生物活性复合膜对大鼠牙周组织缺损的修复作用

Repair effect of complex biomembrane on periodontal tissue defect of rats

目的:联合应用胶原(Co)、聚乳酸/聚羟基乙酸共聚体(PLGA)、牙周膜细胞(PDLCs)和血小板胶(APG)制作生物活性复合膜,评价其对大鼠牙周组织缺损的修复效果。方法:合成厚度约0.5mm的PLGA膜,与PDLCs体外共培养3d。扫描电镜观察PLGA结构及PDLCs生长情况。选取1只成年雄性Wistar大鼠,抽取全血10mL制作富血小板血浆(PRP)1mL,加入含有1 000U·mL-1牛凝血酶的10%CaCl2溶液制成APG。应用酶联免疫吸附法测定全血和PRP中生长因子水平。将60只成年雄性Wistar大鼠随机分为5组,分别制作大鼠下颌牙周骨缺损模型(5mm×2mm,去除根面牙骨质)。各组按置入材料不同命名为对照组、PLGA+Co组、PLGA+APG+Co组、PLGA+PDLCs+Co组和PLGA+PDLCs+APG+Co组。术后2、4和6周每组分别处死4只大鼠,HE染色观察新生牙周骨量,应用Image-Pro Plus测量新生牙周骨面积。结果:PLGA膜平均孔径为(131.80±43.97)μm,孔隙率为60%;PDLCs能在PLGA膜上黏附生长、状态良好。PRP中血小板浓度高达全血的4倍以上,PRP上清液中生长因子水平高于血浆(P<0.05)。术后6周牙周新生骨量PLGA+PDLCs+APG+Co组分别高于PLGA+Co组、PLGA+APG+Co组和PLGA+PDLCs+Co组,差异有统计学意义(P<0.05);PLGA+APG+Co组和PLGA+PDLCs+Co组高于PLGA+Co组(P<0.05);PLGA+Co组、PLGA+APG+Co组、PLGA+PDLCs+Co组和PLGA+PDLCs+APG+Co组均高于对照组(P<0.05);PLGA+APG+Co组与PLGA+PDLCs+Co组间比较差异无统计学意义(P>0.05)。结论:PLGA膜生物相容性良好,孔径大小适宜,具有良好的机械性能;PLGA+PDLCs+APG+Co生物活性复合膜能促进牙周组织生长,对牙周骨组织缺损修复具有显著作用。

Objective To make complex biomembrane with collagen（Co）,polylactic acid/polyglycolic acid copolymer（PLGA）,periodontal ligament cells（PDLCs）and autologous platelet gel（APG）and to evaluate the repair effects on periodontal tissue defect of the rats,and to provide theoretical basis for their clinical application.Methods Synthetic PLGA,human PDLCs and PLGA were co-cultured in vitro for 3d,10mL whole blood of the rats was extracted to make platelet-rich plasma（PRP）,and the growth factor levels in activated PRP and whole blood were detected.10%CaCl2 including 1 000U·mL-1 bovine thrombin was added to make APG.60 Wistar adult male rats were randomly divided into 5groups,and the mandible alveolar bone defect models were established（5mm×2mm,root cementums were struck off）,the 5groups were separately named control group,PLGA＋Co group,PLGA＋APG＋Co group,PLGA＋PDLCs＋Co group,and PLGA＋PDLCs＋APG＋Co group for the stuff placed into mandible alveolar bones＇ defects.Then 4rats in each group were exeacted at the 2nd week,the 4th week and the 6th week after operation,and HE staining was used to observe the quantity of new born bones.Image-Pro Plus was used to obtain the areas of new bones,and SPSS 13.0 was used for statistics analysis.Results The average pore size of PLGA was（131.80±43.97）μm and the porosity was 60%.PDLCs stretched well on PLGA.The number of platelets in PRP were 4times higher than those in whole blood and the growth factor levels in supernatant were higher than those in blood plasma.The quantity of new bones at the 6th week after operation in PLGA＋PDLCs＋APG＋Co group was more than those in PLGA＋Co group,PLGA＋APG＋Co group and PLGA＋PDLCs＋Co group（P0.05）;the quantities of new bones in PLGA＋APG＋Co group and PLGA＋PDLCs＋Co group were more than that in PLGA＋Co group;the quantities of new bones in PLGA＋Co group,PLGA＋APG＋Co group,PLGA＋PDLCs＋Co group and PLGA＋PDLCs＋APG＋Co group were more than that in control group（P0.05）,but there was no significant difference between PLGA＋APG＋Co group and PLGA＋PDLCs＋Co group（P0.05）.Conclusion The pure size PLGA has excellent biocompatibility,suitable high porosity and good mechanical properties.PLGA＋PDLCs＋APG＋Co complex biomembrane plays significant role in promoting periodontal tissue growth.