Lentivirus载体介导hBMP-2基因转染骨髓间质干细胞修复骨缺损的实验研究
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摘要
第一部分大鼠骨髓间质干细胞的分离培养和鉴定
实验一大鼠骨髓间质干细胞的分离和培养
目的:分离和培养大鼠的骨髓间质干细胞。掌握原代细胞的培养技术,了解干细胞的生物学特性。
材料和方法:四月龄的SD大鼠,沿用Friedenstein的方法,利用间质干细胞粘附于组织培养板的特性,首先利用梯度离心分离出单核细胞,再利用干细胞粘附于塑料培养板的特点,获得较纯的贴壁生长的骨髓间质干细胞。
结果:细胞贴壁较快,呈克隆生长,细胞形态为均一的长梭形。在接种24小时后可见成纤维状的细胞以散在的方式贴壁,2—3天就可见一些集落形成,7—10天后集落迅速增多,并且逐渐长大融合成片。随着集落生长的不断扩大而融合为单层。传代培养后细胞不再以成集落的方式生长,而是呈均匀分布生长。原代培养结束时细胞数为10~5左右,P10代细胞数约为10~(12)左右。
结论:大鼠骨髓间质干细胞的分离和培养技术成熟,细胞增殖能力旺盛。
实验二大鼠骨髓间质干细胞的鉴定
目的:通过表面抗原检测确定培养细胞的正确性。为下一步实验确定良好的种子细胞。
材料和方法:通过流式细胞仪检测大鼠骨髓问质干细胞的表面抗原,包括CD29,CD44,CD90,CD105,CD34,CD45,CD11b,组织相容性复合体Ⅰ、Ⅱ。
结果:与对照组相比,大鼠骨髓间质干细胞表面抗原CD29,CD44,CD90,CD105显示阳性,而表面抗原CD34,CD45,CD11b为阴性,组织相容性复合体Ⅰ、Ⅱ均为阴性。
结论:大鼠骨髓间质干细胞表面标志为非单一性,表达间质细胞、内皮细胞和表皮细胞的表面标志,一般认为整合素家族成员CD29,粘附分子CD44、CD105以及胸腺和外周T淋巴细胞标志CD90等是骨髓问质干细胞的重要标志物。
第二部分携带hBMP2基因的慢病毒转染骨體间质干细胞的定向分化
目的:观察慢病毒载体诱导rMSCs后的变化情况,检测rMSCs是否按预期方向向成骨细胞转化。
材料和方法:含有hBMP2基因的慢病毒载体诱导rMSCs,诱导第0、3、6、9、12、15、18、21天测定碱性磷酸酶的活性及细胞染色情况;采用钙的定量测定沉积钙的浓度;免疫组化分析Ⅰ型胶原蛋白表达情况。
结果:诱导后的rMSCs,随着大鼠骨髓间质干细胞诱导时间的延长,其碱性磷酸酶的活性逐渐增加,染色良好,而未经病毒诱导的干细胞,培养21天,其碱性磷酸酶的活性无明显的变化;诱导组Ca~(2+)的浓度持续升高,到第15天后Ca~(2+)的浓度达到最大,此后达到平稳期,未诱导组钙沉积无明显变化;诱导组Ⅰ型胶原蛋白表达明显增加,染色呈阳性,而未诱导组无明显变化。
结论:通过免疫组化和组织化学技术证明,诱导的rMSCs细胞能够产生工型胶原、碱性磷酸酶,并具有矿化能力,证实在慢病毒载体的诱导下rMSCs向成骨细胞分化。
第三部分成骨细胞—β-磷酸三钙复合体的构建
目的:观察大鼠成骨细胞及MSCs在β-磷酸三钙表面的贴附及生长情况。
材料和方法:四块多孔β-磷酸三钙(直径8mm,厚1mm),随机分成实验组和对照组。
实验组β-磷酸三钙在负压条件下用重组人纤维连接蛋白(100μg/ml)浸渍过夜。再将两组β-磷酸三钙分别与rMSCs和大鼠成骨细胞共同培养10天,扫描电镜下观察比较实验组与对照组细胞在β-磷酸三钙表面的贴附数量和形态。
结果:实验组细胞的贴附数量显著高于对照组(P＜0.01)。成骨细胞实验组分泌大量胶原纤维和形成钙化结节。
结论:纤维连接蛋白对β-磷酸三钙的表面修饰能明显提高大鼠成骨细胞和rMSCs的贴附率,有利于成骨细胞的生长及成骨表型。
第四部分自体成骨细胞—β-磷酸三钙修复大鼠颅骨极量骨缺损
目的:评价携带hBMP2基因的慢病毒转染rMSCs与β-磷酸三钙复合构建的组织工程化骨对骨缺损的修复效果。
材料和方法:250～350 g雄性SD大鼠42只,均在全麻及无菌条件下取其左侧股骨及胫骨,行MSCs培养及成骨诱导。实验细胞在培养14d后消化接种于经纤维连接蛋白表面修饰的含孔磷酸钙陶瓷上继续培养10d。所有大鼠均造成颅骨极量缺损(直径8mm)模型,用自体细胞行骨缺损修复。随机分为Lev-BMP2转染细胞+β-TCP组(n=10);rMSC+β-TCP组(n=10);β-TCP组(n=10);Lev-BMP2转染细胞组(n=4);rMSC组(n=4);空白对照组(n=4)。于第4、8、12、20周从形态学及组织学角度观察比较颅骨缺损修复愈合情况。
结果:无论X线摄片及组织切片均表明,实验组颅骨极量缺损修复优于同期对照组。在各时间点Lev-BMP2转染细胞+β-TCP组和rMSC+T-TCP组在植骨区均有新骨形成,而β-TCP组仅见边缘区成骨。
结论:Lev-BMP2转染BMSCs复合β-TCP构建的组织工程化骨可以修复大鼠颅骨极量骨缺损。
PartⅠisolation culture and identification of rat Mesenchymal stem cells(rMSCs)
Experiment 1 isolation and culture of rat Mesenchymal stem cells(rMSCs) Objective:to isolate and culture rat Mesenchymal stem cells(rMSCs).To grasp the techniques of rMSCs culture and investigate the biological characters of rMSCs. Materials and Methods:4-month-old SD rats were used,follow the methods of Friedenstein,because of the characters of rMSCs adhereing to tissue culture flask,(as rMSCs is adherent to tissue petri dish,) monocytes were isolated by gradient centrifugation first;then pure adherent rMSCs were obtained by the property that rMSCs is adherent to plastic petri dish.Results:rMSCs are rapidly adherent,grow in clone,the morphology of cell are homogeneous fusiform,within 24hs fibrous like cells were seen sporadic adherent,some cell clony were seen in 2-3 days,and they increased rapidly in 7-10 days,gradually grow into mixture. As the cell clony grown,they became monolayer at last.After passage,rMSCs no longer growed in clony but in homogeneous distribution,rMSCs number of primary culture is about 10~5,and passage 10 is about 10~(12).
Conclusions:isolation and culture of rMSCs are feasible,and have powerful ability of cell proliferation.
Experiment 2 identification of rat bone marrow Mesenchymal stem cells (rMSCs)
Objective:surface antigen was used to identify the property of cultured cells. to prepare the seed cells of next step.methods and materials:to detect the surface antigen of rat bone marrow MSCs with flow cytometry,include CD29,CD44,CD90,CD105,CD34,CD45,CD11b.results:in contrast to the control group, the surface antigen CD29,CD44,CD90,CD105 of rat bone marrow rMSCs were positive; surface antigen CD34,CD45,CD11b were negative,and major MHC-Ⅰ、Ⅱ(major histocompatibility complex-Ⅰ、Ⅱ) were negative too.
Conclusions:rat bone marrow rMSCs surface markers are multiplicity,which express the surface markers of interstitial cells,endotheliocytes and epidermic cells,generally CD29(the member of integrin family),adhesion molecule CD44,CD105 and CD166,surface marker CD90 of T lymphocytes in thoracic gland and periphery circulation and so on were important markers of bone marrow rMSCs.
partⅡdirectional differentiation of rMSCs infected by lentiviral vector with hBMP-2
Objectives:to observe the change of rMSCs induced by lentivral vector,to detect if rMSCs transform to osteoblast as expectation.Materials and methods:after lentiviral vector induced rMSCs,in the 0,3,6,9,12,15,18 and 21 day,alkaline phosphatase(ALP)activity is evaluated;to determin calcium ion concentration with quantitative assay;immunohistochemistry is used to detect the expression of typeⅠcollagen.Results:as the induced time prolonging,in rMSCs after induction, alkaline phosphatase(ALP) activity is gradually increasing,but ALP activity of rMSCs that were not induced had no obvious change after 21 days culture.After induced,Calciumion concentration is continuous increasing,reached climax after 15 days,then get in stationary phase.Calcium deposition had no marked change in the group that were not induced;typeⅠcollagen expression increased obviously in induced rMSCs and staining is positive,in contrast,which had no change in the control.Conclusions:with immunohistochemistry and histochemistry methods we revealed that induced rMSCs can produce typeⅠcollagen,ALP and had the ability of mineralization,which confirmed rMSCs can be induced to differentiate into osteoblast by lentivral vector.
partⅢosteoblast cultivated with TCP in vitro
Objective:To investigate osteoblast and MSCs grewing and adhension in calciumphosphate bioceram.Materials and methods:Four porous ceramics 8mm×1mm divided into experimental and control groups randomly.The experimental group was soaked in 100g/ml recombinant human fibronectin under negative pressure over night.Then both groups were cultivated in vitro simultaneously with osteoblasts derived from bone marrow MSCs and MSCs of rats respectively for ten days.The morphogenetic pattern and number of the anchored cells were observed under scanning electron microscope.Results:There was statistically significant difference of cell numbers between the experimental and the control groups P＜0.01. Many secretory collagen fiber bundles and mineralized nods were observed in the experimental group under high magnifier.Conclusions:It seems that the fibronectin surface coating is beneficial to osteoblast anchorage and its osteogenic pheno type.
PartⅣRepair of critical size rat calvarial defects with autogenous osteoblasts loading onβ-TCP
Objective:To evaluate osteogenetic effectiveness of lentiviral vector mediated hBMP-2 gene(Lev-hBMP-2) transfected bone marrow derived mesenchymal stem cells for repair of bone defect mixed withβ-TCP in the repair of critical size rat calvarial defects.Materials and Methods:Forty two male adult SD rats(weight: 250-350g) were obtained bone marrow from the left femurs and tibias under general anesthesia and sterile condition.BMSCs were cultured and transfected by Lev-hBMP-2 in vitro.The BMSCs were collected from bone marrow of rats,cultured and transfected by Lev-hBMP-2 in vitro for 14 days.The cells were then seeded and subcultured for another 10 days inβ-TCP that had been subjected to surface porous calcium phosphate ceramic that had been subjected to surface modification via soaking in recombinant human fibronectin.The cells and cell-ceramic compound were used to repair critical size(diameterof 8 mm) calvarial defects in the corresponding rat in the experimental group.The rat were divided into six groups at randomly:1.Lev-hBMP-2 transfected BMSCs+β-TCP(n=10);2.BMSCs+β-TCP(n=10); 3.β-TCP(n=10);4.Lev-hBMP-2 transfected BMSCs(n=4);5.BMSCs(n=4);6. control(n=4).The skull defect reconstruction was observed histomorphometrically at 4th、8th、12th、20th week after operation.
Results:tissue slicing showed that the whole defected areas can found new bone grewing during different stages in the experimental group while osteogenic tissue only appeared in the margin of ceramic in the control group.
Conclusions:The tissue-engineered bone made by Lev-hBMP-2 transfected BMSCs mixed withβ-TCP can repair critical size rat calvarial defects
实验一大鼠骨髓间质干细胞的分离和培养
目的:分离和培养大鼠的骨髓间质干细胞。掌握原代细胞的培养技术,了解干细胞的生物学特性。
材料和方法:四月龄的SD大鼠,沿用Friedenstein的方法,利用间质干细胞粘附于组织培养板的特性,首先利用梯度离心分离出单核细胞,再利用干细胞粘附于塑料培养板的特点,获得较纯的贴壁生长的骨髓间质干细胞。
结果:细胞贴壁较快,呈克隆生长,细胞形态为均一的长梭形。在接种24小时后可见成纤维状的细胞以散在的方式贴壁,2—3天就可见一些集落形成,7—10天后集落迅速增多,并且逐渐长大融合成片。随着集落生长的不断扩大而融合为单层。传代培养后细胞不再以成集落的方式生长,而是呈均匀分布生长。原代培养结束时细胞数为10~5左右,P10代细胞数约为10~(12)左右。
结论:大鼠骨髓间质干细胞的分离和培养技术成熟,细胞增殖能力旺盛。
实验二大鼠骨髓间质干细胞的鉴定
目的:通过表面抗原检测确定培养细胞的正确性。为下一步实验确定良好的种子细胞。
材料和方法:通过流式细胞仪检测大鼠骨髓问质干细胞的表面抗原,包括CD29,CD44,CD90,CD105,CD34,CD45,CD11b,组织相容性复合体Ⅰ、Ⅱ。
结果:与对照组相比,大鼠骨髓间质干细胞表面抗原CD29,CD44,CD90,CD105显示阳性,而表面抗原CD34,CD45,CD11b为阴性,组织相容性复合体Ⅰ、Ⅱ均为阴性。
结论:大鼠骨髓间质干细胞表面标志为非单一性,表达间质细胞、内皮细胞和表皮细胞的表面标志,一般认为整合素家族成员CD29,粘附分子CD44、CD105以及胸腺和外周T淋巴细胞标志CD90等是骨髓问质干细胞的重要标志物。
第二部分携带hBMP2基因的慢病毒转染骨體间质干细胞的定向分化
目的:观察慢病毒载体诱导rMSCs后的变化情况,检测rMSCs是否按预期方向向成骨细胞转化。
材料和方法:含有hBMP2基因的慢病毒载体诱导rMSCs,诱导第0、3、6、9、12、15、18、21天测定碱性磷酸酶的活性及细胞染色情况;采用钙的定量测定沉积钙的浓度;免疫组化分析Ⅰ型胶原蛋白表达情况。
结果:诱导后的rMSCs,随着大鼠骨髓间质干细胞诱导时间的延长,其碱性磷酸酶的活性逐渐增加,染色良好,而未经病毒诱导的干细胞,培养21天,其碱性磷酸酶的活性无明显的变化;诱导组Ca~(2+)的浓度持续升高,到第15天后Ca~(2+)的浓度达到最大,此后达到平稳期,未诱导组钙沉积无明显变化;诱导组Ⅰ型胶原蛋白表达明显增加,染色呈阳性,而未诱导组无明显变化。
结论:通过免疫组化和组织化学技术证明,诱导的rMSCs细胞能够产生工型胶原、碱性磷酸酶,并具有矿化能力,证实在慢病毒载体的诱导下rMSCs向成骨细胞分化。
第三部分成骨细胞—β-磷酸三钙复合体的构建
目的:观察大鼠成骨细胞及MSCs在β-磷酸三钙表面的贴附及生长情况。
材料和方法:四块多孔β-磷酸三钙(直径8mm,厚1mm),随机分成实验组和对照组。
实验组β-磷酸三钙在负压条件下用重组人纤维连接蛋白(100μg/ml)浸渍过夜。再将两组β-磷酸三钙分别与rMSCs和大鼠成骨细胞共同培养10天,扫描电镜下观察比较实验组与对照组细胞在β-磷酸三钙表面的贴附数量和形态。
结果:实验组细胞的贴附数量显著高于对照组(P＜0.01)。成骨细胞实验组分泌大量胶原纤维和形成钙化结节。
结论:纤维连接蛋白对β-磷酸三钙的表面修饰能明显提高大鼠成骨细胞和rMSCs的贴附率,有利于成骨细胞的生长及成骨表型。
第四部分自体成骨细胞—β-磷酸三钙修复大鼠颅骨极量骨缺损
目的:评价携带hBMP2基因的慢病毒转染rMSCs与β-磷酸三钙复合构建的组织工程化骨对骨缺损的修复效果。
材料和方法:250～350 g雄性SD大鼠42只,均在全麻及无菌条件下取其左侧股骨及胫骨,行MSCs培养及成骨诱导。实验细胞在培养14d后消化接种于经纤维连接蛋白表面修饰的含孔磷酸钙陶瓷上继续培养10d。所有大鼠均造成颅骨极量缺损(直径8mm)模型,用自体细胞行骨缺损修复。随机分为Lev-BMP2转染细胞+β-TCP组(n=10);rMSC+β-TCP组(n=10);β-TCP组(n=10);Lev-BMP2转染细胞组(n=4);rMSC组(n=4);空白对照组(n=4)。于第4、8、12、20周从形态学及组织学角度观察比较颅骨缺损修复愈合情况。
结果:无论X线摄片及组织切片均表明,实验组颅骨极量缺损修复优于同期对照组。在各时间点Lev-BMP2转染细胞+β-TCP组和rMSC+T-TCP组在植骨区均有新骨形成,而β-TCP组仅见边缘区成骨。
结论:Lev-BMP2转染BMSCs复合β-TCP构建的组织工程化骨可以修复大鼠颅骨极量骨缺损。
PartⅠisolation culture and identification of rat Mesenchymal stem cells(rMSCs)
Experiment 1 isolation and culture of rat Mesenchymal stem cells(rMSCs) Objective:to isolate and culture rat Mesenchymal stem cells(rMSCs).To grasp the techniques of rMSCs culture and investigate the biological characters of rMSCs. Materials and Methods:4-month-old SD rats were used,follow the methods of Friedenstein,because of the characters of rMSCs adhereing to tissue culture flask,(as rMSCs is adherent to tissue petri dish,) monocytes were isolated by gradient centrifugation first;then pure adherent rMSCs were obtained by the property that rMSCs is adherent to plastic petri dish.Results:rMSCs are rapidly adherent,grow in clone,the morphology of cell are homogeneous fusiform,within 24hs fibrous like cells were seen sporadic adherent,some cell clony were seen in 2-3 days,and they increased rapidly in 7-10 days,gradually grow into mixture. As the cell clony grown,they became monolayer at last.After passage,rMSCs no longer growed in clony but in homogeneous distribution,rMSCs number of primary culture is about 10~5,and passage 10 is about 10~(12).
Conclusions:isolation and culture of rMSCs are feasible,and have powerful ability of cell proliferation.
Experiment 2 identification of rat bone marrow Mesenchymal stem cells (rMSCs)
Objective:surface antigen was used to identify the property of cultured cells. to prepare the seed cells of next step.methods and materials:to detect the surface antigen of rat bone marrow MSCs with flow cytometry,include CD29,CD44,CD90,CD105,CD34,CD45,CD11b.results:in contrast to the control group, the surface antigen CD29,CD44,CD90,CD105 of rat bone marrow rMSCs were positive; surface antigen CD34,CD45,CD11b were negative,and major MHC-Ⅰ、Ⅱ(major histocompatibility complex-Ⅰ、Ⅱ) were negative too.
Conclusions:rat bone marrow rMSCs surface markers are multiplicity,which express the surface markers of interstitial cells,endotheliocytes and epidermic cells,generally CD29(the member of integrin family),adhesion molecule CD44,CD105 and CD166,surface marker CD90 of T lymphocytes in thoracic gland and periphery circulation and so on were important markers of bone marrow rMSCs.
partⅡdirectional differentiation of rMSCs infected by lentiviral vector with hBMP-2
Objectives:to observe the change of rMSCs induced by lentivral vector,to detect if rMSCs transform to osteoblast as expectation.Materials and methods:after lentiviral vector induced rMSCs,in the 0,3,6,9,12,15,18 and 21 day,alkaline phosphatase(ALP)activity is evaluated;to determin calcium ion concentration with quantitative assay;immunohistochemistry is used to detect the expression of typeⅠcollagen.Results:as the induced time prolonging,in rMSCs after induction, alkaline phosphatase(ALP) activity is gradually increasing,but ALP activity of rMSCs that were not induced had no obvious change after 21 days culture.After induced,Calciumion concentration is continuous increasing,reached climax after 15 days,then get in stationary phase.Calcium deposition had no marked change in the group that were not induced;typeⅠcollagen expression increased obviously in induced rMSCs and staining is positive,in contrast,which had no change in the control.Conclusions:with immunohistochemistry and histochemistry methods we revealed that induced rMSCs can produce typeⅠcollagen,ALP and had the ability of mineralization,which confirmed rMSCs can be induced to differentiate into osteoblast by lentivral vector.
partⅢosteoblast cultivated with TCP in vitro
Objective:To investigate osteoblast and MSCs grewing and adhension in calciumphosphate bioceram.Materials and methods:Four porous ceramics 8mm×1mm divided into experimental and control groups randomly.The experimental group was soaked in 100g/ml recombinant human fibronectin under negative pressure over night.Then both groups were cultivated in vitro simultaneously with osteoblasts derived from bone marrow MSCs and MSCs of rats respectively for ten days.The morphogenetic pattern and number of the anchored cells were observed under scanning electron microscope.Results:There was statistically significant difference of cell numbers between the experimental and the control groups P＜0.01. Many secretory collagen fiber bundles and mineralized nods were observed in the experimental group under high magnifier.Conclusions:It seems that the fibronectin surface coating is beneficial to osteoblast anchorage and its osteogenic pheno type.
PartⅣRepair of critical size rat calvarial defects with autogenous osteoblasts loading onβ-TCP
Objective:To evaluate osteogenetic effectiveness of lentiviral vector mediated hBMP-2 gene(Lev-hBMP-2) transfected bone marrow derived mesenchymal stem cells for repair of bone defect mixed withβ-TCP in the repair of critical size rat calvarial defects.Materials and Methods:Forty two male adult SD rats(weight: 250-350g) were obtained bone marrow from the left femurs and tibias under general anesthesia and sterile condition.BMSCs were cultured and transfected by Lev-hBMP-2 in vitro.The BMSCs were collected from bone marrow of rats,cultured and transfected by Lev-hBMP-2 in vitro for 14 days.The cells were then seeded and subcultured for another 10 days inβ-TCP that had been subjected to surface porous calcium phosphate ceramic that had been subjected to surface modification via soaking in recombinant human fibronectin.The cells and cell-ceramic compound were used to repair critical size(diameterof 8 mm) calvarial defects in the corresponding rat in the experimental group.The rat were divided into six groups at randomly:1.Lev-hBMP-2 transfected BMSCs+β-TCP(n=10);2.BMSCs+β-TCP(n=10); 3.β-TCP(n=10);4.Lev-hBMP-2 transfected BMSCs(n=4);5.BMSCs(n=4);6. control(n=4).The skull defect reconstruction was observed histomorphometrically at 4th、8th、12th、20th week after operation.
Results:tissue slicing showed that the whole defected areas can found new bone grewing during different stages in the experimental group while osteogenic tissue only appeared in the margin of ceramic in the control group.
Conclusions:The tissue-engineered bone made by Lev-hBMP-2 transfected BMSCs mixed withβ-TCP can repair critical size rat calvarial defects
引文
1.Croteau S,Rauch F,Silvestri A,et al.Bone morphogenetic proteins in orthopaedics:from basic science to clinical practice.Orthopaedics,1999,22:686-695.
2.杨志明,余希杰,解慧琪,等.不同来源成骨细胞生物学特性的比较研究.中华创伤杂志,2001,17:10-13.
3.Boden SD.Bioactive factor for bone tissue engineering.Clin orthop and Relat Res,1999,(367suppl):s84-89.
4.Lieberman JR,Daluiski A,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.J.Bone Joint Surg Am.1999,81(7):905-17.
5.Sigalla J,David A,Anegon I,et al.Adenovirus-mediated gene transfer into isolatedmouse adult pancreatic islets:normal beta-cell function despite induction ofan anti-adenovirus immune response.Hum Gene Ther 8:1625-1634,1997
6.Level AM,Strappe PM,Zhao J,et al.Lentivirus Vector.J Biomed Sci 2004,11:439-449.
7.Quinonez R,Sutton RE.Lentiviral vectors for gene delivery into cells.DNA Cell Biol 2002,21:937-951.
8.Kafri T.Gene delivery by lentivirus vectors.An overview.Methods Mol Biol 2004,246:367-390.
9.Zhao J,Bolton EM,Pettigrew GJ,et al.Lentivirus-mediated Gene Transfer of Viral Interleukin 10 Prolongs Survival of Cardiac Allografts in Rats.Poster Abstracts Session Ⅱ 2004,9:621-633.
10.Minguell JJ,Erices A,Conget P.Mesenchymal stem cells.Exp Biol Med(Maywood).2001 Jun,226(6):507-20.
11.Kunjachan V,Subramanian A,Hanna M,et al.Comparison of different fabrication techniques used for processing 3-dimensional,porous,biodegradable scaffolds from modified starch for bone tissue engineering.Biomed Sci Instrum,2004,40:129-135.
12.El-Ghannam AR.Advanced bioceramic composite for bone tissue engineering:Design principles and structure-bioactivity relationship.J Biomed Mater Res,2004,69A(3):490-501.
13. Liu X, Ma PX. Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng, 2004, 32(3): 477-486.
14. Walles T, Herden T, Haverich A, et al. Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials, 2003, 24(7): 1233-1239.
1.项鹏,李树浓.骨髓间质干细胞研究进展 中国病理生理杂志 2002,18(11):1438-1442.
2.Bruder SP,Jaiswal N,Ricalton NS,et al.Mesenchymal stem calls in osteobioligy and applied bone regeneration.J Clinical Orthopaedics and Related Research.1998,355:247.
3.雷俊霞,李浩威,黄春浓,等.长期传代的大鼠骨髓间质干细胞的牛物学特性分析 中国病理生理杂志2003,19(10):1325-1330.
4.肖庆中,李浩威,温冠媚等.大鼠骨髓间质干细胞基本生物学特性的研究 中国病理生理杂志 2004,3(20):289-294.
5.Conget PA,Minguell JJ.Phenotypical and functional of human bone marrow mesenchymal progenitor cells.J Cell Physiol.1999,181(1):67.
6、李连达,吴理茂,刘红.大鼠骨髓间质干细胞的培养和牛物学特征 科学技术与工程2003,6(3):547-550.
7.Pittenger MF,Mackay AM,Jsiswal SC,et al.Multilineage potential of adult human mesechymal stem cells.Science.1999,284(5411):143-147.
8.Jiang YH,Balkrishna N,Jahagirdar R,et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature.2002,4(418):41-49.
9.Elena AJ,Sally EK,Anne E,et al.Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells.Arthritis and Rheumatism.2002,12(46):3349-3360.
10.Donald PL,Stephen EH,Douglas MA,et al.Dilution of human mesenchymal stem cells with dermal fibroblasts and the effects on in vitro and in vivo osteochondrogenesis.Developmental Dynamics.2000,219:50-62.
11.Hiroyuki T,Junichi H,Eric C,et al.Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.J Ortho Res.2003,21:44-53.
12.Devine SM,Bartholomew AM,Nelson M,et al.Mesenchymal stem cells are capable of homing to the bone marrow of non-human primares following systemic infusion.Exp Hematol.2001,29:244-255.
13.Shake JG,Gruber PJ,Baumgartner WA,et al.Mesenchymal stem cell implantation in a swine myocardial ingarct model:engraftment and functional effects.Ann Thorac Surg.2002,73:1919-1925.
14.Toma C,Pittenger MF,Cahill KS,et al.Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation.2002,105:93-98.
15.Kinner B,Gerstenfeld LC,Einhorn TA,et al.Expression of smooth muscle actin in connective tissue cells participating in fracture healing in a murine model.Bone.2002,30:738-745.
16.Kinner B,Zaleskas JM,Spector M,et al.Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells.Exp Cell Res.2002,278:72-83.
17.Eckes B,Colucci GE,Smola H,et al.Impaired wound healing in embryonic and adult mice lacking vimentin.J Cell Ther.2000,113:2455-62.
18.Liechty KW,Mackenzie TC,Shaaban AF,et al.Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.Nat Med.2000,6:1282-1286.
19.Millington WS,Allers C,Tuohy G,et al.Validation in mesenchymal progenitor cells of a mutation-independent ex vivo approach to gene therapy for osteogenesis imperfecta.Hum Mol Genet.2002,11:2201-2206.
20.Hutcheson KA,Atkins BZ,Hueman MT,et al.Comparison of benefits on myocardial performance of cellular cardiomyoplasty with skeletal myoblasts and fibroblasts.Cell Transplant.2000,9:359-368.
21.杨志明,余希杰,解慧琪,等.不同来源成骨细胞生物学特性的比较研究[J].中华创伤杂志.2001,17:10-13.
1. Allampallam K, Chakraborty J, Robinson J. Effect of ascorbic acid and growth factors on collagen metabolism of flexor retinaculum cells from individuals with and without carpal tunnel syndrome. Occup Environ Med, 2000, 42(3): 251-259.
2. Maniatopoulos C, Sodek J, Melcher AH , et al. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res, 1988, 254(2): 317-330.
3. Hanada K, Dennis JE, Caplan AI, et al. Stimulatory effects of basic fibroblast growth factor and bone morphogenetic pro-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. J Bone Miner Res, 1997, 12:1606-1614.
4. Cheng BH, Jiang W, Frank MP, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins(BMPs). J Bone J Surg, 2003, 8 (85):1544-1552.
5. Allampallam K, Chakraborty J, Robinson J, et al. Effects of ascorbic acid and growth factors on collgen metabolism of flexor retinaculum cells from individuals with and without carpal tunnel syndrome. Occup Environ Med, 2000, 42(3): 251-259.
6. Maniatopoulos C, Sodek J, Melcher AH, et al. Bone formation in vitro by stromal cells obtained from bone marrow of young adults rats. Cell Tissue Res. 1998, 254(2): 317-330.
7. Cheng SL, Yang JW, Rifas L, et al. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology, 1994, 134(1):277-286.
8. Ebara S, Nakayama K. Mechanism for the action of bone morphogenetic proteins and regulation of their activity. Spine, 2002 , 27(16): 10-15.
9. Wloderski KH. Properties and origin of osteoblasts. Clin Ortho, 1990, 252(3): 276-293.
10. Alborxi A, Mac KG, Lackin CA, et al. Endochondral and intramembranous fetal bone development: osteoblastic cell proliferation and expression of alkaline phosphatase. Cranifac Genet Dev Biol, 1996, 16(2):94-98.
11. Warner GP, Hubbard HL. 32P and 45Ca-metabolism by mareix vesicle-enriched microsomes prepared from chicken epiphyseal cartilage percoll density-gradient fractionation.Calcif Tissue Int,1983,35(3):327-328.
12.杨志明:组织工程皋础与临床:成都:四川科学技术出版社,1999,124-129.
13.Hiroyuki T,Junichi H,Eric C,et al.Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.J Ortho Res,2003,21:44-53.
1. Boyan BD, Hummert TW, Dean DD, et al. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials. 1996, 17(2): 137-46.
2. Kunjachan V, Subramanian A, Hanna M, et al. Comparison of different fabrication techniques used for processing 3-dimensional, porous, biodegradable scaffolds from modified starch for bone tissue engineering. Biomed Sci Instrum, 2004, 40:129-135.
3. El-Ghannam AR. Advanced bioceramic composite for bone tissue engineering: Design principles and structure-bioactivity relationship. J Biomed Mater Res, 2004, 69A(3): 490-501.
4. Liu X, Ma PX. Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng, 2004, 32(3) :477-486.
5. Walles T, Herden T, Haverich A, et al. Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials, 2003, 24(7): 1233-1239.
6. Heymann D, Delecrin J, Deschamps C, et al. In vitro assessment of associating osteogenetic cells with macroporous calcium-phosphate ceramics. RevChir Orthop Reparatrice Appar Mot, 2001, 87:8-17.
7. Steele JG, Johnson G, McLean KM, et al. Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer. J Biomed Mater Res, 2000, 50:475-482.
8. Tsuchida H, Hashimoto J, Crawford E, et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthop Res, 2003, 21:44-53.
9. Hutmacher DW. Scaffold design and fabrication technologies for engineering tissue—state of the art and future perspectives. J Biomater Sci PolymEd, 2001, 12:107-115.
10. Koc N, Timucin M, Korkusuz F, et al. Fabrication and characterization of porous tricalcium phosphate ceramics. Ceramics Int, 2004, (30):205-211.
11. Tsuruga E, Takita H, Itoh H, et al. Porr size of porous hydroxyapatie as the cell-substratum controls BMP-induced osteogenesis. J Biochem, 1997, 121:317-324.
12. Gronthos S, Stewart K, Graves SE, et al. Integrin expression and function on human osteoblast-like cells. J Bone Miner Res, 1997 12(8):1189-97.
13. Suzuki T, Yamamoto T, Toriyama M, et al. Surface instability of calcium phosphate ceramics in tissue medium and the effect on adhesion and growth of anchorage-dependent animal cells.. J Biomed Mater Res, 1997 ,34(4):507-17.
14. Knabe C, Ostapowicz W, Radlanski RJ, et al. In vitro investigation of novel calcium phosphates using osteogenic cultures. J Mater Sci Mater Med, 1998 ,9(6): 337-45.
1.Schmitz JP,Hollinger JO.The critical size defect as an experimental model for craniomandibulofacial nonunions.Clin Orthop Rlat Res.1986,205:299-308.
2.Sweeney TM,Opperman LA,Persing JA,et al.Repair of critical size rat calvarial defects using extracellular matrix protein gels.J Neurosurg.1995,83(4):710-715.
3.Yoshikawa T,Ohgushi H,Nakajima H,et al.In vivo osteogenic durability of cultured bone in porous ceramics.Transplantation,2000,69:128-134.
4.陈希哲,杨连甲,付崇建等.含孔磷酸钙陶瓷复合自体成骨细胞骨组织工程大鼠模型的构建.中华实验外科杂志,2002,19(5):460-461.
5.Gregoire M,Orly I,Menanteau J.The influence of calcium phosphate biomaterials on human bone cell activities:an in vitroa pproach.J Biomed Mater Res,1990,24(2):165-167.
6.Grover LM,Gbureck U,Wright AJ,et al.Biologically mediated resorption of brushite cement in vitro.Biomaterials,2006,27(10):2178-2185.
7.Ooms EM,Wolke JG,van der Waerden JP,et al.Trabecular bone response to injectable calcium phosphate(Ca-P) cement.J Biomed Mater Res,2002,61(1):9-18.
8.Zerbo IR,Bronckers AL,de Lange G,et al.Localisation of osteogenic and osteoclastic cells in porous beta-tricalcium phosphate particles used for human maxillary sinus floor elevation.Biomaterials,2005,26(12):1445-1451.
9.Niemela T.Effect of β -tricalcium phosphate addition on the in vitro degradation of self-reinforced poly-L,D-lactide.Polymer Degrade Stabil,2005,89(3):492-500.
10.Shikinami Y,Matsusue Y,Nakamura T.The complete process of bioresorption and bone replacement using devices made of forged composites of raw hydroxyapatite particles/poly 1-lactide(F-u-HA/PLLA).Biomaterials,2005,26(27):5542-5551.
11.Wiltfang J,Merten HA,Schlegel KA,et al.Degradation characteristics of alpha and beta tri-calcium-phosphate(TCP) in minipigs.J Biomed Mater Res,2002,63(2):165-167.
12.Asanuma K,Masui F,Kamitani K,et al.Clinical application of pure beta-TCP for bone tumors.
13. Okuda T, Ioku K, Yonezawa I, et al. The effect of the microstructure of beta tri-calcium phosphate on the metabolism of subsequently formed bone tissue. Biomaterials, 2007, 28(16):2612-2621.
14. Lieberman JR, Daluiski A, 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. J. Bone Joint Surg Am. 1999, 81(7): 905—17.
15. Virk MS, Conduah A, Park SH et al. Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone, 2008 Jan 5[Epub ahead of print].
16. Hsu WK, Sugiyama o, park SH, et al. Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats. Bone. 2007,40(4): 931-938.
1. Crane GM, Ishaugug SL, Mikos AG, et al. Bone tissue engineering. Nat Med, 1995,1:1332-1334.
2. Croteau S, Rauch F, Silvestri A, et al. Bone morphogenetic proteins in orthopaedics: from basic science to clinical practice. Orthopaedics, 1999, 22: 686-695.
3. Gandolfi MG, Pugnaloni A, Mattioli BM, et al. Osteoblast behaviour in the presence of bisphosphonates: ultrastructural and biochemical in vitro studies. Clin Exp Rheumato, 1999, 17(3): 327-333.
4. Wright V, Peng H, Usas A, et al. BMP4-expressing muscle derived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol Ther, 2002, 6(2): 169—178.
5. Mie M, Ohgushi H, Yanagida Y, et al. osteogenesis coordinated in C3H10TI/2 cells by adipogenesis-dependent BMP-2 expression system. Tissue Eng, 2000, 6(1) :9—18.
6. Krebsbach PH, Gu K, Franceschi RT, et al. Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo. Hum Gene Ther, 2000, 11(8) :1201 —1210.
7. Zuk PA, Zhu M, MizunoH, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng, 2001, 7(2):211-228.
8. Nathan S, Das De S, Thambyah A, et al. Cell-based therapy in the repair of osteochondral defects: a novel use for adipose tissue. Tissue Eng, 2003, 9(4): 733-744.
9. Ogawa R , Mizuno H , Watanabe A , et al. Osteogenic and chondro-genic differentiation by adipose-derived stem cells harvested from GFP transgenic mice. Biochem Biophys Res Commu, 2004, 313(4): 871—877.
10. Yang M, Ma QJ, Dang GT, et al. In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult stem cells expressing BMP-7. Cytotherapy, 2005, 7(3):273 — 281
11.Shimizu T,Sasano Y,Nakajo S,et al.osteoblastic differentiation of periosteum-derived cells is promoted by the physical contact with the bone matrix in vivo.Anat Rec,2001,264(1):72-81.
12.Schantz JT,Hutmacher DW,Chim H,et al.Induction of ectopic bone formation by using human Periosteal cells in combination with a novel scaffold technology:Cell Transplant,2002,11(2):125-138.
13.Friedenstein AJ,Chailakhyan RK,Gerasimov UV,et al.Bon Marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers.Cell Tissue Kinet,1987,20(3):263-272.
14.项鹏,李树浓.骨髓间质干细胞研究进展 中国病理生理杂志 2002,18(11):1438-1442.
15.刘劲松,曾才铭,王宏邦,等.骨髓基质细胞培养和体外成骨研究.中国修复重建外科杂志,1997,11:238-241.
16.杨志明,余希杰,解慧琪,等.不同来源成骨细胞生物学特性的比较研究.中华创伤杂志,2001,17:10-13.
17.Reddi AH,initiation of fracture repair by bone morphogenetic proteins.Clin Orthop,1998,355(suppl):s66-s72.
18.Yang Y,Nunes FA,Berencsi K et al.Cellularimmunity to viral antigens limits El-deleted adenoviruses for gene therapy.Proc Natl Acad Sci U S A 1994,91:4407 - 4411.
19.Hacein-Bey-Abina,S.,et al.LMO2-associatedclonal T cell proliferation in two patients after gene therapy for SCID-X1.Science.2003,302:415-419.
20.Wu.X.Li Y.,Crise B.,and Burgess,S.M.Transcription start regions in the human genomeare favored targets for MLV integration.Science.2003,300:1749 -1751.
21.Sigalla J,David A,Anegon I,et al.Adenovirus-mediated gene transfer into isolatedmouse adult pancreatic islets:normal beta-cell function despite induction ofan anti-adenovirus immune response.Hum Gene Ther 1997,8:1625-1634.
22.Level AM,Strappe PM,Zhao J,et al.Lentivirus Vector.J Biomed Sci 2004, 11:439-449.
23. Quinonez R, Sutton RE. Lentiviral vectors for gene delivery into cells. DNA Cell Biol 2002, 21:937-951.
24. Kafri T: Gene delivery by lentivirus vectors. An overview. Methods Mol Biol 2004, 246:367-390.
25. Zhao J, Bolton EM, Pettigrew GJ, et al. Lentivirus-mediated Gene Transfer of Viral Interleukin 10 Prolongs Survival of Cardiac Allografts in Rats. Poster Abstracts Session II 2004, 9: 621-633.
26. Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest, 2002, 110(6):751 — 759.
27. Kunjachan V, Subramanian A , Hanna M, et al. Comparison of different fabrication techniques used for processing 3-dimensional, porous, biodegradable scaffolds from modified starch for bone tissue engineering. Biomed Sci Instrum, 2004, 40:129-135.
28. El-Ghannam AR. Advanced bioceramic composite for bone tissue engineering: Design principles and structure-bioactivity relationship. J Biomed Mater Res, 2004, 69A(3): 490-501.
29. Liu X, Ma PX. Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng, 2004, 32(3): 477-486.
30. Walles T, Herden T, Haverich A, et al. Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials, 2003, 24(7): 1233-1239.
31. Heymann D, Delecrin J, Deschamps C, et al. In vitro assessment of associating osteogenetic cells with macroporous calcium-phosphate ceramics. RevChir Orthop Reparatrice Appar Mot, 2001,87:8-17.
32. Steele JG, Johnson G, McLean KM, et al. Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer. J Biomed Mater Res,2000, 50:475-482.
33. Tsuchida H, Hashimoto J, Crawford E, et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthop Res, 2003, 21:44-53.
34. Hutmacher DW. Scaffold design and fabrication technologies for engineering tissue—state of the art and future perspectives. J Biomater Sci PolymEd, 2001,12:107-115.
35. Koc N, TimucinM, Korkusuz F, et al. Fabrication and characterization of porous tricalcium phosphate ceramics. Ceramics Int, 2004, (30):205-211.
36. Tsuruga E, Takita H, Itoh H, et al. Porr size of porous hydroxyapatie as the cell-substratum controls BMP-induced osteogenesis. J Biochem, 1997, 121:317-324.
37. Boyan BD, Hummert TW, Dean DD, et al. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials. 1996, 17(2): 137-46.
38. Wang Y, Uemura T, Dong J, et al. Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials. Tissue Eng, 2003, 8:1205-1214.
39. Holtorf HL, Sheffield TL, Ambrose CG, et al. Flow perfusion of marrow stromal cells seeded on porous biphasic calcium phosphateceramics. Ann Biomed Eng. 2005: 33(9):1238-1248.
40. Data N, Pham QP, Sharma U, et al. In vitro generated extracellular matrix and fluid shear stress synergistically edhance 3D osteoblastic differentiation. Proc Natl Acad Sci U S A 2006, 103(8), 2488 - 2493.
41. Baltzer AW, Lattermann C, Whalen JD, et al: A gene therapy approach to accelerating bone healing: Evaluation of gene expression in a New Zealand White rabbit model. Knee Surg Sports Traumatol Arthrosc 1999; 7: 197 - 202.
42. Baltzer AW, 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 Ther. 2000; 7:734-9.
43. Lieberman 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. J Bone Joint Surg Am. 1999; 81: 905-17.
44. 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;78:476-86.
45. Viggeswarapu M, Boden SD, Liu Y et al. Adenoviral delivery of LIM mineralization protein-1 induces new-bone formation in vitro and in vivo. J Bone Joint Surg Am. 2001:83:364-76.
46. Virk MS, Conduah A, Park SH et al. Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone, 2008 Jan 5[Epub ahead of print].
47. Sugiyama O, An DS, Kung SP et al. Lentivirus-Mediated Gene Transfer Induces Long-Term Transgene Expresion of BMP-2 in Vitro and New Bone Formation in Vivo. Mol Ther 2005;Vol 11 No. 3:390-396
2.杨志明,余希杰,解慧琪,等.不同来源成骨细胞生物学特性的比较研究.中华创伤杂志,2001,17:10-13.
3.Boden SD.Bioactive factor for bone tissue engineering.Clin orthop and Relat Res,1999,(367suppl):s84-89.
4.Lieberman JR,Daluiski A,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.J.Bone Joint Surg Am.1999,81(7):905-17.
5.Sigalla J,David A,Anegon I,et al.Adenovirus-mediated gene transfer into isolatedmouse adult pancreatic islets:normal beta-cell function despite induction ofan anti-adenovirus immune response.Hum Gene Ther 8:1625-1634,1997
6.Level AM,Strappe PM,Zhao J,et al.Lentivirus Vector.J Biomed Sci 2004,11:439-449.
7.Quinonez R,Sutton RE.Lentiviral vectors for gene delivery into cells.DNA Cell Biol 2002,21:937-951.
8.Kafri T.Gene delivery by lentivirus vectors.An overview.Methods Mol Biol 2004,246:367-390.
9.Zhao J,Bolton EM,Pettigrew GJ,et al.Lentivirus-mediated Gene Transfer of Viral Interleukin 10 Prolongs Survival of Cardiac Allografts in Rats.Poster Abstracts Session Ⅱ 2004,9:621-633.
10.Minguell JJ,Erices A,Conget P.Mesenchymal stem cells.Exp Biol Med(Maywood).2001 Jun,226(6):507-20.
11.Kunjachan V,Subramanian A,Hanna M,et al.Comparison of different fabrication techniques used for processing 3-dimensional,porous,biodegradable scaffolds from modified starch for bone tissue engineering.Biomed Sci Instrum,2004,40:129-135.
12.El-Ghannam AR.Advanced bioceramic composite for bone tissue engineering:Design principles and structure-bioactivity relationship.J Biomed Mater Res,2004,69A(3):490-501.
13. Liu X, Ma PX. Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng, 2004, 32(3): 477-486.
14. Walles T, Herden T, Haverich A, et al. Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials, 2003, 24(7): 1233-1239.
1.项鹏,李树浓.骨髓间质干细胞研究进展 中国病理生理杂志 2002,18(11):1438-1442.
2.Bruder SP,Jaiswal N,Ricalton NS,et al.Mesenchymal stem calls in osteobioligy and applied bone regeneration.J Clinical Orthopaedics and Related Research.1998,355:247.
3.雷俊霞,李浩威,黄春浓,等.长期传代的大鼠骨髓间质干细胞的牛物学特性分析 中国病理生理杂志2003,19(10):1325-1330.
4.肖庆中,李浩威,温冠媚等.大鼠骨髓间质干细胞基本生物学特性的研究 中国病理生理杂志 2004,3(20):289-294.
5.Conget PA,Minguell JJ.Phenotypical and functional of human bone marrow mesenchymal progenitor cells.J Cell Physiol.1999,181(1):67.
6、李连达,吴理茂,刘红.大鼠骨髓间质干细胞的培养和牛物学特征 科学技术与工程2003,6(3):547-550.
7.Pittenger MF,Mackay AM,Jsiswal SC,et al.Multilineage potential of adult human mesechymal stem cells.Science.1999,284(5411):143-147.
8.Jiang YH,Balkrishna N,Jahagirdar R,et al.Pluripotency of mesenchymal stem cells derived from adult marrow.Nature.2002,4(418):41-49.
9.Elena AJ,Sally EK,Anne E,et al.Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells.Arthritis and Rheumatism.2002,12(46):3349-3360.
10.Donald PL,Stephen EH,Douglas MA,et al.Dilution of human mesenchymal stem cells with dermal fibroblasts and the effects on in vitro and in vivo osteochondrogenesis.Developmental Dynamics.2000,219:50-62.
11.Hiroyuki T,Junichi H,Eric C,et al.Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.J Ortho Res.2003,21:44-53.
12.Devine SM,Bartholomew AM,Nelson M,et al.Mesenchymal stem cells are capable of homing to the bone marrow of non-human primares following systemic infusion.Exp Hematol.2001,29:244-255.
13.Shake JG,Gruber PJ,Baumgartner WA,et al.Mesenchymal stem cell implantation in a swine myocardial ingarct model:engraftment and functional effects.Ann Thorac Surg.2002,73:1919-1925.
14.Toma C,Pittenger MF,Cahill KS,et al.Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation.2002,105:93-98.
15.Kinner B,Gerstenfeld LC,Einhorn TA,et al.Expression of smooth muscle actin in connective tissue cells participating in fracture healing in a murine model.Bone.2002,30:738-745.
16.Kinner B,Zaleskas JM,Spector M,et al.Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells.Exp Cell Res.2002,278:72-83.
17.Eckes B,Colucci GE,Smola H,et al.Impaired wound healing in embryonic and adult mice lacking vimentin.J Cell Ther.2000,113:2455-62.
18.Liechty KW,Mackenzie TC,Shaaban AF,et al.Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.Nat Med.2000,6:1282-1286.
19.Millington WS,Allers C,Tuohy G,et al.Validation in mesenchymal progenitor cells of a mutation-independent ex vivo approach to gene therapy for osteogenesis imperfecta.Hum Mol Genet.2002,11:2201-2206.
20.Hutcheson KA,Atkins BZ,Hueman MT,et al.Comparison of benefits on myocardial performance of cellular cardiomyoplasty with skeletal myoblasts and fibroblasts.Cell Transplant.2000,9:359-368.
21.杨志明,余希杰,解慧琪,等.不同来源成骨细胞生物学特性的比较研究[J].中华创伤杂志.2001,17:10-13.
1. Allampallam K, Chakraborty J, Robinson J. Effect of ascorbic acid and growth factors on collagen metabolism of flexor retinaculum cells from individuals with and without carpal tunnel syndrome. Occup Environ Med, 2000, 42(3): 251-259.
2. Maniatopoulos C, Sodek J, Melcher AH , et al. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res, 1988, 254(2): 317-330.
3. Hanada K, Dennis JE, Caplan AI, et al. Stimulatory effects of basic fibroblast growth factor and bone morphogenetic pro-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. J Bone Miner Res, 1997, 12:1606-1614.
4. Cheng BH, Jiang W, Frank MP, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins(BMPs). J Bone J Surg, 2003, 8 (85):1544-1552.
5. Allampallam K, Chakraborty J, Robinson J, et al. Effects of ascorbic acid and growth factors on collgen metabolism of flexor retinaculum cells from individuals with and without carpal tunnel syndrome. Occup Environ Med, 2000, 42(3): 251-259.
6. Maniatopoulos C, Sodek J, Melcher AH, et al. Bone formation in vitro by stromal cells obtained from bone marrow of young adults rats. Cell Tissue Res. 1998, 254(2): 317-330.
7. Cheng SL, Yang JW, Rifas L, et al. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology, 1994, 134(1):277-286.
8. Ebara S, Nakayama K. Mechanism for the action of bone morphogenetic proteins and regulation of their activity. Spine, 2002 , 27(16): 10-15.
9. Wloderski KH. Properties and origin of osteoblasts. Clin Ortho, 1990, 252(3): 276-293.
10. Alborxi A, Mac KG, Lackin CA, et al. Endochondral and intramembranous fetal bone development: osteoblastic cell proliferation and expression of alkaline phosphatase. Cranifac Genet Dev Biol, 1996, 16(2):94-98.
11. Warner GP, Hubbard HL. 32P and 45Ca-metabolism by mareix vesicle-enriched microsomes prepared from chicken epiphyseal cartilage percoll density-gradient fractionation.Calcif Tissue Int,1983,35(3):327-328.
12.杨志明:组织工程皋础与临床:成都:四川科学技术出版社,1999,124-129.
13.Hiroyuki T,Junichi H,Eric C,et al.Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.J Ortho Res,2003,21:44-53.
1. Boyan BD, Hummert TW, Dean DD, et al. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials. 1996, 17(2): 137-46.
2. Kunjachan V, Subramanian A, Hanna M, et al. Comparison of different fabrication techniques used for processing 3-dimensional, porous, biodegradable scaffolds from modified starch for bone tissue engineering. Biomed Sci Instrum, 2004, 40:129-135.
3. El-Ghannam AR. Advanced bioceramic composite for bone tissue engineering: Design principles and structure-bioactivity relationship. J Biomed Mater Res, 2004, 69A(3): 490-501.
4. Liu X, Ma PX. Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng, 2004, 32(3) :477-486.
5. Walles T, Herden T, Haverich A, et al. Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials, 2003, 24(7): 1233-1239.
6. Heymann D, Delecrin J, Deschamps C, et al. In vitro assessment of associating osteogenetic cells with macroporous calcium-phosphate ceramics. RevChir Orthop Reparatrice Appar Mot, 2001, 87:8-17.
7. Steele JG, Johnson G, McLean KM, et al. Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer. J Biomed Mater Res, 2000, 50:475-482.
8. Tsuchida H, Hashimoto J, Crawford E, et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthop Res, 2003, 21:44-53.
9. Hutmacher DW. Scaffold design and fabrication technologies for engineering tissue—state of the art and future perspectives. J Biomater Sci PolymEd, 2001, 12:107-115.
10. Koc N, Timucin M, Korkusuz F, et al. Fabrication and characterization of porous tricalcium phosphate ceramics. Ceramics Int, 2004, (30):205-211.
11. Tsuruga E, Takita H, Itoh H, et al. Porr size of porous hydroxyapatie as the cell-substratum controls BMP-induced osteogenesis. J Biochem, 1997, 121:317-324.
12. Gronthos S, Stewart K, Graves SE, et al. Integrin expression and function on human osteoblast-like cells. J Bone Miner Res, 1997 12(8):1189-97.
13. Suzuki T, Yamamoto T, Toriyama M, et al. Surface instability of calcium phosphate ceramics in tissue medium and the effect on adhesion and growth of anchorage-dependent animal cells.. J Biomed Mater Res, 1997 ,34(4):507-17.
14. Knabe C, Ostapowicz W, Radlanski RJ, et al. In vitro investigation of novel calcium phosphates using osteogenic cultures. J Mater Sci Mater Med, 1998 ,9(6): 337-45.
1.Schmitz JP,Hollinger JO.The critical size defect as an experimental model for craniomandibulofacial nonunions.Clin Orthop Rlat Res.1986,205:299-308.
2.Sweeney TM,Opperman LA,Persing JA,et al.Repair of critical size rat calvarial defects using extracellular matrix protein gels.J Neurosurg.1995,83(4):710-715.
3.Yoshikawa T,Ohgushi H,Nakajima H,et al.In vivo osteogenic durability of cultured bone in porous ceramics.Transplantation,2000,69:128-134.
4.陈希哲,杨连甲,付崇建等.含孔磷酸钙陶瓷复合自体成骨细胞骨组织工程大鼠模型的构建.中华实验外科杂志,2002,19(5):460-461.
5.Gregoire M,Orly I,Menanteau J.The influence of calcium phosphate biomaterials on human bone cell activities:an in vitroa pproach.J Biomed Mater Res,1990,24(2):165-167.
6.Grover LM,Gbureck U,Wright AJ,et al.Biologically mediated resorption of brushite cement in vitro.Biomaterials,2006,27(10):2178-2185.
7.Ooms EM,Wolke JG,van der Waerden JP,et al.Trabecular bone response to injectable calcium phosphate(Ca-P) cement.J Biomed Mater Res,2002,61(1):9-18.
8.Zerbo IR,Bronckers AL,de Lange G,et al.Localisation of osteogenic and osteoclastic cells in porous beta-tricalcium phosphate particles used for human maxillary sinus floor elevation.Biomaterials,2005,26(12):1445-1451.
9.Niemela T.Effect of β -tricalcium phosphate addition on the in vitro degradation of self-reinforced poly-L,D-lactide.Polymer Degrade Stabil,2005,89(3):492-500.
10.Shikinami Y,Matsusue Y,Nakamura T.The complete process of bioresorption and bone replacement using devices made of forged composites of raw hydroxyapatite particles/poly 1-lactide(F-u-HA/PLLA).Biomaterials,2005,26(27):5542-5551.
11.Wiltfang J,Merten HA,Schlegel KA,et al.Degradation characteristics of alpha and beta tri-calcium-phosphate(TCP) in minipigs.J Biomed Mater Res,2002,63(2):165-167.
12.Asanuma K,Masui F,Kamitani K,et al.Clinical application of pure beta-TCP for bone tumors.
13. Okuda T, Ioku K, Yonezawa I, et al. The effect of the microstructure of beta tri-calcium phosphate on the metabolism of subsequently formed bone tissue. Biomaterials, 2007, 28(16):2612-2621.
14. Lieberman JR, Daluiski A, 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. J. Bone Joint Surg Am. 1999, 81(7): 905—17.
15. Virk MS, Conduah A, Park SH et al. Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone, 2008 Jan 5[Epub ahead of print].
16. Hsu WK, Sugiyama o, park SH, et al. Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats. Bone. 2007,40(4): 931-938.
1. Crane GM, Ishaugug SL, Mikos AG, et al. Bone tissue engineering. Nat Med, 1995,1:1332-1334.
2. Croteau S, Rauch F, Silvestri A, et al. Bone morphogenetic proteins in orthopaedics: from basic science to clinical practice. Orthopaedics, 1999, 22: 686-695.
3. Gandolfi MG, Pugnaloni A, Mattioli BM, et al. Osteoblast behaviour in the presence of bisphosphonates: ultrastructural and biochemical in vitro studies. Clin Exp Rheumato, 1999, 17(3): 327-333.
4. Wright V, Peng H, Usas A, et al. BMP4-expressing muscle derived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol Ther, 2002, 6(2): 169—178.
5. Mie M, Ohgushi H, Yanagida Y, et al. osteogenesis coordinated in C3H10TI/2 cells by adipogenesis-dependent BMP-2 expression system. Tissue Eng, 2000, 6(1) :9—18.
6. Krebsbach PH, Gu K, Franceschi RT, et al. Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo. Hum Gene Ther, 2000, 11(8) :1201 —1210.
7. Zuk PA, Zhu M, MizunoH, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng, 2001, 7(2):211-228.
8. Nathan S, Das De S, Thambyah A, et al. Cell-based therapy in the repair of osteochondral defects: a novel use for adipose tissue. Tissue Eng, 2003, 9(4): 733-744.
9. Ogawa R , Mizuno H , Watanabe A , et al. Osteogenic and chondro-genic differentiation by adipose-derived stem cells harvested from GFP transgenic mice. Biochem Biophys Res Commu, 2004, 313(4): 871—877.
10. Yang M, Ma QJ, Dang GT, et al. In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult stem cells expressing BMP-7. Cytotherapy, 2005, 7(3):273 — 281
11.Shimizu T,Sasano Y,Nakajo S,et al.osteoblastic differentiation of periosteum-derived cells is promoted by the physical contact with the bone matrix in vivo.Anat Rec,2001,264(1):72-81.
12.Schantz JT,Hutmacher DW,Chim H,et al.Induction of ectopic bone formation by using human Periosteal cells in combination with a novel scaffold technology:Cell Transplant,2002,11(2):125-138.
13.Friedenstein AJ,Chailakhyan RK,Gerasimov UV,et al.Bon Marrow osteogenic stem cells:in vitro cultivation and transplantation in diffusion chambers.Cell Tissue Kinet,1987,20(3):263-272.
14.项鹏,李树浓.骨髓间质干细胞研究进展 中国病理生理杂志 2002,18(11):1438-1442.
15.刘劲松,曾才铭,王宏邦,等.骨髓基质细胞培养和体外成骨研究.中国修复重建外科杂志,1997,11:238-241.
16.杨志明,余希杰,解慧琪,等.不同来源成骨细胞生物学特性的比较研究.中华创伤杂志,2001,17:10-13.
17.Reddi AH,initiation of fracture repair by bone morphogenetic proteins.Clin Orthop,1998,355(suppl):s66-s72.
18.Yang Y,Nunes FA,Berencsi K et al.Cellularimmunity to viral antigens limits El-deleted adenoviruses for gene therapy.Proc Natl Acad Sci U S A 1994,91:4407 - 4411.
19.Hacein-Bey-Abina,S.,et al.LMO2-associatedclonal T cell proliferation in two patients after gene therapy for SCID-X1.Science.2003,302:415-419.
20.Wu.X.Li Y.,Crise B.,and Burgess,S.M.Transcription start regions in the human genomeare favored targets for MLV integration.Science.2003,300:1749 -1751.
21.Sigalla J,David A,Anegon I,et al.Adenovirus-mediated gene transfer into isolatedmouse adult pancreatic islets:normal beta-cell function despite induction ofan anti-adenovirus immune response.Hum Gene Ther 1997,8:1625-1634.
22.Level AM,Strappe PM,Zhao J,et al.Lentivirus Vector.J Biomed Sci 2004, 11:439-449.
23. Quinonez R, Sutton RE. Lentiviral vectors for gene delivery into cells. DNA Cell Biol 2002, 21:937-951.
24. Kafri T: Gene delivery by lentivirus vectors. An overview. Methods Mol Biol 2004, 246:367-390.
25. Zhao J, Bolton EM, Pettigrew GJ, et al. Lentivirus-mediated Gene Transfer of Viral Interleukin 10 Prolongs Survival of Cardiac Allografts in Rats. Poster Abstracts Session II 2004, 9: 621-633.
26. Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest, 2002, 110(6):751 — 759.
27. Kunjachan V, Subramanian A , Hanna M, et al. Comparison of different fabrication techniques used for processing 3-dimensional, porous, biodegradable scaffolds from modified starch for bone tissue engineering. Biomed Sci Instrum, 2004, 40:129-135.
28. El-Ghannam AR. Advanced bioceramic composite for bone tissue engineering: Design principles and structure-bioactivity relationship. J Biomed Mater Res, 2004, 69A(3): 490-501.
29. Liu X, Ma PX. Polymeric scaffolds for bone tissue engineering. Ann Biomed Eng, 2004, 32(3): 477-486.
30. Walles T, Herden T, Haverich A, et al. Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials, 2003, 24(7): 1233-1239.
31. Heymann D, Delecrin J, Deschamps C, et al. In vitro assessment of associating osteogenetic cells with macroporous calcium-phosphate ceramics. RevChir Orthop Reparatrice Appar Mot, 2001,87:8-17.
32. Steele JG, Johnson G, McLean KM, et al. Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer. J Biomed Mater Res,2000, 50:475-482.
33. Tsuchida H, Hashimoto J, Crawford E, et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthop Res, 2003, 21:44-53.
34. Hutmacher DW. Scaffold design and fabrication technologies for engineering tissue—state of the art and future perspectives. J Biomater Sci PolymEd, 2001,12:107-115.
35. Koc N, TimucinM, Korkusuz F, et al. Fabrication and characterization of porous tricalcium phosphate ceramics. Ceramics Int, 2004, (30):205-211.
36. Tsuruga E, Takita H, Itoh H, et al. Porr size of porous hydroxyapatie as the cell-substratum controls BMP-induced osteogenesis. J Biochem, 1997, 121:317-324.
37. Boyan BD, Hummert TW, Dean DD, et al. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials. 1996, 17(2): 137-46.
38. Wang Y, Uemura T, Dong J, et al. Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials. Tissue Eng, 2003, 8:1205-1214.
39. Holtorf HL, Sheffield TL, Ambrose CG, et al. Flow perfusion of marrow stromal cells seeded on porous biphasic calcium phosphateceramics. Ann Biomed Eng. 2005: 33(9):1238-1248.
40. Data N, Pham QP, Sharma U, et al. In vitro generated extracellular matrix and fluid shear stress synergistically edhance 3D osteoblastic differentiation. Proc Natl Acad Sci U S A 2006, 103(8), 2488 - 2493.
41. Baltzer AW, Lattermann C, Whalen JD, et al: A gene therapy approach to accelerating bone healing: Evaluation of gene expression in a New Zealand White rabbit model. Knee Surg Sports Traumatol Arthrosc 1999; 7: 197 - 202.
42. Baltzer AW, 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 Ther. 2000; 7:734-9.
43. Lieberman 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. J Bone Joint Surg Am. 1999; 81: 905-17.
44. 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;78:476-86.
45. Viggeswarapu M, Boden SD, Liu Y et al. Adenoviral delivery of LIM mineralization protein-1 induces new-bone formation in vitro and in vivo. J Bone Joint Surg Am. 2001:83:364-76.
46. Virk MS, Conduah A, Park SH et al. Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone, 2008 Jan 5[Epub ahead of print].
47. Sugiyama O, An DS, Kung SP et al. Lentivirus-Mediated Gene Transfer Induces Long-Term Transgene Expresion of BMP-2 in Vitro and New Bone Formation in Vivo. Mol Ther 2005;Vol 11 No. 3:390-396