慢病毒介导骨形态发生蛋白2和血管内皮生长因子165双基因转染骨髓间充质干细胞复合脱钙松质骨治疗兔股骨头坏死
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摘要
背景:骨形态发生蛋白2和血管内皮生长因子165均在骨修复过程中扮演着重要角色。利用慢病毒介导这2种基因转染骨髓间充质干细胞复合脱钙松质骨治疗骨损伤具有重要意义。目的:观察慢病毒介导骨形态发生蛋白2和血管内皮生长因子165基因共转染骨髓间充质干细胞复合脱钙松质骨治疗兔股骨头坏死的效果,以此来探讨双基因在未来骨损伤修复过程中的可行性。方法:采用液氮冰冻联合髓芯减压方法制备股骨头坏死模型,将造模成功的实验兔分为3组(n=12):(1)未转染组:植入骨髓间充质干细胞复合脱钙松质骨;(2)单基因转染组:植入骨形态发生蛋白2转染的骨髓间充质干细胞复合脱钙松质骨;(3)双基因转染组:植入骨形态发生蛋白2和血管内皮生长因子165共转染的骨髓间充质干细胞复合脱钙松质骨。术后3,6,9周各组分别处死4只兔子,Westernblot检测股骨头内骨形态发生蛋白2蛋白的表达量;苏木精-伊红染色观察股骨头坏死修复情况。实验方案经桂林医学院动物实验伦理委员会批准。结果与结论:(1)术后3,6,9周各组均有骨形态发生蛋白2阳性表达,单基因转染组、双基因转染组表达量均高于未转染组(P <0.05),且呈递增趋势;术后3周,单基因转染组、双基因转染组骨形态发生蛋白2表达差异无显著性意义(P> 0.05);术后第6,9周,双基因转染组骨形态发生蛋白2表达量明显高于单基因转染组(P <0.05);(2)术后9周,双基因转染组新生骨痂可填满股骨头缺损处,未转染组、单基因转染组虽有骨痂形成,但均未填满缺损处;苏木精-伊红染色示未转染组有初级骨小梁形成,但骨细胞坏死较多,骨小梁较少;单基因转染组有大量骨小梁形成,形态较好,仅有部分中断,仍不及正常骨组织;双基因转染组形成大量成熟致密骨小梁,形态与正常骨组织无明显差异;(3)结果表明,慢病毒介导骨形态发生蛋白2和血管内皮生长因子165双基因共转染兔骨髓间充质干细胞复合脱钙松质骨治疗股骨头坏死的效果优于骨形态发生蛋白2单基因转染形式,且目的蛋白能在体内长期有效表达。
BACKGROUND: Bone morphogenetic protein 2(BMP-2) and vascular endothelial growth factor 165(VEGF-165) play important roles in bone repair. It is of great significance to treat bone injury by lentivirus-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix. OBJECTIVE: To observe the therapeutic effects of bone marrow mesenchymal stem cells with lentivirus-mediated BMP-2 and VEGF-165 co-transfection and demineralized bone matrix in a rabbit model of osteonecrosis of the femoral head, so as to explore the feasibility of dual genes in the bone repair. METHODS: Femoral head necrosis model was prepared by liquid nitrogen freezing combined with core decompression. Rabbit models successfully prepared were divided into three groups(n=12/group): non-transfection group, treated with bone marrow mesenchymal stem cells combined with demineralized bone matrix; single gene transfection group, treated with BMP-2-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix; dual-gene transfection group, treated with BMP-2 and VEGF-165 co-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix. Four rabbits from each group were sacrificed at 3, 6, and 9 weeks postoperatively. Western blot was used to detect the expression of BMP-2 protein in the femoral head of each group. Hematoxylin-eosin staining was used to observe the repair of femoral head necrosis in each group. The study protocol was approved by the Animal Ethics Committee of Guilin Medical University. RESULTS AND CONCLUSION: BMP-2 positively expressed in all groups at 3, 6, and 9 weeks postoperatively, and the positive expression of target protein was significantly higher in the two transfection groups than the non-transfection group(P < 0.05). The positive expression of BMP-2 in the two transfection groups showed an increasing trend at three time periods. At 3 weeks postoperatively, there was no difference in the BMP-2 expression between the two transfection groups, while the BMP-2 expression was significantly increased in the dual-gene transfection group compared with the single gene transfection group at 6 and 9 weeks postoperatively(P > 0.05). In the dual gene transfection group, new osteophytes could fill in the defect of the femoral head at 9 weeks postoperatively, while in the other two groups, new osteophytes formed were not enough to fill in the defect. Hematoxylin-eosin staining indicated that primary trabeculae were formed in the non-transfection group, but there were more necrotic cells and less bone trabeculae. In the single gene transfection group, a large amount of trabeculae formed with good shape, some of which however were inferior to normal bone tissues because of fractures. In the dual gene transfection group, a mass of mature and dense trabeculae were formed and had normal shape. To conclude, lentivirus-mediated BMP-2 and VEGF-165 co-transfection is superior to single BMP-2 transfection in the combination of bone marrow mesenchymal stem cells and demineralized bone matrix for treating femoral head necrosis. Moreover, in vivo expression of target protein persists for a long time.
BACKGROUND: Bone morphogenetic protein 2(BMP-2) and vascular endothelial growth factor 165(VEGF-165) play important roles in bone repair. It is of great significance to treat bone injury by lentivirus-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix. OBJECTIVE: To observe the therapeutic effects of bone marrow mesenchymal stem cells with lentivirus-mediated BMP-2 and VEGF-165 co-transfection and demineralized bone matrix in a rabbit model of osteonecrosis of the femoral head, so as to explore the feasibility of dual genes in the bone repair. METHODS: Femoral head necrosis model was prepared by liquid nitrogen freezing combined with core decompression. Rabbit models successfully prepared were divided into three groups(n=12/group): non-transfection group, treated with bone marrow mesenchymal stem cells combined with demineralized bone matrix; single gene transfection group, treated with BMP-2-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix; dual-gene transfection group, treated with BMP-2 and VEGF-165 co-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix. Four rabbits from each group were sacrificed at 3, 6, and 9 weeks postoperatively. Western blot was used to detect the expression of BMP-2 protein in the femoral head of each group. Hematoxylin-eosin staining was used to observe the repair of femoral head necrosis in each group. The study protocol was approved by the Animal Ethics Committee of Guilin Medical University. RESULTS AND CONCLUSION: BMP-2 positively expressed in all groups at 3, 6, and 9 weeks postoperatively, and the positive expression of target protein was significantly higher in the two transfection groups than the non-transfection group(P < 0.05). The positive expression of BMP-2 in the two transfection groups showed an increasing trend at three time periods. At 3 weeks postoperatively, there was no difference in the BMP-2 expression between the two transfection groups, while the BMP-2 expression was significantly increased in the dual-gene transfection group compared with the single gene transfection group at 6 and 9 weeks postoperatively(P > 0.05). In the dual gene transfection group, new osteophytes could fill in the defect of the femoral head at 9 weeks postoperatively, while in the other two groups, new osteophytes formed were not enough to fill in the defect. Hematoxylin-eosin staining indicated that primary trabeculae were formed in the non-transfection group, but there were more necrotic cells and less bone trabeculae. In the single gene transfection group, a large amount of trabeculae formed with good shape, some of which however were inferior to normal bone tissues because of fractures. In the dual gene transfection group, a mass of mature and dense trabeculae were formed and had normal shape. To conclude, lentivirus-mediated BMP-2 and VEGF-165 co-transfection is superior to single BMP-2 transfection in the combination of bone marrow mesenchymal stem cells and demineralized bone matrix for treating femoral head necrosis. Moreover, in vivo expression of target protein persists for a long time.
引文
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[3]Zhai L,Sun N,Zhang B,et al.Effects of Focused Extracorporeal Shock Waves on Bone Marrow Mesenchymal Stem Cells in Patients with Avascular Necrosis of the Femoral Head.Ultrasound Med Biol.2016;42(3):753-762.
[4]Yan SG,Huang LY,Cai XZ.Low-intensity pulsed ultrasound:a potential non-invasive therapy for femoral head osteonecrosis.Med Hypotheses.2011;76(1):4-7.
[5]Lozano-Calderon SA,Colman MW,Raskin KA,et al.Use of bisphosphonates in orthopedic surgery:pearls and pitfalls.Orthop Clin North Am.2014;45(3):403-416.
[6]Klumpp R,Trevisan C.Aseptic osteonecrosis of the hip in the adult:current evidence on conservative treatment.Clin Cases Miner Bone Metab.2015;12(Suppl 1):39-42.
[7]Dion NT,Bragdon C,Muratoglu O,et al.Durability of highly cross-linked polyethylene in total hip and total knee arthroplasty.Orthop Clin North Am.2015;46(3):321-327.
[8]Smith AJ,Dieppe P,Howard PW,et al.Failure rates of metal-on-metal hip resurfacings:analysis of data from the National Joint Registry for England and Wales.Lancet.2012;380(9855):1759-1766.
[9]Del Pozo JL,Patel R.Clinical practice.Infection associated with prosthetic joints.N Engl J Med.2009;361(8):787-794.
[10]Ravi B,Pincus D,Wasserstein D,et al.Association of Overlapping Surgery With Increased Risk for Complications Following Hip Surgery:A Population-Based,Matched Cohort Study.JAMA Intern Med.2018;178(1):75-83.
[11]Kim SC,Lim YW,Kwon SY,et al.Effect of leg-length discrepancy following total hip arthroplasty on collapse of the contralateral hip in bilateral non-traumatic osteonecrosis of the femoral head.Bone Joint J.2019;101-B(3):303-310.
[12]Bose S,Roy M,Bandyopadhyay A.Recent advances in bone tissue engineering scaffolds.Trends Biotechnol.2012;30(10):546-554.
[13]Urist MR,Mikulski A,Boyd SD.A chemosterilized antigen-extracted autodigested alloimplant for bone banks.Arch Surg.1975;110(4):416-428.
[14]陈佳滨,李强,茹嘉,等.腺病毒介导BMP-2和EGFP基因转染兔骨髓基质干细胞及种植脱钙骨的体外观察[J].中国骨质疏松杂志,2014,20(8):869-874.
[15]李海冰,瞿向阳,李明,等.应用软骨下钻孔结合液氮冷冻制作兔股骨头缺损坏死模型[J].第三军医大学学报,2014,36(19):2051-2054.
[16]Chan CKF,Gulati GS,Sinha R,et al.Identification of the Human Skeletal Stem Cell.Cell.2018;175(1):43-56.e21.
[17]Lebouvier A,Poignard A,Cavet M,et al.Development of a simple procedure for the treatment of femoral head osteonecrosis with intra-osseous injection of bone marrow mesenchymal stromal cells:study of their biodistribution in the early time points after injection.Stem Cell Res Ther.2015;6:68.
[18]宁寅宽,李强,蔡伟良,等.组织工程骨表面微观形貌和生物矿化的实验研究[J].安徽医科大学学报,2015,50(2):168-171.
[19]李诗鹏,李强,石正松,等.基因重组腺病毒载体与慢病毒载体转染兔骨髓间充质干细胞的对比[J].中国组织工程研究,2017,21(9):1340-1345.
[20]Niikura T,Hak DJ,Reddi AH.Global gene profiling reveals a downregulation of BMP gene expression in experimental atrophic nonunions compared to standard healing fractures.J Orthop Res.2006;24(7):1463-1471.
[21]Govender S,Csimma C,Genant HK,et al.Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures:a prospective,controlled,randomized study of four hundred and fifty patients.J Bone Joint Surg Am.2002;84(12):2123-2134.
[22]Sharma S,Sapkota D,Xue Y,et al.Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis,but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model.Stem Cell Res Ther.2018;9(1):23.
[23]Jiang J,Fan CY,Zeng BF.Experimental construction of BMP2 and VEGF gene modified tissue engineering bone in vitro.Int J Mol Sci.2011;12(3):1744-1755.
[24]Xiao C,Zhou H,Liu G,et al.Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration.Biomed Mater.2011;6(1):015013.
[25]Cai WX,Zheng LW,Li CL,et al.Effect of different rhBMP-2 and TG-VEGF ratios on the formation of heterotopic bone and neovessels.Biomed Res Int.2014;2014:571510.
[26]Peterson B,Zhang J,Iglesias R,et al.Healing of critically sized femoral defects,using genetically modified mesenchymal stem cells from human adipose tissue.Tissue Eng.2005;11(1-2):120-129.
[27]Kujala S,Raatikainen T,Ryh?nen J,et al.Composite implant of native bovine bone morphogenetic protein(BMP)and biocoral in the treatment of scaphoid nonunions--a preliminary study.Scand J Surg.2002;91(2):186-190.
[28]Duan X,Bradbury SR,Olsen BR,et al.VEGF stimulates intramembranous bone formation during craniofacial skeletal development.Matrix Biol.2016;52-54:127-140.
[29]Zhang C,Meng C,Guan D,et al.BMP2 and VEGF165 transfection to bone marrow stromal stem cells regulate osteogenic potential in vitro.Medicine(Baltimore).2018;97(5):e9787.
[2]Zalavras CG,Lieberman JR.Osteonecrosis of the femoral head:evaluation and treatment.J Am Acad Orthop Surg.2014;22(7):455-464.
[3]Zhai L,Sun N,Zhang B,et al.Effects of Focused Extracorporeal Shock Waves on Bone Marrow Mesenchymal Stem Cells in Patients with Avascular Necrosis of the Femoral Head.Ultrasound Med Biol.2016;42(3):753-762.
[4]Yan SG,Huang LY,Cai XZ.Low-intensity pulsed ultrasound:a potential non-invasive therapy for femoral head osteonecrosis.Med Hypotheses.2011;76(1):4-7.
[5]Lozano-Calderon SA,Colman MW,Raskin KA,et al.Use of bisphosphonates in orthopedic surgery:pearls and pitfalls.Orthop Clin North Am.2014;45(3):403-416.
[6]Klumpp R,Trevisan C.Aseptic osteonecrosis of the hip in the adult:current evidence on conservative treatment.Clin Cases Miner Bone Metab.2015;12(Suppl 1):39-42.
[7]Dion NT,Bragdon C,Muratoglu O,et al.Durability of highly cross-linked polyethylene in total hip and total knee arthroplasty.Orthop Clin North Am.2015;46(3):321-327.
[8]Smith AJ,Dieppe P,Howard PW,et al.Failure rates of metal-on-metal hip resurfacings:analysis of data from the National Joint Registry for England and Wales.Lancet.2012;380(9855):1759-1766.
[9]Del Pozo JL,Patel R.Clinical practice.Infection associated with prosthetic joints.N Engl J Med.2009;361(8):787-794.
[10]Ravi B,Pincus D,Wasserstein D,et al.Association of Overlapping Surgery With Increased Risk for Complications Following Hip Surgery:A Population-Based,Matched Cohort Study.JAMA Intern Med.2018;178(1):75-83.
[11]Kim SC,Lim YW,Kwon SY,et al.Effect of leg-length discrepancy following total hip arthroplasty on collapse of the contralateral hip in bilateral non-traumatic osteonecrosis of the femoral head.Bone Joint J.2019;101-B(3):303-310.
[12]Bose S,Roy M,Bandyopadhyay A.Recent advances in bone tissue engineering scaffolds.Trends Biotechnol.2012;30(10):546-554.
[13]Urist MR,Mikulski A,Boyd SD.A chemosterilized antigen-extracted autodigested alloimplant for bone banks.Arch Surg.1975;110(4):416-428.
[14]陈佳滨,李强,茹嘉,等.腺病毒介导BMP-2和EGFP基因转染兔骨髓基质干细胞及种植脱钙骨的体外观察[J].中国骨质疏松杂志,2014,20(8):869-874.
[15]李海冰,瞿向阳,李明,等.应用软骨下钻孔结合液氮冷冻制作兔股骨头缺损坏死模型[J].第三军医大学学报,2014,36(19):2051-2054.
[16]Chan CKF,Gulati GS,Sinha R,et al.Identification of the Human Skeletal Stem Cell.Cell.2018;175(1):43-56.e21.
[17]Lebouvier A,Poignard A,Cavet M,et al.Development of a simple procedure for the treatment of femoral head osteonecrosis with intra-osseous injection of bone marrow mesenchymal stromal cells:study of their biodistribution in the early time points after injection.Stem Cell Res Ther.2015;6:68.
[18]宁寅宽,李强,蔡伟良,等.组织工程骨表面微观形貌和生物矿化的实验研究[J].安徽医科大学学报,2015,50(2):168-171.
[19]李诗鹏,李强,石正松,等.基因重组腺病毒载体与慢病毒载体转染兔骨髓间充质干细胞的对比[J].中国组织工程研究,2017,21(9):1340-1345.
[20]Niikura T,Hak DJ,Reddi AH.Global gene profiling reveals a downregulation of BMP gene expression in experimental atrophic nonunions compared to standard healing fractures.J Orthop Res.2006;24(7):1463-1471.
[21]Govender S,Csimma C,Genant HK,et al.Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures:a prospective,controlled,randomized study of four hundred and fifty patients.J Bone Joint Surg Am.2002;84(12):2123-2134.
[22]Sharma S,Sapkota D,Xue Y,et al.Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis,but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model.Stem Cell Res Ther.2018;9(1):23.
[23]Jiang J,Fan CY,Zeng BF.Experimental construction of BMP2 and VEGF gene modified tissue engineering bone in vitro.Int J Mol Sci.2011;12(3):1744-1755.
[24]Xiao C,Zhou H,Liu G,et al.Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration.Biomed Mater.2011;6(1):015013.
[25]Cai WX,Zheng LW,Li CL,et al.Effect of different rhBMP-2 and TG-VEGF ratios on the formation of heterotopic bone and neovessels.Biomed Res Int.2014;2014:571510.
[26]Peterson B,Zhang J,Iglesias R,et al.Healing of critically sized femoral defects,using genetically modified mesenchymal stem cells from human adipose tissue.Tissue Eng.2005;11(1-2):120-129.
[27]Kujala S,Raatikainen T,Ryh?nen J,et al.Composite implant of native bovine bone morphogenetic protein(BMP)and biocoral in the treatment of scaphoid nonunions--a preliminary study.Scand J Surg.2002;91(2):186-190.
[28]Duan X,Bradbury SR,Olsen BR,et al.VEGF stimulates intramembranous bone formation during craniofacial skeletal development.Matrix Biol.2016;52-54:127-140.
[29]Zhang C,Meng C,Guan D,et al.BMP2 and VEGF165 transfection to bone marrow stromal stem cells regulate osteogenic potential in vitro.Medicine(Baltimore).2018;97(5):e9787.