上海交通大学学报(医学版), 2023, 43(7): 804-813 doi: 10.3969/j.issn.1674-8115.2023.07.002

生物材料与再生医学专题

水凝胶刚度影响髓核细胞表型及其功能的体内外研究

陈泽昊,1,2, 吕振东,3, 张震1, 崔文国,2, 张煜辉,1,3

1.上海大学机电工程与自动化学院,上海 200072

2.上海交通大学医学院附属瑞金医院骨科,上海市伤骨科研究所,上海市中西医结合防治骨与关节病损重点实验室,上海 200025

3.上海交通大学医学院附属仁济医院脊柱外科,上海 200127

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

CHEN Zehao,1,2, LÜ Zhendong,3, ZHANG Zhen1, CUI Wenguo,2, ZHANG Yuhui,1,3

1.School of Mechatronic Engineering and Automation, Shanghai University, Shanghai 200072, China

2.Department of Orthopedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Institute of Traumatology and Orthopedics; Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai 200025, China

3.Department of Spine Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

通讯作者: 崔文国,电子信箱:wgcui80@hotmail.com张煜辉,电子信箱:zhangyuhui@renji.com

编委: 吴洋

收稿日期: 2023-03-05   接受日期: 2023-05-18   网络出版日期: 2023-07-28

基金资助: 国家自然科学基金.  52273133
上海市科学技术委员会项目.  20S31900100

Corresponding authors: CUI Wenguo, E-mail:wgcui80@hotmail.comZHANG Yuhui, E-mail:zhangyuhui@renji.com.

Received: 2023-03-05   Accepted: 2023-05-18   Online: 2023-07-28

作者简介 About authors

陈泽昊(1998—),男,硕士生;电子信箱:844186744@qq.com E-mail:844186744@qq.com

吕振东(1993—),男,住院医师,硕士;电子信箱:lvzhendongyyy@126.com。 E-mail:lvzhendongyyy@126.com

摘要

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、AcanNcam-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。

关键词: 水凝胶 ; 刚度 ; 髓核细胞 ; Yes相关蛋白 ; 椎间盘退变

Abstract

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 live-dead 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)-box transcription factor 9 (Sox9)]. A rat caudal acupuncture intervertebral disc degeneration model was established. Nucleus pulposus cells cultured on different hydrogels were harvested and injected into the degenerated discs separately. Four weeks after surgery, magnetic resonance imaging (MRI) was performed to analyze the water content of the intervertebral discs in each group. Histological tests were performed to examine the disc structure and proteoglycan levels. Results ·The elastic modulus of the hydrogels was 1 kPa and 200 kPa when the concentration of GelMA prepolymerisation solution was at 4% and 15% respectively. SEM observation revealed that the hydrogels showed a loose and porous microstructure, and the porosity of hydrogels decreased significantly with the decrease of their stiffness. In vitro experiments demonstrated that both GelMA hydrogel mediums showed good biocompatibility and the ability to support cell proliferation. Nucleus pulposus cells cultured on the soft matrix (4%GelMA) had a lower elongation and spreading area than those cultured on the stiff matrix (15%GelMA), showing a tendency of YAP concentration in the cytoplasm. The gene expression of 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 and 12.7 times of those in the control group respectively. In vivo experiments on rat disc degeneration showed that the soft hydrogel matrix group had higher disc water content and structural integrity than the stiff hydrogel matrix group. Conclusion ·Compared to stiff GelMA hydrogels, hydrogels with low stiffness better maintain the growth phenotypes in the nucleus pulposus cells and have better therapeutic effect on disc degeneration in vivo.

Keywords: hydrogel ; stiffness ; nucleus pulposus cell ; Yes-associated protein (YAP) ; intervertebral disc degeneration

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陈泽昊, 吕振东, 张震, 崔文国, 张煜辉. 水凝胶刚度影响髓核细胞表型及其功能的体内外研究. 上海交通大学学报(医学版)[J], 2023, 43(7): 804-813 doi:10.3969/j.issn.1674-8115.2023.07.002

CHEN Zehao, LÜ Zhendong, ZHANG Zhen, CUI Wenguo, ZHANG Yuhui. Effect of hydrogel stiffness on nucleus pulposus cell phenotypes in vitro and its repairment of intervertebral disc in vivo. Journal of Shanghai Jiao Tong University (Medical Science)[J], 2023, 43(7): 804-813 doi:10.3969/j.issn.1674-8115.2023.07.002

在椎间盘退变的机制研究中,髓核细胞的表型和功能一直是研究的热点。然而,在体外培养的过程中,髓核细胞的细胞表型易丢失,影响后续研究结果1。天然的髓核是一种柔软的凝胶状组织。然而,在大多数的研究中,培养髓核细胞常用坚硬的塑料或玻璃皿,其弹性模量无法模拟天然组织。另外,由于缺少天然细胞外基质(extracellular matrix,ECM)中的黏附因子2,随着培养时间的延长,在平皿上培养的髓核细胞会逐渐丧失其正常的形态和表型,从原本的多边形向梭形的成纤维细胞转变,并伴随髓核表型因子的丢失3-4。如何在体外培养时尽可能维持髓核细胞的表型,是椎间盘研究中需要解决的问题。

研究5-6显示,体外微环境的刚度会对髓核细胞表型产生显著影响,升高的刚度会加速髓核细胞的凋亡和表型的丢失。为了在体外培养中尽可能地保持细胞表型,有些研究在培养皿上铺一层基质胶或是ECM蛋白7。虽然采用这些方法能一定程度维持髓核细胞表型,但基质胶和预铺ECM蛋白对基质刚度的调节有限,难以达到满意的效果,同时成本高,难以广泛开展。

Yes-相关蛋白(Yes-associated protein,YAP)是细胞的机械传感因子,其转录水平可随着微环境的刚度改变而发生变化。YAP的表达可以在细胞质和细胞核中转移。在天然髓核组织中,髓核细胞的YAP主要在细胞质中表达8。然而,刚性的体外培养环境容易导致YAP在髓核细胞的细胞核中表达,过度激活的YAP可以上调血清应答因子(serum-response factor,SRF)和TEA域(TEA domain,TEAD)的转录活性,抑制髓核细胞合成ECM的能力9

明胶是Ⅰ型胶原蛋白部分变性后的产物,保留了胶原蛋白天然的精氨酸-甘氨酸-天冬氨酸(arginine-glycine-aspartic acid,RGD)肽,有利于细胞黏附10。明胶的氨基可以与甲基丙烯酸酐反应合成光交联GelMA水凝胶,在光引发剂和365 nm蓝光作用下引发自由基聚合交联。GelMA水凝胶有较好的生物相容性和酶降解的特征,是一种常用的细胞培养介质和组织工程支架11。通过改变光交联条件,如预聚物溶液浓度和光照时间等,可以将GelMA水凝胶的刚度在1至数百千帕范围内进行调节12

本研究拟合成光交联GelMA水凝胶,通过调整GelMA预聚物溶液的浓度获得具有不同刚度的水凝胶基质。将髓核细胞接种在不同刚度的水凝胶上进行培养,观察髓核细胞的细胞表型;检测YAP在髓核细胞中的表达定位和相关基因的水平;观察不同刚度的水凝胶基质下培养的髓核细胞体内治疗退变椎间盘的功能差异。

1 材料与方法

1.1 实验动物

5周龄雄性SPF级SD大鼠10只,体质量约200 g,购自上海杰思捷实验动物有限公司,动物生产许可证号为SCXK(沪)2018-0004。所有大鼠在上海交通大学医学院附属仁济医院实验动物中心饲养,动物使用许可证号为SYXK(沪)2018-0013。

1.2 主要材料与仪器

明胶购自美国Sigma-Aldrich公司,甲基丙烯酸酐购自上海阿拉丁生化科技股份有限公司,透析袋(截留相对分子质量3 500)购自上海源叶生物科技有限公司,苯基-2,4,6-三甲基苯甲酰基亚磷酸锂盐(lithium phenyl-2,4,6-trimethylbenzoylphosphinate,LAP)购自上海华夏司印生物技术有限公司,Ⅱ型胶原酶购自上海生工生物工程有限公司,DMEM/F12培养基购自美国Hyclone公司,胎牛血清和胰酶购自美国Gibco公司,活/死细胞染色试剂购自上海碧云天生物技术有限公司,YAP1一抗和Fluor594标记的二抗购自美国Affinity公司,DAPI染液和FITC标记的鬼笔环肽购自上海复申生物科技有限公司,细胞/组织总RNA提取试剂盒购自南京诺唯赞生物科技有限公司,反转录和实时定量PCR(qPCR)试剂盒购自北京宝日医生物技术有限公司,qPCR引物由上海生工生物工程有限公司合成。

动态热机械分析仪Q800和流变仪ARES-G2购自美国TA公司,扫描电子显微镜(scanning electron microscope,SEM)Sirion 200购自美国FEI公司,荧光显微镜购自德国Zeiss公司,激光共聚焦显微镜购自日本Olympus公司,qPCR仪Applied Biosystems 7500购自美国Thermo Scientific公司。

1.3 GelMA合成

称取20 g明胶在60 ℃下搅拌溶解在0.1 mol/L碳酸盐缓冲溶液(pH值9.0)中,以配置10%浓度的明胶溶液。逐滴加入4 mL甲基丙烯酸酐,继续反应3 h。加入100 mL磷酸盐缓冲溶液(phosphate buffer saline,PBS)以停止反应。将溶液倒入透析袋中,透析3 d以去除未反应的杂质。收集透析袋液体,冷冻干燥后得到白色泡沫状的GelMA。

1.4 GelMA水凝胶的制备

称取0.4 g和1.5 g的GelMA泡沫,37 ℃下溶解在10 mL PBS中,配制终浓度分别为4%和15%的GelMA预聚物溶液。加入0.25%质量分数的LAP,溶液通过0.2 μm孔径的针头过滤器灭菌。将溶液加入培养板,暴露于365 nm的蓝光中交联5 min。

1.5 GelMA水凝胶的表征

将完全交联后的水凝胶置于动态热机械分析仪上进行单轴压缩实验,得到应力-应变曲线,取0~30%线性应变区间的直线斜率作为水凝胶的弹性模量。使用流变仪的振荡扫描模式测试水凝胶的剪切储能模量,应变1%,角速度5 rad/s。将水凝胶冻干,使用SEM观察水凝胶的微观形貌。

1.6 髓核细胞的提取与培养

髓核细胞从大鼠尾椎椎间盘中提取。无菌条件下小心分离大鼠尾椎的髓核,剪碎髓核组织并沉浸在含有0.1% Ⅱ型胶原酶的DMEM/F12培养基中,在培养箱中消化4 h。将组织的消化液通过180目的滤网过滤,离心收集髓核细胞,并重新接种在培养瓶中,在含10%胎牛血清和1%双抗的DMEM/F12培养基中培养。当细胞达到80%汇合度的时候消化传代,取第3代髓核细胞用于后续的实验。

1.7 水凝胶生物相容性评价

将髓核细胞重新接种在由4%和15% GelMA溶液交联而成的水凝胶上培养5 d。去除培养板中的培养基,用PBS洗涤细胞。加入活/死细胞染色试剂并在室温中孵育10 min,去除染色剂再次用PBS洗涤。在荧光显微镜下观察细胞。

1.8 髓核细胞的免疫荧光染色

髓核细胞在水凝胶上培养的第2日,观察YAP的定位情况和细胞骨架。使用PBS清洗细胞3次,在4%多聚甲醛中固定30 min,PBS洗涤3次,在0.1% Triton X-100中透化,再次洗涤,并用3%牛血清白蛋白封闭30 min。细胞在YAP1一抗中孵育过夜,PBS洗涤后与Fluor594标记的二抗室温孵育2 h,清洗后的细胞与DAPI染液孵育,再次洗涤后在激光共聚焦显微镜下观察。对于骨架染色,将固定封闭处理后的细胞在鬼笔环肽染液中孵育1 h,细胞使用DAPI复染后在荧光显微镜下观察。通过测量细胞的长轴和短轴计算细胞伸长率13,通过测量细胞的骨架面积评估细胞的扩散水平14

1.9 髓核细胞表型的评估

将髓核细胞分别接种在水凝胶和平皿上,其中平皿培养作为对照组。培养7 d后去除培养基,PBS洗涤1次。为了提取水凝胶组的细胞RNA,加入0.5%的Ⅱ型胶原酶,放入培养箱消化10 min,使黏附的髓核细胞与水凝胶分离。对于在平皿上培养的细胞,直接使用胰酶消化。离心收集细胞,提取细胞RNA,并合成cDNA。使用TB Green法检测神经细胞黏附分子1(neural cell adhesion molecule 1,Ncam-1Y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)及聚集蛋白聚糖(aggrecan,Acan)的mRNA表达,根据2-ΔΔCt法计算mRNA的相对表达水平。引物序列见表1

表1   qPCR引物序列

Tab 1  Primer sequences for qPCR

GenePrimer sequence (5′→3′)
Sox9
ForwardAGCACAAGAAAGACCACCCC
ReverseGACCCTGAGATTGCCCGGAG
Acan
ForwardGACCTGTGTGAGATCGACCA
ReverseGTTGGTTTGGACGCCACTTC
Ncam-1
ForwardTGGTCAAGTACAGAGCGAAGC
ReverseAGGGACTTGAGCATGACGTG
Gapdh
ForwardGACAGCCGCATCTTCTTGTG
ReverseATCCGTTCACACCGACCTTC

新窗口打开| 下载CSV


1.10 髓核细胞治疗对椎间盘退变修复的体内评估

通过注射戊巴比妥(50 mg/kg)麻醉大鼠,使用一根18G细针穿刺大鼠尾椎6~7、7~8和8~9椎体节段间的椎间盘正中央,将细针旋转360°并停留1 min后拔出。尾椎5~6节段间的椎间盘作为空白对照组,不进行针刺退变。如前所述,使用胶原酶分离水凝胶培养物上的髓核细胞,并离心重悬注射进退变模型中。6~7节段间的椎间盘注射10 μL硬水凝胶(15% GelMA)上培养的髓核细胞悬液(硬水凝胶基质组),7~8节段间的椎间盘注射10 μL软水凝胶(4% GelMA)上培养的髓核细胞悬液(软水凝胶基质组)。8~9节段间的椎间盘作为针刺对照组,注射等体积的PBS。术后立即使用磁共振成像(MRI)记录椎间盘含水量。术后4周再次行MRI检测,然后通过过量麻醉处死大鼠,使用苏木精-伊红染色(H-E染色)和番红固绿染色观察椎间盘形态和结构。对各组椎间盘进行组织学分级15,根据纤维环的结构、纤维环与髓核交界处的完整度、髓核细胞的数量以及髓核的基质水平评估椎间盘,级别越高代表退变越严重。

1.11 统计学分析

采用GraphPad Prism 8软件绘制统计图并进行分析。定量资料以x±s表示,组间比较采用t检验,多样本比较采用单因素方差分析。P<0.05表示差异有统计学意义。

2 结果

2.1 GelMA培养基质的物理表征

通过在明胶的氨基侧链上接枝甲基丙烯酸酯基团合成GelMA(图1A)。4% GelMA溶液清澈透明,而15% GelMA溶液的澄清度下降(图1B)。将水凝胶预聚物溶液加入孔板中,2种水凝胶在365 nm下的蓝光下充分交联。4% GelMA水凝胶相比于15% GelMA水凝胶透光度略高。使用移液器标准吸头手动搅拌水凝胶后,2种水凝胶的形态差异更明显,4% GelMA水凝胶的破坏边缘较15% GelMA更加模糊(图1C、D)。

图1

图1   GelMA的合成与表征

Note: A. Schematic representation of GelMA synthesis. B. Photograph of GelMA precursor solutions. C. Crosslinked GelMA hydrogel mediums. D. GelMA hydrogel mediums after mashing with a pipette tip.

Fig 1   Synthesis and characteristics of GelMA


流变实验结果显示:用15%GelMA溶液交联而成的水凝胶剪切储能模量为20 000 Pa,较4% GelMA溶液制成的水凝胶更硬(图2A)。因此,分别将4%和15%GelMA溶液制成的水凝胶命名为软水凝胶基质组和硬水凝胶基质组。单轴压缩实验表明软水凝胶的弹性模量约为1 kPa,而硬水凝胶约为200 kPa(图2B),进一步验证了其刚度的差异。将水凝胶冻干,通过SEM观察(图2C、D),可见2组水凝胶均呈现疏松多孔的结构,其中软水凝胶基质组的孔隙率更高,孔径更大,表明软水凝胶的交联密度更低,力学性能可能更弱。

图2

图2   GelMA水凝胶的物理表征

Note: A. Rheology analysis of hydrogels. B. Compression modulus of hydrogels. C. The SEM image of soft hydrogels. D. The SEM image of stiff hydrogels. P = 0.002, compared with the soft hydrogel group.

Fig 2   Physical characterization of GelMA hydrogels


2.2 髓核细胞在GelMA水凝胶基质上培养时的形态

在软水凝胶和硬水凝胶基质组上培养5 d后,髓核细胞均能黏附在水凝胶表面生长,且伸展良好(图3A、B)。在细胞培养的第2日,通过鬼笔环肽染色观察髓核细胞的骨架形态(图3C、D),可见在软水凝胶基质组上培养的髓核细胞大多呈现出圆形的收缩形态,且软水凝胶基质组培养的髓核细胞伸长率显著低于硬水凝胶基质组,说明细胞的圆度更高(图3E)。软水凝胶基质组上培养的髓核细胞扩散程度也显著低于硬水凝胶基质组(图3F)。

图3

图3   在不同刚度水凝胶基质上培养的髓核细胞形态观察

Note: A. Cell morphology of the soft hydrogel group. B. Cell morphology of the stiff hydrogel group. C. Immunofluorescent image of F-actin in nucleus pulposus cells of the soft hydrogel group. D. Immunofluorescent image of F-actin in nucleus pulposus cells of the stiff hydrogel group. E. Ratio of cell elongation of different groups. F. Quantitative results of cell spreading area cultured on different hydrogels. P=0.002, P=0.000, compared with the soft hydrogel group.

Fig 3   Morphologic observation of nucleus pulposus cells cultured on soft and stiff hydrogel mediums


2.3 GelMA水凝胶基质的生物相容性

在髓核细胞培养的第5日,活/死细胞染色观察结果显示,2种刚度的水凝胶均表现出良好的生物相容性,大部分髓核细胞保持活性,死细胞少见(图4)。2组细胞均能正常增殖,证明2种刚度的水凝胶基质组都具有良好的生物相容性。

图4

图4   GelMA水凝胶基质上培养的髓核细胞的活/死细胞染色观察

Note: A. Live-dead staining of the soft hydrogel group. B. Live-dead staining of the stiff hydrogel group.

Fig 4   Live-dead staining of nucleus pulposus cells cultured on GelMA hydrogels


2.4 髓核细胞的YAP表达定位

免疫荧光染色观察YAP的表达定位,结果显示:软水凝胶基质组上培养的髓核细胞,YAP主要表达在细胞质中,细胞核的YAP荧光强度显著低于细胞质(图5A);而硬水凝胶基质组上髓核细胞的YAP表达情况则相反,在细胞核中观察到了强烈的YAP荧光,其核/质荧光比显著高于软水凝胶基质组(图5B)。以上结果提示软水凝胶基质可能会导致髓核细胞的YAP表达从细胞核的富集转向细胞质,而硬基质则能促进YAP的核内富集。

图5

图5   髓核细胞的YAP表达定位

Note: A. Immunofluorescence images of YAP nuclear localization. B. Quantification of YAP localization. P=0.000, compared with the soft hydrogel group.

Fig 5   Localization of YAP in nucleus pulposus cells


2.5 不同刚度的水凝胶基质对髓核细胞表型的影响

qPCR结果(图6)显示,硬水凝胶基质组的髓核细胞基因水平与对照组相比,差异无统计学意义;而软水凝胶基质组Sox9AcanNcam-1的表达水平分别是对照组的23.7、6.6和12.7倍,差异均有统计学意义(均P<0.05),证明软水凝胶基质组培养的髓核细胞有更强的细胞表型维持能力。

图6

图6   不同刚度的水凝胶基质对髓核细胞表型的影响

NoteP=0.000, P=0.007, compared with the control group.

Fig 6   Effect of hydrogel matrices with different stiffness on the phenotypes of nucleus pulposus cells


2.6 水凝胶刚度对细胞治疗功能的影响

MRI的结果表明,针刺法制作成功的退变椎间盘水信号强度迅速降低,给予细胞治疗后椎间盘的水信号有所恢复,其中,软水凝胶基质组的椎间盘水信号强度最高(图7A、B)。组织学观察结果显示:针刺法成功建立了退变椎间盘模型,其椎间盘结构明显被破坏,髓核组织仅有少量残留,纤维环排列紊乱,很难辨识髓核和纤维环的边界,蛋白聚糖水平显著下降(图7C)。经软水凝胶基质组髓核细胞治疗后的椎间盘结构更完整,核和纤维环边界清晰,大部分的髓核组织得以保留,并且髓核细胞呈现出与空白对照组类似的大液泡状,红色的番红染色区域较多,说明蛋白聚糖水平较高。而经硬水凝胶基质组细胞治疗后,虽然纤维环组织未见明显破坏,但是髓核区域急剧缩小,蛋白聚糖水平也显著下降。进一步的组织学分级结果显示:软水凝胶基质组的组织学分级与空白对照组差异最小(图7D)。以上结果提示水凝胶基质组的髓核细胞在体内均能在不同程度上修复退变的椎间盘,其中软水凝胶基质组能更好地维持椎间盘的健康形态,修复能力更强。

图7

图7   不同刚度水凝胶基质培养的髓核细胞对退变椎间盘的修复作用

Note: A. MRI imaging. B. MRI signal intensity. C. H-E and safranin O/fast green staining of intervertebral discs. Black arrows showing annulus fibrosus and violet arrows showing proteoglycans. D. Histological scores at 4 weeks postoperatively. P=0.003, P=0.017, P=0.000, P=0.050, compared with the control group.

Fig 7   Repair effect of nucleus pulposus cells cultured in hydrogel matrix with different stiffness on intervertebral disc degeneration


3 讨论

生理状态下的髓核是一种凝胶状的软组织。随着椎间盘的退变,柔软的髓核出现纤维化,其微环境的刚度可能也会发生改变。YU等16测量了不同退变阶段大鼠髓核的弹性模量,发现随着椎间盘退变的加剧,髓核的弹性模量显著上升,上升的刚度通过激活PI3K/AKT通路诱导髓核细胞凋亡。常用的细胞培养皿为聚苯乙烯材质,其弹性模量高达3 000 MPa,远超天然软组织的模量范围,会引起在其环境中培养的髓核细胞发生细胞表型的丢失17。较低刚度的基质在体外培养中可能更有助于保持髓核细胞表型。

水凝胶是高度含水的三维网络,具有仿生ECM的特点。明胶是一种天然来源的水凝胶,相比于透明质酸和海藻酸钠等其他的天然水凝胶材料,明胶的优势在于保留了可以促进细胞黏附的RGD肽序列18。因此,明胶及其改性材料适用于细胞培养。GelMA是对明胶进行光基团修饰后合成的材料,保留了明胶良好的生物相容性,且机械性能可以大范围调节。本研究使用碱性碳酸盐缓冲液代替传统PBS合成GelMA,对于甲基丙烯酸酐的添加量要求更低,同时合成后的GelMA溶液的黏度更低19。即使配置15%高浓度的GelMA溶液,也能轻松通过0.2 μm的滤器进行除菌。本研究通过光交联4%和15%的GelMA溶液,制备获得不同刚度的GelMA水凝胶基质。通过流变测试和单轴压缩实验,硬水凝胶基质组的机械性能约为软水凝胶基质组的200倍。水凝胶冻干后的SEM图像显示,硬水凝胶基质的孔隙率更低、孔径显著小于软水凝胶基质,这与高浓度预聚物溶液交联后的网络结构更紧密有关。

髓核细胞接种于不同刚度的GelMA水凝胶基质上均能正常黏附和增殖,显示GelMA具有良好的生物相容性。既往研究20报道,基质刚度会对其培养细胞的大小和形状产生影响,较高的基质刚度更有利于细胞的铺展扩散和骨架形成。本研究发现,在软水凝胶基质上的髓核细胞圆度更高,细胞的铺展面积也小于硬水凝胶基质组。幼年态的髓核细胞通常表现出圆形的形态,随着细胞的衰老,其形态会逐渐向成纤维细胞状转变21。因此,柔软的水凝胶可以维持髓核细胞的圆度,可能有助于髓核细胞的表型维持。

YAP是细胞转导机械信号的调节器,YAP的表达能在细胞核/质之间转移来响应基质刚度22。免疫荧光结果显示软水凝胶基质组中的髓核细胞YAP的表达集中在细胞质中,而硬水凝胶基质组则观察到了明显的YAP核富集。这些结果说明:髓核细胞通过控制形态和扩散来响应基质刚度的机械信号,进一步调控YAP的表达。YAP的定位和表达在组织生长和退行性疾病中发挥重要作用23-24。ZHANG等25发现,骨关节炎的进展与组织硬化和软骨细胞的YAP核积累相关联。WANG等26在体外静水压实验中发现Hippo-YAP/TAZ通路的过度激活会促进髓核细胞的凋亡。硬水凝胶基质组中髓核细胞YAP的细胞核表达可能与该组髓核细胞易丢失细胞表型有关。利用qPCR检测髓核细胞表型相关的基因,结果显示:硬水凝胶基质上生长的髓核细胞表型相关基因显著低于软水凝胶基质组,与细胞骨架荧光染色观察到的硬水凝胶基质组培养的髓核细胞形态发生改变相吻合。XU等27制备了从46 kPa到82 kPa的不同刚度GelMA水凝胶作为基质培养髓核细胞,发现髓核细胞的ECM沉积水平随刚度的上升而下降。然而,该研究中,即使是最柔软的水凝胶,其压缩模量也已处于退变髓核的模量范围之内,难以保持髓核细胞表型。本研究发现接近健康髓核组织特性的1 kPa软水凝胶基质组能显著提高髓核相关基因的水平,可能与YAP在细胞质的富集有关。

本研究发现,利用软水凝胶基质培养的髓核细胞在体治疗大鼠退变椎间盘,椎间盘的含水量和结构完整度均高于硬水凝胶基质组。这种现象可能与软水凝胶基质上培养的髓核细胞维持了更好的髓核细胞固有形态和生理功能有关,其在体内有着更高的存活率或更强的旁分泌能力,有益于椎间盘结构的恢复。有临床研究28显示,髓核细胞移植对于椎间盘退变的疗效不佳。这可能与体外培养髓核细胞的过程中难以保持髓核细胞原有的细胞表型和功能有关。

综上,本研究构建了基于不同刚度GelMA水凝胶基质的髓核细胞体外培养体系,结果显示低刚度的GelMA水凝胶基质可以更好地保持髓核细胞表型和功能,在体治疗退变椎间盘具有更好的疗效。

作者贡献声明

陈泽昊、吕振东参与了实验设计、数据分析和论文写作;张震、崔文国、张煜辉参与了论文的修改。所有作者均阅读并同意了最终稿件的提交。

CHEN Zehao and LÜ Zhendong were responsible for the experimental design, data analysis, and manuscript writing. The manuscript was revised by ZHANG Zhen, CUI Wenguo and ZHANG Yuhui. All the authors have read the last version of paper and consented for submission.

利益冲突声明

所有作者声明不存在利益冲突。

All authors disclose no relevant conflict of interests.

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