人骨髓间充质干细胞复合PLGA的体外灌流培养及成骨细胞分化研究
摘要
骨组织工程为骨缺损修复提供了一种新的疗法。人骨髓间充质干细胞(hMSCs)是一种理想的骨组织工程种子细胞,被广泛用于与支架材料复合构建组织工程骨。聚乳酸-乙醇酸(PLGA)支架在软骨和骨组织工程应用中显示出较好的性能而被广泛接受。灌流式生物反应器常用于培养骨骼组织。本文目的是评价灌流培养条件下人骨髓间充质干细胞在PLGA支架中的生长增殖及分化动态。实验所用支架孔隙率为94.5%-98.0%,孔径大小为280-450μm。首先通过对三个不同接种密度(1×10~6,2×10~6,4×10~6个/ml)接种效率的比较,确定了较为合适的接种密度(2×10~6个/ml)。通过对三个不同流速(0.2,0.4,0.8ml/min)培养效果的比较,确定了较为合适的灌流流速(0.2ml/min)。通过合适的接种密度和合适的灌流流速立体培养hMSCs,研究细胞在支架中的生长动态。MTT法检测细胞活力显示,在整个灌流培养期间,细胞活力持续显著增加,表明细胞增殖旺盛。DNA定量分析表明从接种到培养第6天细胞数量显著增加,之后增殖变缓。用扫描电镜(SEM)来观察细胞在支架中的分布以及细胞外基质的分泌情况,结果显示第5天细胞已经长入支架孔内部,并见有薄层基质合成;第11天,细胞外基质已形成厚厚的一层将支架基本完全覆盖。活细胞荧光染色和苏木素-伊红(H&E)染色可见细胞在支架中均匀分布。通过成骨细胞定向诱导分化实验,检测支架中hMSCs的成骨细胞分化效率。碱性磷酸酶染色和酶活性检测结果表明,成骨诱导第7天细胞已经开始进入早期分化,诱导期间持续分化;诱导组碱性磷酸酶活性显著高于对照组(未诱导组);未诱导组也检测到微量碱性磷酸酶存在,推测是灌流培养产生的剪切力所致。以上结果表明,用该灌流生物反应器系统来进行hMSCs的三维立体扩增和分化是可行的,可以用于骨组织工程研究。
Bone tissue engineering beckons a new frontier for clinical treatment to bone defects. hMSCs, as an ideal cell source, are widely used in the treatment of bony defects. PLGA scaffold has exhibited good biocompatibility in bone tissue engineering. Perfusion bioreactor can provided favorable conditions for the three-dimensional cultivation of bone tissue. The objective of this study was to determine the viability and growth of hMSCs in a three-dimensional porous PLGA scaffold in combination with perfusion bioreactor culture system. The scaffold has a porosity of 94.5%-98.0% and pore size of 280-450μm. hMSCs were seeded at a density of 2×10~6cells/ml, and the perfusion rate was 0.2ml/min. Firstly, three different cell density(1×10~6, 2×10~6, 4×10~6个/ml) were used in the seeding process, and a cell density of 2×10~6个/ml was found to be the more proper seeding density by the comparison of seeding efficiency. Comparison of cell status after 3-days culture under three different perfusion rates (0.2, 0.4, 0.8ml/min) generated the more proper perfusion rate (0.2ml/min). Cell viability by analyzing with MTT colorimetry increased with time, indicating a significant proliferation. The cell quantity based on DNA assay increased in a fast proliferation at first 6 days, and then in a slower proliferation. SEM images showed, at day 5, hMSCs has migrated into the scaffold holes, and a dense layer of extracellular matrix has been formed; and the entire surface of the scaffold was covered by a dense layer of extracellular matrix at day 11. FDA staining and H&E staining showed that the cells spreaded uniformly through the whole scaffold. ALP staining and ALP activity assay showed that the early differentiation began at 7 days after osteogenic differentiation, followed by a continuing differentiation. Moreover, ALP activity of osteogenic medium induced group is much higher than that of the control group.These results demonstrate the feasibility of culturing cell/PLGA in a flow perfusion bioreactor for bone tissue engineering applications.
Bone tissue engineering beckons a new frontier for clinical treatment to bone defects. hMSCs, as an ideal cell source, are widely used in the treatment of bony defects. PLGA scaffold has exhibited good biocompatibility in bone tissue engineering. Perfusion bioreactor can provided favorable conditions for the three-dimensional cultivation of bone tissue. The objective of this study was to determine the viability and growth of hMSCs in a three-dimensional porous PLGA scaffold in combination with perfusion bioreactor culture system. The scaffold has a porosity of 94.5%-98.0% and pore size of 280-450μm. hMSCs were seeded at a density of 2×10~6cells/ml, and the perfusion rate was 0.2ml/min. Firstly, three different cell density(1×10~6, 2×10~6, 4×10~6个/ml) were used in the seeding process, and a cell density of 2×10~6个/ml was found to be the more proper seeding density by the comparison of seeding efficiency. Comparison of cell status after 3-days culture under three different perfusion rates (0.2, 0.4, 0.8ml/min) generated the more proper perfusion rate (0.2ml/min). Cell viability by analyzing with MTT colorimetry increased with time, indicating a significant proliferation. The cell quantity based on DNA assay increased in a fast proliferation at first 6 days, and then in a slower proliferation. SEM images showed, at day 5, hMSCs has migrated into the scaffold holes, and a dense layer of extracellular matrix has been formed; and the entire surface of the scaffold was covered by a dense layer of extracellular matrix at day 11. FDA staining and H&E staining showed that the cells spreaded uniformly through the whole scaffold. ALP staining and ALP activity assay showed that the early differentiation began at 7 days after osteogenic differentiation, followed by a continuing differentiation. Moreover, ALP activity of osteogenic medium induced group is much higher than that of the control group.These results demonstrate the feasibility of culturing cell/PLGA in a flow perfusion bioreactor for bone tissue engineering applications.
引文
[1] Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal and the osteogenic potential of purified huamn mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997, 64:278-294
[2] Pittenger MF, Mackey AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J].Science 1999,284 (5411): 143-147
[3] Caplan AI, Bruder SP. Mesenchymal stem cells: Building blocks for molecular medicine in the 21st century. Trends Mol Med 2001, 7:259-264
[4] Anselme K, Brous O, Noel B, et al. In vitro control of human bone marrow stromal cells for bone tissue engineering. Tissue Eng 2002, 8:941-53
[5] Cancedda R, Mastrogiacomo M, Bianchi G, et al. Bone marrow stromal cells and their use in regenerating bone. Novartis Found Symp 2003, 249:133-43
[6] Kotobuki N, Hirose M, Takakura Y, et al. Cultured autologous huamn cells for hard tissue regeneration:preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs 2004, 28:33-9
[7] Kuo CK, Tuan RS. Tissue engineering with mesenchymal stem cells.IEEE Eng Med Biol Mag 2003, 22:51-56
[8] Shum AWT, Mak AFT. Morphological and biomechanical characterization of poly (glycolic acid) scaffolds after in vitro degradation. Polymer Degradation and Stability 2003, 81:141-149
[9] Agrawal CM, Ray RB. Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. J Biomed Mater Res 2001, 55:141-150
[10] Behravesh E, Yasko AW, Engel PS, et al. Synthetic biodegradable polymers for orthopaedic applications. Clin Orthop Relat Res 1999, 367(Suppl):118-129
[11] Athanasiou KA, Niederauer GG and Agrawal CM. Sterilization, toxicity,biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers. Biomaterials 1996, 17: 93-102
[12] Borden M, EI-Amin SF, Attawia M, et al. Structural and human cellular assessment of a novel micro sphere-based tissue engineered scaffold for bone repair. Biomaterials 2003, 24:597-609
[13] Burg KJ, Delnomdedieu M, Beiler RJ, et al. Application of magnetic resonance microscopy to tissue engineering: A polylactide model. J Biomed Mater Res 2002,61:380-390
[14] Begley CM, Kleis SJ. The fluid dynamic and shear environment in the NASA/J SC rotating-wall perfused-vessel bioreactor. Biotechnol Bioeng 2000, 70: 32-40
[15] Goodwin TJ, Prewett TL, Wolf DA. Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity. Cell Biochem 1993, 51: 301-311
[16] Freed E, Langer R, Martin I. Tissue engineering of cartilage in space. Proc Natl Acad Sci USA 1997, 94: 13885-13890
[17] Mueller SM, Mizuno S, Gerstenfeld LC, et al. Medium perfusion enhances osteogenesis by murine osteosarcoma cells in three-dimensional collagen sponges. Bone Miner Res 1999, 14:2118-2126
[18] 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, 9:1205-1204
[19] Glowacki J, Mizuno S, and Greenberger JS. Perfusion enhances functions of bone marrow stromal cells in three dimensional culture. Cell Transplant 1998,7:319-326
[20] van den Dolder J, Bancroft GN, Sikavitsas VI, et al. Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh. J Biomed Mater Res A 2003,64: 235-241
[21] Goldstein AS, Juarez TM, Helmke CD, et al. Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials 2001,22: 1279-1288
[22] Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering.Trends Biotechnol 2004, 22:80-86
[23] Li Y, Ma T, Kniss D A, et al. Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices. Biotechnol Prog 2001,17:935-944
[24] Burg KJ, Holder WDJ, Culberson CR, et al. Comparative study of seeding methods for three-dimensional polymeric scaffolds. J Biomed Mater Res 2000,51:642-649
[25] Holy CE, Shoichet MS, Davies JE. Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell-seeding density and culture period. J Biomed Mater Res 2000, 51:376-382
[26] Bancroft GN, Sikavitsas VI and Mikos AG. Design of a flow perfusion bioreactor system for bone tissue engineering applications. Tissue Eng 2003, 9:549-554
[27] Burg KJ, Holder WD, Culberson CR, et al. Comparative study of seeding methods for three-dimensional polymeric scaffolds. J Biomed Mater Res 2000,51:642-649
[28] Cartmell SH, Porter BD, Garcia AJ, et al. Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. Tissue Eng 2003,9:1197-1203
[29] Wendt D, Marsano A, Jakob M, et al. Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity. Biotechnol Bioeng 2003, 84:205-21
[30] Hillsley, MV, Frangos, JA. Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow. Calcified Tissue International 1997, 60: 48 - 53.
[31] Jiang GL, White CR, Stevens HY, et al. Temporal gradients in shear stimulate osteoblastic proliferation via ERK 1/2 and retinoblastoma protein. American Journal of Physiology, Endocrinology and Metabolism 2002, 283: E383 - E389.
[32] Klein-Nulend J, Helfrich MH, Sterck JG, et al. Nitric oxide response to shear stress by human bone cell cultures is endothelial nitric oxide synthase dependent.Biochemical and Biophysical Research Communications 1998, 250: 108-114
[33] McAllister TN, Du T, Frangos JA. Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrowderived preosteoclast-like cells. Biochemical and Biophysical Research Communications 2000, 270: 643 - 648.
[34] Reich KM, Frangos JA. Effect of flow on prostaglandin E2 and inositol trisphosphate levels in osteoblasts. American Journal of Physiology 1991, 261(3 Pt 1): C428-C432.
[35] Smalt R, Mitchell FT, Howard RL. Induction of NO and prostaglandin E2 in osteoblasts by wall-shear stress but not mechanical strain. American Journal of Physiology 1997, 273 (4 Pt 1): E751-E758.
[36] Gregory NB, Vassilios IS, Antonios GM. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner. PNAS 2002, 99: 12600-12605
[37] Heidi LH, John AJ, Antonios GM. Flow perfusion culture induces the osteoblastic differentiation of marrow stromal cell-scaffold constructs in the absence of dexamethasone. J Biomed Mater Res A 2005, 72A: 326-334.
[38] Zhao F, Chella R, Ma T. Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: Experiments and hydrodynamic modeling. Biotechnology and Bioengineering 2007, 96:584-595
[39] Cartmell SH, Porter BD, Garcia AJ. Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. Tissue Engineering 2003,9: 1197-1203.
[40] Blaise P, Roger Z, Harlan S, et al. 3-D computational modeling of media flow through scaffolds in a perfusion bioreactor. Journal of Biomechanics 2005, 38:543-549.
[1]杨志明,黄富国,秦廷武等.生物衍生组织工程骨植骨的初步临床应用。中国修复重建外科杂志,2002,16(5):311-314.
[2]Bruder SP,Jaiswal N,Haynesworth SE.Growth kinetics,self-renewal and the osteogenic potential of purified huamn mesenchymal stem cells during extensive subcultivation and following cryopreservation.J Cell Biochem 1997,64:278-294.
[3]Pittenger MF,Mackey AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science 1999,284(5411):143-147.
[4]Caplan AI,Bruder SP.Mesenchymal stem cells:Building blocks for molecular medicine in the 21st century.Trends Mol Med 2001,7:259-264.
[5]Anselme K,Brous O,Noel B,et al.In vitro control of human bone marrow stromal cells for bone tissue engineering.Tissue Eng 2002,8:941-953.
[6] Cancedda R, Mastrogiacomo M, Bianchi G, et al. Bone marrow stromal cells and their use in regenerating bone. Novartis Found Symp 2003, 249: 133-43
[7] Kotobuki N, Hirose M, Takakura Y, et al. Cultured autologous huamn cells for hard tissue regeneration:preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs 2004, 28: 33-9
[8] Stenderup K, Justesen J, Clausen C, et al. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells.Bone 2003, 33: 919-926.
[9] Mauney JR, Kaplan DL, Volloch V. Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion. Biomaterials 2004, 25: 3233-3243.
[10] Walsh S, Jefferiss C, Stewart K, et al. Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells:regulation by fibroblast growth f actor(FGF)-β and relationship to the expression of FGF receptors 1-4. Bone 2000, 27: 185-195
[11] Abdallah BM, Haack SM, Burns JS, et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite of extensive proliferation.Biochem Biophys Res Commun 2005, 326: 527-538.
[12] Ni S, Chang J, Chou L. A novel bioactive porous CaSiO3 scaffold for bone tissue engineering. J Biomed Mater Res A 2006, 76: 196-205.
[13] Xu XL, Tang T, Dai K, et al. Immune response and effect of adenovirus-mediated human BMP-2 gene transfer on the repair of segmental tibial bone defects in goats Acta Orthop 2005, 76: 637-646.
[14] Chim H, Schantz JT. New frontiers in calvarial reconstruction: integrating computer-assisted design and tissue engineering in cranioplasty. Plast Reconstr Surg 2005, 116: 1726-1741.
[15] Bosetti M, Boccafoschi F, Leigheb M, et al . Effect of different growth factors on human osteoblasts activities: A possible application in bone regeneration for tissue engineering. Biomol Eng 2007, 24: 613-618.
[16] James KH, Krshna GG. Tissue engineering with chondrocytes. Facial Plastic Surgery 2002, 18:59-68.
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[22] Goodwin TJ, Prewett TL, Wolf DA. Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity. Cell Biochem 1993, 51: 301-311.
[23] Freed E, Langer R, Martin I. Tissue engineering of cartilage in space. Proc Natl Acad Sci USA 1997, 94: 13885-13890.
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[2] Pittenger MF, Mackey AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J].Science 1999,284 (5411): 143-147
[3] Caplan AI, Bruder SP. Mesenchymal stem cells: Building blocks for molecular medicine in the 21st century. Trends Mol Med 2001, 7:259-264
[4] Anselme K, Brous O, Noel B, et al. In vitro control of human bone marrow stromal cells for bone tissue engineering. Tissue Eng 2002, 8:941-53
[5] Cancedda R, Mastrogiacomo M, Bianchi G, et al. Bone marrow stromal cells and their use in regenerating bone. Novartis Found Symp 2003, 249:133-43
[6] Kotobuki N, Hirose M, Takakura Y, et al. Cultured autologous huamn cells for hard tissue regeneration:preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs 2004, 28:33-9
[7] Kuo CK, Tuan RS. Tissue engineering with mesenchymal stem cells.IEEE Eng Med Biol Mag 2003, 22:51-56
[8] Shum AWT, Mak AFT. Morphological and biomechanical characterization of poly (glycolic acid) scaffolds after in vitro degradation. Polymer Degradation and Stability 2003, 81:141-149
[9] Agrawal CM, Ray RB. Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. J Biomed Mater Res 2001, 55:141-150
[10] Behravesh E, Yasko AW, Engel PS, et al. Synthetic biodegradable polymers for orthopaedic applications. Clin Orthop Relat Res 1999, 367(Suppl):118-129
[11] Athanasiou KA, Niederauer GG and Agrawal CM. Sterilization, toxicity,biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers. Biomaterials 1996, 17: 93-102
[12] Borden M, EI-Amin SF, Attawia M, et al. Structural and human cellular assessment of a novel micro sphere-based tissue engineered scaffold for bone repair. Biomaterials 2003, 24:597-609
[13] Burg KJ, Delnomdedieu M, Beiler RJ, et al. Application of magnetic resonance microscopy to tissue engineering: A polylactide model. J Biomed Mater Res 2002,61:380-390
[14] Begley CM, Kleis SJ. The fluid dynamic and shear environment in the NASA/J SC rotating-wall perfused-vessel bioreactor. Biotechnol Bioeng 2000, 70: 32-40
[15] Goodwin TJ, Prewett TL, Wolf DA. Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity. Cell Biochem 1993, 51: 301-311
[16] Freed E, Langer R, Martin I. Tissue engineering of cartilage in space. Proc Natl Acad Sci USA 1997, 94: 13885-13890
[17] Mueller SM, Mizuno S, Gerstenfeld LC, et al. Medium perfusion enhances osteogenesis by murine osteosarcoma cells in three-dimensional collagen sponges. Bone Miner Res 1999, 14:2118-2126
[18] 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, 9:1205-1204
[19] Glowacki J, Mizuno S, and Greenberger JS. Perfusion enhances functions of bone marrow stromal cells in three dimensional culture. Cell Transplant 1998,7:319-326
[20] van den Dolder J, Bancroft GN, Sikavitsas VI, et al. Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh. J Biomed Mater Res A 2003,64: 235-241
[21] Goldstein AS, Juarez TM, Helmke CD, et al. Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials 2001,22: 1279-1288
[22] Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering.Trends Biotechnol 2004, 22:80-86
[23] Li Y, Ma T, Kniss D A, et al. Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices. Biotechnol Prog 2001,17:935-944
[24] Burg KJ, Holder WDJ, Culberson CR, et al. Comparative study of seeding methods for three-dimensional polymeric scaffolds. J Biomed Mater Res 2000,51:642-649
[25] Holy CE, Shoichet MS, Davies JE. Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell-seeding density and culture period. J Biomed Mater Res 2000, 51:376-382
[26] Bancroft GN, Sikavitsas VI and Mikos AG. Design of a flow perfusion bioreactor system for bone tissue engineering applications. Tissue Eng 2003, 9:549-554
[27] Burg KJ, Holder WD, Culberson CR, et al. Comparative study of seeding methods for three-dimensional polymeric scaffolds. J Biomed Mater Res 2000,51:642-649
[28] Cartmell SH, Porter BD, Garcia AJ, et al. Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. Tissue Eng 2003,9:1197-1203
[29] Wendt D, Marsano A, Jakob M, et al. Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity. Biotechnol Bioeng 2003, 84:205-21
[30] Hillsley, MV, Frangos, JA. Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow. Calcified Tissue International 1997, 60: 48 - 53.
[31] Jiang GL, White CR, Stevens HY, et al. Temporal gradients in shear stimulate osteoblastic proliferation via ERK 1/2 and retinoblastoma protein. American Journal of Physiology, Endocrinology and Metabolism 2002, 283: E383 - E389.
[32] Klein-Nulend J, Helfrich MH, Sterck JG, et al. Nitric oxide response to shear stress by human bone cell cultures is endothelial nitric oxide synthase dependent.Biochemical and Biophysical Research Communications 1998, 250: 108-114
[33] McAllister TN, Du T, Frangos JA. Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrowderived preosteoclast-like cells. Biochemical and Biophysical Research Communications 2000, 270: 643 - 648.
[34] Reich KM, Frangos JA. Effect of flow on prostaglandin E2 and inositol trisphosphate levels in osteoblasts. American Journal of Physiology 1991, 261(3 Pt 1): C428-C432.
[35] Smalt R, Mitchell FT, Howard RL. Induction of NO and prostaglandin E2 in osteoblasts by wall-shear stress but not mechanical strain. American Journal of Physiology 1997, 273 (4 Pt 1): E751-E758.
[36] Gregory NB, Vassilios IS, Antonios GM. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner. PNAS 2002, 99: 12600-12605
[37] Heidi LH, John AJ, Antonios GM. Flow perfusion culture induces the osteoblastic differentiation of marrow stromal cell-scaffold constructs in the absence of dexamethasone. J Biomed Mater Res A 2005, 72A: 326-334.
[38] Zhao F, Chella R, Ma T. Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: Experiments and hydrodynamic modeling. Biotechnology and Bioengineering 2007, 96:584-595
[39] Cartmell SH, Porter BD, Garcia AJ. Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. Tissue Engineering 2003,9: 1197-1203.
[40] Blaise P, Roger Z, Harlan S, et al. 3-D computational modeling of media flow through scaffolds in a perfusion bioreactor. Journal of Biomechanics 2005, 38:543-549.
[1]杨志明,黄富国,秦廷武等.生物衍生组织工程骨植骨的初步临床应用。中国修复重建外科杂志,2002,16(5):311-314.
[2]Bruder SP,Jaiswal N,Haynesworth SE.Growth kinetics,self-renewal and the osteogenic potential of purified huamn mesenchymal stem cells during extensive subcultivation and following cryopreservation.J Cell Biochem 1997,64:278-294.
[3]Pittenger MF,Mackey AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science 1999,284(5411):143-147.
[4]Caplan AI,Bruder SP.Mesenchymal stem cells:Building blocks for molecular medicine in the 21st century.Trends Mol Med 2001,7:259-264.
[5]Anselme K,Brous O,Noel B,et al.In vitro control of human bone marrow stromal cells for bone tissue engineering.Tissue Eng 2002,8:941-953.
[6] Cancedda R, Mastrogiacomo M, Bianchi G, et al. Bone marrow stromal cells and their use in regenerating bone. Novartis Found Symp 2003, 249: 133-43
[7] Kotobuki N, Hirose M, Takakura Y, et al. Cultured autologous huamn cells for hard tissue regeneration:preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs 2004, 28: 33-9
[8] Stenderup K, Justesen J, Clausen C, et al. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells.Bone 2003, 33: 919-926.
[9] Mauney JR, Kaplan DL, Volloch V. Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion. Biomaterials 2004, 25: 3233-3243.
[10] Walsh S, Jefferiss C, Stewart K, et al. Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells:regulation by fibroblast growth f actor(FGF)-β and relationship to the expression of FGF receptors 1-4. Bone 2000, 27: 185-195
[11] Abdallah BM, Haack SM, Burns JS, et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite of extensive proliferation.Biochem Biophys Res Commun 2005, 326: 527-538.
[12] Ni S, Chang J, Chou L. A novel bioactive porous CaSiO3 scaffold for bone tissue engineering. J Biomed Mater Res A 2006, 76: 196-205.
[13] Xu XL, Tang T, Dai K, et al. Immune response and effect of adenovirus-mediated human BMP-2 gene transfer on the repair of segmental tibial bone defects in goats Acta Orthop 2005, 76: 637-646.
[14] Chim H, Schantz JT. New frontiers in calvarial reconstruction: integrating computer-assisted design and tissue engineering in cranioplasty. Plast Reconstr Surg 2005, 116: 1726-1741.
[15] Bosetti M, Boccafoschi F, Leigheb M, et al . Effect of different growth factors on human osteoblasts activities: A possible application in bone regeneration for tissue engineering. Biomol Eng 2007, 24: 613-618.
[16] James KH, Krshna GG. Tissue engineering with chondrocytes. Facial Plastic Surgery 2002, 18:59-68.
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