| Objective1. To observe the biological behavior of cultured CD146+perivascular cells, combined with chondrogenic differentiation, and investigate the feasibility of them for tissue engineering and its relationship with mesenchymal stem cells (MSCs).2. To investigate the effect of laminar shear stress (LSS) on proliferation and apoptosis in MSCs in vitro, and to explore its possible mechanisms.3. To induce MSCs into a nucleus pulposus-like phenotype utilizing simulated microgravity in vitro in order to establish a new cell-based tissue engineering treatment for intervertebral disc degeneration.Methods1. CD146+perivascular cells were isolated from rat adipose tissue. CD146+perivascular cells were isolated and purified using fluorescence activated cell sorter (FACS), and were induced into chondrogenic differentiation. RT-PCR and western blot Assay were employed for chondrogenesis. Furthermore, the migration ability was also detected.2. Here, we examined the effects of laminar shear stress on MSCs in vitro. MSCs from Sprague-Dawley rats bone marrow were isolated, purified and subjected to physiological levels of laminar shear stress. DNA synthesis through [3H]thymidine and cell cycle by flow cytometry were measured to detect cellular proliferation. AnnexinV immunostaining and Bcl-2/Bax mRNA expression were evaluated to determine the effect on MSCs apoptosis.3. For induction of a nucleus pulposus-like phenotype, MSCs were cultured in simulated microgravity in a chemically defined medium supplemented with 0 (experimental group) and 10 ng/mL (positive control group) of transforming growth factorβ1 (TGF-β1). MSCs cultured under conventional condition without TGF-β1 served as blank control group. On the day 3 of culture, cellular proliferation was determined by WST-8 assay. Differentiation markers were evaluated by histology and reverse transcriptase-polymerase chain reaction (RT-PCR). Results1. The CD146+perivascular cells have been successfully cultured in vitro. Like to chondrocytes, the induced CD146+perivascular cells cultured in pellet underwent chondrogenic differentiation, as evidenced by higher expression of Aggrecan and Sox9. The results of migration showed its high ability for control group.2. Results showed that fluid shear stress caused a dose-related reduction of the rate of MSCs proliferation, with the majority of cells being arrested in the Go or G1 phase. Moreover, we found that physiological levels of laminar shear stress exerted a potent suppression effect on MSCs apoptosis.3. TGF-β1 slightly promoted the proliferation of MSCs. The collagen and proteoglycans were detected in both groups after culture for 7 days. The accumulation of proteoglycans was markedly increased. The RT-PCR revealed that the gene expressions of Sox-9, aggrecan and type II collagen, which were chondrocyte specific, was increased in MSCs cultured under simulated microgravity for 3 days. The ratio of proteoglycans/collagen in blank control group was 3.4-fold higher to positive control group, which denoted a nucleus pulposus-like phenotype differentiation.Conclusion1. The results suggest that the CD146+perivascular cells may be a potential cell source for cartilage tissue engineering, and the perivascular site could be the niche for MSCs.2. The results from the cell cycle and apoptosis reveal a critical role for laminar shear stress in maintenance of quiescence to MSCs.3. Independent, spontaneous differentiation of MSCs towards a nucleus pulposus-like phenotype in simulated microgravity occurred without addition of any external bioactive stimulators, namely factors from TGF-βfamily, which were previously considered necessary.
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