聚乙烯亚胺—藻酸盐/BMP-2基因复合物修饰的BMSCs细胞膜片促骨再生作用的研究
|
摘要
由于先天缺陷、肿瘤、外伤及牙周炎等原因造成的骨缺损一直是国内外临床医生所面临的难题。自体骨移植是治疗骨缺损的“金标准”。但是由于自体骨移植物存在供区骨量有限，可能引起供区并发症等缺点，因而限制了它的应用。而异体骨移植物和异种骨移植物具有与宿主骨相似的物理和生物学性质，已经成功应用于许多矫形外科的手术中。然而，它们存在疾病传播和宿主的免疫排斥反应等问题限制了其应用。而合成的骨替代物由于不具有与天然骨相似的机械性能和骨诱导性，因此常常导致移植的失败。骨组织工程学的出现，为解决这些问题提供了一条充满希望的途径。近年来，基于基因和干细胞的治疗已经被用于骨组织工程学的研究，并取得了良好的成果。
骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）是目前骨组织工程学中应用最为广泛的种子细胞，它具有多向分化的潜能，在一定条件下可以分化为骨、软骨、肌肉、脂肪等组织。而骨形态发生蛋白2（bone morphogeneticprotein2, BMP-2）是目前已知的骨诱导能力最强的生长因子，它可以直接诱导间充质干细胞向成骨方向分化。因此，常常利用BMP-2基因转染BMSCs以提高其成骨分化能力。
外源基因进入细胞通常需要基因载体，主要包括病毒载体和非病毒载体两类。病毒载体由于其较高的转染效率是目前应用最多的基因载体，但是作为一种改造的病毒，其价格昂贵，难以大规模生产，且存在安全隐患和免疫原性等问题，因此许多学者把注意力逐步转向对非病毒载体的研究。各种类型的聚乙烯亚胺（polyethylenimine, PEI）是最高效的非病毒载体之一，但是它仍然有许多缺点，如：无组织或细胞靶向性、相对于病毒载体转染效率较低以及具有较高的细胞毒性等。因此，为了克服PEI的这些缺点，需要对PEI进行改造。
支架材料也是骨组织工程学的主要因素之一。常见的支架材料主要包括天然聚合物、陶瓷材料、合成聚合物和复合材料。但是目前已开发或应用的支架材料都存在一些问题，如：引起炎症反应，降解速率不匹配，缺少生物活性等。因此，一种不需要外源性支架的细胞膜片技术的出现了。它以一种无创的方式将细胞以膜状结构收集，完整的保留了细胞和细胞之间的连接和细胞外基质，以及相关蛋白和生物活性因子，为体内骨损伤的愈合提供了适宜的微环境。
基于以上背景，本研究设计为以下三个部分：
一、聚乙烯亚胺-藻酸盐/质粒复合物的制备与表征
利用阳离子聚合物PEI与天然阴离子聚合物海藻酸钠（alginate）通过静电作用构建了PEI-alginate（PEI-al）纳米基因载体，并对其理化性质进行表征。结果显示：PEI-al纳米复合物的平均粒径为73.8nm，平均电势为+19.9mV；扫描电镜观察可见PEI-al纳米复合物为球形的单分散微粒，当PEI-al纳米复合物与BMP-2质粒（BMP-2plasmid, pBMP-2）结合后可发生部分聚集；通过纳米粒度仪测得PEI-al/pBMP-2的平均粒径增加到149.0nm，电势降低到+18.8mV。DNA凝胶阻滞实验结果表明当质量比达到1.5：1时，材料能够完全结合DNA，材料与基因的复合物为中性或是正电性。
二、PEI-al/pBMP-2复合物体内外促成骨的作用的研究
利用PEI-al/pBMP-2复合物转染MC3T3-E1细胞，通过realtime-PCR和茜素红染色研究其体外促成骨的作用；将PEI-al纳米复合物和PEI-al/pBMP-2复合物与明胶海绵支架分别复合后移植到大鼠的颅骨缺损内，观察它们体内促成骨的作用。结果显示：转染PEI-al/pBMP-2后，realtime-PCR检测成骨相关基因的表达都有上调，钙结节的形成量也明显多于转染了PEI-al/pEGFP的细胞；Micro-CT观察到PEI-al/pBMP-2组的大鼠颅骨缺损边缘不规则，内部有大量新生骨样组织，且明显多于PEI-al组；H&E染色结果也与Micro-CT相符， PEI-al/pBMP-2组缺损边缘有新生骨组织，缺损中心处也有一些编织状骨形成，而PEI-al组缺损处全部由纤维结缔组织修复。
三、PEI-al/pBMP-2复合物修饰的BMSCs膜片体内外促成骨作用的研究
利用PEI-al/pBMP-2复合物转染BMSCs后，在含有维生素C的培养基中继续培养10天，获得了能够分泌BMP-2蛋白的BMSCs细胞膜片。通过ELISA检测BMP-2分泌情况；通过realtime-PCR和碱性磷酸酶活性研究其体外促成骨的作用；将分泌EGFP蛋白的BMSCs细胞膜片（EGFP/CS）和分泌BMP-2蛋白的BMSCs细胞膜片（BMP/CS）分别移植到大鼠的颅骨缺损内，观察它们体内促成骨的作用。结果显示：在转染PEI-al/pBMP-2后，BMSCs能够持续分泌BMP-2蛋白达14天以上；realtime-PCR检测表明相关成骨基因的表达均有上调，这一结果说明分泌的BMP-2蛋白能够有效的诱导BMSCs细胞膜片向成骨方向分化；Micro-CT和组织学检测观察到BMP/CS组与其他组相比有效的促进了缺损处骨组织的再生。
以上实验结果表明：PEI-al纳米复合物是一种高效的基因载体；利用PEI-al/pBMP-2修饰的BMSCs细胞膜片能够有效的促进骨缺损的修复。
Bone loss caused by congenital defects, traumatic injury, cancer, reconstructivesurgery or periodontal disease is always the most difficult challenge both at home andabroad. Autologous bone graft is the so-called gold standard in transplantation surgery.But the use of autologous bone may be restricted because the amount of donor bone islimited and the complication in donor site. The strategies of allogenic or xenogenicare associated with problems such as the risk of disease transmission and graftrejection with a concomitant necessity for secondary surgery, and cost much.Synthetic grafts are important substitutes, but they fail to provide similar mechanicalproperties of human bone tissue, often causing failure in bone healing. Bone tissueengineering attempts to solve the referred problems mentioned above. Recently, gene-and stem cell-based therapy have been applied in bone tissue engineering andachieved some good clinical results.
Bone marrow mesenchymal stem cells (BMSCs) have been widely used for bonetissue engineering. They potentially can be used to develop bone，cartilage，muscle oradipose. Bone morphogenetic proteins (BMPs) are well known as osteoinductivegrowth factors which play an important role in bone regeneration process. Theseproteins are capable of inducing the osteogenic differentiation of mesenchymal cells.So BMP-2gene has been frequently used to induce the osteogenic differentiation ofBMSCs.
Gene vectors are required for the transfer of exogenous gene into cells, the mainapproach to gene delivery in gene therapy is viral vector systems due to theirrelatively high transfection efficiency. But viral-based gene vectors are a kind ofengineered viruses, so they have the drawbacks of high cost, immune response againstrepeated administration, oncogenicity and difficulty in large-scale production.Therefore, many scientists have shifted their interest to develop non-viral gene vectors.Different types of polyethyleneimine (PEI) are regarded as a kind of the most effective non-viral gene vectors. However, PEI has the disadvantage of no specifictissues and cells targeting, relative lower transgene efficiency compared with viralvectors and high cytotoxicity. In order to overcome the drawbacks, it is necessary tomodify PEI.
Scaffold is also a main factor of bone tissue engineering, including naturepolymer, ceramics, synthesis polymer and composite material. However, the scaffoldsstill face the problem of causing inflammation, the degradation rate of biomaterial notmatch the regeneration rate of bone and the lack of biological activity. So the newertechnology of cell sheet without scaffold has emerged. The cells are collected as asheet, which maintains the connections between cells and ECM, and the relativeproteins and biological activity factors. It also can provide a suitablemicroenvironment for bone regeneration.
Preparation and characterization of PEI-alginate/plasmid complexes
PEI-al gene carriers were prepared by electrostatic interation between the cationicPEI and polyanionic alginate, and physicochemical properties were characterized. Theresults showed that the average particle size of PEI-al nanocomposites were73.8nm,and the zeta potential distribution was+19.9mV; SEM images showed that PEI-alnanocomposites were spherical and monodispersed particles while some aggregationoccured when PEI-al nanocomposites were blended with pBMP-2; DLS resultsshowed that the average particle size of PEI-al/pBMP-2complexes were increased to149.0nm, and the zeta potential distribution was decreased to+18.8mV; Agarose gelelectrophoresis result suggested the formation of neutral or positive complexesbetween PEI-al and pBMP-2, when the weight ratio reaches1.5.
The effects of PEI-al/pBMP-2on bone formation in vitro and in vivo
PEI-al/pBMP-2complexes were transfected into MC3T3-E1cell line in vitro,and the capability of inducing osteogenic differentiation was evaluated byrealtime-PCR and alizarin red stain. PEI-al/pBMP-2complexes and PEI-alnanocomposites were absorbed into gelatin sponges, respectively, and the gelatinsponges were implanted into the rat calvarial defects in vivo. The effect of boneregeneration was observed by Micro-CT and histologic analysis. The results showedthat the expression of osteogenesis-related gene was increased post-transfected withPEI-al/pBMP-2complexes, and the calcium nodules formation was much more inPEI-al/pBMP-2group than PEI-al/pEGFP group; Micro-CT anlysis indicated that in the PEI-al/pBMP-2group, there are irregular high density thing in the central of thedefect site and the edge of defect is irregular; And more new bone formation wasobserved in PEI-al/pBMP-2group than in the PEI-al group. HE staining showed thatthere are many bone cores in the PEI-al/pBMP-2group, while in the PEI-al groups alldefects were repaired with fibrous connective tissue.
The effects of PEI-al/pBMP-2engineered BMSCs cell sheet on bone formation invitro and in vivo
PEI-al nanocomposites carrying BMP-2gene could efficiently transfect BMSCs,and the BMP-2protein producing cell sheet was made by culturing the cells invitamin C containing medium for10days. To quantify the secretion of BMP-2protein,ELISA assay was performed; the capability of inducing osteogenic differentiation wasevaluated by realtime-PCR and ALP activity. EGFP producing cellsheet (EGFP/CS)and BMP-2producing cellsheet (BMP/CS) were implanted into the rat calvarialdefects in vivo, then the effect of bone regeneration was observed by Micro-CT andhistologic analysis. The results showed that the genetically engineered cells releasedthe BMP-2for at least14days; The expression of osteogenesis-related gene wasincreased, which demonstrated the released BMP-2could effectively induce the cellsheet osteogenic differentiation in vitro. Micro-CT and histologic analysis indicatedthat BMP/CS group was more efficient than other groups on promoting boneformation in the defect area.
Our study demonstrated that PEI-al nanocomposites could efficiently transfectBMSCs and the BMP-2gene engineered cell sheet could effectively enhance boneformation.
骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）是目前骨组织工程学中应用最为广泛的种子细胞，它具有多向分化的潜能，在一定条件下可以分化为骨、软骨、肌肉、脂肪等组织。而骨形态发生蛋白2（bone morphogeneticprotein2, BMP-2）是目前已知的骨诱导能力最强的生长因子，它可以直接诱导间充质干细胞向成骨方向分化。因此，常常利用BMP-2基因转染BMSCs以提高其成骨分化能力。
外源基因进入细胞通常需要基因载体，主要包括病毒载体和非病毒载体两类。病毒载体由于其较高的转染效率是目前应用最多的基因载体，但是作为一种改造的病毒，其价格昂贵，难以大规模生产，且存在安全隐患和免疫原性等问题，因此许多学者把注意力逐步转向对非病毒载体的研究。各种类型的聚乙烯亚胺（polyethylenimine, PEI）是最高效的非病毒载体之一，但是它仍然有许多缺点，如：无组织或细胞靶向性、相对于病毒载体转染效率较低以及具有较高的细胞毒性等。因此，为了克服PEI的这些缺点，需要对PEI进行改造。
支架材料也是骨组织工程学的主要因素之一。常见的支架材料主要包括天然聚合物、陶瓷材料、合成聚合物和复合材料。但是目前已开发或应用的支架材料都存在一些问题，如：引起炎症反应，降解速率不匹配，缺少生物活性等。因此，一种不需要外源性支架的细胞膜片技术的出现了。它以一种无创的方式将细胞以膜状结构收集，完整的保留了细胞和细胞之间的连接和细胞外基质，以及相关蛋白和生物活性因子，为体内骨损伤的愈合提供了适宜的微环境。
基于以上背景，本研究设计为以下三个部分：
一、聚乙烯亚胺-藻酸盐/质粒复合物的制备与表征
利用阳离子聚合物PEI与天然阴离子聚合物海藻酸钠（alginate）通过静电作用构建了PEI-alginate（PEI-al）纳米基因载体，并对其理化性质进行表征。结果显示：PEI-al纳米复合物的平均粒径为73.8nm，平均电势为+19.9mV；扫描电镜观察可见PEI-al纳米复合物为球形的单分散微粒，当PEI-al纳米复合物与BMP-2质粒（BMP-2plasmid, pBMP-2）结合后可发生部分聚集；通过纳米粒度仪测得PEI-al/pBMP-2的平均粒径增加到149.0nm，电势降低到+18.8mV。DNA凝胶阻滞实验结果表明当质量比达到1.5：1时，材料能够完全结合DNA，材料与基因的复合物为中性或是正电性。
二、PEI-al/pBMP-2复合物体内外促成骨的作用的研究
利用PEI-al/pBMP-2复合物转染MC3T3-E1细胞，通过realtime-PCR和茜素红染色研究其体外促成骨的作用；将PEI-al纳米复合物和PEI-al/pBMP-2复合物与明胶海绵支架分别复合后移植到大鼠的颅骨缺损内，观察它们体内促成骨的作用。结果显示：转染PEI-al/pBMP-2后，realtime-PCR检测成骨相关基因的表达都有上调，钙结节的形成量也明显多于转染了PEI-al/pEGFP的细胞；Micro-CT观察到PEI-al/pBMP-2组的大鼠颅骨缺损边缘不规则，内部有大量新生骨样组织，且明显多于PEI-al组；H&E染色结果也与Micro-CT相符， PEI-al/pBMP-2组缺损边缘有新生骨组织，缺损中心处也有一些编织状骨形成，而PEI-al组缺损处全部由纤维结缔组织修复。
三、PEI-al/pBMP-2复合物修饰的BMSCs膜片体内外促成骨作用的研究
利用PEI-al/pBMP-2复合物转染BMSCs后，在含有维生素C的培养基中继续培养10天，获得了能够分泌BMP-2蛋白的BMSCs细胞膜片。通过ELISA检测BMP-2分泌情况；通过realtime-PCR和碱性磷酸酶活性研究其体外促成骨的作用；将分泌EGFP蛋白的BMSCs细胞膜片（EGFP/CS）和分泌BMP-2蛋白的BMSCs细胞膜片（BMP/CS）分别移植到大鼠的颅骨缺损内，观察它们体内促成骨的作用。结果显示：在转染PEI-al/pBMP-2后，BMSCs能够持续分泌BMP-2蛋白达14天以上；realtime-PCR检测表明相关成骨基因的表达均有上调，这一结果说明分泌的BMP-2蛋白能够有效的诱导BMSCs细胞膜片向成骨方向分化；Micro-CT和组织学检测观察到BMP/CS组与其他组相比有效的促进了缺损处骨组织的再生。
以上实验结果表明：PEI-al纳米复合物是一种高效的基因载体；利用PEI-al/pBMP-2修饰的BMSCs细胞膜片能够有效的促进骨缺损的修复。
Bone loss caused by congenital defects, traumatic injury, cancer, reconstructivesurgery or periodontal disease is always the most difficult challenge both at home andabroad. Autologous bone graft is the so-called gold standard in transplantation surgery.But the use of autologous bone may be restricted because the amount of donor bone islimited and the complication in donor site. The strategies of allogenic or xenogenicare associated with problems such as the risk of disease transmission and graftrejection with a concomitant necessity for secondary surgery, and cost much.Synthetic grafts are important substitutes, but they fail to provide similar mechanicalproperties of human bone tissue, often causing failure in bone healing. Bone tissueengineering attempts to solve the referred problems mentioned above. Recently, gene-and stem cell-based therapy have been applied in bone tissue engineering andachieved some good clinical results.
Bone marrow mesenchymal stem cells (BMSCs) have been widely used for bonetissue engineering. They potentially can be used to develop bone，cartilage，muscle oradipose. Bone morphogenetic proteins (BMPs) are well known as osteoinductivegrowth factors which play an important role in bone regeneration process. Theseproteins are capable of inducing the osteogenic differentiation of mesenchymal cells.So BMP-2gene has been frequently used to induce the osteogenic differentiation ofBMSCs.
Gene vectors are required for the transfer of exogenous gene into cells, the mainapproach to gene delivery in gene therapy is viral vector systems due to theirrelatively high transfection efficiency. But viral-based gene vectors are a kind ofengineered viruses, so they have the drawbacks of high cost, immune response againstrepeated administration, oncogenicity and difficulty in large-scale production.Therefore, many scientists have shifted their interest to develop non-viral gene vectors.Different types of polyethyleneimine (PEI) are regarded as a kind of the most effective non-viral gene vectors. However, PEI has the disadvantage of no specifictissues and cells targeting, relative lower transgene efficiency compared with viralvectors and high cytotoxicity. In order to overcome the drawbacks, it is necessary tomodify PEI.
Scaffold is also a main factor of bone tissue engineering, including naturepolymer, ceramics, synthesis polymer and composite material. However, the scaffoldsstill face the problem of causing inflammation, the degradation rate of biomaterial notmatch the regeneration rate of bone and the lack of biological activity. So the newertechnology of cell sheet without scaffold has emerged. The cells are collected as asheet, which maintains the connections between cells and ECM, and the relativeproteins and biological activity factors. It also can provide a suitablemicroenvironment for bone regeneration.
Preparation and characterization of PEI-alginate/plasmid complexes
PEI-al gene carriers were prepared by electrostatic interation between the cationicPEI and polyanionic alginate, and physicochemical properties were characterized. Theresults showed that the average particle size of PEI-al nanocomposites were73.8nm,and the zeta potential distribution was+19.9mV; SEM images showed that PEI-alnanocomposites were spherical and monodispersed particles while some aggregationoccured when PEI-al nanocomposites were blended with pBMP-2; DLS resultsshowed that the average particle size of PEI-al/pBMP-2complexes were increased to149.0nm, and the zeta potential distribution was decreased to+18.8mV; Agarose gelelectrophoresis result suggested the formation of neutral or positive complexesbetween PEI-al and pBMP-2, when the weight ratio reaches1.5.
The effects of PEI-al/pBMP-2on bone formation in vitro and in vivo
PEI-al/pBMP-2complexes were transfected into MC3T3-E1cell line in vitro,and the capability of inducing osteogenic differentiation was evaluated byrealtime-PCR and alizarin red stain. PEI-al/pBMP-2complexes and PEI-alnanocomposites were absorbed into gelatin sponges, respectively, and the gelatinsponges were implanted into the rat calvarial defects in vivo. The effect of boneregeneration was observed by Micro-CT and histologic analysis. The results showedthat the expression of osteogenesis-related gene was increased post-transfected withPEI-al/pBMP-2complexes, and the calcium nodules formation was much more inPEI-al/pBMP-2group than PEI-al/pEGFP group; Micro-CT anlysis indicated that in the PEI-al/pBMP-2group, there are irregular high density thing in the central of thedefect site and the edge of defect is irregular; And more new bone formation wasobserved in PEI-al/pBMP-2group than in the PEI-al group. HE staining showed thatthere are many bone cores in the PEI-al/pBMP-2group, while in the PEI-al groups alldefects were repaired with fibrous connective tissue.
The effects of PEI-al/pBMP-2engineered BMSCs cell sheet on bone formation invitro and in vivo
PEI-al nanocomposites carrying BMP-2gene could efficiently transfect BMSCs,and the BMP-2protein producing cell sheet was made by culturing the cells invitamin C containing medium for10days. To quantify the secretion of BMP-2protein,ELISA assay was performed; the capability of inducing osteogenic differentiation wasevaluated by realtime-PCR and ALP activity. EGFP producing cellsheet (EGFP/CS)and BMP-2producing cellsheet (BMP/CS) were implanted into the rat calvarialdefects in vivo, then the effect of bone regeneration was observed by Micro-CT andhistologic analysis. The results showed that the genetically engineered cells releasedthe BMP-2for at least14days; The expression of osteogenesis-related gene wasincreased, which demonstrated the released BMP-2could effectively induce the cellsheet osteogenic differentiation in vitro. Micro-CT and histologic analysis indicatedthat BMP/CS group was more efficient than other groups on promoting boneformation in the defect area.
Our study demonstrated that PEI-al nanocomposites could efficiently transfectBMSCs and the BMP-2gene engineered cell sheet could effectively enhance boneformation.
引文
[1] Kolar P, Schmidt-Bleek K, Schell H, et al. The early fracture hematoma and itspotential role in fracture healing [J]. Tissue Eng Part B Rev.2010,16:427-434.
[2] Rosset P, Deschaseaux F, Layrolle P. Cell therapy for bone repair [J], Orthopaedics&Traumatology: Surgery&Research.2014,100(1):107-112
[3] Gaber T, Dziurla R, Tripmacher R, et al. Hypoxia inducible factor (HIF) inrheumatology: low O2! See what HIF can do [J]! Ann Rheum Dis.2005,64:971-980.
[4] Wan C, Gilbert SR,Wang Y, et al. Role of hypoxia inducible factor-1alpha pathwayin bone regeneration [J], J Musculoskelet Neuronal Interact.2008,8:323-324.
[5] Wang Y, Wan C, Deng L, et al. The hypoxia-inducible factor alpha pathwaycouples angiogenesis to osteogenesis during skeletal development [J]. J ClinInvest.2007,117:1616-1626.
[6] Wilson CJ, Kasper G, Schutz MA, et al. Cyclic strain disrupts endothelial networkformation on Matrigel [J], Microvasc Res.2009,78:358-363.
[7] Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-nataldevelopmental process: molecular, spatial, and temporal aspects of its regulation[J], J Cell Biochem.2003,88:873-884.
[8] Ponte AL, Marais E, Gallay N, et al. The in vitro migration capacity of humanbone marrow mesenchymal stem cells: comparison of chemokine and growthfactor chemotactic activities [J]. Stem Cells.2007,25:1737-1745.
[9] Gerstenfeld LC, Cho TJ, Kon T, et al. Impaired fracture healing in the absence ofTNF-alpha signaling: the role of TNF-alpha in endochondral cartilage resorption[J]. J Bone Miner Res.2003,18:1584-1592.
[10] Gerstenfeld LC, Cho TJ, Kon T, et al. Impaired intramembranous bone formationduring bone repair in the absence of tumor necrosis factor-alpha signaling [J],Cells Tissues Organs.2001,169:285-294.
[11] Raheja LF, Genetos DC, Wong A, et al. Hypoxic regulation ofmesenchymal stemcell migration: the role of RhoA and HIF-1alpha [J], Cell Biol Int.2011,35(10):981-989.
[12] Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression ofmembers of the transforming growth factor beta superfamily during murinefracture healing [J]. J Bone Miner Res.2002,17:513-520.
[13] Keramaris NC, Calori GM, Nikolaou VS, et al. Fracture vascularity and bonehealing: a systematic review of the role of VEGF [J]. Injury.2008,39:45–57.
[14] Einhorn TA, The cell and molecular biology of fracture healing [J]. Clin OrthopRelat Res.1998,7-21.
[15] Marsh D. Concepts of fracture union, delayed union, and nonunion[J]. ClinOrthop Relat R1998,22-30.
[16] Muscolo DL, Ayerza MA, Aponte-Tinao LA. Massive allograft use in orthopediconcology [J], Orthop Clin N Am.2006,37(1):(65-74).
[17] DeCoster TA, Gehlert R, Mikola EA, et al. Management of posttraumaticsegmental bone defects [J]. J Am Acad Orthop Surg.2004,12:28-38.
[18] Neovius E, Engstrand T. Craniofacial reconstruction with bone and biomaterials:Review over the last11years [J]. J Plast Reconstr Aesthet Surg.2010,63(10):1615-1623.
[19] Bostrom MP, Seigerman DA. The clinical use of allografts, demineralized bonematrices, synthetic bone graft substitutes and osteoinductive growthfactors: asurvey study [J]. HSS J.2005,1(1):9-18.
[20] Aubin JE. Bone stem cells [J]. J Cell Biochem,1998;30-31(suppl):723.
[21] Weissman IL. Stem cells: units of development, units of regeneration, and unitsin evolution [J]. Cell.2000,100:157-168.
[22] Evans MJ and Kaufman MH. Establishment in culture of pluripotential cells frommouse embryos [J]. Nature.1981,292:154-156.
[23] Robertson EJ. Derivation and maintenance of embryonic stem cell cultures [J].Methods Mol Biol.1990,5:223-236.
[24] Lu CC, Brennan J, Robertson EJ. From fertilization to gastrulation: axisformation in the mouse embryo [J]. Curr Opin Genet Dev.2001,11:384-392.
[25] Valdimarsdotter G and Mummery C. Functions of the TGF-b superfamily inhuman embryonic stem cells [J]. APMIS.2005,113:773-789.
[26] Morali OG, Jouneau A, McLaughlin KJ, et al. IGF-II promotes mesodermformation [J]. Dev Biol.2000,227:133-45.
[27] Hwang YS, Chung BG, Ortmann D, et al. Microwell mediated control ofembryoid body size regulates embryonic stem cell fate via differential expressionof WNT5a and WNT11[J]. Proc Natl Acad Sci USA.2009b,106:16978-16983.
[28] Hwang YS, Randle WL, Bielby R, et al. Enhanced derivation of osteogenic cellsfrom murine embryonic stem cells after treatment with HepG2-conditionedmedium and modulation of the embryoid body formation period: application toskeletal tissue engineering [J]. Tissue Eng.2006,12:1381-1392.
[29] Friedenstein AJ, Pettakova KV, Kurolesova AI, et al. Heterotopic transplants ofbone marrow. Analysis of precursor cells for osteogenic and hematopoietictissues [J]. Transplantation.1968,6:230-247.
[30] Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation andhealing by stem cell-expressed VEGF and bone morphogeneticprotein-4[J]. JClin Invest.2002,110:751-759.
[31] Caplan AI. Mesenchymal stem cells: cell-based reconstructive therapy inorthopedics [J]. Tissue Eng.2005,11:1198-1211.
[32] Bosnakovski D, Mizuno M, Kim G, et al. Isolation and multilineagedifferentiation of bovine bone marrow mesenchymal stem cells [J]. Cell TissueRes.2005,319:243-253.
[33] Yamagiwa H, Endo N, Tokunaga K, et al. In vivo bone-forming capacity ofhuman bone marrow-derived stromal cells is stimulated by recombinant humanbone morphogenetic progein-2[J]. J. Bone Miner. Metab.2001,19:20-28.
[34] Miao D, Tong XK, Chan GK, et al. Parathyroid hormone-related peptidestimulates osteogenic cell proliferation through protein kinase C activation of theras/mitogen-activated progein kinase signaling pathway [J]. J Biol Chem.2001,276:32204-32213.
[35] Covas DT, Siufi JLC, Silva ARL, et al. Isolation and culture of umbilical veinmesenchymal stem cells [J]. Braz J Med Biol Res.2003,36:1179-1183.
[36] Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sourceof postnatal human mesenchymal stem cells: candidate MSC-like cells fromumbilical cord [J]. Stem Cells.2003,21:105-110.
[37] Diao Y, Ma Q, Cui F,et al. Human umbilical cord mesenchymal stem cells:osteogenesis in vivo as seed cells for bone tissue engineering [J]. J BiomedMater Res.2009,91:123-131.
[38] Rosada C, Justesen J, Melsvik D, et al. The human umbilical cord blood: apotential source for osteoblast progenitor cells [J]. Calcif Tissue Int.2003,72:135-142.
[39] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng.2001,7(2):221-226.
[40] Bunnell BA, Flaat M, Gagliardi C, et al. Adipose-derived stem cells: isolation,expansion and differentiation [J]. Methods.2008,45:115-120.
[41] Dragoo JL, Choi JY, Lieberman JR, et al. Bone induction by BMP-2transducedstem cells derived from human fat [J]. J Orthop Res,2003,21(4):622-629.
[42] Miyazono K, Maeda S, Imamura T. BMP receptor signaling: transcriptionaltargets, regulation of signals, and signaling cross-talk [J]. Cytokine GrowthFactor Rev.2005,16:251-263.
[43] Ducy P, Zhang R, Geoffroy V, et al. Osf2/Cbfa1: a transcriptional activator ofosteoblast differentiation [J]. Cell.1997,89:747-754.
[44] Ishikawa S, Iwasaki K, Komaki M, et al. Role of ascorbic acid in periodontalligament cell differentiation [J]. J Periodontol.2004,75:709-716.
[45] Sugawara Y, Suzuki K, Koshikawa M, et al. Necessity of enzymatic activity ofalkaline phosphatase for mineralization of osteoblastic cells [J]. Japan JPharmacol.2002,88:262-269.
[46] Li X, Feng Q, Liu X, et al. Collagen-based implants reinforced by chitin fibres ina goat shank bone defect model [J]. Biomaterials.2006,27:1917-1923.
[47] Cruz DM, Gomes M, Reis RL, et al. Differentiation of mesenchymal stem cellsin chitosan scaffolds with double micro and macroporosity [J]. J Biomed MaterRes A.2010,95:1182-1193.
[48] Tanaka K, Goto T, Miyazaki T, et al. Apatite-coated hyaluronan for boneregeneration [J]. J Dent Res.2011,90:906-911.
[49] Fuji T, Anada T, Honda Y, et al. Octacalcium phosphate–precipitated alginatescaffold for bone regeneration [J]. Tissue Eng A.2009,15:3525-3535.
[50] Lichte P, Pape HC, Pufe T, et al. Scaffolds for bone healing: concepts, materialsand evidence [J]. Injury.2011,42:569-573.
[51] Porter JR, Ruckh TT, Popat KC. Bone tissue engineering: a review in bonebiomimetics and drug delivery strategies [J]. Biotechnol Prog.2009,25:1539-1560.
[52] Cancedda R, Giannoni P, Mastrogiacomo M. A tissue engineering approach tobone repair in large animal models and in clinical practice [J]. Biomaterials.2007,28:4240-4250.
[53] Yaszemski MJ, Payne RG, Hayes WC, et al. Evolution of bone transplantation:molecular, cellular and tissue strategies to engineer human bone [J]. Biomaterials.1996,17:175-185.
[54] Okano T, Yamada N, Sakai H, et al. A novel recovery system for cultured cells usingplasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide)[J]. JBiomed Mater Res.1993,27(10):1243-1251.
[55] Yang J, Yamato M, Kohno C, et al. Cell sheet engineering: Recreating tissueswithout biodegradable scaffolds [J]. Biomaterials.2005,26:6415-6422.
[56] Nakamura A, Akahane M, Shigematsu H, et al. Cell sheet transplantation ofcultured mesenchymal stem cells enhances bone formation in a rat nonunionmodel [J]. Bone.2010,46:418-424.
[57] Wei F, Qu C, Song T, et al. Vitamin C treatment promotes mesenchymal stem cellsheet formation and tissue regeneration by elevating telomerase activity [J]. JCell Physiol.2012,227:3216-3224.
[58] Donahue RE, Kessler SW, Bodine D, et al. Helper virus induced T celllymphoma in nonhuman primates after retroviral mediated gene transfer [J]. JExp Med.1992,176:1125-1135.
[59] Schambach A, Zychlinski D, Ehrnstroem B, et al. Biosafety features of lentiviralvectors [J]. Hum Gene Ther.2013,24:132-142.
[60] Marshall E. Gene therapy death prompts review of adenovirus vector [J]. Science.1999,286:2244-2245.
[61] Yan Z，Zhang Y, Duan D，et al. From the cover: trans-splicing vectors expand theutility of adeno-associated virus for gene therapy [J]. Proc Natl Acad Sci U S A.2000,97(12):6716-6721.
[62] Torchilin VP. Recent advances with liposomes as pharmaceutical carriers [J]. NatRev Drug Discov.2005,4:145-160.
[63] Simoes S, Filipe A, Faneca H, et al. Cationic liposomes for gene delivery [J].Expert Opin Drug Deliv.2005,2:237-254.
[64] Felgner PL, Ringold GM. Cationic liposome-mediated transfection [J]. Nature.1989,337:387-388.
[65] El Ouahabi A, Thiry M, Pector V, et al. The role of endosome destabilizingactivity in the gene transfer process mediated by cationic lipids [J]. FEBS Lett.1997,414:187-192.
[66] Nah JW, Yu L, Han SO, et al. Artery wall binding peptide-poly(ethyleneglycol)-grafted-poly(L-lysine)-based gene delivery to artery wall cells [J]. JControl Release.2002,78:273-284.
[67] Fischer D, Bieber T, Li Y, et al. A novel nonviral vector for DNA delivery basedon low molecular weight, branched polyethylenimine: effect of molecular weighton transfection efficiency and cytotoxicity [J]. Pharm Res.1999,16:1273-1279.
[68] Godbey WT, Barry MA, Saggau P, et al. Poly(ethylenimine)-mediatedtransfection: a new paradigm for gene delivery [J]. J Biomed Mater Res.2000,51(3):321-328.
[69] Dunlap DD, Maggi A, Soria MR, et al. Nanoscopic structure of DNA condensedfor gene delivery [J]. Nucleic Acids Res.1997,25(15):3095-3101.
[70] Reschel T, Konak C, Oupicky D, et al. Physical properties and in vitrotransfection efficiency of gene delivery vectors based on complexes of DNA withsynthetic polycations [J]. J Control Release.2002,81(1-2):201-217.
[71] Tang GP, Guo HY, Alexis F, et al. Low molecular weight polyethylenimineslinked by beta-cyclodextrin for gene transfer into the nervous system [J]. J GeneMed.2006,8(6):736-744.
[72] Jiang HL, Kim YK, Arote R, et al. Chitosan-graft-polyethylenimine as a genecarrier [J]. J Control Release.2007,117:273-280.
[73] Gebhart CL, Sriadibhatla S, Vinogradov S, et al. Design and formulation ofpolyplexes based on pluronicpolyethyleneimine conjugates for gene transfer [J].Bioconjug Chem.2002,13:937-944.
[74] Mishra S, Webster P, Davis ME. PEGylation significantly affects cellular uptakeand intracellular trafficking of non-viral gene delivery particles [J]. Eur J CellBiol.2004,83:97-111.
[75] Patnaik S, Aggarwal A, Nimesh S, et al. PEI-alginate nanocomposites as efficientin vitro gene transfection agents [J]. J Control Release.2006,114:398-409.
[76] Grosse S, Aron Y, Honore I, et al. Lactosylated polyethyleneimine for genetransfer into airway epithelial cells: role of the sugar moiety in cell delivery andintracellular trafficking of the complexes [J]. J. Gene Med.2004,6:345-356.
[77] Franceschi RT, Yang S, Rutherford RB, et al. Gene therapy approaches for boneregeneration [J]. Cells Tissues Organs.2004,176:95-108.
[78] Reddi AH. Initiation of fracture repair by bone morphogenetic proteins [J]. ClinOrthop.1998,355(Suppl):66-77.
[79] Rutherford RB, Moalli M, Franceschi RT, et al. Bone morphogeneticprotein-transduced human fibroblast convert to osteoblasts and formation bone invivo [J]. Tissue Eng.2002,8(3):441-452.
[80] Dumont RJ, Dayoub H, Li JZ, et al. Ex vivo bone morphogenetic protein-9genetherapy using human mesenchymal stem cells induces spinal fusion in rodents [J].Neurosugery.2002,51(5):1239-1244.
[81]Lee JY, Peng H, Usas A, et al. Enhancement of bone healing based on ex vivogene therapy using human musclederived cells expressing bone morphogeneticprotein-2[J]. Hum Gene Ther.2002,13(10):1201-1211.
[82] Baltzer AWA, Lattermann C, Whalen JD, et al. Genetic enhancement of fracturerepair: healing of an experimental segmental defect by adenoviral transfer of theBMP-2gene [J]. Gene Ther.2000,7:734-739.
[83] Betz OB, Betz VM, Nazarian A, et al. Direct percutaneous gene delivery toenhance healing of segmental bone defects [J]. J Bone Joint Surg Am.2006,88A:355-365.
[84] Betz OB, Betz VM, Nazarian A, et al. Delayed administration of adenoviralBMP-2vector improves the formation of bone in osseous defects [J]. Gene Ther.2007a,14:1039-1044.
[85] Betz VM, Betz OB, Glatt V, et al. Healing of segmental bone defects by directpercutaneous gene delivery: effect of vector dose [J]. Hum Gene Ther.2007b,18:907-915.
[86] Liu YG, Zhou Y, Hu X, et al. Effect of vascular endothelial growth factor121adenovirus transduction in rabbit model of femur head necrosis. J Trauma.2011,70:1519-1523.
[87] Kimelman-Bleich N, Pelled G, Zilberman Y, et al. Targeted gene-and-hostprogenitor cell therapy for nonunion bone fracture repair [J]. Mol Ther.2011,19:53-59.
[88] Pelled G, Ben-Arav A, Hock C, et al. Direct gene therapy for bone regeneration:gene delivery, animal models, and outcomemeasures [J]. Tissue Eng Part B Rev.2010,16:13-20.
[89] Evans CH, Liu FJ, Glatt V, et al. Use of genetically modified muscle and fatgrafts to repair defects in bone and cartilage [J]. Eur Cell Mater.2009c,18:96-111.
[90] Liu F, Porter RM, Wells J, et al. Evaluation of BMP-2gene-activated musclegrafts for cranial defect repair [J]. J Orthop Res.2012a,30:1095-1102.
[91] Ito H, Koefoed M, Tiyapatanaputi P, et al. Remodeling of cortical bone allograftsmediated by adherent rAAV-RANKL and VEGF gene therapy [J]. Nat Med.2005,11:291-297.
[92] Yazici C, Takahata M, Reynolds DG, et al. Self-complementaryAAV2.5-BMP2-coated femoral allografts mediated superior bone healing versuslive autografts in mice with equivalent biomechanics to unfractured femur [J].Mol Ther.2011,19:1416-1425.
[93] Ben Arav A, Pelled G, Zilberman Y, et al. Adeno-associated virus-coatedallografts: a novel approach for cranioplasty [J]. J Tissue Eng Regen Med.2012,6:43-50.
[94] Lieberman JR, Daluiski A, Stevenson S, et al. The effect of regional gene therapywith bone morphogenetic protein-2-producing bone-marrow cells on the repairof segmental femoral defects in rats [J]. J Bone Joint Surg Am.1999,81:905-917.
[95] Lieberman JR, Le LQ, Wu L, et al. Regional gene therapy with aBMP-2-producing murine stromal cell line induces heterotopic and orthotopicbone formation in rodents [J]. J Orthop Res.1998,16:330–339.
[96] Peterson B, Iglesias R, Zhang J, et al. Genetically modified human derived bonemarrow cells for posterolateral lumbar spine fusion in athymic rats: beyondconventional autologous bone grafting [J]. Spine.2005,30:283-289.
[97] Miyazaki M, Sugiyama O, Tow B, et al. The effects of lentiviral gene therapywith bone morphogenetic protein-2-producing bone marrow cells on spinalfusion in rats [J]. J Spinal Disord Tech.2008,21:372–379.
[98] Miyazaki M, Sugiyama O, Zou J, et al. Comparison of lentiviral and adenoviralgene therapy for spinal fusion in rats [J]. Spine.2008,33:1410-1417.
[99] Peterson B, Zhang J, Iglesias R, et al. Healing of critically sized femoral defects,using genetically modified mesenchymal stem cells from human adipose tissue[J]. Tissue Eng.2005,11:120-129.
[100] Breitbart AS, Grande DA, Mason JM, et al. Gene-enhanced tissue engineering:applications for bone healing using cultured periosteal cells transducedretrovirally with the BMP-7gene [J]. Ann Plast Surg.1999,42:488-495.
[101] Lee JY, Musgrave D, Pelinkovic D, et al. Effect of bone morphogeneticprotein-2-expressing muscle-derived cells on healing of critical-sized bonedefects in mice [J]. J Bone Joint Surg Am.2001,83-A:1032-1039.
[102] Ishihara A, Zekas LJ, Litsky AS, et al. Dermal fibroblast-mediated BMP2therapy to accelerate bone healing in an equine osteotomy model [J]. J OrthopRes.2010,28:403-411.
[102] Shin JH, Kim KH, Kim SH, et al. Ex vivo bone morphogenetic protein-2genedelivery using gingival fibroblasts promotes bone regeneration in rats [J]. J ClinPeriodontol.2010,37:305–311.
[104] Park J, Ries J, Gelse K, et al. Bone regeneration in critical size defects bycellmediated BMP-2gene transfer: a comparison of adenoviral vectors andliposomes [J]. Gene Ther.2003,10(13):1089-1098.
[105] Jinqushi S, Scully SP, Joyce ME, et al. Transforming growth factor-beta1andfibroblast growth factors in rat growth plate [J]. J Orthop Res.1995,13(5):761-768.
[106] Thaller SR, Dart A, Tesluk H. The effects of insulin-like growth factor-1oncritical-size calvarial defects in Sprague–Dawley rats [J]. Ann Plast Surg.1993,31(5):429-433.
[107] Pfeilschifter J, Diel L, Pilz U, et al. Mitogenic responsiveness of human bonecells in vitro to hormones and growth factors decreases with age [J]. J BoneMiner Res,1993,8(6):707-717.
[108] Aebli N, Stich H, Schawalder P, et al. Effects of bone morphogenetic protein-2and hyaluronic acid on the osseointegration of hydroxyapatite-coated implants:an experimental study in sheep [J]. J Biomed Mater Res. A.2005,73(3):295-302.
[109] Saito A, Suzuki Y, Ogata S, et al. Prolonged ectopic calcification induced byBMP-2-derived synthetic peptide [J]. J Biomed Mater Res A.2004;70(1):115-121.
[110] Lucas PA, Laurencin C, Syftestad GT, et al. Ectopic induction of cartilage andbone by water-soluble proteins from bovine bone using a polyanhydridedelivery vehicle [J]. J Biomed Mater Res.1990,24(7):901-911.
[111] Terrell TG, Working PK, Chow CP, et al. Pathology of recombinant humantransforming growth factor-beta1in rats and rabbits [J]. Int Rev Exp Pathol.1993,34(Pt B):43-67.
[112] Bonadio J. Tissue engineering via local gene delivery: update and futureprospects for enhancing the technology [J]. Adv Drug Deliv Rev.2000,44:185-194.
[113] Ryoo HM, Lee MH, Kim YJ. Critical molecular switches involved inBMP-2-induced osteogenic differentiation of mesenchymal cells [J]. Gene.2006,366:51-57.
[114] Boerckel JD, Kolambkar YM, Dupont KM，et al. Effects of protein dose anddelivery system on BMP-mediated bone regeneration [J]. Biomaterials.2011,32(22):5241-5251.
[115] Lee JW, Kang KS, Lee SH, et al. Bone regeneration using amicrostereolithography-produced customized poly (propylene fumarate)/diethylfumarate photopolymer3D scaffold incorporating BMP-2loaded PLGAmicrospheres [J]. Biomaterials.2011,32:744-752.
[116] Sojo K, Sawaki Y, Hattori H, et al. Immunohistochemical study of vascularendothelial growth factor (VEGF) and bone morphogenetic protein-2,-4(BMP-2,-4) on lengthened rat femurs[J]. Journal of Cranio-MaxillofacialSurgery.2005,33(4):238–245.
[117] Luvizuto ER, Tangl S, Zanoni G, Okamoto T, et al. The effect of BMP-2on theosteoconductive properties of b-tricalcium phosphate in rat calvaria defects [J].Biomaterials.2011,32(15):3855-3861.
[118] Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors for fibroblastsin different populations of hematopoietic cells as detected by in vitro colonyassay method [J]. Exp Hematol.1974,2(2):83-92.
[119] Dazzi F, Ramasamy R, Glennie S, et al. The role of mesenchymal stem cells inhaemopoiesis [J]. Blood Rev.2006,20(3):161-171.
[120] Mareschi K, Ferrero I, Rustichelli D, et al. Expansion of mesenchymal stemcells isolated from pediatric and adult donor bone marrow [J]. J Cell Biochem.2006,97(4):744-754.
[121] Prockop DJ. Marrow stromal cells as stem cells for non hematopoietic tissues[J]. Science.1997,276(5309):71-74.
[122] Friedenstein AJ, Ivanov-Smolenski AA, Chajlakjan RK, et al. Origin of bonemarrow stromal mechanocytes in radiochimeras and heterotopic transplants [J].Experimental hematology.1978,6(5):440-444.
[123] Rosen V. BMP2signaling in bone development and repair [J]. Cytokine GrowthFactor Rev.2009,20(5-6):475-480.
[124] Riew KD, Wright NM, Cheng S, et al. Induction of bone formation using arecombinant adenoviral vector carrying the human BMP-2gene in a rabbitspinal fusion model [J]. Calcif Tissue Int.1998,63(4):357-360.
[125] Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge intissue engineering [J]. Advanced Drug Delivery Reviews.2011,63,300-311.
[126] Fang J, Zhu YY, Smiley E, et al. Stimulation of new bone formation by directtransfer of osteogenic plasmid genes [J]. Proc Natl Acad Sci U S A.1996,93:5753-5758.
[127] Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formationand healing by stem cell-expressed VEGF and bone morphogenetic protein-4[J].J Clin Invest.2002,110:751-759.
[2] Rosset P, Deschaseaux F, Layrolle P. Cell therapy for bone repair [J], Orthopaedics&Traumatology: Surgery&Research.2014,100(1):107-112
[3] Gaber T, Dziurla R, Tripmacher R, et al. Hypoxia inducible factor (HIF) inrheumatology: low O2! See what HIF can do [J]! Ann Rheum Dis.2005,64:971-980.
[4] Wan C, Gilbert SR,Wang Y, et al. Role of hypoxia inducible factor-1alpha pathwayin bone regeneration [J], J Musculoskelet Neuronal Interact.2008,8:323-324.
[5] Wang Y, Wan C, Deng L, et al. The hypoxia-inducible factor alpha pathwaycouples angiogenesis to osteogenesis during skeletal development [J]. J ClinInvest.2007,117:1616-1626.
[6] Wilson CJ, Kasper G, Schutz MA, et al. Cyclic strain disrupts endothelial networkformation on Matrigel [J], Microvasc Res.2009,78:358-363.
[7] Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-nataldevelopmental process: molecular, spatial, and temporal aspects of its regulation[J], J Cell Biochem.2003,88:873-884.
[8] Ponte AL, Marais E, Gallay N, et al. The in vitro migration capacity of humanbone marrow mesenchymal stem cells: comparison of chemokine and growthfactor chemotactic activities [J]. Stem Cells.2007,25:1737-1745.
[9] Gerstenfeld LC, Cho TJ, Kon T, et al. Impaired fracture healing in the absence ofTNF-alpha signaling: the role of TNF-alpha in endochondral cartilage resorption[J]. J Bone Miner Res.2003,18:1584-1592.
[10] Gerstenfeld LC, Cho TJ, Kon T, et al. Impaired intramembranous bone formationduring bone repair in the absence of tumor necrosis factor-alpha signaling [J],Cells Tissues Organs.2001,169:285-294.
[11] Raheja LF, Genetos DC, Wong A, et al. Hypoxic regulation ofmesenchymal stemcell migration: the role of RhoA and HIF-1alpha [J], Cell Biol Int.2011,35(10):981-989.
[12] Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression ofmembers of the transforming growth factor beta superfamily during murinefracture healing [J]. J Bone Miner Res.2002,17:513-520.
[13] Keramaris NC, Calori GM, Nikolaou VS, et al. Fracture vascularity and bonehealing: a systematic review of the role of VEGF [J]. Injury.2008,39:45–57.
[14] Einhorn TA, The cell and molecular biology of fracture healing [J]. Clin OrthopRelat Res.1998,7-21.
[15] Marsh D. Concepts of fracture union, delayed union, and nonunion[J]. ClinOrthop Relat R1998,22-30.
[16] Muscolo DL, Ayerza MA, Aponte-Tinao LA. Massive allograft use in orthopediconcology [J], Orthop Clin N Am.2006,37(1):(65-74).
[17] DeCoster TA, Gehlert R, Mikola EA, et al. Management of posttraumaticsegmental bone defects [J]. J Am Acad Orthop Surg.2004,12:28-38.
[18] Neovius E, Engstrand T. Craniofacial reconstruction with bone and biomaterials:Review over the last11years [J]. J Plast Reconstr Aesthet Surg.2010,63(10):1615-1623.
[19] Bostrom MP, Seigerman DA. The clinical use of allografts, demineralized bonematrices, synthetic bone graft substitutes and osteoinductive growthfactors: asurvey study [J]. HSS J.2005,1(1):9-18.
[20] Aubin JE. Bone stem cells [J]. J Cell Biochem,1998;30-31(suppl):723.
[21] Weissman IL. Stem cells: units of development, units of regeneration, and unitsin evolution [J]. Cell.2000,100:157-168.
[22] Evans MJ and Kaufman MH. Establishment in culture of pluripotential cells frommouse embryos [J]. Nature.1981,292:154-156.
[23] Robertson EJ. Derivation and maintenance of embryonic stem cell cultures [J].Methods Mol Biol.1990,5:223-236.
[24] Lu CC, Brennan J, Robertson EJ. From fertilization to gastrulation: axisformation in the mouse embryo [J]. Curr Opin Genet Dev.2001,11:384-392.
[25] Valdimarsdotter G and Mummery C. Functions of the TGF-b superfamily inhuman embryonic stem cells [J]. APMIS.2005,113:773-789.
[26] Morali OG, Jouneau A, McLaughlin KJ, et al. IGF-II promotes mesodermformation [J]. Dev Biol.2000,227:133-45.
[27] Hwang YS, Chung BG, Ortmann D, et al. Microwell mediated control ofembryoid body size regulates embryonic stem cell fate via differential expressionof WNT5a and WNT11[J]. Proc Natl Acad Sci USA.2009b,106:16978-16983.
[28] Hwang YS, Randle WL, Bielby R, et al. Enhanced derivation of osteogenic cellsfrom murine embryonic stem cells after treatment with HepG2-conditionedmedium and modulation of the embryoid body formation period: application toskeletal tissue engineering [J]. Tissue Eng.2006,12:1381-1392.
[29] Friedenstein AJ, Pettakova KV, Kurolesova AI, et al. Heterotopic transplants ofbone marrow. Analysis of precursor cells for osteogenic and hematopoietictissues [J]. Transplantation.1968,6:230-247.
[30] Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation andhealing by stem cell-expressed VEGF and bone morphogeneticprotein-4[J]. JClin Invest.2002,110:751-759.
[31] Caplan AI. Mesenchymal stem cells: cell-based reconstructive therapy inorthopedics [J]. Tissue Eng.2005,11:1198-1211.
[32] Bosnakovski D, Mizuno M, Kim G, et al. Isolation and multilineagedifferentiation of bovine bone marrow mesenchymal stem cells [J]. Cell TissueRes.2005,319:243-253.
[33] Yamagiwa H, Endo N, Tokunaga K, et al. In vivo bone-forming capacity ofhuman bone marrow-derived stromal cells is stimulated by recombinant humanbone morphogenetic progein-2[J]. J. Bone Miner. Metab.2001,19:20-28.
[34] Miao D, Tong XK, Chan GK, et al. Parathyroid hormone-related peptidestimulates osteogenic cell proliferation through protein kinase C activation of theras/mitogen-activated progein kinase signaling pathway [J]. J Biol Chem.2001,276:32204-32213.
[35] Covas DT, Siufi JLC, Silva ARL, et al. Isolation and culture of umbilical veinmesenchymal stem cells [J]. Braz J Med Biol Res.2003,36:1179-1183.
[36] Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sourceof postnatal human mesenchymal stem cells: candidate MSC-like cells fromumbilical cord [J]. Stem Cells.2003,21:105-110.
[37] Diao Y, Ma Q, Cui F,et al. Human umbilical cord mesenchymal stem cells:osteogenesis in vivo as seed cells for bone tissue engineering [J]. J BiomedMater Res.2009,91:123-131.
[38] Rosada C, Justesen J, Melsvik D, et al. The human umbilical cord blood: apotential source for osteoblast progenitor cells [J]. Calcif Tissue Int.2003,72:135-142.
[39] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng.2001,7(2):221-226.
[40] Bunnell BA, Flaat M, Gagliardi C, et al. Adipose-derived stem cells: isolation,expansion and differentiation [J]. Methods.2008,45:115-120.
[41] Dragoo JL, Choi JY, Lieberman JR, et al. Bone induction by BMP-2transducedstem cells derived from human fat [J]. J Orthop Res,2003,21(4):622-629.
[42] Miyazono K, Maeda S, Imamura T. BMP receptor signaling: transcriptionaltargets, regulation of signals, and signaling cross-talk [J]. Cytokine GrowthFactor Rev.2005,16:251-263.
[43] Ducy P, Zhang R, Geoffroy V, et al. Osf2/Cbfa1: a transcriptional activator ofosteoblast differentiation [J]. Cell.1997,89:747-754.
[44] Ishikawa S, Iwasaki K, Komaki M, et al. Role of ascorbic acid in periodontalligament cell differentiation [J]. J Periodontol.2004,75:709-716.
[45] Sugawara Y, Suzuki K, Koshikawa M, et al. Necessity of enzymatic activity ofalkaline phosphatase for mineralization of osteoblastic cells [J]. Japan JPharmacol.2002,88:262-269.
[46] Li X, Feng Q, Liu X, et al. Collagen-based implants reinforced by chitin fibres ina goat shank bone defect model [J]. Biomaterials.2006,27:1917-1923.
[47] Cruz DM, Gomes M, Reis RL, et al. Differentiation of mesenchymal stem cellsin chitosan scaffolds with double micro and macroporosity [J]. J Biomed MaterRes A.2010,95:1182-1193.
[48] Tanaka K, Goto T, Miyazaki T, et al. Apatite-coated hyaluronan for boneregeneration [J]. J Dent Res.2011,90:906-911.
[49] Fuji T, Anada T, Honda Y, et al. Octacalcium phosphate–precipitated alginatescaffold for bone regeneration [J]. Tissue Eng A.2009,15:3525-3535.
[50] Lichte P, Pape HC, Pufe T, et al. Scaffolds for bone healing: concepts, materialsand evidence [J]. Injury.2011,42:569-573.
[51] Porter JR, Ruckh TT, Popat KC. Bone tissue engineering: a review in bonebiomimetics and drug delivery strategies [J]. Biotechnol Prog.2009,25:1539-1560.
[52] Cancedda R, Giannoni P, Mastrogiacomo M. A tissue engineering approach tobone repair in large animal models and in clinical practice [J]. Biomaterials.2007,28:4240-4250.
[53] Yaszemski MJ, Payne RG, Hayes WC, et al. Evolution of bone transplantation:molecular, cellular and tissue strategies to engineer human bone [J]. Biomaterials.1996,17:175-185.
[54] Okano T, Yamada N, Sakai H, et al. A novel recovery system for cultured cells usingplasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide)[J]. JBiomed Mater Res.1993,27(10):1243-1251.
[55] Yang J, Yamato M, Kohno C, et al. Cell sheet engineering: Recreating tissueswithout biodegradable scaffolds [J]. Biomaterials.2005,26:6415-6422.
[56] Nakamura A, Akahane M, Shigematsu H, et al. Cell sheet transplantation ofcultured mesenchymal stem cells enhances bone formation in a rat nonunionmodel [J]. Bone.2010,46:418-424.
[57] Wei F, Qu C, Song T, et al. Vitamin C treatment promotes mesenchymal stem cellsheet formation and tissue regeneration by elevating telomerase activity [J]. JCell Physiol.2012,227:3216-3224.
[58] Donahue RE, Kessler SW, Bodine D, et al. Helper virus induced T celllymphoma in nonhuman primates after retroviral mediated gene transfer [J]. JExp Med.1992,176:1125-1135.
[59] Schambach A, Zychlinski D, Ehrnstroem B, et al. Biosafety features of lentiviralvectors [J]. Hum Gene Ther.2013,24:132-142.
[60] Marshall E. Gene therapy death prompts review of adenovirus vector [J]. Science.1999,286:2244-2245.
[61] Yan Z，Zhang Y, Duan D，et al. From the cover: trans-splicing vectors expand theutility of adeno-associated virus for gene therapy [J]. Proc Natl Acad Sci U S A.2000,97(12):6716-6721.
[62] Torchilin VP. Recent advances with liposomes as pharmaceutical carriers [J]. NatRev Drug Discov.2005,4:145-160.
[63] Simoes S, Filipe A, Faneca H, et al. Cationic liposomes for gene delivery [J].Expert Opin Drug Deliv.2005,2:237-254.
[64] Felgner PL, Ringold GM. Cationic liposome-mediated transfection [J]. Nature.1989,337:387-388.
[65] El Ouahabi A, Thiry M, Pector V, et al. The role of endosome destabilizingactivity in the gene transfer process mediated by cationic lipids [J]. FEBS Lett.1997,414:187-192.
[66] Nah JW, Yu L, Han SO, et al. Artery wall binding peptide-poly(ethyleneglycol)-grafted-poly(L-lysine)-based gene delivery to artery wall cells [J]. JControl Release.2002,78:273-284.
[67] Fischer D, Bieber T, Li Y, et al. A novel nonviral vector for DNA delivery basedon low molecular weight, branched polyethylenimine: effect of molecular weighton transfection efficiency and cytotoxicity [J]. Pharm Res.1999,16:1273-1279.
[68] Godbey WT, Barry MA, Saggau P, et al. Poly(ethylenimine)-mediatedtransfection: a new paradigm for gene delivery [J]. J Biomed Mater Res.2000,51(3):321-328.
[69] Dunlap DD, Maggi A, Soria MR, et al. Nanoscopic structure of DNA condensedfor gene delivery [J]. Nucleic Acids Res.1997,25(15):3095-3101.
[70] Reschel T, Konak C, Oupicky D, et al. Physical properties and in vitrotransfection efficiency of gene delivery vectors based on complexes of DNA withsynthetic polycations [J]. J Control Release.2002,81(1-2):201-217.
[71] Tang GP, Guo HY, Alexis F, et al. Low molecular weight polyethylenimineslinked by beta-cyclodextrin for gene transfer into the nervous system [J]. J GeneMed.2006,8(6):736-744.
[72] Jiang HL, Kim YK, Arote R, et al. Chitosan-graft-polyethylenimine as a genecarrier [J]. J Control Release.2007,117:273-280.
[73] Gebhart CL, Sriadibhatla S, Vinogradov S, et al. Design and formulation ofpolyplexes based on pluronicpolyethyleneimine conjugates for gene transfer [J].Bioconjug Chem.2002,13:937-944.
[74] Mishra S, Webster P, Davis ME. PEGylation significantly affects cellular uptakeand intracellular trafficking of non-viral gene delivery particles [J]. Eur J CellBiol.2004,83:97-111.
[75] Patnaik S, Aggarwal A, Nimesh S, et al. PEI-alginate nanocomposites as efficientin vitro gene transfection agents [J]. J Control Release.2006,114:398-409.
[76] Grosse S, Aron Y, Honore I, et al. Lactosylated polyethyleneimine for genetransfer into airway epithelial cells: role of the sugar moiety in cell delivery andintracellular trafficking of the complexes [J]. J. Gene Med.2004,6:345-356.
[77] Franceschi RT, Yang S, Rutherford RB, et al. Gene therapy approaches for boneregeneration [J]. Cells Tissues Organs.2004,176:95-108.
[78] Reddi AH. Initiation of fracture repair by bone morphogenetic proteins [J]. ClinOrthop.1998,355(Suppl):66-77.
[79] Rutherford RB, Moalli M, Franceschi RT, et al. Bone morphogeneticprotein-transduced human fibroblast convert to osteoblasts and formation bone invivo [J]. Tissue Eng.2002,8(3):441-452.
[80] Dumont RJ, Dayoub H, Li JZ, et al. Ex vivo bone morphogenetic protein-9genetherapy using human mesenchymal stem cells induces spinal fusion in rodents [J].Neurosugery.2002,51(5):1239-1244.
[81]Lee JY, Peng H, Usas A, et al. Enhancement of bone healing based on ex vivogene therapy using human musclederived cells expressing bone morphogeneticprotein-2[J]. Hum Gene Ther.2002,13(10):1201-1211.
[82] Baltzer AWA, Lattermann C, Whalen JD, et al. Genetic enhancement of fracturerepair: healing of an experimental segmental defect by adenoviral transfer of theBMP-2gene [J]. Gene Ther.2000,7:734-739.
[83] Betz OB, Betz VM, Nazarian A, et al. Direct percutaneous gene delivery toenhance healing of segmental bone defects [J]. J Bone Joint Surg Am.2006,88A:355-365.
[84] Betz OB, Betz VM, Nazarian A, et al. Delayed administration of adenoviralBMP-2vector improves the formation of bone in osseous defects [J]. Gene Ther.2007a,14:1039-1044.
[85] Betz VM, Betz OB, Glatt V, et al. Healing of segmental bone defects by directpercutaneous gene delivery: effect of vector dose [J]. Hum Gene Ther.2007b,18:907-915.
[86] Liu YG, Zhou Y, Hu X, et al. Effect of vascular endothelial growth factor121adenovirus transduction in rabbit model of femur head necrosis. J Trauma.2011,70:1519-1523.
[87] Kimelman-Bleich N, Pelled G, Zilberman Y, et al. Targeted gene-and-hostprogenitor cell therapy for nonunion bone fracture repair [J]. Mol Ther.2011,19:53-59.
[88] Pelled G, Ben-Arav A, Hock C, et al. Direct gene therapy for bone regeneration:gene delivery, animal models, and outcomemeasures [J]. Tissue Eng Part B Rev.2010,16:13-20.
[89] Evans CH, Liu FJ, Glatt V, et al. Use of genetically modified muscle and fatgrafts to repair defects in bone and cartilage [J]. Eur Cell Mater.2009c,18:96-111.
[90] Liu F, Porter RM, Wells J, et al. Evaluation of BMP-2gene-activated musclegrafts for cranial defect repair [J]. J Orthop Res.2012a,30:1095-1102.
[91] Ito H, Koefoed M, Tiyapatanaputi P, et al. Remodeling of cortical bone allograftsmediated by adherent rAAV-RANKL and VEGF gene therapy [J]. Nat Med.2005,11:291-297.
[92] Yazici C, Takahata M, Reynolds DG, et al. Self-complementaryAAV2.5-BMP2-coated femoral allografts mediated superior bone healing versuslive autografts in mice with equivalent biomechanics to unfractured femur [J].Mol Ther.2011,19:1416-1425.
[93] Ben Arav A, Pelled G, Zilberman Y, et al. Adeno-associated virus-coatedallografts: a novel approach for cranioplasty [J]. J Tissue Eng Regen Med.2012,6:43-50.
[94] Lieberman JR, Daluiski A, Stevenson S, et al. The effect of regional gene therapywith bone morphogenetic protein-2-producing bone-marrow cells on the repairof segmental femoral defects in rats [J]. J Bone Joint Surg Am.1999,81:905-917.
[95] Lieberman JR, Le LQ, Wu L, et al. Regional gene therapy with aBMP-2-producing murine stromal cell line induces heterotopic and orthotopicbone formation in rodents [J]. J Orthop Res.1998,16:330–339.
[96] Peterson B, Iglesias R, Zhang J, et al. Genetically modified human derived bonemarrow cells for posterolateral lumbar spine fusion in athymic rats: beyondconventional autologous bone grafting [J]. Spine.2005,30:283-289.
[97] Miyazaki M, Sugiyama O, Tow B, et al. The effects of lentiviral gene therapywith bone morphogenetic protein-2-producing bone marrow cells on spinalfusion in rats [J]. J Spinal Disord Tech.2008,21:372–379.
[98] Miyazaki M, Sugiyama O, Zou J, et al. Comparison of lentiviral and adenoviralgene therapy for spinal fusion in rats [J]. Spine.2008,33:1410-1417.
[99] Peterson B, Zhang J, Iglesias R, et al. Healing of critically sized femoral defects,using genetically modified mesenchymal stem cells from human adipose tissue[J]. Tissue Eng.2005,11:120-129.
[100] Breitbart AS, Grande DA, Mason JM, et al. Gene-enhanced tissue engineering:applications for bone healing using cultured periosteal cells transducedretrovirally with the BMP-7gene [J]. Ann Plast Surg.1999,42:488-495.
[101] Lee JY, Musgrave D, Pelinkovic D, et al. Effect of bone morphogeneticprotein-2-expressing muscle-derived cells on healing of critical-sized bonedefects in mice [J]. J Bone Joint Surg Am.2001,83-A:1032-1039.
[102] Ishihara A, Zekas LJ, Litsky AS, et al. Dermal fibroblast-mediated BMP2therapy to accelerate bone healing in an equine osteotomy model [J]. J OrthopRes.2010,28:403-411.
[102] Shin JH, Kim KH, Kim SH, et al. Ex vivo bone morphogenetic protein-2genedelivery using gingival fibroblasts promotes bone regeneration in rats [J]. J ClinPeriodontol.2010,37:305–311.
[104] Park J, Ries J, Gelse K, et al. Bone regeneration in critical size defects bycellmediated BMP-2gene transfer: a comparison of adenoviral vectors andliposomes [J]. Gene Ther.2003,10(13):1089-1098.
[105] Jinqushi S, Scully SP, Joyce ME, et al. Transforming growth factor-beta1andfibroblast growth factors in rat growth plate [J]. J Orthop Res.1995,13(5):761-768.
[106] Thaller SR, Dart A, Tesluk H. The effects of insulin-like growth factor-1oncritical-size calvarial defects in Sprague–Dawley rats [J]. Ann Plast Surg.1993,31(5):429-433.
[107] Pfeilschifter J, Diel L, Pilz U, et al. Mitogenic responsiveness of human bonecells in vitro to hormones and growth factors decreases with age [J]. J BoneMiner Res,1993,8(6):707-717.
[108] Aebli N, Stich H, Schawalder P, et al. Effects of bone morphogenetic protein-2and hyaluronic acid on the osseointegration of hydroxyapatite-coated implants:an experimental study in sheep [J]. J Biomed Mater Res. A.2005,73(3):295-302.
[109] Saito A, Suzuki Y, Ogata S, et al. Prolonged ectopic calcification induced byBMP-2-derived synthetic peptide [J]. J Biomed Mater Res A.2004;70(1):115-121.
[110] Lucas PA, Laurencin C, Syftestad GT, et al. Ectopic induction of cartilage andbone by water-soluble proteins from bovine bone using a polyanhydridedelivery vehicle [J]. J Biomed Mater Res.1990,24(7):901-911.
[111] Terrell TG, Working PK, Chow CP, et al. Pathology of recombinant humantransforming growth factor-beta1in rats and rabbits [J]. Int Rev Exp Pathol.1993,34(Pt B):43-67.
[112] Bonadio J. Tissue engineering via local gene delivery: update and futureprospects for enhancing the technology [J]. Adv Drug Deliv Rev.2000,44:185-194.
[113] Ryoo HM, Lee MH, Kim YJ. Critical molecular switches involved inBMP-2-induced osteogenic differentiation of mesenchymal cells [J]. Gene.2006,366:51-57.
[114] Boerckel JD, Kolambkar YM, Dupont KM，et al. Effects of protein dose anddelivery system on BMP-mediated bone regeneration [J]. Biomaterials.2011,32(22):5241-5251.
[115] Lee JW, Kang KS, Lee SH, et al. Bone regeneration using amicrostereolithography-produced customized poly (propylene fumarate)/diethylfumarate photopolymer3D scaffold incorporating BMP-2loaded PLGAmicrospheres [J]. Biomaterials.2011,32:744-752.
[116] Sojo K, Sawaki Y, Hattori H, et al. Immunohistochemical study of vascularendothelial growth factor (VEGF) and bone morphogenetic protein-2,-4(BMP-2,-4) on lengthened rat femurs[J]. Journal of Cranio-MaxillofacialSurgery.2005,33(4):238–245.
[117] Luvizuto ER, Tangl S, Zanoni G, Okamoto T, et al. The effect of BMP-2on theosteoconductive properties of b-tricalcium phosphate in rat calvaria defects [J].Biomaterials.2011,32(15):3855-3861.
[118] Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors for fibroblastsin different populations of hematopoietic cells as detected by in vitro colonyassay method [J]. Exp Hematol.1974,2(2):83-92.
[119] Dazzi F, Ramasamy R, Glennie S, et al. The role of mesenchymal stem cells inhaemopoiesis [J]. Blood Rev.2006,20(3):161-171.
[120] Mareschi K, Ferrero I, Rustichelli D, et al. Expansion of mesenchymal stemcells isolated from pediatric and adult donor bone marrow [J]. J Cell Biochem.2006,97(4):744-754.
[121] Prockop DJ. Marrow stromal cells as stem cells for non hematopoietic tissues[J]. Science.1997,276(5309):71-74.
[122] Friedenstein AJ, Ivanov-Smolenski AA, Chajlakjan RK, et al. Origin of bonemarrow stromal mechanocytes in radiochimeras and heterotopic transplants [J].Experimental hematology.1978,6(5):440-444.
[123] Rosen V. BMP2signaling in bone development and repair [J]. Cytokine GrowthFactor Rev.2009,20(5-6):475-480.
[124] Riew KD, Wright NM, Cheng S, et al. Induction of bone formation using arecombinant adenoviral vector carrying the human BMP-2gene in a rabbitspinal fusion model [J]. Calcif Tissue Int.1998,63(4):357-360.
[125] Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge intissue engineering [J]. Advanced Drug Delivery Reviews.2011,63,300-311.
[126] Fang J, Zhu YY, Smiley E, et al. Stimulation of new bone formation by directtransfer of osteogenic plasmid genes [J]. Proc Natl Acad Sci U S A.1996,93:5753-5758.
[127] Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formationand healing by stem cell-expressed VEGF and bone morphogenetic protein-4[J].J Clin Invest.2002,110:751-759.