丝素蛋白含量对纳米羟基磷灰石仿生矿化和体外细胞相容性的影响

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朱云荣, 陈玉云, 许国华, 叶晓健, 钟健, 何丹农. 丝素蛋白含量对纳米羟基磷灰石仿生矿化和体外细胞相容性的影响. 无机材料学报, 2012, 27(8): 883-887
ZHU Yun-Rong, CHEN Yu-Yun, XU Guo-Hua, YE Xiao-Jian, ZHONG Jian, HE Dan-Nong. Effect of Silk Fibroin Content on the Bionic Mineralization andIn Vitro Cellular Compatibility of Silk Fibroin/Hydroxyapaptite Nanocomposites . Journal of Inorganic Materials, 2012, 27(8): 883-887 复制到剪切板

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丝素蛋白含量对纳米羟基磷灰石仿生矿化和体外细胞相容性的影响

朱云荣¹, 陈玉云², 许国华¹, 叶晓健¹, 钟健², 何丹农²

1. 长征医院, 上海 200003

2. 纳米技术及应用国家工程研究中心, 上海 200241

叶晓健, 教授. E-mail:yexj1964@hotmail.com; 陈玉云, 硕士. E-mail:jeccica_1982@hotmail.com

朱云荣(1984-), 男, 博士研究生. E-mail:zyr13579@hotmail.com

修回日期:2011-11-28

基金:国家重点基础研究发展计划(2009CB930000); 上海市自然科学基金(0852nm03100);

摘要

通过制备含有不同丝素蛋白(SF)含量的丝素蛋白/纳米羟基磷灰石复合材料, 着重研究丝素蛋白含量对纳米羟基磷灰石(n-HA)仿生矿化过程中晶体成核与生长及体外细胞相容性的影响. 结果表明: SF的加入对n-HA晶体的成核和生长具有明显的调控作用; 丝素含量的大小对n-HA晶体成核和生长没有明显的区别, 但对晶粒在有机大分子中的聚集状态有明显的影响: 当SF含量不超过20wt%时, n-HA晶粒呈现放射状团簇, 当SF含量超过20wt%时, n-HA晶粒无序团聚. 体外细胞相容性结果显示, SF的加入可以促进材料与细胞的界面亲和性, 但SF含量对这种亲和性的影响不明显, 20wt%和30wt%的SF含量对增殖能力具有较强的促进作用.

关键词: 羟基磷灰石; 丝素蛋白含量; 纳米晶; 仿生矿化; 细胞相容性

中图分类号:TB321 文献标志码:A 文章编号:1000-324X(2012)08-0883-05

Effect of Silk Fibroin Content on the Bionic Mineralization andIn Vitro Cellular Compatibility of Silk Fibroin/Hydroxyapaptite Nanocomposites

ZHU Yun-Rong¹, CHEN Yu-Yun², XU Guo-Hua¹, YE Xiao-Jian¹, ZHONG Jian², HE Dan-Nong²

1. Changzheng Hospital, Shanghai 200003, China

2. National Engineering Research Center for Nanotechnology, Shanghai 200241, China

Fund:973 Program (2009CB930000); Natural Science Research Foundation of Shanghai (0852nm03100);

Abstract

Silk fibroin and hydroxyapatite nanocomposites (SF/HA) with various SF content were prepared. Effect of Silk fibroin content on the bionic mineralization and cellular compatibilityin vitroof HA nanocrystals (n-HA) was investigated. The results show that SF content has an obvious effect on the nucleation and growth of n-HA. However, though SF content does not show obvious difference on the nucleation and growth of n-HA, it has obvious effect on the n-HA aggregation. When SF content is less than 20wt%, n-HA orderly disperses in SF matrix. While the SF content is more than 20wt%, the n-HA aggregation becomes disordered. Thein vitro cellular compatibility experiments demonstrate that the SF/HA composites exhibit better cell affinity than pure n-HA. However, SF content has no obvious effect on the cell affinity of n-SF/HA 20wt% SF/HA and 30wt% SF/HA show better osteoblast proliferation.

Keyword: hydroxyapaptite; silk fibrion content; nanocrystals; bionic mineralization; cytocompatibility

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蚕丝丝素蛋白(silk fibroin, SF), 作为一种天然提取蛋白, 以其良好的生物相容性、生物可降解性, 力学性能优良、对细胞的粘附、扩增、分化作用良好等特点, 逐渐应用于生物替代材料和组织工程材料, 丝素蛋白/羟基磷灰石复合材料也被探索用来作为骨组织修复材料^{[ 1, 2]}. 当前对纳米丝素蛋白/羟基磷灰石(n-SF/HA)复合材料的制备与性能的研究集中于制备工艺对HA结晶行为以及SF与HA结合方式的影响^{[ 3, 4, 5]}、制备方法改进^{[ 6, 7]}、以及SF/HA纳米晶^{[ 8, 9, 10]}、在SF纤维上沉积HA^{[ 11, 12]}和在SF薄膜上沉积HA^{[ 13, 14, 15]}等不同的应用形式的研究.

由于自然骨中的无机成分约占70wt%, 有机成分约占30wt%, 很多研究没有对该质量配比进行过论证与核实, 而直接制备出30%SF/70%HA的材料开展后续研究. 针对这个问题, 本研究以丝素蛋白为生物矿化的模板, 采用仿生方法制备不同丝素含量的n-SF/HA, 研究丝素蛋白含量对n-HA的晶体成核与生长行为, 以及材料/细胞界面相容性的作用和影响, 讨论有机大分子SF对无机矿物HA在分子水平上的精细控制过程和有机无机界面处的相互作用.

1 材料与方法

1.1 实验原料

桑蚕茧由湖州市农业科学研究院提供; 骨肉瘤细胞(MG63)购自中科院上海细胞库; 胎牛血清和MEM培养基购自Gibco(加拿大); 二甲基亚砜(DMSO)和噻唑蓝(MTT)购自Sigma(美国); 其他试剂购自国药集团化学试剂有限公司(上海), 并且均为分析纯.

1.2 材料制备与表征

以终产物SF与HA的设计质量比率(设计比率分别为0、10wt%、20wt%、30wt%、40wt%)以及终产物HA中Ca/P为1.67的计量比为原则, 采用文献[16]的制备方法, 合成了一系列不同质量比率的n-SF/HA复合材料. 首先将购买的桑蚕茧用0.5%( w/ v)的Na₂CO₃水溶液进行脱胶处理, 烘干, 得到丝素纤维; 然后称取1.12、2.51、4.3、6.69 g(分别对应设计比率10wt%、20wt%、30wt%、40wt%)的丝素纤维, 置入75℃的CaCl₂-CH₃CH₂OH-H₂O三元溶剂(摩尔比为1:2:8)至完全溶解, 其中三元溶剂中CaCl₂为11.10 g. 然后向该溶解液中逐滴加入(NH₄)₂HPO₄溶液((NH₄)₂HPO₄的质量为7.92 g), 体系中出现白色沉淀, 继续在温度75℃和搅拌条件下反应24 h, 通过加入氨水维持整个体系的pH值为10左右. 最后将白色沉淀反复清洗, 烘干, 得到不同SF含量的n-SF/HA粉末样品. 将上述5组n-SF/HA都采用静电雾化沉积设备^{[ 17]}沉积于钛合金表面形成涂层样品以备作细胞测试, 其中钛合金为φ12 mm× 1.5 mm的片状, 制备出的涂层厚度约50 μm.

用透射电镜TEM(JEM-2100F, Joel, 日本)对n-SF/HA的微观形态进行观察.

1.3 材料的体外细胞相容性检测

1.3.1 细胞增殖检测(四甲基偶氮唑盐微量酶反应比色法(MTT)法)

分别将灭菌处理后的n-SF/HA涂层样品置于24孔板中. 每孔接种细胞悬液700 μL(浓度为1× 10⁴cells/mL), 在37℃和5% CO₂ 环境的培养箱(Thermo, 美国)中孵育, 并分别于第1、4、7 d吸去孔板中的培养液, 每孔加入70 μL MTT溶液(5mg/mL, 即0.5% MTT), 继续培养4 h, 产生蓝紫色结晶物. 每孔加入700 μL二甲基亚砜(DMSO), 置摇床上低速振荡10 min, 使结晶物充分溶解. 取200 μL溶解液转入96孔板中, 在酶联免疫检测仪(infinite F50, TECAN, 瑞士)OD490 nm处测量各孔的吸光值.

根据以下公式计算细胞增长率:

增殖率(%)=(实验-本底)/(对照-本底)×100%

每组样品6个平行样本, 取平均值, T-test法计算标准偏差, p<0.05具有统计学意义.

1.3.2 细胞形态观察

分别将灭菌处理后的n-SF/HA涂层样品置于24孔板中. 每孔接种700μL细胞悬液(1×10⁴cells/mL), 在37℃和5% CO₂ 环境中培养, 并分别于第1、 4、 7 d弃去培养液并加入5%戊二醛溶液, 4℃固定24 h, 梯度乙醇脱水干燥, 并喷金后在扫描电镜SEM(s-4800, Hitachi, 日本)下观察细胞形态.

2 结果与讨论

2.1 n-SF/HA的透射电镜分析(TEM)

图1是5组n-SF/HA颗粒的TEM照片. 由图1(a)、(b)所见, n-HA晶粒形状为棒状, 晶粒尺寸的长度范围为20~100 nm, 宽度为(35±4) nm, 晶粒分布呈明显的随机性. 由图1(c)、(d)所见, 10%SF/HA晶粒形状为针状, 统计分析得出晶粒尺寸长度为(140±10) nm, 宽度为(15±4) nm, n-HA晶粒的形态发生明显变化, 晶粒密集交错团簇并呈现放射状的聚集形态. 图1(e)、(f)中20%SF/HA晶粒形状为针状, 统计分析得出长度为(148±18) nm, 宽度为(12±2) nm, n-HA晶粒团簇成团并呈现放射状聚集形态. 图1(g)、(h)中30% SF/HA晶粒形状为针状, 统计分析得出长度为(143±14) nm, 宽度为(10±2) nm, 晶粒开始团聚. 图1(i)、(j)中40%SF/HA晶粒形状为针状, 统计分析得出长度为(136±10) nm, 宽度为(10±2) nm, 晶粒团聚. 上述现象说明SF的加入对n-HA的晶体形貌和晶体在有机大分子中的聚集状态起到了调控作用: n-HA晶粒形状在SF的调控下由短棒状变化为针状, 长径比增大, 晶粒尺寸在SF的调控下长度增加宽度减少, 晶粒聚集状态由随机分布变化为晶粒密集交错. SF含量的变化对晶粒尺寸的影响不明显, 但是对晶粒的聚集形态的影响较为明显: SF含量不超过20wt%时, 密集交错的晶粒团簇并呈现放射状, SF含量大于20wt%时, 晶粒团聚呈无序状态.

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	图1 n-SF/HA (0 ~ 40wt%)的TEM 照片Fig. 1 TEM imags of five groups of n-SF/HA (0–40wt%) samples(a), (b) HA; (c), (d) 10wt%SF/HA; (e), (f) 20wt%SF/HA; (g), (h) 30wt%SF/HA; (i), (j) 40wt%SF/HA

丝素在三元体系的溶解过程中Ca²⁺与SF中的氨基酸残基以及酰胺基形成静电和共价结合, 大量的Ca²⁺分布于蛋白质表面, 复合时与PO₄^3-通过静电作用彼此互相靠近促进n-HA颗粒的形成, 并且n-HA表面的Ca²⁺与蛋白质中的酰胺基、胺基和羧基等的静电或共价作用, 即Ca²⁺在SF 和n-HA颗粒之间起到了桥架作用^{[ 2, 4, 16]}, Ca²⁺在吸附位上通过静电或者共价作用停留在SF长链上, 才能继续完成成核与矿化的过程.因此, SF含量对n-HA矿化的影响如图2所示, 当SF含量不超过20wt%时, SF提供的吸附位点相对于Ca²⁺不饱和, Ca²⁺通过以SF长链为模板自组装沿长链方向生长, n-HA晶体有序放射状分布; 当SF含量超过20wt%时, SF提供的吸附位点相对于Ca²⁺趋向饱和, Ca²⁺可以和不止一个SF的位点吸附, 导致SF大分子链的牵连缠绕, 因此有序性就有可能降低.

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	图2 HA以SF为模板的生物矿化过程示意图Fig. 2 Schematic models of the biomineralization in the composite of SF/HA

2.2 n-SF/HA的体外细胞相容性评价

图3是通过MTT检测MG63细胞在5组n-SF/HA涂层上培养1、4、7 d的吸光度值. 第1 d和第4 d的吸光度值基本没有差异, 第7 d的吸光度值有较为明显的提高, 第7 d的吸光度值30%SF/HA样品的最高, 其次是20%SF/HA样品组, 其他组差异不显著( p>0.05). 计算第7 d的细胞增殖率, 以同期培养的HA为对照组, 30wt%SF/HA为15.35%, 20wt%SF/HA为13.56%, 说明20wt%和30wt%含量对细胞增殖有促进作用.

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	图3 MG63细胞在五组n-SF/HA (0~40wt%)涂层材料上的增殖性能Fig. 3 Proliferation properties of MG63 on five groups of n-SF/HA (0-40wt%) coating samples

实验还研究了MG63细胞在涂层上贴壁生长情况, 结果显示n-SF/HA(0~40wt%)涂层上细胞都可以良好贴壁并完全伸展, 并且细胞数量接近, 说明SF含量对细胞与材料的界面亲和性没有明显的影响. 本文只列出了n-HA, 20wt%SF/HA涂层上细胞生长粘附1、4、7 d的SEM照片(图4). 结果显示, 在有无丝素加入的表面上, 细胞都可以在材料表面完全铺展, 并与材料紧密结合. 加入丝素与未加入丝素的表面相比, 细胞数量明显增多, 说明SF的加入可以提高MG63细胞与材料的界面相容性. 综合上述MTT与SEM结果分析可知, 与空白组相比, SF的加入提高了细胞与材料的粘附能力, 促进了细胞更快速的增殖, 而20wt%和30wt% SF的含量对增值能力的促进作用略强, 这可能是由于SF含量的增高会引起材料表面粗糙度的增大而导致的.

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	图4 MG63细胞在n-SF/HA (0、20wt%)涂层材料上培养的SEM照片Fig. 4 SEM images of MG63 cultured on n-SF/HA (0, 20wt%) coating samples(a), (b), (c) n-HA controlled caltured of 1, 4, 7 d; (d), (e), (f) 20%SF/HA controlled caltured for 1, 4, 7 d

3 结论

1) SF的加入对n-HA晶体的成核和生长具有明显的调控作用: 促进n-HA晶粒沿 c轴高度择优生长, 长径比增大, 形貌由短棒状调控为针状; 丝素含量对n-HA晶体成核和生长的影响不明显, 但对晶粒在有机大分子中的聚集状态有明显的调控作用: 当SF含量不超过20wt%时, n-HA晶粒呈现放射状团簇, 当SF含量超过20wt%时, n-HA晶粒为无序团聚;

2) SF的加入可以促进材料与细胞的界面亲和性, 但SF含量对这种亲和性的影响不明显, 20wt%和30wt%的含量对增殖能力的促进作用略为明显.

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Biomaterials. 2006, 27(36):6064-6082 DOI:10.1016/j.biomaterials.2006.07.008

Stem cell-based tissue engineering with silk biomaterials

Abstract

Silks are naturally occurring polymers that have been used clinically as sutures for centuries. When naturally extruded from insects or worms, silk is composed of a filament core protein, termed fibroin, and a glue-like coating consisting of sericin proteins. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility, slow degradability and remarkable mechanical properties of the material. In addition, the ability to now control molecular structure and morphology through versatile processability and surface modification options have expanded the utility for this protein in a range of biomaterial and tissue-engineering applications. Silk fibroin in various formats (films, fibers, nets, meshes, membranes, yarns, and sponges) has been shown to support stem cell adhesion, proliferation, and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin. To date fibroin from Bombyx mori silkworm has been the dominant source for silk-based biomaterials studied. However, silk fibroins from spiders and those formed via genetic engineering or the modification of native silk fibroin sequence chemistries are beginning to provide new options to further expand the utility of silk fibroin-based materials for medical applications.

... 蚕丝丝素蛋白(silk fibroin, SF), 作为一种天然提取蛋白, 以其良好的生物相容性、生物可降解性, 力学性能优良、对细胞的粘附、扩增、分化作用良好等特点, 逐渐应用于生物替代材料和组织工程材料, 丝素蛋白/羟基磷灰石复合材料也被探索用来作为骨组织修复材料^[1,2] ...

2004

1.552

0.0

... 丝素在三元体系的溶解过程中Ca²⁺与SF中的氨基酸残基以及酰胺基形成静电和共价结合, 大量的Ca²⁺分布于蛋白质表面, 复合时与PO₄^3-通过静电作用彼此互相靠近促进n-HA颗粒的形成, 并且n-HA表面的Ca²⁺与蛋白质中的酰胺基、胺基和羧基等的静电或共价作用, 即Ca²⁺在SF 和n-HA颗粒之间起到了桥架作用^{[2, 4, 16]}, Ca²⁺在吸附位上通过静电或者共价作用停留在SF长链上, 才能继续完成成核与矿化的过程 ...

2008

0.0

... 当前对纳米丝素蛋白/羟基磷灰石(n-SF/HA)复合材料的制备与性能的研究集中于制备工艺对HA结晶行为以及SF与HA结合方式的影响^[3,4,5]、制备方法改进^[6,7]、以及SF/HA纳米晶^[8,9,10]、在SF纤维上沉积HA^[11,12]和在SF薄膜上沉积HA^[13,14,15]等不同的应用形式的研究 ...

2009

0.531

1.106

Journal of Inorganic Materials. 2009, 24(2):264-268 DOI:10.3724/SP.J.1077.2009.00264

Study on preparation of n-HA and silk fibroin bio-mineral material

<FONT face=Verdana>1. Research Center for NanoBiomaterials,Analytical and Testing center, Sichuan University,Chengdu 610064, China; 2. Schoole of Material and Chemical Engineering, HaiNan University,Danzhou 571737, China</FONT>

A new hydroxyapatite (n-HA)Silk fibroin (SF) bio-mineral material was prepared by using a biomimetic method, with Ca(NO₃)₂ and Na₃PO₄ used as the starting reagent to synthesize the inorganic phase. In details, SF was dissolved in Ca(NO₃)₂ solution without desalting procedure, the compound solution was directly dropped into Na₃PO₄ solution. In this way, HA was synthesized at 37℃ by controlling Ca/P ratio, meanwhile the minerialized HA interacted with SF to form biomineral material. The morphology and structure of this n-HA-SF biomineral material were characterized by XRD, FTIR, XPS and SEM. The results show that there are strong chemical interaction between n-HA and SF. Moreover, in the biomineral material, the inorganic phase is calciumdeficient hydroxyapatite, containing a small amount of carbonated HA with an average crystal size of 37.6nm. In addition, The particle size of biomineral materials range from 50nm to 200nm.It can be deduced that silk fibroin chain can regulate the growth and morphology of HA crystals. The compressive strength of the biomineral material is 32.21MPa， which is a good potential material for bone tissue engineering and unloadedbearing bone defects repairing.

以Ca(NO₃)₂与Na₃PO₄为无机相的前驱体，将丝素蛋白直接溶于Ca(NO₃)₂溶液中，不经过脱盐处理，直接滴入Na₃PO₄溶液中反应，在37℃下丝素蛋白和羟基磷灰石晶体之间相互作用，仿生合成了纳米羟基磷灰石(n-HA)丝素蛋白(SF)生物矿化材料.用FTIR、XRD、XPS和SEM进行表征.结果表明，羟基磷灰石和丝素蛋白两相间具有较强的化学键合，矿化材料中无机相包含少量碳酸根，为缺钙类骨羟基磷灰石并且呈现一定的长轴取向性，说明丝素蛋白大分子对羟基磷灰石晶体的成核和生长起着模板和调控作用.矿化物颗粒尺寸在50~200nm之间，其抗压强度为32.21MPa，可作为非承重部位骨组织缺损修复材料.

2005

2.108

0.0

2006

0.0

2007

1.814

0.0

2008

0.0

2007

0.0

2011

0.531

1.106

Journal of Inorganic Materials. 2011, 26(1):43-48 DOI:10.3724/SP.J.1077.2011.00043

Effect of pretreatment on fabrication of natural fibroin fiber/apatite composites using alternate soaking method

State Key Laboratory of Materials-Oriented ChemicalEngineering, College of Materials Science and Engineering, Nanjing Universityof Technology, Nanjing 210009, China

To develop biomimetic fibroin/apatite composites with bone-like structure byalternate soaking method, the degummed natural fibroin fibers were pretreatedby soaking into CaCl₂:EtOH:H₂O (<>n(CaCl₂):<>n(EtOH):<>n(H₂O)=1:2:8) solution for given time and then soaked into calciumsolution and phosphate solution, respectively, to fabricate fibroin/apatite composites. The effects of pretreatment time andthe alternate soaking times on fibroin microstructure and fibroin/apatitecomposites were examined by SEM, FTIR, XRD and TGA techniques. The pretreatmentusing CaCl₂:EtOH:H₂O(<>n(CaCl₂):<>n(EtOH):<>n(H₂O)=1:2:8) solution makes fibroin fiber exhibit rougher surface and higher internalporosity, which is beneficial to interaction of calcium ion and residue groupssuch as -COOH, –OHand -CONH- from fibroin, and then improvethe deposition of apatite on fibroin fibers. The homogeneous apatites withoriented <>c-axis were formed on the pretreated fibroin fibers. Moreover,there are more than 30wt% apatites deposited on fibroin fibers after alternatesoaking for more than 3 times. The fibroin/apatite composite has potential tobe applied as bone repair materials.

利用氯化钙-乙醇-水(n(CaCl₂):n(EtOH):n(H₂O)=1:2:8)三元溶液对丝素蛋白纤维预处理, 再采用交替浸渍法将其分别浸在钙溶液和磷溶液中交替浸渍、仿生矿化制备丝素蛋白纤维/磷灰石复合材料, 采用SEM、FTIR、XRD和TGA等技术研究了不同预处理时间对丝素蛋白纤维微结构和构建丝素蛋白纤维/磷灰石的影响. 结果表明, 预处理增加了丝素蛋白纤维表面粗糙程度及内部孔隙率, 并使钙离子与丝素蛋白纤维中的羧基、羟基和酰胺基等基团先发生作用, 可以有效地提高磷灰石在丝素蛋白表面的沉积量, 使得预处理后的丝素蛋白纤维与磷灰石的结合更均匀、密实, 获得了磷灰石晶体沿c轴取向生长的仿骨结构的复合材料.

2008

0.869

1.044

2008

2.112

0.0

2009

0.0

2009

0.0

2007

0.869

1.044

2010

0.0

Journal of Material Science. 2010, 45(21):5814-5819 DOI:10.1007/s10853-010-4656-4

Facile fabrication of nano-hydroxyapatite/silk fibroin composite via a simplified coprecipitation route

1.Department of Orthopedic Surgery, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai, 200003 People’s Republic of China<br/>2.National Engineering Research Center for Nanotechnology, Shanghai, 200241 People’s Republic of China<br/>

Nano-hydroxyapatite/silk fibroin (n-HA/SF) composite was successfully fabricated based on a simplified coprecipitation route. In detail, the degummed SF was first dissolved in CaCl₂ aqueous/ethanol solution without desalting procedure, and then (NH₄)₂HPO₄ solution and NH₄OH were dropped into the above solution to form n-HA/SF composite. The structure and morphology of n-HA/SF composite were investigated by Transmission electron microscopy, Fourier transform infrared spectrometry, energy dispersive X-Ray spectrum, X-ray diffraction, and thermogravimetric analyses. Results indicate that the inorganic phase is carbonate-substituted HA with low crystallinity and similar to the crystals of human bone. The HA crystals have diameter of around 20–30 nm and length of about 200–500 nm. The content of SF in the composite is about 30%, and the two phases bonded each other strongly. In addition, a formation mechanism of n-HA/SF was proposed.

2012

0.0

Materials Science and Engineering C. 2012, 32(2):390-394 DOI:10.1016/j.msec.2011.11.002

Micropattern of nano-hydroxyapatite/silk fibroin composite onto Ti alloy surface via template-assisted electrostatic spray deposition

Abstract

The surface topography plays an important role in bone formation, which may present specific physical cues to cells and tissues and further improve the bioactivity or osteoconduction of the implants. In this report, nano-hydroxyapatite/silk fibroin (n-HA/SF) composite were prepared via a simplified coprecipitation method. Then these nanocrystals were deposited onto Ti alloy substrates via a template-assisted electrostatic spraying deposition (ESD) technique. The output voltage and flow rate were investigated to produce a cone-jet spraying of n-HA/SF suspensions. By using different types of precisely-designed templates (stripe, grid, and ring), the Ti alloy substrates with different micropatterns (stripe, square, and ring) composed of n-HA/SF composite were created. Our results demonstrated that the precisely-designed micropatterns of n-HA/SF composite onto Ti and its alloy substrates could be easily achieved by using template-assisted ESD.

Highlights

? Micropattern of nano-hydroxyapatite/silk fibroin (n-HA/SF) composite onto Ti alloy surface. ? Template-assisted electrostatic spray deposition for the micropatterning. ? The output voltage and the flow rate are investigated to produce a cone-jet spraying. ? We use several precisely designed templates for the micropatterning.

... 将上述5组n-SF/HA都采用静电雾化沉积设备^[17]沉积于钛合金表面形成涂层样品以备作细胞测试, 其中钛合金为#cod#x003c6 ...