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Chondrocytes located in hyaline cartilage may maintain phenotype while the chondrocytes situated in calcified cartilage differentiate into hypertrophy. Chondrogenic and hypertrophic differentiation of mesenchymal stem cells (MSCs) are two subsequent processes during endochondral ossification. However, it is necessary for chondrocytes to hold homeostasis and to inhibit hypertrophic differentiation in stem cell-based regenerated cartilage. Dihydroartemisinin (DHA) is derived from artemisia apiacea which has many biological functions such as anti-malarial and anti-tumor. Whereas the effects of DHA on chondrogenic and hypertrophic differentiation are poorly understand. In this study, the cytotoxicity of DHA was determined by CCK8 assay and the cell apoptosis was analyzed by flow cytometry. Additionally, the effects of DHA on chondrogenic and hypertrophic differentiation of MSCs are explored by RT-PCR, western blotting and immunohistochemistry. The results showed that DHA inhibited expression of chondrogenic markers including Sox9 and Col2a1 by activating Nrf2 and Notch signaling. After induced to chondrogenesis, cells were treated with hypertrophic induced medium with DHA. The results revealed that hypertrophic markers including Runx2 and Col10a1 were down-regulated following DHA treatment through Pax6/HOXA2 and Gli transcription factors. These findings indicate that DHA is negative to chondrogenesis and is protective against chondrocyte hypertrophy to improve chondrocytes stability. Therefore, DHA might be not suited for chondogenesis but be potential as a new therapeutic candidate to maintain the biological function of regenerated cartilage., Competing Interests: None.

Objective: To investigate the human tenocyte cytoskeleton under different in vitro stretching conditions and analyze the relations between the changes of tenocytic cytoskeleton and different stretching loads., Methods: Human tenocytes, cultivated for 5 -7 passages, were stretched under 4%, 8% and 12% cyclic mechanical stretching with a duration of 2, 4, 8, 12, 24 hours and a frequency of 0.5 and 1.0 Hz. Laser scanning confocal microscope was used to examine the changes of F-actin and nucleus after immunofluorescent staining at different cyclic mechanical stretching loads on human tenocyte. The uni-cell average fluorescence intensity was measured with an image analysis system by the photos of human tenocyte cytoskeleton and analyzed by the single factor analysis of variance., Results: After cyclic stretching under 4% stretching with a duration of 2 hours at 0.5 Hz, the microfilament of human tenocyte had an irregular and dim alignment. F-actin was thicker and ruptured under 4% stretching with a duration of 4 hours. Under 8% stretching with a duration of 4 hours at 0.5 Hz, all actin microfilaments ruptured, but part of membrane microfilament remained intact. There was a rising trend of actin filament fracturing under 12% stretching with a duration of 2, 4, 8, 12, 24 hours at 1.0 Hz. And all actin filaments fractured at 24 hours. In the control group, the fluorescent intensity of F-actin was at the highest and the filament remained intact. Under the same stretching frequency, the fluorescent intensity of F-actin had a declining trend and significant differences existed under different stretching loads with different durations (P < 0.05). The fluorescent intensity of F-actin increased in all experimental groups, but it was lower than that of the control group with a duration of 8 hours. The expression of F-actin decreased with a longer duration and reached its lowest at 24 hours. The most obvious phenomenon of nuclear condensation and apoptotic body formation was observed under 4% stretching with a duration of 4 hours at 0.5 Hz., Conclusion: Different cyclic mechanical stretching may cause the in vitro breakage and depolymerization of human tenocytic F-actin. Such an effect correlates with stretching force and its duration.