载黄腐酚抗炎支架对羊体内软骨再生的影响研究  

Effect of xanthohumol-loaded anti-inflammatory scaffolds on cartilage regeneration in goats

作　　者：徐松山 赵少华 菅炎鹏 徐勇[3] 刘伟杰 邵欣慰 樊俊 王一公 XU Songshan;ZHAO Shaohua;JIAN Yanpeng;XU Yong;LIU Weijie;SHAO Xinwei;FAN Jun;WANG Yigong(Department of Spine and Spinal Cord Surgery,Xuchang Central Hospital Affiliated to Henan University of Science and Technology,Xuchang Henan,461000,P.R.China;Department of Plastic Surgery,Xuchang Central Hospital Afiliated to Henan University of Science and Technology,Xuchang Henan,461000,P.R.China;Department of Thoracic Surgery,Shanghai Pulmonary Hospital,Shanghai,200433,P.R.China)

机构地区：[1]河南科技大学附属许昌市中心医院脊柱脊髓外科,河南许昌461000 [2]河南科技大学附属许昌市中心医院整形美容科,河南许昌461000 [3]上海市肺科医院胸外科,上海200433

出　　处：《中国修复重建外科杂志》2022年第10期1296-1304,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：2020年河南省部共建青年项目(SBGJ202003054)。

摘　　要：目的通过负载黄腐酚研发一种具有抗炎功能的聚乳酸-羟基乙酸[poly(lactic-co-glycolic acid),PLGA]支架,探讨其在羊体内抗炎及促进软骨再生的效果。方法取PLGA采用致孔剂浸出法制备多孔支架后,将其置于黄腐酚溶液24 h,制备黄腐酚-PLGA支架(以下简称“载药支架”)。取PLGA支架及载药支架以扫描电镜观测支架孔径、液体置换法计算孔隙率、傅里叶变换红外(Fourier transform infrared,FTIR)光谱仪验证支架上黄腐酚负载情况;并与经脂多糖炎症诱导处理的RAW264.7巨噬细胞共培养24 h,RT-PCR和Western blot检测炎症因子(IL-1β、TNF-α)表达,评价其体外抗炎性能。取成年山羊骨髓,采用贴壁法分离培养BMSCs并传代;取第2代细胞分别接种于两种支架构建BMSCs-支架复合物,通过活/死细胞染色及细胞计数试剂盒8(cell counting kit 8,CCK-8)观察支架细胞相容性。将BMSCs-支架复合物体外培养6周后,通过大体观察、组织学染色、Ⅱ型胶原免疫组织化学染色以及生化分析验证BMSCs-载药支架体外再生软骨的可行性。最后,将体外培养6周后的两种BMSCs-支架复合物分别植入6月龄健康雌性山羊皮下,4周后行大体观察、组织学染色、Ⅱ型胶原免疫组织化学染色、生化分析以及RT-PCR检测,综合评估载药支架的体内抗炎效果以及促进软骨再生情况。结果制备的载药支架为白色多孔结构,具有丰富、连续且均匀的孔隙结构,孔径及孔隙率与PLGA支架比较差异均无统计学意义(P>0.05);FTIR光谱仪检测示黄腐酚成功负载至PLGA支架。体外观测示,载药支架炎症因子(IL-1β、TNF-α)基因及蛋白相对表达量均低于PLGA支架(P<0.05);且随培养时间延长活细胞明显增多,各时间点与PLGA支架比较差异无统计学意义(P>0.05)。体外软骨再生评价显示,培养6周后,两种BMSCs-支架复合物呈光滑、半透明淡黄色,并且能基本维持培养前形状;组织学及免疫组织Objective To develop an anti-inflammatory poly(lactic-co-glycolic acid)(PLGA)scaffold by loading xanthohumol,and investigate its anti-inflammatory and cartilage regeneration effects in goats.Methods The PLGA porous scaffolds were prepared by pore-causing agent leaching method,and then placed in xanthohumol solution for 24hours to prepare xanthohumol-PLGA scaffolds(hereinafter referred to as drug-loaded scaffolds).The PLGA scaffolds and drug-loaded scaffolds were taken for general observation,the pore diameter of the scaffolds was measured by scanning electron microscope,the porosity was calculated by the drainage method,and the loading of xanthohumol on the scaffolds was verified by Fourier transform infrared(FTIR)spectrometer.Then the two scaffolds were co-cultured with RAW264.7macrophages induced by lipopolysaccharide for 24 hours,and the expressions of inflammatory factors[interleukin 1β(IL-1β)and tumor necrosis factorα(TNF-α)]were detected by RT-PCR and Western blot to evaluate the anti-inflammatory properties in vitro of two scaffolds.Bone marrow mesenchymal stem cells(BMSCs)was obtained from bone marrow of a6-month-old female healthy goat,cultured by adherent method,and passaged in vitro.The second passage cells were seeded on two scaffolds to construct BMSCs-scaffolds,and the cytocompatibility of scaffolds was observed by live/dead cell staining and cell counting kit 8(CCK-8)assay.The BMSCs-scaffolds were cultured in vitro for6 weeks,aiming to verify its feasibility of generating cartilage in vitro by gross observation,histological staining,collagen typeⅡimmunohistochemical staining,and biochemical analysis.Finally,the two kinds of BMSCs-scaffolds cultured in vitro for 6 weeks were implanted into the goat subcutaneously,respectively.After 4 weeks,gross observation,histological staining,collagen typeⅡimmunohistochemical staining,biochemical analysis,and RT-PCR were performed to comprehensively evaluate the anti-inflammatory effect in vivo and promotion of cartilage regeneration of the drug-loaded sc

关 键 词：软骨组织工程 黄腐酚 支架 抗炎 BMSCS 羊 

分 类 号：R318[医药卫生—生物医学工程]