Objective To investigate the effects of bone morphogenetic protein 2 (BMP-2) on the chondrogenic differentiation of human Achilles tendon-derived stem cells (hATDSCs) in vitro. Methods Achilles tendon was harvested from a voluntary donor with acute Achilles tendon rupture. And nucleated cells were obtained by digesting with collagenase and were cultured to the 3rd passage. The flow cytometry was used to measure the immunophenotyping; and Oil red O staining, alizarin red staining, and Safranin O/fast green staining were used to identify the adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation, respectively. The hATDSCs pellet was cultured in complete culture medium with (experimental group) or without recombinant human BMP-2 (rhBMP-2) (control grup) for 3 weeks. Chondrogenic differentiation of hATDSCs was evaluated by HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II; and the mRNA expressions of SOX9, collagen type II, and Aggrecan were detected by real-time fluorescence quantitative PCR. Results Primary hATDSCs cultured in vitro showed clonal growth; after cell passage, homogeneous spindle fibroblast-like cells were seen. The cells were positive for CD44, CD90, and CD105, while negative for CD34, CD45, and CD146. The results were positive for Oil red O staining at 3 weeks after adipogenic differentiation, for alizarin red staining at 4 weeks after osteogenic differentiation, and for Safranin O/fast green staining at 3 weeks after chondrogenic differentiation. After hATDSCs were induced with rhBMP-2 for 3 weeks, pellets formed in the experimental group, and the size of pellets was significantly larger than that in the control group; the results of HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II were all positive. The results of real-time fluorescence quantitative PCR showed that the mRNA expressions of SOX9, collagen type II, and Aggrecan in the experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion BMP-2 can promote proteoglycan deposition and induce chondrogenic differentiation of hATDSCs in vitro. The effect of BMP-2 on hATDSCs might provide a possible explanation for histopathological changes of tendinopathy.
ObjectiveTo explore the effects of cryopreservation on the cell survival rate, cell viability, early apoptosis, migration ability, and tendon-related marker expression of tendon-derived stem cells (TDSCs) in rat patellar tendons.MethodsThe patellar tendon tissues were harvested from 12 4-month-old male Sprague Dawley rats; 12 patellar tendon tissues from 6 rats were cryopreserved (the experimental group), and the other 12 patellar tendon tissues were not treated (the control group). The patellar tendons were digested with 0.3% type I collagenase to obtain nucleated cells. The survival rate of nucleated cells was detected by trypan blue exclusion assay, and colony-forming ability by crystal violet staining. TDSCs were isolated and cultured to passage 3 (P3). The cell viability of TDSCs was detected by Alamar Blue method, the early apoptosis by Annexin V-FITC/PI assay, the cell migration ability by Transwell method, and the mRNA expressions of tendon-related markers [collagen type I (Col1α1), scleraxis (Scx), and tenomodulin (Tnmd)] by real-time quantitative PCR.ResultsThe survival rate of nucleated cells was 91.00%±3.63% in the control group, and was 61.65%±4.76% in the experimental group, showing significant difference (t=12.010, P=0.000). The formation of the primary nucleated cell clones was observed in 2 groups. At 12 days, the number of colonies forming of the experimental group [(8.41±0.33)/1 000 nucleated cells] was significantly lower than that of the control group [(15.19±0.47)/1 000 nucleated cells] (t=28.910, P=0.000). The percentage of TDSCs in the active nucleated cells in the experimental group (1.37%±0.09%) was significantly lower than that in the control group (1.67%±0.10%) (t=5.508, P=0.003). The growth trend of TDSCs (P3) in the 2 groups was consistent within 14 days. There was no significant difference in absorbance (A) value between 2 groups at each time point (P>0.05). The early apoptotic rate of TDSCs was 1.67%±0.06% in the experimental group and was 1.63%±0.06% in the control group, showing no significant difference (t=0.707, P=0.519). Under microscope, TDSCs adhered to the lower chamber of the Transwell chamber; the number of cells was 445.00±9.70 in the experimental group and was 451.50±12.66 in the control group, showing no significant difference (t=0.998, P=0.342). The relative mRNA expressions of Col1α1, Scx, and Tnmd were 3.498±0.065, 0.062±0.002, and (4.211±0.211)×10–5 in the experimental group and were 3.499±0.113, 0.062±0.001, and (4.341±0.274)×10–5 in the con-trol group, showing no significant difference (t=0.013, P=0.991; t=0.042, P=0.969; t=0.653, P=0.549).ConclusionThe survival rate of nucleated cells in cryopreserved rat tendon tissues is lower, but a large number of active TDSCs, and its cell viability, early apoptosis rate, migration ability in vitro, and cell tenogenic differentiation ability are remained.