The Use Of SHP-2 Gene Transduced Bone Marrow Mesenchymal Stem Cells To Promote Osteogenic Differentiation And Bone Defect Repair In Rat

OBJECTIVES Bone tissue exhibits a powerful regeneration capacity.However,bone repair is severely limited when large bone defects occurs.In bone tissue engineering,growth factor is an important medium to induce osteogenesis and has been widely used in clinical and scientific research with the scaffold.At present the commonly used growth factors includs bone morphogenetic protein,vascular endothelial growth factor,transforming growth factor and so on.But they are is difficult to meet the needs of the development of bone tissue engineering for their shortcomings and disadvantages,.And we,through literature research found that there are a number of studies reported SHP-2 gene can effectively promote osteoclast differentiation,and are involved in the osteogenic cell differentiation pathways,and the gene knockout mice appeared bone stunting disorder,so we predict that the gene can promote osteoblast differentiation and bone formation,and try to confirm the hypothesis by experiment.METHODS We predicted that overexpression of SHP-2 could promote bone marrowderived mesenchymal stem cell(BMSC)osteogenic differentiation and SHP-2 transduced BMSCs could enhance new bone formation,determined using the following study groups:(1)BMSCs transduced with SHP-2 and induced with osteoblast-inducing liquid(BMSCs/SHP-2/OL);(2)BMSCs transduced with SHP-2(BMSCs/-SHP-2);(3)BMSCs induced with osteoblast-inducing liquid(BMSCs/OL)and(4)pure BMSCs.Cells were assessed for osteogenic differentiation by quantitative real-time polymerase chain reaction analysis,western blot analysis,alkaline phosphatase activity and alizarin red S staining.For in vivo assessment,cells were combined with beta-tricalcium phosphate scaffolds and transplanted into rat calvarial defects for 8 weeks.Following euthanasia,skull samples were explanted for osteogenic evaluation,including micro-computed tomography measurement,histology and immunohistochemistry staining.RESULTS Quantitative real-time PCR(qPCR)analysis and Western blot analysis results indicate that important osteogenic factors(ALP,OCN,Runx2)were significantly unregulated by overexpression of SHP-2 in BMSCs.Quantification analysis showed that ALP activity and calcium nodule formation in the BMSCs/SHP-2/OL group was at least 3.4-fold greater than in the BMSCs group.these data indicate that SHP-2 transduced BMSCs effectively promoted new bone formation and the radiographic results indicate that SHP-2 transduced BMSCs promoted new bone formation in ?-TCP scaffolds.Micro-CT measurement indicated that SHP-2 transduced BMSCs effectively promoted new bone formation and the radiographic results indicate that SHP-2 transduced BMSCs promoted new bone formation in ?-TCP scaffolds.Immunohistochemical staining revealed a similar result to H&E and Masson staining with positive expression of OCN and OPN being largely observed in the BMSCs/SHP-2/OL group H&E and Masson trichrome staining of tissue slices confirmed that although new bone formation was observed in all experimental groups,greater bone infill was observed in the BMSCs/SHP-2/OL group.CONCLUSIONS Our study demonstrates that SHP-2 promoted osteogenic differentiation of BMSCs in vitro and that SHP-2 transduced BMSCs combined with ?-TCP scaffolds effectively promoted bone regeneration in critical-sized calvarial defects in rats.This study provides evidence for the use of SHP-2 as a potential growth factor for enhanced bone tissue engineering.