中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (22): 4710-4719.doi: 10.12307/2025.439

• 水凝胶材料Hydrogel materials • 上一篇    下一篇

增强自噬的可注射水凝胶微球改善软骨微环境抗软骨细胞衰老

李文铭,李永航,严才平,王星宽,向  超,张  袁,蒋  科,陈  路   

  1. 川北医学院附属医院,四川省南充市   637000
  • 收稿日期:2024-03-04 接受日期:2024-05-11 出版日期:2025-08-08 发布日期:2024-12-06
  • 通讯作者: 陈路,硕士生导师,主任医师,川北医学院附属医院,四川省南充市 637000 蒋科,博士,副主任医师,硕士生导师,川北医学院附属医院,四川省南充市 637000
  • 作者简介:李文铭,男,1997年生,四川省南充市人,汉族,在读硕士,主要从事软骨修复及细胞衰老相关研究。 李永航,男,1997年生,江苏省宿迁市人,汉族,在读硕士,主要从事骨再生研究。
  • 基金资助:
    南充市2023年市级科技计划专项资金项目(23JCYJPT0036),项目负责人:王星宽;川北医学院2023年校级科研计划项目(CBY23-QNA01),项目负责人:王星宽

Injectable hydrogel microspheres that enhance autophagy can improve cartilage microenvironment and resist chondrocyte senescence

Li Wenming, Li Yonghang, Yan Caiping, Wang Xingkuan, Xiang Chao, Zhang Yuan, Jiang Ke, Chen Lu   

  1. Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China
  • Received:2024-03-04 Accepted:2024-05-11 Online:2025-08-08 Published:2024-12-06
  • Contact: Chen Lu, Master’s supervisor, Chief physician, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China Jiang Ke, PhD, Associate chief physician, Master’s supervisor, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China
  • About author:Li Wenming, Master candidate, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China Li Yonghang, Master candidate, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China
  • Supported by:
    Nanchong City 2023 Municipal Science and Technology Plan Special Fund Project, No. 23JCYJPT0036 (to WXK); 2023 University-Level Scientific Research Project of North Sichuan Medical College, No. CBY23-QNA01 (to WXK)

摘要:


文题释义:

衰老相关分泌表型:衰老细胞分泌的一组促炎、促血管生成和生长刺激因子(包括基质金属蛋白酶、白细胞介素6、白细胞介素8和许多其他炎症细胞因子),可对细胞微环境和周围细胞产生影响。
自噬:是细胞内的一种“自食”现象,细胞通过自噬实现自身的代谢需求和某些细胞器的更新,能降解受损的蛋白质、衰老或损伤的细胞器等细胞结构,从而维持机体细胞的正常功能。 


背景:细胞衰老是骨关节炎的重大危险因素之一,目前尚无广泛认可的针对衰老细胞的抗骨关节炎治疗策略。

目的:制定可行的针对骨关节炎中衰老细胞的治疗策略。 
方法:采用薄膜分散法制备包封雷帕霉素的阳离子脂质体RAPA@Lipo,合成甲基丙烯酰化透明质酸水凝胶,将RAPA@Lipo加入甲基丙烯酰化透明质酸水凝胶水相液中,利用微流控设备制成水凝胶微球,再于紫光光照下交联成固体水凝胶微球(RAPA@Lipo@MS)。将人原代软骨细胞分别与RAPA@Lipo、 RAPA@Lipo@MS共培养,采用CCK-8法、活/死染色评价材料的生物相容性。将大鼠原代软骨细胞分4组培养:正常对照组常规培养48 h,造模组加入H2O2刺激24 h建立衰老细胞模型,RAPA@Lipo组、RAPA@Lipo@MS组建立衰老细胞模型后分别加入RAPA@Lipo、RAPA@Lipo@MS培养24 h,培养结束后,采用免疫荧光观察p62及Ⅱ型胶原蛋白的表达,RT-PCR检测白细胞介素6、基质金属蛋白酶13、Ⅱ型胶原、聚集蛋白聚糖及ADAMTS-5的mRNA表达。

结果与结论:①CCK-8检测、活/死染色结果显示,RAPA@Lipo与RAPA@Lipo@MS均具有良好的生物相容性;②与正常对照组相比,造模组细胞p62蛋白表达升高(P < 0.05),Ⅱ型胶原蛋白表达降低(P < 0.05),白细胞介素6、基质金属蛋白酶13及ADAMTS-5的mRNA表达升高(P < 0.05),Ⅱ型胶原及聚集蛋白聚糖mRNA表达降低(P < 0.05);与造模组比较,RAPA@Lipo@MS组p62蛋白表达降低(P < 0.05),Ⅱ型胶原蛋白表达升高(P < 0.05),白细胞介素6、基质金属蛋白酶13及ADAMTS-5的mRNA表达降低(P < 0.05),Ⅱ型胶原及聚集蛋白聚糖mRNA表达升高(P < 0.05);③结果表明,RAPA@Lipo@MS通过增强自噬控制体内细胞质量、减少体内的衰老细胞,局部清除骨关节炎中的衰老细胞和衰老相关分泌表型因子,以此减缓骨关节炎的进展,创造一个促再生的软骨微环境。 

https://orcid.org/0009-0008-1808-9871 (李文铭) 

中国组织工程研究杂志出版内容重点:生物材料;骨生物材料;口腔生物材料;纳米材料;缓释材料;材料相容性;组织工程

关键词: 骨关节炎, 水凝胶微球, 自噬, 衰老细胞, 衰老相关分泌表型, 雷帕霉素

Abstract: BACKGROUND: Cell senescence is one of the major risk factors for osteoarthritis, but there is no widely accepted anti-osteoarthritis therapy targeting senescent cells.
OBJECTIVE: To develop a feasible treatment strategy targeting senescent cells in osteoarthritis.
METHODS: The cationic liposome containing rapamycin, RAPA@Lipo, was prepared by thin film dispersion method. Methylallylated hyaluronic acid hydrogel was synthesized, and RAPA@Lipo was added to the methylallylated hyaluronic acid hydrogel aqueous phase solution. The hydrogel microspheres were prepared by microfluidic equipment. Solid hydrogel microspheres (RAPA@Lipo@MS) were crosslinked under violet light. Primary human chondrocytes were co-cultured with RAPA@Lipo and RAPA@Lipo@MS, respectively. The biocompatibility of the materials was evaluated by CCK-8 assay and live/dead staining. Primary rat chondrocytes were cultured in four groups. Normal control group was cultured for 48 hours. The model group was stimulated with H2O2 for 24 hours to establish senescent cell model. RAPA@Lipo group and RAPA@Lipo@MS group were cultured for 24 hours after establishing senescent cell model with RAPA@Lipo and RAPA@Lipo@MS, respectively. After culture, immunofluorescence was used to observe the expression of p62 and type II collagen. RT-PCR was used to detect the mRNA expression of interleukin 6, matrix metalloproteinase 13, type II collagen, aggrecan, and ADAMTS-5.
RESULTS AND CONCLUSION: (1) The results of CCK-8 assay and live/dead staining showed that RAPA@Lipo and RAPA@Lipo@MS had good biocompatibility. (2) Compared with the normal control group, the protein expression of p62 was increased (P < 0.05); the expression of type II collagen was decreased (P < 0.05), and the mRNA expression levels of interleukin 6, matrix metalloproteinase 13, and ADAMTS-5 were increased (P < 0.05); mRNA expression levels of type II collagen and aggrecan were decreased (P < 0.05) in the model group. Compared with the model group, the expression of p62 protein was decreased (P < 0.05); the expression of type II collagen was increased (P < 0.05), and the mRNA expression levels of interleukin 6, matrix metalloproteinase 13, and ADAMTS-5 were decreased (P < 0.05); mRNA expression of type II collagen and aggrecan increased (P < 0.05) in the RAPA@Lipo@MS group. (3) These findings indicate that RAPA@Lipo@MS can control the quality of cells in vivo by enhancing autophagy, reduce senescent cells in vivo, and locally eliminate senescent cells and senescence-associated secretory phenotype factors in osteoarthritis, thereby slowing the progression of osteoarthritis and creating a cartilage microenvironment that promotes regeneration.

Key words: osteoarthritis, hydrogel microsphere, autophagy, senescent cell, senescence-associated secretory phenotype, rapamycin

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