纳米梯度支架复合BMSCs体外构建骨—软骨的初步研究
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摘要
制备Ⅰ型胶原/纳米羟基磷灰石(nano-HAp)复合物,TEM、IR、XRD表征；盐滤沥法制备分别由PLGA/I型胶原/nano-HAp及PLGA/透明质酸钠组成的一体化双层骨-软骨支架,SEM观察支架表面形貌,MTT法测细胞毒性及与BMSCs复合培养检测该支架生物相容性。
用静电纺丝的方法制备不同纳米HAp含量的PLGA/Hap复合电纺膜,分别接种大鼠BMSCs复合培养。14d和21d后,进行碱性磷酸酶、茜素红染色,观察BMSCs成骨分化情况。
静电纺丝法制备PLGA/HAp/Zein复合纤维膜,FT-IR、DSC分别检测；采用多层沉积静电纺丝方法,制备HAP、透明质酸分别呈梯度分布的三维支架,植入兔膝关节骨-软骨缺损处,对支架自身的变化及缺损修复效果进行初步的研究。
结果表明,一体化双层骨-软骨支架具有良好的微观结构,无细胞毒性,细胞与支架生物相容性良好,但细胞生长缓慢；不同HAp含量的PLGA能电纺成微米/纳米纤维,BMSCs能够良好地粘附、生长和增殖,碱性磷酸酶和茜素红染色表明BMSCs具有向骨细胞分化的趋势；PLGA/Hap/Zein能获得纳米/微米级电纺纤维。通过多层沉积方式,成功制备纳米梯度骨-软骨支架。植入兔骨软骨缺损4W后,该支架生物相容性良好,与宿主骨-软骨整合较好,支架与宿主界面有粘连并有少量的新生组织长入,支架本身暂无溶胀、收缩及分层等现象。
Complex of collagen I and nano-HAp was prepared and characterized by TEM, IR and XRD, and the integrated bilayed osteochondral scaffold was manufactured by combining nano-HAp, collagen I, PLGA and sodium hyaluronate(HA) and PLGA with sodium leaching method. The surface topography, biocompatibility and cytoxicity of the scaffold were assessed by SEM, MTT assay when seeded with bone marrow stem cells (BMSCs).
Complex of different concentration of nano-HAp and PLGA was prepared by electrospinning. With alkaline phosphatase and alizarin red staining after co-culturing with BMSCs and PLGA/HAp electrospun membrane for 14 and 21 days, the osteogenic differentiation of BMSCs and osteoblasts activity expression were observed.
PLGA/HAp/Zein co-electrospun fibers were prepared and evaluated by FT-IR and DSC separately. With multi-layer deposit under electrospinning, PLGA/HAp/Zein and PLGA/HA electrospun to 3-D scaffolds, while the density of HAp and HA in gradient, and was transplanted into osteochondral defects of rabbits. The change of the scaffold and the repair status of defects were also evaluated preliminarily.
The results indicated that the integrated bilayed osteochondral scaffold had good micro-structure and biocompatibility for rat BMSCs, BMSCs were able to adhere to the surface of the nano- or microfibrous PLGA/HAp electrospun scaffold with different HAp contents and good proliferation. The results of alkaline phosphatase and alizarin red staining indicated that BMSCs has the tendency of osteogenic differentiation. Nanofibrous gradient osteochondral scaffold was successfully prepared by multi-layed deposit electrospinning. After 4 weeks of scaffold transplation in rabbits articular cartilage in vivo, the results indicated that the scaffold had good biocompatibility, and integrated with osteochondral defects well, neo-tissue adhesion and growth were found in the interface of scaffold and osteochondral defects, as well, the swelling, shrink and delamination of implants were not found for all animals.
用静电纺丝的方法制备不同纳米HAp含量的PLGA/Hap复合电纺膜,分别接种大鼠BMSCs复合培养。14d和21d后,进行碱性磷酸酶、茜素红染色,观察BMSCs成骨分化情况。
静电纺丝法制备PLGA/HAp/Zein复合纤维膜,FT-IR、DSC分别检测；采用多层沉积静电纺丝方法,制备HAP、透明质酸分别呈梯度分布的三维支架,植入兔膝关节骨-软骨缺损处,对支架自身的变化及缺损修复效果进行初步的研究。
结果表明,一体化双层骨-软骨支架具有良好的微观结构,无细胞毒性,细胞与支架生物相容性良好,但细胞生长缓慢；不同HAp含量的PLGA能电纺成微米/纳米纤维,BMSCs能够良好地粘附、生长和增殖,碱性磷酸酶和茜素红染色表明BMSCs具有向骨细胞分化的趋势；PLGA/Hap/Zein能获得纳米/微米级电纺纤维。通过多层沉积方式,成功制备纳米梯度骨-软骨支架。植入兔骨软骨缺损4W后,该支架生物相容性良好,与宿主骨-软骨整合较好,支架与宿主界面有粘连并有少量的新生组织长入,支架本身暂无溶胀、收缩及分层等现象。
Complex of collagen I and nano-HAp was prepared and characterized by TEM, IR and XRD, and the integrated bilayed osteochondral scaffold was manufactured by combining nano-HAp, collagen I, PLGA and sodium hyaluronate(HA) and PLGA with sodium leaching method. The surface topography, biocompatibility and cytoxicity of the scaffold were assessed by SEM, MTT assay when seeded with bone marrow stem cells (BMSCs).
Complex of different concentration of nano-HAp and PLGA was prepared by electrospinning. With alkaline phosphatase and alizarin red staining after co-culturing with BMSCs and PLGA/HAp electrospun membrane for 14 and 21 days, the osteogenic differentiation of BMSCs and osteoblasts activity expression were observed.
PLGA/HAp/Zein co-electrospun fibers were prepared and evaluated by FT-IR and DSC separately. With multi-layer deposit under electrospinning, PLGA/HAp/Zein and PLGA/HA electrospun to 3-D scaffolds, while the density of HAp and HA in gradient, and was transplanted into osteochondral defects of rabbits. The change of the scaffold and the repair status of defects were also evaluated preliminarily.
The results indicated that the integrated bilayed osteochondral scaffold had good micro-structure and biocompatibility for rat BMSCs, BMSCs were able to adhere to the surface of the nano- or microfibrous PLGA/HAp electrospun scaffold with different HAp contents and good proliferation. The results of alkaline phosphatase and alizarin red staining indicated that BMSCs has the tendency of osteogenic differentiation. Nanofibrous gradient osteochondral scaffold was successfully prepared by multi-layed deposit electrospinning. After 4 weeks of scaffold transplation in rabbits articular cartilage in vivo, the results indicated that the scaffold had good biocompatibility, and integrated with osteochondral defects well, neo-tissue adhesion and growth were found in the interface of scaffold and osteochondral defects, as well, the swelling, shrink and delamination of implants were not found for all animals.
引文
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[2]Ahsan T, Sah RL. Biomechanics of integrative cartilage repair[J]. Osteoarthritis and Cartilage,1999,7(1):29-40.
[3]Solchaga LA, Yoo JU, L undberg M, et al. Hyaluronan-based poleymers in the treatment of osteochondral defects[J]. J Orthop Res,2000,18(5):773-780.
[4]Schaefer D, Martin I, Shastri P, et al. In vitro generation of osteochondral composites[J]. Biomaterials,2000,21(24):2599-2606.
[5]Malafaya PB, Reis RL. Bilayered chitosan-based scaffolds for osteochondral tissue engineering:Influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor[J]. Acta Biomater.2009,5(2):644-660.
[6]Guo X, Park H, Liu G, et al. In vitro generation of an osteochondral construct using injectable hydrogel composites encapsulating rabbit marrow mesenchymal stem cells[J]. Biomaterials,2009,30(14):2741-2752.
[7]Guo X, Park H, Young S, et al. Repair of osteochondral defects with biodegradable hydrogel composites encapsulating marrow mesenchymal stem cells in a rabbit model[J]. Acta Biomater.2010,6(8):2920-2931.
[8]Schaefer D, Martin I, Jundt G, et al. Tissue-engineered composites for the repair of large osteochondral defects. Arthritis Rheum,2002,46(9):2524-2534.
[9]Mano J F, Reis R L. Osteochondral defects:present situation and tissue engineering approaches[J]. Tissue Engineering Regen Med,2007,1(4):261-273.
[10]Martin I, Miot S, Barbero A, et al. Osteochondral tissue engineering[J]. J Biomech,2007,40(4):750-765.
[11]Guo XM, Wang C Y, Duan C M, et al. Repair of osteochondral defects with autologous chondrocytes seeded onto bioceramic scaffold in sheep[J]. Tissue Engineering,2004,10(11-12):1830-1840.
[12]Waldman SD, GrynpasMD, Pilliar RM, et al. Characterizati on of catilagenous tissue formed on calcium polyphosphate substrates in vitro[J]. J Biomed Mater Res, 2002,62(3):323-330.
[13]Robertson WB, Fick D, Wood DJ, et al. MRI and clinical evaluation of collagen-covered autologous chondrocyte implantation (CACI) at two years[J]. The Knee,2007,14(2):117-127.
[14]Fukuda A., Kato K., Hasegawa M., et al. Enhanced repair of large osteochondral defects using a combination of artificial cartilage and basic fibroblast growth factor[J]. Biomaterials,2005,26 (20):4301-4308.
[15]许波,董启榕,伏治国,等.同种异体骨髓基质细胞移植修复兔膝关节骨软骨缺损[J].苏州大学学报(医学版),2008,28(1)：56-58.
[16]Fan H., Hu Y., Qin L., et al. Porous gelatin-chondroitin-hyaluronate tri-copolymer scaffold containing microspheres loaded with TGF[J]. J. Biomed Mater Res A,2006,77(4):785-794.
[17]Hunziker EB, Kapfinger E, Martin J, et al. Insulin-like growth factor (IGF)-binding protein3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage[J]. Osteoarthritis Cartilage,2008,16(2): 185-194.
[18]Tamai N, Myoui A, Hirao M, et al. A new biotechnology for articular cartilage repair:subchondral implantation of a composite of interconnected porous hydroxyapatite, synthetic polymer (PLA-PEG), and bone morphogenetic protein-2 (rhBMP-2)[J]. Osteoarthritis Cartilage,2005,13(5):405-417.
[19]Huang X, Yang D, Yan W, et al. Osteochondral repair using the combination of fibroblast growth factor and amorphous calcium phosphate poly(1-lactic acid) hybrid materials[J]. Biomaterials,2007,28(20):3091-3100.
[20]Cao T, Ho KH, Teoh SH. Scaffold design and in vitro study of osteochondral coculture in a three-dimensional porous polycap rolactone scaffold fabricated by fused deposition modeling[J]. Tissue Engineering,2003,9 supply 1:S103-112.
[21]Sherwood JK, Riley SL, palazzolo R, et al. A three-dimensional osteochondral composite scaffold for articular cartilage repair[J]. Biomaterials,2002,23(24): 4739-4751.
[22]S. Ghosh, J. C. Viana, R. L. Reis, et al. Bi-layered constructs based on poly (L-lactic acid) and starch for tissue engineering of osteochondral defects [J]. Materials Science and Engineering C,2008,28(1):80-86.
[23]Martin I, Miot s, Barbero A, et al. osteochondral tissue engineering[J]. Journal of Biomechanics,2007,40(4):750-765.
[24]Wakitani S, Goto T, Pineda SJ. Mesenehymal cell-based repair of larg, full-thickness defeets of artieular cartilage [J]. J Bone Joint Surg,1994,76(4): 579-592.
[25]Muschler GF, Midura RJ. Connective tissue progenitors:practical concepts for clinical applications[J]. Clin Orthop Relat Res,2002, Feb (395):66-80.
[26]Noe D, Djouad F, Jorgensen C. Regenerative medicine through mesenehymal stem cells for bone and cartilage repair[J]. Curr OPin Investig Drugs,2002,3(7): 1000-1004.
[27]Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science,1999,284(5411):143-147.
[28]Cooch KJ, Blunk T, Courter DL, et al. Bone morphogenetic proteins2,-12 and-13 modulate in vitro development of engineered cartilage[J]. Tissue Engeneering,2006, 8(4):591-601.
[29]Martin I, Suetterlin R, BaschongW, et al. Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 druing 2D expansion and BMP-2 during 3D cultivation[J]. J Cell Biochem,2005,83(1):121-128.
[30]Holland TA, Bodde EWH, Cuijpers V, et al. Degradable hydrogel scaffolds for in vivo delivery of single and dual growth factors in cartilage repair[J]. Osteoarthr Cartilage,2007,15(2):187-197.
[31]Wakitani S. Present status and perspective of articular cartilage regeneration[J]. Yakugaku Zasshi,2007,127(5):857-863.
[32]Wakitani S, Imoto K, Yamamoto T, et al. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees[J]. Osteoarthritis Cartilage,2002,10(3):199-206.
[33]Kramer J, Hegert C, Guan K. Embryonic stem cell-derived chondrogenic differentiation in vitro:activation by BMP-2 and BMP-4[J]. Mechanisms of Development.2000,92(2):193-205
[34]Jose MV, Thomas V, Johnson KT, et al. Aligned PLGA/HA nanofibrous nanocomposite scaffolds for bone tissue engineering[J]. Acta Biomater,2009,5(1):305-315.
[35]F Yang, JGC Wolke, JA Jansen. Biomimetic calcium phosphate coating on electrospun poly( ε-caprolactone) scaffolds for bone tissue engineering[J]. Chemical Engineering Journal,2008,137(1):154-161.
[36]Zhang Y, Venuqopal JR, EI-Turki A, et al. Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering[J]. Biomaterials, 2008,29(32):4314-4322.
[37]何创龙,黄争鸣,张彦中等.静电纺丝法制备组织工程纳/微米纤维支架[J].自然科学进展.2005,15(10)：1175-1182.
[38]Wannatong L, Sirivat A. Electrospun fibers of polypyrrole/polystyrene blend for gas sensing applications[J]. PMSE Preprints,2004,91(35):692-703.
[39]Kim C, Park SH, Lee WJ, et al. Characteristics of supercapacitor electrodes of PBI-based carbon nanofiber web prepared by electrospinning[J]. Electrochimi Acta,2004,50(39):877-881.
[40]Jiang HL, Fang DF, Hsiao BJ, et al. Preparation and characterization of ibuprofen-loaded poly (lactide-co-glycolide)/poly(ethylene glycol)-g-chitosan electrospun membranes[J]. J Biomat Sci-Polym E,2004,15(19):279-296.
[4l]Khil MS, Cha DI, Kim HY, et al. Electrospun nanofibrous polyurethane membrane as wound dressing[J]. J Biomed Mater Res B,2003,67B(25):675-679.
[42]Li W J, Laurencin C T, Caterson E J, et al. Electrospun nanofibrous structure: A novel scaffold for tissue engineering[J]. J Biomed Mater Res,2002,60(4): 613-621.
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