羟基磷灰石复合二氧化锆泡沫陶瓷的理化性能与细胞化性能
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摘要
背景:研究已证实羟基磷灰石/二氧化锆泡沫陶瓷材料具有良好的促骨再生能力,但关于其遗传毒性还未见报道。目的:检测羟基磷灰石/二氧化锆泡沫陶瓷材料的孔隙率与抗压强度,以及遗传毒性与细胞毒性,探索人工诱导多能干细胞来源间充质干细胞在羟基磷灰石/二氧化锆泡沫陶瓷材料上的成骨分化能力。方法:在添加造孔剂的情况下,采用高温烧结法制备羟基磷灰石/二氧化锆泡沫陶瓷材料,检测其孔隙率与抗压强度。采用鼠伤寒沙门氏菌回复突变实验检测羟基磷灰石/二氧化锆泡沫陶瓷材料的遗传毒性。分别采用羟基磷灰石/二氧化锆泡沫陶瓷材料浸提液(实验组)、苯酚溶液(阳性对照组)、生理盐水(阴性对照组)培养L929细胞,24h后,采用CCK-8法检测细胞毒性。将人工诱导多能干细胞来源间充质干细胞接种于羟基磷灰石/二氧化锆泡沫陶瓷材料上,培养第3,7,10,14天,检测细胞碱性磷酸酶分泌量;培养第14天,免疫组织化学法检测复合体细胞Ⅰ型胶原表达,扫描电镜观察细胞黏附情况。结果与结论:①羟基磷灰石/二氧化锆泡沫陶瓷材料的孔隙率为(76.72±0.75)%,抗压强度为(11.60±1.35) MPa;②鼠伤寒沙门氏菌回复突变实验显示,羟基磷灰石/二氧化锆泡沫陶瓷材料的代谢产物无致突变、无致癌作用;③CCK-8实验显示,实验组的细胞存活率与阴性对照组比较差异无显著性意义(P> 0.05),但明显高于阳性对照组(P <0.05);④随着培养时间的延长,细胞碱性磷酸酶分泌量逐渐增加,各时间点间两两比较差异均有显著性意义(P <0.05);⑤培养第14天,大量人工诱导多能干细胞来源间充质干细胞均匀分布于羟基磷灰石/二氧化锆泡沫陶瓷材料表面,细胞形态及生长良好,并且Ⅰ型胶原表达较多;⑥结果表明,羟基磷灰石/二氧化锆泡沫陶瓷材料具备一定的抗压能力,无致突变作用和细胞毒性,可促进人工诱导多能干细胞来源间充质干细胞的成骨分化。
BACKGROUND: Studies have confirmed that hydroxyapatite/zirconium dioxide foam ceramic materials have the ability to promote bone regeneration, but their genotoxicity has not been reported. OBJECTIVE: To detect the porosity, compressive strength, genotoxicity and cytotoxicity of hydroxyapatite/zirconium dioxide foam ceramics and to explore the osteogenic differentiation of artificially induced pluripotent stem cells cultured on the hydroxyapatite/zirconium dioxide foam ceramics. METHODS: The hydroxyapatite/zirconium dioxide foam ceramics was prepared by high temperature sintering method with addition of pore-forming agent. The porosity and compressive strength of hydroxyapatite/zirconium dioxide foam ceramics were detected, and the genotoxicity of the material were tested using Salmonella typhimurium reverse mutation test. L929 cells were cultured with hydroxyapatite/zirconium dioxide foam ceramics extract(experimental group), phenol solution(positive control group), and normal saline(negative control group). After 24 hours of culture, the cytotoxicity was detected using cell counting kit-8 toxicity test. Artificially induced pluripotent stem cells-derived mesenchymal stem cells were inoculated onto the hydroxyapatite/zirconium dioxide foam ceramics. On the 3~(rd), 7~(th), 10~(th) and 14~(th) days of culture, the amount of alkaline phosphatase secreted by the cells was detected. On the 14~(th) day of culture, the expression of type I collagen in the cells was detected by immunohistochemistry, and cell adhesion was observed by scanning electron microscope. RESULTS AND CONCLUSION:(1) The porosity and compressive strength of hydroxyapatite/zirconium dioxide foam ceramics were(76.72±0.75)% and(11.60±1.35) MPa, respectively.(2) In the Salmonella typhimurium reverse mutation test, the metabolites of hydroxyapatite/zirconium dioxide foam ceramics were not mutagenic or carcinogenic.(3) In the cell counting kit8 test, the survival rate of the cells in the experimental group was close to that in the negative control group(P > 0.05), but still significantly higher than that in the positive control group(P < 0.05).(4) With the extension of culture time, the secretion amount of alkaline phosphatase was increased gradually, and there was significant difference at different observational time points(P < 0.05).(5) On the 14~(th) day of culture, a great amount of artificially induced pluripotent stem cells-derived mesenchymal stem cells evenly distributed and grew well on the surface of hydroxyapatite/zirconium dioxide foam ceramics, and these cells had good morphology and highly expressed type I collagen. To conclude, the hydroxyapatite/zirconium dioxide foam ceramics has a certain compressive capacity and does not exhibit mutagenic effects and cytotoxicity, and promote the osteogenic differentiation of artificially induced pluripotent stem cells-derived mesenchymal stem cells cultured on its surface.
BACKGROUND: Studies have confirmed that hydroxyapatite/zirconium dioxide foam ceramic materials have the ability to promote bone regeneration, but their genotoxicity has not been reported. OBJECTIVE: To detect the porosity, compressive strength, genotoxicity and cytotoxicity of hydroxyapatite/zirconium dioxide foam ceramics and to explore the osteogenic differentiation of artificially induced pluripotent stem cells cultured on the hydroxyapatite/zirconium dioxide foam ceramics. METHODS: The hydroxyapatite/zirconium dioxide foam ceramics was prepared by high temperature sintering method with addition of pore-forming agent. The porosity and compressive strength of hydroxyapatite/zirconium dioxide foam ceramics were detected, and the genotoxicity of the material were tested using Salmonella typhimurium reverse mutation test. L929 cells were cultured with hydroxyapatite/zirconium dioxide foam ceramics extract(experimental group), phenol solution(positive control group), and normal saline(negative control group). After 24 hours of culture, the cytotoxicity was detected using cell counting kit-8 toxicity test. Artificially induced pluripotent stem cells-derived mesenchymal stem cells were inoculated onto the hydroxyapatite/zirconium dioxide foam ceramics. On the 3~(rd), 7~(th), 10~(th) and 14~(th) days of culture, the amount of alkaline phosphatase secreted by the cells was detected. On the 14~(th) day of culture, the expression of type I collagen in the cells was detected by immunohistochemistry, and cell adhesion was observed by scanning electron microscope. RESULTS AND CONCLUSION:(1) The porosity and compressive strength of hydroxyapatite/zirconium dioxide foam ceramics were(76.72±0.75)% and(11.60±1.35) MPa, respectively.(2) In the Salmonella typhimurium reverse mutation test, the metabolites of hydroxyapatite/zirconium dioxide foam ceramics were not mutagenic or carcinogenic.(3) In the cell counting kit8 test, the survival rate of the cells in the experimental group was close to that in the negative control group(P > 0.05), but still significantly higher than that in the positive control group(P < 0.05).(4) With the extension of culture time, the secretion amount of alkaline phosphatase was increased gradually, and there was significant difference at different observational time points(P < 0.05).(5) On the 14~(th) day of culture, a great amount of artificially induced pluripotent stem cells-derived mesenchymal stem cells evenly distributed and grew well on the surface of hydroxyapatite/zirconium dioxide foam ceramics, and these cells had good morphology and highly expressed type I collagen. To conclude, the hydroxyapatite/zirconium dioxide foam ceramics has a certain compressive capacity and does not exhibit mutagenic effects and cytotoxicity, and promote the osteogenic differentiation of artificially induced pluripotent stem cells-derived mesenchymal stem cells cultured on its surface.
引文
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[26]Jin QM,Takita H,Kohgo T,et al.Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation.J Biomed Mater Res.2000;51(3):491-499.
[27]王春燕,全仁夫,汪宏斌,等.HA粉末与烧结体的高温分解及再生特性[J].武汉理工大学学报,2010,32(21):18-21.
[28]全仁夫,杨迪生,苗旭东,等.二氧化锆梯度复合羟基磷灰石的生物相容性研究[J].中国修复重建外科杂志,2006,20(5):569-573.
[29]Azqueta A,Lorenzo Y,Collins AR.In vitro comet assay for DNArepair:a warningconcerning application to cultured cells.Mutagenesis.2009;24:379-381.
[30]Yamarik TA.Safety assessment of dichlorophene and chlorophene.Int J Toxicol.2004;23 Suppl 1:1-27.
[31]Krucińska I,?ywicka B,Komisarczyk A,et al.Biological Properties of Low-Toxicity PLGA and PLGA/PHB Fibrous Nanocomposite Implants for Osseous Tissue Regeneration.Part I:Evaluation of Potential Biotoxicity.Molecules.2017;22(12):2092.
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[33]Szymonowicz M,Korczynski M,Dobrzynski M,et al.Cytotoxicity Evaluation of High-Temperature Annealed Nanohydroxyapatite in Contact with Fibroblast Cells.Materials(Basel).2017;10(6):590.
[34]孙开瑜,徐铭恩,周永勇.基于3D打印的Ⅰ型胶原涂覆β-TCP骨组织工程支架研究[J].中国生物医学工程学报,2018,37(3):335-343.
[35]董溪溪,张彤,曹均凯.关于诱导性多潜能干细胞成骨分化的研究进展[J].中华老年口腔医学杂志,2013,11(4):239-243.
[36]Strong M,Farrugia A,Rebulla P.Stem cell and cellular therapy developments.Biologicals.2009;37(2):103-107.
[37]Nasu A,Ikeya M,Yamamoto T,et al.Genetically matched human i PS cells reveal that propensity for cartilage and bone differentiation differs with clones,not cell type of origin.Plo SOne.2013;8(1):e53771-e53771.
[38]Chijimatsu R,Ikeya M,Yasui Y,et al.Characterization of Mesenchymal Stem Cell-Like Cells Derived From Human i PSCs via Neural Crest Development and Their Application for Osteochondral Repair.Stem Cells Int.2017;2017:1960965.
[2]Salih V,Georgiou G,Knowles JC,et al.Glass reinforced hydroxyapatite for hard tissue surgery--part II:in vitro evaluation of bone cell growth and function.Biomaterials.2001;22(20):2817-2824.
[3]Kim HW,Georgiou G,Knowles JC,et al.Calcium phosphates and glass composite coatings on zirconia for enhanced biocompatibility.Biomaterials.2004;25(18):4203.
[4]Bhowmick A,Pramanik N,Manna PJ,et al.Development of porous and antimicrobial CTS-PEG-HAP-Zn Onano-composites for bone tissue engineering.Rsc Adv.2015;5(120):99385-99393.
[5]Bhowmick A,Saha A,Pramanik N,et al.Novel magnetic antimicrobial nanocomposites for bone tissue engineering applications.Rsc Adva.2015;5(32):25437-25445.
[6]Chou BY,Chang E.Influence of deposition temperature on mechanical properties of plasma-sprayed hydroxyapatite coating on titanium alloy with Zr O2,intermediate layer.JTherm Spray Technol.2003;12(2):199-207.
[7]Chou BY,Chang E.Interface investigation of plasma-sprayed hydroxyapatite coating on titanium alloy with Zr O2,intermediate layer as bond coat.Scripta Mater.2001;45(4):487-493.
[8]Müller U,Imwinkelried T,Horst M,et al.Do Human Osteoblasts Grow Into Open-Porous Titanium?Eur Cells and Mater.200611:8-15.
[9]Chang CH,Lin FH,Kuo TF,et al.“Cartilage tissue engineering,”Biomedical Engineering:Applications.Basis Communicat.2005;17:1-11.
[10]Tamai N,Myoui A,Tomita T,et al.Novel hydroxyapatite ceramics with an interconnective porous structure exhibit superior osteoconduction in vivo.J Biomed Mater Res.2002;59:110-117.
[11]An SH,Matsumoto T,Miyajima H,et al.Porous zirconia/hydroxyapatite scaffolds for bone reconstruction.Dent Mater.2012;28(12):1221-1231.
[12]Kim HW,Shin SY,Kim HE,et al.Bone formation on the apatite-coated zirconia porous scaffolds within a rabbit calvarial defect.J Biomater Appl.2008;22(6):485-504.
[13]Quan RF,Tang YH,Huang ZM,et al.Difference of adherence,proliferation and osteogenesis of mesenchymal stem cells cultured on different HA/Zr O2 composites Chin J Traumatol.2012;15(3):131-139.
[14]Shao RX,Quan RF,Wang T,et al.Effects of a bone graft substitute consisting of porous gradient HA/Zr O2 and gelatin/chitosan slow-release hydrogel containing BMP-2 and BMSCs on lumbar vertebral defect repair in rhesus monkey.JTissue Eng Regen Med.2017;96(42):e8309.
[15]Franco-Bernardes MF,Rocha OP,Pereira LC,et al.The herbicides trifluralin and tebuthiuron have no genotoxic or mutagenic potential as evidenced by genetic tests.Environ Sc Pollut Res Int.2017;(1):1-9.
[16]Li R,Ma Y,Zhang Y,et al.Potential of rh BMP-2 and dexamethasone-loaded Zein/PLLA scaffolds for enhanced in vitro osteogenesis of mesenchymal stem cells.Colloids Surf B Biointerfaces.2018;169:384.
[17]Ressler A,Ródenas-Rochina A,Ivankovi?J,et al.Injectable chitosan-hydroxyapatite hydrogels promote the osteogenic differentiation of mesenchymal stem cells.Carbohydr Polym.2018;197:469-477.
[18]Yu X,Liu S,Chen H,et al.CGRP gene-modified r BMSCs show better osteogenic differentiation capacity in vitro.J Mol Histol.2018;49(4):357-367.
[19]Ao HY,Xie YT,Yang SB,et al.Covalently immobilised type Icollagen facilitates osteoconduction and osseointegration of titanium coated implants.J Orthop Translat.2016;5(5):16-25.
[20]Greil P.Active‐Filler‐Controlled Pyrolysis of Preceramic Polymers.J Am Ceram Soc.2010;78(4):835-848.
[21]Hench LL,Polak JM.Third-Generation Biomedical Materials.Science.2002;295(5557):1014-1017.
[22]Feng B,Weng J,Yang BC,et al.Characterization of surface oxide films on titanium and adhesion of osteoblast.Biomaterials.2003;24(25):4663.
[23]Anselme K,Ponche A,Bigerelle M.Relative influence of surface topography and surface chemistry on cell response to bone implant materials.Part 2:biological aspects.Proc Inst Mech Eng H.2010;224(12):1487-507.
[24]Zinger O,Anselme K,Denzer A,et al.Time-dependent morphology and adhesion of osteoblastic cells on titanium model surfaces featuring scale-resolved topography.Biomaterials.2004;25(14):2695-2711.
[25]Jiang L,Li Y,Wang X,et al.Preparation and properties of nano-hydroxyapatite/chitosan/carboxymethyl cellulose composite scaffold.Carbohydr Polym.2008;74(3):680-684.
[26]Jin QM,Takita H,Kohgo T,et al.Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation.J Biomed Mater Res.2000;51(3):491-499.
[27]王春燕,全仁夫,汪宏斌,等.HA粉末与烧结体的高温分解及再生特性[J].武汉理工大学学报,2010,32(21):18-21.
[28]全仁夫,杨迪生,苗旭东,等.二氧化锆梯度复合羟基磷灰石的生物相容性研究[J].中国修复重建外科杂志,2006,20(5):569-573.
[29]Azqueta A,Lorenzo Y,Collins AR.In vitro comet assay for DNArepair:a warningconcerning application to cultured cells.Mutagenesis.2009;24:379-381.
[30]Yamarik TA.Safety assessment of dichlorophene and chlorophene.Int J Toxicol.2004;23 Suppl 1:1-27.
[31]Krucińska I,?ywicka B,Komisarczyk A,et al.Biological Properties of Low-Toxicity PLGA and PLGA/PHB Fibrous Nanocomposite Implants for Osseous Tissue Regeneration.Part I:Evaluation of Potential Biotoxicity.Molecules.2017;22(12):2092.
[32]蒋媛,杨杨.二氧化锆的临床研究进展[J].临床口腔医学杂志,2017,33(2):125-127.
[33]Szymonowicz M,Korczynski M,Dobrzynski M,et al.Cytotoxicity Evaluation of High-Temperature Annealed Nanohydroxyapatite in Contact with Fibroblast Cells.Materials(Basel).2017;10(6):590.
[34]孙开瑜,徐铭恩,周永勇.基于3D打印的Ⅰ型胶原涂覆β-TCP骨组织工程支架研究[J].中国生物医学工程学报,2018,37(3):335-343.
[35]董溪溪,张彤,曹均凯.关于诱导性多潜能干细胞成骨分化的研究进展[J].中华老年口腔医学杂志,2013,11(4):239-243.
[36]Strong M,Farrugia A,Rebulla P.Stem cell and cellular therapy developments.Biologicals.2009;37(2):103-107.
[37]Nasu A,Ikeya M,Yamamoto T,et al.Genetically matched human i PS cells reveal that propensity for cartilage and bone differentiation differs with clones,not cell type of origin.Plo SOne.2013;8(1):e53771-e53771.
[38]Chijimatsu R,Ikeya M,Yasui Y,et al.Characterization of Mesenchymal Stem Cell-Like Cells Derived From Human i PSCs via Neural Crest Development and Their Application for Osteochondral Repair.Stem Cells Int.2017;2017:1960965.