Promotion Of Osteogenic Differentiation Through Lentivirus-mediated Smad6Gene RNA Interference In Rat Bone Marrow Mesenchymal Stem Cells

Background: The repair of bone defects is one of the most common problems toorthopedic surgery. In recent years, with the development of tissue engineering technology,which offers possible to repair bone defect. The ideal bone tissue engineering mustpossess the following three elements: the seed cells, cytokines and scaffolds. Bone marrowmesenchymal stem cells (BMSCs) are adult stem cells with multi-differentiation potential.Thay can differentiate into bone cells, fat cells, and cartilage cells under different inducingconditions.. BMSCs are the most commonly used seed cells for bone tissue engineeringbecause of relatively easy to obtain from the bone marrow and in vitro amplificationtechnology is relatively mature. The osteogenic differentiation process of BMSCssubject to the regulation of several signaling pathways, the BMP signaling pathway is oneof the most studies signaling pathway. BMPs signaling pathway can be reinforced throughthe activation of cell surface BMP receptors (BMPR). The activated BMPR then activates avariety of Smads proteins and conducts signal in the cells. Up to now, at least nine Smadproteins have been found. They can be divided into receptor modulators Smads (R-Smads,Smad1,2,3,5,8,9), co-regulated Smads (Co-Smads, Smad4), and inhibitory Smads(I-Smads, Smad6,7) according to the functions. The I-Smads, including Smad6and Smad7,plays a negative feedback regulation in BMP/TGF-β signaling pathway. Studies haveshown that Smad6and Smad7selective blocking effect on TGF-and BMP signalingpathways: Smad7have blocking effects on both TGF-β and BMP pathways, but Smad6seems to block the BMP signaling pathway only. Therefore, we believe that endogenousSmad6can suppress osteogenic differentiation of BMSCs. Objective: First, we design,build and screen out the effective lentiviral RNA interference (RNAi) carrier according tothe rat Smad6gene. Then, the rat BMSCs were cultured in vitro and transfected withrecombined lentiviral RNAi carrier. The interference efficiency of Smad6gene and proteinof the BMSCs were observed after infection, and the biological behavior of BMSCs wasobserved while Smad6gene was inhibited. Then the osteogenic differentiation of Smad6 gene interferenced BMSCs was evaluated in vitro. Finally, we constructed tissueengineering bone with the gene modified BMSCs and valuated the effects of restoration therat radial bone effect. Methods:(1) Construction of Smad6gene RNAi lentiviral vector:Two pairs of shRNA fragment were designed and synthesized according to rat Smad6gene,then connected to pLKO.1plasmid, transformed E. coli. The plasmid were identified withagarose gel electrophoresis and gene sequencing. Finally lentiviral RNAi vector werepackaged in HEK-293T cells. In the meantime negative control lentiviral vector wasconstructed.(2) Screening of the Smad6gene lentiviral vector: The rat BMSCs wereseparated and cultured in vitro, and added a different number of lentiviral particlesovernight infection according to the gradient dilution method. The multiplicity of infection(MOI) were decided by achieve an effective infection efficiency (80%) the lowest viralload. The rat BMSCs were infected with recombinant lentiviral vector in accordance withthe MOI. The inhibition efficiency of Smad6gene and protein were detected byquantitative PCR and Western-Blot. The proliferation were observed by drawing a cellulargrowth curve and MTS.(3) Osteogenic differentiation of gene modified BMSCs in vitro:BMSCs were divided into the following two groups: shSmad6group and negativecontrol(NC) group. The BMSCs were cultured in osteoggenic medium withdexamethasone, β-glyceronephosphate, and L-ascorbic acid. Detection methods includealkaline phosphatas(eALP)staining, alkaline phosphatase quantitative detection,alizarinred staining, Von Kossa staining RT-PCR and quantitative PCR detection of osteogenicgene expression (including Runx2, ALP, bone sialoprotein, and osteocalcin).(4) The effectof genetically modified BMSCs repair rat radial bone defect: Thirty SD rats were dividedinto three groups: shSmad6+β-tricalcium phosphate (β-TCP) group (n=10), NC+β-TCPgroup (n=10), and β-TCP group (n=10). The observation methods including: X-rayobservation, HE histological examination and bone histomorphometry analysis. Results:(1)Two pairs of shRNA oligonucleotides were designed and synthetized according to the ratSmad6gene, and successfully connected to the pLKO.1carrier. The right recombinedvector was identified by gene sequencing. The recombinant shRNA lentiviral particles were packaged in in HEK-293T cells.(2) After the rat BMSCs were infected withrecombinant lentivirus, the endogenous Smad6gene and protein were significantly reducedby quantitative PCR and Western-Blot detection. The results showed the effective shSmad6lentiviral vector was successfully constructed. Further observed by drawing a cellulargrowth curve and MTS found that the cell proliferation wasn't substantiallyaffected.(3)The osteogenic differentiation experiments in vitro showed that alkalinephosphatase expression of shSmad6group was significantly higher than that of the NCgroup (p <0.05), and the calcium nodules formation of shSmad6group was significantlymore than that of NC group. The Runx2, ALP, bone sialoprotein (BSP), and osteocalcin(OCN) gene expression of shSmad6group was significantly higher than that of the NCgroup (p<0.05).(4) The repair rat radius bone defects in vivo:①The X-ray results:Twelve weeks after surgery,8of10bone defect was repaired in theshSmad6+β-TCP group and3of10bone defect in the shSmad6+β-TCP group. While inthe the beta-TCP group there have no obvious bone healing. The resultsof X-ray score showed that shSmad6+β-TCP group was significantly higher than that ofthe NC+β-TCP group (P<0.05).②histology and histomorphometry analysis: A largenumber of new bone was formated12weeks after surgery and most of the bone defectswere repaired in shSmad6+β-TCP group. While in the NC+β-TCP group, the quantityof new bone callus was less than that of shSmad6+β-TCP group. Newborn trabecularbone area measurement showed that the quantity of new bone callus of shSmad6+β-TCPgroup was significantly higher than that of NC+β-TCP group (P <0.05). Conclusion: Therecombinant lentiviral RNAi vector was successfully designed and constructed accordingto rat Smad6gene. The inhibitory efficiency to rat BMSCs endogenous Smad6was morethan95%. After the endogenous Smad6gene of BMSCs been restrained, the osteogenicdifferentiation of BMSCs were significant increased in vitro and the efficiency of repairbone defects was enhanced significantly in vivo.