| The injury of bone, particularly the defect of large bone is very common. Inaddition to sport injuries, many diseases such as trauma, tumor or infection also cancause damages to some or all of bone tissue. The damages can lead to the lack ofphysiological functions, affect the normal life seriously and threat the health. Bonetissue engineering is the most effective way to re-establishing bone currently. Bonemarrow mesenchymal stem cells (MSCs) are ideal seed cells for bone tissue engineering.Either MSCs were large-scale cultured in vitro or transplanted into body, both ofimproper fluid shear stress and hypoxia can cause some damages to cells. In order topromote the tolerability of MSCs to damages of fluid shear stress and hypoxia, thehypothesis that mechano growth factor (MGF) E peptide pretreatment can protect MSCsagainst the damages of fluid shear stress and hypoxia was proposed. In this study, MSCsfrom Sprague Dawley (SD) rats were the seed cells, a parallel-plate flow chambersupplied fluid shear stress and compound CoCl2simulated hypoxia. We investigated theeffect of MGF E peptide pretreatment (24h) protects MSCs against damages of fluidshear stress (30min) and hypoxia (24h), and we focused on the activity andproliferation of MSCs. The main contents and results are as follows:①MGF E peptide pretreatment protects MSCs against damage of fluid shearstress (72dyn/cm2)1) Effects of three different magnitudes of fluid shear stress on the activity of MSCs.The results showed that, compare to the control group,8dyn/cm2fluid shear stresshas no obvious effect on the activity of MSCs.24dyn/cm2fluid shear stress (in therange of physiological level in vivo) improved the activity of MSCs by55.29%.When the fluid shear stress was increased to72dyn/cm2, the cellular activity wassignificantly decreased by54.47%.2) Effect of MGF E peptide pretreatment on the activity of MSCs under72dyn/cm2fluid shear stress. Compared to the group without MGF E peptide pretreatment (0ng/ml) under72dyn/cm2fluid shear stress,25,50and100ng/ml MGF E peptidepretreatment increased the cellular activity by98.07%,155.07%and68.66%,respectively. The results also implied that the efficiency of50ng/ml MGF E peptidepretreatment was higher than those of25and100ng/ml. The activity of MSCspretreated with50ng/ml MGF E peptide under72dyn/cm2fluid shear stress has been recovered to the same level in static condition without MGF E peptidepretreatment.3) The long-term effect of50ng/ml MGF E peptide pretreatment. Compared to thegroup without MGF E peptide pretreatment under72dyn/cm2fluid shear stress, atthe same time point (24,48and96h), the cellular activity of MSCs which werepretreated with50ng/ml MGF E peptide was increased by155.07%,34.33%and34.83%, respectively. It seems that50ng/ml MGF E peptide pretreatment had thehighest efficiency after the MSCs were further been incubated for24h.4) Effect of MGF E peptide pretreatment on the proliferation of MSCs under72dyn/cm2fluid shear stress. The EdU results showed that, compared to the controlgroup, less cells presented a red fluorescence indicating EdU labeling in the injurygroup. The proliferation rate of MSCs decreased from8.80±0.06%to5.12±0.02%(p<0.05). Compared to the injury group,50ng/ml MGF E peptide pretreatmentimproved the number of EdU-labeled MSCs significantly. The proliferation rate ofMSCs was increased to7.06±0.15%,10.19±0.52%,12.51±0.7%at24,48and96h(p<0.05). In the experimental groups (50ng/ml MGF E peptide pretreatment), theactivity of MSCs cultured for48h has been recovered to the same level in staticcondition without MGF E peptide pretreatment.5) Effect of MGF E peptide pretreatment on the activity of MSCs under8dyn/cm2fluid shear stress.25~100ng/ml MGF E peptide pretreatment has no influence onthe activity of MSCs in static condition. Compare to the static groups,25and50ng/ml MGF E peptide pretreatment under8dyn/cm2fluid shear stress improved theactivity of MSCs by62.21%and80.69%, respectively.6) Compare to the static group, without and with50ng/ml MGF E peptidepretreatment under24dyn/cm2fluid shear stress improved the cellular activity ofMSCs by55.29%and105.12%, respectively. The result may imply that the fluidshear stress and MGF E peptide pretreatment have synergistic effect on the activityof MSCs.②MGF E peptide pretreatment protects MSCs against damage of hypoxia (CoCl2:500μmol/l)1) Effects of different concentrations of CoCl2on the activity of MSCs. Compare to thecontrol group,100and200μmol/l CoCl2improved the activity of MSCs by12.31%and11.97%, respectively. However,500、1000and2000μmol/l CoCl2declined thecellular activity by31.59%，46.35%and72.98%, respectively. 2) Effect of MGF E peptide pretreatment on the activity of MSCs under500μmol/lCoCl2. Compared to the group without MGF E peptide pretreatment under500μmol/l CoCl2,5,10,25,50and100ng/ml MGF E peptide pretreatment increasedthe cellular activity by37.28%,54.26%,47.38%,39.86%and41.50%, respectively.The activity of MSCs has been recovered to the same level with the group withoutMGF E peptide pretreatment and CoCl2.3) The long-term effect of5ng/ml MGF E peptide pretreatment. Compared to thegroup without MGF E peptide pretreatment under500μmol/l CoCl2, at the sametime point (0,24,48and96h), the cellular activity of MSCs which were pretreatedwith5ng/ml MGF E peptide was increased by37.28%,33.01%,82.85%and43.11%, respectively. It seems that5ng/ml MGF E peptide pretreatment had thehighest efficiency after the MSCs were further been incubated for48h.4) Effect of MGF E peptide pretreatment on the proliferation of MSCs under500μmol/l CoCl2. The EdU results showed that, compared to the control group, lesscells presented a red fluorescence indicating EdU labeling in the injury group. Theproliferation rate of MSCs decreased from6.76±0.39%to3.00±0.03%(p<0.05).Compared to the injury group,5ng/ml MGF E peptide pretreatment improved thenumber of EdU-labeled MSCs significantly. The proliferation rate of MSCs wasincreased to6.17±0.16%(p<0.05). The proliferation of MSCs has been recovered tothe same level with the group without MGF E peptide pretreatment and CoCl2.In summary, the results of this study indicated that the activity and proliferation ofMSCs were damaged when the cells were exposed to improper fluid shear stress (highenough:72dyn/cm2) and hypoxia (low enough:500μmol/l CoCl2simulated hypoxia).Pretreatment with appropriate concentration of MGF E peptide can promote thetolerability of MSCs to damages of fluid shear stress and hypoxia. And the activity andproliferation of MSCs were increased significantly. However, the effects of MGF Epeptide showed a time-dependent manner. |
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