载骨碎补总黄酮磷酸钙骨水泥对骨缺损模型大鼠诱导膜中成骨细胞分化的影响及其机制研究
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摘要
目的:探究载骨碎补总黄酮磷酸钙骨水泥对骨缺损模型大鼠诱导膜成骨分化的作用及其机制。方法:分别以磷酸钙骨水泥(CPC)、聚甲基丙烯酸甲酯(PMMA)骨水泥为载体,以强骨胶囊内容物(有效成分为骨碎补总黄酮)制备载药CPC和载药PMMA骨水泥。选取64只雄性SD大鼠,随机分为载药CPC组、载药PMMA骨水泥组、未载药CPC组、未载药PMMA骨水泥组,每组16只。分离大鼠股骨并行截骨,以制备骨缺损模型,然后分别植入相应骨水泥。造模4周后,各组大鼠切开并保护好诱导膜,取出骨水泥,植入自体松质骨。于造模后第4周对大鼠后肢骨进行X射线拍片;于造模后第4周和植骨后第6周时分别取大鼠骨缺损区诱导膜和新生骨,采用苏木精-伊红染色法观察诱导膜组织形态学,并测定新生骨组织的骨小梁宽度及成骨细胞数;采用免疫组化法检测诱导膜中人骨成型蛋白2(BMP-2)、血管内皮生长因子(VEGF)的蛋白表达水平;采用Western blotting法检测新生骨组织中Smad1、Smad4、Smad7的蛋白表达水平。结果:与其余3组比较,载药CPC组大鼠骨缺损区的骨水泥降解更明显,且可见诱导膜组织形成,骨缺损区域更小;诱导膜中毛细血管内皮细胞丰富、排列有序;新生骨组织中骨小梁宽度及成骨细胞数均显著增加(P<0.05);诱导膜中BMP-2、VEGF的蛋白表达水平和新生骨组织中Smad1、Smad4、Smad7的蛋白表达水平均显著升高(P<0.05)。结论:载骨碎补总黄酮CPC具有促进骨缺损模型大鼠诱导膜成骨的作用。这可能是通过激活BMP-2/Smad通路,从而调节成骨细胞分化;同时通过提高VEGF表达,促进血管内皮细胞分化,加快形成毛细血管网,从而促进骨愈合。
OBJECTIVE:To investigate the effects and its mechanism of calcium phosphate bone cement(CPC)loading total flavonoids of Davallia mariesii on osteogenic differentiation of induced membrane in rats. METHODS:Drug-loading CPC and drugloading polymethyl methacrylate(PMMA) cement were prepared with the contents of Qianggu capsules(total flavonoids of D.mariesii as active ingredient)using CPC and PMMA cement as carrier. Totally 64 male SD rats were randomly divided into drugloading CPC group,drug-loading PMMA cement group,no-drug CPC group,no-drug PMMA cement group,with 16 rats in each group. The femur of rats was separated and osteotomized to prepare bone defect model,and then the corresponding bone cement was implanted. Four weeks after modeling,the induced membranes of rats were cut and protected. Bone cement was taken out and autogenous cancellous bone was implanted. At the 4 th week after modeling,X-ray photographs were taken on the hind limb bones of rats. At the 4 th week after modeling and 6 th week after bone grafting,induced membranes and new bone were taken from the bone defect area of rats respectively. HE staining was used to observe the morphology of induced membrane,and the width of bone rabecular and the number of osteoblasts of new bone tissue were measured. Immunohistochemistry was used to detect the protein expression of BMP-2 and VEGF in induced membrane. Western blotting assay was used to detect the protein expression of Smad1,Smad4 and Smad7 in new bone. RESULTS:Compared with other 3 groups, the degradation of bone cement in drugloading CPC group was more obvious in the bone defect areas,which showed that the formation of induced membrane was observed and the bone defect areas were smaller;capillary endothelial cells were abundant and orderly arranged in the induced membranes,and the width of bone trabeculae and the number of osteoblasts in the new bone tissue increased significantly(P<0.05);the protein expression of BMP-2 and VEGF in the induced membrane,the protein expression of Smad1,Smad4 and Smad7 in new bone were increased significantly(P<0.05). CONCLUSIONS:CPC loading total flavonoids of D.mariesii promotes the formation of induced membrane osteoblast in bone defect model rats, which may be associated with regulating osteoblast differentiation by activating BMP-2/Smad pathway;at the same time,it can promote bone healing by promoting the differentiation of vascular endothelial cells,accelerating the formation of capillary network and increasing the expression of vascular endothelial cells.
OBJECTIVE:To investigate the effects and its mechanism of calcium phosphate bone cement(CPC)loading total flavonoids of Davallia mariesii on osteogenic differentiation of induced membrane in rats. METHODS:Drug-loading CPC and drugloading polymethyl methacrylate(PMMA) cement were prepared with the contents of Qianggu capsules(total flavonoids of D.mariesii as active ingredient)using CPC and PMMA cement as carrier. Totally 64 male SD rats were randomly divided into drugloading CPC group,drug-loading PMMA cement group,no-drug CPC group,no-drug PMMA cement group,with 16 rats in each group. The femur of rats was separated and osteotomized to prepare bone defect model,and then the corresponding bone cement was implanted. Four weeks after modeling,the induced membranes of rats were cut and protected. Bone cement was taken out and autogenous cancellous bone was implanted. At the 4 th week after modeling,X-ray photographs were taken on the hind limb bones of rats. At the 4 th week after modeling and 6 th week after bone grafting,induced membranes and new bone were taken from the bone defect area of rats respectively. HE staining was used to observe the morphology of induced membrane,and the width of bone rabecular and the number of osteoblasts of new bone tissue were measured. Immunohistochemistry was used to detect the protein expression of BMP-2 and VEGF in induced membrane. Western blotting assay was used to detect the protein expression of Smad1,Smad4 and Smad7 in new bone. RESULTS:Compared with other 3 groups, the degradation of bone cement in drugloading CPC group was more obvious in the bone defect areas,which showed that the formation of induced membrane was observed and the bone defect areas were smaller;capillary endothelial cells were abundant and orderly arranged in the induced membranes,and the width of bone trabeculae and the number of osteoblasts in the new bone tissue increased significantly(P<0.05);the protein expression of BMP-2 and VEGF in the induced membrane,the protein expression of Smad1,Smad4 and Smad7 in new bone were increased significantly(P<0.05). CONCLUSIONS:CPC loading total flavonoids of D.mariesii promotes the formation of induced membrane osteoblast in bone defect model rats, which may be associated with regulating osteoblast differentiation by activating BMP-2/Smad pathway;at the same time,it can promote bone healing by promoting the differentiation of vascular endothelial cells,accelerating the formation of capillary network and increasing the expression of vascular endothelial cells.
引文
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[15]姜晓鑫.载骨碎补磷酸钙骨水泥的研究[D].重庆:西南交通大学,2011.
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[17]王雷,王孝辉,张海龙,等.不同剂量骨碎补总黄酮对大鼠Masquelet技术诱导膜中BMP-2和VEGF表达的影响[J].中国中医骨伤科杂志,2018,26(4):1-4.
[18]QUITTET MS,TOUZANI O,SINDJI L,et al.Effects of mesenchymal stem cell therapy,in association with pharmacologically active microcarriers releasing VEGF,in an ischaemic stroke model in the rat[J].Acta Biomater,2015.DOI:10.1016/j.actbio.2014.12.017.
[19]KUAMR Y,BISWAS T,THACKER G,et al.BMP signaling driven osteogenesisis critically dependent on Prdx-1expression-mediated maintenance of chondrocyte prehypetrophy[J].Free Radic Biol Med,2018,2(16):2980-2993.
[20]周强,朱嘉绮,孙鑫,等.左归丸对去卵巢致绝经后骨质疏松症大鼠骨组织中BMP2、Smad1表达的影响[J].辽宁中医杂志,2016,43(12):2659-2661.
[21]高璐,郑洪新,陈谊敬,等.淫羊藿苷、补骨脂素、齐墩果酸、二苯乙烯苷正交配伍调控BMP2、Smad1、4诱导BM-SCs成骨分化的影响[J].世界科技技术:中医药现代化,2014,16(5):1094-1102.
[22]ABDOLLAH S,MACIAS-SILVA M,TSUKAZAKI T,et al.TβRl phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling[J].J Biol Chem,1997,272(44):27678-27685.
[23]莘晓陶,李曦光,毕贤峰,等.Smad7在兔下颌骨牵张成骨过程中的表达[J].中国组织工程研究与临床康复,2010,14(20):3633-3636.
[24]孙效棠,赵黎,胡蕴玉,等.磷酸钙骨水泥载药核心的块型重组合异种骨体内缓释及修复兔长段感染性骨缺损的研究[J].中国修复重建外科杂志,2005,19(3):165-169.
[2]DONEGAN DJ,SCOLARO J,MATUSZEWSKI PE,et al.Staged bone grafting following placement of an antibiotic spacer block for the management of segmental long bone defects[J].Orthopedics,2011,34(11):e730-e735.
[3]TAYLOR BC,FRENCH BG,FOWLER TT,et al.Induced membrane technique for reconstruction to manage bone loss[J].J Am Acad Orthop Surg,2012,20(3):142-150.
[4]梁佩清,全昌云,康婷,等.改性PMMA骨水泥的临床研究进展[J].功能材料,2017,48(2):2048-2054.
[5]宋炎成,裴福兴,万昌秀.磷酸钙骨水泥:骨骼系统理想的药物载体[J].中国临床康复,2006,10(41):117-120.
[6]LEE K,WEIR MD,LIPPENS E,et al.Bone regeneration via novel macroporous CPC scaffolds in critical-sized cranial defects in rats[J].Dent Mater,2014,30(7):e199-e207.
[7]CHENG SW,LIN ZQ,WANG W,et al.Osteogenic capability of autologous rabbit adipose-derived stromal cells in repairing calvarial defects[J].Chin J Traumatol,2011,14(5):288-292.
[8]杨洲,唐德志,姚长风,等.健腰密骨片对成骨细胞增殖分化和BMP信号通路报导基因活性的影响[J].上海中医药大学学报,2014,28(5):80-83.
[9]姜自伟,曾景奇,李悦,等.骨碎补总黄酮对大鼠胫骨牵张末期BMP-2与Smad-1表达的影响[J].辽宁中医杂志,2018,45(1):166-168、227.
[10]王泰龄,黄受方,李维华,等.《全国免疫组织化学技术与诊断标准化专题研讨会》会议纪要[J].中华病理学杂志,1996,25(6):326-328.
[11]叶向阳,甄平,李晓飞,等.磷酸钙骨水泥药物缓释体系的研究应用[J].中国组织工程研究与临床康复,2009,13(47):9317-9320.
[12]李娟莹,黄剑锋,曹丽云.磷酸钙骨水泥药物缓释载体的研究进展[J].陕西科技大学学报(自然科学版),2008,26(3):25-29.
[13]WOO KM,JUN JH,CHEN VJ,et al.Nano-fibrous scaffolding promotes osteoblast differentiation and biomineralization[J].Biomaterials,2007,28(2):335-343.
[14]李明,李君,付昆.骨碎补总黄酮对膝骨关节炎模型兔HIF-1α和VEGF表达的影响[J].中国药房,2018,29(18):2484-2488.
[15]姜晓鑫.载骨碎补磷酸钙骨水泥的研究[D].重庆:西南交通大学,2011.
[16]李晋玉,赵学千,孙旗,等.骨碎补总黄酮的实验及临床研究概况[J].中国骨质疏松杂志,2018,24(10):1357-1364.
[17]王雷,王孝辉,张海龙,等.不同剂量骨碎补总黄酮对大鼠Masquelet技术诱导膜中BMP-2和VEGF表达的影响[J].中国中医骨伤科杂志,2018,26(4):1-4.
[18]QUITTET MS,TOUZANI O,SINDJI L,et al.Effects of mesenchymal stem cell therapy,in association with pharmacologically active microcarriers releasing VEGF,in an ischaemic stroke model in the rat[J].Acta Biomater,2015.DOI:10.1016/j.actbio.2014.12.017.
[19]KUAMR Y,BISWAS T,THACKER G,et al.BMP signaling driven osteogenesisis critically dependent on Prdx-1expression-mediated maintenance of chondrocyte prehypetrophy[J].Free Radic Biol Med,2018,2(16):2980-2993.
[20]周强,朱嘉绮,孙鑫,等.左归丸对去卵巢致绝经后骨质疏松症大鼠骨组织中BMP2、Smad1表达的影响[J].辽宁中医杂志,2016,43(12):2659-2661.
[21]高璐,郑洪新,陈谊敬,等.淫羊藿苷、补骨脂素、齐墩果酸、二苯乙烯苷正交配伍调控BMP2、Smad1、4诱导BM-SCs成骨分化的影响[J].世界科技技术:中医药现代化,2014,16(5):1094-1102.
[22]ABDOLLAH S,MACIAS-SILVA M,TSUKAZAKI T,et al.TβRl phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling[J].J Biol Chem,1997,272(44):27678-27685.
[23]莘晓陶,李曦光,毕贤峰,等.Smad7在兔下颌骨牵张成骨过程中的表达[J].中国组织工程研究与临床康复,2010,14(20):3633-3636.
[24]孙效棠,赵黎,胡蕴玉,等.磷酸钙骨水泥载药核心的块型重组合异种骨体内缓释及修复兔长段感染性骨缺损的研究[J].中国修复重建外科杂志,2005,19(3):165-169.
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