国产多孔钽对兔成骨细胞生物学行为及功能的影响
|
摘要
背景:国产多孔钽植入材料具备三维立体空间结构,而材料的三维立体结构可能会影响细胞的生长及分泌。目的:观察与国产多孔钽复合培养后成骨细胞増殖、细胞周期、成骨性细胞因子分泌功能的变化。方法:提取第3代兔成骨细胞,分3组培养:正常组常规培养,对照组加入多孔钽材料浸提液,实验组加入多孔钽支架材料。培养3,5,7 d,CCK-8法检测各组细胞增殖,扫描电镜观察实验组成骨细胞在多孔钽支架上的生长情况,Elisa试剂盒检测各组成骨细胞内骨钙素与Ⅰ型胶原的分泌;培养7 d时,流式细胞仪检测各组细胞周期。结果与结论:(1)细胞增殖:3组培养不同时间点的细胞增殖无差异;(2)扫描电镜:复合培养第3天,细胞在多孔钽支架表面和孔隙内黏附生长,排列较稀疏,突起较少;复合培养第5天,细胞边缘有突起向四周伸展,相邻细胞间突起相互连接横跨孔隙,连成片状;复合培养第7天,细胞突起融合汇合成片状,分泌的基质覆盖大部分支架表面;(3)Elisa检测:随着培养时间的延长,3组成骨细胞骨钙素分泌逐渐增加,Ⅰ型胶原分泌先升高后降低,实验组培养不同时间点的骨钙素分泌均高于正常组、对照组;(4)细胞周期:3组成骨细胞均是正常的二倍体细胞,无异倍体细胞出现,细胞DNA含量正常,3组细胞周期分布相似;(5)结果表明:多孔钽支架材料具有良好的生物相容性,可能促进成骨细胞的矿化与成骨。
BACKGROUND: The domestic porous tantalum implant material has a three-dimensional spatial structure that may affect the growth and secretion of cells. OBJECTIVE: To investigate the proliferation, cell cycle, osteogenic secretion of osteoblasts co-cultured with domestic porous tantalum scaffolds. METHODS: Passage 3 rabbit osteoblasts were extracted and divided into three groups: normal culture group, control group added with the porous tantalum extract, and experimental group co-cultured with the porous tantalum scaffold. The proliferation of cells in each group was detected by cell counting kit-8 method at 3, 5, 7 days of culture. The growth of osteoblasts on the porous tantalum scaffold was observed by scanning electron microscopy. ELISA kit assay was applied to detect the levels of osteocalcin and type Ⅰ collagen in osteoblasts at 3, 5, 7 days of culture. The cell circle of osteoblasts was detected by flow cytometry at 7 days of culture. RESULTS AND CONCLUSION:(1) Cell proliferation: There was no difference in cell proliferation among the three groups at different time points(P > 0.05).(2) Scanning electron microscopy: On the 3~(rd) day of compound culture, the cells adhered to the scaffold surface and pores, and arranged sparsely with few protrusions. On the 5~(th) day, the cells began to extend, and the adjacent cells were interconnected to form a flaky growth. On the 7~(th) day, the cells merged into pieces, and the secreted matrix covered most of the scaffold surface.(3) ELISA detection: With the cultivation time, the osteocalcin secretion from the osteoblasts was gradually increased in all the three groups, while the secretion of type Ⅰ collagen was increased first and then decreased. The secretion of osteocalcin and type Ⅰ collagen in the experimental group at different time points were higher than that in normal culture and control groups.(4) Cell cycle: The osteoblasts in the three groups were normal diploid cells, no aneuploid cells appeared, and the cell DNA content was normal. The cell cycle distribution was similar in the three groups. To conclude, the domestic porous tantalum scaffolds have good biocompatibility and may promote osteoblast mineralization and osteogenesis.
BACKGROUND: The domestic porous tantalum implant material has a three-dimensional spatial structure that may affect the growth and secretion of cells. OBJECTIVE: To investigate the proliferation, cell cycle, osteogenic secretion of osteoblasts co-cultured with domestic porous tantalum scaffolds. METHODS: Passage 3 rabbit osteoblasts were extracted and divided into three groups: normal culture group, control group added with the porous tantalum extract, and experimental group co-cultured with the porous tantalum scaffold. The proliferation of cells in each group was detected by cell counting kit-8 method at 3, 5, 7 days of culture. The growth of osteoblasts on the porous tantalum scaffold was observed by scanning electron microscopy. ELISA kit assay was applied to detect the levels of osteocalcin and type Ⅰ collagen in osteoblasts at 3, 5, 7 days of culture. The cell circle of osteoblasts was detected by flow cytometry at 7 days of culture. RESULTS AND CONCLUSION:(1) Cell proliferation: There was no difference in cell proliferation among the three groups at different time points(P > 0.05).(2) Scanning electron microscopy: On the 3~(rd) day of compound culture, the cells adhered to the scaffold surface and pores, and arranged sparsely with few protrusions. On the 5~(th) day, the cells began to extend, and the adjacent cells were interconnected to form a flaky growth. On the 7~(th) day, the cells merged into pieces, and the secreted matrix covered most of the scaffold surface.(3) ELISA detection: With the cultivation time, the osteocalcin secretion from the osteoblasts was gradually increased in all the three groups, while the secretion of type Ⅰ collagen was increased first and then decreased. The secretion of osteocalcin and type Ⅰ collagen in the experimental group at different time points were higher than that in normal culture and control groups.(4) Cell cycle: The osteoblasts in the three groups were normal diploid cells, no aneuploid cells appeared, and the cell DNA content was normal. The cell cycle distribution was similar in the three groups. To conclude, the domestic porous tantalum scaffolds have good biocompatibility and may promote osteoblast mineralization and osteogenesis.
引文
[1]杨化娟,杨柯,张炳春.医用不锈钢的发展及展望[J].材料导报,2005,19(6):56-59.
[2]Bermudez MD,Carrion FJ,Lopez R,et al.Erosion corrosion of stainless steels,titanium,tantalum and zirconium.Wear.2005;258(1-4):693-700.
[3]Zhang M,Wang GL,Zhang HF,et al.Repair of segmental long bone defect in a rabbit radius nonunion model:comparison of cylindrical porous titanium and hydroxyapatite scaffolds.Artif Organs.2014;38(6):493-502.
[4]于思荣.生物医学钛合金的研究现状及发展趋势[J].材料科学与工程,2000,18(2):131-134.
[5]Bertrand E,Gloriant T,Gordin DM,et al.Synthesis and characterization of a new superelastic Ti-25Ta-25Nb biomedical alloy.J Mech Behav Biomed.2010;3:559-564.
[6]赵晋,闫振宇,张立智.骨性关节炎软骨和软骨下骨之间信号通路[J].中华骨质疏松和骨矿盐疾病杂志,2016,9(2):193-198.
[7]单鹏程,曹永平.软骨下骨在骨关节炎发病机制中的作用[J].中国矫形外科杂志,2009,17(23):1792-1794.
[8]Hanzlik JA,Day JS,Clare M,et al.Is There A Difference in Bone Ingrowth in Modular Versus Monoblock Porous Tantalum Tibial Trays.J Arthroplasty.2015;30(6):1073-1078.
[9]Li X,Lin Z,Duan Y,et al.Repair of large segmental bone defects in rabbits using BMP and FGF composite xenogeneic bone.Genet Mol Res.2015;14(2):6395-6400.
[10]贺瑞,张文志,尚希福,等.钽金属椎间融合器在腰椎间盘突出症减压术后复发融合术中的应用[J].颈腰痛杂志,2013,34(5):387-390.
[11]刘永庆,李琪佳,王志强.多孔金属骨科内植物改良策略的研究进展[J].医学综述,2016,22(20):4021-4024.
[12]Wang H,Li Q,Wang Q,et al.Enhanced repair of segmental bone defects in rabbit radius by porous tantalum scaffolds modified with the RGD peptide.J Mater Sci Mater Med.2017;28:50.
[13]张大鹏,刘永庆,王志强.医用多孔钽的特性及其表面改性的研究进展[J].中国煤炭工业医学杂志,2017,20(2):236-239.
[14]于晓明,谭丽丽,杨柯.医用金属表面的钽涂层制备及其临床应用趋势[J].中国骨科临床与基础研究杂志,2013,5(2):115-119.
[15]赖振权,崔逸爽,陈超,等.新型骨植入材料多孔钽-骨结合界面成骨以及相关成骨因子的表达及意义[J].中国组织工程研究,2017,21(18):2789-2795.
[16]Sagomonyants KB,Hakim-Zargar M,Jhaveri A,et al.Porous tantalum stimulates the proliferation and osteogenesis of osteoblasts from elderly female patients.J Orthop Res.2011;29(4):609-616.
[17]Bobyn JD,Stackpool GJ,Hacking SA,et al.Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomatcrial.J Bone Joint Surg Br.1999;81(5):907-914.
[18]Wang Q,Zhang H,Li Q,et al.Biocompatibility and osteogenic properties of porous tantalum.Exp Ther Med.2015;9(3):780-786.
[19]王辉,王茜,张辉,等.带蒂筋膜瓣包裹国产多孔钽修复兔桡骨节段性骨缺损实验研究[J].中国修复重建外科杂志,2017,31(10):1200-1207.
[20]李琪佳,王茜,甘洪全,等.多孔钽材料细胞毒性、生物相容性及体内成骨性研究[J].中华骨科杂志,2014,34(9):954-961.
[21]Findlay DM,Welldon K,Atkins GJ,et al.The proliferation and phenotypic expression of human osteoblasts on tantalum metal.Biomaterials.2004;25(12):2215-2227.
[22]ANSI/AAMI.ISO 10993-5.Biological evaluation of medical devices Part5:Tests for cytotoxicity:in vitro methods[S].Arlington,VA.
[23]ANSI/AAMI.ISO 10993-12.Biological evaluation of medical devices Part 12:Sample preparation and reference materials[S].Arlington,VA.
[24]武垚森,池永龙.小梁金属在骨科中的应用[J].中国骨科杂志,2007,27(12):939-941.
[25]董伟,刘洪臣.钽及多孔钽表面改性技术在组织工程学及口腔医学的研究进展[J].中华老年口腔医学杂志,2017,15(2):113-116.
[26]欧阳建安,王大平.多孔钽骨科临床应用概况[J].国际骨科杂志,2011,32(5):314-315.
[27]武垚森,池永龙.小梁金属(多孔钽)在骨科的应用现状[J].中华骨科杂志,2007,27(12):939-941.
[28]初殿伟,王玮,张华亮,等.骨软骨组织工程支架的研究现状及发展趋势[J].国际生物医学工程杂志,2009,32(2):105-116.
[29]Guldberg RE,Duvall CL,Peister A,et al.3D imaging of tissue integration with porous biomaterials.Biomaterials.2008;29(28):3757-3761.
[30]张彦博,李瑞延,刘贯聪,等.多孔钽的理化/生物特性及其骨整合能力研究进展[J].生物骨科材料与临床研究,2017,14(5):7-11.
[31]Cardaropoli F,Alfieri V,Caiazzo F,et al.Manufacturing of porous biomaterials for dental implant applications through selective laser melting.Adv Mater Res.2012;6(535-537):1222-1229.
[32]Wan Y,Wang Y,Liu Z,et al.Adhesion and proliferation of OCT-1osteoblast-like cells on micro-and nano-scale topography structured poly(L-lactide).Biomaterials.2005;26(21):4453-4459.
[33]Mustafa K,Wroblewski J,Hultenby K,et al.Effect of titanium surface blasted with Ti02 particles on the initial attachment of cells derived from human mandibular bone.Clin Oral Implants Res.2000;11(2):116-128.
[34]Marelli B,Ghezzi CE,Barralet JE,et al.Three-dimensional mineralization of dense nanofibrillar collagen-bioglass hybrid scaffolds.Biomacromolecules.2010;11(6):1470-1479.
[35]张辉,李亮,王茜,等.骨形成蛋白-7对多孔钽-软骨细胞复合物分泌功能以及COL-II、AGG和Sox9基因表达的影响[J].北京大学学报(医学版),2015,47(1):216-222.
[36]王茜,张辉,耿丽鑫,等.国产多孔钽复合MG63细胞培养Col-1、OC和OPN蛋白表达及意义[J].中国矫形外科杂志,2015,23(5):441-449.
[37]史伟,甘洪全,王茜,等.不同程度低氧培养对多孔钽-人成骨细胞复合物细胞形态及COL-1、OC蛋白表达的影响[J].中国矫形外科杂志,2017,25(8):729-736.
[38]Pagani F,Francucci CM,Moro L.Markers of bone turnover:biochemical and clinical perspectives.J Endocrinol Invest.2005;28(Suppl 10):8-13.
[39]Lin Z,Solomon KL,Zhang X,et al.In vitro Evaluation of Natural Marine Sponge Collagen as a Scaffold for Bone Tissue Engineering.Int J Biol Sci.2011;7(7):968-977.
[40]Hauschka PV,Lian JB,Cole DEC,et al.Osteocalcin and matrix GLA protein:vitamin K-dependent proteins in bone.Physiol Rev.1989;69(3):990-1047.
[41]丁思阳,夏露,陈宁,等.微弧氧化纯钛表面对成骨细胞蛋白合成功能的影响[J].口腔医学研究,2012,28(2):125-128.
[2]Bermudez MD,Carrion FJ,Lopez R,et al.Erosion corrosion of stainless steels,titanium,tantalum and zirconium.Wear.2005;258(1-4):693-700.
[3]Zhang M,Wang GL,Zhang HF,et al.Repair of segmental long bone defect in a rabbit radius nonunion model:comparison of cylindrical porous titanium and hydroxyapatite scaffolds.Artif Organs.2014;38(6):493-502.
[4]于思荣.生物医学钛合金的研究现状及发展趋势[J].材料科学与工程,2000,18(2):131-134.
[5]Bertrand E,Gloriant T,Gordin DM,et al.Synthesis and characterization of a new superelastic Ti-25Ta-25Nb biomedical alloy.J Mech Behav Biomed.2010;3:559-564.
[6]赵晋,闫振宇,张立智.骨性关节炎软骨和软骨下骨之间信号通路[J].中华骨质疏松和骨矿盐疾病杂志,2016,9(2):193-198.
[7]单鹏程,曹永平.软骨下骨在骨关节炎发病机制中的作用[J].中国矫形外科杂志,2009,17(23):1792-1794.
[8]Hanzlik JA,Day JS,Clare M,et al.Is There A Difference in Bone Ingrowth in Modular Versus Monoblock Porous Tantalum Tibial Trays.J Arthroplasty.2015;30(6):1073-1078.
[9]Li X,Lin Z,Duan Y,et al.Repair of large segmental bone defects in rabbits using BMP and FGF composite xenogeneic bone.Genet Mol Res.2015;14(2):6395-6400.
[10]贺瑞,张文志,尚希福,等.钽金属椎间融合器在腰椎间盘突出症减压术后复发融合术中的应用[J].颈腰痛杂志,2013,34(5):387-390.
[11]刘永庆,李琪佳,王志强.多孔金属骨科内植物改良策略的研究进展[J].医学综述,2016,22(20):4021-4024.
[12]Wang H,Li Q,Wang Q,et al.Enhanced repair of segmental bone defects in rabbit radius by porous tantalum scaffolds modified with the RGD peptide.J Mater Sci Mater Med.2017;28:50.
[13]张大鹏,刘永庆,王志强.医用多孔钽的特性及其表面改性的研究进展[J].中国煤炭工业医学杂志,2017,20(2):236-239.
[14]于晓明,谭丽丽,杨柯.医用金属表面的钽涂层制备及其临床应用趋势[J].中国骨科临床与基础研究杂志,2013,5(2):115-119.
[15]赖振权,崔逸爽,陈超,等.新型骨植入材料多孔钽-骨结合界面成骨以及相关成骨因子的表达及意义[J].中国组织工程研究,2017,21(18):2789-2795.
[16]Sagomonyants KB,Hakim-Zargar M,Jhaveri A,et al.Porous tantalum stimulates the proliferation and osteogenesis of osteoblasts from elderly female patients.J Orthop Res.2011;29(4):609-616.
[17]Bobyn JD,Stackpool GJ,Hacking SA,et al.Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomatcrial.J Bone Joint Surg Br.1999;81(5):907-914.
[18]Wang Q,Zhang H,Li Q,et al.Biocompatibility and osteogenic properties of porous tantalum.Exp Ther Med.2015;9(3):780-786.
[19]王辉,王茜,张辉,等.带蒂筋膜瓣包裹国产多孔钽修复兔桡骨节段性骨缺损实验研究[J].中国修复重建外科杂志,2017,31(10):1200-1207.
[20]李琪佳,王茜,甘洪全,等.多孔钽材料细胞毒性、生物相容性及体内成骨性研究[J].中华骨科杂志,2014,34(9):954-961.
[21]Findlay DM,Welldon K,Atkins GJ,et al.The proliferation and phenotypic expression of human osteoblasts on tantalum metal.Biomaterials.2004;25(12):2215-2227.
[22]ANSI/AAMI.ISO 10993-5.Biological evaluation of medical devices Part5:Tests for cytotoxicity:in vitro methods[S].Arlington,VA.
[23]ANSI/AAMI.ISO 10993-12.Biological evaluation of medical devices Part 12:Sample preparation and reference materials[S].Arlington,VA.
[24]武垚森,池永龙.小梁金属在骨科中的应用[J].中国骨科杂志,2007,27(12):939-941.
[25]董伟,刘洪臣.钽及多孔钽表面改性技术在组织工程学及口腔医学的研究进展[J].中华老年口腔医学杂志,2017,15(2):113-116.
[26]欧阳建安,王大平.多孔钽骨科临床应用概况[J].国际骨科杂志,2011,32(5):314-315.
[27]武垚森,池永龙.小梁金属(多孔钽)在骨科的应用现状[J].中华骨科杂志,2007,27(12):939-941.
[28]初殿伟,王玮,张华亮,等.骨软骨组织工程支架的研究现状及发展趋势[J].国际生物医学工程杂志,2009,32(2):105-116.
[29]Guldberg RE,Duvall CL,Peister A,et al.3D imaging of tissue integration with porous biomaterials.Biomaterials.2008;29(28):3757-3761.
[30]张彦博,李瑞延,刘贯聪,等.多孔钽的理化/生物特性及其骨整合能力研究进展[J].生物骨科材料与临床研究,2017,14(5):7-11.
[31]Cardaropoli F,Alfieri V,Caiazzo F,et al.Manufacturing of porous biomaterials for dental implant applications through selective laser melting.Adv Mater Res.2012;6(535-537):1222-1229.
[32]Wan Y,Wang Y,Liu Z,et al.Adhesion and proliferation of OCT-1osteoblast-like cells on micro-and nano-scale topography structured poly(L-lactide).Biomaterials.2005;26(21):4453-4459.
[33]Mustafa K,Wroblewski J,Hultenby K,et al.Effect of titanium surface blasted with Ti02 particles on the initial attachment of cells derived from human mandibular bone.Clin Oral Implants Res.2000;11(2):116-128.
[34]Marelli B,Ghezzi CE,Barralet JE,et al.Three-dimensional mineralization of dense nanofibrillar collagen-bioglass hybrid scaffolds.Biomacromolecules.2010;11(6):1470-1479.
[35]张辉,李亮,王茜,等.骨形成蛋白-7对多孔钽-软骨细胞复合物分泌功能以及COL-II、AGG和Sox9基因表达的影响[J].北京大学学报(医学版),2015,47(1):216-222.
[36]王茜,张辉,耿丽鑫,等.国产多孔钽复合MG63细胞培养Col-1、OC和OPN蛋白表达及意义[J].中国矫形外科杂志,2015,23(5):441-449.
[37]史伟,甘洪全,王茜,等.不同程度低氧培养对多孔钽-人成骨细胞复合物细胞形态及COL-1、OC蛋白表达的影响[J].中国矫形外科杂志,2017,25(8):729-736.
[38]Pagani F,Francucci CM,Moro L.Markers of bone turnover:biochemical and clinical perspectives.J Endocrinol Invest.2005;28(Suppl 10):8-13.
[39]Lin Z,Solomon KL,Zhang X,et al.In vitro Evaluation of Natural Marine Sponge Collagen as a Scaffold for Bone Tissue Engineering.Int J Biol Sci.2011;7(7):968-977.
[40]Hauschka PV,Lian JB,Cole DEC,et al.Osteocalcin and matrix GLA protein:vitamin K-dependent proteins in bone.Physiol Rev.1989;69(3):990-1047.
[41]丁思阳,夏露,陈宁,等.微弧氧化纯钛表面对成骨细胞蛋白合成功能的影响[J].口腔医学研究,2012,28(2):125-128.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |