ObjectiveTo investigate the effect of transforming growth factor β3 (TGF-β3) at different concentrations on the differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated with TGF-β3 at the concentrations of 5.0, 2.5, 1.0, and 0 ng/mL. At 1, 3, and 5 days, the mRNA expressions of tendogenic differentiation related genes[collagen type Ⅰ,tenascin C (TNC), tenomodulin (TNMD), scleraxis (Scx)], osteogenic differentiation related genes[Runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP)], chondrogenic differentiation genes (Sox9 and collagen type Ⅱ),and adipogenic differentiation genes[AP2 and peroxisome proliferator-activated receptor γ (PPARγ)] were measured by real-time quantitative PCR (qRT-PCR). ResultsTSCs could differentiated in different directions after treated with TGF-β3 at different concentrations at different time points. TGF-β3 was able to induce TSCs differentiated into tenocytes, which was related to the concentration and time of duration, and the two factors have interaction. Stimulation of TGF-β3 at low concentration and for short time could inhibit non-tendogenic differentiation of TSCs, but at high concentration and for long time, TGF-β3 enhanced TSCs differented into osteocytes or chondrocytes. ConclusionEffects of TGF-β3 on TSCs differentiation are complicated and depend on the concentration and time of duration, which may be a key factor between tendogenic and non-tendogenic differentiations of TSCs.
ObjectiveTo investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor γ (PPARγ), 1ipoprotein lipase (LPL), and fatty acid binding protein (aP2). ResultsThe final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P<0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P<0.05). ConclusionInhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.
ObjectiveTo investigate the effect of different mechanical tensions on the expressions of RhoA/Rho associated protein kinases (ROCK) in rat tendon stem cells (TSCs). MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged, 2-3 months; weighing, 200-250 g) by enzymatic digestion method and cultured for 2-3 passages, then seeded on micro groovdishes. The 4% (4% stretch group) and 8% (8% stretch group) mechanical stretching was performed for 4 hours every day at 1 Hz. After 1, 2, and 3 days, the protein and mRNA expressions of RhoA and ROCK were measured by Western blot and real-time quantitative PCR. The cell proliferation was measured by cell counting kit 8. The cells were not stretched as control group. ResultsThe TSCs at passage 2 showed a cobble-stone shape and aggregation growth; TSCs seeded on micro groovdishes showed random growth, and the cells grew along the stretching direction after mechanical stretching. The mRNA expressions of RhoA and ROCK in control group, 4%, and 8% stretch groups showed an increasing tendency at 1, 2, and 3 days, showing significant difference between groups (P<0.05). The protein expressions of RhoA and ROCK in 4% and 8% stretch groups were similar to those in control group at 1 day (P>0.05), but the expressions in 4% and 8% stretch groups showed an increasing tendency at 2 and 3 days, which were significantly higher than those in control group (P<0.05). The cell proliferation of 8% stretch group was significantly lower than that of 4% stretch group and control group at each time point (P<0.05), but no significant difference was found between 4% stretch group and control group (P>0.05). ConclusionThe expressions of RhoA and ROCK of rat TSCs are positively correlated with stretch intensity. So RhoA/ROCK may be an important molecule in TSCs after mechanical stretching.