具有抗炎作用的载双氯芬酸钠明胶多孔支架促进体内软骨再生研究  

Research of diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity for promoting in vivo cartilage regeneration

作　　者：赵少华 菅炎鹏 王一公 徐勇[3] 刘伟杰 邵欣慰 樊骏 徐松山 ZHAO Shaohua;JIAN Yanpeng;WANG Yigong;XU Yong;LIU Weijie;SHAO Xinwei;FAN Jun;XU Songshan(Department of Plastic Surgery,Xuchang Central Hospital Affiliated to Henan University of Science and Technology,Xuchang Henan,461000,P.R.China;Department of Spine and Spinal Cord Surgery,Xuchang Central Hospital Afiliated to Henan University of Science and Technology,XuchangHenan,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整形美容科,河南许昌461000 [2]河南科技大学附属许昌市中心医院脊柱脊髓科,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》2023年第1期91-100,共10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 研发一种具有抗炎作用的载双氯芬酸钠明胶多孔支架,为缓解再生软骨组织植入体内后的炎症反应及促进体内软骨再生提供新的思路。方法 将双氯芬酸钠与明胶混匀,通过冻干法制备载双氯芬酸钠明胶多孔支架作为实验组,单纯明胶多孔支架作为对照组。观察支架大体形貌,扫描电镜观测支架孔径,排水法计算支架孔隙率,傅里叶变换红外光谱仪和X射线衍射仪检测双氯芬酸钠负载情况,体外缓释实验检测实验组双氯芬酸钠释放曲线。将两组支架材料与脂多糖预激活的RAW264.7巨噬细胞体外共培养后,行RT-PCR、ELISA、Western blot检测IL-1β和TNF-α的表达,评价支架体外抗炎性能。将新西兰大白兔第2代软骨细胞种植于两组支架上进行体外培养,活/死细胞染色及细胞计数试剂盒8法检测支架细胞相容性,行大体观察、HE染色、番红O染色、Ⅱ型胶原免疫组织化学染色及生化成分定量分析验证支架体外再生软骨的可行性。最后将两组软骨细胞-支架复合物植入新西兰大白兔皮下,4周后行大体观察、HE染色、番红O染色、Ⅱ型胶原免疫组织化学染色及生化成分定量分析验证体内软骨再生情况,以及RT-PCR法检测炎症相关基因CD3和CD68的表达,综合评估支架体内抗炎性能。结果 两组支架均具有相似的外观、微孔结构、孔径和孔隙率,差异无统计学意义(P>0.05);双氯芬酸钠作为药物分子成功分散于明胶支架中;体外抗炎结果显示,与单纯明胶多孔支架比较,载双氯芬酸钠明胶多孔支架IL-1β和TNF-α的mRNA和蛋白相对表达量明显降低(P<0.05)。体外软骨再生评价显示,随培养时间延长活细胞明显增多,各时间点两组支架比较差异无统计学意义(P>0.05);两组支架均再生出白色软骨样组织,组织学观察呈典型的软骨陷窝结构和特异性软骨细胞外基质分泌,软骨特异性糖胺聚糖(glycosaminoglycan,GAG)和Ⅱ型Objective To develop a diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity and provide a new avenue for alleviating the inflammatory response and enhancing cartilage regeneration in vivo.Methods Diclofenac sodium was homogeneously mixed with gelatin to prepare a diclofenac sodium-loaded porous gelatin scaffold by freeze-drying method as the experimental group, and a pristine porous gelatin scaffold was served as a control group. The general morphology of the scaffold was observed, the pore size of the scaffold was measured by scanning electron microscopy, the porosity of the scaffold was calculated by drainage method, the loading of diclofenac sodium into the gelatin scaffold was detected by fourier transform infrared spectrometer and X-ray diffraction examinations, and the release kinetics of diclofenac sodium from gelatin scaffold was tested using an in vitro release assay.The two scaffolds were co-cultured with lipopolysaccharide-predisposed RAW264.7 in vitro, and the expressions of interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) were detected by reverse transcription polymerase chain reaction(RT-PCR), enzyme-linked immuno sorbent assay, and Western blot, to detect the in vitro anti-inflammatory effect of the drug-loaded scaffold. Thereafter, the second generation chondrocytes of New Zealand white rabbits were inoculated on the two groups of scaffolds for in vitro culture, and the cytocompatibility of the scaffold was tested by live/dead staining and cell counting kit 8 assay, the feasibility of in vitro cartilage regeneration of the scaffold was evaluated via gross observation, HE staining, Safranin-O staining, and immunohistochemical collagen type Ⅱ staining, as well as biochemical quantitative analyses. Finally, the two groups of chondrocyte-scaffolds were implanted subcutaneously into New Zealand white rabbits, and after 4 weeks, the general observation, HE staining, safranin O staining,immunohistochemical collagen type Ⅱ staining, and biochemical quantitative analyse

关 键 词：双氯芬酸钠 明胶 抗炎 体内软骨再生 组织工程 支架 

分 类 号：R318.08[医药卫生—生物医学工程]