Materials Science and Engineering C 49 (2015) 526–533

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Surface controlled calcium phosphate formation on three-dimensional bacterial cellulose-based nanofibers Honglin Luo a, Guangyao Xiong b, Chen Zhang c, Deying Li b, Yong Zhu d, Ruisong Guo a, Yizao Wan a,⁎ a

School of Materials Science and Engineering, Tianjin University, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072, PR China School of Mechanical and Electrical Engineering, East China Jiaotong University, Nanchang, Jiangxi 330013, PR China School of Optometry and Ophthalmology, Tianjin Medical University Eye Hospital, Tianjin 300384, PR China d School of Chemical Engineering, Tianjin University, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072, PR China b c

a r t i c l e

i n f o

Article history: Received 5 April 2014 Received in revised form 21 December 2014 Accepted 14 January 2015 Available online 15 January 2015 Keywords: Calcium phosphate Bacterial cellulose Nanofiber X-ray absorption near-edge structure spectroscopy Surface chemistry

a b s t r a c t Studies on the early calcium phosphate (Ca-P) formation on nanosized substrates may allow us to understand the biomineralization mechanisms at the molecular level. In this work, in situ formation of Ca-P minerals on bacterial cellulose (BC)-based nanofibers was investigated, for the first time, using the X-ray absorption near-edge structure (XANES) spectroscopy. In addition, the influence of the surface coating of nanofibers on the formation of CaP minerals was determined. Combined with XRD analysis, XANES results revealed that the nascent precursor was ACP (amorphous calcium phosphate) which was converted to TCP (β-tricalcium phosphate), then OCP (octacalcium phosphate), and finally to HAP (hydroxyapatite) when phosphorylated BC nanofibers were the templates. However, the formation of nascent precursor and its transformation process varied depending on the nature of the coating material on nanofibrous templates. These results provide new insights into basic mechanisms of mineralization and can lead to the development of novel bioinspired nanostructured materials. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The fascinating properties and sophisticated structures of natural minerals such as bones, teeth, diatoms, and shells have attracted much interest around the world. The hierarchical self-assembly and orientation of the calcium phosphate (Ca-P) crystals in the nanosized collagen matrix is believed to contribute to the high toughness of bones [1]. Therefore, many nanofibers (such as peptide–amphiphile nanofibers [2], collagen fibrils [3], viruses [4] and carbon nanofibers [5]) have been used as templates for the deposition of Ca-P minerals in an attempt to understand the mechanism of natural biomineralization process as well as to seek industrial and technological applications. Understanding the evolution of minerals from initial precursors to a thermodynamically stable form is of paramount importance in controlling the final structure and properties of the minerals [6–8]. However, the determination of initial Ca-P and its growth during biomineralization is still a big challenge since the in situ observation of the growth process is difficult [9]. Numerous techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), time-of-flight cluster static secondary ion mass spectra (ToFSSIMS), in situ transmission electron microscopy (TEM), and in situ atomic force microscopy (AFM) [9–14] have been employed for the in

⁎ Corresponding author. E-mail address: [email protected] (Y. Wan).

http://dx.doi.org/10.1016/j.msec.2015.01.053 0928-4931/© 2015 Elsevier B.V. All rights reserved.

situ observation of Ca-P formation in solutions rather than on threedimensional (3D) nano-scaled fibrous templates. It has been documented that X-ray absorption near-edge structure (XANES) spectroscopy is a useful technique that can be used to determine the valence, oxidation state, coordination number of individual elements as well as the chemical structure of compounds [15]. The most significant advantage of XANES is that this technique is specific for elements. The patterns of XANES spectra are like fingerprints, representing only specific elements. Previous studies by various groups have suggested that XANES can be used to examine the phase transformation, such as the transformation characteristics of biogenic Ca-P [16,17] and transformation of amorphous calcium carbonate [18]. However, to the best knowledge of the authors, XANES has not been used for the investigation of Ca-P formation on a nanofibrous template. From a biomimetic point of view, investigation of Ca-P deposition on a 3D nanofibrous template may discover the biomineralization mechanisms similar to those involved in nature. In this context, using a natural nanofibrous template, namely bacterial cellulose (BC), produced by Gram-negative, acetic acid bacteria Acetobacter xylinum (or Gluconaacetobacter xylinus), is promising. In addition to such appealing properties as ultrahigh mechanical strength and modulus, high water holding capability and porosity, and good biocompatibility, BC displays intrinsic 3D network structure, and, in particular, the diameter of BC fibers is in the nanometer scale. Therefore, studies regarding the Ca-P deposition on 3D BC nanofibrous templates are of vital importance to the understanding of the biomineralization

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mechanisms in nature. To this end, Ca-P formation on pristine and surface-modified BC nanofibers has been reported by using conventional techniques such as XRD, TEM, and FTIR [19–25]. For many years, it has been believed that octacalcium phosphate (OCP) is the precursor of hydroxyapatite (HAP) on BC nanofibers regardless of surface nature [24, 26]. However, that finding is inconsistent with the common notion that amorphous calcium phosphate (ACP) is the first-formed solid CaP phase which evolves into crystalline phases [13,27,28] and that the surface nature of templates can significantly alter the nucleation and transformation of the mineralizing crystals [29–31]. The existing controversy means little is known as to how Ca-P deposition proceeds on a 3D nanofibrous template and how the surface chemistry of fibrous templates in nanoscale affects the formation and transformation process of minerals. Accordingly, more investigation is needed for the understanding of the biomineralization mechanisms and the construction of organic–inorganic hybrids and bone scaffolds. Unlike previous studies regarding Ca-P formation on BC [19–26], for the first time we systemically investigate the structure and chemical states of newly formed Ca-P deposits on BC-based nanofibers by XANES spectroscopy in an attempt to understand the early (as short as 2 min) formation of nascent precursors and thereafter the evolution of Ca-P minerals and determine the influence of surface coating on Ca-P formation and evolution of Ca-P minerals. To this end, phosphorylated BC (P-BC), gelatin-immobilized BC (Gel-BC), and εpolylysine-immobilized BC (PLL-BC) were used as the nanofibrous templates (either negative or positive surface) for the deposition of Ca-P minerals. 2. Experimental 2.1. Materials The materials used in the present work included bovine gelatin (Gel, purchased from Sigma, USA), ε-polylysine (PLL, 99%, a generous gift from Tianjin University of Science and Technology, Tianjin, China), procyanidin (PA, crosslinker, purchased from Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China). All other reagents were purchased from ACROS® unless otherwise indicated. All reagents were of analytical grade and used as received. 2.2. Preparation of BC and P-BC The preparation and cleansing procedures of BC pellicles were identical to those described in our previous work [20–22]. The phosphorylation of BC was conducted by the same procedure as reported earlier [22, 32]. Briefly, BC pellicles in the form of disc with a diameter of 35 mm and a thickness of 8 mm were placed into a round-bottomed flask equipped with a condenser, thermometer, and nitrogen gas inlet. 200 mL of dimethyl formamide (DMF) and 15 g of urea were added to the flask. The flask was then heated to 110 °C and immediately a solution of 19 mL of 98% phosphoric acid (H3PO4) in 50 mL DMF was added to the flask. The suspension was further heated to 136 °C and left to reflux for an hour under gentle stirring using a magnetic stirrer and a nitrogen stream. The reaction mixture was cooled under flowing nitrogen gas. The resulting P-BC pellicles were removed and thoroughly rinsed with deionized water to wash out the excess H3PO4.

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2.4. Ca-P deposition on nanofibrous templates In order to induce Ca-P formation, P-BC, Gel-BC, and PLL-BC pellicles were immersed in a 1.5 SBF (simulated body fluid) at 37 °C for predetermined periods from as short as 2 min to 7 days. As reported previously [33], the 1.5 SBF solution was prepared by dissolving reagent grade NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2·H2O, and Na 2 SO4 in deionized water with the ion concentrations of Na + 213.0 mM, K+ 7.5 mM, Ca2 + 3.8 mM, Mg2 + 2.3 mM, HCO− 3 6.3 mM, HPO 24 − 1.5 mM, SO 24 − 0.75 mM, and Cl− 223.0 mM. The solution was buffered at pH 7.4 with tris(hydroxymethyl) aminomethane ((CH2OH)3CNH2) and 1 M HCl at 37 °C. The supersaturation condition in the 1.5 SBF solution was maintained by periodic replacement with a fresh solution. Upon completion of immersion, samples were taken out from the solution at predetermined time points, thoroughly rinsed with deionized water, and finally air dried for XANES and XRD measurements. 2.5. SEM observation SEM was employed to examine the morphology and structure of various samples. Prior to SEM observation using a Nano 430 FE-SEM (FEI, USA) at 10 kV, lyophilized Ca-P coated P-BC, Gel-BC, and PLL-BC samples were affixed to a copper flake with carbon adhesive and sputter-coated with gold at 10 mA for 3 min. 2.6. Zeta potential measurement The zeta potential analyzer (Model ELS-Z Otsuka Electronics Co., Ltd., Japan) was used to measure the zeta potential of P-BC, Gel-BC, and PLLBC samples. 2.7. XANES spectroscopy analysis The phosphorus (P) K-edge XANES measurements of air-dried Ca-Pcoated BC samples were performed at the beamline 4B7A of Beijing Synchrotron Radiation Facility (BSRF). P K-edge XANES spectra were obtained on a double-crystal monochromator (DCM) covering energy range of 2140–2180 eV with an energy resolution of 0.2 eV and the spectra were recorded in partial fluorescence yield (PFY) mode using a solid state Si(Li) detector (PGT, USA). The electron storage ring was operated at 2.5 GeV and the current was 150–250 mA. The chamber pressure was kept at 10−6 Torr during the measurement. Data analysis of the experimental XANES spectra was performed using the WinXAS3.1 and the reported spectra were obtained after normalization. In addition, four high-purity Ca-P minerals including ACP, TCP (β-tricalcium phosphate), OCP, and HAP purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) were used as P standards for comparison with the sample spectra. The use of standard samples was essential for the identification of the obtained XANES spectra [16,17]. 2.8. X-ray diffraction (XRD) analysis The crystalline structure was studied using a Rigaku D/max 2500 Xray diffractometer with a Cu Kα radiation (λ = 0.15405 nm). The lyophilized P-BC, Gel-BC, and PLL-BC samples were scanned from 5 to 50° at a scan speed of 2°/min. The data were obtained through a MDI/JADE6 software package attached to the Rigaku XRD instrument.

2.3. Immobilization of gelatin and ε-polylysine on BC

3. Results and discussion

BC pellicles (35 mm in diameter and 8 mm in thickness) were immersed in either gelatin solution (0.25 wt.%) or ε-polylysine solution (0.25 wt.%) at 37 °C for 24 h. Afterwards, samples were immersed in 200 mL PA solution (0.5%, in PBS) under constant stirring (140 rpm) at 37 °C for 2 h for crosslinking. The obtained Gel-BC and PLL-BC samples were thoroughly rinsed with deionized water.

3.1. Zeta potential Table 1 lists the zeta potential of various BC-based nanofibrous templates. Note that the zeta potential of P-BC was negative and so was GelBC while the zeta potential of PLL-BC changed to positive from negative. Moreover, Gel-BC showed a lower zeta potential than P-BC did. Our

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H. Luo et al. / Materials Science and Engineering C 49 (2015) 526–533 Table 1 Zeta potential of various BC-based nanofibrous templates. Samples

Zeta potential (mV)

P-BC Gel-BC PLL-BC

−21.8 ± 4.2 −38.1 ± 1.1 7.3 ± 0.10

previous work demonstrated that a thin layer of gelatin [34] and εpolylysine [35] was coated onto the surface of BC nanofibers for GelBC and PLL-BC, respectively. The structure of P-BC, Gel-BC, and PLL-BC is schematically illustrated in Fig. 1. The presence of gelatin (with an isoelectric point of ca. 5.0) on BC imparted a negatively charged surface for Gel-BC and, on the contrary, the existence of ε-polylysine imparted the nanofiber surface of PLL-BC with positive charges due to the amine groups (–NH+ 3 ) [35]. As shown in Fig. 1, the negatively charged surface of P-BC was due to the existence of large amount of hydroxyl groups (–OH) and phosphate groups (–PO3− 4 ). The lower zeta potential of Gel-BC than P-BC indicated that the charge density on Gel-BC was larger compared to P-BC. 3.2. Morphology of Ca-P minerals on BC-based nanofibers Fig. 2 shows selected SEM micrographs of Ca-P minerals on P-BC, Gel-BC, and PLL-BC. Fig. 2(a) revealed that aggregated Ca-P minerals were deposited on P-BC nanofibers after 12 h immersion in 1.5 SBF, but some surfaces of P-BC nanofibers are not covered with Ca-P minerals. In the case of Gel-BC, continuous Ca-P coating was observed on nanofibers after 12 h immersion in 1.5 SBF (Fig. 2(b) and its inset), indicating a better ability of inducing Ca-P formation as compared to P-BC. Interestingly, no Ca-P aggregate could be observed on PLL-BC after 12 h immersion (Fig. 2(c)). Fig. 2(d) revealed that, after 24 h immersion, only some sparsely deposited Ca-P minerals were noticed on PLL-BC, suggesting its poor ability of triggering Ca-P formation. 3.3. XANES analysis There are two ways to interpret XANES spectra. One is to compare the XANES of the materials of interest with the results of molecular

orbital density of state calculations. Another most widely used one is the fingerprint method in which the XANES of the material of interest is compared with the XANES of other materials of known structures [36]. Therefore, in this work, the P K-edge XANES spectra of four Ca-P standards (ACP, TCP, OCP, and HAP) were obtained (Fig. 3). These spectral features agreed well with those reported in literature [16,37]. In all spectra, a peak was found at around 2168.0 eV, which corresponds to oxygen oscillation and is a characteristic feature for Ca-P minerals [16, 17]. In addition, the four spectra exhibited a shoulder at around 2153.5 eV, which is an indication of Ca-P [16] and consistent with the value reported previously [37], but the shoulder seemed to be more distinctive in the more crystalline Ca-Ps and increased in the order of ACP b TCP b OCP b HAP. Note that these Ca-P minerals could be distinguished by the presence and absence of a peak at around 2162.0 eV [37] (although a slight shift of this peak was observed in TCP, as reported by Sato and co-workers [17]). For instance, the ACP spectrum showed a less distinctive shoulder and the peak at 2162.0 eV disappeared, and OCP and HAP had more pronounced shoulder at 2153.5 eV as compared to TCP and ACP. However, the difference in the spectral pattern between OCP and HAP was subtle and practically it was almost impossible to distinguish OCP from HAP with XANES technique alone due to a lack of distinctive features to separate them [37]. Fig. 4 shows the P K-edge XANES spectra of Ca-P minerals deposited on the surfaces of BC-based nanofibers as a function of immersion time. Fig. 4(a) revealed that, as expected, no shoulder at 2153.5 eV could be detected before P-BC was immersed in 1.5 SBF. Note that the shoulder at 2153.5 eV was observed after only 2 min immersion in 1.5 SBF. By comparing this spectrum with the standard Ca-P spectra (Fig. 3), the presence of ACP could be confirmed. This suggests that the induction period for the formation of ACP was only 2 min. The rapid formation of ACP in solution was previously reported by other researchers [13]. However, this is the first time to report the short induction period of ACP formation on BC-based nanofibers. As can be seen from Fig. 4(a), the freshly deposited nascent mineral was short-lived and disappeared after 3.5 h immersion when a peak at around 2162.0 eV was observed. This indicates the conversion of ACP to TCP. It was noted that the shoulder at 2153.5 eV became more pronounced after 12 h immersion, suggesting the conversion of OCP and/or HAP from TCP. Therefore, XANES results suggest that the nascent phase of Ca-P was ACP,

Fig. 1. Schematic diagram showing the structure of P-BC, Gel-BC, and PLL-BC nanofibers.

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Fig. 2. SEM images of Ca-P coated P-BC (a), Gel-BC (b) and PLL-BC (c and d) after immersion in 1.5 SBF for 12 h (a–c) and 24 h (d).

Fig. 3. P K-edge XANES spectra of four Ca-P standards (ACP, TCP, OCP, and HAP).

which experienced subsequent transitions to TCP and then to OCP and/or HAP. In our previous work, SEM, XRD, and FTIR were applied to explore the early growth (starting from 4 h) of Ca-P minerals on the surface of BC nanofibers and it was found that OCP was the precursor of HAP [26]. Similarly, Nge et al. declared that the initial precursor of HAP on BC was OCP [24]. However, to the best knowledge of the authors, none of the previous studies regarding Ca-P formation on BC nanofibers [21,22,24,26,38,39] has observed the formation of ACP and TCP probably due to the fact that such techniques as XRD and SEM are unable to capture the information of nascent Ca-P deposits. It is believed that studies on the early Ca-P formation on nano-sized substrates may possibly allow us to understand the formation mechanisms of biominerals at the molecular level [26] and thus enable us to develop synthetic biomaterials which would resemble natural ones more closely. In this regard, the employment of XANES is of critical importance to the investigation of Ca-P formation on various templates, in particular during the very early stages. Fig. 4(b) shows the XANES spectra of the Ca-P minerals deposited on Gel-BC. Surprisingly, we found that, after 2 min soaking in 1.5 SBF, formation of TCP could be detected without any presence of ACP, which differed from P-BC. Immediately afterwards, TCP began to transfer into OCP and/or HAP and the transition ended at 6 h immersion when complete OCP and/or HAP was formed. A comparison between Fig. 4(a) and (b) revealed that the formation of TCP on Gel-BC and its transition to

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Fig. 4. P K-edge XANES spectra of Ca-P coated P-BC (a), Gel-BC (b), and PLL-BC (c) after immersion in 1.5 SBF for different time.

OCP and/or HAP were earlier as compared to P-BC. In other words, these transformations were accelerated as compared to those on P-BC. Notably, a different trend was observed for the Ca-P formation on PLL-BC (Fig. 4(c)). After 2 min soaking in 1.5 SBF, the shoulder at 2153.5 eV was not detected, indicating no existence of ACP at this stage. Furthermore, the shoulder at 2153.5 eV was not observed in the XANES spectra until the PLL-BC sample was immersed in 1.5 SBF for 1 h. Moreover, the transition of ACP to TCP was finalized after 1 day immersion in 1.5 SBF and the transition of TCP to OCP and/or HAP was noted after 3 days precipitation on PLL-BC. This indicates that these transformations on PLL-BC were slowed down as compared to those on P-BC and Gel-BC. 3.4. XRD analysis As mentioned above, XANES is unable to tell OCP from HAP while XRD is unable to obtain any information of ACP and TCP due to the fact that the amount of Ca-P minerals is very limited during the very

early stage of Ca-P deposition. However, we found that a combination of XRD and XANES could distinguish HAP from OCP. The XRD results obtained in this work are shown in Fig. 5. Fig. 5(a) showed that, in addition to the peaks at 14.6, 16.2, and 22.6°, which could be assigned to (110), (110), and (200) planes of cellulose I, respectively [40], the phase of OCP was also identified after 12 h immersion in 1.5 SBF (three characteristic peaks (010), (002), and (320) at 9.3, 27.6, and 31.6°, respectively [41], were detected). After 1 day immersion, peak (211) at 31.7° of HAP was observed, indicating that OCP has converted to HAP. The (211) peak sharpened and another peak at 25.9°, corresponding to (002) diffraction of HAP, appeared after immersion in 1.5 SBF for 3 days, which were consistent with our previous reports [21,22]. Therefore, as illustrated in Fig. 6, the transformation process of Ca-P on P-BC was ACP → TCP → OCP → HAP. This transition pattern indicates that the Ca/P ratio experienced an unusual changing trend, decreasing from around 1.5 (for ACP and TCP) to 1.33 (for OCP) and then going up to 1.67 (for HAP). Although this changing pattern in Ca/P ratio was also reported by Oliveira et al. who observed Ca/P ratios of 1.34, 1.33, 1.44,

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1.22, and 1.57 at immersion times of 6, 12, 24, 96, and 192 h, respectively [42], the underlying mechanism is not fully understood yet. Similarly, OCP was identified after Gel-BC was immersed in 1.5 SBF for 12 h (Fig. 5(b)). Therefore, by combining the XRD and XANES results, we could conclude that the conversion of OCP from TCP started at 6 h immersion in 1.5 SBF. Fig. 5(b) also revealed that the peaks at 25.9° and 31.7° of HAP were observed after 18 h immersion, suggesting the conversion of OCP to HAP. Therefore, the transformation process on Gel-BC was TCP → OCP → HAP, as can be seen in Fig. 6. In the case of PLL-BC, no OCP peak could be detected and only HAP could be found after 3 days of immersion in 1.5 SBF (Fig. 5(c)), indicating that the formation of OCP was skipped and thus the conversion of ACP → TCP → HAP was confirmed (Fig. 6), agreeing well with the transformations occurred in a supersaturated calcium phosphate solution [13]. 3.5. Mechanisms of Ca-P formation on BC-based nanofibrous templates

Fig. 5. Typical XRD patterns of Ca-P coated P-BC (a), Gel-BC (b), and PLL-BC (c) after immersion in 1.5 SBF for different time.

Fig. 6. Induction period and transformation process of Ca-P minerals on various BC-based nanofibrous templates.

Although the phase transformation of Ca-P minerals has been extensively studied by many researchers [13,27,43,44], a consistent conclusion has yet to be obtained due to the complexity of the transformation processes. For instance, Sugiura and co-workers reported that the initial ACP first transformed into TCP, and then into OCP [13], which is consistent with the processes observed on P-BC. However, some groups found that ACP converted directly into HAP [12,45], while others stated that the transformation took place through an intermediate like OCP [46] and subsequent HAP crystallization [9]. Although it is still a big challenge to fully understand these phase transformations observed in this work, which is beyond the scope of this work, the findings presented here suggest that the initial formation of precursors is dependent on the surface chemistry of the templates. Fig. 6 shows the comparisons in induction period and transition time point of various Ca-P minerals on BC-based nanofibers. It was clearly seen in Fig. 6 that the coating materials with varying surface chemistry had an important impact on the formation of Ca-P minerals and their transitions. Specifically, P-BC and Gel-BC showed shorter induction period and quicker transformation than PLL-BC did, which was consistent with SEM results shown in Fig. 2 and agreed with the common notion that negatively charged substrates can accelerate the nucleation and growth of HAP as compared with positively charged substrates [47, 48]. The results presented in this work further confirm that amine groups are very weak in inducing Ca-P formation, which is consistent with the finding reported by Tanahashi and Matsuda [49]. The poor induction capability of the aminated surfaces was also confirmed by other researchers [50]. Tachibana et al. reported that the rapid chelation of calcium ion with carboxyl groups induced quicker formation of Ca-P than the trapping of phosphate ion by amino groups [51]. These previous reports can be used to interpret the difference between PLL-BC and Gel-BC. However, explanations for the differences in the Ca-P formation between –COOH-terminated Gel-BC and –PO3− 4 -terminated PBC cannot be found in literature. For instance, previous work confirmed that the phosphate groups on the surface of P-BC favored Ca-P formation as compared to unphosphorylated BC where only hydroxyl groups were present on the surface of BC [22]. Likewise, previous studies demonstrated that carboxyl group could greatly promote Ca-P formation, while nonionic groups (such as OH) were very weak Ca-P-inducing surfaces [49,52]. Interestingly, when the zeta potential of P-BC and Gel-BC (Table 1) is taken into consideration, we may conclude that the charge density is responsible for the difference between the two negatively charged samples, which is consistent with the theory that surface charges determine the solid/cluster interfacial energies involved in the nucleation of Ca-P particles from Ca2+ and PO3− 4 species in supersaturation [50]. Moreover, it has been well accepted that an important initial step in Ca-P formation is the attraction of Ca 2 + [53] and electrically negative groups act as nucleation sites for the deposition and growth of Ca-P minerals [50]. Accordingly, the following mechanisms are

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Fig. 7. Schematic illustration of Ca-P formation on the surface of P-BC (a) and Gel-BC (b) nanofibers.

proposed as illustrated in Fig. 7. In the first step, abundant carboxyl groups on the surface of Gel-BC and phosphate groups on P-BC surface strongly absorb Ca2+ ions by electrostatic interaction. Therefore, the accumulation of Ca2+ ions owing to electrostatic attraction increases the supersaturation near the negatively charged surfaces, and as a result, the initial nucleation is preferentially triggered. It is reasonable to believe that more Ca2+ ions are adsorbed near the surface of Gel-BC as compared to P-BC since the charge density of Gel-BC is higher than PBC. Namely, Gel-BC possesses more adsorption sites than P-BC does (see Fig. 7). A higher concentration of Ca2 + ions near the surface of Gel-BC means quicker adsorption of phosphate ions and thus faster Ca-P precipitation from 1.5 SBF as compared to P-BC. As a result, the rate of Ca-P nucleation on the surface of Gel-BC is faster than on P-BC. However, understanding how the charge density affects the transformation rate is still a big challenge and this will be the focus of our future investigation. 4. Conclusions In this study, we prepared 3D nanofibrous BC-based templates with varying surface chemistry by coating various materials on the surface of BC nanofibres and investigated the Ca-P formation on these templates by XANES technique together with XRD method. We proved that surface chemistry of 3D nanofibrous templates had a significant effect on the formation of nascent Ca-P minerals and their transitions. The nascent Ca-P deposited on P-BC and PLL-BC was ACP whereas TCP was the initial phase on Gel-BC. The transition process of Ca-P minerals on P-BC, Gel-BC, and PLL-BC was ACP → TCP → OCP → HAP, TCP → OCP → HAP, and ACP → TCP → HAP, respectively. Moreover, the rate of transformation of Ca-P minerals followed the order of Gel-BC N P-BC N PLL-BC. These differences are attributed to the different surface functional groups and charge density of the BC-based nanofibers. The transformation process evidenced here may be extended to the studies of Ca-P and other minerals on various substrates, to the understanding of the phase transformation in living organisms, and thus present a new strategy for mimicking biological materials. Acknowledgments This work is supported by the National Natural Science Foundation of China (Grant Nos. 51172158 and 81200663) and the Science and Technology Support Program of Tianjin (Grant No. 11ZCKFSY01700). References [1] Q. Fu, E. Saiz, M.N. Rahaman, A.P. Tomsia, Adv. Funct. Mater. 23 (2013) 5461–5476. [2] J.D. Hartgerink, E. Beniash, S.I. Stupp, Science 294 (2001) 1684–1688.

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Surface controlled calcium phosphate formation on three-dimensional bacterial cellulose-based nanofibers.

Studies on the early calcium phosphate (Ca-P) formation on nanosized substrates may allow us to understand the biomineralization mechanisms at the mol...
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