ARTICLE pubs.acs.org/Langmuir
Crystallization of Calcium Carbonate on Chitosan Substrates in the Presence of Regenerated Silk Fibroin Yaodong Wu, Cheng Cheng,† Jinrong Yao, Xin Chen, and Zhengzhong Shao* Key Laboratory of Molecular Engineering of Polymers of Ministry of Education, Laboratory of Advanced Materials, Department of Macromolecular Science, Fudan University, Shanghai 200433, China
bS Supporting Information ABSTRACT: The crystallization of calcium carbonate (CaCO3) was investigated using a mineralization system composed of a chitosan membrane and regenerated silk fibroin (RSF). Such a system may resemble the mineralization in molluscs, where chitosan is a derivative of chitin and RSF an analogue of nacreous protein. It was found that the vaterite disks generally formed on the chitosan membrane while the aragonite disks also appeared with changes of pH value or temperature of the solution. The crystallization of CaCO3 in the vicinity of the chitosan membrane was much more affected by the environment of crystallization, compared to that in bulk solution. Detailed observation from high-resolution scanning and transmission electron microscopy (HRSEM and TEM) showed that these disks consisted of nanoparticles about 20 nm in size, thus suggesting that the accumulation of hybrid CaCO3/RSF nanoparticles induced the formation of crystalline disks on the chitosan membrane.
1. INTRODUCTION Recently, the structure of biominerals and the physicochemical properties of the organics involved in biomineralization processes have been extensively studied, leading to remarkable findings regarding their highly specialized mechanical and optical properties.1,2 Now the attention is focused the mechanisms behind the formation of biominerals and biomimetic synthesis of organic/ inorganic hybrid materials endowed with fine structures and even specific functions.3 Generally, organic molecules employed in biomineralization can be categorized into two groups, i.e., insoluble matrix and soluble additives. The polysaccharide chitin is one of universal insoluble templates, which is associated with different kinds of proteins, other polysaccharides, and minerals depending on the organism, such as arthropods and molluscs.4 In these organisms, the interactions between chitin and other biomolecules are considered to play an imperative role in calcification.4,5 In the shell of mollusk, the formation of nacre layers is closely related to the templating effect of organics during the crystallization of calcium carbonate; therefore, investigation into the roles of different organics involved in the crystallization is crucial in understanding biomineralization.5 Lavi-Kalisman et al.6 proposed a structural model of the nacreous organics, i.e., silk-like gel interspersed with soluble acidic proteins sandwiched in the network of chitin fibers. This kind of “sandwich” structure is becoming the guideline of biomimetic synthesis of artificial nacre materials.7-9 Meanwhile, it was reported that crystallization of calcium carbonate on insoluble chitin matrix represented oriented selective growth of particular faces of crystals to some extent.10,11 As a derivative of chitin, chitosan is adjustable and r 2011 American Chemical Society
frequently employed as an insoluble substrate to mimic biomineralization. In these processes, the soluble additives are usually synthesized polymers or peptides with acidic groups, such as poly(acrylic acid) (PAA),12-18 poly(glutamic acid) (PGA),14 and polyaspartate (PAsp),19 etc. In natural nacre, acidic proteins provide nucleation sites and the nacreous silk-like protein gel (with a small amount of acidic groups itself20,21) represents antiparallel structure which is considered to be geometrically commensurate with the arrangement of Ca2þ ions in the (001) face of aragonite.1,6 Furthermore, acidic proteins extracted from nacre layers induced aragonite formation in vitro when preadsorbed on a substrate of β-chitin and natural silk fibroin while acidic proteins extracted from prismatic layers led to mainly calcite formation under the same conditions. This suggests that the cooperation of different organic components plays an imperative role in polymorphism determination of biominerals.22 It has been found that regenerated silk fibroin (RSF) is much more similar to silk-like proteins in nacre in the amino acid sequence and secondary structure20,23 compared to conventional synthesized acidic polymers.12-19 Indeed, RSF23 and its segment24,25 specifically regulated the crystallization of calcium carbonate in solutions. Moreover, silk fibers as insoluble matrix with the assistance of RSF also displayed a templating effect on the oriented deposition of aragonite.26 Thus, from an in vitro study of biomineralization, silk fibroin in abundant supply may replace both of silk-like protein and acidic proteins to play the regulating role. Received: November 26, 2010 Revised: January 7, 2011 Published: February 16, 2011 2804
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To address the novel coeffect of chitosan and silk fibroin on mineralization, we investigated the growth of CaCO3 in a system containing insoluble chitosan matrix and soluble regenerated silk fibroin, which is very close to the mineralization system of molluscs. Through controlling the assembly of the substances in the solution via changing the solvent environment, e.g., solution pH and temperature, CaCO3 crystals with different morphologies and polymorphs were obtained on the chitosan membrane in the presence of dissolved silk fibroin. We propose that the interaction of silk fibroin and small CaCO3 particles may be imperative in crystallization process of certain CaCO3 polymorphs.
2. EXPERIMENTAL SECTION 2.1. Preparation of Chitosan Matrix and RSF Solution. Chitosan, with a weight-average molecular weight of 1100 kDa and a degree of deacetyl (DD) of 75%, was purchased from Jinan Haidebei Marine Biological Product Co. Ltd. (Jinan, China). Chitosan was dissolved in 2% (v/v) acetic acid aqueous solution to prepare a 2% (w/w) chitosan solution.27 Then glutaraldehyde with amount of 0.5% molar of the amino groups on chitosan was added into the solution under stirring. After complete mixing for 2 h, the mixture was poured into a polystyrene dish and dried at room temperature. After that, the dried films were immersed in 0.5% (w/w) sodium hydroxide aqueous solution to remove the remaining acetic acid and then washed repeatedly with deionized water. Finally, chitosan membranes were prestretched with 50% of elongation if needed. RSF aqueous solution was obtained through dissolving degummed Bombyx mori silk following established procedures.28 After being dissolved in 9.5 mol L-1 LiBr aqueous solution, RSF solution was dialyzed against deionized water for 3 days and then centrifuged at 6000 rpm for about 5 min. The supernatant with about 4% (w/w) of RSF was collected and stored at 4 °C. Calcium chloride and sodium bicarbonate stored solutions were prepared with analytical chemicals and deionized water. 2.2. Growth of CaCO3 on Chitosan Membrane. The supersaturated calcium carbonate solutions containing appropriate amounts of RSF were prepared with different pH values according to previous paper.23 The supersaturated CaCO3 solution, which contained 0.01 mol L-1 Ca2þ and 0.01 mol L-1 HCO3-, was prepared by mixing calcium chloride and sodium bicarbonate stored solutions directly. Chitosan membrane fixed on frame was placed vertically in 150 mL supersaturated solution at temperatures of 5, 25, or 45 °C. After different mineralizing time, chitosan membrane was brought out from the solution and rinsed with deionized water three times to remove silk fibroin and precipitates formed in bulk solution which were adsorbed on the membranes. At last, all samples were dried at room temperature. 2.3. Characterization. The surface morphology of the mineralized membrane was observed with a TS 5136MM scanning electron microscope (SEM) after sputtered with gold. High-magnification images and energydispersive X-ray spectroscopy (EDX) were obtained on a Hitachi S4800 field emission SEM equipped with a Bruker Quantax 400. The ultrathin sections were loaded on carbon-coated copper grids for transmission electron microscope (TEM) observation and selective area electron diffraction (SAED) on a JEM-2100F. Birefringence of crystalline disks was observed on an Olympus BX51 polarizing optical microscope (POM). The crystallographic polymorphs of minerals were detected on a Raman spectrometer (Renishaw inVia Reflex equipped with a Leica 2500 optical microscope and a laser of 785 nm with CCD detector) and an X-ray diffractometer (XRD) (X’pert Pro with Cu KR radiation). Membrane samples were employed for POM, Raman, and XRD characterization.
3. RESULTS AND DISCUSSION 3.1. Effect of RSF during Crystallization of CaCO3 on Chitosan Membrane. CaCO3 can exist in six different forms,
Figure 1. CaCO3 products grown on chitosan membranes with a range of concentrations of RSF at initial pH 7.9 for 1 day. (a) 0% (w/w), (b) 0.01% (w/w), (c) 0.1% (w/w), (d) 1% (w/w), and (e) XRD patterns of CaCO3 products above. “c” = calcite; “v” = vaterite.23
i.e., three anhydrous crystalline, two hydrated crystalline, and an amorphous state.16 Among the three anhydrous polymorphs, calcite is the most stable, aragonite is less stable, while vaterite is the most unstable form and is likely to transform into calcite in aqueous solution. As additive to solution, RSF may affect the morphology and polymorphism of CaCO3 crystals through its assembling behavior; e.g., “rice-like” particle with a polymorph of calcite was synthesized at lower pH value while spherical particle with a blending polymorph of calcite and vaterite was obtained at higher pH value.23 The results show that, without RSF in the solution, a large number of laminated vaterite crystals precipitated on both sides of the chitosan membrane accompanied by sporadic calcite at first (Figure 1a,e) and subsequently transformed into thermodynamically stable rhombic calcite within 7 days (Figure s1 in the Supporting Information). However, in the presence of a small amount of RSF, e.g., 0.01% (w/w), ellipsoidal vaterite and irregular rhombic calcite grew on the membrane (Figure 1b,e). When the concentration of RSF increased to 0.1% (w/w), only vaterite disks were observed on chitosan membrane 2805
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Figure 2. Micrographs of disk-like CaCO3 developed on chitosan membranes by the crystallization for 3 days in the presence of 1% (w/w) RSF. (a) SEM image, (b) HRSEM image of the edge of crystalline disks, (c) HRSEM of the center of crystalline disks, (d) polarized optical micrograph, (e) TEM image of the cross section of the crystalline disk, and (f) corresponding ED pattern of nanoparticles in (e). The parameters of the crystal are referenced to JCPDS card 04-0844.
Figure 3. CaCO3 disks grown on chitosan membranes in the presence of 1% (w/w) RSF at pH 7.9 for 21 days. (a) Top view and (b) side view. Black arrows indicate “the growth line” on the crystalline disk.
(Figure 1c,e) at initial pH value of 7.9. As a soluble additive, analogous to many synthesized polymers29,30 and other biopolymers,31 RSF can inhibit fast crystallization of minerals.23,25 The higher RSF concentration led to lower nucleation density of CaCO3 and caused each nucleation site to share more calcium sources.16 Therefore, the size of crystalline disk with obvious concentric circles on the surface in 1% (w/w) RSF solution (Figure 1d) was slightly larger than that in 0.1% (w/w) RSF solution (Figure 1c). The results suggested that RSF had significant influence on the crystallization and morphology of CaCO3 on chitosan membrane. 3.2. Microstructure of Crystalline Disks. HRSEM images showed that the center of the disk-like CaCO3 was composed of a number of angular nanoparticles around 20 nm in size, while the edge of the crystalline disk consisted of particles fused with each other (Figure 2). Although the morphologies of the center and edge of disk were different, the results of Raman and EDX indicated that the chemical composition seemed invariable
(Figure s2 in the Supporting Information). According to Scherrer’s equation,32 the crystallite size estimated from the half-peak width of (112) peak of vaterite at 27.5° in the XRD pattern (Figure 1e) was around 17 nm, which is consistent with the observation of HRSEM. The POM image (Figure 2d) displayed Maltese cross patterns in crystalline disks, suggesting that the disk-like crystals nucleated at the center of disks and grew radially.13,16,17,19 TEM images also revealed that the crystalline disk was composed of nanoparticles (less electron lucent phase with diameters varying from 20 to 30 nm in Figure 2e) cemented by organic components (electron lucent thread-like phase in Figure 2e), and the ED patterns exhibited vaterite of varying orientation (Figure 2f). During mineralization, the chitosan membrane was covered entirely by a layer of mineral deposits, and impingement occurred between adjacent disks (Figure 3). The vaterite disks that formed on the chitosan membrane remained stable for 21 days in the solution (Figure s3 in the Supporting Information), 2806
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Langmuir which suggested that the addition of silk fibroin inhibited the transformation of the crystal from vaterite to calcite. From the SEM work, we found that the diameter and thickness of disks increased with time, where the growth of crystalline disk was quite rapid during the first 3 days but slowed down in the following days (Figure 4). It was notable that the growth of CaCO3 disks was achieved by continuous accumulation of nanoparticles, in both radius and height, as reflected by the growth line (concentric circles indicated by black arrows in Figure 3). This is probably due to the nucleation and growth of a new mineral layer on the forming/formed layer under the regulation of RSF. Furthermore, oval-shaped crystalline disks where the long axes were along with the stretching direction of prestretched chitosan membranes were achieved (Figure s4 in the Supporting Information), suggesting that the directional adsorption of RSF by arranged functional groups of chitosan
Figure 4. Growth of CaCO3 disk with the mineralization. The square and triangular symbols represent diameter and thickness of the disk, respectively. Noticed that boundaries of crystalline disks became indefinite in later period due to the impingement; the statistics of size were only taken within 7 days.
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induced oriented growth of the disk.33 However, there was no difference between the polymorph of the disk-like crystals on prestretched membranes and that on unstretched membranes. 3.3. Effect of pH and Temperature on the Polymorph of Disk-like Crystal. The growth of disk-like CaCO3 crystals on the chitosan membrane could be achieved with different pH values and temperatures (Figure 5). However, when the pH was between 8.5 and 9.5, in addition to crystalline disks, a number of particles formed in bulk solution and deposited on the membrane (Figure 5b-d). We believe this may be due to competition between the aggregates of RSF inducing CaCO3 particle in such pH values23 and the interaction of chitosan with RSF34,35 or RSF/CaCO3 hybrid leading to disk-like crystal. Moreover, when pH value of the solution was 8.5 or 9.0, two kinds of disk with diameters around 100 and 20 μm appeared on the membrane. Results from a Raman spectrometer attached to an optical microscope showed that the polymorphs of large disk, small disk, and particle of CaCO3 were vaterite, aragonite, and calcite, respectively (Figure 6). As the pH increased to 9.5, the supersaturation of the system was so high that only thermodynamically unstable vaterite disks grew on the membrane, accompanied by a huge amount of precipitated calcite particles. In the meantime, the crystallization of CaCO3 was also varied with temperature. The polymorphs of CaCO3 produced on chitosan membrane at various conditions, determined by Raman spectroscopy, are summarized in Table 1. It seemed that the aragonite disks preferred to form at relatively high temperature in pH of 7.9 and 8.5, and the minimum temperature for the formation of aragonite decreased with the increasing of pH value. 3.4. Formation Mechanism of Crystalline Disks. The crystallization of minerals is controlled by both thermodynamic and kinetic factors. Generally, a slow thermodynamically controlled crystallization is likely to yield the stable polymorph of calcite, and kinetically controlled crystallization from high supersaturated solutions tends to produce metastable crystals such as vaterite.30 In the supersaturated solution, amorphous phase of CaCO3 forms first and transforms to anhydrous crystals with polymorphs dependent on the supersaturation.30,36 In our case, at the moment of mixing of Ca2þ and HCO3- ions, numerous
Figure 5. SEM images of CaCO3 grown on chitosan membranes at a range of pH values and temperatures in the presence of 1% (w/w) RSF. (a) pH 7.9, 25 °C; (b) pH 8.5, 25 °C; (c) pH 9.0, 25 °C; (d) pH 9.5, 25 °C; (e) pH 8.5, 5 °C; (f) pH 7.9, 45 °C. White arrows indicate aragonite disks, and the inset in (b) shows a magnified image of “rice-like” particles. 2807
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amorphous nanoparticles formed for the high supersaturation of the solution (Figure s5a in the Supporting Information).37 Meanwhile, a large number of silk protein molecules in the solution were bound to these particles through the interactions between the hydrophilic segments and CaCO3 to form hybrids.23 Part of these particles may remain in amorphous state (Figure s5b in the Supporting Information) due to the stabilization effect
Figure 6. Raman spectroscopy of CaCO3 on chitosan membrane at pH 8.5, 25 °C. (a) Crystalline disks with polymorph of aragonite (small disks indicated by white arrows in Figure 5b). (b) Crystalline disks with polymorph of vaterite (large disks in Figure 5b). (c) Calcite particles forming in bulk solution adsorbed onto chitosan membrane.15,30
Table 1. Crystallographic Polymorphs of the CaCO3 Products for Various Conditions Determined by Raman Spectra pH temperature (°C)
a
7.9
8.5
9.0
9.5
5
va
v
v þ a,b cc
v, c
25
v
v þ a, c
v þ a, c
v, c
45
vþa
v þ a, c
-d
-d
Vaterite disks. b Aragonite disks. c Calcite particles. d No data.
of adsorbed RSF,25 while others maybe transformed into deficient nanocrystals (Figure s5c in the Supporting Information)29 containing silk fibroin. The homogeneous nucleation of crystals was inhibited to slow down the crystallization in the bulk solution. That means the fast crystallization from CaCO3 nanoparticles was hindered, thus shifting the product to thermodynamically stable calcite. As the nucleation occurred by means of the hydrophobic aggregation of RSF, the calcite particles precipitated in the bulk solution.23 On the other hand, RSF and hybrid CaCO3/RSF nanoparticles associated by electrostatic interactions were adsorbed on the surface of the chitosan membrane via hydrogen bonds mainly between -CONHand -NH2,34,35 which increased the local supersaturation in the vicinity of membrane (Scheme 1a). In this case, kinetic factors became predominant and metastable vaterite nucleated and grew. As a result, Ca2þ ions and hybrid CaCO3/RSF nanoparticles accumulated to the nucleation sites continuously to make the crystals grow radially and ultimately yielded disk-like crystals (Scheme 1b). HRSEM observations on the center of the surface (Figure 2c) and cross section (Figure s6 in the Supporting Information) of disk-like crystals as well as TEM image of the cross section (Figure 2e) certified the aggregation model of nanoparticles. Additionally, from the TEM image, it can be found that nanoparticles were cemented by an organic component. Through the calculation of the XRD results and the HRSEM images of the disk-like crystals grown over different times, we found there was no change in the size of nanoparticles comprising the disk (data not shown), suggesting a great deal of RSF surrounding the CaCO3 particles inhibited the growth of these nanoparticles and maintained their stability. Whether the polymorph of the crystallized CaCO3 is aragonite or vaterite, it is kinetically determined by supersaturation which strongly depends on the solution pH.16,17,36 The high pH led to the increase of the conversion of CO32- from HCO3- as well as the negative charge of RSF. The isoelectric point of RSF is about 4.2 (heavy chain), and the dissociation constant of chitosan is about 6.3.16,38 Hence, within the pH range adopted in our investigation (from 7.9 to 9.5), RSF is negatively charged while chitosan is close to neutral. The eventual CaCO3 supersaturation in the membrane vicinity was dominated by the competition between increasing the negative charge of RSF on the membrane (which attracted Ca2þ to promote local supersaturation) and
Scheme 1. Mechanism of the Formation of Disk-like CaCO3: (a) Nucleation of CaCO3 Due to the Adsorption of RSF and CaCO3/RSF Hybrid Nanoparticles; (b) Accumulation of CaCO3/RSF Hybrid Nanoparticles Inducing Growth of the Disk-like Crystal
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Langmuir that in bulk solution (which bound Ca2þ to limit the transportation of it from bulk solution to membrane surface).16 Therefore, the local supersaturation in the membrane vicinity at the certain pH values, e.g., 8.5 or 9.0, may be suitable for the production of both aragonite and vaterite. On the other hand, aragonite was inclined to precipitate at relatively high temperature at the certain pH due to its higher surface energy compared with that of calcite or vaterite.36,39 Our previous work has indicated that the initial pH values of solution and the temperatures which were varied in the same range had no obvious effect on the polymorphs of CaCO3 produced in the system only contained soluble RSF.23 Thus, our results show that the influence of pH and temperature on the crystallization of CaCO3 was much greater in the vicinity of chitosan membrane than that in bulk solution.
4. CONCLUSIONS In this study, disk-like CaCO3 was formed on an insoluble chitosan matrix surrounded by the supersaturated CaCO3 solution containing RSF. The growth of the crystalline disk was achieved by continuous accumulation of hybrid CaCO3/RSF nanoparticles associated by electrostatic interactions. The polymorph of the disk-like crystals was determined by the local supersaturation and dominated by the competition between the changes of negative charge of RSF in bulk solution and that on the membrane dependent on pH. Furthermore, higher temperature favors the crystallization of the polymorph with a higher surface energy, e.g., aragonite. We suggest that the influence of solution pH and temperature on the polymorphs of CaCO3 was much greater on the membrane than that in bulk solution. The results may provide insight into not only the formation of natural biomaterials but also the design of novel organic/inorganic composite materials in vitro. ’ ASSOCIATED CONTENT
bS
Supporting Information. SEM images of CaCO3 on the chitosan membrane in the absence of RSF, CaCO3 grown on prestretched chitosan membrane, and cross section of crystalline disk; TEM images and ED patterns of nanoparticles in the bulk solution; XRD pattern and Raman spectra of disk-like vaterite. This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Present Addresses †
DWI an der RWTH Aachen e.V., Institut f€ur Technische and Makromolekulare Chemie der RWTH Aachen University, Pauwelsstr. 8, D-52056 Aachen, Germany.
’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (NSFC 20974024) and 973 Project of Chinese Ministry of Science and Technology (No. 2009CB930000). The authors thank Dr. Yan Chen and Dr. Qingsong Wu for their kind help on TEM observation. Special thanks to Dr. C. Holland (University of Oxford, UK) for his critical reading and polishing of the English.
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