The Influence Of BMP-9 Gene Transfection Combined With Bisphosphonate Therapy On Implant Osseointegration

Part I:Separation, Culture and Identification of Rat Bone Marrow Mesenchymal Stem CellsObjective: To successfully separate and culture rat bone marrow mesenchymal stem cells to provide seed cells for subsequent researches.Methods: The method of whole bone marrow adherent was employed to separate, culture and purify rat bone marrow mesenchymal stem cells in vitro. The growth curve was drawn through morphological observation. Cell surface markers and identified osteogenic and adipogenic abilities were detected by flow cytometry.Results: After 1-2 passages, the rat bone marrow mesenchymal stem cells obtained with the method of whole bone marrow adherent grew radially or spirally in a line, showing consistent morphology and stability.CD29, CD44 and CD90 on cell surface displayed positive expression,while CD11 B and CD45 showed negative expression. Induced under certain conditions, oil red O staining and alkaline phosphatase stainingbecame positive.Conclusions: BMSCs obtained through experiment have general biological characteristics of mesenchymal stem cells and multi-directional differentiation potential after induction and culture.BMSCs are successfully obtained.Part II:BMSCs Cell Transfection by Recombinant Adenovirus BMP-9 AmplificationObjective: To obtain high-titer recombinant adenovirus BMP-9 and transfect BMSCs and to monitor the expression level of BMP-9 after transfection.Methods: High-titer recombinant adenovirus BMP-9 was obtained by HEK293 cell amplification. Rat BMSCs were transfected with different titers. The growth curve of transfected BMSCs was drawn after observing the transfection rate of BMP-9 under fluorescent inverted microscope. The expression of BMP-9 proteins in BMSCs in different periods after transfection was detected with Western blotting.Results: The titer of virus infection fluid collected through 2ul virus transfection and amplification was 1×108pfu/ml. When MOI was 100, the transfection rate of Ad-BMP-9 adenovirus to rat BMSCs was 80%-90%.During the determination of cell growth curve with MTT method, no influence of transfection on cell amplification was observed, showing no significant difference when compared to the un-transfected group. The expression level of BMP-9 proteins could be detected at 3d after transfection, which peaked at 1w, gradually weakened after 1w and still exhibited a high level after 4w.Conclusions: When transfecting BMSCs with BMP-9, BMP-9shows a stable expression and meets the requirements of the experiment.Part III:Influence of BMP-9 Gene Transfection Combined with Bisphosphonate Therapy on Osteogenic DifferentiationObjective: To transfect the BMSCs with BMP-9 gene after stimulation with alendronate at different concentrations and observe the influence on its osteogenesis in vitro.Methods: 10-5, 10-6, 10-7, 10-8 and 10-9Mol/L of Aln were used to stimulate rat BMSCs. Applied alkaline phosphatase(ALP) staining and quantitative monitoring at 5d and 10 d. 10-7, 10-8 and 10-9Mol/L of Aln were used to stimulate the transfection of BMSCs with BMP-9 gene.Alkaline phosphatase(ALP) staining and quantitative monitoring were performed at 5d and 10 d. The experiment was divided into10-7Aln+BMP-9, 10-9Aln +BMP-9, BMP-9, 10-7Aln, 10-9Aln, GFP and control groups before applying alizarin red, tartrate-resistant acid phosphatase staining at 7d and 14 d and testing OCN, OPN, Rankl expression with fluorescence quantitative PCR and Western blot.Results: After stimulating BMSCs with Aln at different concentrations, ALP activity increased with the rise in Aln concentration,reaching the highest at 10-7Mol/L and then gradually declining. For Aln at the corresponding concentration, the activity at 10 d was weaker than that at 5d. After the action of 10-7-10-9Mol/L Aln along with BMP-9 on BMSCs, ALP activity showed a more significant increase. With the increase of Aln concentration, this synergistic effect was further strengthened with no significant statistical difference(P>0.01). According to the results of alizarin red staining, calcium salt deposits increased significantly in BMP-9 group. The combined use of Aln and BMP-9 at different concentrations showed an obvious increase in calcium salt deposits in comparison to BMP-9 group. The deposits increased as the concentration of Aln rose, but the statistical difference between different concentration levels was not significant(P>0.01). As shown by tartrate-resistant acid phosphatase(TRAP) staining, BMP-9 could induce the expression of cells with positive TRAP staining. The staining intensity at 14 d was greater than that at 7d, but Control, GFP and Aln groups had no cells with positive TRAP staining, which decreasedsignificantly with the addition of Aln. The results of Westemblot and Real-time RT-PCR suggested that OPN and OCN expression after the combined use of BMP-9 and Aln at 7d and 14 d was significantly higher than the separate use of them(P<0.01). This synergistic effect increased with the rise of Aln concentration, but the statistical difference between10-7Aln+BMP-9 and 10-9Aln+BMP-9 was not significant(P>0.01). Aln could obviously inhibit the expression of RANKL(P<0.01), but the inhibition difference between Aln at two concentrations was not significant(P>0.01). Using BMP-9 alone increased RANKL expression(P<0.01), which was inhibited after Aln was added. In comparison to BMP-9 group, RANKL expression in 10-7Aln+BMP-9 group and10-9Aln+BMP-9 group reduced significantly(P<0.01), but the difference between two concentrations was not significant(P>0.01).Conclusions: BMP-9 can promote BMSCs osteoblast differentiation in both early and late osteogenesis while inducing the expression of factors related to osteoclastogenesis. Aln can inhibit the positive role of BMP-9 in osteoclast regulation and the induction of BMSCs osteoclast-like cells differentiation by BMP-9. There were interaction between them in osteogenic induction, thus providing basis for BMP-9and Aln therapy at cellular level.Part IV: In vivo Study on the Influence of BMP-9 Gene Transfection Combined with Bisphosphonate Therapy on Implant osseointegrationObjective: To study the influence of BMP-9 gene transfection combined with bisphosphonate therapy on bone internal fixation within implants in vivo.Methods: 64 six-month-old SD rats were randomly divided into four groups, with 16 rats in each one: pure screw fixation group(Control group), alendronate group(Aln group), BMP-9 transfection group(BMP-9 group) and BMP-9 transfection + alendronate group(BMP-9+Aln group). Screws were implanted into the proximal end of bilateral tibia three months after bilateral oophorectomy. BMSCs were simultaneously implanted and transfected with BMP-9 in BMP-9 group and BMP-9+Aln group. 1 ml/kg Aln was subcutaneously injected twice a week in Aln group and BMP-9+Aln group, and subcutaneous injection of the same volume of normal saline was administrated in the rest groups immediately after screw implant. 8 rats were sacrificed in each group at4 w and 8w after screw implant for biomechanical testing, Micro-CT scanning, hard tissue slicing V-G staining and decalcified bone BMP-9immumohistochemical staining.Results: screw pull-out strength in BMP-9+Aln group at 4w and 8wwas 1 time higher than that of Control group and significantly higher than that of BMP-9 group and Aln group(P<0.01). According to the results of Micro-CT, the increase in BV/TV, TB.TH and Tb.N were respectively,about five times, one and one times that of the Control group, but TB.Sp was less than half of the Control group(P<0.01). BV/TV, TB.TH and Tb.N also increased significantly in Aln group and BMP-9 group, but had poorer effect than BMP-9+Aln group. Tb.Sp decrease was also lower than BMP-9+Aln group. As revealed by hard tissue slicing V-G staining,massive bone growth could be observed between screw threads in BMP-9+Aln group at 4w and bone formation appeared around. Its screw-bone contact ratio was almost three times that of Control group(P<0.01). Bone growth could also be seen between threads in Aln group and BMP-9 group, with much more in the latter(P<0.01). But the bone tissues around screws were less than the Aln group. Many bone tissues formed in groups and more bone grew between threads at 8w when compared to Control group, with BMP-9+Aln group being the most obvious, whose screw-bone contact ratio was almost 2 time that of Control group(P<0.01). Bone growth between threads increased in BMP-9 group with a significantly higher bone contact rate than Aln group but less surrounding bone tissues than Aln group. BMP-9immumohistochemical staining showed intracytoplasmic positive BMP-9expression in osteoblasts and mature bone cells at 4w in BMP-9 groupand BMP-9+Aln group, weaker expression at 8w and no obviously positive BMP-9 expression in Control group and Aln group.Conclusions: In addition to significantly improving the stability of implants in bones, BMP-9 gene transfection combined with bisphosphonate therapy plays an interactive role in increasing the surrounding bone tissues of implants and bone contact.