Cultispher微载体构建组织工程软骨修复关节软骨缺损
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摘要
背景:纤维蛋白胶和微载体均可作为软骨组织工程的良好载体,但因力学性能差和可塑性差等缺点限制其广泛应用。目的:以负载软骨细胞的Cultispher微载体为基础,复合纤维蛋白胶,构建Cultispher微载体/纤维蛋白胶复合支架,观察其用于修复兔膝关节软骨缺损的效果。方法:将兔软骨细胞与Cultispher微载体置于搅拌式生物反应器中三维悬浮共培养,待细胞帖附至微载体表面并大量扩增后,将负载有软骨细胞的Cultispher微载体与纤维蛋白胶复合,构建Cultispher微载体/纤维蛋白胶复合支架,并用于修复兔膝关节股骨滑车软骨缺损。实验按不同的软骨缺损处植入物分为3组:MCF组以负载软骨细胞的Cultispher微载体/纤维蛋白胶复合支架修复软骨缺损;MF组以单纯Cultispher微载体/纤维蛋白胶复合支架修复软骨缺损;空白对照组旷置软骨缺损,不作任何处理。术后3,6个月取材。检测并记录大体观、大体观评分、病理学染色、病理学评分、Micro-CT扫描等指标,评估软骨修复效果。结果与结论:①大体观显示MCF组的软骨修复效果明显优于MF组和空白对照组;②番红"O"、天狼猩红病理染色结果显示MCF组的修复组织主要以透明软骨为主,而MF组和空白对照组的修复组织主要以纤维组织为主;③Micro-CT扫描结果显示,MCF组较MF组和空白对照组获得更好的软骨下骨重建;④MCF组的大体观评分与病理学评分均明显高于MF组和空白对照组;⑤结果证实,负载软骨细胞的Cultispher微载体/纤维蛋白胶复合支架能成功修复兔股骨滑车软骨缺损。
BACKGROUND: Both Cultispher microcarrier and fibrin can act as carriers in cartilage tissue engineering, but their application is limited by poor mechanical properties and poor plasticity.OBJECTIVE: To combine Cultispher microcarrier carrying chondrocytes with fibrin glue to construct Gultispher/fibrin composite scaffold, and to investigate the effect of this composite scaffold in the articular cartilage repair in a rabbit model. METHODS: Rabbit chondrocyte and Cultispher microcarriers were co-cultured in a stirred bioreactor until the chondrocytes adhered to and proliferated quickly on the microcarrier surface. Chondrocytes-seeded microcarries were then combined with fibrin glue to construct microcarrier/fibrin glue composite scaffolds, to repair trochlear cartilage defects of the knee joint in the rabbit model. In the experiment, three different treatments were respectively done for repair of cartilage defects, including implantation of chondrocytes-seeded microcarries/fibrin glue composite scaffold(MCF group), implantation of Gultispher/fibrin composite scaffold(MF group), and no treatment(blank control group). At 3 and 6 months after surgery, gross observation, histological evaluation, pathological evaluation and Micro-CT scanning were conducted to evaluate the cartilage repair effects. RESULTS AND CONCLUSION: Gross observation showed that the MCF group achieved better effect on cartilage repair, compared to the other two groups. Histopathological evaluation revealed hyaline-like cartilage tissues in the MCF group while fibrocartilage tissues were seen in the other two groups, shown by safranin O staining and sirius red staining. Micro-CT scanning results showed better subchondral bone remodeling was found in the MCF group than the other two groups. Gross observation and pathological observation showed better outcomes in the MCF group than the MF and blank control groups. To conclude, the chondrocyte-seeded Cultispher microcarrier/fibrin glue composite scaffold succeeds in the articular cartilage repair.
BACKGROUND: Both Cultispher microcarrier and fibrin can act as carriers in cartilage tissue engineering, but their application is limited by poor mechanical properties and poor plasticity.OBJECTIVE: To combine Cultispher microcarrier carrying chondrocytes with fibrin glue to construct Gultispher/fibrin composite scaffold, and to investigate the effect of this composite scaffold in the articular cartilage repair in a rabbit model. METHODS: Rabbit chondrocyte and Cultispher microcarriers were co-cultured in a stirred bioreactor until the chondrocytes adhered to and proliferated quickly on the microcarrier surface. Chondrocytes-seeded microcarries were then combined with fibrin glue to construct microcarrier/fibrin glue composite scaffolds, to repair trochlear cartilage defects of the knee joint in the rabbit model. In the experiment, three different treatments were respectively done for repair of cartilage defects, including implantation of chondrocytes-seeded microcarries/fibrin glue composite scaffold(MCF group), implantation of Gultispher/fibrin composite scaffold(MF group), and no treatment(blank control group). At 3 and 6 months after surgery, gross observation, histological evaluation, pathological evaluation and Micro-CT scanning were conducted to evaluate the cartilage repair effects. RESULTS AND CONCLUSION: Gross observation showed that the MCF group achieved better effect on cartilage repair, compared to the other two groups. Histopathological evaluation revealed hyaline-like cartilage tissues in the MCF group while fibrocartilage tissues were seen in the other two groups, shown by safranin O staining and sirius red staining. Micro-CT scanning results showed better subchondral bone remodeling was found in the MCF group than the other two groups. Gross observation and pathological observation showed better outcomes in the MCF group than the MF and blank control groups. To conclude, the chondrocyte-seeded Cultispher microcarrier/fibrin glue composite scaffold succeeds in the articular cartilage repair.
引文
[1]Yin H,Wang Y,Sun Z,et al.Induction of mesenchymal stem cell chondrogenic differentiation and functional cartilage microtissue formation for in vivo cartilage regeneration by cartilage extracellular matrix-derived particles.Acta Biomater.2016;33:96-109.
[2]Zhang L,Hu J,Athanasiou KA.The role of tissue engineering in articular cartilage repair and regeneration.Crit Rev Biomed Eng.2009;37:1-57.
[3]Gaut C,Sugaya K.Critical review on the physical and mechanical factors involved in tissue engineering of cartilage.Regen Med.2015;22:1-15.
[4]Matsunaga D,Akizuki S,Takizawa T,et al.Repair of articular cartilage and clinical outcome after osteotomy with microfracture or abrasion arthroplasty for medial gonarthrosis.Knee.2007;14(6):465-471.
[5]Hunziker EB.Articular cartilage repair:basic science and clinical progress.A review of the current status and prospects.Osteoarthr Cartil.2002;10:432-463.
[6]Eyrich D,Brandl F,Appel B,et al.Long-term stable fibrin gels for cartilage engineering.Biomaterials.2007;28(1):55-65.
[7]Mercier NR,Costantino HR,Tracy MA,et al.A novel injectable approach for cartilage formation in vivo using PLGmicrospheres.Ann Biomed Eng.2004;32:418-429.
[8]Chun KW,Yoo HS,Yoon JJ,et al.Biodegradable PLGAmicrocarriers for injectable delivery of chondrocytes:effect of surface modification on cell attachment and function.Biotechnol.Prog.2004;20:1797-1801.
[9]Newman KD,Mc Burney MW.Poly(D-,L-lactic-co-glycolic acid)microspheres as biodegradable microcarriers for pluripotent stem cells.Biomaterials.2004;25:5763-5771.
[10]Li YY,Cheng HW,Cheung KM,et al.Mesenchymal stem cell-collagen microspheres for articular cartilage repair:cell density and differentiation status.Acta Biomater.2014;10:1919-1929.
[11]Wakitani S,Goto T,Pineda SJ,et al.Mesenchymal cell-based repair of large,full-thickness defects of articular cartilage.JBone Joint Surg Am.1994;76(4):579-592.
[12]Asik M,Ciftci F,Sen C,et al.The microfracture technique for the treatment of full-thickness articular cartilage lesions of the knee:midterm results.Arthroscopy.2008;24(11):1214-1220.
[13]Lin YM,Lim JF,Lee J,et al.Expansion in microcarrier spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells.Cytotherapy.2016;18(6):740-753.
[14]Pettersson S,Wetter?J,Tengvall P,et al.Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds-impact ofmicrocarrier selection on cell proliferation.Biomed Mater.2011;6(6):065001.
[15]Kang H,Lu S,Peng J,et al.Chondrogenic differentiation of human adipose-derived stem cells using microcarrier and bioreactor combination technique.Mol Med Rep.2015;11(2):1195-1199.
[16]Goepfert C,Lutz V,Lünse S,Kittel S,et al.Evaluation of cartilage specific matrix synthesis of human articular chondrocytes after extended propagation on microcarriers by image analysis.Int J Artif Organs.2010;33(4):204-218.
[17]Beris AE,Lykissas MG,Kostas-Agnantis I,et al.Treatment of full-thickness chondral defects of the knee with autologous chondrocyte implantation:a functional evaluation with long-term follow up.Am J Sports Med.2012;40:562-567.
[18]Marlovits S,Tichy B,Truppe M,et al.Collagen expression in tissue engineered cartilage of aged human articular chondrocytes in a rotating bioreactor.Int J Artif Organs.2003;26(4):319-330.
[19]Schulze-Tanzil G,de Souza P,Villegas Castrejon H,et al.Redifferentiation of dedifferentiated human chondrocytes in high-density cultures.Cell Tissue Res.2002;308:371-379.
[20]Hong Y,Gao C,Xie Y,et al.Collagen-coated polylactide microspheres as chondrocyte microcarriers.Biomaterials.2005;26:6305-6313.
[21]Cochis A,Grad S,Stoddart MJ,et al.Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel.Sci Rep.2017;23:45018.
[22]Chen J,Yuan Z,Liu Y,et al.Improvement of In Vitro Three-Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor.Stem Cells Transl Med.2017;6(3):982-991.
[23]Nazempour A,Quisenberry CR,Abu-Lail NI,et al.Combined effects of oscillating hydrostatic pressure,perfusion and encapsulation in a novelbioreactor for enhancing extracellular matrix synthesis by bovine chondrocytes.Cell Tissue Res.2017 Jul 7.
[24]Chung HJ,Go DH,Bae JW,et al.Synthesis and characterization of Pluronic grafted chitosan copolymer as a novel injectable biomaterial.Curr Appl Phys.2005;5:485-488.
[25]Formica FA,?ztürk E,Hess SC,et al.A Bioinspired Ultraporous Nanofiber-Hydrogel Mimic of the Cartilage Extracellular Matrix.Adv Healthc Mater.2016;25:450-488.
[26]Pescador D,Ibá?ez-Fonseca A,Sánchez-Guijo F,et al.Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.J Mater Sci Mater Med.2017;28(8):115.
[27]Yao Y,Huang Y,Qian D,et al.Effect of Various Ratios of Co-Cultured ATDC5 Cells and Chondrocytes on the Expression of Cartilaginous Phenotype in Microcavitary Alginate Hydrogel.JCell Biochem.2017 Jun 15.
[28]Olubamiji AD,Zhu N,Chang T,et al.Traditional Invasive and Synchrotron-Based Noninvasive Assessments of Three-Dimensional-Printed Hybrid Cartilage Constructs In Situ.Tissue Eng Part C Methods.2017;23(3):156-168.
[2]Zhang L,Hu J,Athanasiou KA.The role of tissue engineering in articular cartilage repair and regeneration.Crit Rev Biomed Eng.2009;37:1-57.
[3]Gaut C,Sugaya K.Critical review on the physical and mechanical factors involved in tissue engineering of cartilage.Regen Med.2015;22:1-15.
[4]Matsunaga D,Akizuki S,Takizawa T,et al.Repair of articular cartilage and clinical outcome after osteotomy with microfracture or abrasion arthroplasty for medial gonarthrosis.Knee.2007;14(6):465-471.
[5]Hunziker EB.Articular cartilage repair:basic science and clinical progress.A review of the current status and prospects.Osteoarthr Cartil.2002;10:432-463.
[6]Eyrich D,Brandl F,Appel B,et al.Long-term stable fibrin gels for cartilage engineering.Biomaterials.2007;28(1):55-65.
[7]Mercier NR,Costantino HR,Tracy MA,et al.A novel injectable approach for cartilage formation in vivo using PLGmicrospheres.Ann Biomed Eng.2004;32:418-429.
[8]Chun KW,Yoo HS,Yoon JJ,et al.Biodegradable PLGAmicrocarriers for injectable delivery of chondrocytes:effect of surface modification on cell attachment and function.Biotechnol.Prog.2004;20:1797-1801.
[9]Newman KD,Mc Burney MW.Poly(D-,L-lactic-co-glycolic acid)microspheres as biodegradable microcarriers for pluripotent stem cells.Biomaterials.2004;25:5763-5771.
[10]Li YY,Cheng HW,Cheung KM,et al.Mesenchymal stem cell-collagen microspheres for articular cartilage repair:cell density and differentiation status.Acta Biomater.2014;10:1919-1929.
[11]Wakitani S,Goto T,Pineda SJ,et al.Mesenchymal cell-based repair of large,full-thickness defects of articular cartilage.JBone Joint Surg Am.1994;76(4):579-592.
[12]Asik M,Ciftci F,Sen C,et al.The microfracture technique for the treatment of full-thickness articular cartilage lesions of the knee:midterm results.Arthroscopy.2008;24(11):1214-1220.
[13]Lin YM,Lim JF,Lee J,et al.Expansion in microcarrier spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells.Cytotherapy.2016;18(6):740-753.
[14]Pettersson S,Wetter?J,Tengvall P,et al.Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds-impact ofmicrocarrier selection on cell proliferation.Biomed Mater.2011;6(6):065001.
[15]Kang H,Lu S,Peng J,et al.Chondrogenic differentiation of human adipose-derived stem cells using microcarrier and bioreactor combination technique.Mol Med Rep.2015;11(2):1195-1199.
[16]Goepfert C,Lutz V,Lünse S,Kittel S,et al.Evaluation of cartilage specific matrix synthesis of human articular chondrocytes after extended propagation on microcarriers by image analysis.Int J Artif Organs.2010;33(4):204-218.
[17]Beris AE,Lykissas MG,Kostas-Agnantis I,et al.Treatment of full-thickness chondral defects of the knee with autologous chondrocyte implantation:a functional evaluation with long-term follow up.Am J Sports Med.2012;40:562-567.
[18]Marlovits S,Tichy B,Truppe M,et al.Collagen expression in tissue engineered cartilage of aged human articular chondrocytes in a rotating bioreactor.Int J Artif Organs.2003;26(4):319-330.
[19]Schulze-Tanzil G,de Souza P,Villegas Castrejon H,et al.Redifferentiation of dedifferentiated human chondrocytes in high-density cultures.Cell Tissue Res.2002;308:371-379.
[20]Hong Y,Gao C,Xie Y,et al.Collagen-coated polylactide microspheres as chondrocyte microcarriers.Biomaterials.2005;26:6305-6313.
[21]Cochis A,Grad S,Stoddart MJ,et al.Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel.Sci Rep.2017;23:45018.
[22]Chen J,Yuan Z,Liu Y,et al.Improvement of In Vitro Three-Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor.Stem Cells Transl Med.2017;6(3):982-991.
[23]Nazempour A,Quisenberry CR,Abu-Lail NI,et al.Combined effects of oscillating hydrostatic pressure,perfusion and encapsulation in a novelbioreactor for enhancing extracellular matrix synthesis by bovine chondrocytes.Cell Tissue Res.2017 Jul 7.
[24]Chung HJ,Go DH,Bae JW,et al.Synthesis and characterization of Pluronic grafted chitosan copolymer as a novel injectable biomaterial.Curr Appl Phys.2005;5:485-488.
[25]Formica FA,?ztürk E,Hess SC,et al.A Bioinspired Ultraporous Nanofiber-Hydrogel Mimic of the Cartilage Extracellular Matrix.Adv Healthc Mater.2016;25:450-488.
[26]Pescador D,Ibá?ez-Fonseca A,Sánchez-Guijo F,et al.Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.J Mater Sci Mater Med.2017;28(8):115.
[27]Yao Y,Huang Y,Qian D,et al.Effect of Various Ratios of Co-Cultured ATDC5 Cells and Chondrocytes on the Expression of Cartilaginous Phenotype in Microcavitary Alginate Hydrogel.JCell Biochem.2017 Jun 15.
[28]Olubamiji AD,Zhu N,Chang T,et al.Traditional Invasive and Synchrotron-Based Noninvasive Assessments of Three-Dimensional-Printed Hybrid Cartilage Constructs In Situ.Tissue Eng Part C Methods.2017;23(3):156-168.