国产多孔钽对MG63细胞中Ⅰ型胶原、谷氨酰转氨酶2和钙结合蛋白A4表达的影响
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摘要
背景:目前有证据已证实国产多孔钽具有良好的生物相容性及促成骨性能,但具体的成骨机制及对成骨因子的影响尚不明确。目的:观察国产多孔钽材料对MG63细胞Ⅰ型胶原、谷氨酰转氨酶2及钙结合蛋白A4表达的影响。方法:将对数生长期的MG63细胞接种于24孔板,分3组培养:空白组加入常规培养基,钽浸提液组加入多孔钽材料浸提液,钽支架组加入多孔钽材料与常规培养基。培养第1,3,5,7,9天,CCK-8法检测各组细胞增殖;培养第5天,采用ELISA酶联免疫吸附法检测各组细胞分泌Ⅰ型胶原、谷氨酰转氨酶2和钙结合蛋白A4的水平,Western-blot法检测各组细胞中钙结合蛋白A4、Ⅰ型胶原、谷氨酰转氨酶2蛋白的表达。结果与结论:(1)随着培养时间的延长,各组细胞数量呈逐渐增加趋势,3组不同时间点的细胞增殖比较无差异(P> 0.05);(2)钽支架组Ⅰ型胶原、谷氨酰转氨酶2分泌高于空白组、钽浸提液组(P <0.05),钽浸提液组Ⅰ型胶原、谷氨酰转氨酶2分泌高于空白组(P <0.05);钽支架组钙结合蛋白A4分泌低于其他两组(P <0.05);(3)钽支架组中Ⅰ型胶原、谷氨酰转氨酶2蛋白表达均高于空白组、钽浸提液组(P <0.05),钽浸提液组中Ⅰ型胶原、谷氨酰转氨酶2蛋白表达高于空白组(P <0.05);钽支架组钙结合蛋白A4蛋白表达均低于空白组、钽浸提液组(P <0.05);(4)结果表明,国产多孔钽材料可促进MG63细胞分泌Ⅰ型胶原、谷氨酰转氨酶2,抑制其分泌钙结合蛋白A4。
BACKGROUND: At present, there is evidence that domestic porous tantalum has good biocompatibility and osteogenic properties, but the specific osteogenic mechanism and its effect on osteogenic factors are still unclear. OBJECTIVE: To observe the effects of domestic porous tantalum materials on the expression of collagen type I, tissue transglutaminase-2 and calcium-binding protein A4 in MG63 cells. METHODS: MG63 cells in logarithmic growth phase were inoculated onto 24-well plates and cultured in three groups: in blank group, conventional medium was added; in tantalum extract group, porous tantalum material extract was added; and in tantalum scaffold group, porous tantalum material and conventional medium were added. On 1, 3, 5, 7 and 9 days of culture, the cell proliferation of each group was detected by cell counting kit-8 method. On 5 days of culture, the levels of collagen type I, tissue transglutaminase-2 and calcium-binding protein A4 secreted by MG63 cells in each group were detected by ELISA. Western blot assay was used to detect the expression of three proteins in each group. RESULTS AND CONCLUSION:(1) With the prolongation of culture time, the number of cells in each group increased gradually. There was no difference in cell proliferation among the three groups at different time points (P > 0.05).(2) The secretory levels of collagen type I and tissue transglutaminase-2 in the tantalum scaffold group were significantly higher than those in the blank group and tantalum extract group (P < 0.05), while the secretion of collagen type I and tissue transglutaminase-2 in the tantalum extract group was significantly higher than that in the blank group (P < 0.05). The secretion of calcium-binding protein A4 in the tantalum scaffold group was significantly lower than that in the other two groups (P < 0.05).(3) The expression of collagen type I and tissue transglutaminase-2 protein in the tantalum scaffold group was significantly higher than that in the blank group and tantalum extract group (P < 0.05), while the expression of collagen type I and tissue transglutaminase-2 protein in the tantalum extract group was significantly higher than that in the blank group (P < 0.05). The expression of calcium-binding protein A4 in the tantalum scaffold group was significantly lower than that in the blank group and tantalum extract group (P < 0.05). To conclude, domestic porous tantalum materials could promote the secretion of collagen type I and tissue transglutaminase-2 by MG63 cells, and inhibit the secretion of calcium-binding protein A4.
BACKGROUND: At present, there is evidence that domestic porous tantalum has good biocompatibility and osteogenic properties, but the specific osteogenic mechanism and its effect on osteogenic factors are still unclear. OBJECTIVE: To observe the effects of domestic porous tantalum materials on the expression of collagen type I, tissue transglutaminase-2 and calcium-binding protein A4 in MG63 cells. METHODS: MG63 cells in logarithmic growth phase were inoculated onto 24-well plates and cultured in three groups: in blank group, conventional medium was added; in tantalum extract group, porous tantalum material extract was added; and in tantalum scaffold group, porous tantalum material and conventional medium were added. On 1, 3, 5, 7 and 9 days of culture, the cell proliferation of each group was detected by cell counting kit-8 method. On 5 days of culture, the levels of collagen type I, tissue transglutaminase-2 and calcium-binding protein A4 secreted by MG63 cells in each group were detected by ELISA. Western blot assay was used to detect the expression of three proteins in each group. RESULTS AND CONCLUSION:(1) With the prolongation of culture time, the number of cells in each group increased gradually. There was no difference in cell proliferation among the three groups at different time points (P > 0.05).(2) The secretory levels of collagen type I and tissue transglutaminase-2 in the tantalum scaffold group were significantly higher than those in the blank group and tantalum extract group (P < 0.05), while the secretion of collagen type I and tissue transglutaminase-2 in the tantalum extract group was significantly higher than that in the blank group (P < 0.05). The secretion of calcium-binding protein A4 in the tantalum scaffold group was significantly lower than that in the other two groups (P < 0.05).(3) The expression of collagen type I and tissue transglutaminase-2 protein in the tantalum scaffold group was significantly higher than that in the blank group and tantalum extract group (P < 0.05), while the expression of collagen type I and tissue transglutaminase-2 protein in the tantalum extract group was significantly higher than that in the blank group (P < 0.05). The expression of calcium-binding protein A4 in the tantalum scaffold group was significantly lower than that in the blank group and tantalum extract group (P < 0.05). To conclude, domestic porous tantalum materials could promote the secretion of collagen type I and tissue transglutaminase-2 by MG63 cells, and inhibit the secretion of calcium-binding protein A4.
引文
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[16]Lee HJ,Chang HL.Transglutaminase-2 Is Involved in Expression of Osteoprotegerin in MG-63 Osteosarcoma Cells Biomol Ther(Seoul).2013;21(3):204-209.
[17]Zhou M,Li ZQ,Wang ZL.S100A4 upregulation suppresses tissue ossification and enhances matrix degradation in experimental periodontitis models.Acta Pharmacol Sin.2015;36(11):1388-1394.
[18]Mankani MH,Afghani S,Franco J,et al.Lamellar spacing in cuboid hydroxyapatite scaffolds regulates bone formation by human bone marrow stromal cells.Tissue Eng Part A.2011;17(11-12):1615-1623.
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[23]Lachiewicz PF,Bolognesi MP,Henderson RA,et al.Can tantalum cones provide fixation in complex revision knee arthroplasty?Clin Orthop Relat Res.2012;470(1):199-204.
[24]Hanzlik JA,Day JS.Bone ingrowth in well-fixed retrieved porous tantalum implants.J Arthroplasty.2013;28(6):922-927.
[25]Howard JL,Kudera J,Lewallen DG,et al.Early results of the use of tantalum femoral cones for revision total knee arthroplasty.J Bone Joint Surg Am.2011;93(5):478-484.
[26]刘洪臣,路荣建.新型多孔钽人工种植牙[J].中华老年口腔医学杂志,2016,14(1):41-44.
[27]Nakashima Y,Mashima N,Imai H,et al.Clinical and radiographic evaluation of total hip arthroplasties using porous tantalum modular acetabular components:5-year follow-up of clinical trial.Mod Rheumatol.2013;23(1):112-118.
[28]钟建鑫,节云峰,罗金英,等.多孔钽颗粒在下颌骨缺损修复中的作用[J].第三军医大学学报,2015,37(12):1277-1280.
[29]Aeschlimann D,Thomazy V.Protein crosslinking in assembly and remodelling of extracellular matrices:the role of transglutaminases.Connect Tissue Res.2000;41(1):1-27.
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[32]Fortunati D,Chau DY,Wang Z,et al.Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing.Amino Acids.2014;46(7):1751-1761.
[33]Strutz F,Okada H,Lo CW,et al.Identification and characterization of a fibroblast marker:FSP1.J Cell Biol.1995;130(2):393-405.
[34]Wang Z,Griffin M.The role of TG2 in regulating S100A4-mediated mammary tumour cell migration.Plos One.2013;8(3):e57017.
[2]Drosse I,Volkmer E,Capanna R,et al.Tissue engineering for bone defect healing:an update on a multi-component approach.Injury.2008;39(2):S9-S20.
[3]Laurencin C,Khan Y,Elamin SF.Bone graft substitutes.Skeletal Radiol.2007;36(12):1105-1107.
[4]邓子文,黄东.同种异体骨移植修复骨缺损的应用进展[J].山东医药,2017,57(32):98-100.
[5]Kato H,Nakamura T,Nishiguchi S,et al.Bonding of alkaliand heat-treated tantalum implants to bone.J Biomed Mater Res B Appl Biomater.2015;53(1):28-35.
[6]Bermúdez MD,Carrión FJ,Martínez-Nicolás G,et al.Erosion-corrosion of stainless steels,titanium,tantalum and zirconium.Wear.2005;258(1):693-700.
[7]Balla VK,Bodhak S,Bose S,et al.Porous tantalum structures for bone implants:fabrication,mechanical and in vitro biological properties.Acta Biomaterialia.2010;6(8):3349-3359.
[8]Hacking SA,Bobyn JD,Toh K,et al.Fibrous tissue ingrowth and attachment to porous tantalum.J Biomed Mater Res.2015;52(4):631-638.
[9]Matsuno H,Yokoyama A,Watari F,et al.Biocompatibility and osteogenesis of refractory metal implants,titanium,hafnium,niobium,tantalum and rhenium.Biomaterials.2001;22(11):1253-1262.
[10]Li X,Wang L,Yu X,et al.Tantalum coating on porous Ti6Al4Vscaffold using chemical vapor deposition and preliminary biological evaluation.Mater Sci Eng C Mater Biol Appl.2013;33(5):2987-2994.
[11]Wauthle R,Vand SJ,Amin YS,et al.Additively manufactured porous tantalum implants.Acta Biomaterialia.2015;14:217-225.
[12]王茜.国产多孔钽对成骨生物学效应影响机制的体外研究[D].广州:南方医科大学,2015.
[13]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,201721(18):2789-2795.
[14]沈小辉,杨华清,唐文娟,等.1α,25-二羟维生素D_3对成骨细胞增殖分化、I型胶原及核心结合因子α-1基因表达的影响[J].实用老年医学,2014,28(6):500-505.
[15]刘刚,胡蕴玉,廉凯,等.Ⅰ型胶原对骨髓基质干细胞粘附及分化的影响[J].中国骨与关节损伤杂志,2002,17(5):361-363.
[16]Lee HJ,Chang HL.Transglutaminase-2 Is Involved in Expression of Osteoprotegerin in MG-63 Osteosarcoma Cells Biomol Ther(Seoul).2013;21(3):204-209.
[17]Zhou M,Li ZQ,Wang ZL.S100A4 upregulation suppresses tissue ossification and enhances matrix degradation in experimental periodontitis models.Acta Pharmacol Sin.2015;36(11):1388-1394.
[18]Mankani MH,Afghani S,Franco J,et al.Lamellar spacing in cuboid hydroxyapatite scaffolds regulates bone formation by human bone marrow stromal cells.Tissue Eng Part A.2011;17(11-12):1615-1623.
[19]Cai F,Wu XT,Xie XH,et al.Evaluation of intervertebral disc regeneration with implantation of bone marrow mesenchymal stem cells(BMSCs)using quantitative T2 mapping:a study in rabbits.Int Orthop.2015;39(1):149-159.
[20]王茜,张辉,耿丽鑫,等.MG63细胞与国产多孔钽材料共培养后成骨相关因子的表达研究[J].中国修复重建外科杂志,2014,28(11):1422-1427.
[21]Zardiackas LD,Parsell DE,Dillon LD,et al.Structure,metallurgy,and mechanical properties of a porous tantalum foam.J Biomed Mater Res.2015;58(2):180-187.
[22]Shimko DA,Shimko VF,Sander EA,et al.Effect of porosity on the fluid flow characteristics and mechanical properties of tantalum scaffolds.J Biomed Mater Res B Appl Biomater.2005;73(2):315-324.
[23]Lachiewicz PF,Bolognesi MP,Henderson RA,et al.Can tantalum cones provide fixation in complex revision knee arthroplasty?Clin Orthop Relat Res.2012;470(1):199-204.
[24]Hanzlik JA,Day JS.Bone ingrowth in well-fixed retrieved porous tantalum implants.J Arthroplasty.2013;28(6):922-927.
[25]Howard JL,Kudera J,Lewallen DG,et al.Early results of the use of tantalum femoral cones for revision total knee arthroplasty.J Bone Joint Surg Am.2011;93(5):478-484.
[26]刘洪臣,路荣建.新型多孔钽人工种植牙[J].中华老年口腔医学杂志,2016,14(1):41-44.
[27]Nakashima Y,Mashima N,Imai H,et al.Clinical and radiographic evaluation of total hip arthroplasties using porous tantalum modular acetabular components:5-year follow-up of clinical trial.Mod Rheumatol.2013;23(1):112-118.
[28]钟建鑫,节云峰,罗金英,等.多孔钽颗粒在下颌骨缺损修复中的作用[J].第三军医大学学报,2015,37(12):1277-1280.
[29]Aeschlimann D,Thomazy V.Protein crosslinking in assembly and remodelling of extracellular matrices:the role of transglutaminases.Connect Tissue Res.2000;41(1):1-27.
[30]Gelse K,P?schl E,Aigner T.Collagens-structure,function,and biosynthesis.Adv Drug Deliv Rev.2003;55(12):1531-1546.
[31]卫爱林,叶小丰,刘世清,等.磷酸三钙/透明质酸/Ⅰ型胶原/骨髓基质胞复合物修复骨缺损与自体骨的比较[J].中国组织工程研究与临床康复,2007,11(9):1779-1782.
[32]Fortunati D,Chau DY,Wang Z,et al.Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing.Amino Acids.2014;46(7):1751-1761.
[33]Strutz F,Okada H,Lo CW,et al.Identification and characterization of a fibroblast marker:FSP1.J Cell Biol.1995;130(2):393-405.
[34]Wang Z,Griffin M.The role of TG2 in regulating S100A4-mediated mammary tumour cell migration.Plos One.2013;8(3):e57017.
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