ARTICLE pubs.acs.org/Biomac
State of Water, Molecular Structure, and Cytotoxicity of Silk Hydrogels Keiji Numata,*,† Takuya Katashima,‡ and Takamasa Sakai‡ † ‡
Enzyme Research Team, RIKEN Biomass Engineering Program, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
bS Supporting Information ABSTRACT: A novel technique was developed to regulate the bulk water content of silk hydrogels by adjusting the concentrations of silk proteins, which is helpful to investigate the effects of the state of water in polymeric hydrogel on its biological functions, such as cytotoxicity. Gelation of the silk hydrogel was induced with ethanol and its gelation behavior was analyzed by rheometry. The silk hydrogels prepared at various silk concentrations were characterized with respect to their water content, molecular and network structures, state of water, mechanical properties, and cytotoxicity to human mesenchymal stem cells. The network structure of silk hydrogel was heterogeneous with β-sheet and fibrillar structures. The influence of the state of water in the silk hydrogel on the cytotoxicity was recognized by means of differential scanning calorimetry and cell proliferation assay, which revealed that the bound water will support cell-adhesion proteins in the cellular matrix to interact with the surface of the silk hydrogels.
’ INTRODUCTION Hydrogel is an attractive biomaterial for regenerative medicine and tissue engineering because of its excellent biocompatibility, which is attributed to its high water content of over 90%. The role of water molecules in hydrogels has been investigated by many researchers, with the result that bound (nonfreezing), bulk (freezing), and intermediate (freezing bound) water have been shown to exist in hydrogels.13 Differential scanning calorimetry (DSC) and NMR relaxometry have been used as typical methods to characterize and distinguish the state of water molecules in polymeric hydrogels.4,5 The difference in state of water in organisms has been suggested to affect various aspects of biological structures, including their size, in bacterial and vegetative cells.68 The bulk water is not crucial for enzymatic hydration, whereas the bound water plays an important role in enzymatic catalysis.9,10 The biological activities of enzymes and proteins have been reported to depend on how the water molecules associate with these bioactive molecules, that is, the activities depend on the bound water content of the enzymes and proteins.11 The bound water content has been considered a significant factor in the control of drug release rate as well as enzymatic activity in hydrogel-based biomaterials.12 Although these previous studies have indicated that the state of water in hydrogels used as biomaterials therefore must influence their biocompatibility and biological response, the relationship between the state of water and the cytotoxicity of hydrogels has not been completely clarified until now. Silk fibroins have been successfully used in the biomedical field as sutures for several decades, and have also been explored as biomaterials for cell culture, tissue engineering, and drug delivery r 2011 American Chemical Society
systems, earning Food and Drug Administration approval for such expanded utility because of their excellent mechanical properties, versatility in processing, and low cytotoxicity.1315 Virgin silkworm silk with the associated contaminant sericin proteins was reported as a potential allergen causing a Type I allergic reaction due to upregulated IgEs in response to the sericins; however, once the sericins are properly removed, there is minimal response from the core fibroin structural proteins.16,17 Further, the degradation products of silk proteins with β-sheet structures, when exposed to R-chymotrypsin, have recently been identified and shown to have no cytotoxicity to in vitro neuron cells.18,19 The gelation mechanism of silk solution was studied based on the β-sheet structure content and pH of the silk solution, and the results indicated that the gelation is dependent on the β-sheet content.20,21 Silk-based hydrogels have also been investigated by Kaplan and co-workers, who found that the gelation of silk solution was induced by pH change, ultrasonication, or vortex.2226 Upon the gelation, a random-coil structure of silk molecules was shown to transform into a β-sheet structure, and subsequently, the βsheet structures aggregated to form molecular network structures, namely, hydrogels.24,26 Human mesenchymal stem cells (hMSC) grew and proliferated over 21 days in the sonication-induced hydrogel prepared with 4% silk solution, indicating that the silk hydrogel is biocompatible and low-cytotoxic enough to be used in cell encapsulation.24,26 The silk hydrogel has therefore been considered as a candidate for a noncytotoxic biomaterial, but it Received: February 16, 2011 Revised: April 12, 2011 Published: April 26, 2011 2137
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Biomacromolecules is still necessary to clarify the effects of the state of water and the effects of the average molecular weights between cross-links of silk hydrogels on various biological activities, such as cellular viability and adhesiveness. In the present study, we developed a facile and quick method to prepare silk hydrogel using ethanol, and also analyzed the gelation behavior, state of water, secondary structure and mechanical properties of the resulting hydrogel. The average molecular weight between cross-links of the silk hydrogel prepared at different polymer concentrations was calculated and compared with the network structure of the silk hydrogels. Further, the cell proliferation and viability of hMSC on the silk hydrogels was characterized, and then the relationship among the state of water, molecular structure, and cytotoxicity of the silk hydrogel was discussed.
’ MATERIALS AND METHODS Preparation of Silk Solution. Silk solution was prepared by reference to the method reported previously.27 Silkworm cocoons of Bombyx mori were cut and boiled for 30 min in a 0.02 M NaCO3 solution and then washed with Milli-Q water to remove sericin proteins and wax. The extracted silk proteins were dried and dissolved in a 9.3 M LiBr solution at 60 °C for 2 h at a concentration of 20 wt %. The silk solution was dialyzed with Milli-Q for at least 4 days using a dialysis membrane (Pierce Snake Skin MWCO 3500; Thermo Fisher Scientific, Waltham, MA). The dialysis was completed when the conductivity of the dialysis solution was identical to that of Milli-Q. The silk solution of 1 mL was dried at 60 °C for 24 h, and then the resultant silk film was weighed to determine a concentration of the silk solution. The final concentration of the silk solution was approximately 6 wt %. Gelation of Silk Hydrogel. The silk solution and ethanol were mixed in a 15 mL Falcon tube, and then the resultant solution was poured in the interstice of the double cylinder of a rheometer (MCR501; Anton Paar, Austria). The measurement limit of the rheometer was approximately 0.1 Pa. The oscillatory shear rheological properties, that is, the storage modulus (G0 ) and the loss modulus (G00 ), during gelation were measured at 37 °C with a double cylinder geometry with 26.7 and 28.9 mm diameter cylinders. The sample volume was 6 mL and was sealed by the sample cap to inhibit the evaporation of water. The strain and the frequency were 1% and 1 Hz, respectively. The measurements were performed at least three times. All oscillatory tests were performed within the linear viscoelastic region of the gelled material (Figure S1). The gelation point (tgel) in rheological curves was defined simply as the crossover of the storage modulus G0 and the loss modulus G00 .28 Preparation of Silk Hydrogel. The silk solution was mixed and slightly vortexed with various amounts of ethanol at 37 °C. The ratios of the silk solution/ethanol were 1/9, 2/8, 3/7, 4/6, 5/5, 6/4, 7/3, 8/2, and 9/1, resulting in silk concentrations of 7, 13, 19, 25, 32, 38, 44, 50, and 57 g/L. The silk gels prepared by the addition of ethanol were left in a sealed tube for 24 h after their gelation with ethanol. The silk gels were washed with Milli-Q for 24 h, and silk hydrogel without ethanol (equilibrium swollen state) was obtained. The absence of ethanol in the silk hydrogel was confirmed by differential scanning calorimetry (DSC, Pyris 1; Perkin-Elmer, Waltham, MA). Characterization of Silk Hydrogel. The water contents of silk hydrogels were determined gravimetrically by lyophilization for 1 week. The water content (X) was calculated as follows:
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10 mg of hydrogels was transferred into a DSC aluminum pan, and subsequently the pan was first cooled to 50 °C and then heated to 30 °C at a rate of 5 °C/min, according to the literature.3,4,2932 The linear baseline to integrate peaks was determined, and then the melting temperature (Tm) and enthalpy of fusion (ΔH) were calculated from the DSC thermograms using Perkin-Elmer Pyris Software. An attenuated total reflectance Fourier transform infrared spectroscope (ATR-FTIR; IR Prestige-21; Shimadzu, Tokyo, Japan) equipped with a multiple-reflection, horizontal MIRacle ATR attachment (using a Ge crystal, from Pike Tech, Madison, WI) and a DLATGS detector with temperature control for Middle/Far IR was used to evaluate the secondary structure of silk proteins composing the hydrogels. For each measurement, 128 scans were accumulated with a resolution 4 cm1, and the wavenumber ranged from 400 to 4000 cm1. ATR-FTIR spectra in the amide I region were deconvoluted to determine the fraction of the β sheet structures formed during gelation with ethanol. The deconvolution and quantitative evaluation of the ATR-FTIR spectra were carried out using IR solution 1.50 (Shimadzu), according to the previous method.22,33,34 WAXD patterns of the silk hydrogels prepared on glass sample holders with a large volume (2 mm deep) enough to ignore dehydration of samples during the measurement were recorded at 25 °C on a Rigaku RINT 2500 system (Rigaku Corporation, Tokyo, Japan) using nickelfiltered Cu KR radiation (λ = 0.154 nm; 40 kV; 110 mA) in the 2θ range from 4 to 60° at a scan speed of 2.0°/min. Degrees of crystallinity (Xc) of the polymeric films were calculated from diffracted intensity data according to Vonk’s method.35 The silk hydrogels were also prepared in an acryl cylinder that was 15 mm in diameter and 7.5 mm in height for compression tests of the hydrogels. The compression tests were carried out to obtain stress strain curve, elastic modulus, and elastic limit of the silk hydrogel using a mechanical testing apparatus (INSTRON 3365; Instron Corp., Norwood, MA) at a velocity of 0.75 mm/min. Average molecular weights between cross-links were calculated based on the elastic modulus and the equation as follows: E ¼ ðF=MÞRT where E, F, R, T, and M are the elastic modulus, concentration of polymer, gas constant, temperature (K), and theoretical molecular weight between cross-links.36 The silk hydrogel was cast on a slide glass with cover glass and then used as a sample. The networks of silk molecules in the hydrogels were observed by differential interference contrast microscopy (Axio Observer Z1, 100 objective; Carl Zeiss, Oberkochen, Germany). Images were obtained using a CCD camera with AxioVision Rel 4.8 software (Carl Zeiss). Cell Culture and In Vitro Cell Viability. Human mesenchymal stem cells (hMSC) were purchased (Lonza Walkersville Inc., Walkerville, MD) and cultured in growth medium containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 1 ng/mL basic fibroblast growth factor (bFGF) in the presence of 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL fungizone at 37 °C in a 5% CO2 incubator. For cell viability, hMSC (8000 cells/well) were seeded into 96-well plates coated with silk hydrogels and cultured for 48 h in the media (100 μL). The cell viability of hMSC on the silk hydrogels was characterized by a standard 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, WI) according to the manufacturer’s instructions (n = 4). The cell viability was calculated as follows:
X, % ¼ ½1 ðweight of silk proteins after the lyophilizationÞ=
½cell viability, % ¼ ½absorbance at 490 nm of the cell culture incubated on the silk hydrogel=½absorbance at 490 nm of the
ðweight of silk hydrogel before the lyophilizationÞ 100 DSC measurements were performed using a Perkin-Elmer Pyris 1 (Waltham, MA) equipped with a cooling accessory to quantitatively characterize the bound and bulk water of the hydrogel. Approximately
cull culture incubated on a 96-well cell culture plateða positive controlÞ 100 2138
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Table 1. Gelation Points (tgel) of a Mixture of Silk Solution and Ethanol
0
00
Figure 1. Storage modulus (G ) and the loss modulus (G ) during gelation of the silk solution (38 g/L) induced with ethanol were measured at 37 °C. The value of G0 was under the measurement limit of the rheometer below 570 s. Statistical differences in cell viability were determined by unpaired t-test with a two-tailed distribution and differences were considered statistically significant at p < 0.05.
’ RESULTS Gelation of Silk Solution Induced with Ethanol. The silk protein aqueous solution with a concentration of 63 g/L was mixed with ethanol at various ratios (10/0, 9/1, 8/2, 7/3, 6/4, 5/ 5, 4/6, 3/7, 2/8, and 1/9), resulting in the silk solutions with different concentrations (57, 50, 44, 38, 32, 25, 19, 13, and 7 g/ L). The time evolution of storage modulus G0 and loss modulus G00 of the samples were measured at 37 °C to determine the gelation point (tgel). The storage modulus G0 and the loss modulus G00 of a mixture of silk solution and ethanol (7/3 ratio) with a final silk concentration of 44 g/L are shown in Figure 1. The G0 and G00 increased with time and crossover occurred at around 1150 s. Because the gelation threshold is defined as the intermediate point between sol and gel, the tgel is estimated as this point. The values of tgel of all samples are listed in Table 1. The samples prepared at silk concentrations of 32 and 25 g/L showed relatively shorter tgel, while the samples with relatively higher (57 g/L) and lower concentration (13 and 7 g/L) of silk proteins demonstrated longer tgel. Secondary Structures Determined by ATR-FTIR and WAXD. For following analyses, the silk gels obtained from the mixture of the silk solution and ethanol were washed with Milli-Q for 24 h, and then ethanol in the silk gels was completely removed, which was confirmed by the disappearance of a peak originating from ethanol in the DSC profile (Figure S2). ATR-FTIR spectra of the silk hydrogels and silk solution in the amide I region were measured and characterized as shown in Figures 2 and S3. The dashed lines mark the center of the adsorption bands at 1650 and 1625 cm1, which are characteristic of the random-coil and β-sheet structures of silk proteins, respectively, according to previous reports.3742 The amide I band was deconvoluted to determine the beta-sheet contents of the silk hydrogels. The β-sheet contents of the silk hydrogels increased with an increase in silk concentration from 14 to 57% (Figure 3). The secondary structure of silk proteins in their hydrogels prepared at high concentrations such as 50 and 44 g/L was predominantly a β-sheet structure
final concentration of silk
silk solution/ethanol
gelation points
proteins, g/L
ratio, (v/v)%
(tgel), sec
57 50
9/1 8/2
8.6 104 ( 4.0 103b 2.2 104 ( 1.7 103b
44
7/3
1.2 103 ( 1.2 102b
38
6/4
5.6 102 ( 50b
32
5/5
∼5
25
4/6
∼4
19
3/7
∼15
13
2/8
6.6 103 ( 1.5 102b
1/9
-a
7 a
b
Not detected. Average and standard deviation (n = 3).
(Figure 2b,c), whereas random-coil structures predominated in the hydrogels prepared at the relatively low concentrations of 25 and 19 g/L (Figure 2f,g). The silk solution with a concentration of 63 g/L, which demonstrated no gelation, showed a β-sheet content of 6%, suggesting the secondary structure of silk proteins in their solution was predominantly a random-coil structure. The secondary structure of the silk molecules was also characterized by WAXD. The WAXD data of the silk hydrogels showed a shift of the peak from 21 to 27°, which indicates that conformational transition from a random-coil to a beta-sheet structure was induced with an increase in the concentration of silk (Figure 4), according to the previous reports.21,43,44 Even by the low β-sheet content, the gelation of silk solution could be induced, for example, gelation of 19 g/L silk/ethanol solution. This result supports the ATR-FTIR data on the secondary structure of the silk hydrogels, namely, that gelation of the silk solution was accompanied by formation of β-sheet structure of silk molecules. Mechanical Properties and Molecular Networks. The silk hydrogels prepared using various silk concentrations were characterized with respect to the elastic modulus and elastic limit. Figure 5 shows a typical stressstrain curve of the silk hydrogel prepared with a silk concentration of 25 g/L. The elastic property of the silk hydrogel disappeared over the elastic limit (arrow in Figure 5) and, at the same time, the hydrogel released water. This release of water from the silk hydrogel indicated that the released water was essential to maintain silk hydrogel as an elastic material. In addition, the release of water from the other conventional hydrogels, such as agarose and polyacrylamide gels, was not observed, demonstrating that the state of water of the silk hydrogel differed from that of the other hydrogels. Both mechanical properties, which were obtained from the stressstrain curves, increased with an increase in silk concentration (Figure 6). The average molecular weights between cross-links of the silk hydrogel were calculated from the determined elastic modulus (Figure 7). In addition, the average distances between cross-links were calculated according to the backbone structure of poly(amino acid), that is, one amino acid unit with a molecular weight of 110 g/mol (one monomer unit) is approximately 0.39 nm in length along its backbone (Figure 7). The theoretical molecular weight and distance between cross-links were based on the assumption that the molecular network is homogeneous. However, the actual molecular network of the silk hydrogel was observed by 2139
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Figure 4. WAXD patterns of silk hydrogels prepared at various concentrations of silk proteins.
Figure 2. ATR-FTIR spectra in the Amide I region for silk solution and silk hydrogels prepared at various concentrations of silk proteins.
Figure 5. Typical stressstrain curve of the silk hydrogel prepared at a silk concentration of 25 g/L. The arrow denotes the limit point of elastic behavior as described in the text.
Figure 3. β-Sheet contents of silk solution and silk hydrogels determined from ATR-FTIR measurements. Error bars represent the standard deviation of samples (n = 3).
differential interference contrast microscopy (Figure 8), and the results showed that the sizes of the molecular network of the silk hydrogel prepared at a silk concentration of 32 g/L ranged from roughly 5 to 50 μm, namely, that the network structure of the silk hydrogel was heterogeneous. The differential interference contrast microscopy results implied that the network sizes of the silk hydrogel roughly decreased with silk concentration, even though the network was heterogeneous (Figure S4). The theoretical distance between cross-links of the silk hydrogel prepared at a silk concentration of 32 g/L was approximately 180 nm, which was significantly lower than the actual values. A comparison of the theoretical and the actual values of the distance between cross-links therefore demonstrated that the molecular network structure of silk hydrogels is not a single molecular network as in the case of chemically synthesized polymeric hydrogels, but rather was composed of fibrillar network structures that contained silk fibers with a diameter of approximately 1 μm. Bound and Bulk Water Contents in Silk Hydrogel. The resulting silk hydrogels prepared at various silk concentrations were characterized with respect to their water contents
(Figure 9). The water content in the equilibrium swollen state was almost same with that in as-prepared state, indicating the water contents of the silk hydrogels decreased with an increase in silk concentration. The highest water content of the silk hydrogels was nearly 99%; this content was observed at a silk concentration was 13 g/L. A series of DSC profiles of the silk hydrogel with various concentrations of silk proteins was recorded (Figure 10). Bound (nonfreezing) and bulk (freezing) water in hydrogels have been investigated by combination of DSC and the other complementary approaches.13 According to the literature regarding the DSC curves of hydrogels,3,2932 the melting peaks of water at relatively lower temperature, around 0 °C, were assigned to bound water, whereas the melting peaks of water at relatively high temperature, around 410 °C, were assigned to bulk water. Two major peaks originating from bound (lower temperature) and bulk water (higher temperature) in the silk hydrogels were observed, as shown in Figure 10 (panels af). The melting temperature Tm of the bulk water decreased with an increase in silk concentration, and the peak of the bulk water finally merged with the peak of the bound water around 4 °C at a silk concentration of 57 g/L. The melting temperature Tm and enthalpy of fusion ΔH of the bound and bulk water in the silk hydrogels were further characterized quantitatively (Table 2). The Tm and ΔH of the bulk water in the silk hydrogels significantly decreased with an increase in the concentration of silk proteins, while those of bound water were almost invariable throughout the range of concentrations, indicating that the concentration of silk protein in the 2140
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Figure 9. Water contents in equilibrium swollen state and as-prepared state of silk hydrogels prepared at various concentrations of silk proteins.
Figure 6. Elastic modulus (A) and elastic limit (B) of silk hydrogels prepared at various concentrations of silk proteins. Error bars represent the standard deviation of samples (n = 3).
Figure 10. DSC endothermic profiles of silk hydrogels prepared at various concentrations of silk proteins.
viability of 100% was calculated from the cell culture seeded on a cell culture plate after incubation for 48 h as a positive control. The cell viability of hMSC, namely, the noncytotoxicity of the silk hydrogel, significantly increased with an increase in silk concentration. Figure 7. Theoretical molecular weights (g/mol) and distances (nm) between cross-links of the silk hydrogels calculated from the elastic modulus shown in Figure 6A. Error bars represent the standard deviation of samples (n = 3).
Figure 8. Differential interference contrast images of the silk hydrogel prepared at a silk concentration of 32 g/L. (A) A homogeneous network structure. (B) A heterogeneous structure. Scale bars denote 10 μm.
silk hydrogel prepared using ethanol was capable of controlling the content as well as the state, from bulk to bound via intermediate, of water molecules in the silk hydrogel. Cell Viability (Cytotoxicity). The cell viability of hMSC on the silk hydrogels prepared at various concentrations of silk solution was evaluated using an MTS assay (Figure 11). A cell
’ DISCUSSION The gelation of the silk solution can be described as a ternary phase-separation system of silk polymer, water (solvent), and ethanol (nonsolvent), as schematically shown in Figure 12.45,46 The conditions used in this study are represented as the bold arrow in Figure 12. As increasing the solution/ethanol ratio, the phase is expected to change from solution phase (I) to aggregation of silk polymer phase (III) by way of gel phase (II). The retardation of tgel at the higher (57 g/L, silk solution/ethanol = 9/1) and lower concentration (13 g/L, silk solution/ethanol = 2/ 8) are corresponding to the boundary between phases (I) and (II) and phases (II) and (III), respectively. The sample prepared at a silk concentration of 7 g/L (silk solution/ethanol = 1/9), which showed no gelation (Table 1), is in phase (III). The slightly decrease in loss modulus G00 just before the gelation (Figure 1), which is unusual and different from that of synthetic polymers,47 also indicates the aggregation of silk polymers with the formation of the β-sheet structure and subsequent gelation from aggregated silk polymers. Based on the structural and morphological characterizations of the silk hydrogels by ATRFTIR (Figures 2 and 3), WAXD (Figure 4), and differential interference contrast microscopy (Figure 8), the silk molecules were assembled to form fibrillar and heterogeneous networks with β-sheet structures, which are prerequisite for the present gelation. Also, the 2141
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Table 2. Melting Temperatures and ΔH of Bound and Bulk Water in the Silk Hydrogels bound water
bulk water
ΔH,
ΔH,
J/g Tm, °C
J/g
final concentration silk solution/ethanol
a
of silk proteins, g/L
ratio, (v/v)%
Tm, °C
57
9/1
4
171
a
a
50 44
8/2 7/3
4 4
234 197
1 0
36 56
38
6/4
3
201
2
45
32
5/5
3
197
4
59
25
4/6
3
222
6
140
19
3/7
1
222
14
156
Not detected by DSC, as shown in Figure 10.
peaks originated from the bound water seemed to contain two peaks (Figure 10), implying that there might be two types of bound water in the silk hydrogel, namely, water bound to microscale networks (microfibrils) and water intercalated in the β-sheet assembly. Natural polymers, including protein polymers, are known to form supermolecular structures with a high degree of inhomogeneities during their gelations.48,49 The elastic modulus and tensile strength of natural dragline silk was reported to be 10 and 1.1 GPa, whereas Kim et al. reported that the highest compressive strength and elastic modulus of silk hydrogel prepared at 37 °C showed approximately 2 and 4 MPa.44,50 A combination of these data and the present results implies that the strength of silk fibers has not been applied to reconstituted silk materials. On the contrary, a hydrogel with an ideally homogeneous molecular network, tetra-PEG gel, was recently reported to show relatively superior mechanical properties.47,51,52 It is therefore possible to fabricate an extremely strong hydrogel if the network structure of the silk molecules is regulated to be ideally homogeneous, because natural silk fibers originally show high mechanical properties. The silk concentration influenced the water content, state of water, beta-sheet content, mechanical properties, network structure and cytotoxicity of the silk hydrogel. The water content of the silk hydrogel was tuned from approximately 95 to 98% by changing the solution/ethanol ratio (Figure 9). The increase in β-sheet structure shown in Figure 2 is due to the removal of water molecules between the β-strand molecules.53 The state of water in the silk hydrogel was dependent on the concentration of silk; the bulk water in the silk hydrogel decreased with an increase in silk concentration (Figure 10 and Table 2). Figure 10 further shows the shift of the peak originating from bulk water, which means that the mobility of bulk water decreased with an increase in silk concentration. The silk hydrogels prepared at silk concentrations of 19, 25, and 32 g/L obviously contained the bulk water, but those prepared at 38, 44, and 50 g/L contained water in a state intermediate between bulk and bound water. Furthermore, the silk hydrogel (57 g/L) seemed to contain only bound water (Figure 10g). This demonstrates that the bulk water content of the silk hydrogel can be readily regulated by regulating the concentration of silk proteins, which is helpful to investigate the effects of the state of water of polymeric hydrogel on the other properties. The cell viability of hMSC cultured on the silk hydrogel increased significantly with an increase in silk concentration, which may have been due to increase in the β-sheet content, elastic modulus, network size, and bound water content. In the previous study,24 silk hydrogel-based encapsulation with lower β-sheet content and lower elastic moduli were reported to show higher cell
Figure 11. Cell viability of hMSC seeded on the silk hydrogels, which was determined from the absorbance at 490 nm measured using the cell cultures after incubation for 48 h. A cell viability of 100% was calculated from a positive control, namely, the cell culture seeded on a cell culture plate after incubation for 48 h. Error bars represent the standard deviation of samples (n = 3). *Significant difference between two groups at p < 0.05.
Figure 12. Schematic ternary phase diagram of the system: silk polymerwater (solvent)ethanol (nonsolvent). Solutions in region I are a homogeneous liquid state. Silk polymer solutions in region II separated into two phases and their gelation occurs. In region III, silk polymer aggregates. Silk solutions in region IV are in a glass state. A bold arrow denotes the state studied in this study.
viability. Additionally, the previous studies were lacking in investigations on neither the state of water nor the network structure in the silk hydrogels.2325,44 According to the present results, the cell viability increased monotonically with an increase in silk concentration (Figure 11), whereas the elastic moduli showed no monotonical dependence on silk concentrations. Also, bound water in hydrogels has been reported to play an important role in enzymatic catalysis.9,10,12 Further, ionic-surface substrates are considered to be more cell-adhesive materials than hydrophobic-surface substrate, due to the presence of bound water at the surface of the ionic-surface substrates.54,55 Thus, the present data suggest the bound water contents of silk hydrogels may play a more important role in cytotoxicity of silk hydrogels rather than the β-sheet contents or the elastic modulus of silk hydrogels. The present study is the first to quantitatively characterize the state of water of silk hydrogels, including a determination of the ratio of bulk to bound water (Figure 10 and Table 2). The DSC data exhibited that the amount as well as the mobility of the bulk water in the silk hydrogel decreased with an increase in silk concentrations, while the amount of the bulk water decreased, namely, the amount of 2142
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Biomacromolecules the bound water relatively increased. Based on the studies mentioned above and the present results on the state of water of the silk hydrogel,9,10,12,54,55 the cell viability of hMSC on the silk hydrogels was suggested to be under the influence of the ratio of the bulk and bound water. In other words, the improvement of the cell viability of the silk hydrogel implies that the cells as well as the cell-adhesion proteins in the extracellular matrix preferentially expand and adhere on a substrate containing more bound water. The cell-adhesion proteins also may need bound water to exhibit their functions, similar to the other proteins.9,10,12 The bound water in the silk hydrogel will therefore accelerate cell-adhesion proteins, such as fibronectins, in the cellular matrix to interact with the surface of the silk hydrogels, whereas the bulk water would disturb the cell-adhesion proteins to adhere on the surface of the silk hydrogels, due to the relatively higher mobility of water. This new insight into the state of water of hydrogels provides options to design hydrogel-based biomaterials to form cellinteractive biointerfaces.
’ CONCLUSIONS A silk hydrogel prepared using ethanol was characterized with respect to its gelation behavior, network and molecular structures, state of water, and cytotoxicity to hMSC. The network structure of the silk hydrogel was heterogeneous with β-sheet structures. The bulk water content of the silk hydrogel was found to be readily regulated by the concentration of silk proteins, which is helpful to investigate the effects of the state of water of polymeric hydrogel on the other properties. The influence of the state of water in the silk hydrogel on the cytotoxicity was recognized by means of DSC and MTS assay. Based on the results, the bound water is considered to support cell-adhesion proteins in the cellular matrix to interact with the surface of the silk hydrogels. On the other hand, the bulk water would disturb the celladhesion proteins to adhere on the surface of the silk hydrogels, due to the relatively higher mobility of water. Thus, the present study was the first to discuss the relationship between the state of water and the viability of cells cultured on polymeric hydrogels. ’ ASSOCIATED CONTENT
bS
Supporting Information. Figure S1, the storage and loss moduli (G0 and G00 ) as a function of strain, the DSC endothermic profiles of silk hydrogels before and after Milli-Q wash; Figure S2, the DSC endothermic profiles of silk hydrogels before and after Milli-Q wash; Figure S3, ATR-FTIR spectra for the silk solution; and Figure S4, the differential interference contrast images of the silk hydrogels. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Tel.: þ81-48-467-9525. Fax: þ81-48-462-4664. E-mail: keiji.
[email protected].
’ ACKNOWLEDGMENT This work was supported by grants from The Sumitomo Foundation (K.N.).
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