改良低温机械物理研磨法制备纳米脱钙骨基质
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摘要
背景:前期研究证实纳米脱钙骨基质为一种无毒、组织相容性良好、生物利用度高、炎症反应轻、成骨能力强的骨移植替代物,但其制备耗时较长,需多次间断取样行粒度分析,研磨效率低。目的:采用低温物理研磨法制备纳米脱钙骨基质,分析纳米脱钙骨基质的结构特征及制备效率。方法:采用改良Urist法制备同种异体脱钙骨基质,通过二次低温物理研磨法,应用Cryo Mill全自动冷冻研磨仪先将脱钙骨基质初步研磨至微米级别颗粒,再通过E-Max高能水冷球磨仪进行纳米尺度研磨,最后通过扫描电镜观测研磨效果及材料形貌。结果与结论:采用二次低温物理研磨法制备的纳米脱钙骨基质底层致密,表面充满不规则纳米颗粒,颗粒直径20-50 nm,纳米颗粒相互团聚,表面密布纳米级别凹槽,纳米纤维结构间相互连接,内部形成大量相互连通的微米级别孔隙,其微观结构符合纳米生物材料范畴;二次低温物理研磨法仅需25 min,提高了研磨效率;结果说明,二次低温物理研磨法制备纳米脱钙骨基质的研磨结果更确切、材料粒度分布均匀性更高。
BACKGROUND: Previous studies have confirmed that nano-demineralized bone matrix is an alternative to an autograft with non-toxicity,good tissue compatibility, high bioavailability, mild inflammatory reaction, strong osteogenic ability. But its preparation takes long time.Intermittent sampling is required for particle size analysis.OBJECTIVE: To prepare nano-demineralized bone matrix by low temperature mechanical trituration and analyze its structure characteristics and preparation efficacy.METHODS: Allogeneic decalcified bone matrix was prepared by the modified Urist method. The Cryo Mill automatic freezing grinder was used to initially grind the decalcified bone matrix to the micron-sized particles. Then the E-Max high-energy water-cooled ball mill was used to grind the micro-sized particles into nano-sized particles. Finally, the grinding effect and material appearance were observed by scanning electron microscopy.RESULTS AND CONCLUSION: After two-step grinding, the prepared decalcified bone matrix had nanostructure composed by irregular20-50 nm-sized nanoparticles through agglomeration. The surface of nano-particles was densely covered with nano-scale grooves. A large number of interconnected micro-pores formed between nanofiber structures, the microstructure of which conformed to the category of nanobiomaterials. Compared with the Micro superfine mill, the new trituration process only took 25 minutes, which increases grinding efficacy.These results suggest that two steps of low temperature mechanical trituration for preparation of nano-demineralized bone matrix produces more uniform particle size distribution.
BACKGROUND: Previous studies have confirmed that nano-demineralized bone matrix is an alternative to an autograft with non-toxicity,good tissue compatibility, high bioavailability, mild inflammatory reaction, strong osteogenic ability. But its preparation takes long time.Intermittent sampling is required for particle size analysis.OBJECTIVE: To prepare nano-demineralized bone matrix by low temperature mechanical trituration and analyze its structure characteristics and preparation efficacy.METHODS: Allogeneic decalcified bone matrix was prepared by the modified Urist method. The Cryo Mill automatic freezing grinder was used to initially grind the decalcified bone matrix to the micron-sized particles. Then the E-Max high-energy water-cooled ball mill was used to grind the micro-sized particles into nano-sized particles. Finally, the grinding effect and material appearance were observed by scanning electron microscopy.RESULTS AND CONCLUSION: After two-step grinding, the prepared decalcified bone matrix had nanostructure composed by irregular20-50 nm-sized nanoparticles through agglomeration. The surface of nano-particles was densely covered with nano-scale grooves. A large number of interconnected micro-pores formed between nanofiber structures, the microstructure of which conformed to the category of nanobiomaterials. Compared with the Micro superfine mill, the new trituration process only took 25 minutes, which increases grinding efficacy.These results suggest that two steps of low temperature mechanical trituration for preparation of nano-demineralized bone matrix produces more uniform particle size distribution.
引文
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[30] Vieira S,Vial S,Reis RL,et al.Nanoparticles for bone tissue engineering. Biotechnol Prog.2017; 33(3):590-611.
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[33] Rouwkema J,Rivron NC,van Blitterswijk CA.Vascularization in tissue engineering.Trends Biotechnol.2008;26(8):434-441.
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[35] Wang X,Zhang G,Qi F,et al.Enhanced bone regeneration using an insulin-loaded nano-hydroxyapatite/collagen/PLGA composite scaffold.Int J Nanomedicine.2017;13:117-127.
[2] Bhattacharjee P,Kundu B,Naskar D,et al.Silk scaffolds in bone tissue engineering:An overview. Acta Biomater.2017;63:1-17.
[3] Blokhuis TJ, Arts JJ.Bioactive and osteoinductive bone graft substitutes:definitions, facts and myths.Injury.2011;42 Suppl2:S26-29.
[4] Gong T, Xie J, Liao J, et al.Nanomaterials and bone regeneration. Bone Res.2015;3:15029.
[5] Betz RR.Limitations of autograft and allograft:new synthetic solutions.Orthopedics.2002;25(5 Suppl):s561-570.
[6] Zimmermann G, Moghaddam A.Allograft bone matrix versus synthetic bone graft substitutes.Injury. 2011;42 Suppl 2:S16-21.
[7] Aryaei A,Jayatissa AH,Jayasuriya AC.Mechanical and biological properties of chitosan/carbon nanotube nanocomposite films. J Biomed Mater Res A. 2014;102(8):2704-2712.
[8] Touri R,Moztarzadeh F,Sadeghian Z,et al.The use of carbon nanotubes to reinforce 45S5 bioglass-based scaffolds for tissue engineering applications.Biomed Res Int. 2013;2013:465086.
[9] Chae T,Yang H,Leung V,et al.Novel biomimetic hydroxyapatite/alginate nanocomposite fibrous scaffolds for bone tissue regeneration.J Mater Sci Mater Med. 2013;24(8):1885-1894.
[10] Laschke MW,Strohe A,Menger MD,et al.In vitro and in vivo evaluation of a novel nanosize hydroxyapatite particles/poly(ester-urethane)composite scaffold for bone tissue engineering. Acta Biomater.2010;6(6):2020-2027.
[11]房雷,陈雄生,黄凯,等.人纳米脱钙骨基质复合物的理化性质和安全性[J].中国组织工程研究, 2013,17(38):6701-6708.
[12]黄凯,陈雄生,贾连顺,等.纳米脱钙骨基质促进骨愈合[J].中国组织工程研究,2013,17(21):3862-3869.
[13]黄凯,陈雄生,贾连顺,等.纳米脱钙骨基质的制备及其性能检测[J].中国矫形外科杂志, 2009,17(13):1017-1019.
[14] Grgurevic L,Pecina M, Vukicevic S,et al.Urist and the discovery of bone morphogenetic proteins.Int Orthop. 2017;41(5):1065-1069.
[15] Chen X,Wang W,Cheng S,et al.Mimicking bone nanostructure by combining block copolymer self-assembly and 1D crystal nucleation.ACS Nano.2013;7(9):8251-8257.
[16] Song SH,Kim SG,Kim SE,et al.Is DBM beneficial for the enhancement of bony consolidation in distraction osteogenesis? A randomized controlled trial. Biomed Res Int.2015;2015:281738.
[17] Drosos GI,Touzopoulos P,Ververidis A,et al.Use of demineralized bone matrix in the extremities. World J Orthop.2015;6(2):269-277.
[18] Stevens MM,George JH.Exploring and engineering the cell surface interface.Science.2005; 310(5751):1135-1138.
[19] Hasani-Sadrabadi MM,Hajrezaei SP,Emami SH,et al.Enhanced osteogenic differentiation of stem cells via microfluidics synthesized nanoparticles. Nanomedicine. 2015;11(7):1809-1819.
[20] Dan Y,Liu O,Liu Y,et al.Development of Novel Biocomposite Scaffold of Chitosan-Gelatin/Nanohydroxyapatite for Potential Bone Tissue Engineering Applications. Nanoscale Res Lett.2016;11(1):487.
[21]钱鋆,沈尊理.冻干脱钙骨表面纳米结构对细胞行为的影响[J].中国矫形外科杂志,2005,13(12):911-914.
[22]李香琴,宋克东.成骨细胞在纳米材料表面上粘附特性[J].大连理工大学学报,2005,45(5):653-657.
[23] Gao X,Song J,Ji P,et al.Polydopamine-Templated Hydroxyapatite Reinforced Polycaprolactone Composite Nanofibers with Enhanced Cytocompatibility and Osteogenesis for Bone Tissue Engineering. ACS Appl Mater Interfaces.2016;8(5):3499-3515.
[24] Hasani-Sadrabadi MM,Hajrezaei SP,Emami SH,et al.Enhanced osteogenic differentiation of stem cells via microfluidics synthesized nanoparticles. Nanomedicine.2015;11(7):1809-1819.
[25] Lavernia EJ, Han BQ,Schoenung JM.Cryomilled nanostructured materials:Processing and properties.Mater Sci Eng A.2008;493(1-2):207-214.
[26]董亮.低温粉碎技术[J].电子质量,2008,29(3):87-88.
[27]董亮,张赞蓉.粉碎技术在样品前处理中的应用—利用RETSCH研磨粉碎仪器获得有代表性的样品[J].食品安全导刊, 2011,5(11):34-35.
[28]佚名.德国Retsch(莱驰)高能纳米球磨仪E_(max)全新上市[J].中国粉体工业,2014,11(5):53-53.
[29] Wahajuddin, Arora S.Superparamagnetic iron oxide nanoparticles:magnetic nanoplatforms as drug carriers.Int J Nanomedicine.2012;7:3445-3471.
[30] Vieira S,Vial S,Reis RL,et al.Nanoparticles for bone tissue engineering. Biotechnol Prog.2017; 33(3):590-611.
[31] Fernandez-Urrusuno R,Fattal E,Rodrigues JM Jr,et al.Effect of polymeric nanoparticle administration on the clearance activity of the mononuclear phagocyte system in mice. J Biomed Mater Res.1996;31(3):401-408.
[32] Verdun C,Brasseur F,Vranckx H,et al.Tissue distribution of doxorubicin associated with polyisohexylcyanoacrylate nanoparticles.Cancer Chemother Pharmacol. 1990;26(1):13-18.
[33] Rouwkema J,Rivron NC,van Blitterswijk CA.Vascularization in tissue engineering.Trends Biotechnol.2008;26(8):434-441.
[34] Woodard JR,Hilldore AJ,Lan SK,et al.The mechanical properties and osteoconductivity of hydroxyapatite bone scaffolds with multi-scale porosity. Biomaterials. 2007;28(1):45-54.
[35] Wang X,Zhang G,Qi F,et al.Enhanced bone regeneration using an insulin-loaded nano-hydroxyapatite/collagen/PLGA composite scaffold.Int J Nanomedicine.2017;13:117-127.