BMP-2基因治疗的组织工程骨联合显微外科血供重建修复兔桡骨缺损的实验研究
|
摘要
目前大量研究证实，BMP-2在体内、体外均可促进细胞增殖，诱导细胞向成骨细胞转化，促进骨形成。其与种子细胞和支架材料复合后构建的组织工程骨，可通过骨引导和骨诱导方式共同完成骨修复和重建。然而BMP-2外源性植入效价低且移植骨血运问题没有得到有效解决。因此本研究采用基因治疗的策略，将BMP-2基因导入骨髓基质干细胞，并种植到去抗原异种骨支架中，构建BMP-2基因治疗的组织工程骨，期望利用细胞运载BMP-2到特定的损伤部位并稳定释放。在裸鼠皮下（异位）及兔桡骨节段性骨缺损处（骨损伤部位），观察转基因细胞分泌的BMP-2促进成骨及血管化的效果。并且采用显微外科技术，分别将带血管蒂的骨膜和血管束植入与BMP-2基因治疗的组织工程骨联合移植来修复骨缺损，观察额外增加血运对加速骨愈合的影响，为探索适合临床应用的组织工程骨修复的最佳方式奠定实验基础。
一、BMP-2腺病毒载体转染hBMSC及基因治疗的组织工程骨体外构建
BMP-2重组腺病毒表达载体的构建、扩增和滴度测定
利用携带人BMP-2基因的腺病毒穿梭质粒pAdCMV-BMP-2，在脂质体介导下，与腺病毒基因组质粒pJM17共转染293细胞，经同源重组获得BMP-2腺病毒表达载体（Ad-BMP-2）。PCR扩增及基因测序鉴定，证明BMP-2基因被成功构建进腺病毒载体中。通过感染293细胞大量扩增重组病毒，测定病毒滴度为2×1010 pfu/ml。
2．Ad-BMP-2体外转染人骨髓基质干细胞及转染后BMP-2表达检测
体外培养人骨髓基质干细胞（hBMSC），诱导其向成骨细胞、脂肪细胞转化以鉴定其多向分化潜能。然后将Ad-BMP-2转染hBMSC，通过免疫细胞化学、原位杂交染色检测细胞内BMP-2表达；Western blot检测转染后细胞培养液中BMP-2分泌蛋白表达。同等条件下Ad-Lacz对照腺病毒载体转染细胞，X-gal免疫染色观察转染效率。证实BMP-2基因可高效导入细胞（转染效率达90%以上），在mRNA水平和蛋白水平均呈高表达，且可分泌到外周环境中。
3．BMP-2基因转染对hBMSC增殖、分化及VEGF表达的影响
流式细胞仪检测表明，BMP-2基因转染后S期细胞比例增加，G1期细胞减少，说明细胞DNA的合成以及细胞的增殖活动加强。骨钙素免疫荧光、Ⅰ型胶原免疫细胞化学染色和碱性磷酸酶活性测定结果显示，转基因表达的BMP-2促进了hBMSC向成骨细胞转化。
VEGF原位杂交染色，图像分析系统测定平均染色灰度值，统计学结果表明BMP-2基因转染可上调细胞VEGF的表达，提示BMP-2可间接诱导血管再生。
4．BMP-2基因治疗的组织工程骨体外构建
制备去抗原牛松质骨（BCB）支架，植入小鼠双侧股四头肌肌袋内。免疫学MTT法检测表明BCB无明显抗原性；组织学观察见淋巴细胞浸润较少，证实其体内植入的安全性和良好的组织相容性。
将Ad-BMP-2转染后的hBMSC种植到BCB支架中复合培养，扫描电镜见转基因细胞在材料表面分布均匀，贴附生长良好。能量谱仪检测到细胞部位含钙量较空白处明显增高。Western blot 检测到培养液上清中BMP-2的存在。表明转基因细胞可以在支架中生长增殖，并继续外源性分泌BMP-2，促进自身矿化成骨。用于体内植入的BMP-2基因治疗的组织工程骨构建成功。
二、BMP-2基因治疗的组织工程骨在裸鼠背部异位成骨
将BMP-2基因治疗的组织工程骨移植到裸鼠背部皮下，实验分为5组：①Ad-BMP-2转染hBMSC＋BCB支架；②Ad-Lacz转染hBMSC＋BCB；③未转染细胞＋rh-BMP-2＋BCB；④未转染细胞＋BCB；⑤单纯BCB。4、8周处死取材。组织切片HE染色、扫描电镜和透射电镜观察新骨形成，骨钙素免疫组化染色检测宿主间充质细胞向成骨细胞的诱导分化。Y染色体探针荧光原位杂交法（FISH）追踪植入hBMSC的命运，观测其是否参与成骨。
结果表明：（1）BMP-2基因治疗方式诱导成骨速率较快，而整合BMP-2蛋白组新骨形成则相对迟缓，血运相对较差，成骨受到明显影响。BMSC虽具有骨诱导潜能，然而成人来源的细胞单独应用，成骨作用极其微弱，表明BMP-2对于刺激hBMSC分化和诱导异位成骨起着至关重要的作用。（2）BMP-2诱导成骨方式以软骨化骨为主。新形成的骨组织含钙量较低，成熟的骨细胞栖居于骨陷窝内，并沿新形成的哈佛氏管伸出突起，而细胞外基质表达Ⅰ型胶原，从结构和功能上证明新形成的组织为骨组织。（3）BMP-2基因转染的hBMSC不仅作为生长因子的载体，而且直接参与成骨。（4）BMP-2可以诱导宿主间质细胞趋化、聚集，向成骨细胞、成软骨细胞转化。
三、腺病毒介导的BMP-2体外基因治疗修复兔桡骨缺损
分离培养兔BMSC，经BMP-2腺病毒载体转染后，与BCB支架复合，自体移植到1.5cm桡骨缺损处。实验分组同上。术后4、8、12周X光片和组织切片HE、甲苯胺蓝染色观察骨缺损修复，并同时采用图像分析系统测定新骨密度及新骨形成面积；免疫组化染色检测BMP-2分布及表达情况；墨汁灌注观测新生骨血运；生物力学测试骨修复12周后的机械强度。
结果显示BMP-2基因治疗组：（1）4周X线片见移植骨大半呈密度增高的成骨影。8周，骨折线模糊，骨缺损初步修复。12周骨缺损修复完善，骨皮质连续，髓腔再通。（2）组织学观察见4周时支架降解吸收的同时被新生
骨同步取代，可见软骨内骨化，骨软骨岛相互?
Bone morphogenetic protein-2 (BMP-2) has been demonstrated to be a potential growth factor in facilitating cell proliferation and differentiation into cells of the osteoblast lineage, and then accelerate bone formation both in vitro and in vivo. BMP-2 combined with many kinds of seed cells and scaffolds has been widely used in bone tissue engineering procedure, aiming to achive bone repair and reconstruction via osteoconduction and osteoinduction. Unfortunately it is hard to maintain BMP-2 to the necessary concentration in situ and revascularization of the transplanted bone remains to be an obstacle for clinical application of tissue engineering bone. According to the gene therapy technique, we transferred the human BMP-2 gene into the bone mesenchymal stem cells (BMSC) and then seeded the gene-modified cells into a free-antigen allogenetic bone scaffold in this study. As we expected, BMP-2 was carried special anatomic site using the MSC as delivery vehicle and stably was secreted. In both ectopic (subcutanously transplanted on the back of nude mice) and orthotopic (radial segmental defecit in rats) sites, the effects of BMSC expressing BMP-2 on bone formation and vascularization were evaluated. Furthermore we transplanted vascularized periosteum and vessel bundle into the BMP-2 gene modified engineered bone graft to repair bone defect to discuss the effect of an additional blood supply on bone hailing, in order to find the best method to bridge bone
defect, and to look for an most applicable style of engineered bone repair.
Part I: Adenovirus vector carrying hBMP-2 gene (Ad-BMP-2) infected human bone mysenchymal stem cell (hBMSC) and construction of gene therapeutic tissue engineering bone in vitro
1. Construction, purification and titration of adenoviral vector Ad-BMP-2
The recombinant adenoviral shuttle plasmid pAdCMV-BMP-2 was co-transfected into 293 cells with pJM17 using lipofectamine, and adenoviral vector Ad-BMP-2 was produced by in vivo homologous recombination. Identified by PCR and gene sequencing, construction of adenoviral vectors Ad-BMP-2 were successful achieved. After propagation of 293 cells transfected with the vector and purification of the virus, the titer of the adenovirus detected by CPE assay was 2×1010 pfu/ml.
2. Ad-BMP-2 transfacted hBMSC in vitro and detection of BMP-2 expression
hBMSC was cultured in vitro and identified by it’s potential to differentiate into multi-lineages like osteoblast and lipid cell et al. then Ad-BMP-2 was transferred into hBMSC and BMP-2 expression inside hBMSC was verified by immunocytochemistry and in situ hybrydization, while Western blot analysis of the cell culture medium of BMP-2 gene transferred hBMSC showed that BMP-2 was expressed in level of mRNA and protein and secreted into the medium by the gene modified cells. The recombinant adenovirus containing E. Coli lacZ gene [Adv- (beta) gal] was transduced by same method and used as control in the study. X-gal immunostaining was tested to assess the transfection efficiency that was higher than 90%.
3. Effects of BMP-2 gene transfaction on hBMSC proliferation,
differentiation and VEGF expression in hBMSC
Flow cytometer (FCM) examination showed the percentage of S phase cells in BMP-2 gene transferred hBMSC increased, while that of G1 phase cells decreased, which indicated that DNA synthesis and cell proliferation was promoted after gene transfaction. Osteocalcin immunofluorencence, type I collagen immunocytochemistry staining and ALP activity examination showed that BMP-2 gene transfection facilitate hBMSC differentiate into osteoblast. VEGF in situ hybridization, which was assessed by the mean staining gray value showed that BMP-2 gene transfaction up-regulated VEGF expression and then induce vessel regeneration indirectly.
4. Construction of BMP-2 gene modified tissue-engineered bone in vitro
Free-antigen bovine cancellous bone (BCB) was prepared according to the related references and transplanted into a muscle bag of the quadricept musle in rats, immunological examinatio
一、BMP-2腺病毒载体转染hBMSC及基因治疗的组织工程骨体外构建
BMP-2重组腺病毒表达载体的构建、扩增和滴度测定
利用携带人BMP-2基因的腺病毒穿梭质粒pAdCMV-BMP-2，在脂质体介导下，与腺病毒基因组质粒pJM17共转染293细胞，经同源重组获得BMP-2腺病毒表达载体（Ad-BMP-2）。PCR扩增及基因测序鉴定，证明BMP-2基因被成功构建进腺病毒载体中。通过感染293细胞大量扩增重组病毒，测定病毒滴度为2×1010 pfu/ml。
2．Ad-BMP-2体外转染人骨髓基质干细胞及转染后BMP-2表达检测
体外培养人骨髓基质干细胞（hBMSC），诱导其向成骨细胞、脂肪细胞转化以鉴定其多向分化潜能。然后将Ad-BMP-2转染hBMSC，通过免疫细胞化学、原位杂交染色检测细胞内BMP-2表达；Western blot检测转染后细胞培养液中BMP-2分泌蛋白表达。同等条件下Ad-Lacz对照腺病毒载体转染细胞，X-gal免疫染色观察转染效率。证实BMP-2基因可高效导入细胞（转染效率达90%以上），在mRNA水平和蛋白水平均呈高表达，且可分泌到外周环境中。
3．BMP-2基因转染对hBMSC增殖、分化及VEGF表达的影响
流式细胞仪检测表明，BMP-2基因转染后S期细胞比例增加，G1期细胞减少，说明细胞DNA的合成以及细胞的增殖活动加强。骨钙素免疫荧光、Ⅰ型胶原免疫细胞化学染色和碱性磷酸酶活性测定结果显示，转基因表达的BMP-2促进了hBMSC向成骨细胞转化。
VEGF原位杂交染色，图像分析系统测定平均染色灰度值，统计学结果表明BMP-2基因转染可上调细胞VEGF的表达，提示BMP-2可间接诱导血管再生。
4．BMP-2基因治疗的组织工程骨体外构建
制备去抗原牛松质骨（BCB）支架，植入小鼠双侧股四头肌肌袋内。免疫学MTT法检测表明BCB无明显抗原性；组织学观察见淋巴细胞浸润较少，证实其体内植入的安全性和良好的组织相容性。
将Ad-BMP-2转染后的hBMSC种植到BCB支架中复合培养，扫描电镜见转基因细胞在材料表面分布均匀，贴附生长良好。能量谱仪检测到细胞部位含钙量较空白处明显增高。Western blot 检测到培养液上清中BMP-2的存在。表明转基因细胞可以在支架中生长增殖，并继续外源性分泌BMP-2，促进自身矿化成骨。用于体内植入的BMP-2基因治疗的组织工程骨构建成功。
二、BMP-2基因治疗的组织工程骨在裸鼠背部异位成骨
将BMP-2基因治疗的组织工程骨移植到裸鼠背部皮下，实验分为5组：①Ad-BMP-2转染hBMSC＋BCB支架；②Ad-Lacz转染hBMSC＋BCB；③未转染细胞＋rh-BMP-2＋BCB；④未转染细胞＋BCB；⑤单纯BCB。4、8周处死取材。组织切片HE染色、扫描电镜和透射电镜观察新骨形成，骨钙素免疫组化染色检测宿主间充质细胞向成骨细胞的诱导分化。Y染色体探针荧光原位杂交法（FISH）追踪植入hBMSC的命运，观测其是否参与成骨。
结果表明：（1）BMP-2基因治疗方式诱导成骨速率较快，而整合BMP-2蛋白组新骨形成则相对迟缓，血运相对较差，成骨受到明显影响。BMSC虽具有骨诱导潜能，然而成人来源的细胞单独应用，成骨作用极其微弱，表明BMP-2对于刺激hBMSC分化和诱导异位成骨起着至关重要的作用。（2）BMP-2诱导成骨方式以软骨化骨为主。新形成的骨组织含钙量较低，成熟的骨细胞栖居于骨陷窝内，并沿新形成的哈佛氏管伸出突起，而细胞外基质表达Ⅰ型胶原，从结构和功能上证明新形成的组织为骨组织。（3）BMP-2基因转染的hBMSC不仅作为生长因子的载体，而且直接参与成骨。（4）BMP-2可以诱导宿主间质细胞趋化、聚集，向成骨细胞、成软骨细胞转化。
三、腺病毒介导的BMP-2体外基因治疗修复兔桡骨缺损
分离培养兔BMSC，经BMP-2腺病毒载体转染后，与BCB支架复合，自体移植到1.5cm桡骨缺损处。实验分组同上。术后4、8、12周X光片和组织切片HE、甲苯胺蓝染色观察骨缺损修复，并同时采用图像分析系统测定新骨密度及新骨形成面积；免疫组化染色检测BMP-2分布及表达情况；墨汁灌注观测新生骨血运；生物力学测试骨修复12周后的机械强度。
结果显示BMP-2基因治疗组：（1）4周X线片见移植骨大半呈密度增高的成骨影。8周，骨折线模糊，骨缺损初步修复。12周骨缺损修复完善，骨皮质连续，髓腔再通。（2）组织学观察见4周时支架降解吸收的同时被新生
骨同步取代，可见软骨内骨化，骨软骨岛相互?
Bone morphogenetic protein-2 (BMP-2) has been demonstrated to be a potential growth factor in facilitating cell proliferation and differentiation into cells of the osteoblast lineage, and then accelerate bone formation both in vitro and in vivo. BMP-2 combined with many kinds of seed cells and scaffolds has been widely used in bone tissue engineering procedure, aiming to achive bone repair and reconstruction via osteoconduction and osteoinduction. Unfortunately it is hard to maintain BMP-2 to the necessary concentration in situ and revascularization of the transplanted bone remains to be an obstacle for clinical application of tissue engineering bone. According to the gene therapy technique, we transferred the human BMP-2 gene into the bone mesenchymal stem cells (BMSC) and then seeded the gene-modified cells into a free-antigen allogenetic bone scaffold in this study. As we expected, BMP-2 was carried special anatomic site using the MSC as delivery vehicle and stably was secreted. In both ectopic (subcutanously transplanted on the back of nude mice) and orthotopic (radial segmental defecit in rats) sites, the effects of BMSC expressing BMP-2 on bone formation and vascularization were evaluated. Furthermore we transplanted vascularized periosteum and vessel bundle into the BMP-2 gene modified engineered bone graft to repair bone defect to discuss the effect of an additional blood supply on bone hailing, in order to find the best method to bridge bone
defect, and to look for an most applicable style of engineered bone repair.
Part I: Adenovirus vector carrying hBMP-2 gene (Ad-BMP-2) infected human bone mysenchymal stem cell (hBMSC) and construction of gene therapeutic tissue engineering bone in vitro
1. Construction, purification and titration of adenoviral vector Ad-BMP-2
The recombinant adenoviral shuttle plasmid pAdCMV-BMP-2 was co-transfected into 293 cells with pJM17 using lipofectamine, and adenoviral vector Ad-BMP-2 was produced by in vivo homologous recombination. Identified by PCR and gene sequencing, construction of adenoviral vectors Ad-BMP-2 were successful achieved. After propagation of 293 cells transfected with the vector and purification of the virus, the titer of the adenovirus detected by CPE assay was 2×1010 pfu/ml.
2. Ad-BMP-2 transfacted hBMSC in vitro and detection of BMP-2 expression
hBMSC was cultured in vitro and identified by it’s potential to differentiate into multi-lineages like osteoblast and lipid cell et al. then Ad-BMP-2 was transferred into hBMSC and BMP-2 expression inside hBMSC was verified by immunocytochemistry and in situ hybrydization, while Western blot analysis of the cell culture medium of BMP-2 gene transferred hBMSC showed that BMP-2 was expressed in level of mRNA and protein and secreted into the medium by the gene modified cells. The recombinant adenovirus containing E. Coli lacZ gene [Adv- (beta) gal] was transduced by same method and used as control in the study. X-gal immunostaining was tested to assess the transfection efficiency that was higher than 90%.
3. Effects of BMP-2 gene transfaction on hBMSC proliferation,
differentiation and VEGF expression in hBMSC
Flow cytometer (FCM) examination showed the percentage of S phase cells in BMP-2 gene transferred hBMSC increased, while that of G1 phase cells decreased, which indicated that DNA synthesis and cell proliferation was promoted after gene transfaction. Osteocalcin immunofluorencence, type I collagen immunocytochemistry staining and ALP activity examination showed that BMP-2 gene transfection facilitate hBMSC differentiate into osteoblast. VEGF in situ hybridization, which was assessed by the mean staining gray value showed that BMP-2 gene transfaction up-regulated VEGF expression and then induce vessel regeneration indirectly.
4. Construction of BMP-2 gene modified tissue-engineered bone in vitro
Free-antigen bovine cancellous bone (BCB) was prepared according to the related references and transplanted into a muscle bag of the quadricept musle in rats, immunological examinatio
引文
1 Khan SN, Bostrom MP, Lane JM. Bone growth factors. Orthop Clin Nor Am, 2000, 31(3): 375-388.
2 Wozney JM, Rosen V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop, 1998, 346(1): 26-37.
3 Bonadio J, Smiley E, Patil P, et al. Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration. Nat Med, 1999, 5(7): 753-759.
4 Laurencin CT, Attawia MA, Lu LQ, et al. Poly(lactide-co-glycolide) /hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration. Biomaterials, 2001, 22(11): 1271-1277.
5 Liberman JR, Daluiski A, Stevenson S, et al. The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats. Bone Joint Surg Am, 1999, 81(7): 905-917.
6 Lieberman JR, Le LQ, Wu L, et al. Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents. J Orthop Res, 1998, 16(3): 330-339.
7 Breitbart AS, Grande DA, Mason JM, et al. Gene enhanced tissue engineering: applications for bone healing using cultured periosteal cells transduced retrovirally with the BMP-7 gene. J Ann Plast Surg, 1999, 42(5): 488-495.
8 Lou J, Xu F, Merkel K, et al. Gene therapy: adenovirus-mediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and differentiation in vitro and bone formation in vivo. J Orthop Res, 1999, 17(1): 43-50.
9 Fang J, Zhu YY, Smiley E, et al. Stimulation of new bone formation by direct transfer of osteogenic plasmid genes. Proc Natl Acad Sci U S A, 1996, 93(12): 5753-5758.
10 Franceschi RT, Wang D, Krebsbach PH, et al. Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7. J Cell Biochem. 2000 Jun 6; 78(3):476-486.
11 Musgrave DS, Bosch P, Ghivizzani S, et al. Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone. Bone, 1999, 24(6): 541-547.
12 Boden SD, Titus L, Hair G, et al. Lumbar spine fusion by local gene therapy with a cDNA encoding a novel osteoinductive protein (LMP-1). Spine, 1998, 23(23): 2486-2492.
金科，林晨，张学艳，等. 应用于基因治疗的重组体腺病毒的构建方法. 遗传, 1997; 19: 36-38.
lden TD, Pittman DD, Hankins GR, et al. In vivo endochondral bone formation using a bone morphogenetic protein-2 adenoviral vector. Hum Gene Ther, 1999; 10(13): 2245-2253.
陈希哲，杨连甲，杨维东. 组织工程与基因工程联合应用于骨缺损修复的研究. 现代康复, 2001; 5: 59-60.
Musgrave DS, Pruchnic R, Bosch P, et al. Human skeletal muscle cells in ex vivo gene therapy to deliver bone morphogenetic protein-2. J Bone Joint Surg Br, 2002; 84(1): 120-127.
Tsuchida H, Hashimoto J, Crawford E, et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthop Res, 2003; 21(1): 44-53.
Mendes SC, Tibbe JM, Veenhof M, et al. Bone Tissue-Engineered Implants Using Human Bone Marrow Stromal Cells: Effect of Culture Conditions and Donor Age. Tissue Eng, 2002; 8(6): 911-920.
Robert F. Tissue engineers build new bone. Science, 2000; 289(5384): 1498-1500.
Lee JY, Qu-Petersen Z, Cao B, et al. Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol, 2000; 150:1085-1100.
Baltzer AWA, Lattermann C, Whalen JD, et al. Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene. Gene Therapy, 2000; 7: 734-739.
22 罗卓荆, 胡蕴玉, 王茜. 块状重组合异种骨修复犬桡骨骨缺损. 中华骨科杂志,1998; 18: 363-366.
2 Wozney JM, Rosen V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop, 1998, 346(1): 26-37.
3 Bonadio J, Smiley E, Patil P, et al. Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration. Nat Med, 1999, 5(7): 753-759.
4 Laurencin CT, Attawia MA, Lu LQ, et al. Poly(lactide-co-glycolide) /hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration. Biomaterials, 2001, 22(11): 1271-1277.
5 Liberman JR, Daluiski A, Stevenson S, et al. The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats. Bone Joint Surg Am, 1999, 81(7): 905-917.
6 Lieberman JR, Le LQ, Wu L, et al. Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents. J Orthop Res, 1998, 16(3): 330-339.
7 Breitbart AS, Grande DA, Mason JM, et al. Gene enhanced tissue engineering: applications for bone healing using cultured periosteal cells transduced retrovirally with the BMP-7 gene. J Ann Plast Surg, 1999, 42(5): 488-495.
8 Lou J, Xu F, Merkel K, et al. Gene therapy: adenovirus-mediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and differentiation in vitro and bone formation in vivo. J Orthop Res, 1999, 17(1): 43-50.
9 Fang J, Zhu YY, Smiley E, et al. Stimulation of new bone formation by direct transfer of osteogenic plasmid genes. Proc Natl Acad Sci U S A, 1996, 93(12): 5753-5758.
10 Franceschi RT, Wang D, Krebsbach PH, et al. Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7. J Cell Biochem. 2000 Jun 6; 78(3):476-486.
11 Musgrave DS, Bosch P, Ghivizzani S, et al. Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone. Bone, 1999, 24(6): 541-547.
12 Boden SD, Titus L, Hair G, et al. Lumbar spine fusion by local gene therapy with a cDNA encoding a novel osteoinductive protein (LMP-1). Spine, 1998, 23(23): 2486-2492.
金科，林晨，张学艳，等. 应用于基因治疗的重组体腺病毒的构建方法. 遗传, 1997; 19: 36-38.
lden TD, Pittman DD, Hankins GR, et al. In vivo endochondral bone formation using a bone morphogenetic protein-2 adenoviral vector. Hum Gene Ther, 1999; 10(13): 2245-2253.
陈希哲，杨连甲，杨维东. 组织工程与基因工程联合应用于骨缺损修复的研究. 现代康复, 2001; 5: 59-60.
Musgrave DS, Pruchnic R, Bosch P, et al. Human skeletal muscle cells in ex vivo gene therapy to deliver bone morphogenetic protein-2. J Bone Joint Surg Br, 2002; 84(1): 120-127.
Tsuchida H, Hashimoto J, Crawford E, et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthop Res, 2003; 21(1): 44-53.
Mendes SC, Tibbe JM, Veenhof M, et al. Bone Tissue-Engineered Implants Using Human Bone Marrow Stromal Cells: Effect of Culture Conditions and Donor Age. Tissue Eng, 2002; 8(6): 911-920.
Robert F. Tissue engineers build new bone. Science, 2000; 289(5384): 1498-1500.
Lee JY, Qu-Petersen Z, Cao B, et al. Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol, 2000; 150:1085-1100.
Baltzer AWA, Lattermann C, Whalen JD, et al. Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene. Gene Therapy, 2000; 7: 734-739.
22 罗卓荆, 胡蕴玉, 王茜. 块状重组合异种骨修复犬桡骨骨缺损. 中华骨科杂志,1998; 18: 363-366.