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BACKGROUND:Mechanical stimulation has been confirmed to promote osteogenic differentiation of bone marrow stromal stem cells,but the mechanism is unknown.Primary cilia are important mechanoreceptors and regulate various signaling pathways such as TGF-β1/BMP-2/SMAD.They are likely to be important targets for mechanical regulation of bone marrow stromal stem cells. OBJECTIVE:To investigate the effect and mechanism of fluid shear stress on osteogenic differentiation of bone marrow stromal stem cells. METHODS:Rat bone marrow stromal stem cells were divided into control group,mechanical stimulation group(fluid shear mechanics intervention by shaking table),mechanical stimulation + IFT88 silencing group(mechanical stimulation + silencing IFT88 expression with siRNA).After 24 hours of intervention,qRT-PCR was utilized to determine the expression of transforming growth factor β1 and bone morphogenetic protein 2.Western blot assay was used to detect the expression of phosphorylated SMAD2/3 protein.Immunofluorescent staining of primary cilia was conducted and morphology was analyzed. RESULTS AND CONCLUSION:Shear stress stimulation could promote the transcriptional activity of transforming growth factor β1 and bone morphogenetic protein 2 genes,and increase the expression of phosphorylated SMAD2/3 protein.After siRNA interfered with primary cilia,this mechanical response effect was significantly reduced.There was a Spearman correlation between the change ratio of the primary cilium area of bone marrow stromal stem cells and the increased ratio of transforming growth factor β1 and bone morphogenetic protein 2 gene transcription.These findings indicate that primary cilia/intraflagellar transport mediates the activation of fluid shear stress-responsive transforming growth factor β1/bone morphogenetic protein 2/SMAD signaling pathway and promotes osteogenic differentiation of bone marrow stromal stem cells.
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Objective To construct bone marrow stem cell sheets and to investigate its effects in the process of osteogenesis.Methods BMSCs were differentiated into osteoblasts and then seeded into a temperature responsive culture dish to construct BMSC sheets.PLGA scaffolds in which both BMSC suspension and BMSC sheets were added,were implanted into the left side of the dogs' mandible.In the other side,PLGA scaffolds that were not wapped with BMSC sheets were implanted as control.At 16 weeks,the samples were processed for radiological analysis and histological examination.Results Cells in the BMSC sheets grew well.In the experimental side,the optical density of the samples was higher than that of the control side (P＜0.05) and plenty of lamellar bones and Haversian system were observed.Conclusions The formation of lamellar bones can be promoted by PLGA scaffolds and BMSC sheets in the process of tissue engineering bone reconstrution.
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[Objective]To investigate the effects of bone marrow stromal stem cells(MSCs) on the bone defect around the implant and the osteointegration of implant-bone interface.[Method]Fifteen healthy clean New Zealand rabbits were studied.MSCs were separated,cultured and induced to osteoblasts.Cancellous bone defects(0.6?1.2 cm)were created in bilateral femur condyle in rabbits.Titanium alloy columns(0.3?1.0 cm)were implanted into both defects.Tissue engineering bone was implanted around the left titanium alloy implant,but only hydroxyapatite ceramic in the right.The biological characteristics were evaluated by X-ray examination,SEM,energy dispersive X-ray analysis and histologic studies at 4,8,and 12 weeks postoperatively.[Result] X-ray examination showed the density of bone tissue around the implant was aequalis.The interspace between implant and bone was filled with lots of bone trabeculae in the experimental group at 12 weeks postoperatively.The density of bone tissue was inhomogeneous,and low-density umbra existed in the interspace in the control group.Energy dispersive X-ray analysis showed the content of the Ca and P in the experimental group was statistically higher than that in the control group at different time points(P0.05.The ratio of Ca to P became higher and reaching the peak level at 8 week.Histological study showed the bone defects around the implant were repaired by new mature bone in the left at 12 weeks postoperatively,while in the right no new bone was found at any time point.[Conclusion]The compounds of hydroxyapatite ceramic and osteoblasts induced by MSCs can repair the bone defect around the implant and improve the osteointegration of implant-bone interface.
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Aim To investigate the effects of osthol on bone marrow stromal stem cells in vitro under the con-ditions of the ability to differentiate into osteoblasts and the case of proliferation. Methods The rat bone marrow sample was obtained,and the all bone marrow cell culture methods were used to separate and collect the stratum of mononuclear cells. The cells were cultured in DMEM containing 10% fetal bovine serum. Three days later the culture medium was changed for the first time. Nine days later,serial subcultivation proceeded. The final concentration of osthol was 1 ? 10 -4,1 ? 10 -5,1 ? 10 -6,1 ? 10 -7 mol?L -1 respectively. MTT method was adopted in proliferation analysis. Under the induced condition,the Alkaline phosphatase activity, calcium salt sediment yield and osteocalcin were meas-ured on 4th,8th,12th,16th day. On the fifteenth day,his-tochemistry dyeing proceeded for calcified tubercle. Total RNA was isolated and the gene expression of bFGF, IGF-1,Osterix and Runx-2 was investigated by RT-Real Time PCR. Results The BMSCs proliferation was refrained by osthol dose-dependently. But it evidently led to osteogenesis. The ALP activity,calcium salt sediment yield and osteocalcin were raised,and calcified tubercle amount was increased. Besides,it also could enhance the mRNA level of bFGF,IGF-1,Osterix and Runx-2. Conclusion The osthol with final concentra-tion of 1 ? 10 -5 mol?L -1 can markedly promote BM-SCs differentiation to osteogenesis. which proves osthol is an active constituent of the traditional Chinese medi-cine Common Cnidium Fruit.
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Objective: To study the effects of tissue engineered bone in the repair of mandibular defect.Methods:The experiments were conducted in 12 dogs.Bone marrow stromal stem cells(BMSCs) of dog were cultured in DMEM containing 100 ml/L fetal bovine serum and induced to differentiate towards osteoblasts.Then the cells were seeded onto absorbable polylactic acid(PLA) compounded with rhBMP-2(0.16 mg for each implant), the composite was implanted into the oval-shaped mandibular bone defect in the size of 30 mm?12 mm on one side,another side was used as blank control.The dogs were divided into 4 grups with 3 in each group.PLA/rhBMP/BMSCs,PLA/rhBMP,PLA/BMSCs and PLA were used as the implants in group A,B,C and D respectively. 2, 4 and 8 weeks after implantation,the effectiveness of bone formation was evaluated by means of gross observation,histological and scanning electronic microscope ( SEM) examination. Results: In group A new bone formation in the implanted defects was observed 4 weeks after operation, the defects were replaced by muture bone tissue 8 weeks after operation. In group B a little new bone was found in the implanted area 4 weeks after operation and fibrous bone was observed 8 weeks after operation. In group C chondral ossification was found and in group D fibrous tissue was found in the bone defects 8 weeks after operation.Conclusion:PLA/rhBMP/BMSCs may be feasible in the repair of bone defect.