氨基酸對(duì)水熱合成羥基磷灰石纖維形貌的影響

齊美麗，肖桂勇，呂宇鵬

(1山東大學(xué) 材料液固結(jié)構(gòu)演變與加工教育部重點(diǎn)實(shí)驗(yàn)室，濟(jì)南250061；2山東大學(xué) 材料科學(xué)與工程學(xué)院，濟(jì)南250061；3 山東大學(xué) 蘇州研究院，江蘇 蘇州215123)

氨基酸對(duì)水熱合成羥基磷灰石纖維形貌的影響

齊美麗1,2,3，肖桂勇1,2,3，呂宇鵬1,2,3

(1山東大學(xué) 材料液固結(jié)構(gòu)演變與加工教育部重點(diǎn)實(shí)驗(yàn)室，濟(jì)南250061；2山東大學(xué) 材料科學(xué)與工程學(xué)院，濟(jì)南250061；3 山東大學(xué) 蘇州研究院，江蘇 蘇州215123)

以Ca(NO3)2·4H2O和(NH4)2HPO4的水溶液為前驅(qū)體，基于生物礦化的基本原理，利用水熱法制備結(jié)晶度較高的羥基磷灰石(HA)纖維，重點(diǎn)研究酸性氨基酸L-谷氨酸(Glu)、中性氨基酸L-苯丙氨酸(Phe)和堿性氨基酸L-賴氨酸(Lys)的添加對(duì)產(chǎn)物物相和形貌的影響。結(jié)果表明：添加這3種氨基酸均對(duì)產(chǎn)物的物相影響不大，制得樣品的主要組成相都是HA，部分樣品含有少量碳酸鈣。3種氨基酸的加入均改變纖維沿c軸生長(zhǎng)的趨勢(shì)：加入Glu后得到球狀形貌的HA，Lys的加入使得產(chǎn)物形貌變得不均勻，而加入Phe后得到分散性較好的棒狀纖維。

羥基磷灰石纖維；氨基酸；水熱法；形貌

羥基磷灰石(Hydroxyapatite，HA)是組成人體骨骼和牙齒主要的無機(jī)成分，因具有優(yōu)異的生物相容性、骨傳導(dǎo)性、生物活性及安全無毒性等諸多優(yōu)點(diǎn)，被廣泛應(yīng)用于硬組織缺損的修復(fù)與再生以及載藥釋藥等領(lǐng)域[1-4]。眾所周知，HA屬于生物陶瓷，其脆性低這一致命缺陷極大地限制了其應(yīng)用。通過控制HA基生物材料的尺寸和形貌進(jìn)而改變材料的柔韌性等力學(xué)性能是一種可行的方法。但是，如何攻克尺寸瓶頸，制備出具有較好柔韌性且均勻連續(xù)的HA超長(zhǎng)纖維是一個(gè)具有很大挑戰(zhàn)性的難題。目前，制備HA纖維的方法主要有固相法[5]、熔鹽法[6]、水熱法[7]、溶劑熱法[8]、靜電紡絲法[9]等。其中，水熱法在得到結(jié)晶度高、粒度均一、形態(tài)規(guī)則的粒子上具有其他方法無可比擬的優(yōu)越性[10]，材料的尺寸和形貌的“人為控制”一直是材料研究者的理想目標(biāo)。除了調(diào)控水熱物化參數(shù)(溫度、壓力、反應(yīng)物濃度、pH值等)形成合適反應(yīng)環(huán)境來合成不同結(jié)晶形態(tài)的粒子外[11]，表面活性劑的添加為實(shí)現(xiàn)HA晶粒尺寸及形貌等的調(diào)控提供了一種有效途徑[12-15]。然而，表面活性劑分子對(duì)HA表面的特定吸附引入了一些有毒物相，另外有機(jī)溶劑的添加產(chǎn)生了如環(huán)保、健康等問題；因此，適宜的調(diào)節(jié)劑可以從生物體內(nèi)尋找啟發(fā)。

采用氨基酸調(diào)控HA晶體的生長(zhǎng)是一種自然選擇，因?yàn)榘被崾侨梭w的生理物質(zhì)，能夠通過血液循環(huán)在人體的所有組織中自由運(yùn)動(dòng)[16]，是HA成核生長(zhǎng)的有效調(diào)節(jié)劑。一般認(rèn)為離子通過擴(kuò)散、吸附等形式在晶體的表面沉積從而實(shí)現(xiàn)晶體的生長(zhǎng)，而有機(jī)/無機(jī)的界面給生物礦化創(chuàng)造了十分重要的活化點(diǎn)，活化點(diǎn)的存在使得生物分子控制晶體生長(zhǎng)成為可能[17]。前期研究表明：氨基酸通過靜電引力和氫鍵作用可以對(duì)HA晶體表面產(chǎn)生特定的吸附，從而阻礙HA的生長(zhǎng)[18-22]。然而，系統(tǒng)地研究不同種類的氨基酸對(duì)HA晶體形貌影響的報(bào)道較少。

本工作旨在研究酸性氨基酸L-谷氨酸(Glu)、中性氨基酸L-苯丙氨酸(Phe)和堿性氨基酸L-賴氨酸(Lys)對(duì)水熱法合成HA纖維形貌的影響，在反應(yīng)時(shí)間與反應(yīng)溫度為定值的情況下，分別觀察在加入不同量的同一種氨基酸大分子物質(zhì)時(shí)合成的HA晶體的形貌特點(diǎn)，為不同形貌和尺寸的HA晶體的制備以及更好地滿足不同性能要求提供了實(shí)驗(yàn)依據(jù)。

1 實(shí)驗(yàn)材料與方法

Ca(NO3)2·4H2O，(NH4)2HPO4，尿素，Glu，Phe和Lys均購(gòu)自國(guó)藥集團(tuán)化學(xué)試劑有限公司，分析純。按照Ca/P=1.8配制Ca(NO3)2·4H2O和(NH4)2HPO4兩種溶液后與1mol/L的尿素溶液混合，分別加入Glu，Phe和Lys后用體積比為1∶1的硝酸調(diào)節(jié)溶液的pH后磁力攪拌30min。Glu的加入量為0.2060，0.6180，1.0299g，Phe的加入量為0.2313，0.6938，1.5363g，Lys的加入量為0.2047，0.6140，1.0233g。將上述前驅(qū)體溶液倒入聚四氟乙烯內(nèi)襯的不銹鋼反應(yīng)釜中，控制填充度為80%，反應(yīng)溫度設(shè)定為180℃，保溫時(shí)間5h后自然冷卻至室溫。最后將產(chǎn)物離心，得白色沉淀，用去離子水清洗1次、無水乙醇清洗兩次后放入鼓風(fēng)干燥箱中干燥，得到最終產(chǎn)物。

采用Rigaku D/max-γB型X射線衍射儀(XRD)對(duì)產(chǎn)物的相組成進(jìn)行檢測(cè)分析，測(cè)試條件：銅靶(CuKα)；管電壓和管電流分別為40kV和100mA，掃描速率為4(°)/min，掃描范圍10°～60°。采用場(chǎng)發(fā)射掃描電鏡(FE-SEM)對(duì)產(chǎn)物的表面形貌進(jìn)行分析，加速電壓和加速電流分別設(shè)定為5kV和10μA，測(cè)試前進(jìn)行噴金處理。

2 結(jié)果與分析

2.1 物相分析

2.2 微觀形貌分析

本工作用到的氨基酸的等電點(diǎn)以及反應(yīng)前后溶液的pH值總結(jié)見表1，可知反應(yīng)前后溶液的pH均為堿性(8.4～9.1)，氨基酸的加入對(duì)反應(yīng)后溶液的pH值影響不大。

2.2.1 Glu對(duì)產(chǎn)物形貌的影響

圖2是添加不同量的Glu后制備的產(chǎn)物SEM照片，其中圖2(a)為無添加劑的原始試樣形貌，圖2(b)，(c),(d)分別為添加0.2060，0.6108g和1.0299g的Glu后得到的產(chǎn)物形貌?？梢钥闯?，未添加任何種類氨基酸后反應(yīng)得到的產(chǎn)物從一個(gè)形核點(diǎn)出發(fā)生長(zhǎng)，微觀形貌呈帶狀；添加Glu后部分帶狀纖維從形核點(diǎn)處脫落；隨著Glu添加量的增加，產(chǎn)物形貌變化較大，趨近于花瓣?duì)钗⑶?，這與XRD測(cè)試結(jié)果相一致。

Glu能夠?qū)A形貌產(chǎn)生明顯的影響，特別是當(dāng)濃度增加時(shí)，其影響的效果尤為顯著，其主要原因是Glu能夠影響HA晶體的生長(zhǎng)過程。通常認(rèn)為，酸性氨基酸對(duì)結(jié)晶的影響與靜電作用和氨基酸的螯合作用有關(guān)。在HA成核初期，Glu就會(huì)對(duì)其產(chǎn)生影響。亞穩(wěn)態(tài)的磷酸八鈣OCP(Ca8H2(PO4)6·5H2O)首先形核，然后OCP水解轉(zhuǎn)化為HA晶體[27]。含有羧基基團(tuán)的Glu優(yōu)先吸附在片狀OCP晶體的(100)面，是一種固定吸附，在OCP的(100)面轉(zhuǎn)化成HA的(100)面后，這種吸附作用仍然存在，致使其在很大程度上影響了OCP的形貌以及后續(xù)向HA的轉(zhuǎn)變[16]。隨著水熱反應(yīng)的進(jìn)行，尿素不斷分解，溶液成堿性，在靜電引力的作用下，Glu將主要吸附在(100)面，阻礙HA晶體在a，b面的生長(zhǎng)。此外，Glu與HA表面強(qiáng)烈的化學(xué)鍵作用也是Glu阻礙HA生長(zhǎng)的一大因素，二者最終導(dǎo)致產(chǎn)物呈花瓣?duì)畹奈⑶颉?/p>

圖1 未添加及分別添加不同量的氨基酸后制備產(chǎn)物的XRD圖 (a)無添加劑；(b)添加0.2060，0.6180g和1.0299g谷氨酸；(c)添加0.2313,0.6938g和1.1563g苯丙氨酸；(d)添加0.2047,0.6140g和1.0233g賴氨酸Fig.1 XRD patterns of the products synthesized with and without the addition of different amounts of amino acids (a)without any addition;(b) with addition of Glu at 0.2060, 0.6180g and 1.0299g;(c)with addition of Phe at 0.2313, 0.6938g and 1.1563g;(d)with addition of Lys at 0.2047, 0.6140g and 1.0233g

表1 3種氨基酸的等電點(diǎn)pI及反應(yīng)前后溶液的pH值
Table 1 Isoelectric point of three amine acids and the pH values of three kinds of solutions under different hydrothermal treatment by the three pH regulators

AminoacidpIpHvalueBeforereactionAfterreactionGlu3.223.0308.9043.0488.4553.0158.748Phe5.483.5158.8963.4988.9453.4858.806Lys9.743.5159.0673.5168.9703.5188.891

2.2.2 Phe對(duì)產(chǎn)物形貌的影響

圖3分別為添加0.2313(圖3(a),(b)),0.6938g(圖3(c))和1.5363g(圖3(d))Phe后得到的產(chǎn)物SEM照片?？梢钥闯?，加入0.2313g的Phe反應(yīng)得到的HA部分保留了團(tuán)聚體形貌，纖維從形核點(diǎn)處脫落，隨Phe濃度的增加，團(tuán)聚現(xiàn)象逐漸減弱，纖維呈棒狀，分散性較好。

2.2.3 Lys對(duì)產(chǎn)物形貌的影響

圖4(a)，(b),(c),(d)分別為添加0.2047，0.6140g和1.0233g的Lys后得到的產(chǎn)物SEM照片。可以看出，加入0.2047g的Lys反應(yīng)得到的HA部分保留了團(tuán)聚體形貌。隨Lys濃度的增加，團(tuán)聚體的形貌逐漸消失，帶狀纖維有斷裂的現(xiàn)象，纖維尺寸不均勻。

圖2 添加不同量的Glu后制備的產(chǎn)物SEM照片 (a)0g;(b)0.2060g;(c)0.6108g;(d)1.0299g Fig.2 SEM images of the products with addition of different amounts of Glu (a)0g;(b)0.2060g;(c)0.6108g;(d)1.0299g

圖3 添加不同量的Phe后制備的產(chǎn)物SEM照片 (a),(b)0.2313g;(c)0.6938g;(d)1.5363gFig.3 SEM images of the products with addition of different amounts of Phe (a),(b)0.2313g;(c)0.6938g;(d)1.5363g

圖4 添加不同量的Lys后制備的產(chǎn)物SEM照片 (a)，(b)0.2047g;(c)0.6140g;(d)1.0233gFig.4 SEM images of the products with addition of different amounts of Lys (a)，(b)0.2047g;(c)0.6140g;(d)1.0233g

3 結(jié)論

(1)采用酸性的L-谷氨酸(Glu)、中性的L-苯丙氨酸(Phe)和堿性的L-賴氨酸(Lys)作為調(diào)節(jié)劑，調(diào)控水熱反應(yīng)過程中HA纖維的生長(zhǎng)。

(2)添加3種氨基酸后得到的樣品主晶相都是HA，部分樣品含少量碳酸鈣；不同氨基酸的加入導(dǎo)致晶體擇優(yōu)取向發(fā)生了變化，從Glu，Phe到Lys的加入，晶體從(211)晶面逐漸轉(zhuǎn)變?yōu)?300)晶面擇優(yōu)生長(zhǎng)。

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(本文責(zé)編：高 磊)

Effect of Amino Acids on Morphology of Hydrothermally Synthesized Hydroxyapatite Fibers

QI Mei-li1,2,3,XIAO Gui-yong1,2,3,LYU Yu-peng1,2,3

(1 Key Laboratory for Liquid-Solid Structural Evolution and Processing of Materials (Ministry of Education),Shandong University,Jinan 250061, China;2 School of Materials Science and Engineering,Shandong University,Jinan 250061,China;3 Suzhou Institute,Shandong University,Suzhou 215123,Jiangsu,China)

Based on the basic principle of biomineralization, hydroxyapatite fiber (HAF) with high crystallinity was fabricatedviaa hydrothermal route with Ca(NO3)2·4H2O and (NH4)2HPO4as the resources, respectively. Effects of the addition of acidic amino acid L-glutamic acid (Glu), neutral amino acid L-phenylalanine (Phe) and basic amino acid L-lysine (Lys) on the phase composition and morphology of the obtained products were laid special emphasis on. The results show that the products obtained by using the three amino acids are all hydroxyapatite (HA) phase with minor CaCO3in some samples. Meanwhile, all of the amino acids inhibit the growth of the fibers. Spherical morphology exists when Glu is added, the homogeneity of the fibers deteriorates with the addition of Lys. However, rod-like fibers with good uniformity can be obtained with the addition of Phe.

hydroxyapatite fiber;amino acid;hydrothermal method;morphology

山東大學(xué)基本科研業(yè)務(wù)費(fèi)專項(xiàng)資金(2015CJ018)；江蘇省青年科學(xué)基金(BK20140412)；山東省優(yōu)秀中青年科學(xué)家科研獎(jiǎng)勵(lì)基金項(xiàng)目(BS2013CL030)

2016-08-22；

2017-03-15

呂宇鵬(1970-)，男，教授，博士，主要研究方向?yàn)樯锊牧霞敖饘俨牧媳砻娓男?，?lián)系地址：山東省濟(jì)南市經(jīng)十路17923號(hào)山東大學(xué)千佛山校區(qū)(250061)，E-mail:biosdu@sdu.edu.cn

10.11868/j.issn.1001-4381.2016.000994

TB321

A

1001-4381(2017)05-0046-06