Effect of polydopamine on the biomimetic mineralization of mussel-inspired calcium phosphate cement in vitro

Materials Science and Engineering C 44 (2014) 44–51

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Materials Science and Engineering C

j ourna l homepage: www.e lsev ie r .com/ locate /msec

Effect of polydopamine on the biomimetic mineralization ofmussel-inspired calcium phosphate cement in vitro

Zongguang Liu a, Shuxin Qu a,⁎, Xiaotong Zheng a, Xiong Xiong a, Rong Fu b, Kuangyun Tang b,Zhendong Zhong b, Jie Weng a

a Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Chinab Department of Plastic Surgery, Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu 610041, China

⁎ Corresponding author. Tel.: +86 28 87601897; fax: +E-mail address: [email protected] (S. Qu).

http://dx.doi.org/10.1016/j.msec.2014.07.0630928-4931/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

Article history:Received 10 April 2014Received in revised form 27 June 2014Accepted 27 July 2014Available online 12 August 2014

Keywords:Calcium phosphate cementPolydopamineBiomimetic mineralization

Inspired by the excellent adhesive property of mussel adhesive protein, we added polydopamine (PDA) tocalcium phosphate cement (PDA–CPC) to enhance its compressive strength previously. The mineralizationand mechanism on PDA–CPC were investigated by soaking it in simulated body fluid in this study. The resultsindicated that PDA promoted the conversion of dicalcium phosphate dihydrate and α-tricalcium phosphate tohydroxyapatite (HA) in the early stage but inhibited this conversion subsequently. PDA promoted the rapidmineralization on PDA–CPC to form a layer of nanoscale calcium phosphate (CaP) whereas there was no CaPformation on the control-CPC after 1 d of soaking. This layer of nanoscale CaP was similar to that of naturalbone, which was always observed during soaking. X-ray photoelectron spectroscopy showed that the peak ofC_O of PDA existed in the newly formed CaP on PDA–CPC, indicating the co-precipitation of CaP with PDA.Furthermore, the newly formed CaP on PDA–CPC was HA confirmed by transmission electron microscopy,which the newly formedHAwas in associationwith PDA. Therefore, PDA increased the capacity ofmineralizationof CPC and induced the formation of nanoscale bone-like apatite on PDA–CPC. Thus, this provides the feasibleroute for surface modification on CPC.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Calcium phosphate cement (CPC) has been an alternative to autolo-gous and allogenic bone grafting for bone defects due to its similar com-position to those of natural bone [1]. The inadequate mechanicalproperties of CPC result in its inefficient application in load-bearingbone defects unfortunately [2]. Studies focus on improving themechan-ical property of CPC by admixing additiveswith powder or liquid of CPC,i.e., polymer fibers [3], cellulose [4], carbon nanotubes [5], strontium [6],and magnesium [7]. The additives are usually randomly distributed inthe CPC, resulting in composites with relatively isotropic properties orlimited CPC mechanical strength improvement [8].

Many studies inspired by the adhesion of mussels to ships or rocksunder wet conditions have reported that the adhesive proteins secretedbymussels mainly contain dihydroxyphenylalanine (DOPA) and lysine.Similarly, dopamine (DA) contains the same catechol functional groupas that of the side chain of DOPA residues and the same amine functionalgroup of lysine residues [9], which proves that DA is a strong adhesivewith a wide range of inorganic and organic materials due to its self-polymerization to form polydoapmine (PDA) films [10]. The extraordi-nary adhesive property of PDA is due to its abundant catechol moieties

86 28 87601371.

[11], which form covalent or strong non-covalent interactions (hydrogenbonds or stacking interactions) with substrates [12]. PDA has been ap-plied in surface modification, typically in biomaterials, because it is lesstime-consuming than other chemical techniques and does not requireorganic solvents [13]. PDA coating significantly promotes the adhesionand proliferation of osteoblasts (MC3T3-E1) [14] and human umbilicalvein endothelial cells, whereas it remarkably decreases those of humanumbilical artery smooth muscle cells [15]. Hong et al. [9] reported thatPDA is nontoxic, and can reduce the in vivo toxicity of poly-L-lactic andcadmium selenide quantum dots in contact with tissue or blood. Theseshow that PDA is biocompatible and can be used in biomaterials. PDAhas been recently used as the intermediate layer to immobilize silver[16,17], or HA nanoparticles and RGD [18], or heparin [19], or growthfactors [20,21] on a biomedical metal.

PDA obtained through the oxidation of DA in Tris–HCl buffer solution(pH = 8.5) was added into CPC (PDA–CPC) in our previous study [22],which significantly increased the compressive strength of PDA–CPC.PDA promoted the dicalcium phosphate dihydrate (DCPD) conversioninto HA after setting for 24 h. In addition, it is reported that PDA hasthe capacity to concentrate Ca2+ using its catechol moieties, whichresults in the local supersaturation of Ca2+ and the formation of HAcrystals on the substrate [23,24]. Several biomaterials (i.e., titanium,polyester fibers and carbon nanotubes) are easily covered by apatitelayers with the aid of PDA after soaking in simulated body fluid (SBF)

Fig. 1. XRD patterns of PDA–CPC (a) and the control-CPC (b) surfaces before and aftersoaking in SBF for 1 d, 3 d, 5 d, 7 d, 10 d, and 14 d. PDA–CPC and the control-CPC sampleswere suspended in SBF to avoid the deposition of CaP sediment on their surfaces.

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[11,23]. In addition, PDA coating has also been used in dentistry to pro-mote dentin remineralization, such that all dentin tubules are filled bydensely packed hydroxyapatite crystals [25]. Nonetheless, the effect ofPDA on PDA–CPC mineralization in terms of its Ca2+ concentration hasnot been explored.

In this study, PDA–CPC was prepared according to our previousstudy, and was then soaked in SBF to study its in vitro mineralization.The morphology, chemical composition, crystal structure, and mineral-ization mechanism of PDA–CPC were investigated.

2. Materials and methods

2.1. Calcium phosphate cement

CPC, which consists of 58 wt.% α-tricalcium phosphate (α-TCP),25 wt.% DCPD, 8.5 wt.% HA, and 8.5 wt.% analytical grade CaCO3, wasused in this study, with minor modifications [26]. HA and α-TCP werepurchased from the National Engineering Research Center in Biomate-rials, Sichuan University, Chengdu, China. CaCO3 was purchased fromKelong Chemical Inc., Chengdu, China. DCPDwas synthesized in the lab-oratory [27]. All starting powders were mixed and dried overnight at60 °C. The cement solution was prepared by mixing Tris–HCl buffer(10 mM, pH 8.5) with 40 mg/mL DA (Sigma-Aldrich, Germany) [22].The cement solution was exposed to air for 2 d to oxidize and crosslink.The cement solution without DAwas used as control. The experimentalCPC and the control CPC are referred to as PDA–CPC and the control-CPC, respectively.

CPC was prepared by the mixing cement solution with startingpowder at the ratio of 0.3 mL:1 g. After mixing, the pastes were placedin cylindrical molds to form specimens with dimensions of 15 mm indiameter and 5 mm in height. The disk specimens were removed fromthe mold to an atmosphere of 100% relative humidity at 37 °C for 24 h.

2.2. Biomimetic mineralization of CPC

All CPC specimens were cleaned using ultrasound for 30 min andsuspended in SBF [28] in a shaking (100 rpm) water bath at 37 °C for1 d, 3 d, 7 d, 10 d, and 14 d. CPCs were rinsed with distilled water anddried in a desiccator without heating.

2.3. Characterization of biomimetic mineralization of CPC

2.3.1. X-ray diffractionX-ray diffraction analysis (XRD; X'Pert Pro, Philips, TheNetherlands)

was performed to identify the crystalline phases on the surfaces of allthe CPC specimens after soaking in SBF. The diffraction patterns weredetermined with a scanning angle 2θ ranging from 4° to 50° in step-scan intervals of 0.02°, with Cu Kα radiation at 40 kV and 40 mA.

2.3.2. Attenuated total reflectance-Fourier transform infrared spectroscopyAttenuated total reflectance (ATR) Fourier-transform infrared spec-

troscopy (FTIR; 5700, Nicolet, USA) was used to identify the functionalgroups on the surfaces of all the CPC specimens. The spectra werecollected over 4000 cm−1 to 400 cm−1, and a reflection attachment(Spectra-Tech, FT80 RAS) at the incident angle of 80° was used.

2.3.3. Scanning electron microscopyScanning electron microscopy (SEM; Quanta 200, FEI, The

Netherlands) and energy-dispersive spectroscopy (EDS; 7760/68ME,EDAX, USA) were used to observe the morphology and identify of theCa/P ratios of the deposited phases on the CPC surface. The sampleswere sputter-coated with gold prior to examination.

2.3.4. X-ray photoelectron spectroscopyX-ray photoelectron spectroscopy (XPS; XSAM800, Kratos, UK) was

performed to evaluate the relative contents of different elements on the

CPC surface, using Al Kα radiation (1486.6 eV) as excitation source, andusing voltage and current values of 12 kV and 11 mA, respectively.

2.3.5. Transmission electron microscopyTransmission electron microscopy (TEM; JEM-2100F, Jeol, Japan)

and EDS (832, Oxford, UK) were used to characterize the calcium phos-phate precipitation on the surface of PDA–CPC soaked in SBF for 14 d.PDA–CPC was embedded in ethoxyline resin, and was cut into 100 nmsections using a microtome (UC7, Leica, Germany). The sections wereanalyzed through selected area electron diffraction (SAED) and high-resolution TEM (HRTEM).

3. Results and discussion

In our previous study, the mechanical strength of PDA–CPC was sig-nificantly improved compared to that of the control CPC. Further studyshowed that PDA promoted the conversion of the more soluble DCPDto HA during setting [22]. In addition, the catechol in PDA concentratedcalcium ions and affected themineralization of calcium-basedmaterials[23,29]. Therefore, the effect of PDA on the subsequent CPC conversionand mineralization in SBF in vitro was studied in this study.

Fig. 1 shows the XRD patterns of PDA–CPC and the control-CPC sur-faces before and after soaking in SBF for 1 d, 3 d, 5 d, 7 d, 10 d, and 14 d.Fig. 1a and b shows that themain phaseswere DCPD, CaCO3,α-TCP, andHA before soaking. The relative intensities of the diffraction peaks ofDCPD in PDA–CPC were obviously reduced compared to that of thecontrol-CPC. This was because of the accelerated conversion of DCPD

Fig. 3. Photographs of PDA–CPC and the control-CPC after soaking in SBF for 1 d, 3 d, 5 d,7 d, 10 d, and 14 d. The top and bottom rows represent the control-CPC and PDA–CPC,respectively. A change in color of the PDA–CPC surface from bright to dark withsoaking time indicates that polydopamine (PDA) gradually formed on the PDA–CPCsurface. (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

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during PDA–CPC setting [22]. The DPCD diffraction peaks of both PDA–CPC and the control-CPC disappeared after soaking in SBF for 1 d. The in-tensities of the CaCO3 and α-TCP diffraction peaks gradually decreasedduring the soaking time. After 7 d of soaking in SBF, the primary diffrac-tion maximum of CaCO3 disappeared both in PDA–CPC and in thecontrol-CPC. The intensities of the HA diffraction peaks graduallyincreased. The crystalline phases of the surfaces on both PDA–CPC andthe control-CPC soaked in SBF for 14 d were mainly α-TCP and poorlycrystalline HA.

Fig. 2 shows the variation of the quantitative phase percentages onPDA–CPC and the control-CPC vs. the soaking time. The percentage ofeach phase (Px) was calculated according to the following equationbased on XRD patterns:

Px ¼IxIsum

where Ix represents the intensity of the diffraction primarymaximumofthe X phase, and Isum represents the summation of the DCPD, CaCO3, α-TCP, and HA intensities of the diffraction primary maximum of thepresent phase.

Fig. 2 shows that theDCPDpercentages on PDA–CPCwere lower thanthose of the control-CPC, whereas the HA percentages on PDA–CPCwerehigher than those of the control-CPC before soaking. The CaCO3 and HApercentages of PDA–CPC were higher than those of the control-CPCbefore and after soaking for 1 d, 3 d, and 5 d for CaCO3, and for 1 d, 3 d,5 d, and 7 d for HA. The α-TCP percentages on PDA–CPC were lowerthan those of the control-CPC after soaking for 1 d, 3 d, 5 d, and 7 d. Incontrast, the α-TCP percentages of PDA–CPC were higher than those ofthe control CPC, whereas the HA percentages of PDA–CPC were lowerthan those of the control CPC after soaking for 10 d and 14 d. Thissuggested that PDA promotes the conversions of DCPD and α-TCP toHA at an early stage (before soaking, 1 d, 3 d, 5 d, and 7 d), but impededthe conversions afterwards.

PDA has been recently used for the surface modification of metal orpolymer biomaterials to improve their bioactivity and hydrophilicity[14,23]. PDA-modified substrate accumulated Ca2+ and established alocal Ca2+ supersaturation, facilitating HA crystal formation on thesurface [23,24].

In this study, non-polymerized DAwas released from PDA–CPC intoSBF and polymerized to PDA on the surface of PDA–CPC, similar to thesurface modification of metals or polymer biomaterials [14,23]. ThePDA–CPC images showed that the color of PDA–CPC obviously changedfrom bright to dark during the soaking in SBF for 1 d, 3 d, 5 d, 7 d, 10 d,

Fig. 2. The variation of the quantitative phase percentages on PDA–CPC and the control-CPC vs. the soaking time in SBF. PDA promoted the conversion of DCPD and α-TCP to HAat early stages (before soaking, 1 d, 3 d, 5 d, and 7 d), but subsequently impeded the con-version (10 d and 14 d).

and 14 d (Fig. 3). The color change indicates the formation of PDA,which was in a manner of melanin formation of DA [30]. The hydroxylgroups of PDA easily deprotonate in alkaline solutions to form phenoxyanions, which is due to the reduction of electron density of the p-πconjugated O-benzene ring system, and results in the affinity of PDAfor metal ions [31]. The formed phenoxy anion concentrated Ca2+

from the SBF on the surface of PDA–CPC and promoted HA mineralnucleation. The formed HA, accompanied by polymerized DA coveringthe α-TCP reaction sites in PDA–CPC after soaking in SBF in the laterstage, e.g., 10 d and 14 d, reduced the conversion of α-TCP to HA.

Fig. 4 shows the FTIR spectra of PDA–CPC and the control-CPC beforeand after soaking in SBF for 7 d and 14 d. Fig. 4a shows that the peaks at526 cm−1 were assigned to the HPO4

2− of DCPD [32], the peaks at710 cm−1, 872 cm−1, and 1420 cm−1 were assigned to the CO3

2− groupof CaCO3 [33,34], and the peaks at around 472 cm−1, 563 cm−1,602 cm−1, 1026 cm−1, and 1062 cm−1 were ascribed to the vibrationsof PO4

3− [35–37]. After soaking in SBF for 7 d and 14 d (Fig. 4b and c), thebands of the v3 vibration of P\O shifted from 1062 cm−1 to 1030 cm−1

1026 cm−1 for PDA–CPC and 1044 cm−1 and 1042 cm-1 for the control-CPC, whereas other bands did not obviously shift. This differencemightbe associated with HA precipitation with the polymerization of the re-leased DA.

The incorporated PDA in the newly precipitated CaP may alter thechemical environment of P\O, resulting in the obvious red-shift ofthe v3 vibration of P\O. The peak at 1030 cm−1 was reported to corre-spond to the v3 vibration of P\O in cancellous bone [38], whichwas theconsequence of in vivo mineralization and was associated with theregulation of proteins, enzymes, etc. The adsorption peak of P\O at1030 cm−1 only appeared in the FTIR spectra of PDA–CPC after soakingin SBF for 7 d and 14 d. The FTIR spectra suggested that the formed CaPon the surface of PDA–CPC has increasing similarity to the CaP of naturalbone with increasing time, owing to the influence of PDA.

The SEM images of PDA–CPC and the control-CPC surfaces beforeand after soaking in SBF for 1 d, 7 d, and 14 d are shown in Fig. 5. Themorphologies of both PDA–CPC and the control-CPC were determinedby the particles of the CPC powder before soaking. The PDA–CPC surfaceappeared denser than that on the control-CPC (Fig. 5a0 and b0) due tothe excellent adhesive property of PDA. In this study, PDAwas obtainedthrough the oxidation of the liquid phase of the DA-containing cement,and binding with the initial CPC powder particles during the settingprocess.

After 1 d of soaking in SBF, a layer of spherical precipitations withmicro-scales formed on PDA–CPC (Fig. 5a1). EDS showed that thislayer precipitates to be CaP with the molar ratio of 1.17. The sphericalCaP consisted of short wire-like CaP at the nanoscale (inset ofFig. 5a1), which resembles that of natural bone. In contrast, the surface

Fig. 4.ATR-FTIR spectra of PDA–CPC and the control-CPC surfaces before and after soakingin SBF for 7 d and 14 d. a: before soaking; b and c: after soaking for 7 d, and 14 d,respectively.

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on the control-CPC after 1 d still consisted of the initial particles, but theparticle sizes were less than those before soaking (Fig. 5b1). In thisstudy, all specimens were suspended in SBF to avoid the pure sedimen-tation of CaP on the surfaces. In general, the term bioactivity has beenattributed to materials which form a superficial layer of bone-like apa-tite during soaking in SBF [39]. The accelerated mineralization capacityof PDA–CPC indicated that PDA increased its bioactivity at an earlystage. After soaking for 7 d, the surface of PDA–CPC (Fig. 5a2) was alsocovered with a layer of spherical CaP, but the size of spherical CaP wasbigger compared to those after 1 d. In detail, the precipitation CaP wasstill wire-like with nanoscale (inset of Fig. 5a2). The surface of thecontrol-CPC (Fig. 5b2) was also covered with a spherical CaP layer, butthe detail of the morphology of this spherical CaP layer was differentfrom that on the PDA–CPC surface, showing nanoscale plate-like CaP(inset of Fig. 5b2). Fig. 5a3 shows the PDA–CPC surface after soaking in

Fig. 5. SEM images of the surface on PDA–CPC and the control-CPC before and after soaking in Srespectively; b0, b1, b2, and b3: the surface on the control-CPC before and after soaking for 1 d,soaking for 14 d, respectively.

SBF for 14 d, on which bigger spherical CaPs without significant bound-aries were observed. The mineralization was suggested to graduallyproceed with time. The inset of Fig. 53 shows spherical CaP agglomer-ates consisting of short rod-like particles with nanoscale on PDA–CPC,which were similar to those of natural bone [40]. Plate-like CaPs onthe micro-meter scale were observed on the control-CPC, as shown inFig. 5b3, which were obviously different from those on PDA–CPC after14 d of soaking.

Fig. 5a4 and b4 show cross-sections of PDA–CPC and the control-CPCafter soaking for 14 d, respectively. PDA–CPC was covered with hemi-spherical CaP, whereas the surface of the control-CPC was coveredwith plate-like CaP. The internal morphologies of PDA–CPC and thecontrol-CPC were different. The former showed dense integrated bulkdue to the adhesive property of PDA, whereas the latter showed looseparticles. The transition between the newly formed CaP layer and thebulk of PDA–CPC was more continuous compared to that of thecontrol-CPC.

The SEM images showed that PDA significantly affected themineral-ization of CPC because it promoted rapid mineralization and the forma-tion of a CaP layer with a nanoscale structure on PDA–CPC during thesoaking in SBF. The rapid mineralization on PDA–CPC was probably as-sociated with the ability of the catechol-containing PDA to concentrateCa2+ and to induce CaP crystallization [11,23]. The CaP layer with thenanoscale structure changed its morphology from wire-like to shortrod-like over time, which resembles natural bone [40]. The nanostruc-tured CaP in the CPC–bone interface may improve the biocompatibilityand characteristics, such as surface area, roughness, protein adsorbance,and osteoinductivity [41], which are advantageous for the subsequentadhesion of osteoblasts.

The EDS of PDA–CPC soaked in SBF for 1 d and 7 d indicated that theCa/P molar ratios of the newly formed crystals on the surface of PDA–CPC were 1.17 and 1.03, respectively. This was lower than that of HA(Ca/P = 1.67), indicating calcium-deficient apatite. The Ca/P molarratio of the newly formed CaP after 7 d of soaking was lower than thatafter soaking for 1 d. This might be because the newly formed CaPlayer on the surface of PDA–CPC was too thin after soaking for 1 d,and partly because PDA–CPC substrate material was detected by theelectron beam of EDS. However, the newly formed CaP layer was thickenough for the detection depth of EDS after soaking for 7 d, resultingin an apparently lower Ca/P molar ratio compared to that soaked inSBF for 1 d.

Fig. 6a shows the XPS spectra of PDA–CPC and the control-CPC aftersoaking in SBF for 14 d. C (C1s), O (O1s), N (N1s), and Ca (Ca2s, Ca2p,Ca3s and Ca3p) were on PDA–CPC and the control-CPC surfaces. The in-tensities of Ca and O on PDA–CPCwere lower than those on the control-CPC, whereas its intensities of C and N were higher than those on thecontrol-CPC. Allied to the XRD patterns of the surface, the high intensi-ties of Ca andOon the control-CPCwere attributed to theHA andα-TCP.The high intensities of C and N on PDA–CPC surface were attributed toPDA. XPS has a sensitivity of only few nanometers in the depth, so PDAwas hypothesized to have co-precipitated with CaP during mineraliza-tion. Fig. 6b–e show the high-resolution XPS spectra of the selectedC1s, O1s, N1s, and Ca2p. In terms of Gauss fitting, the C1s peaksof PDA–CPC could be divided into four main peaks (Fig. 6b). Peak I(284.7 eV) is assigned to the carbon–hydrogen (C\H), peak II(286.4 eV) is corresponded to the typical single bond of carbon in con-junctionwith oxygen or nitrogen (C\N/C\OH), peak III (287.9 eV) is at-tributed to carbonate-type carbon from impurities in CaP, and peak IV(288.7 eV) is owing to C_O [30,42–45]. Peaks I, II, and III of the C1speakswere also observed in the XPS of the control-CPC at the same bind-ing energies, but peak IVwas not found. The C_O bond exists in quinineafter the polymerization of DA, resulting in the peak IV of C1s in XPS.

BF. a0, a1, a2, and a3: the surface on PDA–CPC before and after soaking for 1 d, 7 d, and 14 d,7 d, and 14 d, respectively; a4 and b4: cross sections of PDA–CPC and the control-CPC after

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The difference of the Gauss fitting of C1s peaks indicated that DA poly-merized on the surface with CaP precipitation during soaking in SBF,whichwas consistent with the XRD and EDS. Fig. 6c shows the XPS spec-tra of O1s on PDA–CPC and the control-CPC. A new peak (III) at 533.3 eVwas found at the O1s XPS spectra of PDA–CPC, which corresponds toC_O [46], which demonstrated that the DA polymerization accompa-nied the CaP layer formation. In addition, the O1s spectrum on thecontrol-CPC surface could be deconvoluted into two peak components.Peak I located at 531.2 eV corresponds to the O1s in the P_O groups

Fig. 6. XPS spectra of surface on PDA–CPC and the control-CPC after soaking in SBF for 14 d. a: sCa2p, respectively.

from α-TCP and HA, and peak II at 532.7 eV is attributed to the basic\OH of the O1s from HA [47]. The major N1s peak near 399.5 eV(shown as Fig. 6d) is due to the N\H of the amine group of PDA [48]and to a minor extent, to the adsorption of Tris-buffer on the surfaces.Fig. 6e shows that the major Ca2p contribution deconvoluted into twopeaks, at 350.7 (Ca2p 3/2) and 347.2 eV (Ca2p 1/2), which are attributedto HA and α-TCP, respectively [49,50]. The high-resolution XPS spectraindicated that the peaks for the C1s and N1s species of PDA–CPC weresubstantially higher than those on the control CPC, due to the existence

urvey scan XPS spectra; b, c, d, and e: the high-resolution XPS spectra of C1s, O1s, N1s, and

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of the benzene ring groups,methyl, and amine groups in PDA,which alsocaused the changes of the color of the surface (Fig. 3). PDAwas shown toco-precipitate with newly formed CaP during themineralization. The in-tensities of the Ca2p peaks of PDA–CPC were lower than those of thecontrol-CPC due to the formation of PDA on the surface, which coveredthe newly formed CaP, and only a few signals of Ca2p could be detectedby XPS due to the penetration depth in nanometer scale.

Fig. 7a–e shows the TEM micrographs of PDA–CPC after 14 d ofsoaking. Three different regions (marked (1), (2), and (3)) were dis-tinguished by the difference of electron density and contrast, show-ing newly formed nanoscale precipitates on the surface of PDA–CPC.Regions (1) and (3) show dark contrast, resulting from the biggeratomic number of the newly formed CaP and PDA–CPC substrate,respectively. Region (2) appeared as a lighter contrast, resultingfrom the lighter atomic number of C, N, O and H of PDA that co-precipitated with CaP on PDA–CPC. Regions (1) and (2) were ana-lyzed by EDS, as shown in Fig. 7b. Themain elements in region (1) in-cluded Cl, K, Na, Mg, S, O, and C, which were components of SBF,whereas, the region (2) included N and C as major components be-sides the elements found in region (1), which supported PDA andCaP co-precipitation.

Fig. 7. TEM images of PDA–CPC surface after 14 d soaking. a: TEM bright-field images, the regirespectively; b: EDS spectra of areas (1) and (2); c: SAEDpattern of region (1); d: HRTEM imageFFT patterns of the HRTEM fringes of regions I, II, and III, respectively. The bubble areas (markeother areas all exhibit typical crystalline fringes. TEM images demonstrate that the newly form

Fig. 7c shows the SAED pattern of region (1). The distance betweenthe diffraction center and diffraction spotswasmeasured and calculatedreferred to the bar (5 1/nm). This distance was the interplanar spacing(d) corresponding to a group of crystal planes. Then, the crystal phaseand crystal indexes were determined compared these “d” data tothose of standard PDF (the Powder Diffraction File) cards of calciumphosphates. As a result, the series of “d” data were consistent withthose of HA. The newly formed crystal is determined as HA. Finally,the different diffraction planes, (111), (210), (002), (311), (310),(213), and (004) of HA were determined and marked in SAED pattern.The lattice spacing calculated from HRTEM (Fig. 7d) and fast Fouriertransform (FFT) patterns (Fig. 7e) was 0.25 nm, 0.26 nm, 0.27 nm,and 0.28 nm, which correspond to crystal planes (301), (202), (112),and (211) of HA, respectively. The bubble areas (marked B in themicrograph) did not have fringe patterns like crystalline materials,whereas the other areas exhibited typical crystalline fringes. PDA thatco-precipitated with HA can be attributed to this bubble area.

Summarizing the above results and discussion, and considering pre-vious studies [11,23,24], the mineralization of CaP on PDA–CPC in SBFcan be described as the following. Non-ploymerized DA in PDA–CPCwas quickly released into SBF. Then DA oxidized and polymerized to

ons (1), (2), and (3) represent the newly formed CaP, PDA layer, and PDA–CPC substrate,of region (1), and regions I, II, and III were chosen for the FFT studies; e: the correspondingd B in the micrograph) did not have fringe patterns like crystalline materials, whereas theed nanoscale CaP on PDA–CPC was HA, and the precipitation of HA is associated with PDA.

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PDA, with CaP precipitation on the surface of PDA–CPC in SBF. Mean-time, the catechol-containing PDA accumulated Ca2+ from the SBF tothe surface, which resulted in the local supersaturation of Ca2+ andthe nucleation and growth of CaP minerals [24]. However, the furthertransformation of α-TCP to HA in PDA–CPC was inhibited by the syner-gistic effect of PDA and the newly formed CaP [51]. Therefore, thisstudy provides a simple way to increase the mineralization capacityand the formation of nano to micro-scale structure on CPC surface atan early stage, which is useful in improving early osseointegration inclinical applications. PDA integration may be a useful in modifying thesurfaces of other CaP-based biomaterials, such as CaP ceramics, CaP coat-ing and bioglass. Moreover, the rapid biomimetic mineralization onPDA–CPC is a potentially useful for the adhesion of proteins and cellsand the reduction of adverse reactions of CPC at the early stages afterimplantation.

4. Conclusion

In this study, PDA–CPCmineralization after soaking in SBF for differ-ent durations was investigated using XRD, ATR-FTIR, SEM, XPS, andTEM. PDA promoted the conversion of DCPD and α-TCP to HA at theearlier stages (before soaking, 1 d, 3 d, 5 d, and 7 d), but inhibited thisconversion afterwards (10 d and 14 d). Catechol-containing PDAconcentrated Ca2+ from the SBF at the surface and promoted rapidmin-eralization on PDA–CPC to form a nanoscale HA layer, which occurredlater on the control CPC. The nanoscale HA layer had characteristics sim-ilar to those of natural bone. The HA layer co-precipitated with PDA, asconfirmed by XPS and TEM. This layer possesses a high specific surfacearea, which beneficially influences the adhesion and proliferation ofproteins and cells. Therefore, this method provides a feasible route tosurface modification of CaP-based biomaterials.

Acknowledgments

The present study was supported by the National Basic ResearchProgram of China (973 Program, 2012CB933602), the NationalNatural Science Foundation of China (51372210, 51203130), the Re-search Fund for the Doctoral Program of Higher Education of China(20130184110023), and the Construction Program for InnovativeResearch Team of University in Sichuan Province (14TD0050).

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