ABSTRACT
Objective To study the roles of icariin and cyclic tensile strain (CTS) in promoting the osteogenic differentiation of adipose-derived stem cells (ASCs) and the molecular mechanisms involved.Methods ASCs were isolated from Sprague-Dawley rats and treated either with icariin (10-7 mol/L) or with 1000 μ,2000 μ or 3000 μ of CTS for 7 days,or with icariin plus CTS at 2000 μ for three days.Alkaline phosphatase (ALP) activity was detected after 3 and 7 days of intervention.Western blotting was performed to detect the expression of Runt-related transcriptional factor 2 (Runx2),Yes-associated protein (YAP) and connective tissue growth factor (CTGF) after the third day of the intervention.A reverse transcription polymerase chain reaction was performed at 7 days to detect the expression of osteopontin (OPN) and collagen la and after 3 days to detect the expression of the YAP target gene,CTGF and ankyrin repeating domain 1 (Ankrd1).Results Icariin and CTS at 1000 μ,2000 μ or 3000 μ could all significantly promote the expression of ALP protein.CTS at 2000 μ was the nost effective.The co-treatment with icariin and CTS significantly promoted ALP protein expression compared with icariin or CTS treatment alone.It also significantly promoted the expression of Runx2 and CTGF protein.Icariin or CTS (2000 μ) alone could not promote the expression of YAP protein,but icariin combined with CTS (2000 μ) promoted it significantly.Either icariin or CTS (2000 μ) could significantly promote ALP activity after 3 and 7 days,but icariin combined with CTS had the most obvious effect.Both icariin and CTS (2000 μ) could also significantly promote the expression of the osteogenesis-related genes OPN and collagen la,as well as the YAP targeted genes CTGF and Ankrdl.However,the combination of icariin and CTS had the greatest effect in promoting the expression of OPN mRNA,collagen la mRNA,CTGF mRNA and ankrdl mRNA.Conclusion Icariin and CTS co-treatment may promote osteogenic differentiation of ASCs via activating YAP expression.
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Objective To investigate the effects of different cyclic tensile strains on the proliferation and expression of bone marrow stromal cells (BMSCs)-cocultured human degenerated anulus fibrosus (AF) cells. Methods AF cells were isolated from a patient with degenerated intervertebral disc degeneration (IVD), which were co-cultured with BMSCs. The solely cultured AF cells were used as control group. The two groups of cells were expanded in monolayer, and cyclically strained for 3 hours, which were applied 0, 5%, 10%, 15%and 20%strains at a frequency of 0.25 Hz using BioDynamic test instrument. A flow cytometry method was used to examine the AF cell proliferation at 24 hours followed the application of cyclic tensile strains. After the total RNA was extracted, real-time PCR technology was used to detect the gene expression of collagenⅠand aggrecan. Results Under the same appropriate stress, the proliferative index (PI), the proportion of cells in the period of DNA synthesis, the expression of collagenⅠand aggrecan were significantly higher in the co-cultured group than those of control group (P<0.05). However, the best mechanical stimulation was different in the two groups. For the AF cells, the peaks of PI, the proportion of cells in the period of S period, the expression of collagenⅠand aggrecan were found in the 10% strain group, while for the co-cultured cells, they were found in the 15% strain group. Conclusion Co-culturing with BMSCs has a positive effect on the proliferation and expression of human degenerative fibrous ring cells, which can protect AF cells from bad stress stimulation.
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Objective To investigate the effect of mechanical loading with different magnitudes on the proliferation, differentiation and activity of preosteoclasts and osteoclasts. Methods One group of RAW264.7 preosteoclastic cells cultured in osteoclast inductive medium were subjected to the cyclic tensile strain for three days, and then cultured for four days; the other group of RAW264.7 cells were induced in osteoclast inductive medium for four days to be osteoclasts, then subjected to the cyclic tensile strain for three days. Results Under the tensile strain at different magnitudes, the proliferation variations in two groups of RAW264.7 cells were approximately identical, but changes in the activities of tartrate-resistant acid phosphatage (TRAP) and numbers of TRAP-positive multinucleated cells (osteoclasts) in the two groups were significantly different. Under the moderate tensile strain (2 500 με), the reduction of TRAP activity and osteoclasts number were both the highest in the first group, and both the lowest in the second group. Conclusions The influence of different tensile strain on osteoclast differentiation and osteoclastic activity of preosteoclasts in early differentiation is different to that of the preosteoclasts already differentiated into osteoclasts.
ABSTRACT
Objective To investigate the effects of different cyclic tensile strain on the proliferation of human anulus fibrosus cells from degenerated discs.Methods Anulus fibrosus(AF) cells were isolated from a degenerated human IVD,expanded in monolayer,and cyclically strained for 3 hours,applying 0,5％,10％,15％ and 20％ strains at a frequency of 0.25 Hz with the use of the DioDynamic test instrument.The flow cytometry method was used to examine the Af cells proliferation at 24 hours following application of the cyclic tensile strains.Results The proliferative index (PI) increased with the magnitude value of cyclic tensile strain except 20％ group.The most significant increase of proliferation index were found in 15％ group.Conclusion There might be some corelationships between magnitude of cyclic tensile strain and the proliferation of the degenerative AF cells.