两种可吸收胶原膜小鼠皮下植入后降解速率及对宿主细胞功能影响的比较研究

Comparative study on the degradation rate and regulatory effects of two resorbable collagen membranes during the in vivo implantation

目的比较脱细胞猪小肠黏膜下层可吸收生物膜(SIS膜)及猪胶原可吸收生物膜(Bio-Gide膜)植入小鼠皮下后的降解速率及对周围宿主细胞的调控效果,探讨SIS膜和Bio-Gide膜在性能和促修复效果上的差异。方法体内研究采用6~8周龄雄性C57BL/6小鼠,通过小鼠皮下植入SIS膜和Bio-Gide膜后不同时间点(1、3、5、7、14及28 d)取材(每个时间点每组3只)进行组织学HE染色,分析比较两种膜的降解速率,通过免疫荧光检测F4/80及CD31表达评估两种膜对局部巨噬细胞及新生血管的影响,通过免疫组化检测Ki67及CD146评估两种膜对干细胞的动员效果。体外研究将小鼠牙周膜干细胞与两种膜材料共培养后,利用扫描电镜观察细胞形态和黏附情况,用实时荧光定量PCR(RT-qPCR)探讨两组细胞的增殖指标Ki67、趋化因子Cxcl1、Ccl1、炎症因子Tnfa的基因表达情况。结果小鼠体内研究表明,两种膜植入后28 d时,SIS膜降解率[(16.84±4.00)%]与Bio-Gide膜降解率[(24.07±3.97)%]差异无统计学意义(P=0.090),两种膜材料均能保持局部屏障效果。植入早期(3 d)两种膜材料周围的F4/80阳性巨噬细胞浸润数量[SIS膜组:(20.67±5.69)个/视野,Bio-Gide膜组:(25.33±2.52)个/视野]差异无统计学意义(P=0.292),但SIS膜相较Bio-Gide膜可显著促进局部组织中血管再生[CD31阳性细胞数量:SIS膜组为(4.67±1.15)个/视野,Bio-Gide膜组为(1.00±1.00)个/视野](P=0.015)及CD146阳性干细胞募集[阳性细胞数量:SIS膜组为(22.33±4.16)个/视野,Bio-Gide膜组为(11.33±2.52)个/视野](P=0.025)。体外RT-qPCR结果显示,与Bio-Gide膜相比,SIS膜可显著促进小鼠牙周膜干细胞的趋化因子基因Cxcl1的表达(P<0.001),但并不影响炎症因子基因Tnfa的表达(P=0.885)。结论SIS膜在体内外实验中与Bio-Gide膜降解速率相当,两种膜材料对植入局部的炎症反应及巨噬细胞的影响未见显著差异,均能满足引导性组织再生术的屏障效果要求。

ObjectiveTo explore the differences in the performance and tissue repair promotion effects of small intestinal submucosa membrane(SIS membrane)and Bio-Gide resorbable collagen membrane(Bio-Gide membrane)by performing the subcutaneous implantation models in mice.MethodsFor in vivo studies,we stablished membrane implantation models using 6-8 week-old male C57BL/6 mice.The degradation rates were explored through HE staining analysis at different time points(1,3,5,7,14 and 28 d,3 mice/group/time point).The influences of the two membranes on local macrophages and neovasculum were evaluated by immunofluorescence detection of F4/80 and CD31,and the mobilization effects of the two membranes on local stem cells were evaluated by immunohistochemical detection of Ki67 and CD146.For in vitro studies,mice periodontal ligament stem cells(mPDLSCs)were co-cultured with these two membrane materials,and the cell morphologies were observed by scanning electron microscopy.In addition,the gene expressions of Ki67,Cxcl1,Ccl1,Tnfa were investigated by real-time fluorescence quantitative PCR(RT-qPCR).ResultsThe results of in vivo studies showed that by day 28,there was no significant difference in degradation rate between these two membrane materials[SIS degradation rate:(16.84±4.00)%,Bio-Gide degradation rate:(24.07±3.97)%,P=0.090],illustrating that both of them could maintain the barrier effects for more than one month.In addition,there was no significant difference in the infiltration number of local F4/80 positive macrophages between these two groups by the day 3 after implantation[SIS:(20.67±5.69)cells/visual field,Bio-Gide:(25.33±2.52)cells/visual field,P=0.292].However,compared with the Bio-Gide membrane,SIS membrane significantly promoted local CD31+vascular regeneration[SIS:(4.67±1.15)cells/visual field,Bio-Gide:(1.00±1.00)cells/visual field,P=0.015]and CD146+stem cell recruitment[SIS:(22.33±4.16)cells/visual field,Bio-Gide:(11.33±2.52)cells/visual field,P=0.025].The RT-qPCR results also showed that SIS membrane promoted the gene expression of Cxcl1(SIS vs Bio-Gide P<0.001)in mPDLSCs,but had no effect on the gene expression of Tnfa(SIS vs Bio-Gide P=0.885).ConclusionsSIS membrane showed a similar degradation rate compared with Bio-Gide membrane,and there was no significant difference in the effects of these two membranes on local inflammation or macrophages.Therefore,both of these membranes could meet the barrier effects required by guided tissue regeneration.