甲基丙烯酸酯化硫酸软骨素/脱细胞华通胶/姜黄素复合水凝胶调控巨噬细胞极化与BMSCs成软骨分化的研究

Study on the methylacrylated chondroitin sulfate/decellularized wharton's jelly/curcumin composite hydrogel regulating macrophage polarization and chondrogenic differentiation of BMSCs

  • 摘要: 背景 关节软骨损伤后自我修复能力有限,易进展为骨关节炎。传统修复方法存在局限,而组织工程为软骨再生提供了新策略。目的 本研究旨在构建并评估一种负载姜黄素的甲基丙烯酸酯化硫酸软骨素/脱细胞华通胶(methacrylated chondroitin sulfate/Wharton/Curcumin,CMSA/Wharton/Curcumin,CWC)复合仿生水凝胶,系统探究其理化性能、生物相容性、免疫调节能力及在炎症条件下促进骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成软骨分化的潜能。方法 采用405 nm紫外光交联技术制备CWC复合水凝胶,通过扫描电镜、溶胀实验对其形貌与性能进行表征。采用CCK-8 法、活/死细胞染色及鬼笔环肽染色,评价CSMA、CW(CSMA/Wharton)及CWC三组水凝胶浸提液对大鼠BMSCs的生物相容性、增殖的影响。通过qRT-PCR和免疫荧光实验检测巨噬细胞M1/M2 表型相关基因(CD86,CD206 等)表达,评估材料的免疫调节作用。在白细胞介素1β(interleukin-1 beta,IL1β)诱导的炎症环境中,通过qRT-PCR 检测BMSCs成软骨分化关键基因(Sox9,Col2,Acan,Col1)的表达,评估不同水凝胶对干细胞的成软骨分化能力。结果 CWC水凝胶呈现稳定、均匀且相互连通的三维多孔结构,姜黄素成功负载,且华通胶的加入使网络结构更为致密。生物学评价表明,三组材料均无明显细胞毒性,并能支持细胞增殖与铺展,其中负载姜黄素的CWC组促增殖效果最为显著(P<0.05)。在免疫调节方面,CWC水凝胶可显著下调巨噬细胞M1 型标志物(IL1β、CD86),上调M2 型标志物(Arg1、CD206)的mRNA表达,有效诱导其向抗炎表型极化。在炎症条件下,CWC水凝胶能够显著逆转IL1β 对成软骨分化的抑制,提升软骨特异性基因(Sox9、Col2、Acan)的表达,并同时抑制Col1 的表达,其保护与促成软骨分化的效果优于CSMA及CW组。结论 本研究成功构建了负载姜黄素的CSMA/Wharton 光交联复合水凝胶。该材料具有良好的生物相容性与显著的免疫调节能力,并在炎症环境中可有效保护并促进BMSCs的成软骨分化。姜黄素的负载赋予了材料明确的抗炎与促成软骨活性,结合华通胶提供的仿生微环境,实现了“抗炎-促再生”的协同作用,为软骨修复提供了一种新型的功能化支架策略。

     

    Abstract: Background Articular cartilage exhibits limited self-repair capability after injury and is prone to progressing toward osteoarthritis. Conventional repair approaches face inherent limitations, while tissue engineering offers a novel strategy for cartilage regeneration.Objective This study aims to construct and evaluate a curcumin loaded methacrylated chondroitin sulfate/ decellularized Wharton's jelly (CSMA/Wharton/Curcumin, CWC)composite hydrogel, and systematically investigate its physicochemical properties, biocompatibility,immunomodulatory capacity, and potential to promote chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)under inflammatory conditions. Methods The CWC composite hydrogel was fabricated via 405 nm ultraviolet light-induced crosslinking. Its morphology and physicochemical properties were characterized by scanning electron microscopy (SEM) and swelling experiments. The biocompatibility and proliferative effects of hydrogel extracts from the three groups—CSMA, CW (CSMA/Wharton's jelly), and CWC—on rat bone marrow-derived mesenchymal stem cells (BMSCs) were evaluated using the CCK-8 assay,live/dead staining, and phalloidin staining. The immunomodulatory function of the materials was assessed by detecting the expression of M1/M2 macrophage phenotype-related markers (e.g., CD86,CD206) through quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence. Under an inflammatory microenvironment induced by interleukin 1β (IL1β), the chondrogenic differentiation capacity of BMSCs was evaluated by measuring the expression levels of key chondrogenic genes, including Sox9,Col2a1,Acan,and Col1, via qRT-PCR.Results The CWC hydrogel exhibited a stable,homogeneous,and interconnected three-dimensional porous structure.Curcumin was successfully loaded,and the incorporation of Wharton's jelly resulted in a more compact network architecture. Biological evaluation indicated that all three groups of materials showed no obvious cytotoxicity and could support cell proliferation and spreading.Among them, the curcumin-loaded CWC group demonstrated the most significant pro‑proliferative effect (P<0.05). In terms of immunomodulation,the CWC hydrogel significantly downregulated the mRNA expression of M1 macrophage markers (IL‑1β,CD86) and upregulated that of M2 markers (Arg1,CD206), effectively polarizing macrophages toward an anti‑inflammatory phenotype. Under inflammatory conditions, the CWC hydrogel markedly reversed the IL‑1β‑induced suppression of chondrogenic differentiation, enhanced the expression of cartilage‑specific genes (Sox9,Col2,Acan),and concurrently suppressed Col1 expression.Its protective and pro‑chondrogenic effects were superior to those of the CSMA and CW groups. Conclusion In this study, a curcumin-loaded, photocrosslinked CSMA/Wharton's jelly composite hydrogel was successfully fabricated. The material demonstrated favorable biocompatibility and significant immunomodulatory capacity, and it effectively protected and promoted the chondrogenic differentiation of BMSCs under inflammatory conditions.The incorporation of curcumin endowed the scaffold with distinct anti-inflammatory and pro‑chondrogenic activities. Combined with the biomimetic microenvironment provided by Wharton's jelly, the system achieved a synergistic"anti‑inflammatory and pro‑regenerative"effect,offering a novel functionalized scaffold strategy for cartilage repair.

     

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