An Electrophoretic Approach Provides Tunable Mineralization Inside Agarose Gels Junji Watanabe†,‡ and Mitsuru Akashi*,†,‡ Department of Applied Chemistry, Graduate School of Engineering, Osaka UniVersity, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan, and 21st COE “Center for Integrated Cell and Tissue Regulation”, Osaka UniVersity, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan
CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 2 478–482
ReceiVed April 9, 2007; ReVised Manuscript ReceiVed October 14, 2007
ABSTRACT: Mineralization inside agarose gels was examined using an electrophoretic approach. Effective ion migration for mineralization, for example, of calcium carbonate and hydroxyapatite, was shown to be a crucially important factor. Calcium, carbonate, and phosphate ions easily migrated into the gel interior from ionic solutions when an electric field was applied to both terminals of the gel. The time to reach complete mineral formation was only 3 min, and 10-50 µg of calcium carbonate and 50-100 µg of hydroxyapatite were formed in 1 mg of dry gel. The concentration of the ion species regulated the total amount of mineral formation. These ionic movements into the gel interior were achieved by a novel electrophoretic migration method. The resulting minerals were characterized by infrared spectroscopy, X-ray diffraction, and scanning electron spectroscopy, and then the minerals were assigned as calcite and hydroxyapatite. Moreover, the dissolution of these minerals was evaluated in phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane hydrochloride, and citric acid buffer solutions. The results showed that the minerals could dissolve at pH 5.8 immediately. Alternatively, the minerals could transform into hydroxyapatite in PBS at pH 7.4. This result indicated that the minerals in the composite could undergo further mineralization or transformation under physiological conditions. In summary, mineralization of an agarose gel interior could proceed using an electrophoretic approach.
1. Introduction Minerals evolved in living systems from ancient times. A volcano produces volcanic ashes, and subsequently a river, lake, or sea could be formed. In particular, the oceans are well-known as the origin of life. The most primitive living thing is a magnetic bacterium, which contains nanoscale magnetic particles. Furthermore, a diatom contains silica compounds, a coral reef is made from calcium carbonate, and a vertebrate contains bones as its skeleton. Minerals such as a calcium phosphate and a hydroxyapatite are easily attached onto heated steel walls, the surfaces of which are covered with milk proteins, in a milk processing industry.1 This is a major problem in the dairy industry. In most cases, the minerals in nature are sophisticated and hierarchical microstructures, and the minerals are generally formed on an organic material as a template. In particular, the hydrogel interior is an attractive space that exhibits interconnectivity within their three-dimensional (3D) reactive space. In general, the hydrogel interior has frequently been used to load and release drugs in a stimuli-response fashion.2 On the other hand, the hydrogel interior is also utilized for analytical chemistry, and agarose gels have often been used in electrophoresis to separate DNA molecules, because of the large 3D space in their interior. In this paper, we focused on the interior space in hydrogels and designed a tunable mineralization method using an electrophoretic approach. Calcium carbonate and hydroxyapatite were the target minerals in terms of interactive organic–inorganic biocomposites. In general, mineral formation was studied by using template materials not only in the nanosized reactive space3,4 but also at the molecular level.5,6 Calcium carbonate shows several polymorphs such as calcite, aragonite, and vaterite. The stability of these polymorphs is regulated by * To whom correspondence should be addressed. Tel.: +81-6-6879-7356. Fax: +81-6-6879-7359. E-mail:
[email protected]. † Department of Applied Chemistry, Graduate School of Engineering. ‡ 21st COE “Center for Integrated Cell and Tissue Regulation”.
thermodynamics.7 Vaterite does not exist in nature due to its lower thermodynamic stability. However, we can control their polymorphs by the addition of divalent cations such as Mg2+, Fe2+, and Ba2+.8 On the other hand, the stoichiometry of hydroxyapatite is known to be Ca10(PO4)6(OH)2. However, slight imbalances in the stoichiometric ratio of calcium and phosphorus ions in hydroxyapatite were generally observed due to its adaptable crystalline structure. Koutsopoulos summarized a relationship between the preparation methods and the resulting crystalline structure for hydroxyapatite.9 Biomimetic crystallization of hydroxyapatite was discovered by Kokubo in 1990, and his pioneering work was based on simulated body fluids.10 Moreover, a double diffusion method was also investigated within porous polymer matrices.11,12 Akashi et al. proposed an alternate soaking process to prepare calcium carbonate and hydroxyapatite on/in hydrogels and used them as injectable biomaterials for bone regeneration.13–18 The alternate soaking process was recently improved by changing the composition of the ionic solutions.19 These mineralization processes were subject to ionic diffusion into the substrates from ionic solutions. Therefore, it consumed a great deal of time to prepare these mineral composites. To improve this timeconsuming process, we have already reported a new, preliminary paradigm of mineralization using an electrophoretic approach.20 In the first report, we presented a technique using electrophoresis to deliver ions into the interior of hydrogels. Moreover, the resulting hydroxyapatite was examined by an in vivo study in terms of bone conductivity.21 In this study, we have further improved our electrophoretic approach for mineralization in terms of diversity and stability. In our previous report, the stability of the hydroxyapatite formed, and the small amount of hydroxyapatite formed was a crucial problem. These issues were caused by changing the pH during electrophoresis mineralization. Therefore, calcium hydroxide was added to the agarose gels to neutralize the pH of the gel interior. Moreover, the additive was a source of calcium ions, and an improvement in the amount of product could be expected.
10.1021/cg0703487 CCC: $40.75 2008 American Chemical Society Published on Web 01/19/2008
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Calcium carbonate and hydroxyapatite formation in the agarose gel interior would be a good candidate not only as a scaffold for regenerative medicine but also as a protein reservoir for controlled release.
2. Materials and Methods 2.1. Materials. Agarose (SeaKem Gold) was purchased from Cambrex Bio Science Rockland, Inc., Rockland, ME, USA. The gel strength is over 3500 g/cm2 at a 1.5 wt% gel concentration. Sodium carbonate and calcium hydroxide were purchased from Nakalai Tesque Inc., Kyoto, Japan. Sodium chloride, disodium hydrogenphosphate tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), citric acid, and sodium citrate were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Dulbecco’s Phosphate Buffered Saline (PBS) (10×) was purchased from Invitrogen Corp., CA, USA, and was used after dilution. All other reagents were of extra pure grade and were used as received. Ultrapure water was used throughout the experiments. 2.2. Mineralization in the Agarose Gel Interior by an Electrophoretic Approach. A submarine-typed electrophoresis apparatus (Mupid, Advance Co., Ltd., Tokyo, Japan) with a Pt electrode was used for the mineralization. First, calcium hydroxide was dissolved in ultrapure water to a concentration of 10 mmol/L by heating, and then agarose powder was added to the solution. The agarose solution was molded by the gel tray (6 × 11 × 0.8 cm). Sodium chloride was also added to the solution at the given concentrations if necessary. The resulting agarose gel was then set in the electrophoresis apparatus. Calcium chloride, sodium carbonate, and disodium hydrogenphosphate were dissolved in ultrapure water, and the final concentration was adjusted to 10 or 5 mmol/L. To prepare calcium carbonate in the gel interior, each calcium chloride and sodium carbonate aqueous solution was poured into the anode and cathode tanks, respectively. Similarly, calcium chloride and disodium hydrogen phosphate aqueous solutions were also poured into the appropriate tanks to form hydroxyapatite. After the setup, the electrophoresis was performed for only 3 min at 100 V. The resulting agarose gel was immersed into ultrapure water to remove surplus ions from the gel interior. 2.3. Quantification of Minerals in the Agarose Gel Interior. The resulting minerals contained calcium ions and therefore mineral formation was evaluated in terms of the total amount of calcium. First, the resulting agarose gel containing minerals was sliced into small blocks (roughly 50 mg in the swollen state). The sliced blocks were immersed into 1 mL of hydrochloric acid aqueous solution (1 mol/L) overnight to dissolve the minerals in the agarose gel interior. Twenty microliters of the supernatant was used for further evaluation, and the concentration of calcium ion was evaluated using Calcium E-Test Wako (Wako Pure Chemical Industries, Ltd.). The calcium ions in the supernatant were captured by the chelating reagent (methyl xylenol blue), which was contained in the detection kit. After the formation of the chelate compound, the change in absorbance at 610 nm was monitored by a UV–vis spectrophotometer (U-3010, Hitachi Ltd., Japan). A calcium standard solution (10 mg/dL, Wako Pure Chemical Industries, Ltd.) was used to generate a calibration curve. The total amount of calcium carbonate and hydroxyapatite was calculated from the results of the calcium ion concentration and the molecular weights of CaCO3 (MWCA)100) and Ca10(PO4)6(OH)2 (MWHA ) 1004) using the following equations. Amount of CaCO3 (µg) ) conc of Ca2+ (µg/mg-dry gel) × gel weight (mg) × MWCA/40 (1) Amount of hydroxyapatite (µg) ) conc of Ca2+ (µg/mg-dry gel) × gel weight (mg) × MWHA/(40×10) (2)
2.4. Characterization of Mineral Composites. The resulting composite gel was lyophilized, and then Fourier transform infrared (FTIR) spectra were recorded by the attenuated total reflection (ATR) method using a FT-IR spectrometer (Spectrum 100, Perkin-Elmer Japan Co., Ltd.) ranging from 2000 to 380 cm-1. The morphology of the minerals was observed using a scanning electron microscope (SEM,
JSM-6700FE, JEOL, Tokyo, Japan) after staining with osmium tetraoxide. The SEM observation was carried out at 3000× magnification. The mineral composite was also characterized in terms of the X-ray diffraction patterns of its crystals (XRD, RINT In Plane ultraX18, Rigaku, Tokyo, Japan). The X-ray source was Cu KR, and 40 kV and 200 mA was used for the measurements. The scan speed was 2°/min, and 10° to 50° was monitored. 2.5. Dissolution of Minerals in Buffer Solution. The mineral composites were immersed in a buffer solution to estimate the mineral dissolution. The composites were sliced and then immersed in a given buffer solution. In the present study, PBS (pH 5.8 and 7.4), Tris-HCl (pH 7.4), and citric acid buffers (pH 5.8) were used. The dissolution study was performed at 37 °C. The supernatant was sampled, and the concentration of calcium ion was quantified using the Calcium E-Test Wako at adequate intervals. The dissolution rate was normalized by the initial calcium ion content.
3. Results and Discussion 3.1. Preparation of Mineral Composites by Electrophoresis. In the present study, we focused on calcium carbonate and hydroxyapatite formation in the agarose gel interior. We converted the mineralization of agarose gel from an alternate soaking process to an electrophoretic approach. This electrophoresis approach was first reported in 2006.20 However, the methodology provided only preliminary results, and in particular the pH shift (below pH 5) in the gel interior and the elapsed time (roughly 30 min) required further improvement. Therefore, we selected calcium hydroxide as the ionic source for the mineralization as well as the electric conductive source. Calcium hydroxide is a typical, poorly water soluble inorganic compound. However, 10 mmol/L could be dissolved in hot water. Thus, a homogeneous agarose gel containing calcium hydroxide was prepared. Moreover, the calcium hydroxide showed an alkaline pH, and therefore a neutralizing function could be expected through the mineralization by an electrophoretic approach. Table 1 shows a summary of the preparative conditions of the mineralization. In the case of calcium carbonate, CA is used as an abbreviation. CA10/5 means calcium carbonate with 10 mmol/L sodium chloride in the agarose gel and 5 mmol/L ionic solutions for mineralization. Hydroxyapatite formation is abbreviated as HA. Sodium chloride was added to the agarose gel by changing the concentration from 0 to 10 mmol/L. The purpose of the addition of sodium chloride was an enhancement of the electric conductivity. Figure 1 shows the typical setup and mineral composites. Each ionic solution was completely separated by the agarose gel, because the ionic solutions formed a precipitate (calcium carbonate or hydroxyapatite) if they were mixed together. The calcium ion should be set in the anode side to migrate to the cathode side through the agarose gel. Alternatively, carbonate or phosphate ions should be placed in the cathode side. When calcium and carbonate ions were mixed together in the agarose gel, calcium carbonate was formed. Mineral formation was observed after 3 min of electrophoresis, and the transparent agarose gel changed color to a whitish turbid color (Figure 1b). The mineralization proceeded for 3 min, and then mineral was formed at the edge of the agarose gel (5 mm). The rate of mineral formation was 1.67 mm/min, which was much higher than that of our previous report (0.67 mm/min).20 The enhancement of the formation rate was roughly 2.5-fold faster. Moreover, the pH of the gel interior was slightly alkaline due to the calcium hydroxide. Therefore, the formed minerals were stable in the agarose gel interior. 3.2. Mineral Formation in the Agarose Gel Interior. Figure 2 shows the total amount of calcium ion in the dry gel and its mineral content (calcium carbonate or hydroxyapatite). In the
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Table 1. Preparative Conditions of Mineralization by Electrophoresis Approach conc of solutions (mmol/L) in agarose gel
a
a
in aqueous solution
abbreviation
minerals
Ca(OH)2
NaCl
CaCl2
Na2CO3
CA10/10 CA10/5 CA5/10 CA5/5 CA0/10 CA0/5 HA10/10 HA10/5 HA5/10 HA5/5 HA0/10 HA0/5
calcium carbonate
10 10 10 10 10 10 10 10 10 10 10 10
10 10 5 5 0 0 10 10 5 5 0 0
10 5 10 5 10 5 10 5 10 5 10 5
10 5 10 5 10 5
hydroxyapatite
Na2HPO4
10 5 10 5 10 5
Agarose gel was prepared at 1.5 wt%.
Figure 3. ATR-IR spectra of mineral composites; (a) calcium carbonate/ agarose composite and (b) hydroxyapatite/agarose composite.
Figure 1. Mineralization of agarose gel interior by electrophoresis approach; (a) setup of conventional electrophoresis apparatus and (b) formed calcium carbonate in the gel interior (turbid region was observed on the right side (5 mm)).
Figure 2. Total amount of calcium ions in dry gel (n ) 3); (a) calcium carbonate/agarose composite and (b) hydroxyapatite/agarose composite.
case of calcium carbonate formation, 20 µg of calcium ions were detected in 1 mg of dry gel (CA10/10). This calcium content corresponded to 50 µg of calcium carbonate in 1 mg of dry gel. The mineralization ratio was roughly 5% relative to 1 mg of dry gel. On the other hand, CA0/5 showed a small amount of calcium carbonate formation in the agarose gel, with 10 µg of calcium carbonate in 1 mg of dry gel (roughly 0.1%). This could be changed by the preparative concentrations. Thus,
mineral formation was well correlated with the preparative concentrations of not only sodium chloride but also the ionic solutions (calcium and carbonate). In particular, the concentration of sodium chloride was important for ionic migration, which was subject to electric conductivity. In this study, the time to reach complete mineralization was all regulated to 3 min. Therefore, the total amount of mineral formed was subject to ion migration, and the mineral formation was easily regulated by altering the total amount of ion migration. In the case of hydroxyapatite (HAp) formation, HA10/10 resulted in 40 µg of calcium ions in 1 mg of dry gel, translating into 100 µg of hydroxyapatite (roughly 10%). The resulting hydroxyapatite formation was much larger (10 times) than that of our previous report (roughly 1%).20 The higher hydroxyapatite formation was caused by a stabilization of the pH in the gel interior. The pH was crucial in forming minerals in the gel interior, particularly under acidic conditions, which could easily dissolve the formed minerals. The stabilization of the pH was achieved by using calcium hydroxide. The effects of the preparative concentrations regarding sodium chloride and the ionic solution were not clearly observed for hydroxyapatite formation. HA0/10 and HA0/5 were significantly lower than the other concentrations, but a significant trend could not be observed. 3.3. Characterization of Mineral Formation. Figure 3 shows the ATR-IR spectra of the composites, which were prepared from 10 mmol/L of each ionic solution. In the case of calcium carbonate, typical carbonate peaks (CO32-) were observed at 712 (υ4), 848 (υ2), and 872 cm-1 (υ2). The assigned peaks were not observed from the bare agarose gel. Therefore, calcium carbonate was formed inside the agarose gel interior by an electrophoretic approach. The ATR-IR characterization
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Figure 6. Dissolution rate of calcium carbonate from CA10/10 composite at 37 °C; (a) PBS (pH 7.4 and 5.8) and (b) Tris-HCl (pH 7.4) and citric acid buffer (pH 5.8). Each square and circle symbol means pH 7.4 and pH 5.8, respectively.
Figure 4. X-ray diffraction patterns of mineral composites; (a) calcium carbonate/agarose composite (CA10/10) and (b) hydroxyapatite/agarose composite (HA10/10).
provided a similar trend, even if the total amount of calcium carbonate was variable (CA10/10, CA5/10, and CA0/10). On the other hand, hydroxyapatite was also detected from the ATRIR spectra (Figure 3b). Typical phosphate peaks (PO43-) were observed at 560 (υ3) and 1088 cm-1 (υ4). Similarly, an OH peak at 600 cm-1 was observed. In this case, the IR transmittance did not correlate with the hydroxyapatite content in the composite gels. The mineral composites were characterized in terms of their crystalline structure by X-ray diffraction (XRD) (Figure 4). A typical calcite pattern of calcium carbonate was observed as shown in Figure 4a, and each peak was observed at 23.0° (102), 29.4° (104), 36.0° (110), 39.4° (113), 43.1° (202), 47.5° (108), and 48.5° (202). Moreover, trace peaks from vaterite were observed at 24.9° and 32.8°, but the resulting peaks were too weak to assign to their crystalline structure. Using this electrophoretic approach, calcium carbonate (calcite) was specifically formed inside the agarose gel. Figure 4b shows the XRD pattern of the hydroxyapatite composites, and five peaks were observed at 25.8° (002), 28.2° (102), 29.1° (210), 31.7° (211), and 39.8° (310). The observed peaks were not large, even when the total hydroxyapatite content was roughly 10% in the agarose gel composite. In general, hydroxyapatite has many polymorphs such as β-tricalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate. Although these polymorphs also show a typical XRD pattern, no peaks were observed in the hydroxyapatite composites. Taking this result into account, we concluded that hydroxyapatite was deposited in the gel interior by electrophoretic ion migration.
Figure 5 shows the microstructure of the minerals using SEM. In the case of calcium carbonate, the crystals were dispersed within agarose gel networks. The size of each crystal was roughly 2 µm per side. The morphology of calcium carbonate was typical calcite. Moreover, the size of the calcite crystals was monomodal, indicating homogeneous mineralization in the gel interior. Alternatively, a spherical morphology was observed in the hydroxyapatite agarose gel composite (Figure 5b). The hydroxyapatite had a monosized distribution around 1 µm, and the particles formed their aggregates and were located on/in the agarose gel polymer network. From these results, we concluded that the electrophoretic approach provided homogeneous mineralization in the gel interior, and the resulting minerals showed a well-defined size distribution. 3.4. Dissolution Properties of the Mineral Composites. The mineral composites were immersed in a buffer solution. In general, minerals are stable at neutral or alkaline pH conditions but are easily dissolved in an acidic environment. Therefore, the resulting mineral composites were evaluated on their dissolution rate upon changing the pH. PBS, Tris-HCl, and citric acid buffer solutions were used as the media. All of the ionic concentrations were adjusted to 0.15 by sodium chloride to simulate physiological conditions. Figure 6 shows the dissolution rate of calcium carbonate from the agarose gel composite. In the case of PBS, the calcium carbonate was immediately dissolved at pH 5.8. Eighty percent of the calcium carbonate was dissolved after 5 h, and the release of the remaining calcium carbonate was not significantly observed from the composite gels. On the other hand, only 40% of the calcium carbonate was dissolved in pH 7.4 PBS, and further dissolution was not observed. This result indicated that calcium carbonate would be stabilized in PBS at pH 7.4. What is the driving force to stabilize the calcium carbonate? Ratner et al. reported that a nacre surface transformed to hydroxyapatite in PBS.22 Nacre is known as mother-of-pearl and is composed of 95% calcium carbonate and 5% organic materials. The transformation was
Figure 5. Scanning electron microscope observations of mineral composites; (a) calcium carbonate/agarose composite (CA 10/10) and (b) hydroxyapatite/agarose composite (HA10/10).
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4. Conclusions
Figure 7. Dissolution rate of hydroxyapatite from HA10/10 composite at 37 °C; (a) PBS (pH 7.4 and 5.8) and (b) Tris-HCl (pH 7.4) and citric acid buffer (pH 5.8). Each square and circle symbol means pH 7.4 and pH 5.8, respectively.
observed after 5 days of incubation. The fundamental mechanism responsible for the surface transformation was believed to be the ionic balance between dissolution and precipitation. Taking this report into account, the calcium carbonate composite gel could transform from calcium carbonate to hydroxyapatite in PBS at pH 7.4. This transformation proceeded after the initial dissolution (over 5 h). Therefore, the dissolution rate was maintained at 40% after 5 h. In this transformation, the buffer solution is crucial, so a similar dissolution test was carried out using Tris-HCl (pH 7.4) and citric acid buffer (pH 5.8) as shown in Figure 6b. This result was clear; the calcium carbonate in the agarose gel composite could be dissolved not only at pH 5.8 but also at pH 7.4. In this study, the ionic strength was adjusted to 0.15 using sodium chloride throughout the experiment. However, the dissolution rate at pH 7.4 was completely different between PBS and Tris-HCl. This result indicated that the ion species are crucial factors to dissolve and transform the minerals. This significant transformation was observed in agarose gel composites containing hydroxyapatite. Figure 7 shows the dissolution rate of hydroxyapatite. In the case of PBS at pH 7.4, the hydroxyapatite was dissolved within the first period (within 5 h), and then the dissolution rate decreased upon increasing the incubation periods. This result indicated that the hydroxyapatite would be transformed in PBS after 5 h. We hypothesized that poorly crystallized hydroxyapatite was a candidate to be dissolved for the first time; the poorly crystallized hydroxyapatite could be completely dissolved at pH 5.8. Moreover, the time to reach complete dissolution was roughly 20 h. The effect of the buffer solution is shown in Figure 7b. The hydroxyapatite could easily dissolve in Tris-HCl (pH 7.4) and citric acid (pH 5.8), and the time to complete dissolution was similar to calcium carbonate. Taking these results into account, we conclude that the mineral dissolution was regulated by the buffer solutions and pH. The resulting mineral composite gels are good candidates as implantable materials for tissue regeneration, and these minerals will be stable under physiological conditions in spite of a local acidic environment caused by an inflammatory response.
Mineralization within an agarose gel was demonstrated by an electrophoretic approach. Calcium carbonate and hydroxyapatite were formed in the agarose gel interior within 3 min. This electrophoretic approach resulted in mineral formation not only at the surface but also inside the hydrogel interior. In addition, minerals in the composite gels could dissolve under acidic conditions and transform to hydroxyapatite in phosphate buffered saline. These mineral composites are of great importance in terms of providing injectable soft biomaterials. Acknowledgment. Part of this study was financially supported by a Grant-in-Aid for 21st Century COE Program for “Center for Integrated Cell and Tissue Regulation” from The Ministry of Education, Culture, Sports, Science and Technology, Japan, and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (19655082).
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