Objective: To prepare a novel hyaluronic acid methacrylate (HAMA) hydrogel microspheres loaded polyhedral oligomeric silsesquioxane-diclofenac sodium (POSS-DS) patricles, then investigate its physicochemical characteristics and in vitro and in vivo biological properties.
Methods: Using sulfhydryl POSS (POSS-SH) as a nano-construction platform, polyethylene glycol and DS were chemically linked through the "click chemistry" method to construct functional nanoparticle POSS-DS. The composition was analyzed by nuclear magnetic resonance spectroscopy and the morphology was characterized by transmission electron microscopy. In order to achieve drug sustained release, POSS-DS was encapsulated in HAMA, and hybrid hydrogel microspheres were prepared by microfluidic technology, namely HAMA@POSS-DS. The morphology of the hybrid hydrogel microspheres was characterized by optical microscope and scanning electron microscope. The in vitro degradation and drug release efficiency were observed. Cell counting kit 8 (CCK-8) and live/dead staining were used to detect the effect on chondrocyte proliferation. Moreover, a chondrocyte inflammation model was constructed and cultured with HAMA@POSS-DS. The relevant inflammatory indicators, including collagen type Ⅱ, aggrecan (AGG), matrix metalloproteinase 13 (MMP-13), recombinant A disintegrin and metalloproteinase with thrombospondin 5 (Adamts5), and recombinant tachykinin precursor 1 (TAC1) were detected by immunofluorescence staining and real-time fluorescence quantitative PCR, with normal cultured chondrocytes and the chondrocyte inflammation model without treatment as control group and blank group respectively to further evaluate their anti-inflammatory activity. Finally, by constructing a rat model of knee osteoarthritis, the effectiveness of HAMA@POSS-DS on osteoarthritis was evaluated by X-ray film and Micro-CT examination.
Results: The overall particle size of POSS-DS nanoparticles was uniform with a diameter of about 100 nm. HAMA@POSS-DS hydrogel microspheres were opaque spheres with a diameter of about 100 μm and a spherical porous structure. The degradation period was 9 weeks, during which the loaded POSS-DS nanoparticles were slowly released. CCK-8 and live/dead staining showed no obvious cytotoxicity at HAMA@POSS-DS, and POSS-DS released by HAMA@POSS-DS significantly promoted cell proliferation (P<0.05). In the chondrocyte anti-inflammatory experiment, the relative expression of collagen type Ⅱ mRNA in HAMA@POSS-DS group was significantly higher than that in control group and blank group (P<0.05). The relative expression level of AGG mRNA was significantly higher than that of blank group (P<0.05). The relative expressions of MMP-13, Adamts5, and TAC1 mRNA in HAMA@POSS-DS group were significantly lower than those in blank group (P<0.05). In vivo experiments showed that the joint space width decreased after operation in rats with osteoarthritis, but HAMA@POSS-DS delayed the process of joint space narrowing and significantly improved the periarticular osteophytosis (P<0.05).
Conclusion: HAMA@POSS-DS can effectively regulate the local inflammatory microenvironment and significantly promote chondrocyte proliferation, which is conducive to promoting cartilage regeneration and repair in osteoarthritis.
目的: 制备负载低聚倍半硅氧烷-双氯芬酸钠（polyhedral oligomeric silsesquioxane-diclofenac sodium，POSS-DS）纳米颗粒的甲基丙烯酰透明质酸（hyaluronic acid methacrylate，HAMA）水凝胶微球，对其进行表征并探究体内外生物学特性。.
方法: 以八巯基POSS（sulfhydryl POSS，POSS-SH）为纳米构筑平台，采用 “点击化学”法将聚二乙醇和DS以化学键接枝其上，构建功能化纳米颗粒POSS-DS，并使用核磁共振波谱仪分析成分，透射电镜对形貌进行表征。为实现药物长期缓慢释放，将POSS-DS包载于HAMA中，通过微流控技术制备多功能水凝胶微球，即HAMA@POSS-DS；利用光镜及扫描电镜对其形态进行表征，观察体外降解及药物释放效率，采用细胞计数试剂盒8（cell counting kit 8，CCK-8）试剂盒及活/死染色法检测对软骨细胞增殖的影响；并构建软骨细胞炎症模型后用HAMA@POSS-DS进行处理，通过免疫荧光染色及实时荧光定量PCR检测相关炎症指标，即Ⅱ型胶原、聚集蛋白聚糖（aggrecan，AGG）、基质金属蛋白酶13（matrix metalloproteinase 13，MMP-13）、解聚蛋白样金属蛋白酶5（recombinant A disintegrin and metalloproteinase with thrombospondin 5，Adamts5）、速激肽1（recombinant tachykinin precursor 1，TAC1），以正常培养软骨细胞及未作处理的炎症模型分别作为对照组及空白组，进一步评估其抗炎性能。最后，通过构建大鼠膝关节骨关节炎模型，经X线片及Micro-CT检查，验证HAMA@POSS-DS对骨关节炎的治疗效果。.
结果: POSS-DS纳米颗粒总体粒径均一，约100 nm。HAMA@POSS-DS为不透明球体，粒径约100 μm，呈多孔结构；体外降解周期为9周，期间缓释负载的POSS-DS。CCK-8试剂盒及活/死染色法检测显示HAMA@POSS-DS无明显细胞毒性，且其释放的POSS-DS对细胞增殖有促进作用（P<0.05）。软骨细胞抗炎实验中，HAMA@POSS-DS组Ⅱ型胶原mRNA相对表达量高于对照组和空白组、AGG mRNA相对表达量高于空白组、MMP-13、Adamts5以及TAC1 mRNA相对表达量低于空白组，上述差异均有统计学意义（P<0.05）。体内实验显示术后大鼠骨关节炎关节间隙宽度减小，但HAMA@POSS-DS可延缓关节间隙变窄进程，并改善关节周围骨赘增生情况（P<0.05）。.
结论: HAMA@POSS-DS可有效改善局部炎症微环境，并显著促进软骨细胞增殖，有利于促进骨关节炎的软骨再生与修复。.
Keywords: Polyhedral oligomeric silsesquioxane; anti-inflammation; cartilage regeneration; hydrogel microsphere; osteoarthritis.