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BACKGROUND:Piezo1,a mechanosensitive protein,is tightly connected to osteogenic differentiation,and it has been demonstrated that TAZ has a role in regulating osteogenic differentiation.It is unclear whether TAZ participates in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells by Piezo1,so it is crucial to investigate its unique mechanism to prevent osteonecrosis of the femoral head. OBJECTIVE:To elucidate what function Piezo1 plays in osteogenic differentiation and TAZ expression in human bone marrow mesenchymal stem cells. METHODS:The siRNA targeting Piezo1 was constructed and transfected into 293T cells.The silencing efficiency was detected by RT-qPCR.The selected Piezo1-Home-2337 was packaged according to the silencing efficiency,and its optimal multiplicity of infection value was assayed by immunofluorescence staining.The packaged Piezo1 silencing recombinant lentivirus was transfected into human bone marrow mesenchymal stem cells,and its silencing effect was detected by RT-qPCR and western blot assay.Alizarin red staining,alkaline phosphatase activity analysis,immunofluorescence staining,RT-qPCR and western blot assay were utilized to analyze the effect of silencing Piezo1 on the osteogenic differentiation of human bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION:(1)The mRNA and protein levels of Piezo1 in human bone marrow mesenchymal stem cells transfected by si-Piezo1 were decreased significantly,with a statistically significant difference compared with normal and negative control groups.(2)The alkaline phosphatase activity in the si-Piezo1 group was much lower and the calcium deposition in the si-Piezo1 group was significantly reduced compared with the negative control group.(3)The mRNA levels of osteogenesis-related genes including Runt-related transcription factor 2(Runx2),osteopontin(OPN),distal-less homeobox 5(DLX5),osteocalcin,β-catenin and Tafazzin(TAZ)in the si-Piezo1 group were significantly decreased compared with the negative control group.Afterward,the expression levels of TAZ and β-catenin protein in the si-Piezo1 group were down-regulated significantly compared with the negative control group,whereas the expression levels of p-TAZ and p-β-catenin protein in the si-Piezo1 group had the opposite condition.(4)The results of immunofluorescence staining showed that the expression of TAZ and β-catenin in human bone marrow mesenchymal stem cells in the si-Piezo1 group was less compared with the negative control group.(5)These findings indicate that Piezo1 can promote the osteogenic differentiation of human bone marrow mesenchymal stem cells.The osteogenic ability of human bone marrow mesenchymal stem cells is significantly reduced after silencing Piezo1,and the expression of TAZ is also reduced.
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BACKGROUND:Studies have found that activation of nuclear factor-erythroid 2-related factor 2/heme oxidase-1(Nrf2/HO-1)pathway can alleviate oxidative stress caused by cerebral ischemia-reperfusion injury,but whether human bone marrow mesenchymal stem cells(hBMSC)can activate Nrf2/HO-1 pathway to alleviate cerebral ischemia-reperfusion injury is still lacking relevant studies. OBJECTIVE:To investigate whether intracranial transplantation of hBMSC alleviates oxidative stress injury in cerebral ischemia-reperfusion animal models by activating Nrf2/HO-1 pathway. METHODS:Totally 40 male SPF SD rats were randomly divided into sham operation group,model group,hBMSC transplantation group,hBMSC+solvent group and hBMSC+Nrf2 inhibitor group.Each group consisted of eight animals.In the model group and the hBMSC transplantation group,middle cerebral artery occlusion model was prepared by thread embolization method.The thread embolization was removed 1 hour later,and 30 μL PBS or hBMSC cultured to at least passage 5 was injected into the right cortex and striatum of rats.In the hBMSC+Nrf2 inhibitor group and hBMSC+solvent group,the left ventricle was injected with Nrf2 inhibitor Brusatol and its solvent dimethyl sulfoxide respectively 24 hours before model establishment,then the middle cerebral artery occlusion model was prepared,and hBMSC was injected.Relevant indexes were detected 3 days after transplantation. RESULTS AND CONCLUSION:(1)CT and TTC staining showed the same area and volume of cerebral infarction:model group>hBMSC+Nrf2 inhibitor group>hBMSC+solvent group>hBMSC transplantation group>sham operation group.(2)Hematoxylin-eosin staining and Nissl's staining showed that the ischemic brain tissue was intact and the neurons were normal in the sham operation group.Compared with the model group,the pathological morphology and neuronal injury of the hBMSC transplantation group and the hBMSC+solvent group were significantly improved.Compared with the hBMSC+solvent group,the hBMSC+Nrf2 inhibitor group had more serious pathological morphology and neuronal damage.(3)Western blot assay and oxidative stress index detection results showed that compared with the sham operation group,Nrf2 and HO-1 proteins were decreased(all P<0.05),malondialdehyde was increased and superoxide dismutase was decreased(all P<0.05)in the model group.Compared with the model group,the expression levels of Nrf2 and HO-1 proteins were increased(all P<0.05),malondialdehyde was decreased and superoxide dismutase was increased(all P<0.05)in the hBMSC transplantation group and the hBMSC+solvent group.Compared with the hBMSC+solvent group,the expression levels of Nrf2 and HO-1 proteins were simultaneously decreased(all P<0.05),and malondialdehyde was increased and superoxide dismutase was decreased(all P<0.05)in the hBMSC+Nrf2 inhibitor group.(4)These results indicate that hBMSC can alleviate cerebral ischemia-reperfusion injury possibly by activating Nrf2/HO-1 pathway.
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OBJECTIVE@#To examine the morphology and biocompatibility of a native acellular porcine pericardium (APP) in vitro and to evaluate its barrier function and effects on osteogenesis when used in guided bone regeneration (GBR) in vivo.@*METHODS@#First, the morphology of APP (BonanGenⓇ) was detected using a scanning electron microscope (SEM). Next, for biocompatibility test, proliferation of human bone marrow mesenchymal stem cells (hBMSCs) were determined using cell counting kit-8 (CCK-8) after being seeded 1, 3 and 7 days. Meanwhile, the cells stained with phalloidine and 4, 6-diamidino-2-phenylindole (DAPI) were observed using a confocal laser scanning microscopy (CLSM) to view the morphology of cell adhesion and pattern of cell proliferation on day 5. A 3-Beagle dog model with 18 teeth extraction sockets was used for the further research in vivo. These sites were randomly treated by 3 patterns below: filled with Bio-OssⓇand coverd by APP membrane (APP group), filled with Bio-OssⓇand covered by Bio-GideⓇmembrane (BG group) and natural healing (blank group). Micro-CT and hematoxylin-eosin (HE) were performed after 4 and 12 weeks.@*RESULTS@#A bilayer and three-dimensional porous ultrastructure was identified for APP through SEM. In vitro, APP facilitated proliferation and adhesion of hBMSCs, especially after 7 days (P < 0.05). In vivo, for the analysis of the whole socket healing, no distinct difference of new bone ratio was found between all the three groups after 4 weeks (P>0.05), however significantly more new bone regeneration was detected in APP group and BG group in comparison to blank group after 12 weeks (P < 0.05). The radio of bone formation below the membrane was significantly higher in APP group and BG group than blank group after 4 and 12 weeks (P < 0.05), however, the difference between APP group and BG group was merely significant in 12 weeks (P < 0.05). Besides, less resorption of buccal crest after 4 weeks and 12 weeks was observed in APP group of a significant difference compared in blank group (P < 0.05). The resorption in BG group was slightly lower than blank group (P>0.05).@*CONCLUSION@#APP showed considerable biocompatibility and three-dimentional structure. Performing well as a barrier membrane in the dog alveolar ridge preservation model, APP significantly promoted bone regeneration below it and reduced buccal crest resorption. On the basis of this study, APP is a potential osteoconductive and osteoinductive biomaterial.
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Animales , Perros , Humanos , Materiales Biocompatibles , Regeneración Ósea , Osteogénesis , Pericardio , Porcinos , Extracción Dental , Alveolo DentalRESUMEN
Objective: To explore the effects of the microenvironment of rabbit bladder acellular matrix graft (BAMG) on proliferation, cell surface markers, and molecular protein level of human bone marrow mesenchymal stem cells (hBMSCs). Methods: We prepared BAMG immersion fluid medium and detected its effect on the proliferation of hBMSCs by MTT method. The expressions of CD44, CD45, CD73 and PDGFRβ were detected by flow cytometry. The expressions of PPAR, OCN and α-SMA were detected by RT-PCR, and the expression of OCT was detected by Western blot. Results: hBMSCs had good compatibility with BAMG. The MTT method showed that BAMG and BAMG immersion medium did not affect the proliferation capacity of hBMSCs. The surface of hBMSCs cells cultured with immersion fluid still expressed CD44, CD73 and PDGFRβ, but not CD45. RT-PCR showed that OCN, PPAR, and α-SMA were all expressed. Western blot test also showed the positive expression of OCT-4. Conclusion: hBMSCs can still keep their original biological characteristics in the microenvironment of rabbit BAMG. It can be the seed cells and combined substrate materials for urinary system tissue engineering.
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Objective To investigate whether human bone marrow mesenchymal stem cells (hBC-MSCs) have the immune-suppression ability on the proliferation of peripheral blood mononuclear cells (PBMCs) and their pro-inflammatory cytokine production in rats,to explore what kinds of human cytokines are required for the induction of hBM-MSCs to become immune-suppressive,and to observe the effect of intravenous delivery of hBM-MSCs on tumor necrosis factor (TNF)-α transcription and expression in the core infarct areas of rats after cerebral ischemia.Methods The fetal-originated hBC-MSCs and rat PBMCs were extracted;the rat PBMCs were activated by adding 10 μg/mL concanavalin A (ConA).(1) The first experiment was divided into hBM-MSCs+PBMCs group,hBM-MSCs+PBMCs+ConA group,PBMCs group and hBM-MSCs group;CCK-8 assay was employed to detect the proliferation of these cells.(2) The second experiment was divided into hBM-MSCs+PBMCs+ConA group,PBMCs+ConA group;ELISA was used to detect the TNF-α,interferon-γ (IFN-γ) and intedeukin (IL)-10 expressions.(3) The third experiment was divided into hBM-MSCs+PBMCs (IFN-γ+IL-1α) group,hBM-MSCs+PBMCs (IFN-γ+IL-1β) group,hBM-MSCs+PBMCs (IFN-γ+TNF-α) group and hBM-MSCs+PBMCs group;CCK-8 assay was used to detect the proliferation of these cells.(4) Thirty SD rats were randomly divided sham-operated group,control group (giving normal saline after ischemia) and hBM-MSCs group (giving hBM-MSCs after ischemia,n=10);on the third d of ischemia,the TNF-α mRNA and protein expressions at the infarct areas was detected by real time PCR and Western blotting,respectively.Results (1) The optical density (OD) in the hBM-MSCs+PBMCs group was significantly increased as compared with that in the PBMCs group and hBM-MSCs group (P＜0.05);OD in the hBM-MSCs+PBMCs group was significantly increased as compared with that in the hBM-MSCs+PBMCs+ConA group (P＜0.05).(2) The TNF-α,IFN-γand IL-10 levels in the PBMCs+ConA group were (1030±196) pg/mL,(2880±250) pg/mL and (330±45) pg/mL;the TNF-α and IFN-γlevels in hBM-MSCs+PBMCs+ConA group were (160±10) pg/mL and (240±55) pg/mL,which were significantly lower than those in the PBMCs+ConA group (P＜0.05);the IL-10 level in hBM-MSCs+PBMCs+ConA group was (750±110) pg/mL,which was significantly higher than that in the PBMCs+ConA group (P＜0.05).(3) The OD in the hBM-MSCs+PBMCs(IFN-γ+IL-1α)group,hBM-MSCs+PBMCs (IFN-γ+IL-1 β) group and hBM-MSCs+PBMCs (IFN-γ+TNF-α) group was significantly decreased as compared with that in the hBM-MSCs+PBMCs group (P＜0.05).(4) The TNF-α mRNA expression in the sham-operated group,control group and hBM-MSCs group was 0.490±0.128,2.369±0.788 and 1.002±0.408;the TNF-α protein expression in the sham-operated group,control group and hBM-MSCs group was 0.144±0.028,0.314±0.029,0.240±0.029;the TNF-α protein and mRNA expressions in the hBM-MSCs group were significantly decreased as compared with those in the control group (P＜0.05).Conclusions The allogeneic transplantation of hBC-MSCs is competent in suppressing the inflammation of rats in vitro and in vivo.Furthermore,this immune-suppression ability is not innate,but cytokine stimulation dependent.The immune-suppression ability of hBM-MSCs on rat PBMCs are at least partly responsible for the therapeutic effect of hBM-MSCs transplantation into the rat models,such as ischemia stroke.