大鼠脂肪干细胞在纳米羟基磷灰石-壳聚糖-果胶/壳聚糖-明胶支架的成骨向分化研究  

Osteogenic differentiation of rat adipose-derived stem cells cultured in nano-hydroxyapatite-chitosan-pectin/chitosan-gelatin scaffold in vitro

作　　者：韩瑞金 张琪 郝彤[3] 李俊杰[4] 王常勇[3] HAN Rui-jin;ZHANG Qi;HAO Tong;LI Jun-jie;WANG Chang-yong(Graduate School,Jinzhou Medical University Jinzhou 121001;Department of Stomatology,the People's Hospital of Qiannan,Duyun 558000;Department of Advanced Interdisciplinary Studies,Institute of Basic Medical Science,Academy of Military Medical Sciences,Beijing 100850;School of Chemical Engineering and Technology,Tianjin University,Tianjin 300350)

机构地区：[1]锦州医科大学研究生学院,辽宁锦州121001 [2]黔南州人民医院口腔科,贵州都匀558000 [3]军事医学科学院基础医学研究所前沿交叉学科研究室,北京100850 [4]天津大学化工学院,天津300350

出　　处：《生物医学工程与临床》2023年第1期7-14,共8页Biomedical Engineering and Clinical Medicine

摘　　要：目的探索纳米羟基磷灰石-壳聚糖-果胶/壳聚糖-明胶(nHCP/CG)支架对大鼠脂肪干细胞(r ADSC)定向成骨分化的影响,为其临床应用提供理论支持。方法选择SD成年大鼠5只,体质量60~80 g,雌雄不限。从SD成年大鼠两侧腹股沟脂肪组织原代分离、培养并鉴定r ADSC,然后将其接种在nHCP/CG支架材料上,并以壳聚糖-明胶(CG)支架为对照。通过四甲基偶氮唑盐(MTT)法、Live/Dead及苏木精-伊红(HE)染色法评价支架的细胞相容性;在添加和不添加成骨诱导剂培养条件下,以碱性磷酸酶(ALP)活性及多种免疫组织化学染色测定r ADSC在nHCP/CG支架中的成骨分化行为。结果r ADSC具有间充质干细胞的特性,其在nHCP/CG复合支架材料上活性较好。nHCP/CG支架较CG支架更利于r ADSC的黏附和增殖;r ADSC在nHCP/CG支架中培养1 d、3 d、7 d时细胞数量明显高于CG支架中的细胞数量(0.432±0.028 vs 0.174±0.004、0.532±0.017 vs 0.291±0.023、0.595±0.014 vs 0.754±0.038)且差异有显著统计学意义(P<0.01)。r ADSC在nHCP/CG支架和CG支架中培养14 d和21 d均表现出较高的ALP活性;第14天时,在CG支架诱导培养细胞的ALP活性大于常规培养(0.298±0.029 vs 0.224±0.008),差异有统计学意义(P<0.05);在nHCP/CG支架中培养14 d和21 d的细胞加入诱导剂(0.224±0.006 vs 0.271±0.010、0.181±0.012 vs 0.209±0.009),ALP活性小于常规培养且差异有统计意义(P<0.05)。无论是否添加成骨诱导剂,接种在nHCP/CG支架上的r ADSC茜素红染色、Von Kossa染色、Ⅰ型胶原及Ⅷ因子免疫组化染色均表达为阳性,说明nHCP/CG支架能够自发促进r ADSC向成骨细胞分化。结论r ADSC可作为骨组织工程的种子细胞;nHCP/CG支架材料能够提供支持r ADSC黏附和生长的微环境,具有良好的生物相容性,更为重要的是能够自发诱导r ADSC定向成骨分化,有望成为一种应用于临床的骨组织工程支架材料。Objective To explore the effect of nano-hydroxyapatite-chitosan-pectin/chitosangelatin(nHCP/CG)scaffolds on directional osteogenic differentiation of rat adipose stem cells(rADSC),and provide theoretical support for its clinical applica tion.Methods Five male or female adult SD rats with body mass of 60-80 g were sacrificed.The primary r ADSC was isolated,cultured and identified from bilateral inguinal adipose tissues of the rats,and then seeded on nHCP/CG scaffolds,with chitosan-gelatin(CG)scaffolds as control.The cytocompatibility of scaffolds was evaluated by methyl thiazolyl tetrazolium salt(MTT)assay,Live/Dead and hematoxylin-eosin(HE)staining.The osteogenic differentiation behavior of r ADSC in nHCP/CG scaffolds was determined by alkaline phosphatase(ALP)activity and various immunohistochemical staining with or without osteogenic inducers.Results r ADSC showed the characteristics of mesenchymal stem cells with better activity on nHCP/CG composite scaffold materials,and nHCP/CG scaffold was more conducive to r ADSC adhesion and proliferation than CG scaffold.The number of r ADSC cells cultured in nHCP/CG scaffolds for 1 day,3 days and 7 days was significantly higher than those in CG scaffolds(0.432±0.028 vs 0.174±0.004,0.532±0.017 vs 0.291±0.023,0.595±0.014 vs 0.754±0.038),and the differences were statistically significant(P<0.01).r ADSC showed higher ALP activity in nHCP/CG scaffold and CG scaffold after cultured for 14 days and 21 days.At the 14th day,the ALP activity of cells induced on CG scaffold was higher than that of conventional cultured cells(0.298±0.029 vs 0.224±0.008),and the difference was statistically significant(P<0.05).The cells cultured in nHCP/CG scaffolds for 14 days and 21 days were added with inducers(0.224±0.006 vs 0.271±0.010,0.181±0.012 vs 0.209±0.009),and ALP activity was lower than that of conventional cultured cells and the difference was statistically significant(P<0.05).With or without osteogenic inducer,alizarin red staining,Von Kossa staining,type I collagen and fa

关 键 词：纳米羟基磷灰石-壳聚糖-果胶/壳聚糖-明胶(nHCP/CG)支架 脂肪干细胞 成骨分化 血管化 鼠 

分 类 号：R318.08[医药卫生—生物医学工程] R-33[医药卫生—基础医学]