负载蛋白聚糖4的温度敏感性聚己内酯-聚乙二醇-聚己内酯可注射水凝胶对软骨修复的影响

Effect of proteoglycan 4-loaded thermosensitive poly(caprolactone)-b-poly(ethylene glycol)-b-poly(caprolactone)injectable hydrogel on cartilage repair

目的探讨负载蛋白聚糖4(PRG4)的温度敏感性聚己内酯-聚乙二醇-聚己内酯共聚物(PCEC)可注射水凝胶材料表征及修复新西兰兔软骨缺损的效果。方法2022年3月至2023年6月,利用PCEC温敏性水凝胶负载了PRG4效应因子(PRG4@PCEC),使用扫描电子显微镜观察冷冻干燥的PRG4@PCEC水凝胶的形貌,测量PCEC和PRG4@PCEC水凝胶的最低共溶温度;使用活/死细胞染色技术评估水凝胶的生物安全性;采用雄性新西兰大白兔18只,36个膝关节缺损样本,随机分为3组,其中12个缺损组用PRG4@PCEC水凝胶修复,12个缺损组用PCEC水凝胶修复,12个缺损组为空白对照组,观察水凝胶的软骨修复能力;通过微型计算机断层扫描(Micro-CT)分析软骨下骨再生,标本组织切片苏木精-伊红(HE)、马松(Masson)染色及荧光定量聚合酶链反应(PCR)评估水凝胶对软骨缺损的修复作用。组间比较采用t检验。结果水凝胶具有明显的多孔微观结构,PCEC和PRG4@PCEC水凝胶分别在35℃和38℃表现出最低共溶温度;激光共聚焦观察活/死细胞染色结果证明PRG4@PCEC水凝胶对于兔软骨细胞具有良好的生物相容性;载有PRG4的水凝胶组在8周时,缺损已经完全被高含量的新生软骨组织填充,表明软骨缺损修复最佳;Micro-CT显示在8周时PCEC组[(37.33±0.39)mm^(3)]和PRG4@PCEC组[(25.33±0.21)mm^(3)]软骨下骨缺损体积均小于对照组[(51.57±0.49)mm^(3),t=32.19、69.56,P<0.01];切片染色显示PRG4@PCEC水凝胶组软骨下骨有效形成;荧光定量PCR结果显示在4周和8周PCEC组(2.79±0.07、2.18±0.10)与PRG4@PCEC组白细胞介素-1β(IL-1β)表达水平(1.54±0.07、0.80±0.10)均低于对照组(3.21±0.08、2.54±0.07,t=0.68、4.97、27.63、24.43,P<0.01),同时PCEC组(0.95±0.24、1.45±0.34)和PRG4@PCEC组Ⅱ型胶原蛋白(COL2)基因表达水平(1.19±0.45、1.53±0.26)高于对照组(0.64±0.07、1.01±0.14,t=7.47、0.08、10.70、3.12,P<0.05)。结论负载PRG4的温度敏感性PCEC可注射水凝胶具有良好的温度敏感性和生物安全性,对关节软骨缺损有明显修复作用。

Objective To investigate the characterization and repair effect of proteoglycan 4(PRG4)-loaded temperature-sensitive oly(caprolactone)-b-poly(ethylene glycol)-b-poly(caprolactone)(PCEC)injectable hydrogel material in cartilage defects of New Zealand rabbits.Methods From March 2022 to June 2023,the PCEC temperature-sensitive hydrogel loaded with PRG4 effector was used in this study.The morphology of freeze-dried PRG4@PCEC hydrogel was observed by scanning electron microscopy,the minimum co-solubility temperature of PCEC and PRG4@PCEC hydrogel was measured.Live/dead cell staining was used to evaluate the biological safety of the hydrogel.Eighteen male New Zealand white rabbits with 36 knee joint defects were randomly divided into three groups,12 defect groups were repaired with PRG4@PCEC hydrogel,12 defect groups were repaired with PCEC hydrogel,and 12 defect groups were blank control group.The cartilage repair ability of hydrogel was observed.Subchondral bone regeneration was analyzed by micro-computed tomography(Micro-CT).Tissue sections of the specimens were stained with hematoxylin and eosin(HE)and Masson staining and fluorescence quantitative polymerase chain reaction(PCR)to evaluate the repair effect of the hydrogel on cartilage defects.The t-test was used to compare the measurement data.Results The hydrogel had obvious porous microstructure.PCEC and PRG4@PCEC hydrogels showed the lowest co-solubility temperature at 35℃and 38℃;Laser confocal observation of live/dead cell staining showed that PRG4@PCEC hydrogel had good biocompatibility for rabbit chondrocytes.In the PRG4-loaded hydrogel group,the defect was completely filled with a high content of new cartilage tissue at 8 weeks,indicating that the cartilage defect was repaired optimally.Micro-CT showed that the subchondral bone defect volume in the PCEC group[(37.33±0.39)mm^(3)]and PRG4@PCEC group[(25.33±0.21)mm^(3)]was smaller than that in the control group[(51.57±0.49)mm^(3),t=32.19,69.56,P<0.01]at 8 weeks.Section staining showed that the subchondral bone was effectively formed in the PRG4@PCEC hydrogel group.The results of real-time PCR showed that the expression levels of IL-1βin PCEC group(2.79±0.07,2.18±0.10)and PRG4@PCEC group(1.54±0.07,0.80±0.10)were lower than those in control group(3.21±0.08,2.54±0.07,t=0.68,4.97,27.63,24.43,P<0.01),PCEC group(0.95±0.24,1.45±0.34)and PRG4@PCEC group(1.19±0.45,1.53±0.26)The expression level of COL2 gene in the control group(0.64±0.07,1.01±0.14)was significantly higher than that in the control group(t=7.47,0.08,10.70,3.12,P<0.05).Conclusion PRG4-loaded thermosensitive PCEC injectable hydrogel has good temperature sensitivity and biological safety,and has obvious repair effect on articular cartilage defects.