Silk fibroin sodium alginate fibrous hydrogels regulated hydroxyapatite crystal growth

Silk fibroin/sodium alginate fibrous hydrogels regulated hydroxyapatite crystal growthJinfa Ming a ,b ,Zhijuan Jiang b ,Peng Wang b ,Shiyu Bie b ,Baoqi Zuo b ,⁎a The College of Textiles &Fashion,Qingdao University,Qingdao 266071,ChinabNational Engineering Laboratory for Modern Silk,College of Textile and Clothing Engineering,Soochow University,Suzhou 215123,Chinaa b s t r a c ta r t i c l e i n f o Article history:Received 9November 2014Received in revised form 29January 2015Accepted 9March 2015Available online 11March 2015Keywords:Silk fibroinSodium alginate Hydroxyapatite Fibrous hydrogelUse of organic templates for controlling the growth of inorganic crystals is one of the research topics in biomimetic field.In particular,oriented growth of hydroxyapatite (HAp)in organic fibrous matrix is provided a new view angle to study biomineralization of bone and its potential biomedical applications.The crystallization of HAp in fibrous hydrogels could mimic such biomineralization.In this paper,we report HAp nanorod crystal synthe-sized successfully by a biomimetic method using calcium chloride and ammonium dihydrogen phosphate as reagents in the presence of silk fibroin/sodium alginate (SF/SA)fibrous hydrogels.The effects of in fluence factors such as mineral times,pH,and temperature on controlling HAp nanorod crystals are discussed.The elongated HAp nanorods with rectangular column are grown with the increase of mineral times in biomimetic process.By changing pH,HAp nanorod crystals are obtained at alkaline condition in fibrous hydrogels.Moreover,compared to other temperatures,rod-shaped HAp crystals were formed at 20°C.The results imply this to be an effective method for preparing HAp crystals with controllable morphology for bone repair application.©2015Elsevier B.V.All rights reserved.1.IntroductionNatural mineral organization such as bone and tooth is a hierarchi-cally structured composite material containing biological macromole-cules (protein,polysaccharide,etc.)and inorganic minerals,which has been well studied by the materials engineering community because of its unique structure and mechanical properties [1,2].Hydroxyapatite (HAp),the mineral constituents of human bones and tooth,has been studied in biomedical applications for many years [3,4].Many chemical methods have been used for the preparation of HAp crystals with con-trollable size and morphology,such as solution –precipitation [5],sol –gel synthesis [6],solid-state reaction [7],and hydrothermal method [8].However,these methods mostly prepare irregular forms of powder [9].Presently,the preparation of HAp crystals is developed by biomi-metic approach,which is a research topic in the field of bone tissue engineering [10].In biomimetic preparation process,organic templates are used to control the morphology and structure of HAp crystals [11].Masanori Kikuchi and co-workers reported a bone-like HAp/collagen nanocomposite prepared by self-organization [12].The results showed that the c -axes of blade-shaped HAp nanocrystals 50–100nm in size were aligned along collagen fibers up to 20μm in length.At the same time,the mechanical properties of composites had40MPa in bending strength and 2.5GPa in Young's modulus.Wei et al.studied HAp crystals depositing on regenerated silk fibroin (SF)nano fibers by a biomimetic Ca –P method [13].The results exhibited HAp crystals with 30nm diameter distributing on the sur-face of SF nano fibers.Recently,HAp has been intensely studied with the aim of under-standing how crystal polymorph and structural features can be con-trolled by natural polymers [14].Some natural polymers containing polysaccharides (alginate,chitosan,and cellulose)and proteins (colla-gen,gelatin,and silk),are used to control the crystallization of HAp in recent research works [15–17].However,the mineral growth envi-ronment rarely occurred in gel state [9,18].In our previous study,we reported silk fibroin/sodium alginate (SF/SA)fibrous hydrogels to regulate and control the growth of HAp crystals.This fibrous hydrogels containing SF protein and SA polysaccharides can better mimic the real mineralization system of bone more than a single protein system [9,14,19,20].At the same time,rectangular column and size-controllable HAp nanorods were controlled in SF/SA fibrous hydrogels at room temperature.In this study,we aim to analyze the effects of in fluence factors on controlling HAp nanorod growth in SF/SA fibrous hydrogels.By study-ing the crystallization process in fibrous hydrogels,the probable mech-anism of HAp nanorod crystal growth is analyzed.Through this study,it provides clues to understand HAp biomineralization process as it occurs in bone formation and suggests a pathway for the biomimetic fabrica-tion of biomaterials with controllable morphology and structure.Materials Science and Engineering C 51(2015)287–293⁎Corresponding author.E-mail addresses:mingjinfa@ ,jinfa.ming@ (J.Ming),bqzuo@ (B.Zuo)./10.1016/j.msec.2015.03.0140928-4931/©2015Elsevier B.V.All rightsreserved.Contents lists available at ScienceDirectMaterials Science and Engineering Cj ou r n a l h o m e p a g e :w ww.e l s e v i e r.c om /l o c a t e /ms e c2.Experimental2.1.MaterialsBombyx mori silk was bought from Zhejiang province,China.Sodium alginate was purchased from Sinopharm Chemical Reagent Co.,Ltd. (Shanghai,China),and used without any further purification.All chem-ical reagents(lithium bromide,sodium carbonate,calcium chloride, formic acid,ammonium dihydrogen phosphate,ethanol,etc.)were bought from Sinopharm Chemical Reagent Co.,Ltd.(Shanghai,China), which were analytical grade,and also used without any further purification.2.2.Preparation of regenerated SF solutionB.mori silkfibers were boiled in0.05wt.%Na2CO3solution for 30min and then rinsed thoroughly with deionized water to extract the glue-like sericin proteins.Each step was repeated twice.Degummed silk was then dissolved in formic acid/CaCl2(FA/CaCl2,5wt.%concentra-tion)solvent at room pared to traditional dissolved method(LiBr aqueous solution and CaCl2/ethanol/water solution),the FA/CaCl2dissolved silk by breaking hydrogen bonds in the crystalline region while preserving the nanofibril structures[21].During the disso-lution process,the dissolution behavior of silk fromfiber to nanofibrilswas regulated by CaCl2concentration.This solution(SF–FA–CaCl2) was used forfilm formation.The formedfilms were redissolved in 9.3M LiBr solution at room temperature for4h,yielding a10%(w/v) solution.This solution was dialyzed against distilled water using Slide-A-Lyzer dialysis cassettes(Sigma,USA,molecular weight cut-off3500) for72h to remove salt ions.Thefinal concentration of aqueous silk solution was~1.0wt.%,determined by weighing the remaining solid after drying.This preparation process was two-step dissolution method, and its obtained aqueous solution was stored at5°C for use.2.3.Preparation of SF/SAfibrous hydrogelSA(2g)was dissolved in deionized water at room temperature.The solution was mixed under constant stirring in a blender for1h,standing 24h at5°C to obtain a uniformly0.5wt.%SA solution.And then,SF and SA aqueous solutions with70/30ratio were mixed by stirring,and the concentration of the mixture solution was controlled at1.0wt.%.The mixed solution was stored overnight at5°C to achieve homogeneity and to avoid any premature precipitation of the protein,which occurred at room temperature.Finally,hydrogels were prepared by adding5mL of blend solution in24well plates(Coring,USA).The solutions were allowed to gel in an incubator at37°C for obtainingfibrous hydrogel (Fig.1a).SF/SAfibrous hydrogels had nanofiber network morphology withβ-sheet structure(Fig.1b,c).According to our published proce-dures[22],the porosity offibrous hydrogels was97.0±1.3%,which its compressive stress was31.9±3.6kPa(Fig.1d).In addition,SA hydrogels were gelled by adding10mM Ca2+solution.The morphology of SA hydrogels was interconnected nanofiber networks[9].2.4.Preparation of HAp crystals in SF/SAfibrous hydrogelsThe mineral process was used to grow HAp crystals in SF/SAfibrous hydrogels.Firstly,SF/SAfibrous hydrogels were treated in75%(v/v) ethanol aqueous solution for30min to prepare the water-insoluble hydrogels.The water-insoluble hydrogels were immersed directly in 0.1M CaCl2supersaturated solution for1h at room temperature and washed twice with deionized water to remove free ionic calcium. Then,the samples were placed in0.1M(NH4)2HPO4supersaturated solution.After soaking for different conditions(mineral time,pH,tem-perature),cycles were repeated every2h.The HAp-deposited SF/SA fibrous hydrogels were rinsed with deionized water and freeze-dried.As control,HAp crystals were prepared according to our previous procedures[23].HAp crystals synthesized by solution–precipitation approach exhibited needle-like morphology with25.7±2.9nm.2.5.Characterization2.5.1.Scanning electron microscopy(SEM)The morphology of SF/SAfibrous hydrogels and its HAp crystal growth was examined by SEM(S4800,Hitachi,Japan).Samples for SEM experiment were observed with gold coating.At the same time, energy dispersive X-ray spectroscopy(EDX)was employed to deter-mine the elemental composition.2.5.2.X-ray diffraction(XRD)The experiment was recorded on X Pert-Pro MPD(PANalytical, Netherlands)with CuKαradiation working at40kV and40mA in the interval range from10°to60°with a scan rate2°min−1.2.5.3.Fourier transform infrared(FTIR)FTIR spectra were obtained using Nicolet5700(Thermal Nicolet Company,USA)in absorbance mode at the wave number ranging from400to4000cm−1.3.Results and discussion3.1.HAp crystal growth infibrous hydrogelsBone,natural organic–inorganic ceramic composite,consists of col-lagenfibrils containing embedded,well-aligned nanocrystalline,rod-like HAp crystals[24].In order to mimic the growth process of HAp crys-tals in bone formation,SF/SAfibrous hydrogels were used to control HAp crystal growth.Fig.2showed the morphology and structure of HAp crystals regulated by different organic templates for a mineral time of1h at room temperature.At blank template,needle-like HAp crystals with25.7±2.9nm were obtained by solution–precipitation (Fig.2d)and its crystalline structure was characterized by XRD (Fig.2e).Compared to JCPDS-ICDD database,the main peaks at(002), (211),(300),(202),(310),(222),(213),and(004)were appeared, attributing to typical HAp characteristic peaks.At the same preparation condition,crystals withflower-shaped morphology were obtained by using SFfibrous hydrogels as template(Fig.2a).SEM results suggested that hydroxyl,carboxyl,and carbonyl groups in SF moleculeshad Fig.1.SEM images(a,b),FTIR(c),and mechanical properties(d)of SF/SAfibrous hydrogels prepared through70/30proportions of SF/SA aqueous solution at37°C.288J.Ming et al./Materials Science and Engineering C51(2015)287–293in fluenced in inducing the mineralization of HAp crystals.Besides these groups,the polar amino acids would affect the mineral process.These polar amino acid residues such as aspartic,arginine and glutamic were not uniformly distributed along the peptide chains but were arranged in polar clusters,which would be capable of binding the Ca 2+cations to nucleation [25].And then,the aggregation of HAp crystals was directed by SF molecular chains in fibrous hydrogels [26].Finally,HAp crystals with flower-shaped morphology were formed in fibrous hydrogels.In SA fibrous hydrogels,HAp nucleation took place because of the strong interaction of the carboxylic groups present on the poly-meric backbone of SA with multivalent cations such as Ca 2+in the form of “egg-box ”[27,28].When the phosphate solution was added tothe Ca 2+–SA fibrous hydrogels,there was an interaction of PO 43−ions on Ca 2+–SA complexes to nucleate HAp due to the effects of supersatu-ration.The con fined crystallization of HAp was grown in the SA fibrous hydrogels,and then these aggregates gathered into a spherical-like shape (Fig.2c).However,rectangular nanorod HAp crystals grew in SF/SA fibrous hydrogels,which was con firmed by EDX (Fig.2f).In the nanorod crystal growth process,the stereo-chemical geometry and the charge distribution in fibrous hydrogels were supposed to endow SF and SA molecules with the capability to control the crystallization process.When fibrous hydrogels were treated by Ca 2+ion solution,Ca 2+ions on the SF and SA molecules get attached to PO 43−and OH −groups by electrostatic interaction as a driving force for nanocrystal growth.Finally,the coalescence of nanorods with the same directionformed the partially oriented bundles [9].Therefore,it was thus clear that the morphology of HAp crystals was controlled by different fibrous hydrogels.3.2.Effect of mineral times on the formation of HAp crystalsMineral times are one of the important factors to in fluence the HAp crystal growth in fibrous hydrogels.Fig.3a –d showed the morphology of HAp crystals which grew in SF/SA fibrous hydrogels at room temper-ature with various mineral times.For a mineral time of 1h at room tem-perature,Fig.3(A,a)depicted that the crystal sample was composed of many regular nanorods.The crystal structure of nanorods was analyzed by FTIR (Fig.4A).Compared to FTIR spectra of pure HAp crystals (Fig.4A –e),the characteristic bands at 563,602,and 1028cm −1appeared at a mineral time of 1h (Fig.4A-a),attributing to PO 43−v 4mode,PO 43−v 4mode,and PO 43−v 3mode,respectively [25,29].At the same time,this nanorod crystal growth in fibrous hydrogels was HAp crystals,which were con firmed by EDX (Fig.3e).When the mineraliza-tion time was 4h,HAp nanorods with about 439.9±74.5nm in width were grown (Fig.3b).Following the mineralization time increasing to 48h,the elongated nanorod crystals with more than 20μm lengths were obtained in SF/SA fibrous hydrogels (Fig.3c,d).The crystal struc-ture of nanorod crystals was also examined by FTIR (Fig.4A).The peaks at 560–610cm −1and 1000–1100cm −1appeared,attributing to phos-phate groups in HAp crystals.In addition,XRD results also showedHApFig.2.SEM images and EDX (f)of HAp crystal growth controlled by different mineralization templates:(a)SF fibrous hydrogel,(b)SF/SA fibrous hydrogel,(c)SA fibrous hydrogel,and (d)blank;(e)XRD of HAp crystals prepared by solution –precipitation.289J.Ming et al./Materials Science and Engineering C 51(2015)287–293nanorod crystal growth with increasing mineral pared to XRD results of pure HAp crystals (Fig.4B-e),the main characteristic peaks of all samples appeared at (002),(211),(310),(222),(213),and (004),respectively (Fig.4B).3.3.Effect of pH on the formation of HAp crystalsIn hydrogel system,the growth process of HAp crystals was affected by various factors such as temperature,pH,and additives [4,30].Fig.5showed the morphology of HAp crystals which grew in SF/SA fibrous hydrogels with different pH values from 6to 10.Irregular blocky-shaped particles grew in SF/SA fibrous hydrogels at pH =6(Fig.5A,a).The XRD pattern of these irregular particles appeared at 26.2°,32.3°,34.0°,39.8°,46.9°,49.7°,and 53.5°,corresponding to the diffraction planes (002),(211),(202),(310),(222),(213),and (004),respectively,attributing to typical characteristic peaks of HAp crystals (Fig.6a).For pH =7,many regularly-shaped HAp crystals appeared (Fig.5B,b).Increasing the pH values,the solution was from acidic to alkalineinFig.3.SEM images and EDX (e)of HAp crystals prepared by different mineral times:(A,a)1h,(b)4h,(c)12h,and (d)48h,respectively.Fig.4.FTIR (A)and XRD (B)of HAp crystals prepared by different mineral times:(a)1h,(b)4h,(c)12h,(d)48h,and (e)pure HAp crystals prepared by solution –precipitation.290J.Ming et al./Materials Science and Engineering C 51(2015)287–293fibrous hydrogels.HAp crystals with rod-shaped morphology were observed in SF/SA fibrous hydrogels (Fig.5C,D),and its crystalline structure was also characterized by XRD (Fig.6c,d).The main charac-teristic peaks of these crystals also appeared at (002),(211),(202),(310),(222),(213),and (004),respectively.Although no apparent difference was seen from XRD results of HAp crystals which grew at different pH values of 6to 10,the morphology of all samples above changed obviously.This indicates that pH value is a signi ficant pa-rameter variable in altering the morphology.3.4.Effect of temperature on the formation of HAp crystalsTo obtain rod-shaped HAp crystals in SF/SA fibrous hydrogels at lower feasible temperature,a series of experiments was designed.The pH values of solution were all adjusted to 8by ammo-nium hydroxide,and temperature ranged from 5to 60°C for 12h.Fig.7showed the morphology of HAp crystals which grew in SF/SA fibrous hydrogels with different mineral temperatures.The crystal aggregation had been obtained at 5°C (Fig.7a).At 20°C,the rod-shaped crystals with uniform size were formed in SF/SA fibrous hydrogels (Fig.7b).However,with the reaction temperature going up,granular crystals were obtained (Fig.7c,d).At the same time,the crystalline structure of crystal samples prepared at different temperatures was characterized by XRD (Fig.7e).The XRD pattern for all crystal samples exhibited the characteristic peaks at (002),(211),(310),(222),(213),and (004),respectively,attributing to HApcrystals.Fig.5.Morphology of HAp crystals which grew in SF/SA fibrous hydrogels with different pH conditions:(A,a)pH =6,(B,b)pH =7,(C,c)pH =8,and (D,d)pH =10,respectively.291J.Ming et al./Materials Science and Engineering C 51(2015)287–2933.5.HAp crystal crystallization process in SF/SA fibrous hydrogelOrganic templates for controlling single crystal growth have been employed as a means to prepare the morphology,structure,and purity of the resulting crystals.More recently,crystal growth in hydrogelsystem has emerged as a popular platform for modeling biomineraliza-tion processes [31].This interest is motivated by hydrogel matrices identi fied in association with mineralization by biological organisms.Therefore,in this present study,HAp nanorod crystals were controlled in SF/SA fibrous hydrogels.To learn the detail of HAp nanorod crystal growth process in hydro-gel system,crystal growth in SF/SA fibrous hydrogels with different in-fluence factors was carried out.Firstly,the water-insoluble SF/SA fibrous hydrogels were immersed directly in 0.1M CaCl 2supersaturated solution.The aim of this process was to provide nucleated sites for facilitating the crystal growth.The reasons were SF and SA molecules were all easily coordinated with Ca 2+ions in fibrous hydrogels.In SF molecules,the –C –O –and –N –H –groups had been preferentially coor-dinated with Ca 2+ions [9].At the same time,SA,one of the polyanionic copolymers,was easily contacted with solutions of divalent cations such as Ca 2+,forming the so-called “egg-box ”model.This “egg-box ”model was con firmed by Morris et al.[14,32].After it was treated by Ca 2+solution,the fibrous hydrogel samples were soaked in 0.1M (NH 4)2HPO 4supersaturated solution for HAp crystal growth.At the early stage of crystal growth,PO 43−ions were quickly diffused into fibrous hydrogels.According to double-layer theory,Ca 2+ionson the SF and SA molecules got attached to the PO 43−and OH –groups by electrostatic interaction as a driving force to further gather more Ca 2+ions,forming the HAp precursor.Above all,the HApnanorodFig.6.XRD results of HAp crystals grew in SF/SA fibrous hydrogels with different pH con-ditions:(a)pH =6,(b)pH =7,(c)pH =8,and (d)pH =10,respectively.Fig.7.Morphology and XRD (e)of HAp crystals which grew in SF/SA fibrous hydrogels with different mineral temperatures;the temperatures were as follows:(a)5°C,(b)20°C,(c)37°C,and (d)60°C,respectively.292J.Ming et al./Materials Science and Engineering C 51(2015)287–293crystallization microenvironment in fibrous hydrogels is distinguished from that in solution by the con finement of solutes to within the hydro-gel networks.This microenvironment of fibrous hydrogels presented several advantages in HAp crystal growth studies:Brownian motion,laminar flow,and convective currents were suppressed,making diffu-sion the dominant mass transport mechanism available to solutes in gel media [31].With the continuous reaction,gel fibrous networks were capable of supporting the growing crystals,preventing sedimen-tation [31].Nanocrystals were grown and attached along these organic chains to form nanorods (Fig.8).At the same time,the diffusion-limited conditions presented in fibrous hydrogels causing a modi fication to the HAp crystal growth regimes and resulting different morphologies were shown in this research.The morphology of solution-grown crystals was controlled by the driving force alone;however,the morphology of gel-grown crystals was in fluenced by a balance between the driving force and the diffusion rate of ions to the reaction front [31].Due to this diffu-sive transport,ion concentration gradients were formed at the interface of HAp nanocrystals.As the ion concentration threshold was reached,HAp nanocrystals grew larger along the SF/SA nano fibers in hydrogels.In the HAp crystal growth process,experimental parameters such as temperature,mineral times,and pH were in fluenced by crystal growth in fibrous hydrogels through the dependence of diffusion coef ficients and crystal solubilities.4.ConclusionSF/SA fibrous hydrogels were prepared and served as templates to control HAp crystal mineralization.In the mineralization process,the effect of in fluence factors such as mineral times,pH,and temperature was discussed.Time-dependent experiment showed the growth of elongated HAp nanorod crystals in mineralization process.The result exhibited fibrous networds in hydrogels supported the crystal growth along the SF/SA nano fibers to form nanorod crystals.By changing pH,the morphology of HAp crystals was changed from blocky-shaped to rod-shaped at acid –alkaline transition of hydrogels.Finally,HApnanorod crystals were obtained at alkaline condition in fibrous pared to other temperatures,rod-shaped HAp crystals were easily formed at 20°C.The controllable morphology of HAp nano-rods in fibrous hydrogels was in fluenced by a balance between the driv-ing force and the diffusion rate of ions to the reaction front.However,detailed crystallographic data of HAp nanorods were not available from this present study.Above all,the experimental results demon-strated a HAp nanorod crystal growth in SF/SA fibrous hydrogels.Therefore,this research provided clues for a deeper understanding of HAp biomimetic process and offered a novel pathway to fabricate novel biomaterials with controllable morphology for bone repair application.AcknowledgmentWe gratefully acknowledged the support of the Second Phase of Jiangsu Universities'Distinctive Discipline Development Program for Textile Science and Engineering of Soochow University,National Sci-ence Foundation of China (No.81271723),and National Engineering Laboratory for Modern Silk.References[1]S.Busch,Angew.Chem.Int.Ed.43(2004)1428–1431.[2]M.J.Olszta,X.G.Cheng,S.S.Jee,R.Kumar,Y.Y.Kim,M.J.Kaufman,E.P.Douglas,L.B.Gower,Mater.Sci.Eng.R.Rep.58(2007)77–116.[3]J.R.Woodard,A.J.Hilldore,n,C.J.Park,A.W.Morgan,J.A.C.Eurell,S.G.Clark,M.B.Wheeler,R.D.Jamison,A.J.W.Johnson,Biomaterials 28(2007)45–54.[4]J.B.Liu,X.Y.Ye,H.Wang,M.K.Zhu,B.Wang,H.Yan,Ceram.Int.29(2003)629–633.[5]K.McLeod,G.I.Anderson,N.K.Dutta,R.St.C.Smart,N.H.Voelcker,R.Sekel,S.Kumar,J.Biomed.Mater.Res.A 79A (2006)271–281.[6] D.M.Liu,T.Troczynski,W.J.Tseng,Biomaterials 22(2001)1721–1730.[7]S.Pramanik,A.K.Agarwal,K.N.Rai,A.Garg,Ceram.Int.33(2007)419–426.[8]R.L.Xin,K.F.Yu,J.Mater.Sci.44(2009)4205–4209.[9]J.F.Ming,S.Y.Bie,Z.J.Jiang,P.Wang,B.Q.Zuo,Mater.Lett.126(2014)169–173.[10]J.J.Li,Y.P.Chen,Y.J.Yin,F.L.Yao,K.D.Yao,Biomaterials 28(2007)781–790.[11] A.Ethirajan,U.Ziener,ndfester,Chem.Mater.21(2009)2218–2225.[12]M.Kikuchi,S.Itoh,S.Ichinose,K.Shinomiya,J.Tanaka,Biomaterials 22(2001)1705–1711.[13]K.Wei,Y.Li,K.O.Kim,Y.Nakagawa,B.S.Kim,K.Abe,G.Q.Chen,I.S.Kim,J.Biomed.Mater.Res.A 97A (2011)272–280.[14]J.F.Ming,B.Q.Zuo,J.Cryst.Growth 386(2014)154–161.[15]L.Chen,J.X.Hu,J.B.Ran,X.Y.Shen,H.Tong,Int.J.Biol.Macromol.65(2014)1–7.[16]K.Sangeetha,A.Thamizhavel,E.K.Girija,Mater.Lett.91(2013)27–30.[17]K.N.Li,J.N.Wang,X.Q.Liu,X.P.Xiong,H.Q.Liu,Carbohydr.Polym.90(2012)1573–1581.[18]Y.F.Ma,Q.L.Feng,X.Bourrat,Mater.Sci.Eng.C 33(2013)2413–2420.[19] F.Zhang,Q.Lu,J.F.Ming,H.Dou,Z.Liu,B.Q.Zuo,M.D.Qin,F.Li,D.L.Kaplan,X.G.Zhang,J.Mater.Chem.B 2(2014)3879–3885.[20]J.F.Ming,B.Q.Zuo,Polym.Eng.Sci.54(2014)129–136.[21] A.Mata,Y.B.Geng,K.Henrikson,C.Aparicio,S.Stock,R.L.Satcher,S.I.Stupp,Bioma-terials 31(2010)6004–6012.[22]L.C.Palmer,C.J.Newcomb,S.R.Kaltz,E.D.Spoerke,S.I.Stupp,Chem.Rev.108(2008)4754–4783.[23]J.F.Ming,B.Q.Zuo,Mater.Chem.Phys.137(2012)421–427.[24]H.J.Zhou,J.Lee,Acta Biomater.7(2011)2769–2781.[25]X.D.Kong,F.Z.Cui,X.M.Wang,M.Zhang,W.Zhang,J.Cryst.Growth 270(2004)197–202.[26] C.Cheng,Z.Z.Shao,F.Vollrath,Adv.Funct.Mater.18(2008)2172–2179.[27]M.Rajkumar,N.Meenakshisundaram,V.Rajendran,Mater.Charact.62(2011)469–479.[28]Y.F.Ma,Q.L.Feng,J.Solid State Chem.184(2011)1008–1015.[29]M.C.Chang,J.Tanaka,Biomaterials 23(2002)4811–4818.[30]J.H.Tao,H.H.Pan,Y.W.Zeng,X.R.Xu,R.K.Tang,J.Phys.Chem.B 111(2007)13410–13418.[31] E.Asenath-Smith,H.Y.Li,E.C.Keene,Z.W.She,L.A.Estroff,Adv.Funct.Mater.22(2012)2891–2914.[32] E.R.Morris,D.A.Rees,D.Thom,Carbohydr.Res.66(1978)145–154.Fig.8.Simulation diagram of HAp nanorod crystal growth in SF/SA fibrous hydrogels.293J.Ming et al./Materials Science and Engineering C 51(2015)287–293。