Schnurri-3 Participate In Regulating BMP-induced Osteogenic Differentiation And Angiogenesis Of Human Amniotic Mesenchymal Stem Cells

Background/Aims: At present,the role of blood vessel formation in the construction of bone tissue engineering has attracted more and more attention,but in the construction of bone tissue engineering,the molecular mechanism between blood vessel formation and osteogenic differentiation has not yet been elucidated.Human amniotic mesenchymal stem cells(hAMSC)are a type of pluripotent stem cells derived from the surface of human waste placenta.They have the ability to differentiate into osteoblasts,chondrocytes and adipocytes.HAMSCs also Has good angiogenic capacity;Schnurri-3(Shn3),also known as human immunodeficiency virus type I enhancer binding protein 3(HIVEP3),belongs to the BMP / TGF-?homolog family.Due to the lack of Shn3 in osteoblasts,the level of Runx2 protein is increased to enhance bone formation;bone morphogenetic protein 9(Bone morphogenetic protein 9)has a significant upregulation of various mesenchymal stem cells(MSCs).The expression of osteogenesis-related factors during bone differentiation can also stimulatethe expression of angiogenesis-related factors.However,it is unclear whether Shn3 can regulate and how BMP9-induced hAMSCs induce osteogenic differentiation and angiogenesis in vivo and in vitro.Based on this,we investigate whether Shn3 is involved in regulating the effects of BMP9 on osteogenic differentiation and angiogenesis in vivo and in vitro,and its signaling pathways and molecular mechanisms.Methods:(1)Pancreatin / collagenase combined digestion method was used to isolate and culture hAMSCs,and the phase change microscope was used to observe the morphological changes;the third-generation hAMSCs were used to detect the cell surface molecular markers by flow cytometry(FCM)Expression;Immunofluorescence staining was used to identify the expression of vimentin and CK-19 in hAMSCs,and to detect the differentiation potential of third-generation hAMSCs into osteoblasts,cartilage and adipocytes.(2)By constructing recombinant adenovirus(Ad)exogenously overexpressing BMP9(Ad-BMP9),Shn3(Ad-Shn3)and silent Shn3adenovirus(Ad-Sim-Shn3)and red fluorescent protein(Red fluorescent protein)protein,RFP)(Ad-RFP)after transfecting the third-generation hAMSCs,observe the transfection efficiency,and use qRT-PCR to detect the expression of Shn3 gene in cells;use alkaline phosphatase staining and biochemical quantitative detection At 5 and 7 days,Shn3 had an early osteogenic differentiation effect on BMP9-induced third-generationhAMSCs;alizarin red S staining and bone mineralization were used to quantitatively observe the late stages of Shn3 on BMP9-induced third-generation hAMSCs on days 14 and 21.The role of bone mineralization,and qRT-PCR was used to detect Runx2(Runt related transcription factor 2),bone sialoprotein(BSP),type I collagen Collagen type I(COL-I),and mRNA expression levels of OSX(Osterix),a transcription factor associated with bone formation.(3)Through the use of exogenous recombinant adenovirus overexpression or silent Shn3 gene transfection technology,set up BMP9 group,Sim-Shn3 group,BMP9 + Sim-Shn3 group and BMP9 + Shn3 group,and transfect the third-generation hAMSCs in After 24 h,the cells were injected into the skin of nude mice to form a model of heterotopic ossification in nude mice.The ectopic bone block samples were collected at about 4 weeks,and the bone block scans were analyzed in parallel by Micro-CT(?-CT).Fixation,embedding and section staining of bone blocks were performed.Masson's Trichrome trichrome staining,H & E staining(hematoxylin and eosin staining)were used to analyze the number and distribution of bone trabeculae,and Safranin O-fast staining green staining)to observe the formation of cartilage in heterotopic bone formation.Immunohistochemical staining was used to observe the ectopic bone fragments of each experimental group.The main indicators of ectopic bone fragments including OCN and osteopontin(OPN)and angiogenesis-relatedmolecules such as angiopoietin-1(Angiopoietin 1,ANGPT1)and vascular endothelial growth factor(Vascular endothelial growth factor,VEGF)in ectopic bone mass and distribution.(4)The expression and distribution of VEGF,the third-generation hAMSCs vascular differentiation indicator induced by BMP9,after overexpressing or silencing Shn3 adenovirus transfected cells were observed using cellular immunohistochemistry.After transfecting BMP9-induced third-generation hAMSCs with overexpression or silenced Shn3 adenovirus,they were co-cultured with human umbilical vein endothelial cells(HUVECs)in the Transwell chamber,with adenovirus-transfected hAMSCs in the upper,HUVECs cells are in the lower layer,using Matrigel to lay on the bottom of the plate,to observe the tubule forming ability of HUVECs cells,introduction to detect the overexpression or silence of Shn3 on BMP9-induced hAMSCs angiogenesis ability(5)Using electrospun poly(lactic-co-glycolic acid,PLGA)copolymer scaffold material,after overexpressing or silencing Shn3 adenovirus transfected with BMP9-induced third-generation hAMSCs and other groups After the cells were mounted on the PLGA scaffold material and cultured for 24 hours,the scanning electron microscope(SEM)was used to observe and analyze the scaffold material mechanism and the cell growth after the cell was mounted.The transfected adenovirus was mounted byusing the above scaffold material After culturing the cells in the experimental groups for 48 h,the PLGA-cell compl00 ex was transplanted into the subcutaneous tissue of the mouse back.After 5 weeks,the subcutaneous graft samples of the mouse back were collected,and the samples were observed in general.The growth of blood vessels in the body,and then immunofluorescence staining of the graft samples to observe the expression and distribution of von Willebrand Factor(vWF)in the graft.(6)Western blot was used to detect the protein expression levels of OCN,OPN,and Runx2,which are related to osteogenic differentiation of the third-generation hAMSCs induced by BMP9 overexpression or silencing of Shn3 adenovirus;Western blot detection of BMP / Smads classic signal(Smad-1 / 5/8)after overexpression or silence of Shn3 adenovirus transfected with BMP9-induced third-generation hAMSCs;expression was detected by chromatin immunoprecipitation(Chromatin immunoprecipitation,ChIP)experiment was used to detect the direct effects of overexpression or silence of Shn3 adenovirus on BMP9-induced third-generation hAMSCs on osteogenic transcription factors Runx2 and VEGF.Results:(1)Using pancreatin and type II collagenase combined digestion method can obtain a large number of hAMSCs with uniform morphology at one time;after passage of cells to the third generation of hAMSCs,hAMSCs can stably express vimentin,while low expression ofCK-19;Flow cytometry 3rd generation hAMSCs cell surface molecular markers conform to the phenotype identified by MSCs.The third-generation hAMSCs have the potential to differentiate into osteoblasts,cartilage and adipocytes.(2)Exogenous overexpression of recombinant adenovirus BMP9(Ad-BMP9),Shn3(Ad-Shn3)and silent Shn3 adenovirus(Ad-Sim-Shn3)and red fluorescent protein(Ad-RFP)successfully transfected the third After substituting hAMSCs,and qRT-PCR results showed that Shn3 peaked around 72 h.ALP staining and alkaline phosphatase activity results showed that exogenous overexpression of Shn3 attenuated BMP9-induced early osteogenic differentiation of hAMSCs on days 3,5,and 7,while silencing Shn3 expression enhanced BMP9-induced early development of hAMSCs.Bone differentiation effect;alizarin red S staining and quantitative results of bone mineralization suggest that exogenous overexpression of Shn3 inhibited BMP9-induced late bone mineralization and calcium nodule formation in hAMSCs on days 14 and 21;qRT-PCR results It was shown that on day 7,exogenous overexpression of Shn3 down-regulated BMP9-induced hAMSCs osteogenic differentiation-related factors including Runx2,BSP,COL-I and OSX mRNA expression.(3)On the basis of the above-mentioned Shn3 inhibiting BMP9-induced hAMSCs osteogenic differentiation in vitro,further study the effect of Shn3 on BMP9-induced osteogenic differentiation ofsubcutaneous heterotopic bone in nude mice.Exogenous recombinant adenovirus overexpression or silenced Shn3 gene transfected with BMP9-induced third-generation hAMSCs cells in nude mice ectopic osteogenesis model,gross observation results and micro-CT results showed that compared with the BMP9 group,BMP9 + Shn3 group ectopic osseous bone volume decreased.Compared with other groups,the BMP9 +Sim-Shn3 group had stronger ectopic osteogenesis ability.Micro-CT analysis results show that exogenous Shn3 expression increases the average bone density of BMP9-induced ectopic bone mass in hAMSCs.Conversely,exogenous overexpression Shn3 decreases the average bone density of BMP9-induced ectopic bone formation in hAMSCs.Quantitative analysis of bone tissue morphology revealed bone volume / total volume(BV / TV),trabecular number(Tb.N),trabecular thickness(Tb.Th)and Bone mineral density(BMD)increased significantly in the BMP9 + Sim-Shn3 group compared to the BMP9 group.On the contrary,compared with the BMP9 group,the above parameters of the BMP9 + Shn3 group decreased to varying degrees,but the bone was small There is no significant difference in beam separation in each group.Histochemical staining results showed that compared with BMP9 group,Sim-Shn3 can increase the number of trabecular bone and the formation of bone matrix in BMP9 induced heterotopic osteogenesis.Exogenous overexpression of Shn3 inhibited BMP9-induced bone matrix formation,and immunohistochemical stainingshowed that silencing Shn3 gene expression can enhance BMP9-induced ectopic ossification-related osteogenic factors OCN and OPN and angiogenesis-related factor ANGPT1 And VEGF expression,but exogenous overexpression of the Shn3 gene suppresses this effect.(4)The results of cell immunohistochemical staining showed that exogenously silenced Shn3 expression enhanced the expression of VEGF-related indicators of vascular differentiation of hAMSCs induced by BMP9 on the 7th day;the results of immunofluorescence staining showed that HUVECs cells expressed endothelial mucin(Endomucin),EMC31),CD31,VEGF,and vWF molecules;the use of Transwell co-culture with HUVECs cells to induce tubule formation experiments also showed that exogenously silenced Shn3 expression enhances the ability of BMP9-induced hAMSCs to indirectly form tubules.(5)After overexpression or silencing of Shn3 adenovirus transfected with BMP9-induced third-generation hAMSCs and other groups of cells were mounted on PLGA scaffold materials and cultured for 24 hours,the results of scanning electron microscopy showed that the cells were in PLGA electrospinning scaffolds The growth condition is good,most cells can adhere to the wall;PLGA-cell complex transplanted into the subcutaneous angiogenesis test sample of mice,the general observation results show that silencing the expression of the Shn3 gene can enhance BMP9-induced subcutaneous blood vessel growth,and the sample'simmunofluorescence The staining results suggest that silencing the expression of Shn3 gene can enhance the expression of vWF,a BMP9-induced angiogenesis-related molecule,compared with other groups.(6)Western blot results showed that on the 7th day,compared with other groups,silencing the expression of Shn3 gene could enhance the expression of BMP9-induced hAMSCs osteogenic differentiation related protein levels including OCN,OPN and Runx2;exogenous The expression of BMP9 enhanced the phosphorylation level of Smad1 / 5/8,while Sim-Shn3 significantly enhanced the phosphorylation expression of Smad1/ 5/8 induced by BMP9.ChIP analysis results showed that the transcription factor Runx2 can bind to the VEGF promoter region in hAMSC induced by BMP9.The results of immunoprecipitation and immunoblotting showed that exogenously silenced Shn3 expression can enhance the activity of transcription factor Runx2 in BMP9-induced cells to its downstream molecule VEGF promoter,proving the direct interaction between transcription factor Runx2 and VEGF molecule.Conclusion:(1)Human amniotic mesenchymal stem cells have the basic phenotype of MSCs,and have the ability to differentiate into multiple lines,including the potential of osteoblasts,cartilage and adipocytes.(2)Exogenously silenced Shn3 gene expression can enhance BMP9-induced early osteogenic differentiation and late bonemineralization of hAMSCs,while exogenous overexpression of Shn3 gene expression weakens MP9-induced early osteogenic differentiation and late bone mineralization of hAMSCs Change.After exogenously silenced Shn3 expression,it can enhance BMP9-induced angiogenesis of hAMSCs in vitro.(3)By silencing the expression of Shn3 gene,it can promote the early and late osteogenic differentiation and blood vessel formation induced by BMP9 in vivo and in vivo,which may be mediated by enhancing BMP /Smads signal,and Shn3 is involved in regulating BMP9 In the two-way effect of induced hAMSCs osteogenic differentiation and angiogenesis,Shn3 plays an important role in regulating the osteogenic transcription factor Runx2 and activating its downstream molecule VEGF to promote BMP9-induced hAMSCs osteogenesis and angiogenesis.