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型胶原含量差异均无统计学意义(P>0.05)。体内实验表明,实验组样本呈瓷白色软骨样形貌,组织学染色呈明显的软骨陷窝结构和软骨特异性细胞外基质,GAG和Ⅱ型胶原含量明显高于对照组,CD3和CD68的蛋白和mRNA相对表达量明显低于对照组,差异均有统计学意义(P<0.05)。结论 载双氯芬酸钠明胶多孔支架具有合适的孔径、孔隙率、细胞相容性和良好抗炎性能,为缓解再生软骨组织植入体内后的炎症反应并促进体内软骨再生提供了可靠方案。
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 analyses were performed to verify the cartilage regeneration in vivo, and the expression of inflammation-related genes CD3 and CD68 was detected by RT-PCR to comprehensively evaluate the anti-inflammatory performance of the scaffolds in vivo. Results The two scaffolds exhibited similar gross, microporous structure, pore size, and porosity, showing no significant difference(P>0.05).Diclofenac sodium was successfully loaded into gelatin scaffold. Data from in vitro anti-inflammatory assay suggested that diclofenac sodium-loaded gelatin scaffold showed alleviated gene and protein expressions of IL-1β and TNF-α when compared with gelatin scaffold(P<0.05). The evaluation of cartilage regeneration in vitro showed that the number of living cells increased significantly with the extension of culture time, and there was no significant difference between the two groups at each time point(P>0.05). White cartilage-like tissue was regenerated from the scaffolds in both groups,histological observation showed typical cartilage lacuna structure and specific cartilage extracellular matrix secretion.There was no significant difference in the content of cartilage-specific glycosaminoglycan(GAG) and collagen type Ⅱbetween the two groups(P>0.05). In vivo experiments showed that the samples in the experimental group had porcelain white cartilage like morphology, histologic staining showed obvious cartilage lacuna structure and cartilage specific extracellular matrix, the contents of GAG and collagen type Ⅱ were significantly higher than those in the control group,and the protein and mRNA expressions of CD3 and CD68 were significantly lower than those in the control group, with significant differences(P<0.05). Conclusion The diclofenac sodium-loaded gelatin scaffold presents suitable pore size,porosity, and cytocompatibility, as well as exhibited satisfactory anti-inflammatory ability, providing a reliable scheme for alleviating the inflammatory reaction of regenerated cartilage tissue after in vivo implantation and promoting cartilage regeneration in vivo.
作者
赵少华
菅炎鹏
王一公
徐勇
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)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
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)。
关键词
双氯芬酸钠
明胶
抗炎
体内软骨再生
组织工程
支架
Diclofenac sodium
gelatin
anti-inflammatory
in vivo cartilage regeneration
tissue engineering
scaffold
