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 24 hours 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 RAW264.7 macrophages induced by lipopolysaccharide for 24 hours, and the expressions of inflammatory factors [interleukin 1β (IL-1β) and tumor necrosis factor α (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 a 6-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 for 6 weeks, aiming to verify its feasibility of generating cartilage in vitro by gross observation, histological staining, collagen type Ⅱ immunohistochemical 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 type Ⅱ immunohistochemical 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 scaffolds.
Results: The prepared drug-loaded scaffold had a white porous structure with abundant, continuous, and uniform pore structures. Compared with the PLGA scaffold, there was no significant difference in pore size and porosity ( P>0.05). FTIR spectrometer analysis showed that xanthohumol was successfully loaded to PLGA scaffolds. The in vitro results demonstrated that the gene and protein expressions of inflammatory cytokines (IL-1β and TNF-α) in drug-loaded scaffold significantly decreased than those in PLGA scaffold ( P<0.05). With the prolongation of culture, the number of live cells increased significantly, and there was no significant difference between the two scaffolds ( P>0.05). The in vitro cartilage regeneration test indicated that the BMSCs-drug-loaded scaffolds displayed smooth and translucent appearance with yellow color after 6 weeks in vitro culture, and could basically maintained its original shape. The histological and immunohistochemical stainings revealed that the scaffolds displayed typical lacunar structure and cartilage-specific extracellular matrix. In addition, quantitative data revealed that the contents of glycosaminoglycan (GAG) and collagen type Ⅱ were not significantly different from BMSCs-PLGA scaffolds ( P>0.05). The evaluation of cartilage regeneration in vivo showed that the BMSCs-drug-loaded scaffolds basically maintained their pre-implantation shape and size at 4 weeks after implantation in goat, while the BMSCs-PLGA scaffolds were severely deformed. The BMSCs-drug-loaded scaffolds had typical cartilage lacuna structure and cartilage specific extracellular matrix, and no obvious inflammatory cells infiltration; while the BMSCs-PLGA scaffolds had a messy fibrous structure, showing obvious inflammatory response. The contents of cartilage-specific GAG and collagen type Ⅱ in BMSCs-drug-loaded scaffolds were significantly higher than those in BMSCs-PLGA scaffolds ( P<0.05); the relative gene expressions of IL-1β and TNF-α were significantly lower than those in BMSCs-PLGA scaffolds ( P<0.05).
Conclusion: The drug-loaded scaffolds have suitable pore size, porosity, cytocompatibility, and good anti-inflammatory properties, and can promote cartilage regeneration after implantation with BMSCs in goats.
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；组织学及免疫组织化学染色示支架中均出现典型的软骨陷窝结构和软骨特异性细胞外基质表达；软骨特异性糖胺聚糖（glycosaminoglycan，GAG）和Ⅱ型胶原含量与BMSCs-PLGA支架复合物差异均无统计学意义（ P>0.05）。体内软骨再生评价显示，植入羊体内4周后BMSCs-载药支架复合物基本维持植入前形状和大小，而BMSCs-PLGA支架复合物严重变形；BMSCs-载药支架复合物具有典型的软骨陷窝结构和软骨特异性细胞外基质，且未出现明显炎症细胞浸润；而BMSCs-PLGA支架复合物为杂乱的纤维状结构，可见明显炎症反应。BMSCs-载药支架复合物软骨特异性GAG和Ⅱ型胶原含量均明显高于BMSCs-PLGA支架复合物（ P<0.05）；IL-1β、TNF-α基因相对表达量均低于BMSCs-PLGA支架复合物（ P<0.05）。.
结论: 载药支架具有合适孔径、孔隙率、细胞相容性和良好抗炎性能，联合BMSCs植入羊体内后能促进软骨再生。.
Keywords: Cartilage tissue engineering; anti-inflammation; bone marrow mesenchymal stem cells; goat; scaffold; xanthohumol.