摘要
目的探讨负载人脐带间充质干细胞外泌体(hUCMSC⁃Exos)的可注射光固化高孔隙率甲基丙烯酸明胶(Porous GelMA)/甲基丙烯酰化丝素蛋白(SilMA)复合水凝胶(PSE)促进膝关节软骨再生的效果及其机制。方法将60 g/L Porous GelMA与200 g/L SilMA溶液按6∶1体积比混合,紫外照射30 s固化,制得Porous GelMA/SilMA水凝胶(P/S6)。hUCMSC⁃Exos通过差速离心联合超滤法提取,以200μg/ml与Porous GelMA/SilMA复合溶液混合后紫外固化30 s形成负载Exos的PSE。将大鼠软骨细胞(P1代)分为对照组、P/S6组和PSE组,检测PSE的孔隙率、抗压强度及Exos缓释特性。软骨细胞分为对照组、白细胞介素⁃1β(IL⁃1β)组、P/S6组、PSE组;IL⁃1β组、P/S6组和PSE组细胞经10 ng/ml IL⁃1β处理24 h构建体外软骨缺损模型,分别与完全培养基、P/S6提取液和PSE提取液共培养3 d,对照组细胞不作处理;通过Western blot和qRT⁃PCR检测软骨蛋白聚糖抗体(ACAN)、性别决定区Y框蛋白9(SOX9)、基质金属蛋白酶13(MMP13)、Ⅱ型胶原(COLⅡ)的表达水平。小鼠单核巨噬细胞白血病细胞(RAW264.7)分为对照组、P/S6组、PSE组,分别用完全培养基、P/S6、PSE提取物培养基培养3 d,通过qRT⁃PCR检测精氨酸酶1重组蛋白(ARG1)、甘露糖受体(CD206)、诱导型一氧化氮合酶(iNOS)的表达水平。通过转录组测序筛选PSE促进软骨细胞再生的差异基因,富集分析关键信号通路。选取24只SD大鼠建立软骨缺损模型,按随机数字表法分为损伤对照组、P/S6组、PSE组(每组8只)。暴露大鼠右膝关节,随后用钻头在大鼠股骨髌骨沟中心钻一个直径2 mm、高1 mm的孔。损伤对照组用磷酸盐缓冲液处理,P/S6组和PSE组注入对应水凝胶后光固化,随后逐层缝合切口,伤后6、10周取标本行HE染色、番红固绿染色观察各组软骨缺损再生情况,免疫组化染色检测各组软骨损伤区域COLⅡ、MMP13、ARG1和CD206的阳性细胞面积。结果PSE具有与天然软骨匹配的抗压强度(0.41 MPa)、高孔隙率(85%)及Exos缓释能力(14 d释放率约85%)。软骨细胞修复实验中,与IL⁃1β损伤组相比,PSE组显著上调软骨合成代谢标志物表达COLⅡ提升2.1倍,ACAN提升1.8倍,SOX9提升1.5倍(P<0.01),同时显著抑制分解代谢标志物表达(MMP13降低52%)(P<0.01)。巨噬细胞极化实验中,相较于对照组,PSE组诱导巨噬细胞ARG1表达量提高68%(P<0.01),促进巨噬细胞的M2极化。转录组分析结果表明,PSE通过激活磷脂酰肌醇3⁃激酶(PI3K)/蛋白激酶B(Akt)通路和细胞外基质(ECM)受体互作通路促进ECM合成,并抑制炎症相关基因表达。动物实验中,组织学显示PSE组缺损区形成表面光滑连续的透明软骨。PSE组术后10周新生软骨标志物COLⅡ阳性细胞面积为(9.94±0.26)%,大于损伤对照组的(1.67±0.11)%(P<0.01),同时M2巨噬细胞标志物CD206阳性细胞面积达(14.44±0.23)%,大于损伤对照组的(3.41±0.36)%(P<0.01)。结论成功构建的PSE通过“ECM合成代谢增强⁃炎症微环境重塑”双重作用机制,显著促进膝关节软骨缺损的再生修复。其核心机制在于PSE负载的Exos特异性激活PI3K/Akt信号通路(促进软骨细胞增殖与存活)和ECM⁃受体互作通路(驱动ECM合成与组装),同时有效诱导巨噬细胞向M2抗炎表型极化,从而协同调控软骨ECM代谢并抑制炎症反应。
Objective To investigate the effect and mechanism of injectable photopolymerizable porous gelatin methacrylate anhydride(Porous GelMA)/methacrylated silk fibroin(SilMA)composite hydrogel(PSE)loaded with human umbilical cord mesenchymal stem cell⁃derived exosomes(hUCMSC⁃Exos)in promoting knee joint cartilage regeneration.Methods The porous GelMA solution(60 g/L)was mixed with SilMA solution(200 g/L)at a volume ratio of 6∶1.The mixture was ultraviolet⁃irradiated for 30 seconds to form a cured Porous GelMA/SilMA hydrogel(P/S6).The hUCMSC⁃Exos was isolated via differential centrifugation coupled with ultrafiltration and then was incorporated into the Porous GelMA/SilMA composite solution at 200μg/ml,followed by ultraviolet irradiation for 30 seconds to generate Exos-loaded PSE.Primary rat chondrocytes(P1)were divided into control group,P/S6 group,and PSE group to characterize the porosity,compressive strength,and sustained exosome release kinetics of PSE hydrogel.Chondrocytes were allocated to control group,interleukin⁃1β(IL⁃1β)group,P/S6 group,and PSE group,among which the last three groups were preconditioned with 10 ng/ml IL⁃1βfor 24 hours,and then cultured in complete medium,P/S6 extract and PSE extract for 3 days,respectively,to establish in vitro cartilage defect models,while the control group remained untreated.Western blot and qRT⁃PCR analysis were conducted to quantify the expression levels of antibody to aggrecan core protein(ACAN),sex⁃determining region Y⁃box transcription factor 9(SOX9),matrix metalloproteinase⁃13(MMP13)and collagen type II(COL II).Murine monocyte⁃macrophage leukemia cells(RAW264.7)were divided into control group,P/S6 group,and PSE group,which were then cultured in complete medium,PSE extract,and PSE extract medium for 3 days,respectively.qRT⁃PCR was employed to detect the expression levels of recombinant arginase⁃1 protein(ARG1),mannose receptor(CD206),and inducible nitric oxide synthase(iNOS).Transcriptomic sequencing was used to identify differentially expressed genes during PSE⁃mediated chondrocyte regeneration,followed by functional enrichment analysis of key signaling pathways.Twenty⁃four SD rats were selected to establish cartilage defect models and assigned to injury control group,P/S6 group,and PSE group according to the random number table(8 rats per group).The right knee joints of the rats were surgically exposed,and cylindrical osteochondral defects(a diameter of 2.0 mm×a depth of 1.0 mm)were surgically created in the center of the femoral trochlear groove using a drill bit.The injury control group received phosphate⁃buffered saline,while the P/S6 group and PSE group were injected with corresponding hydrogels followed by photo⁃crosslinking.Incisions then were closed in layers.At 6 and 10 weeks after injury,specimens were harvested for HE staining and safranin O⁃fast green staining to evaluate cartilage regeneration and immunohistochemistry staining to quantify the positive area fractions for COL II,MMP13,ARG1,and CD206 in the defect areas.Results PSE hydrogel exhibited compressive strength matching native cartilage(0.41 MPa),high porosity(85%),and sustained exosome release capacity(cumulative release rate of approximately 85%over 14 days).In chondrocyte repair experiments,compared to the IL⁃1βgroup,the PSE group demonstrated significantly upregulated expression of anabolic markers of cartilage(COL II expression increased by 2.1-fold,ACAN by 1.8-fold,and SOX9 by 1.5-fold)(P<0.01)as well as significantly suppressed expression of catabolic markers(MMP13 expression decreased by 52%)(P<0.01).In macrophage polarization assays,the PSE group exhibited ARG1 expression increased by 68%when compared to the control group(P<0.01),thus promoting M2 polarization of macrophages.Transcriptomic analysis revealed that PSE enhanced extracellular matrix(ECM)synthesis by activating the phosphatidylinositol 3⁃kinase/protein kinase B(PI3K/Akt)pathway and ECM⁃receptor interaction pathway,as well as by suppressing inflammation⁃related gene expression.Histological evaluation in animal experiments revealed regeneration of hyaline cartilage with smooth,continuous surfaces in the defect areas in the PSE group.At 10 weeks after surgery,the neocartilage⁃positive area in the PSE group was(9.94±0.26)%,significantly larger than(1.67±0.11)%in the injury control group(P<0.01).Besides,the CD206⁺M2 macrophage⁃positive area reached(14.44±0.23)%in the PSE group,significantly larger than(3.41±0.36)%in the injury control group(P<0.01).Conclusions The PSE hydrogel successfully engineered in the study can significantly promote regenerative repair of knee cartilage defects through a dual mechanism of enhanced ECM anabolism and remodeled inflammatory microenvironment.The core mechanisms involve specific activation of the PI3K/Akt pathway(boosting chondrocyte proliferation and survival)and ECM⁃receptor interaction pathway(driving ECM synthesis and assembly)by exosome⁃loaded PSE,while effectively polarizing macrophages toward an anti⁃inflammatory M2 phenotype so as to coordinately regulate cartilage ECM metabolism and suppress inflammatory responses.
作者
毕研驰
袁丁
朱亮
赵海波
江帆
肖晓
于腾波
Bi Yanchi;Yuan Ding;Zhu Liang;Zhao Haibo;Jiang Fan;Xiao Xiao;Yu Tengbo(Qingdao University Qingdao Medical College,Qingdao 266073,China;Department of Grassroots Medical Management,Qingdao Municipal Hospital,Qingdao 266071,China;Qingdao Hospital Central Laboratory,Rehabilitation University,Qingdao 266071,China;Department of Orthopedics,Qingdao Hospital,Rehabilitation University,Qingdao 266071,China)
出处
《中华创伤杂志》
北大核心
2025年第8期778-788,共11页
Chinese Journal of Trauma
基金
国家自然科学基金(82472431,82401552)
山东省自然科学基金(ZR2024QH590)
山东省2021年重点研发计划(科技示范工程)(2021SFGC0502)
青岛市2022年科技计划项目科技惠民示范引导工程(22⁃3⁃7⁃smjk⁃5⁃nsh)。
关键词
水凝胶
软骨
关节
再生
组织工程
外泌体
Hydrogel
Cartilage,articular
Regeneration
Tissue engineering
Exosomes
