Study Of The Promotion Of Osteogenic Differentiation From Adipose-derived Stem Cells Modified By VEGF Gene

IntroductionBone repair has been a basic problem in Orthopaedics. In fact, it is a very troublesome disease of deficient in bone defects and Bone nonunion associated with the process of bone repair and bone formation. There are two procession involved in bone formation that osteoblast differentiation factor induced osteoblastic differentiation of BMSCs and oseoblastic bone regeneration. It has been reported that osteoblast differentiation factors included BMP, IGF, FGF, PDGF etc, and clarity their signal transduct pathway. Vascular endothelial growth factor, VEGF promoted proliferation of the endothelial cells and angiogenesis. Thereby it is useful for enhancing blood supplement to bone fracture region. VEGF applied might provide a new pathway for bone fracture in the clinical treatment. Adipose-derived stem cells (ADSCs) has been worked as seeding cells for clinical application to treat bone defects, osteoporosis and bone facture. In addition, ADSCs also worked as gene vector to deliver many kinds of bone growth inducing factor to stimuli bone growth and accelerated bone repair processes. It has been reported that hypoxia-prestimulated bone marrow stromal cells are capable of differentiating into osteoblasts in vitro. We guesses whether that ADSCs modified by VEGF gene carried out double functions of bone formation and angiogenesis in vivo. In this study, Adenoviral vector were used to deliver VEGF gene into ADSCs and mechanism of otseoblastic differentiation from ADSCs were observed. These results are cable to provide experimental data for construct od artificial bone to treat bone defects.Material and MethodsRecombinant adenoviral vector with VEGF were constructed using the second generation adenoviral vector constructed system, pAdEasy-1. Human VEGF gene were inserted pAdshutle vector by enzyme cut and ligation. Positive clonies were screened and proliferation. Recombinant plasmids were isolated and cotransformed into E. coli BJ5183, and then kanamycin resistant recombinant plasmids were transfected into 293 cells for production of recombinant adenoviruses expressing VEGF. AV-VEGF was transfected into COS-7 cells and western blots carried out to confirm the expression of VEGF.Adult ADSCs were isolated from adult rat fat tissues in vitro. Cell proliferation was observed by drafting cell growth curve. Labeling protein of stem cells was detected by immunofluorescence technique.ADSCs wih Ad-VEGF transferred were cultured and detected the VEGF concentration by ELISA assay in different time to confirm AV-VEGF transfected efficiency to ADSCs. MTT assay were used for ADSCs proliferation, conceptive radiommunoassays (RIA) were used for Osteocalcin, enzyme activity detection for ALP. The formation process of mineralization nodes were observed with microscope and calcified nodules stain was made, and Collagen I were analyzed by immunofluorescence technique. Finally, RT-PCR assay were used to detecte Cbfa1 and Osterix mRNA level in osteoblatic differentiation.ResultsRecombinant adenovirus with VEGF were produced by pADEasy-1 system had a high transferred efficiency to COS-7 cells and expressed the VEGF protein by Western Blot confirmation. ADSCs were transfected with 1×10¹⁰ VP/ml of AV-VEGF and expressed VEGF positive cells was up to 86%. The concentration of VEGF in culture medium increased in post-transfected 24h than control and reach to peak at post-transfected 72h. However it still was detected VEGF level at 21 day after adenovirus transfected.ADSCs were obtained from adult rat fat tissues and cultured with the DMEM medium containedβ-glycerophosphate sodium and ascorbic acid. Individual cells grew adherent to the wall of the bottle at post-24h. There are very few cells grew adherent to the wall of bottle. At post-72h, assumed fiber-like cells, mainly in long streak shape and few in polygonal shape were seen. These cells showed a clone like growth. These results indicated that in 3 generation time the capability of ADSCs proliferation there are no significant difference (P<0.05) compared with 6th generation. It was decreased after 9th generation. The amplified times of differ generation is 1.38 day for 3rd generation, 1.66 day for 6th generation and 2.27 day for 9th generation by calculated cell growth curve. ADScs of the 3rd generation were detected by immunofluorescence staining for antigen of stem cells. The results showed that these cells were positive for CD29, CD44, CD99 and CD105, but negative for CD31, CD45 and HLA-DR.ADSCs transfected with AV-VEGF were cultured in osteogenic conditional medium. Effects of VEGF to osteogenic differentiation were observed. The results showed that there were mineralization nodes emerged at 7th day after BMP-2 protein treatment group, at 14th day after VEGF transfected. The number of the mineralization nodes was calculated random at 5 different regions. It found that mineralization nodes were no difference at AV-VEGF transfected group compared with combine of BMP-2 and Av-VEGF group. Theses results suggested that VEGF be able to induce mineralization nodes formation, but the capability of mineralization is weak than BMP-2 (P<0.01).Alkaline phosphatase (ALP) enzyme activity and osteocalcin were analyzed in ADSCs at 2, 4, 8, 14d post-transfected of AV-VEGF. The results showed that low level osteocalcin was detected in post-transfected, and higher than untreated group, but lower than BMP-2 treated group. ADSCs with VEGF treatment emerged increased ALP activity at 8th day, and no significant high at combine of VEGF and BMP-2 treated group than only BMP-2 treatment at 21st day. It suggested that there is visible up-regulation effect of VEGF on ALP in the processes of osteogenic differentiation.ADSCs were induced by BMP-2 and then AV-VEGF transfected at 1d, 7d and 14d. The mineralization nodes, ALP activity and osteocalcin appeared an increase trend with time delay (P<0.05). However no difference was found in ALP activity and osteocalcin at 7d and 14d.To determine the change of osteogenic inducing factors gene transcription in he processes. RT-PCR assay were used to detect mRNA level of Cbfa1 and Osterix. The results showed low level of Cbfa1 mRNA was observed at 14th day post-transfected of AV-VEGF, and less than BMP-2 treated group. No significant increase of Cbfa1 transcription at combine of VEGF and BMP-2 treated group than only BMP-2 treatment group. Osterix were reported that worked as down-stream gene under the Cbfa1 regulation. In VEGF group, Osterix mRNA level was evidently lower than BMP-2 group. There is a different pattern with Cbfa1 gene transcription, Osterix mRNA level increase in combined of VEGF and BMP-2 group. It suggested that it is possibility there are Cbfa1 independent signal pathway involve in the early stage of osteogenic differentiation by VEGF induced. ConclusionRecombinant adenovirus with VEGF were produced by pADEasy-1 system had a high transferred efficiency to ADSCs cells and expressed the VEGF protein. ADSCs were capable to osteogenic differentiation in conditional medium by VEGF induced,but the osteogenic capability is weak than BMP-2 induced. It suggested there are different pathway to promote osteogenic differentiation between VEGF and BMP-2.The main effect of VEGF in the processes of osteogenic differentiation maybe enhance osteoblasts functions.