Biomimetic synthesis of enamel-like hydroxyapatite on self-assembled monolayers

Materials Science and Engineering C 27 (2007) 756 – 761 www.elsevier.com/locate/msec

Biomimetic synthesis of enamel-like hydroxyapatite on self-assembled monolayers Hong Li a , Weiya Huang b , Yuanming Zhang b,⁎, Mei Zhong c a

Department of Materials Science and Engineering, Jinan University, Guangzhou, 510632, PR China b Department of Chemistry, Jinan University, Guangzhou, 510632, PR China c Department of Stomatology, Affiliated Hospital of Jinan University, Guangzhou, 510632, PR China Received 25 January 2006; received in revised form 25 July 2006; accepted 2 August 2006 Available online 20 September 2006

Abstract Hydroxyapatite (HAp) crystals mimicking tooth enamel in chemical composition and morphology were formed on sulfonic-terminated selfassembled monolayer (SAM) in 1.5SBF with F− at 50 °C for 7 days. F− ions showed a marked effect on the composition and morphology of deposited HAp crystals. In the absence of F− ions, HAp containing CO2− 3 were formed on SAM, and worm-like crystals of 200–300 nm in length − aggregated to form a spherical morphology. When F− was added, HAp crystals containing both CO2− 3 and F were formed on SAM. Needleshaped crystals of high aspect ratio and 1–2 μm in length grew elongated along the c-axial direction. In addition, these needle-shaped crystals grew in bundles, mimicking HAp crystals in tooth enamel. After the process of ripening, the needles in bundle grew to large size of up to 10 μm in length, and still kept no crystal–crystal fusion like enamel HAp crystals. The formation of enamel-like HAp can be attributed to the substitute of F− for OH− by disturbing the normal progress of HAp formation on SAM. The results suggest potential applications in preparing a novel dental material by a simple method. © 2006 Elsevier B.V. All rights reserved. Keywords: Biomimetic synthesis; Hydroxyapatite; Self-assembled monolayer; Enamel-like

1. Introduction Hydroxyapatite (HAp) has excellent biocompatibility and bioactivity, and is considered as one of the most promising biomaterials for clinical use such as bone replacement material in restorative dental and orthopedic implants [1,2]. However, the body of sintered HAp alone is not sufficient to be applied as bone or tooth repair devices because of its weakness in mechanical properties [2,3]. Natural hard tissues usually own a good combination of stiffness and toughness due to their detailed composite micro-architectures, arising from the precisely controlled nucleation and growth of inorganic mineral crystals by organic matrices through specific protein-mediated processes [4–7]. For example, the development of the unique structure in tooth enamel is believed to be modulated by proteins in enamel matrix, principally amelogenins which is supposed to ⁎ Corresponding author. Tel.: +86 20 33334209; fax: +86 20 85226262. E-mail address: [email protected] (Y. Zhang). 0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.08.002

play a key role as a template for controlling the growth of enamel crystals [6]. Therefore, understanding of these biosynthesized systems and development of biomimetic strategies to design new materials are expected to improve biological and mechanical performance for biomaterials [8–14]. Mature mammalian tooth enamel is the hardest, most highly mineralized tissue in the vertebrate body [6,15,16]. Although it has the same chemical composition as the prototype materials such as carbonated hydroxyapatite (CHA) and fluoridated hydroxyapatite (F-HAp), tooth enamel crystal shows some unique characteristics: extremely elongated in the c-axial direction [17], high aspect ratio of at least 1000 [18] and bundled together to form enamel prisms which constitute tooth enamel. These micro-architectures greatly attribute to the stiffness and toughness of tooth enamel. Such properties are hardly obtainable for synthesized prototype hydroxyapatite which is similar to natural tooth enamel both in chemical composition and morphology. Unlike bone, once destroyed, tooth enamel can not be reconstructed no matter how small the damage is. Therefore, research

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on preparing enamel-like HAp is of great importance in order to develop better dental biomaterial for potential restorative application. A biomimetic way has been used for preparing so-called bone-like apatite, which is carbonate-containing with small crystallites and defective structure. A mimicking physiological condition, simulated body fluid (SBF) has been widely used as a soaking medium since it was first prepared by Kokubo et al. [19]. Wen and co-workers also reported deposition of long and thin needle-shaped crystals of enamel-like calcium phosphate onto a bioactive glass (45S5 type Bioglass) in a supersaturated calcifying solution (SCS) containing recombinant porcine amelogenins rP172 [20–22]. It has been realized that nucleation and growth of calcium phosphate crystals in vivo are modulated by specific proteins in mineralizing tissues, intrinsically by functional groups in proteins. Some functional groups like PO4H2−, COO−, SO3H− and OH− [22–25] were confirmed to have ability to induce bone-like apatite nucleation via a biomimetic way. The deposition of bone-like apatite could improve the biological properties for potential restorative application. Therefore, it is of great importance to study the ability of functional group to induce enamel-like structures and properties via a biomimetic way. Self-assembled monolayer (SAM) technique is an effective way to fabricate charged surface terminated with polar head groups [23,24]. Sulfonic group showed a relative strong ability to induce apatite deposition in the previous study [26]. In the present study, sulfonic groups (–SO3Na) were fabricated onto Si (100) substrates using SAM technique and would provide a negative charged surface while presenting in their dissociated forms (–SO3−). Si (100) was selected as a substrate because the Si(100) surface could be easily modified with a SAM [24,26– 28] for our efforts aiming to test the possibility of formation of enamel-like HAp via a biomimetic way. According to previous reports [29–32], the mineralization of tooth enamel is highly sensitive to free F− ions, which drive for the growth of fluoridated hydroxyapatite mineral. In order to prepare enamel-like HAp, KF solution was added to 1.5SBF to achieve a concentration of F− ions at 5 ppm, which was similar to the concentration of F− in developing tooth enamel fluid [33]. In this study, bundled crystals of HAp needles with high aspect ratio mimicking enamel crystals were formed on SAM surfaces via a biomimetic way. The obtained apatite crystals were partially substituted by carbonate and fluoride. Morphology as well as chemical nature of the synthesized materials was characterized by scanning electron microscope (SEM), X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) spectroscopy. Possible mechanisms for functional groups to direct structure and morphology were discussed. 2. Experimental procedures Si (100) wafers used as substrates were commercially available and cut into appropriate size (square, 10 by 10 mm). Sulfonic-terminated SAM was prepared according to reported procedures [28]. Thin Au films were formed briefly by vacuum evaporation of Au onto Si (100), followed by rinsing with

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deionized water and absolute ethanol immediately prior to use. Then the slides were immersed in 1 mM ethanol solution containing 3-mercapto-1-propanesulfonic acid (sodium salt, Alfa Aesar) for 24 h at room temperature. Upon removal from solution, the substrates were rinsed extensively with absolute ethanol and finally deionized water before use. The contact angle of the starting Si (100) wafer was about 50 ± 2°, while it decreased to 26 ± 2° after preparation of sulfonic-terminated SAM. The decrease in contact angle was due to the fabrication of hydrophilic sulfonic terminal groups onto the Si (100) substrates [34]. The growth medium, 1.5SBF solution which had concentrations of Ca2+ and PO43− ions 1.5 times as much as those of 1.0SBF was prepared by dissolving raw materials of NaCl, NaHCO3, KCl, K2HPO4, MgCl2, CaCl2 and Na2SO4 in deionized water according to concentrations showed in Table 1. It should be noted that concentrations of other ions were the same for the 1.5SBF and 1.0SBF. The pH of solution was carefully adjusted to 7.25 by trishydroxymethylaminomethane ((CH2OH)3CNH2) and HCl. A calculated amount of KF solution (1000 ppm F−) was added to the prepared 1.5SBF to achieve 5 ppm F− concentration. All chemicals used were analytical grade and all solutions were prepared with deionized water. The prepared SAM substrates were floated upside down in a vessel containing 100 ml 1.5SBF with and without 5 ppm F− at 50 °C. The temperature was maintained at 50 °C to promote nucleation since higher temperature would give rise to a lower-solubility HAp product [28]. After soaking for 7 days, the SAM slides were removed from the solution, rinsed with deionized water for several times and then dried at room temperature. Static contact angle of water on sulfonic-terminated SAM surfaces was measured at 25 °C using a water-drop contactangle goniometer (TANTEC Company, CAM-PLUS). Three parallel measurements were performed on each sample. Surfaces of SAM after soaking were examined by X-ray powder diffraction (XRD, MSAL XD-2) using CuKα at 40 kV and 20 mA. Crystals formed after soaking were carefully scraped from the SAM surfaces and mixed with KBr to form a pellet for Fourier transform infrared spectra (FTIR, Bruker) analysis. Morphology of these crystals was also examined under scanning electron microscope (SEM, Philips XL-30E) operated at 30kV. The SEM samples were coated with a thin platinum film. 3. Results and discussion FTIR spectra of the synthesized samples are presented in Fig. 1. Both Fig. 1a and b show typical apatite bands. The Table 1 Concentration of ions in 1.0SBF and 1.5SBF in comparison with those in human blood plasma Concentration (mM)

Blood plasma 1.0SBF 1.5SBF

Na+

K+

Ca2+

Mg2+

Cl−

HCO−3

HPO2− 4

SO2− 4

142.0 142.0 142.0

5.0 5.0 5.0

2.5 2.5 3.75

1.5 1.5 1.5

103.0 148.8 148.8

27.0 4.2 4.2

1.0 1.0 1.5

0.5 0.5 0.5

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doublet appeared at about 565 cm− 1 and 603 cm− 1 and the band at 1040 cm− 1are due to asymmetrical v4 and v3 stretching vibrations of PO43− , while the band at around 875 cm− 1 and the doublet at 1420 cm− 1 and 1460 cm− 1 arise from CO32− [35]. The presence of CO32− bands indicates that the crystals formed on the SAM in 1.5SBF with or without F− both contain carbonate. Low-intensity bands at 630 and 3570 cm− 1 that are attributed to OH− [36] are clearly visible only in the stoichiometric HAp powder as demonstrated in Fig. 1a. The band observed at 742 cm− 1 in Fig. 1b is due to the presence of fluoride [37], confirming that the HAp crystals grown in 1.5SBF with F− contain F−. XRD patterns of the samples are presented in Fig. 2. Both samples show characteristic peaks of HAp at about 2θ = 26° for (002) diffraction and 32° for (211). In Fig. 2b, the peaks are stronger than those in Fig. 2a and the (002) peak is higher than its (211) diffraction peak, which indicate that the crystals with F− have better crystallinity and might grow preferentially along the c-axial direction [38]. The better crystallinity of HAp crystal containing F− can be explained by its structure. Usually the HAp structure can be viewed as the isolated PO43− tetrahedral oriented toward Ca2+ ions along the a-axis, along with the anions X− (OH− or F− or Cl−) of the electronegative elements placed around Ca2+ ions in the direction perpendicular to the caxis [39]. Once the OH− are partially substituted by the F−, the existing hydrogen atoms of the OH− group are tightly bound to the nearby F− anions because of the higher affinity of the fluorine in respect to the oxygen, producing a quite well-ordered apatite structure, which leads to increasing crystallinity [39]. The naturally occurring HAp phase in tooth enamel contains approximately 3.5 wt.% of carbonate and 0.04–0.07 wt.% of fluoride [40]. It is widely accepted that mature mammalian tooth enamel materials can be simulated with prototypes of fluoridated carbonate-containing hydroxyapatite. From the IR and XRD results, carbonate-containing HAp crystals were formed on the SAM after soaking in 1.5SBF with and without F−. Addition of 5 ppm F− promotes formation of HAp containing

Fig. 1. FTIR spectra of HAp formed on SAM after soaking in 1.5SBF without (a) and with (b) F − at 50 °C for 7 days.

Fig. 2. XRD patterns of HAp formed on SAM after soaking in 1.5SBF without (a) and with (b) F − at 50 °C for 7 days.

both F− and CO32− in its crystal lattice, which mimics the tooth enamel in chemical composition. Fig. 3 shows the SEM images of samples on the SAMs terminated with –SO3H groups after soaking 1.5SBF at 50 °C for 7 days. A few particles are observed on the SAM with – SO3H as shown in Fig. 3a. A highly magnified SEM image of apatite morphology on the SAM with –SO3H is shown in Fig. 3b. The spherical aggregates of worm-like structure, shown in Fig. 3c and d, are typical of apatite morphology obtained via a biomimetic way [14]. Fig. 4 shows the SEM images of samples on SAM surface after soaking in 1.5SBF with 5 ppm F− at the same conditions. SEM results reveal that this morphology is different to that of the deposited apatite on a substrate through biomimetic processing utilizing SBF [40]. A crystalline layer of about 2.5 μm thickness is formed on SAM. The surface of this layer is composed of a lot of densely packed crystalline particles. The magnified images are shown in Fig. 4c and d. The HAp crystals grow in bundles of needles of approximately 1–2 μm in length. The longitudinal direction of the needles is generally believed to be in the caxis of the apatite crystal [39]. This is further confirmed by the XRD patterns shown in Fig. 2. The morphology of the bundled needle-shaped HAp crystals with high aspect ratio are reminiscent of the long and thin HAp crystals seen in the early stage of tooth enamel formation [41]. It has been reported that Si–OH [42], Ti–OH [43] and COOH [39] groups can induce apatite formation in a biomimetic solution. In the body fluid environment, these functional anionic groups bind positively charged calcium ions (Ca2+) to form complexes between these species at initial stage inducing apatite nucleation. The complexes further incorporate phosphate ions to form apatite nuclei. This initial stage may govern the heterogeneous nucleation of hydroxyapatite on the substrate after exposure to a solution mimicking body fluid environment. Consequently, functional groups with negative charge act as an inductive factor on apatite nucleation.

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Fig. 3. Scanning electron microscope images of HAp formed on a SAM terminated with –SO3H groups after soaking in 1.5SBF at 50 °C for 7 days: (a) a few particles; (b) spherical aggregates; (c) and (d) highly magnified SEM image of worm-like apatite morphology.

In 1.5SBF of neutral condition used in the present experiments, the –SO3Na groups are presented in their dissociated forms (–SO3− ) and thus a negatively charged surface is

provided. It is presumed that complexes such as –SO3Ca+ and (–SO3)2Ca are formed in the induction period for apatite nucleation [25]. The excess positive charge of the –SO3/Ca

Fig. 4. Scanning electron microscope images of HAp formed on a SAM terminated with –SO3H groups after soaking in 1.5SBF with 5 ppm F− at 50 °C for 7 days in (a) and (b); bundles of crystals in (c) and (d).

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Fig. 5. Morphology of needle-shaped HAp after ripening for 50 days at room temperature.

complexes builds up an adjacent layer of negative charge (phosphate groups). The residual charge of this layer in turn attracts other calcium ions. When the local degree of super saturation is high enough, the critical radius for nucleation are formed. Further growth of crystals takes place when the lattice sites predetermined by the initially formed nuclei are filled. This outward growth from a point leads to spherical morphology of the deposit when high affinity binding of calcium ions by carboxymethyl groups [44], or sulfonic groups [25]. As the ions are sterically prevented from moving into periodic lattice sites, the spherical aggregates with worm-like morphology are formed. The growth from a point can also lead to radial morphology. In the previous study on biomimetic crystallization of calcium carbonate [45], crystals were reported to organize radically and grown radiating from the center along the crystallographic [001] direction. However, to the best of our knowledge, the radial growth of apatite via a biomimetic way is not found.

It is evident that morphological changes occurred when F− ions was added to the 1.5SBF. The presence of F− ions might modify the process of crystal growth by substitution OH− ions in the HAp lattice and lead to formation of needle-shaped crystals. A possible intrinsic mechanism for the effect of F− is that substitution of F− for OH− disturbs the progress of a step on the (001) plane by changing the interfacial free energy between the solution and the HAp crystal [46]. The substitution of OH− by F− might accelerate the incorporation of constituent ions at surface growth sites of (001) plane, which results in the speedup in accretion along the c-axis direction. This in turn leads to increasing crystal aspect ratio. Needle-shaped radial apatite is formed in a microenvironment where Ca2+ and PO43− ions are in super saturation in the vicinity of –SO3− sites and then is bundled together in a way similar to natural apatite crystals in tooth enamel. Further overgrowth by the ripening process can lead to big radial crystals with sizes up to 10–15 μm in length, as shown in Fig. 5. The most interesting phenomena are that the needleshaped crystals do not fuse together, and maintain independent organized-growth mostly along the longitudinal direction for even higher aspect ratio. During the early stage of enamel formation, the HAp nuclei further grow by uptaking inorganic ions in body fluid. The negative surface of amelogenins nanospheres then interacts electrostatically with the specific faces of enamel HAp crystal, inhibiting pre-mature crystal–crystal fusion [6]. Therefore, enamel HAp crystals are characteristic of organized nano-sized needle shape. In vitro experiment, the organized long and thin crystals observed after SCSrP172 immersion closely resemble the apatite crystals observed in the early stage of enamel biomineralization [22]. The modulation of rP172 on apatite crystals growth confirms the elongating effect of recombinant amelogenins on the calcium phosphate crystals formed in solution and gel [21,22]. It is verified to a large extent the hypothesis on the function of amelogenins assemblies in preventing premature lateral crystal–crystal fusions while promoting the c-axial crystal growth of the crystals throughout the full thickness of the secretory matrix of the developing enamel. In our synthetic study, it is speculated that modulation on calcium phosphate crystals growth is performed by the functional group and biomimetic environment. Considering the clinical acceptance, the biomimetic way, as discussed here, might provide a new methodology for constructing enamel-like biomaterials. The results from this study may provide a new thought to understand biominerilization and a new method to tailor biosynthesis by biomacromolecular, polypeptide and functional group assembly. Further study is currently under way. 4. Conclusions Hydroxyapatite (HAp) crystals mimicking tooth enamel in chemical composition and morphology were formed on sulfonicterminated self-assembled monolayer (SAM). F− ions showed a marked effect on the composition and morphology of deposited HAp crystals. In the absence of F− ions, HAp containing CO32− of worm-like crystals with 200–300 nm length aggregated to form a spherical morphology. When F− was added, HAp crystals

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containing both CO32− and F− were formed. Bundled needleshaped crystals of high aspect ratio and 1–2 μm in length grew along the c-axial direction, mimicking HAp crystals in tooth enamel. After the process of ripening, the needles in bundles grew to large size of up to 10 μm in length, and still maintain no crystal–crystal fusion like enamel HAp crystals. The formation of enamel-like HAp can be attributed to the substitute of F− for OH− by disturbing the normal progress of HAp formation. Results from this study suggest potential applications in preparing a novel dental material by a simple method. Acknowledgments The authors would like to express their gratitude to Dr. Shawn Xiang Zhang for his help. References [1] L.L. Hench, J. Am. Ceram. Soc. 74 (1991) 1487. [2] R.G.T. Geesink, M.T. Manley (Eds.), Calcium Phosphate Biomaterials: A Review of the Literature, Raven Press, New York, 1993, p. 1. [3] B.R. Constantz, I.C. Ison, M.T. Fulmer, R.D. Poser, S.T. Smith, M. Vanwagoner, J. Ross, S.A. Goldstein, J.B. Jupiter, D.I. Rosenthal, Science 267 (1995) 1796. [4] A.H. Heuer, D.J. Fink, V.J. Laraia, J.L. Arias, P.D. Calver, K. Kendall, G.L. Messing, Science 255 (1992) 1098. [5] P. Calvert, MRS Bull. 17 (1992) 37. [6] A.G. Fincham, J. Moradian-Oldak, J.P. Simmer, J. Struct. Biol. 126 (1999) 270. [7] C. Du, G. Falini, S. Fermani, C. Abbott, J. Moradian-Oldak, Science 307 (2005) 1450. [8] E. Dalas, K.J. Kallitsis, P.G. Koutsoukos, Langmuir 7 (1991) 1822. [9] B.C. Bunker, P.C. Rieke, B.J. Tarasevich, A.H. Campbell, G.E. Fryxell, G.L. Graft, L. Song, J. Liu, J.W. Virden, G.L. McVay, Science 264 (1994) 48. [10] J. Weng, B. Feng, M. Wang, X. Zhang, Key Eng. Mater. 288–289 (2005) 277. [11] A. Takeuchi, C. Ohtsuki, T. Miyazaki, H. Tanaka, M. Yamazaki, M. Tanihara, J. Biomed. Mater. Res. 65A (2003) 283. [12] E.K. Girija, Y. Yokogawa, F. Nagata, Chem. Lett. 7 (2002) 702. [13] X.Y. Yuan, A.F.T. Mak, J.L. Li, J. Biomed. Mater. Res. 57 (2001) 140. [14] T. Kokubo, Thermochim. Acta 280 (1996) 479. [15] A.G. Fincham, J. Moradian-Oldak, T.G.H. Diekwisch, D.M. Lyaruu, J.T. Wright, P. Bringas, H.C. Slavkin, J. Struct. Biol. 115 (1995) 50.

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