多巴胺表面修饰/负载软骨源性形态发生蛋白1的3D打印聚己内酯-羟基磷灰石三维多孔支架促进人BMSCs成软骨分化的实验研究

Dopamine modified and cartilage derived morphogenetic protein 1 laden polycaprolactone-hydroxyapatite composite scaffolds fabricated by three-dimensional printing improve chondrogenic differentiation of human bone marrow mesenchymal stem cells

目的探讨利用3D打印技术制备、经多巴胺表面修饰及负载软骨源性形态发生蛋白1(cartilage derived morphogenetic protein 1,CDMP1)的聚己内酯(polycaprolactone,PCL)-羟基磷灰石(hydroxyapatite,HA)三维多孔支架,体外诱导人BMSCs(human BMSCs,hBMSCs)成软骨分化的可行性。方法采用3D打印技术制备PCL-HA支架,经多巴胺表面修饰后,将CDMP-1负载于支架上,扫描电镜观察支架表面微结构,并检测孔隙率和水静态接触角。体外成软骨分化实验:分为A、B、C 3组,A组为PCL-HA支架,B组为多巴胺表面修饰的PCL-HA支架,C组为多巴胺表面修饰及负载CDMP-1的PCL-HA支架;将h BMSCs植入3组支架,成软骨诱导培养后比较细胞黏附率、细胞增殖(MTT法)和细胞活性(Live/Dead染色法),并采用实时荧光定量PCR检测Ⅱ型胶原和聚集蛋白聚糖(Aggrecan)的基因表达。结果 3组支架均呈三维多孔圆柱体状,孔洞相互联通,孔径为400~500μm,孔隙率为56%,材料纤维走向为0°/90°。经多巴胺表面修饰后,支架颜色由初始的白色变为棕色;水静态接触角也由76°降为0°。体外培养24 h,A、B、C组细胞黏附率分别为34.3%±3.5%、48.3%±1.5%、57.4%±2.5%,比较差异均有统计学意义(P<0.05)。Live/Dead染色显示3组细胞均有较好的细胞活性。MTT检测显示各组h BMSCs均生长良好,吸光度(A)值随培养时间延长而增大;培养4、7、14、21 d时,C组A值显著高于A、B组,B组高于A组,差异均有统计学意义(P<0.05)。随培养时间延长,3组Ⅱ型胶原mRNA和Aggrecan mRNA表达均持续增加;培养7、14、21 dⅡ型胶原mRNA相对表达量及培养14、21 d Aggrecan mRNA相对表达量,C组均显著高于A、B组,B组高于A组,比较差异有统计学意义(P<0.05)。结论采用3D打印技术制备、经多巴胺表面修饰及负载CDMP-1的PCL-HA三维多孔支架,体外与hB MSCs共培养,可促进细胞黏附、增殖及成软骨分化。

Objective To prepare dopamine modified and cartilage derived morphogenetic protein 1 （CDMP1） laden polycaprolactone-hydroxyapatite （PCL-HA） composite scaffolds by three-dimensional （3D） printing and evaluate the effect of 3D scaffolds on in vitro chondrogenic differentiation of human bone marrow mesenchymal stem cells （hBMSCs）. Methods A dimensional porous PCL-HA scaffold was fabricated by 3D printing. Dopamine was used to modify the surface of PCL-HA and then CDMP-1 was loaded into scaffolds. The surface microstructure was observed by scanning electron microscope （SEM） and porosity and water static contact angle were also detected. The cytological experiment in vitro were randomly divided into 3 groups： group A （PCL-HA scaffolds）, group B （dopamine modified PCL-HA scaffolds）, and group C （dopamine modified and CDMP-1 laden PCL-HA scaffolds）. The hBMSCs were seeded into three scaffolds, in chondrogenic culture conditions, the cell adhesive rate, the cell proliferation （MTT assay）, and cell activity （Live-Dead staining） were analyzed; and the gene expressions of collagen type II and Aggrecan were detected by real-time fluorescent quantitative PCR. Results The scaffolds in 3 groups were all showed a cross-linked and pore interconnected with pore size of 400-500 ~tm, porosity of 56%, and fiber orientation of 00/90~. For dopamine modification, the scaffolds in groups B and C were dark brown while in group A was white. Similarly, water static contact angle was from 76~ of group A to 0~ of groups B and C. After cultured for 24 hours, the cell adhesion rate of groups A, B, and C was 34.3%±3.5%, 48.3%±1.5%, and 57.4%±2.5% respectively, showing significant differences between groups （P〈0.05）. Live/Dead staining showed good cell activity of cells in 3 groups. MTT test showed that hBMSCs proliferated well in 3 groups and the absorbance （A） value was increased with time. The A value in group C was significantly higher than that in groups B and A, and in group B than in group A after cultured for 4, 7, 14, and 21 days, all showing significant differences （P〈0.05）. The mRNA relative expression of collagen type II and Aggrecan increased gradually with time in 3 groups. The mRNA relative expression of collagen type II after cultured for 7, 14, and 21 days, and the mRNA relative expression of Aggrecan after cultured for 14 and 21 days in group C were significantly higher than those in groups A and B, and in group B than in group A, all showing significant differences （P〈0.05）. Conclusion Co-culture of dopamine modified and CDMP1 laden PCL-HA scaffolds and hBMSCs in vitro can promote hBMSCs＇ adhesion, proliferation, and chondrogenic differentiation.