Objective To study the changes in the biological behavior of bone marrow mesenchymal stem cells (BMSCs) transfected with red fluorescent protein by lentivirus (RFP-BMSCs) seeded on in poly-D, L-lactide acid (PDLLA) scaffolds with bioactive modification by ammonia plasma and Gly-Arg-Gly-Asp-Ser (GRGDS) in vitro. Methods Circular sheets of PDLLA scaffolds (8 mm in diameter and 1 mm in thickness) were prepared and aminated with PDLLA (group A) or modified with the peptide conjugate A/PDLLA (group PA), with untreated PDLLA as the control (group P). The RFP-BMSCs were seeded on the scaffold materials and their proliferation and metabolic activity were detected using CyQuant NF and Alamar blue staining. The mineralization on the scaffolds was observed using calcein fluorescent dye under a fluorescent microscope. The adhesion and proliferation of RFP-BMSCs were observed by fluorescent microscope, and scanning electron microscope (SEM) was used to confirm the observed adhesion of the seed cells. Results The RFP-BMSCs seeded on the 3 scaffolds all showed proliferative activity at different time points after cell seeding, and the cell numbers decreased significantly in the order of PA>A>P (P0.001). The cell number was significantly greater in group PA than in group A at all the time points except for days 10 (P=0.077) and 12 (P=0.491), and gradually became similar with the passage of time. The metabolic changes of the cells follow a similar pattern of cell proliferation. RFP-BMSCs showed more active proliferation in group A and group PA than in group P. On days 14 and 21, the intensity of green fluorescence decreased in the order of group PA, A and P. The RFP-BMSCs showed better adhesion in group PA than in group A, and the cells in group P appeared more scattered under scanning electron microscope. Conclusion Bioactive modification of PDLLA by ammonia treatment and conjugation withGRGDS peptides may promotes the adhesion, proliferation, metabolism and mineralization of RFP-BMSCs seeded on PDLLA scaffolds.%目的 探讨氨等离子体改性、酰胺键接枝甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(GRGDS)短肽的活性修饰方法对消旋聚乳酸(PDLLA)组织工程骨上慢病毒转染红色荧光蛋白的骨髓间充质干细胞生物学行为的影响.方法 制备圆片状直径8 mm、厚1 mm的PDLLA三维多孔支架,分为3组:表面氨基化PDLLA(A组),接枝肽A/PDLLA(PA组),以未经处理的PDLLA(P组)作为对照组.CyQuantNF和AlamarBlue分别检测支架上细胞的增殖和代谢活性;钙黄绿素进行矿化荧光染色,荧光显微镜观察支架上钙盐沉积以及RFP-BMSCs的贴附与增殖;扫描电镜观察细胞的贴附.结果 接种细胞后各时间点,3组材料上细胞均能增殖,组间细胞数量差别均有统计学意义(P＜0.001),PA组＞A组＞P组.除第10天(P=0.077)和第12天(P=0.491)PA组和A组间细胞数量差异无统计学意义外,其余各时间点,PA组数量均明显高于A组.随着时间的延长,两组间细胞数量逐渐接近.细胞代谢活性检测与增殖检测结果近似,骨支架上细胞增殖程度越高,其代谢越活跃.荧光显微镜观察,A组和PA组材料上RFP-BMSCs较P组增殖活跃;钙盐沉积荧光染色显示,第14天和第21天,绿色荧光强度PA组＞A组＞P组.扫描电镜显示,PA组材料能获得较A组更好的细胞贴附,P组细胞较稀疏.结论 氨等离子体改性接枝GRGDS肽的新型活性修饰PDLLA骨支架,能明显促进RFP-BMSCs为种子细胞的贴附、增殖、代谢与矿化.
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