基底膜基质/壳聚糖诱导骨髓间充质干细胞成软骨分化
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摘要
背景:课题组前期研究发现基底膜基质能够定向诱导骨髓间充质干细胞向软骨方向分化,但其力学性能与实际应用有较大差距,还需要进一步研究。目的:制备兼具适宜力学性能和优异生物相容性的壳聚糖/基底膜基质水凝胶支架用于软骨修复。方法:以京尼平为交联剂,将壳聚糖溶液与基底膜基质按2︰1,1︰1,1︰3比例混合制成水凝胶,接种大鼠骨髓间充质干细胞,培养14 d。经材料力学测试、细胞增殖、活细胞染色、酶联免疫吸附测试以及阿尔新蓝染色等方法评价材料诱导细胞成软骨分化能力。结果与结论:在基底膜基质内添加壳聚糖后,材料力学性能从0.48 k Pa上升到1.78 k Pa。标志性蛋白分泌结果显示,纯壳聚糖组早期诱导成软骨活性高于其他组,但后期诱导能力减弱,而含基底膜基质各组在后期能够保持较好的诱导活性,其中壳聚糖/基底膜基质=1︰1组材料具有一定的力学强度,且Ⅱ型胶原和Ⅹ型胶原的表达量在14 d较其他组高。结果表明实验制备的壳聚糖/基底膜基质水凝胶具有良好的力学性能,并能够促进骨髓间充质干细胞向软骨方向分化,可用于软骨组织工程研究。
BACKGROUND: Previous studies have found that the basement membrane matrix can induced the chondrogenic differentiation of bone marrow mesenchymal stem cells, but there is a wide gap between its mechanical properties and practical application, and further research is needed.OBJECTIVE: To prepare a suitable matrigel/chitosan scaffold that has appropriate mechanical properties and remarkable bioactivity for cartilage repair.METHODS: We selected genipin as cross-linking agent, and mixed Matrigel with cross-linked chitosan at different ratios(2:1, 1:1, 1:3). Then rat bone marrow mesenchymal stem cells were seeded on different scaffolds and cultured for 14 days. The mechanical properties of materials were measured by DMA. Cell counting kit-8, FDA staining, ELISA kits and Alcian blue staining were used to measure the bioactivity of materials.RESULTS AND CONCLUSION: The storage modulus of scaffolds was raised from 0.48 k Pa to 1.78 k Pa with increase ratio of chitosan. Cells spread well in the early period on all scaffolds, and then the cells on the chitosan scaffold showed reduced chondrogenic activity, but cells on the scaffolds with matrigel could maintain chondrogenic differentiation. The matrigel/chitosan scaffold at a ratio of 1:1 had appropriate mechanical properties and higher levels of collagen II and collagen X at 14 days. The prepared matrigel/chitosan scaffold with decent mechanical performance can promote the differentiation of bone marrow mesenchymal stem cells into chondrogenic lineages, which can be used in cartilage tissue engineering.
BACKGROUND: Previous studies have found that the basement membrane matrix can induced the chondrogenic differentiation of bone marrow mesenchymal stem cells, but there is a wide gap between its mechanical properties and practical application, and further research is needed.OBJECTIVE: To prepare a suitable matrigel/chitosan scaffold that has appropriate mechanical properties and remarkable bioactivity for cartilage repair.METHODS: We selected genipin as cross-linking agent, and mixed Matrigel with cross-linked chitosan at different ratios(2:1, 1:1, 1:3). Then rat bone marrow mesenchymal stem cells were seeded on different scaffolds and cultured for 14 days. The mechanical properties of materials were measured by DMA. Cell counting kit-8, FDA staining, ELISA kits and Alcian blue staining were used to measure the bioactivity of materials.RESULTS AND CONCLUSION: The storage modulus of scaffolds was raised from 0.48 k Pa to 1.78 k Pa with increase ratio of chitosan. Cells spread well in the early period on all scaffolds, and then the cells on the chitosan scaffold showed reduced chondrogenic activity, but cells on the scaffolds with matrigel could maintain chondrogenic differentiation. The matrigel/chitosan scaffold at a ratio of 1:1 had appropriate mechanical properties and higher levels of collagen II and collagen X at 14 days. The prepared matrigel/chitosan scaffold with decent mechanical performance can promote the differentiation of bone marrow mesenchymal stem cells into chondrogenic lineages, which can be used in cartilage tissue engineering.
引文
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[2]Lancaster MA,Renner M,Martin CA,et al.Cerebral organoids model human brain development and microcephaly.Nature.2013;501(7467):373-379.
[3]Huch M,Dorrell C,Boj SF,et al.In vitro expansion of single Lgr5+liver stem cells induced by Wnt-driven regeneration.Nature.2013;494(7436):247-250.
[4]Takebe T,Sekine K,Enomura M,et al.Vascularized and functional human liver from an i PSC-derived organ bud transplant.Nature.2013;499(7459):481-484.
[5]Mammoto A,Connor KM,Mammoto T,et al.A mechanosensitive transcriptional mechanism that controls angiogenesis.Nature.2009;457(7233):1103-1108.
[6]Tibarewal P,Zilidis G,Spinelli L,et al.PTEN protein phosphatase activity correlates with control of gene expression and invasion,a tumor-suppressing phenotype,but not with AKT activity.Sci Signal.2012;5(213):ra18.
[7]Taravel MN,Domard A.Collagen and its interactions with chitosan,III some biological and mechanical properties.Biomaterials.1996;17(4):451-455.
[8]Li Z,Zhang M.Chitosan-alginate as scaffolding material for cartilage tissue engineering.J Biomed Mater Res A.2005;75(2):485-493.
[9]Wang T,Xu Z,Jiang W,et al.Cell-to-cell contact induces mesenchymal stem cell to differentiate into cardiomyocyte and smooth muscle cell.Int J Cardiol.2006;109(1):74-81.
[10]梁源,隋珂,尚冯青,等.血管内皮祖细胞/骨髓间充质干细胞复合细胞膜片的构建[J].中国组织工程研究,2014,18(41):6561-6566.
[11]Yamada Y,Hozumi K,Aso A,et al.Laminin active peptide/agarose matrices as multifunctional biomaterials for tissue engineering.Biomaterials.2012;33(16):4118-4125.
[12]Hadley MA,Byers SW,Suárez-Quian CA,et al.Extracellular matrix regulates Sertoli cell differentiation,testicular cord formation,and germ cell development in vitro.J Cell Biol.1985;101(4):1511-1522.
[13]Kleinman HK,Martin GR.Matrigel:basement membrane matrix with biological activity.Semin Cancer Biol.2005;15(5):378-386.
[14]Engler AJ,Sen S,Sweeney HL,et al.Matrix elasticity directs stem cell lineage specification.Cell.2006;126(4):677-689.
[15]Stolz M,Gottardi R,Raiteri R,et al.Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy.Nat Nanotechnol.2009;4(3):186-192.
[16]Subramanian A,Lin HY.Crosslinked chitosan:its physical properties and the effects of matrix stiffness on chondrocyte cell morphology and proliferation.J Biomed Mater Res A.2005;75(3):742-753.
[17]Schuh E,Kramer J,Rohwedel J,et al.Effect of matrix elasticity on the maintenance of the chondrogenic phenotype.Tissue Eng Part A.2010;16(4):1281-1290.