Jaw Defects Of Mini Pig Repaired By Gingival Stem Cell Induced With SB431542

[Objective]To explore the feasibility of gingival mesenchymal stem cells(GMSCs)as tissue engineering seed cells in reconstruction of maxillofacial region large bone defect,and the effect of TGF-? inhibitor SB431542 on proliferation and osteogenic differentiation of GMSCs in vivo and in vitro.[Methods]GMSCs were isolated and cultured in vitro from gingival tissues resected in the stage II surgery of implants or in the surgery of the third molar extraction.The expression of surface markers of mesenchymal stem cells(CD73,CD90)and hematopoietic stem cells(CD34,CD45),the ability of osteogenic differentiation were evaluated by flow cytometry(FCM)to biologically identify mesenchymal stem cells.The proliferation ability and effect of 1?M SB431542 on the proliferation of GMSCs were detected by CCK-8 proliferation assay.The cells cultured in this experiment were randomly divided into 3 groups:the blank group cells were cultured with mesenchymal stem cell medium,the control group cells were cultured with osteogenic induction medium,and the experimental group cells were cultured with osteogenic induction medium supplemented with 1 ?M SB431542 for osteogenesis.After 21 days of 3 groups culturing in vitro,mineralized nodule formation was examined by alizarin red staining.Photographs were taken and were graphic analyzed by Image-Pro Plus 6.0.Data analysis was also conducted by GraphPad Prism 7.Osteogenic-related gene expression of alkaline phosphatase(ALP),osteocalcin(OCN),Runt-related transcription factor-2(Runx2),Osteopontin(OPN)and COL-I,was examined by Real-Time Polymerase Chain Reaction(RT-PCR).Data analysis was conducted byGraphPad Prism 7.Each mini pig has 4 groups in vivo:golden standard group(replantation of bone block removed by ring drill),Bio-Oss group(implantation of Bio-Oss bone material),GMSCs group(implantation of Bio-Oss bone material mixed with pig GMSCs),SB group(implantation of Bio-Oss bone material mixed with pig GMSCs induced by SB).In operation,4 bone defect areas(diameter of 12mm,depth of 5mm)were prepared on maxilla of each mini pig by ring drill and the space between each defect area was 8mm.4 groups of bone materials were respectively implanted into 4 bone defect areas and covered by heal-all membrane.2 months after operation,the mini pigs were executed and samples were taken from bone regeneration areas.Micro CT,CBCT,HE staining was conducted to observe and analysis osteogenesis in vivo and to evaluate the effect of SB431542 on proliferation and osteogenic differentiation of GMSCs.[Results]Primary cells were obtained by improved tissue block method.MSCs were observed to adherent after passaging.The cells were basically the same size,with a long spindle shape,a homogeneous cytoplasm,a round nucleus in the middle of the cell and a typical fibroblast state,and the proliferation rate is fast.They had the ability of colony formation and the characteristics of adult stem cells.FCM showed that primary cultured DMSCs expressed surface markers CD73(99.4%),CD90(99.7%)positively,hematopoietic stem cells expressed surface markers CD34(0.2%),CD45(0.1%)negatively,which means that it conformed to the feature that the stem cells were the source of mesenchymal.CCK-8 assay showed that there was no significant difference in proliferation between GMSCs supplemented with ?M SB431542 and control group(P>0.05).Alizarin red staining showed that the mineralized nodules in the experimental group were higher than control group and there was significant difference(P<0.05).The expression of ALP,OCN,Runx2,OPN and COL-I in experimental group was higher than that in control group and there was significant difference(P<0.05).CBCT and Micro CT images showed that all bone defect areas had different degrees of bone regeneration.Bone density(BD)showed that golden standard group was closest to normal bone tissue and the BD of SB group was higher than GMSCs group.The Bio-Oss group had the worst osteogenesis effect.HE staining showed that newly formed bone-like tissue in golden standard group were close to normal bones;there was big new bone-like tissue in the SB group and a small amount of new bone-like tissue in the GMSCs group;the Bio-Oss group showed homogenous red stained bone matrix;the SB group showed more mineralized nodules compared with the GMSCs group and Bio-Oss group.[Conclusions]By using in vitro proliferation,osteogenic differentiation staining,evaluation of osteogenic gene expression level,HE staining in vivo,micro CT and CBCT,this study suggested that the small molecule drug SB431542,specific inhibitor of TGF-?pathway,could significantly improve the osteogenic potential of gingival mesenchymal stem cells in vitro and in vivo,yet have no evident influence on proliferation.It suggested that gingival mesenchymal stem cells can be a reliable source of tissue engineering seed cells while it can provide a new idea for the tissue engineering regeneration.