水凝胶刚度影响髓核细胞表型及其功能的体内外研究

Effect of hydrogel stiffness on nucleus pulposus cell phenotypes in vitro and itsrepairment of intervertebral disc in vivo

目的·探究水凝胶刚度对髓核细胞表型及其治疗大鼠椎间盘退变功能的影响。方法·构建不同浓度甲基丙烯酸酯明胶(methacrylate gelatin,GelMA)的水凝胶,使用流变分析和单轴压缩实验检测其刚度,扫描电子显微镜(scanning electron microscope,SEM)观察其微观结构和形态。将具有正常表型的髓核细胞接种于GelMA水凝胶表面,活/死细胞染色评价水凝胶的生物相容性,鬼笔环肽染色观察髓核细胞在不同刚度培养基质上的生长形态。免疫荧光染色追踪髓核细胞Yes相关蛋白(Yes-associated protein,YAP)的核定位,实时定量PCR检测髓核细胞相关基因[神经细胞黏附分子1(neural cell adhesion molecule 1,Ncam-1)、聚集蛋白聚糖(aggrecan,Acan)和Y染色体性别决定区(sex-determing region of Y chromosome,SRY)-盒转录因子9(SRY-box transcription factor 9,Sox9)]的表达水平。建立大鼠尾椎针刺椎间盘退变模型,获取不同培养基上的髓核细胞并分别注射到退变椎间盘中,4周后行磁共振成像检测,分析各实验组椎间盘含水量,组织学方法检测椎间盘结构和蛋白聚糖水平。结果·当GelMA预聚溶液浓度为4%和15%时,所得水凝胶的弹性模量分别为1 kPa和200 kPa。SEM显示水凝胶均呈疏松多孔结构,且水凝胶的孔隙率随其刚度增加而显著降低。体外实验显示2种刚度的GelMA水凝胶基质均有良好的生物相容性。相较于硬水凝胶基质(15%GelMA),软水凝胶基质(4%GelMA)上培养的髓核细胞伸长率更低、扩散面积更小,并表现出YAP在细胞质聚集的趋势。髓核细胞相关基因表达检测显示:软水凝胶基质组Sox9、Acan和Ncam-1的水平分别是对照组的23.7、6.6和12.7倍。体内实验显示,用不同刚度基质培养的髓核细胞治疗退变椎间盘,软水凝胶基质组的椎间盘含水量及结构完整度均高于硬水凝胶基质组。结论·相比于高刚度GelMA水凝胶,低刚度水凝胶基质能更好地维持髓核细胞的生长表型,并使其发挥更好的治疗椎间盘退变的功效。

Objective·To investigate the effect of hydrogel stiffness on nucleus pulposus cell phenotype and its function in repairing intervertebral disc degeneration in rats.Methods·Methacrylate gelatin(GelMA)hydrogels with different concentrations were constructed.The stiffness of the hydrogels was investigated by using rheological analysis and uniaxial compression test.The microstructure and morphology of the hydrogels were observed by scanning electron microscopy(SEM).Nucleus pulposus cells with normal phenotype were inoculated on the surface of GelMA hydrogels.The biocompatibility of the hydrogel was evaluated by livedead cell staining and the growth pattern of nucleus pulposus cells on hydrogels with different stiffness was observed with phalloidin staining under microscope.Immunofluorescence staining was performed to examine the nuclear localization of Yes-associated protein(YAP)and real-time quantitative reverse transcription PCR(qRT-PCR)was used to detect the expression levels of nucleus pulposus cell-associated genes[neural cell adhesion molecule 1(Ncam-1),aggrecan(Acan),sex-determing region of Y chromosome(SRY)-boxtranscription factor 9(Sox9)].A rat caudal acupuncture intervertebral disc degeneration model was established.Nucleus pulposus cellscultured on different hydrogels were harvested and injected into the degenerated discs separately.Four weeks after surgery,magneticresonance imaging(MRI)was performed to analyze the water content of the intervertebral discs in each group.Histological tests wereperformed to examine the disc structure and proteoglycan levels.Results·The elastic modulus of the hydrogels was 1 kPa and 200 kPawhen the concentration of GelMA prepolymerisation solution was at 4%and 15%respectively.SEM observation revealed that thehydrogels showed a loose and porous microstructure,and the porosity of hydrogels decreased significantly with the decrease of theirstiffness.In vitro experiments demonstrated that both GelMA hydrogel mediums showed good biocompatibility and the ability tosupport cell proliferation.Nucleus pulposus cells cultured on the soft matrix(4%GelMA)had a lower elongation and spreading areathan those cultured on the stiff matrix(15%GelMA),showing a tendency of YAP concentration in the cytoplasm.The gene expressionof nucleus pulposus cells was examined and the levels of Sox9,Acan and Ncam-1 in the soft matrix hydrogel group were 23.7,6.6 and12.7 times of those in the control group respectively.In vivo experiments on rat disc degeneration showed that the soft hydrogel matrixgroup had higher disc water content and structural integrity than the stiff hydrogel matrix group.Conclusion·Compared to stiffGelMA hydrogels,hydrogels with low stiffness better maintain the growth phenotypes in the nucleus pulposus cells and have bettertherapeutic effect on disc degeneration in vivo.