{"title":"沃顿果冻间充质基质细胞衍生的细胞外囊泡在体外三维藻酸盐珠培养模型中促进髓核细胞新陈代谢","authors":"Veronica Tilotta, Gianluca Vadalà, Luca Ambrosio, Giuseppina Di Giacomo, Claudia Cicione, Fabrizio Russo, Adas Darinskas, Rocco Papalia, Vincenzo Denaro","doi":"10.1002/jsp2.1274","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Background</h3>\n \n <p>Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin–eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (<i>MMP1, MMP13, ADAMTS5, IL6, NOS2</i>), anabolic (<i>ACAN</i>), and hNPC marker (<i>SOX9, KRT19</i>) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.</p>\n </section>\n </div>","PeriodicalId":14876,"journal":{"name":"JOR Spine","volume":"7 1","pages":""},"PeriodicalIF":3.4000,"publicationDate":"2023-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jsp2.1274","citationCount":"0","resultStr":"{\"title\":\"Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate-bead culture model\",\"authors\":\"Veronica Tilotta, Gianluca Vadalà, Luca Ambrosio, Giuseppina Di Giacomo, Claudia Cicione, Fabrizio Russo, Adas Darinskas, Rocco Papalia, Vincenzo Denaro\",\"doi\":\"10.1002/jsp2.1274\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n \\n <section>\\n \\n <h3> Background</h3>\\n \\n <p>Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods</h3>\\n \\n <p>After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin–eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (<i>MMP1, MMP13, ADAMTS5, IL6, NOS2</i>), anabolic (<i>ACAN</i>), and hNPC marker (<i>SOX9, KRT19</i>) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Results</h3>\\n \\n <p>Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Conclusions</h3>\\n \\n <p>WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.</p>\\n </section>\\n </div>\",\"PeriodicalId\":14876,\"journal\":{\"name\":\"JOR Spine\",\"volume\":\"7 1\",\"pages\":\"\"},\"PeriodicalIF\":3.4000,\"publicationDate\":\"2023-10-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jsp2.1274\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"JOR Spine\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/jsp2.1274\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ORTHOPEDICS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"JOR Spine","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/jsp2.1274","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ORTHOPEDICS","Score":null,"Total":0}
引用次数: 0
摘要
背景间充质干细胞（MSCs）的椎间盘内移植已成为治疗椎间盘退行性病变（IDD）的一种很有前景的疗法。然而，椎间盘（IVD）恶劣的微环境可能会影响植入细胞的存活。有趣的是，有研究报告称，间充质干细胞释放的细胞外囊泡（EVs）等旁分泌因子可使 IVD 再生。本研究旨在利用体外三维藻酸盐珠培养模型，研究沃顿果冻间充质干细胞（WJ-MSC）衍生的EVs对人髓核细胞（hNPCs）的治疗作用。 方法 在对 EV 进行分离和表征后，将从手术标本中分离出的 hNPCs 封装在藻酸盐珠中，并用 10、50 和 100 μg/mL WJ-MSC-EVs 进行处理。细胞增殖和活力通过流式细胞术和活/死染色进行评估。藻酸盐珠中的 hNPCs 经石蜡包埋并染色后进行组织学分析（苏木精-伊红和阿尔新蓝），以评估细胞外基质（ECM）的组成。通过 qPCR 分析分解代谢基因（MMP1、MMP13、ADAMTS5、IL6、NOS2）、合成代谢基因（ACAN）和 hNPC 标记基因（SOX9、KRT19）的基因表达水平。通过 Western 印迹分析评估 I 型和 II 型胶原蛋白的含量。 结果 用 WJ-MSC-EVs 处理退化的 hNPCs 后，细胞含量增加，细胞死亡减少。与对照组相比，EV 处理大大减少了亚硝酸盐的产生。此外，蛋白多糖含量也得到了提高，并通过阿尔新蓝组织学染色得到了证实。通过抑制分解代谢和炎症基因的表达水平，EV 刺激减轻了 ECM 降解和炎症反应。此外，EV 处理还能维持 NPC 表型标记基因。 结论 WJ-间充质干细胞衍生的 EV 可改善 hNPC 的生长和存活率，减轻 ECM 降解和氧化应激，为 IVD 再生提供了新的机会，是一种极具吸引力的细胞疗法替代策略。
Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate-bead culture model
Background
Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.
Methods
After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin–eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.
Results
Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.
Conclusions
WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.
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