聚羟基烷酸酯与绵羊骨髓基质干细胞相容性的研究

目的评价聚羟基烷酸酯(PHBV)作为组织工程支架与绵羊骨髓基质干细胞(BMSCs)的生物相容性。方法原代培养绵羊BMSCs,传至2～3代后,接种至PHBV膜和泡沫样三维支架上,光镜和扫描电镜观察细胞形态,计数1、2、6 h时的细胞黏附率;并以接种至培养板上细胞为对照组,每日细胞计数,绘制生长曲线;按培养液量与支架体积10 mL／cm3为标准浓度制备浸提液,并制备标准浓度1／16～16倍的浸提液,以MTT法检测细胞毒性;流式细胞仪分析接种到材料上的细胞周期,计算增殖指数;BMSCs接种于PHBV三维支架上4、8、12 d,以Hoechst33258荧光法定量测定细胞内DNA含量, BCA法测定蛋白质含量。结果第3代BMSCs接种至PHBV膜上2 h后即大部黏附,黏附率75．6％,与对照组相比差异无统计学意义,绘制生长曲线见细胞生长与对照组无差异;MTT法检测见9个浓度梯度的浸提液毒性均为0级;光镜和扫描电镜观察见细胞接种于PHBV膜上2 h后大部分黏附,3 d后伸展良好,呈纺锤形或梭形,在三维支架的孔隙内立体生长,1周开始细胞间连接,3周广泛连接,分泌大量基质;流式细胞分析见接种于材料上的细胞周期无变化;接种至PHBV三维支架上的细胞内DNA、蛋白质浓度与对照组比较无差异。结论PHBV作为BMSCs的组织工程支架材料,具有良好的生物相容性。

Biocompatibility between copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate and bone marrow stromal cells

Objective To evaluate the biocompatibility between copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) and bone marrow stromal cells (BMSCs). Methods Sheep BMSCs were isolated and cultured. The passage 3 cells were seeded onto the PHBV films and three-dimensional foams. The seeded cells were observed with light microscopy and scanning electron microscopy (SEM) for their morphology and their attachment onto the PHBV films 1 h, 2 h, 6 h after seeding. Those seeded onto the culture plates served as control and their numbers were counted every 24 h. The cell toxicity was measured with MTT (tetrazolium salt) assay. The cycle of seeded cells was detected with flow cytometry. The protein content in seeded cells was determined by bicinchoninic acid assay(BCA). The content of DNA was quantified using the Hoechst33258 assay. Results The ratio of cell attachment onto PHBV films at 2 h after seeding was 75. 6% , which was not statistically different from that in the control group. There was either no significant difference in proliferation of seeded cells between the experiment group and the control group. MTT assays indicated extract solutions of every gradient concentration were not toxic. Flow cytometry showed no significant difference in cycle of seeded cells between the experiment and the control. Microscopy showed most cells attached onto the PHBV films 2 h after seeding, and extended well and acquired a spindle fibrocyte-like morphology 3 days later. Moreover, on the three-dimensional foams, the seeded cells lay in micro pores and grew tridimensionally. The conjunction of cells appeared at 1 week, and extended at 3 weeks, with a large amount of extra cellular matrix around cells. Conclusion As a kind of tissue engineering material for BMSCs seeding, PHBV has an excellent biocompatibility.