clematis chinensis Osbeck and low-intensity pulsed ultrasound on cartilage damage.Methods:Chondrocytes were isolated, digested and harvested from knee articular of New Zealand rabbits, then the phenotype was identified by methods of toluidine blue and immunocytochemical staining. The cells of second generation in logarithmic growth phase were cultured in DMEM with concentration of0.01、0.1、0.5mg/ml Clematis chinensis Osbeck. CCK-8assay method was adopted to observe proliferation of chondrocytes. The cells of second generation in logarithmic growth phase were stimulated for20min each day for3days. An ultrasound signal consisting of a50ms burst sine wave of1.0MHz repeatingat1kHz, with an intensity of40,50,80mv spatial and temporal average, was applied. Cell proliferation were evaluated by CCK-8assay. The cells of second generation in logarithmic growth phase were cultured under four different sets of conditions:control group, LIPUS group, Clematis chinensis Osbeck, and Clematis chinensis Osbeck+LIPUS group. They were evaluated by measuring cell proliferation, synthesis of collagen type Ⅱ and expression of TGF-β1.Results:(1)Primary cultured chondrocytes was small round shape, and most adhered after24h whose appearance was polygonal and irregular-shaped; After4days, chondrocytes growed and fused into flaky distribution, with a typical slabstone-like appearance. Activity of the chondrocytes become stronger after first passage and long spindle-shaped cells were observed after four generations;after six generations,most cells showed long spindle-shaped fibroblast-like appearance, Activity becomed decreased and the rate of growth also slowed down.(2) Results demonstrated positive effects on the proliferation of chondrocytes was at concentration of0.1mg/ml Clematis chinensis Osbeck.(3) Results showed positive effects on the proliferation, followinglow-intensity pulsed ultrasound stimulation with intensities of50mv.(4) There were significant differences in proliferation and collagen type Ⅱ secretion and TGF-β1expression increased after cultured under the condition of Clematis chinensis Osbeck and LIPUS.Conclusion:The study demonstrated a synergistic effect between Clematis chinensis Osbeck and LIPUS. Because LIPUS is safe and noninvasive, the results of the present study suggest that it would be a promising new therapy for prevention of osteoarthritis. |
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