米醇溶蛋白及壳聚糖共混合膜对骨髓间充质干细胞成骨分化的影响
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摘要
背景:有学者通过壳聚糖与玉米醇溶蛋白共混改性,制备了玉米醇溶蛋白/壳聚糖复合膜,初步评价了其理化性能,表明壳聚糖在一定程度上改善了玉米醇溶蛋白的力学性能和亲水性能,由此,作者预期玉米醇溶蛋白/壳聚糖复合膜具有良好细胞相容性,益于骨髓间充质干细胞的成骨分化。目的:探讨玉米醇溶蛋白/壳聚糖复合膜对骨髓间充质干细胞成骨分化的影响,探索其作为骨组织工程材料的可行性。方法:以60%醋酸为溶剂,通过共混和流延法制备玉米醇溶蛋白/壳聚糖(ZC-n)复合膜。采用傅里叶红外光谱分析、力学性能测试、吸水率及扫描电镜等方法来表征其结构与理化性能。利用体外细胞培养方法评价复合膜的细胞相容性。通过贴壁分离筛选法分离SD大鼠骨髓间充质干细胞,利用扫描电镜、荧光标记、碱性磷酸酶测定等方法评价玉米醇溶蛋白?壳聚糖复合膜对骨髓间充质干细胞成骨分化的影响。结果与结论:(1)随着壳聚糖含量的增加,ZC-n复合膜的拉伸强度得到提高,吸水率及亲水性得到增强;(2)壳聚糖具有促进细胞黏附、生长和增殖的作用,即ZC-n膜表现出良好的细胞相容性;(3)ZC-n膜与骨髓间充质干细胞结合并将其诱导分化为成骨细胞,且随着壳聚糖含量的增加,其诱导分化的效果越明显;(4)结果表明,玉米醇溶蛋白?壳聚糖复合膜在骨组织工程领域中具有一定的应用潜能。
BACKGROUND: Some scholars have prepared zein/chitosan composite membrane based on blending methods, and preliminary evaluation of its physical and chemical properties shows that chitosan partly improves the mechanical properties and hydrophilic properties of zein. Therefore, zein/chitosan composite membrane presumably has good cytocompatibility, which is beneficial to osteogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To explore the effect of zein/chitosan composite membrane on the differentiation of bone marrow mesenchymal stem cells into osteoblasts and its feasibility as a bone tissue-engineered material. METHODS: With 60% acetic acid as solvent, zein/chitosan composite membrane was prepared by blending and casting method. The structure and physicochemical properties of the composite membrane were investigated by Fourier transform infrared spectroscopy, tensile testing, water absorption testing and scanning electron microscopy. And the cytocompatibility of the membrane was evaluated by in vitro cell cufture. Besides, bone marrow mesenchymal stem cells from Sprague-Dawley rat were isolated via adherence screening method, and the effects of the composite membrane on the osteogenic differentiation of these cells were observed by scanning electron microscopy, fluorescent labeling and alkaline phosphatase assay. RESULTS AND CONCLUSION: The tensile strength, water absorption and hydrophilicity of the films were improved with the chitosan increased; chitosan could promote cell proliferation indicating the good cytocompatibility of the composite films. Moreover, osteogenic induction occurred in bone marrow mesenchymal stem cells cultured on the composite membrane, and with an increase of chitosan, the induction was promoted. In conclusion, zein/chitosan composite membrane can be applied widely in the field of bone tissue engineering.
BACKGROUND: Some scholars have prepared zein/chitosan composite membrane based on blending methods, and preliminary evaluation of its physical and chemical properties shows that chitosan partly improves the mechanical properties and hydrophilic properties of zein. Therefore, zein/chitosan composite membrane presumably has good cytocompatibility, which is beneficial to osteogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To explore the effect of zein/chitosan composite membrane on the differentiation of bone marrow mesenchymal stem cells into osteoblasts and its feasibility as a bone tissue-engineered material. METHODS: With 60% acetic acid as solvent, zein/chitosan composite membrane was prepared by blending and casting method. The structure and physicochemical properties of the composite membrane were investigated by Fourier transform infrared spectroscopy, tensile testing, water absorption testing and scanning electron microscopy. And the cytocompatibility of the membrane was evaluated by in vitro cell cufture. Besides, bone marrow mesenchymal stem cells from Sprague-Dawley rat were isolated via adherence screening method, and the effects of the composite membrane on the osteogenic differentiation of these cells were observed by scanning electron microscopy, fluorescent labeling and alkaline phosphatase assay. RESULTS AND CONCLUSION: The tensile strength, water absorption and hydrophilicity of the films were improved with the chitosan increased; chitosan could promote cell proliferation indicating the good cytocompatibility of the composite films. Moreover, osteogenic induction occurred in bone marrow mesenchymal stem cells cultured on the composite membrane, and with an increase of chitosan, the induction was promoted. In conclusion, zein/chitosan composite membrane can be applied widely in the field of bone tissue engineering.
引文
[1]尹华月,王典,鲁传华,等.高分子材料α-玉米醇溶蛋白(α-Zein)结构研究进展[J].化工新型材料,2013,41(6):178-180.
[2]刘健,翟文静,吴景景,等.牙龈成纤维细胞-玉米醇溶蛋白-中药双黄补复合体对犬牙周组织再生的实验性研究[J].医学研究生学报,2013,26(9):916-920.
[3]罗钧秋,陈代文,余冰,等.不同蛋白源对生长大鼠骨骼肌蛋白质代谢相关基因表达的影响[J].中国畜牧杂志,2012,48(15):37-40.
[4]GB/T 16886.5-2003/ISO 10993-5:1999.医疗器械的生物学评价第5部分:体外细胞毒性试验[S].北京:中国标准出版社,2004.
[5]Zheng ZH,Wei YJ,Wang G,et al.In vitro biocompatibility of three chitosan/polycation composite materials for nerve regeneration.Neural Regeneration Research.2008;3(8):837-842.
[6]Park SJ,Yu SM,Chun HJ,et al.Effect of Hyaluronic acid on attachment and proliferation of chondrocyte on chitosan/hyaluronic acid bead scaffolds.Tissue Engineering and Regenerative Medicine.2009;6(4-11):438-444.
[7]Anderson TJ,Lamsal BP.Zein extraction from corn,corn products,and coproducts and modifications for various applications:a review.Cereal Chemistry.2011;88(2):159-173.
[8]Sousa FF,Luzardo-álvarez A,Blanco-Méndez J,et al.NMR techniques in drug delivery:application to zein protein complexes.Int J Pharm.2012;439(1-2):41-48.
[9]Salerno A,Zeppetelli S,Oliviero M,et al.Microstructure,degradation and in vitro MG63 cells interactions of a new poly(epsilon-caprolactone),zein,and hydroxyapatite composite for bone tissue engineering.Journal of Bioactive and Compatible Polymers.2012;27(3):210-226.
[10]Torres-Giner S,Ocio MJ,Langaron JM,et al.Novel antimicrobial ultrathin structures of zein/chitosan blends obtained by electrospinning.Carbohydrate Polymers.2009;77(2):261-266.
[11]Ibl V,Stoger E.The formation,function and fate of protein storage compartments in seeds.Protoplasma.2012;249(2):379-392.
[12]Patel AK,Michaud P,Baynast HD,et al.Preparation of chitosan-based adhesives and assessment of their mechanical properties.Bioresources.2013;127(5):3869-3876.
[13]于音,赵刚,许侃,等.兔骨髓间充质干细胞体外培养向成骨和成脂方向的诱导分化[J].中国组织工程研究与临床康复,2008,12(8):1449-1452.
[14]Balloni S,Calvi EM,Damiani F,et al.Effects of titanium surface roughness on mesenchymal stem cell commitment and differentiation signaling.Int J Oral Maxillofac Implants.2009;24(4):627-635.
[15]Tang M,Chen W,Weir MD,et al.Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering.Acta Biomater.2012;8(9):3436-3445.
[2]刘健,翟文静,吴景景,等.牙龈成纤维细胞-玉米醇溶蛋白-中药双黄补复合体对犬牙周组织再生的实验性研究[J].医学研究生学报,2013,26(9):916-920.
[3]罗钧秋,陈代文,余冰,等.不同蛋白源对生长大鼠骨骼肌蛋白质代谢相关基因表达的影响[J].中国畜牧杂志,2012,48(15):37-40.
[4]GB/T 16886.5-2003/ISO 10993-5:1999.医疗器械的生物学评价第5部分:体外细胞毒性试验[S].北京:中国标准出版社,2004.
[5]Zheng ZH,Wei YJ,Wang G,et al.In vitro biocompatibility of three chitosan/polycation composite materials for nerve regeneration.Neural Regeneration Research.2008;3(8):837-842.
[6]Park SJ,Yu SM,Chun HJ,et al.Effect of Hyaluronic acid on attachment and proliferation of chondrocyte on chitosan/hyaluronic acid bead scaffolds.Tissue Engineering and Regenerative Medicine.2009;6(4-11):438-444.
[7]Anderson TJ,Lamsal BP.Zein extraction from corn,corn products,and coproducts and modifications for various applications:a review.Cereal Chemistry.2011;88(2):159-173.
[8]Sousa FF,Luzardo-álvarez A,Blanco-Méndez J,et al.NMR techniques in drug delivery:application to zein protein complexes.Int J Pharm.2012;439(1-2):41-48.
[9]Salerno A,Zeppetelli S,Oliviero M,et al.Microstructure,degradation and in vitro MG63 cells interactions of a new poly(epsilon-caprolactone),zein,and hydroxyapatite composite for bone tissue engineering.Journal of Bioactive and Compatible Polymers.2012;27(3):210-226.
[10]Torres-Giner S,Ocio MJ,Langaron JM,et al.Novel antimicrobial ultrathin structures of zein/chitosan blends obtained by electrospinning.Carbohydrate Polymers.2009;77(2):261-266.
[11]Ibl V,Stoger E.The formation,function and fate of protein storage compartments in seeds.Protoplasma.2012;249(2):379-392.
[12]Patel AK,Michaud P,Baynast HD,et al.Preparation of chitosan-based adhesives and assessment of their mechanical properties.Bioresources.2013;127(5):3869-3876.
[13]于音,赵刚,许侃,等.兔骨髓间充质干细胞体外培养向成骨和成脂方向的诱导分化[J].中国组织工程研究与临床康复,2008,12(8):1449-1452.
[14]Balloni S,Calvi EM,Damiani F,et al.Effects of titanium surface roughness on mesenchymal stem cell commitment and differentiation signaling.Int J Oral Maxillofac Implants.2009;24(4):627-635.
[15]Tang M,Chen W,Weir MD,et al.Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering.Acta Biomater.2012;8(9):3436-3445.