水凝胶刚度影响髓核细胞表型及其功能的体内外研究

doi:10.3969/j.issn.1674-8115.2023.07.002

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

• 生物材料与再生医学专题 • 上一篇

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

收稿日期:2023-03-05 接受日期:2023-05-18 出版日期:2023-07-28 发布日期:2023-07-28
通讯作者: 崔文国,张煜辉 E-mail:844186744@qq.com;lvzhendongyyy@126.com;wgcui80@hotmail.com;zhangyuhui@renji.com
作者简介:陈泽昊（1998—），男，硕士生；电子信箱：844186744@qq.com
吕振东（1993—），男，住院医师，硕士；电子信箱：lvzhendongyyy@126.com。
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)

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

CHEN Zehao¹^,²(), LÜ Zhendong³(), ZHANG Zhen¹, CUI Wenguo²(), ZHANG Yuhui¹^,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

Received:2023-03-05 Accepted:2023-05-18 Online:2023-07-28 Published:2023-07-28
Contact: CUI Wenguo,ZHANG Yuhui E-mail:844186744@qq.com;lvzhendongyyy@126.com;wgcui80@hotmail.com;zhangyuhui@renji.com
Supported by:
National Natural Science Foundation of China(52273133);Foundation of Science and Technology Commission of Shanghai Municipality(20S31900100)

摘要/Abstract

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、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。

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.

Key words: hydrogel, stiffness, nucleus pulposus cell, Yes-associated protein (YAP), intervertebral disc degeneration

中图分类号:

R681.5

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[J]. 上海交通大学学报（医学版）, 2023, 43(7): 804-813.

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[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(7): 804-813.

图/表 8

表1 qPCR引物序列

Tab 1 Primer sequences for qPCR

Gene	Primer sequence (5′→3′)
Sox9
Forward	AGCACAAGAAAGACCACCCC
Reverse	GACCCTGAGATTGCCCGGAG
Acan
Forward	GACCTGTGTGAGATCGACCA
Reverse	GTTGGTTTGGACGCCACTTC
Ncam-1
Forward	TGGTCAAGTACAGAGCGAAGC
Reverse	AGGGACTTGAGCATGACGTG
Gapdh
Forward	GACAGCCGCATCTTCTTGTG
Reverse	ATCCGTTCACACCGACCTTC

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

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. ①P = 0.002, compared with the soft hydrogel group.

Fig 2 Physical characterization of GelMA hydrogels

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. ①P=0.002, ②P=0.000, compared with the soft hydrogel group.

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

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

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. ①P=0.000, compared with the soft hydrogel group.

Fig 5 Localization of YAP in nucleus pulposus cells

图6 不同刚度的水凝胶基质对髓核细胞表型的影响Note： ①P=0.000, ②P=0.007, compared with the control group.

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

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. ①P=0.003, ②P=0.017, ③P=0.000, ④P=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

参考文献 28

1	SCHUBERT A K, SMINK J J, PUMBERGER M, et al. Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells[J]. J Orthop Surg Res, 2018, 13(1): 209.
2	GUIMARAES C F, GASPERINI L, MARQUES A P, et al. The stiffness of living tissues and its implications for tissue engineering[J]. Nat Rev Mater, 2020, 5(5): 351-370.
3	HE J, CHEN C, CHEN L, et al. Honeycomb-like hydrogel microspheres for 3D bulk construction of tumor models[J]. Research (Wash D C), 2022, 2022: 9809763.
4	YIP C H, CHEN A D, WONG Y H, et al. Multiphoton microfabrication and micropatternining (MMM)-based screening of multiplex cell niche factors for phenotype maintenance-Bovine nucleus pulposus cell as an example[J]. Biomaterials, 2022, 281: 121367.
5	WANG B, KE W, WANG K, et al. Mechanosensitive ion channel Piezo1 activated by matrix stiffness regulates oxidative stress-induced senescence and apoptosis in human intervertebral disc degeneration[J]. Oxid Med Cell Longev, 2021, 2021: 8884922.
6	LIANG T, ZHANG L L, XIA W, et al. Individual collagen fibril thickening and stiffening of annulus fibrosus in degenerative intervertebral disc[J]. Spine, 2017, 42(19): E1104-E1111.
7	CHON B H, LEE E J, JING L, et al. Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system[J]. Stem Cell Res Ther, 2013, 4(5): 120.
8	ZHANG C, WANG F, XIE Z, et al. The hippo pathway orchestrates cytoskeletal organisation during intervertebral disc degeneration[J]. Acta Histochem, 2021, 123(6): 151770.
9	FEARING B V, JING L F, BARCELLONA M N, et al. Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape[J]. FASEB J, 2019, 33(12): 14022-14035.
10	KLOTZ B J, GAWLITTA D, ROSENBERG A J W P, et al. Gelatin-methacryloyl hydrogels: towards biofabrication-based tissue repair[J]. Trends Biotechnol, 2016, 34(5): 394-407.
11	CAI C D, ZHANG X S, LI Y G, et al. Self-healing hydrogel embodied with macrophage-regulation and responsive-gene-silencing properties for synergistic prevention of peritendinous adhesion[J]. Adv Mater, 2022, 34(5): e2106564.
12	SCHUURMAN W, LEVETT P A, POT M W, et al. Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs[J]. Macromol Biosci, 2013, 13(5): 551-561.
13	LIU Z, ZHAO B, ZHANG L, et al. Modulated integrin signaling receptors of stem cells via ultra-soft hydrogel for promoting angiogenesis[J]. Compos B Eng, 2022, 234: 109747.
14	ZHANG T, LIN S Y, SHAO X R, et al. Regulating osteogenesis and adipogenesis in adipose-derived stem cells by controlling underlying substrate stiffness[J]. J Cell Physiol, 2018, 233(4): 3418-3428.
15	MASUDA K, AOTA Y, MUEHLEMAN C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration[J]. Spine, 2005, 30(1): 5-14.
16	YU Z H, JI Y C, LI K, et al. Stiffness of the extracellular matrix affects apoptosis of nucleus pulposus cells by regulating the cytoskeleton and activating the TRPV2 channel protein[J]. Cell Signal, 2021, 84: 110005.
17	VINING K H, MOONEY D J. Mechanical forces direct stem cell behaviour in development and regeneration[J]. Nat Rev Mol Cell Biol, 2017, 18(12): 728-742.
18	DALY A C, RILEY L, SEGURA T, et al. Hydrogel microparticles for biomedical applications[J]. Nat Rev Mater, 2020, 5(1): 20-43.
19	SHIRAHAMA H, LEE B H, TAN L P, et al. Precise tuning of facile one-pot gelatin methacryloyl (GelMA) synthesis[J]. Sci Rep, 2016, 6: 31036.
20	BELL S, REDMANN A L, TERENTJEV E M. Universal kinetics of the onset of cell spreading on substrates of different stiffness[J]. Biophys J, 2019, 116(3): 551-559.
21	BARCELLONA M N, SPEER J E, FEARING B V, et al. Control of adhesive ligand density for modulation of nucleus pulposus cell phenotype[J]. Biomaterials, 2020, 250: 120057.
22	SCOTT K E, FRALEY S I, RANGAMANI P. A spatial model of YAP/TAZ signaling reveals how stiffness, dimensionality, and shape contribute to emergent outcomes[J]. Proc Natl Acad Sci USA, 2021, 118(20): e2021571118.
23	SEDOV E, KOREN E, CHOPRA S, et al. THY1-mediated mechanisms converge to drive YAP activation in skin homeostasis and repair[J]. Nat Cell Biol, 2022, 24(7): 1049-1063.
24	JANG M, AN J, OH S W, et al. Matrix stiffness epigenetically regulates the oncogenic activation of the Yes-associated protein in gastric cancer[J]. Nat Biomed Eng, 2021, 5(1): 114-123.
25	ZHANG X, CAI D, ZHOU F, et al. Targeting downstream subcellular YAP activity as a function of matrix stiffness with Verteporfin-encapsulated chitosan microsphere attenuates osteoarthritis[J]. Biomaterials, 2020, 232: 119724.
26	WANG Y, BAI B, HU Y, et al. Hydrostatic pressure modulates intervertebral disc cell survival and extracellular matrix homeostasis via regulating hippo-YAP/TAZ PATHWAY[J]. Stem Cells Int, 2021, 2021: 5626487.
27	XU P P, GUAN J J, CHEN Y, et al. Stiffness of photocrosslinkable gelatin hydrogel influences nucleus pulposus cell properties in vitro[J]. J Cell Mol Med, 2021, 25(2): 880-891.
28	BINCH A L A, FITZGERALD J C, GROWNEY E A, et al. Cell-based strategies for IVD repair: clinical progress and translational obstacles[J]. Nat Rev Rheumatol, 2021, 17(3): 158-175.

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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 its repairment of intervertebral disc in vivo

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