Objective To investigate the effect of local delivery of delta 12-prostaglandinJ2-loaded poly(lactic-co-glycolic acid) (△ 12-PGJ22-NC) on growth factors expression and bone formation.Methods △ 12-PGJ2-NC was prepared by the emulsion solvent diffusion method.The physical and chemical properties of the nanoparticles were evaluated by particle size analysis,transmission electron microscopy,drug-loading ratio and the in vitro release study.Then standardized transcortical defect(5.0 mm × 1.5 mm) was conducted in the femur of 48 male Wistar rats which were randomly divided into four groups(n=12),S,K,F,and N.Thirty microliter of saline(S),unloaded nanoparticles(K),△ 12-PGJ2(F) and △ 12-PGJ2-NC(N) in a collagen vehicle were delivered inside a titanium chamber fixed over the defect.Then,four subgroups were randomly divided in each group named as D3,D7,D14,and D28(n=3) according to the days 3,7,14,and 28 after the surgery.At days 3,7,14,and 28,the mRNA expression of the bone morphogenetic protein-6(BMP-6),platelet-derived growth factor-B(PDGF-B) in defect aera was analyzed by real time quantitive-polymerase chain reaction(qRT-PCR).The protein expression of the BMP-6 and Ephrin-B2 was evaluated by Western blotting.HE staining was employed to reveal new bone formation in weeks 2 and 4.Results △ 12-PGJ2-NC appeared opalescent white and remained relatively stable,with an average particle size of (135.2±0.85) nm.The images from transmission electron microscopy showed that △ 12-PGJ2-NC was spherical in shape and homogeneously distributed.The encapsulation efficiency of △ 12-PGJ2 with the poly(lactic-co-glycolic acid) (PLGA) nanocapsules was about 92％.The in vitro release of △ 12-PGJ2-NC at 37 ℃ showed a sustained fashion and the average accumulated amount was 30％,52％,77％,91％,and 98％ respectively,at 0.5,1,2,4 and 6 h.Compared with the animals treated with saline,after dose of 100 mg/L △ 12-PGJ2 and △ 12-PGJ2-NC apllication,the mRNA expression level of BMP-6,PDGF-B increased significantly(P＜0.05,P＜ 0.001).The protein expression of BMP-6,Ephrin-B2 also was up-regulated.Histomorphometry revealed that new bone formation increased at the same dose of 100 mg/L.But the unloaded nanoparticles did not have the same effect(P＞0.05).Conclusions A stable△ 12-PGJ2 loaded nanoparticle was successfully prepared.△ 12-PGJ2-NC may upregulate the expression of BMP-6,PDGF-B and Ephrin-B2,and promote new bone formation in bone defect area.%目的 探讨delta12-前列腺素J2(△12-prostaglandin J2,△12-PGJ2)以聚乳酸-羟基乙酸共聚物为载体的纳米粒[△12-PGJ2-loaded poly(lactic-co-glycolic acid),△12-PGJ2-NC]对骨生长因子表达及骨再生的影响,为其应用于牙周病治疗提供依据.方法 用乳化溶剂蒸发法制备△12-PGJ2-NC,通过粒径分析、透射电镜观察、包封率测定及体外释放试验评价△12-PGJ2-NC的理化性质;将48只Wistar雄性大鼠按随机数字表法随机分为4组,在大鼠双后肢股骨中段制备5.0 mm×1.5 mm骨皮质缺损,以可吸收性胶原海绵为载体分别将30μl生理盐水(S组)、空白聚乳酸-羟基乙酸共聚物[poly (lactic-coglycolic acid),PLGA]纳米粒(K组)、△12-PGJ2(F组)和△12-PGJ2-NC(N组)分别作用于骨缺损区,每组12只.建模结束后3、7、14、28 d收集左侧缺损区骨组织(每个时间点3只),提取mRNA和蛋白质,通过实时定量PCR法检测各组骨形成蛋白6(bone morphogenetic protein-6,BMP-6)、血小板源性生长因子B(platelet-derived growth factor-B,PDGF-B)mRNA表达量;用蛋白质印迹法检测BMP-6、Ephrin-B2的蛋白表达量;收集右侧缺损区骨组织并行HE染色,半定量分析术后14、28 d的新生骨量.结果 △12-PGJ2-NC呈乳光白色的均质悬浮液,测得粒径为(135.24±0.85)nm,透射电镜下呈圆形,分布较均匀,无大量聚集现象,载药率高达92％.在0.5、1、2、4及6h时,△12-PGJ2-NC的平均累计释放量分别为30％、52％、77％、91％和98％.动物实验实时定量PCR结果显示,与S组相比,N组缺损区骨组织中BMP-6、PGDF-B的mRNA表达显著增高(P＜0.05或P＜0.01);蛋白质印迹法结果显示,与S、K、F组相比,N组缺损区骨组织中BMP-6和Ephrin-B2的蛋白表达水平显著增高;HE染色结果显示,N组缺损区新骨形成量也显著升高,差异均有统计学意义;K组与S组相比,实时定量PCR、蛋白质印迹、HE染色结果差异均无统计学意义(P＞0.05).结论 成功制备了性质稳定的△12-PGJ2-NC;△12-PGJ2-NC可以上调局部骨缺损区BMP-6、PGDF-B和Ephrin-B2的基因和(或)蛋白表达水平,促进骨缺损修复.
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