静电纺丝聚乳酸聚乙醇酸／聚乙二醇纳米纤维作为组织工程支架的体外细胞相容性研究

In vitro biological compatibility of co-electrospun poly （lactide.co-glycolide）/polyethylene glycol nanofibrous membrane in tissue engineering scaffolds for spinal cord injury

目的探讨静电纺丝聚乳酸聚乙醇酸（PLGA）／聚乙二醇（PEG）共聚物纳米纤维作为组织工程支架的可行性，及其与大鼠骨髓基质干细胞（BMSCs）的体外相容性。方法静电纺丝法分别制备PLGA／PEG和PLGA纳米纤维支架，扫描电镜（SEM）观察材料结构；分离培养大鼠BMSCs，取第3代BMSCs分别接种于PLGA／PEG纳米纤维支架和PLGA纳米纤维支架进行培养，噻唑蓝（MTr）法测定其细胞毒性及细胞增殖；接种后2、4、6h血球计数板计数法测定其黏附率；DAPI荧光染色观察细胞核形态；SEM观察细胞和支架的形态、黏附及生长情况。结果SEM观察显示两组支架呈相互交联的多孔网状无纺结构。PLGA／PEG组和PLGA组纤维直径分别为（655±57）nm和（539±48）nm；孔隙率分别为86．8％±1．5％和84．7％±1．2％。MTr检测BMSCs在两组支架中生长良好，OD值均随时间延长而增大，两组各时间点比较差异均有统计学意义（P〈0．05）。各时间段PLGA／PEG组的细胞黏附率明显高于PLGA组，差异均有统计学意义（P〈0．05）。DAPI染色示各组细胞核形态正常，核质均染，未见明显凋亡及坏死细胞；PLGA／PEG组细胞较PLGA组明显增多。SEM观察显示，PLGA／PEG组BMSCs在支架上生长良好，基质分泌、生长睛况优于PLGA组。结论采用静电纺丝法制备的PLGA／PEG纳米纤维支架安全无毒，具备合适的孔径和孔隙率，适合BMSCs生长，细胞相容性良好，是一种组织工程良好的支架载体。

Objective To study the feasibility of a new type of tissue engineering scaffolds for spinal cord injury made of electrospun poly （lactide-co-glycolide） -polyethylene glycol （PLGA-PEG） nanofiber membrane and its biocompatibility with bone marrow stem ceils （BMSCs） . Methods Electrostatic spinning was used to prepare porous nanofiber scaffolds of PLGA-PEG and porous nanofiber scaffolds of polylactic glycolic acid （PLGA）. The scaffolds were observed by scanning electron microscopy （SEM）. BMSCs were separated from male SD rats and cultured. BMSCs of the third passage were seeded and cultured onto the scaffolds made of electrospun PLGA-PEG nanofiber membrane （the experimental group） and the scaffolds made of the electrospun PLGA nanofiber membrane （the control group）, respectively. MTT （methyl thiazolyl tetrazolium） method was used to determine the toxicity and proliferation of the cells. The blood corpuscle counting plate was used to measure the adhesion rate 2, 4 and 6 hours postinoculation. DAPI （4＇ 6-diamidino-2-phenylidole） dyeing was used to observe the nucleal morphology. The morphology, adhesion and growth of the cells after co-culture were observed by SEM. Results The scaffolds in the 2 groups had an interconnected porous network structure, with a fiber diameter of 655 ± 57 nm in the experimental group and of 539 ＋ 48 nm in the control group respectively. The pores in the scaffolds were interconnected, with a porosity of 86. 8% ± 1.5% and of 84. 7% ± 1.2% respectively. MTT detection showed BMSCs grew well in both groups and the absorbance （OD） value increased over time with significant between-group differences at different time points （P 〈0. 05）. At each time point, the cell adhesion rate was significantly higher in the experimental group than in the control group （ P 〈 0. 05） . DAPI fluorescence staining showed normal morphology and quality of the nuclei, no obvious cellular apoptosis or necrosis in both groups, and significantly more cells in the experimental group. SEM observed that BMSCs grew much better on the scaffolds in the experimental group, proliferating massively and secreting matrix, than those in the control group. Conclusion The porous nanofiber scaffolds of PLGA / PEG prepared by electrostatic spinning can be a new type of tissue engineering carrier for spinal cord injury research because they are safe, free of toxicity, and suitable for BMSCs growth with fine cyto-compatibility and a proper aperture and porosity.