新型三元复合材料纳米羟基磷灰石/聚酰胺66/氧化锆的制备及体外生物相容性
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摘要
背景:纳米羟基磷灰石/聚酰胺66复合材料具有高仿生特性,可通过与宿主骨直接结合来发挥生物活性作用,但其缺乏足够的力学强度。目的:制备新型三元复合材料纳米羟基磷灰石/聚酰胺66/氧化锆(nano-hydroxyapatite/polyamide66/yttria-stabilized tetragonal zirconia,nH A/PA66/YTZ),验证其力学特性及体外生物相容性。方法:采用两步法制备三元复合材料n HA/PA66/YTZ,其中纳米羟基磷灰石与氧化锆的质量比分别100∶0、90∶10、80∶20、60∶40。扫描电镜观察复合材料表征,力学测试仪测试其抗弯强度、抗张强度、抗压强度、弹性模量、断裂伸长率等力学参数,评价其力学性能,筛选最佳质量比复合材料,用于以下实验。分别采用细胞培养液(空白对照组)、纳米羟基磷灰石/聚酰胺66材料浸提液(对照组)、nHA/PA66/YTZ材料浸提液(实验组)培养小鼠成骨细胞MC3T3-E1,CCK-8法检测细胞增殖;将纳米羟基磷灰石/聚酰胺66材料(对照组)、nHA/PA66/YTZ材料(实验组)分别与小鼠成骨细胞MC3T3-E1共培养,24h后采用激光共聚焦显微镜观察MC3T3-E1细胞在复合材料表面的黏附、增殖情况。结果与结论:(1)扫描电镜显示,氧化锆晶粒填充了原本纳米羟基磷灰石晶粒之间的空隙,纳米羟基磷灰石/氧化锆均匀分散在聚酰胺66基体中;(2)生物力学测试显示,纳米羟基磷灰石与氧化锆质量比为60∶40nHA/PA66/YTZ材料的抗压强度、抗弯强度、抗张强度、断裂伸长率及弹性模量最高,力学性能最优,选择其进行细胞相容性实验;(3)CCK-8检测显示,随着时间的延长,3组细胞数量逐渐增加,3组间细胞增殖比较无差异;(4)激光共聚焦显微镜显示,实验组复合材料上的细胞呈现融合、团聚及分层现象,细胞内肌动蛋白丝更多;对照组复合材料上的细胞呈现单层及分散现象,细胞数量与细胞内的肌动蛋白丝较实验组少;(5)结果表明,三元复合材料nH A/PA66/YTZ在体外实验中表现出良好的力学性能、生物安全性及生物相容性。
BACKGROUND: Nano-hydroxyapatite/polyamide 66(nHA/PA66) composite materials possess high bionic properties and exert biological activity by directly combining with host bone, but it lacks sufficient mechanical strength. OBJECTIVE: To prepare a novel ternary biomaterial composed of nHA/PA66/yttria-stabilized tetragonal zirconia(YTZ), and to investigate its mechanical properties and biocompatibility. METHODS: The biomaterial was prepared by two-step approach, and the mass ratio of nano-hydroxyapatite to yttria-stabilized tetragonal zirconia was 100:0, 90:10, 80:20, and 60:40, respectively. The characterization of nHA/PA66/YTZ was observed by scanning electron microscope. The mechanical parameters of nHA/PA66/YTZ including bending strength, tensile strength, compressive strength, elastic modulus and breaking elongation were tested to evaluate its mechanical properties. The mechanical properties were evaluated by the mechanical tester to select composite materials with the optimum mass ratio applied to the following experiments. The MC3 T3-E1 cells were cultured by the cell-culture medium(blank control group), nHA/PA66 material extract(control group), nHA/PA66/YTZ material extract(experimental group). The cell proliferation was detected by cell counting kit-8 assay. The nHA/PA66 material(control group) and nHA/PA66/YTZ material(experimental group) were respectively co-cultured with MC3 T3-E1 cells. The adhesion and proliferation of MC3 T3-E1 cells on the surface of composite materials were observed by the laser scanning confocal microscope after 24 hours. RESULTS AND CONCLUSION: The scanning electron microscope showed that YTZ grains filled the gaps between the original nano-hydroxyapatite grains and the nHA/YTZ was evenly dispersed in the matrix of polyamide 66. The biomechanical test revealed that the compressive strength, bending strength, tensile strength, elongation at break and elastic modulus were the highest at 60:40 of the mass ratio of nHA and YTZ, while the mechanical properties were optimal, which could be chosen for the cell compatibility experiments. Cell counting kit-8 assay showed that there was no significant difference in the cell proliferation among groups as the time expended and the number of cells in each group increased. The laser scanning confocal microscope displayed that the cells showed fusion, agglomeration and stratification and more actin filaments in themselves on nHA/PA66/YTZ composite material. The cells had monolayer and dispersion on nHA/PA66 composite material. The number of cells and actin filaments in cells were less than those in the group of nHA/PA66/YTZ. These results showed that the ternary composite materials of nHA/PA66/YTZ exhibit good mechanical properties, biological safety, and biocompatibility in vitro experiment.
BACKGROUND: Nano-hydroxyapatite/polyamide 66(nHA/PA66) composite materials possess high bionic properties and exert biological activity by directly combining with host bone, but it lacks sufficient mechanical strength. OBJECTIVE: To prepare a novel ternary biomaterial composed of nHA/PA66/yttria-stabilized tetragonal zirconia(YTZ), and to investigate its mechanical properties and biocompatibility. METHODS: The biomaterial was prepared by two-step approach, and the mass ratio of nano-hydroxyapatite to yttria-stabilized tetragonal zirconia was 100:0, 90:10, 80:20, and 60:40, respectively. The characterization of nHA/PA66/YTZ was observed by scanning electron microscope. The mechanical parameters of nHA/PA66/YTZ including bending strength, tensile strength, compressive strength, elastic modulus and breaking elongation were tested to evaluate its mechanical properties. The mechanical properties were evaluated by the mechanical tester to select composite materials with the optimum mass ratio applied to the following experiments. The MC3 T3-E1 cells were cultured by the cell-culture medium(blank control group), nHA/PA66 material extract(control group), nHA/PA66/YTZ material extract(experimental group). The cell proliferation was detected by cell counting kit-8 assay. The nHA/PA66 material(control group) and nHA/PA66/YTZ material(experimental group) were respectively co-cultured with MC3 T3-E1 cells. The adhesion and proliferation of MC3 T3-E1 cells on the surface of composite materials were observed by the laser scanning confocal microscope after 24 hours. RESULTS AND CONCLUSION: The scanning electron microscope showed that YTZ grains filled the gaps between the original nano-hydroxyapatite grains and the nHA/YTZ was evenly dispersed in the matrix of polyamide 66. The biomechanical test revealed that the compressive strength, bending strength, tensile strength, elongation at break and elastic modulus were the highest at 60:40 of the mass ratio of nHA and YTZ, while the mechanical properties were optimal, which could be chosen for the cell compatibility experiments. Cell counting kit-8 assay showed that there was no significant difference in the cell proliferation among groups as the time expended and the number of cells in each group increased. The laser scanning confocal microscope displayed that the cells showed fusion, agglomeration and stratification and more actin filaments in themselves on nHA/PA66/YTZ composite material. The cells had monolayer and dispersion on nHA/PA66 composite material. The number of cells and actin filaments in cells were less than those in the group of nHA/PA66/YTZ. These results showed that the ternary composite materials of nHA/PA66/YTZ exhibit good mechanical properties, biological safety, and biocompatibility in vitro experiment.
引文
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[25]Walker J,Shadanbaz S,Woodfield TB,et al.Magnesium biomaterials for orthopedic application:a review from a biological perspective.J Biomed Mater Res B Appl Biomater.2014;102(6):1316-1331.
[26]Ding Y,Tian R,Yang Z,et al.Effects of serum albumin on the degradation and cytotoxicity of single-walled carbon nanotubes.Biophys Chem.2017;222:1-6.
[27]Bojar W,Ciach T,Kucharska M,et al.Cytotoxicity Evaluation and Crystallochemical Analysis of a Novel and Commercially Available Bone Substitute Material.Adv Clin Exp Med.2015;24(3):511-516.
[28]Li H,Gong M,Yang A,et al.Degradable biocomposite of nano calcium-deficient hydroxyapatite-multi(amino acid)copolymer.Int J Nanomedicine.2012;7:1287-1295.
[29]Liu X,Shen H,Song S,et al.Accelerated biomineralization of graphene oxide-incorporated cellulose acetate nanofibrous scaffolds for mesenchymal stem cell osteogenesis Colloids Surf BBiointerfaces.2017;159:251-258.
[30]Zhang X,Zhang Y,Zhang X,et al.Mechanical properties and cytocompatibility of carbon fibre reinforced nano-hydroxyapatite/polyamide66 ternary biocomposite.J Mech Behav Biomed Mater.2015;42:267-273.
[31]Anselme K.Biomaterials and interface with bone.Osteoporos Int.2011;22(6):2037-2042.
[2]Shokrollahi P,Mirzadeh H,Scherman OA,et al.Biological and mechanical properties of novel composites based on supramolecular polycaprolactone and functionalized hydroxyapatite.J Biomed Mater Res A.2010;95(1):209-221.
[3]Zhang X,Zhang Y,Zhang X,et al.Mechanical properties and cytocompatibility of carbon fibre reinforced nano-hydroxyapatite/polyamide66 ternary biocomposite.J Mech Behav Biomed Mater.2015;42:267-273.
[4]Li J,Man Y,Zuo Y,et al.In Vitro and In Vivo Evaluation of a nHA/PA66 Composite Membrane for Guided Bone Regeneration.J Biomater Sci Polym Ed.2011;22(1-3):263-275.
[5]Xiong Y,Ren C,Zhang B,et al.Analyzing the behavior of a porous nano-hydroxyapatite/polyamide 66(n-HA/PA66)composite for healing of bone defects.Int J Nanomedicine.2014;9:485-494.
[6]Zhang Y,Deng X,Jiang D,et al.Long-term results of anterior cervical corpectomy and fusion with nano-hydroxyapatite/polyamide 66 strut for cervical spondylotic myelopathy.Sci Rep.2016;6:26751.
[7]Yang P,Bian C,Huang X,et al.Core decompression in combination with nano-hydroxyapatite/polyamide 66 rod for the treatment of osteonecrosis of the femoral head.Arch Orthop Trauma Surg.2014;134(1):103-112.
[8]Yang X,Song Y,Liu L,et al.Anterior reconstruction with nano-hydroxyapatite/polyamide-66 cage after thoracic and lumbar corpectomy.Orthopedics.2012;35(1):e66-73.
[9]Zhao Z,Jiang D,Ou Y,et al.A hollow cylindrical nano-hydroxyapatite/polyamide composite strut for cervical reconstruction after cervical corpectomy.J Clin Neurosci.2012;19(4):536-540.
[10]Im SM,Huh YH,Cho LR,et al.Comparison of the fracture resistances of glass fiber mesh-and metal mesh-reinforced maxillary complete denture under dynamic fatigue loading.J Adv Prosthodont.2017;9(1):22-30.
[11]Lewicki JP,Rodriguez JN,Zhu C,et al.3D-Printing of Meso-structurally Ordered Carbon Fiber/Polymer Composites with Unprecedented Orthotropic Physical Properties.Sci Rep.2017;7:43401.
[12]Manicone PF,Rossi Iommetti P,Raffaelli L.An overview of zirconia ceramics:basic properties and clinical applications.JDent.2007;35(11):819-826.
[13]Bergschmidt P,Bader R,Mittelmeier W.Metal hypersensitivity in total knee arthroplasty:revision surgery using a ceramic femoral component-a case report.Knee.2012;19(2):144-147.
[14]Bergschmidt P,Bader R,Ganzer D,et al.Ceramic femoral components in total knee arthroplasty-two years follow-up results of an international prospective multicentre study.Open Orthop J.2012;6:172-178.
[15]Sumitomo N,Noritake K,Hattori T,et al.Experiment study on fracture fixation with low rigidity titanium alloy:plate fixation of tibia fracture model in rabbit.J Mater Sci Mater Med.2008;19(4):1581-1586.
[16]Morwood MP,Garrigues GE.Shoulder arthroplasty in the patient with metal hypersensitivity.J Shoulder Elbow Surg.2015;24(7):1156-1164.
[17]Yao MZ,Huang-Fu MY,Liu HN,et al.Fabrication and characterization of drug-loaded nano-hydroxyapatite/polyamide66 scaffolds modified with carbon nanotubes and silk fibroin.Int JNanomedicine.2016;11:6181-6194.
[18]Liao J,Zhang Y,Guan X,et al.Synthesis and characterization of nano-hydroxyapatite/polyamide 66 biocomposites reinforced with multi-walled carbon nanotubes.J Biomater Sci Polym Ed.2016;27(16):1674-1684.
[19]You F,Li Y,Zuo Y,et al.The influence ofγ-ray irradiation on the mechanical and thermal behaviors of nHA/PA66 composite scaffolds.ScientificWorldJournal.2013;2013:162384.
[20]Asgharzadeh Shirazi H,Ayatollahi MR,Asnafi A,et al.To reduce the maximum stress and the stress shielding effect around a dental implant-bone interface using radial functionally graded biomaterials.Comput Methods Biomech Biomed Engin.2017;13:1-10.
[21]Gyorgyey A,Ungvari K,Kecskemeti G,et al.Attachment and proliferation of human osteoblast-like cells(MG-63)on laser-ablated titanium implant material.Mater Sci Eng C Mater Biol Appl.2013;33(7):4251-4259.
[22]Ma R,Tang S,Tan H,et al.Preparation,characterization,and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as orthopedic implant material.Int J Nanomedicine.2014;9:3949-3961.
[23]Meyer U,Buchter A,Wiesmann HP,et al.Basic reactions of osteoblasts on structured material surfaces.Eur Cell Mater.2005;9:39-49.
[24]Kilian KA,Bugarija B,Lahn BT,et al.Geometric cues for directing the differentiation of mesenchymal stem cells.Proc Natl Acad Sci USA.2010;107(11):4872-4877.
[25]Walker J,Shadanbaz S,Woodfield TB,et al.Magnesium biomaterials for orthopedic application:a review from a biological perspective.J Biomed Mater Res B Appl Biomater.2014;102(6):1316-1331.
[26]Ding Y,Tian R,Yang Z,et al.Effects of serum albumin on the degradation and cytotoxicity of single-walled carbon nanotubes.Biophys Chem.2017;222:1-6.
[27]Bojar W,Ciach T,Kucharska M,et al.Cytotoxicity Evaluation and Crystallochemical Analysis of a Novel and Commercially Available Bone Substitute Material.Adv Clin Exp Med.2015;24(3):511-516.
[28]Li H,Gong M,Yang A,et al.Degradable biocomposite of nano calcium-deficient hydroxyapatite-multi(amino acid)copolymer.Int J Nanomedicine.2012;7:1287-1295.
[29]Liu X,Shen H,Song S,et al.Accelerated biomineralization of graphene oxide-incorporated cellulose acetate nanofibrous scaffolds for mesenchymal stem cell osteogenesis Colloids Surf BBiointerfaces.2017;159:251-258.
[30]Zhang X,Zhang Y,Zhang X,et al.Mechanical properties and cytocompatibility of carbon fibre reinforced nano-hydroxyapatite/polyamide66 ternary biocomposite.J Mech Behav Biomed Mater.2015;42:267-273.
[31]Anselme K.Biomaterials and interface with bone.Osteoporos Int.2011;22(6):2037-2042.