可评价注射式再生支架修复效果的小鼠骨缺损模型的构建研究

Establishment of a mouse bone defect model to evaluate the repaireffect of injectable regenerative scaffolds

目的建立小鼠股骨缺损模型并探索甲基丙烯酰化明胶(gelatin methacryloyl,GelMA)对缺损后骨再生的影响。方法将40只8周龄的雌性C57BL/6N小鼠随机分成4组:骨缺损组(n=10),5%GelMA组(n=10),10%GelMA组(n=10),15%GelMA组(n=10)。造模2周后,使用HE染色观察股骨组织结构;使用Masson染色观察股骨胶原纤维形态;使用OCN免疫组化染色分析骨特异性蛋白表达水平。结果GelMA具有良好的可注射性,可经PCR Pipettes移液器注入骨缺损区。HE染色结果表明10%GelMA组相较于骨缺损组、5%GelMA组和15%GelMA组具有更好的骨修复效果,缺损部位组织结构更加完整;Masson染色结果表明10%GelMA组有更多成骨相关的胶原纤维形成;RT-qPCR分析显示,10%GelMA组小鼠骨缺损部位OCN表达显著高于骨缺损组(P<0.001)、5%GelMA组(P<0.01)和15%GelMA组(P<0.01)。10%GelMA组小鼠骨缺损部位Osterix表达显著高于骨缺损组(P<0.001)、5%GelMA组(P<0.001)和15%GelMA组(P<0.01);OCN免疫组化染色结果表明10%GelMA组的修复区骨特异性蛋白表达显著高于骨缺损组(P<0.01)、5%GelMA组(P<0.01)和15%GelMA组(P<0.05)。结论构建了可评价注射式再生支架修复效果的小鼠骨缺损模型,并应用于基于GelMA的可注射再生支架筛选和治疗效果评价,为开展相关领域工作提供实验基础。

Objective To establish a mouse femoral bone defect model and explore the effect of a GelMA hydrogel on bone regeneration and repair.Methods Forty 8-week-old female C57BL/6N mice were randomly divided into four groups:bone defect group(n=10),5%GelMA group(n=10),10%GelMA group(n=10),and 15%GelMA group(n=10).At 2 weeks after modeling,HE staining was used to observe the femur structure.Masson staining was used to observe the morphology of femoral collagen fibers.OCN immunohistochemical staining was used to analyze bone�specific protein expression levels.Results GelMA was well injectable and injected into bone defects using PCR pipettes.HE staining showed that the 10%GelMA group had a better bone repair effect than bone defect,5%GelMA,and 15%GelMA groups,and the tissue structure of the defect site was more complete.Masson staining showed more osteogenic collagen fibers in the 10%GelMA group.RT-qPCR analysis showed that OCN expression in the bone defect site in the 10%GelMA group was significantly higher than that in the bone defect group(P<0.001),5%GelMA group(P<0.01),and 15%GelMA group(P<0.01).Osterix expression in the bone defect site in the 10%GelMA group was significantly higher than that in the bone defect group(P<0.001),5%GelMA group(P<0.001),and 15%GelMA group(P<0.01);OCN immunohistochemical staining showed that bone-specific protein expression in the repair area in the 10%GelMA group was significantly higher than that in the bone defect group(P<0.01),5%GelMA group(P<0.01),and 15%GelMA group(P<0.05).Conclusions A mouse bone defect model to evaluate the repair effect of injectable regenerative scaffolds was established and applied to the screening and therapeutic effect evaluation of injectable regenerative scaffolds based on GelMA,which provides an experimental basis to carrying out studies in related fields.