Studies on the Formation Mechanism and the Structure of the

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Studies on the Formation Mechanism and the Structure of the Anisotropic Collagen Gel Prepared by Dialysis-Induced Anisotropic Gelation Kazuya Furusawa,*,† Shoichi Sato,‡ Jyun-ichi Masumoto,‡ Yohei Hanazaki,‡ Yasuyuki Maki,§ Toshiaki Dobashi,§ Takao Yamamoto,§ Akimasa Fukui,† and Naoki Sasaki† †

Faculty of Advanced Life Science, Hokkaido University, Kita-ku Kita 10 Nishi 8, Sapporo, Hokkaido, Japan Transdisciplinary Life Science Course, Graduate School of Life Science, Hokkaido University, Kita-ku Kita 10 Nishi 8, Sapporo, Hokkaido, Japan § Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Tenjincho 1-5-1, Kiryu, Gunma, Japan ‡

S Supporting Information *

ABSTRACT: We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 256 mM at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.

1. INTRODUCTION Connective tissue cells adhere on extracellular matrices (ECMs), which consist of the following: structural proteins: collagen, elastin, fibronectin, and so on; glycosaminoglycans: hyaluronan, chondroitin sulfate, heparin sulfate, and so on; and proteoglycans, which are complexes of the structural proteins and the glycosaminoglycans.1 Interactions between the cells and the ECMs affect various cell behaviors, such as locomotion, proliferation, differentiation, and morphology.2−6 To study the relationship between the cell behaviors and the cell−ECM interaction in vitro7−11 and in vivo,12 various functional cell scaffolds have been developed. Collagen gel is widely used as a cell scaffold to study the relationship. Recently, collagen scaffolds with ordered structures have been prepared as cell scaffolds.13−21 For example, highly ordered collagen scaffolds have been developed by stabilizing cholesteric liquid crystal phases of a concentrated collagen solution.19−21 The ordered structure of collagen scaffolds affects cell morphology, proliferation, and differentiation.14,15 Connective tissues have gradient properties, such as stiffness gradient, that are due to their inhomogeneous structures. The gradient properties of cell scaffolds also affect various cell behaviors.22−24 For example, it has been suggested that cell migration is guided by the direction of the stiffness gradient of scaffolds.22,23 Therefore, cell scaffolds mimicking both the anisotropy and the gradient © 2011 American Chemical Society

property of native tissues are useful for investigating the cell− ECM interaction and can be utilized as a novel cell scaffold to provide engineered tissues. We believe that the anisotropic cell scaffolds with the gradient properties achieve control over spatial distribution of cell morphology, population, proliferation, and differentiation on single engineered tissues. Recently, we found that the dialysis of semiflexible and rigid biopolymer solutions into multivalent cation solutions results in gels with a birefringence.25−30 The birefringence of gels is attributed to an anisotropic 3D network structure. Thus, we call the gels “anisotropic gel”. Other authors have observed that a flow of calcium cations into synthetic rigid polymer solution results in a gel with an ordered structure.31 The anisotropic gels also have gradient properties, such as a birefringence gradient.32,33 We previously demonstrated that an anisotropic collagen gel can be prepared by dialysis of collagen solutions into phosphate solutions with neutral pH values.27 Because the anisotropic collagen gel is easily prepared and has a unique structure and biocompatibility, it can be utilized as a novel scaffold. However, the hierarchical structure and formation mechanism of the anisotropic collagen gel are not well understood. Received: June 25, 2011 Revised: November 22, 2011 Published: November 22, 2011 29

dx.doi.org/10.1021/bm200869p | Biomacromolecules 2012, 13, 29−39

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prepared using a chamber A, which had glass windows and plexiglass spacers 0.5 or 1.5 mm thick (see Figure 1A). The dimensions of the

The final goal of this study is to provide a novel cell scaffold for investigating the relationship among the cell behaviors, the structure and properties of the cell scaffolds. For this purpose, it is essential first to elucidate the hierarchical structure and formation mechanism of the anisotropic collagen gel and to establish the method for controlling the structure and properties of the anisotropic collagen gel. In the present report, we investigated the hierarchical structure and formation mechanism of an anisotropic collagen gel. The macroscopic morphology and microscopic structure of the gel were investigated. The macroscopic morphology was observed under natural light and polarized light. The spatial distribution of collagen concentration was visualized by immunofluorescence staining and confocal laser scanning microscopy (CLSM). The 3D network of collagen gel consisted of collagen fibrils with submicrometer sizes. The small-angle light scattering (SALS) patterns of the anisotropic collagen gel were obtained to elucidate the microscopic structure. Previously, we found that a spatial distribution of the structure and properties of anisotropic gels prepared by the dialysisinduced anisotropic gelation can be controlled by changing the dynamics of the anisotropic gelation.32,33 Therefore, we also investigated the dynamics of the anisotropic gelation of collagen in controlling the spatial distribution of the structure and properties of the anisotropic collagen gel. To examine the dynamics of the anisotropic gelation of collagen, the time course of the thickness of the collagen gel layer was measured and analyzed by applying the theory for dialysis-induced anisotropic gelation known as the “moving boundary picture”.34 It has been reported that pH, ionic strength, and temperature affect the structure and properties of collagen gels.21,35 To elucidate the effects of the pH, ionic strength, and temperature on the anisotropic gelation of collagen, the changes in state of the collagen solution added dropwise to phosphate solutions with various pHs and ionic strengths at different temperatures were examined.

Figure 1. (A) Schematic illustration of the anisotropic collagen gels for studying the hierarchical structure in the cross-section A. The sample with a thickness of 1.5 mm was used for observing the macroscopic morphology. The sample with a thickness of 0.5 mm was used for measuring the small-angle light scattering. (B) Schematic illustration of the anisotropic collagen gels for studying the hierarchical structure in the cross-section B. The red arrows indicate the growth direction of the anisotropic collagen gel. window for the transmission of light were 9.0 × 13.5 mm, and the dimensions of the inlet for injecting the collagen solution were 9.0 × 0.5 mm or 9.0 × 1.5 mm. For preparing the anisotropic collagen gel, the atelo-collagen solution was injected into a chamber A, and then it was immersed in the phosphate solution with a pH of 7.1 and an ionic strength of 256 mM at room temperature (25 °C) for 24 h. The anisotropic collagen gel prepared using the chamber A was used to investigate the hierarchical structure for the cross-section parallel to the growth direction of the gel (cross-section A). The anisotropic collagen gel with a thickness of 1.5 mm was used to observe the macroscopic morphology and to measure the birefringence, and a gel 0.5 mm thick was used to measure the SALS pattern. In the second method, an anisotropic collagen gel was prepared using chamber B, which had a flat glass bottom and a silicon rubber wall (see Figure 1B). The dimensions of chamber B were 5.0 (W) × 5.0 (L) × 1.0 mm (D). After the collagen solution had been injected into chamber B, a dialysis membrane was placed on the chamber. A glass ring with a diameter of 22 mm and a height of 20 mm was placed on the dialysis membrane. To prepare the anisotropic collagen gel, an appropriate amount of the phosphate solution with a pH of 7.1 and an ionic strength of 256 mM was poured into the glass ring at room temperature (25 °C) for 1 h. The anisotropic collagen gel was used to investigate the hierarchical structure for the cross-section perpendicular to the growth direction of the gel (cross-section B). To observe the spatial distribution of the collagen concentration in the cross-section B, the collagen molecules in the anisotropic collagen gel were visualized by immunofluorescence staining. The anisotropic collagen gel was fixed with formaldehyde for 30 min at 4 °C. The sample was then incubated at room temperature for 1 h in anticollagen alpha I (SC-8784-R, Santa Cruz Biotechnology, Inc.) diluted 1:200 in PBS, followed by incubation at room temperature for 1 h in Alexa Fluor 633-conjugated antirabbit IgG (Molecular Probes) diluted 1:1000 in PBS. The sample was washed twice with PBS to remove nonassociated antibodies. The sample was

2. EXPERIMENTAL SECTION An “atelo-collagen” was employed for this study. Here, the atelocollagen is enzymatically extracted from the bovine dermis. Because both terminal peptide regions of native collagen, which are called “telopeptide”, were excised by the enzymatic extraction, the atelo-collagen has only a triple helical region of native collagen. The atelo-collagen solution was purchased from Koken Co. Ltd. (Tokyo, Japan) and was used without further purification. The concentration of atelo-collagen was 5 mg/mL. The solvent of atelo-collagen solution was 1 mM HCl aqueous solution at a pH of 3.0. According to the manufacturer’s specification, approximately 95% of the collagen molecules in the atelo-collagen solution is type I collagen and the remaining 5% is type III collagen. KH2PO4 and Na2HPO4 were purchased from Wako Pure Chemical Co. Ltd. The desired amounts of KH2PO4 and Na2HPO4 were dissolved in Milli-Q water at various concentrations of KH2PO4 (6.5−26 mM) and Na2HPO4 (10−40 mM). The pH values of the phosphate solutions were adjusted by adding 4 N HCl and were measured using a pH meter (HM-25R TOADKK) at the following temperatures: 10, 20, 30, and 40 °C. A degree of dissociation of phosphate ion is dependent on a pH value and on a temperature. Therefore, we estimate the degree of dissociation of phosphate ions at various pH values and at various temperatures to determine the ionic strength of the phosphate solution. To estimate the degree of dissociation, we employed a second dissociation constant of phosphate ions at various temperatures determined by H. Fukada et al.36 To investigate the macroscopic morphology and the microscopic structure of the anisotropic collagen gel, samples were prepared using two methods. In the first method, anisotropic collagen gel was 30


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The time course of x(t) was analyzed by applying the theory based on the “moving boundary picture”.34 We introduce the scaledthickness of gel layer (x̃) and the scaled-immersion time (t)̃ as

mounted on a coverslip and imaged using a confocal laser scanning microscope (CLSM; Leica TCS SP5). The fluorescence from the collagen fibrils was monochromated by a dichroic filter, and it was detected by a CCD area image sensor. CSLM images were obtained as 8-bit grayscale images. The fluorescence intensities from the collagen fibrils were in arbitrary units (a.u.), with higher numbers corresponding to more antibodies. The fluorescence intensity was scaled by the gray levels ranging from 0 a.u. (black) to 255 a.u. (white). The gray levels were converted to pseudocolor levels ranging from 0 a.u. (black) to 255 a.u. (red) by using Leica LAS AF software equipped with Leica TCS SP5. To obtain Z-stack CSLM images, XY-optical sections were stacked to the Z-direction with the thickness of 1 μm. A schematic illustration of the apparatus for measuring the SALS pattern of the anisotropic collagen gel is shown in Figure 2. The light

x̃ =

x R

(1)

and

t̃ =

t R2

(2)

where R is the radius of the cover glass. According to the “moving boundary picture”, the relationship between x̃ and t ̃ is expressed as

1 1 1 (1 − x)̃ 2 ln(1 − x)̃ − x 2̃ + x ̃ = Kt ̃ 4 2 2

(3)

where the coefficient K is the growth rate of the gel layer, which is related to the diffusion coefficient of ions in the phosphate solution. Next, we define the left-hand side of eq 3 as a universal function describing the dynamics of gelation (ỹ(x̃(t)̃ )),

̃ ≡ y ̃(x(̃ t ))

1 1 1 (1 − x)̃ 2 ln(1 − x)̃ − x 2̃ + x ̃ 4 2 2

(4)

Thus, we have the relationship between ỹ(x̃(t)̃ ) and t,̃

̃ = Kt ̃ y ̃(x(̃ t ))

(5)

The relationship between the diffusion coefficient (D) and K is approximately expressed as

D≈

ρg ρs

K (6)

Here, ρg and ρs are the critical concentration of ions in the phosphate solution for forming gel and the concentration of ions in the phosphate solution. The details of the theory were provided in a previous report.34 To examine the effects of pH, ionic strength, and temperature on changes in state of collagen solutions in phosphate solutions, 20 μL of the collagen solution was added dropwise to 10 mL of phosphate solutions with various pHs and ionic strengths at different temperatures: 10, 20, 30, and 40 °C. The changes in state of collagen solutions added dropwise to the phosphate solutions were observed under polarized light and unpolarized light.

Figure 2. Schematic illustration of the apparatus for measuring the small-angle light scattering pattern of the anisotropic collagen gel. source was a 2 mW He−Ne laser with a wavelength of 632.8 nm (Hughes Aircraft, Helium−Neon Laser Head, 3222H-PC). The incident beam directions were perpendicular to the cross-section A for investigating the microscopic structure in the cross-section A (denoted by incident beam direction A) and to the cross-section B for investigating the microscopic structure in the cross-section B (denoted by incident beam direction B). The diameter of the beam spot was 1 mm at the sample. The distance from the sample to the screen (L) was 117 mm. The SALS pattern projected onto the screen was acquired in the RAW image format using a digital camera (Nikon D3100). The SALS patterns for the incident beam direction A were obtained as a function of a distance from the top of the gel (d), whereas the SALS patterns for incident beam direction B were obtained at four distinct positions on the sample. To obtain the 1D SALS profile, the sector average of the 2D SALS pattern was calculated using the sector with an angle of 10°. The 1D scattering profiles were corrected by subtracting a background profile obtained by measuring a SALS pattern without the sample from that obtained from a pattern with samples. The anisotropic collagen gel with the dimensions of 9.0 (W) × 8.0 (H) × 1.5 mm (D) was prepared using a chamber, similar to chamber A, to measure the birefringence as a function of the distance from the top of the gel (d) using a laboratory-made device.32 For simplicity, a circular geometry was chosen as the shape of the collagen gel for investigating the dynamics of the anisotropic gelation of collagen. The 5 mg/mL collagen solution was sandwiched between a set of circular cover glasses with the radius of 6.0 mm and then immersed in a phosphate solution with a pH of 7.1 and an ionic strength of 256 mM at 20 °C. The front line of the gel layer was easily determined by a sharp increase in turbidity. To investigate the growth process of the collagen gel layer, we measured the time course of the thickness (x(t)) between the front line of the gel layer and the circumference of the gel, where t is immersion time.

3. RESULTS AND DISCUSSION 3.1. Hierarchical Structure of Anisotropic Collagen Gel. 3.1.1. Birefringence Pattern of Anisotropic Collagen Gel. To observe the macroscopic morphology in the crosssection A of the anisotropic collagen gel, an aliquot of collagen solutions was injected to the chamber A and it was immersed into the phosphate solution. A gel membrane was immediately formed at the interface between the collagen solution and the phosphate solution. The gel layer grew from the gel membrane to the bottom of the chamber A. Figure 3A shows the macroscopic morphology in the cross-section A of the anisotropic collagen gel observed under natural light. Alternating turbid stripes and transparent layers were observed, and they aligned parallel to the growth direction of the gel layer. The turbid stripes coalesced with increasing d; the thickness of the turbid stripes increased with increasing d. The collagen gel showed birefringence, as shown in Figure 3B. The light intensity transmitted through the sample increased and decreased by rotating the sample (Figure 3C). Figure 3D shows the macroscopic morphology in the crosssection B of the anisotropic collagen gel observed under natural light. A turbid network and transparent pores were observed. The turbid stripes and the transparent layer observed in the 31


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Figure 3. Macroscopic morphology in the cross-section A of the anisotropic collagen gel observed under natural light (A) and polarized light (B and C). The white arrows indicate the orientation directions of the polarizer and analyzer. The macroscopic morphology in the cross-section B of the anisotropic collagen gel observed under natural light (D) and polarized light (E).

Figure 4. (A) Low resolution CLSM image of the cross-section B of the anisotropic collagen gel. The magnification is 40× (including digital zoom factor of 4.0×). (B) High resolution CLSM image of the cross-section B of the anisotropic collagen gel. The magnification is 292× (including digital zoom factor of 4.64). The collagen molecules are pseudocolored according to color scales shown at the bottom of figures. (C) Fast Fourier transformation (FFT) pattern of (A).

Figure 4A is shown in Figure 4C. A ring-like pattern was observed in Figure 4C. From the position of the ring-like pattern in Figure 4C, a characteristic length was estimated to be about 100 μm. The characteristic length was approximately equal to the average distance between centers of adjacent pore regions observed in Figure 4A (∼98 ± 4 μm). The result suggests that the ring-like pattern is attributed to a semiperiodic array of pores. The formation of the semiperiodic structure might be attributed to the formation of pores with the roughly monodisperse sizes. This characteristic is generally observed in systems undergoing phase separation through the spinodal decomposition.37,38 Thus, the formation of the characteristic macroscopic morphology of the anisotropic collagen gel could be attributed to a phase separation of the collagen solution. However, the collagen solution does not separate into two macroscopic phases completely, because the gelation of the collagen solution pins the phase separation structure, as observed in the systems with gelation temperatures near phase separation temperatures.39,40 Figure 4B shows the high resolution image of the crosssection B of the anisotropic collagen gel. In Figure 4B, collagen

cross-section A correspond to the turbid network and the transparent pores observed in the cross-section B, respectively. Figure 3E shows the birefringence pattern in the cross-section B of the anisotropic collagen gel. The strong birefringence intensity was observed in the interface region between the turbid network and the transparent pores. The birefringence intensity in the bulk region of the turbid network is lower than that in the interface region, and no birefringence was observed in the transparent pores. The results show that anisotropic and isotropic structures coexist in the anisotropic collagen gel. 3.1.2. Confocal Laser Scanning Microscopy of Anisotropic Collagen Gel. The spatial distribution of the collagen concentration in the cross-section B imaged by immunofluorescence staining and confocal laser scanning microscopy is shown in Figure 4. A continuous network and pores were observed in Figure 4A. The fluorescent intensity of the continuous network region was significantly higher than that of the pore region. The result shows that the concentration of collagen in the continuous network region was higher than that in the pore region. The fast Fourier transform pattern of 32


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fibrils are clearly observed both in the concentrated phase and in the diluted phase. The result shows that the 3D network structure of the anisotropic collagen gel consists of the collagen fibrils. The concentrated phase consists of a high-density collagen fibril network. On the other hand, a low-density collagen fibril network is observed in the diluted phase. The result shows that the collagen gel network is formed in not only the concentrated phase but also the diluted phase. Phase separation of a rod-like polymer solution induces an isotropic−anisotropic phase transition.38,41−43 On the other hand, it has been reported that concentrated collagen solutions exhibit lyotropic crystal phases.18−21,44 Therefore, it is interesting to compare the concentration of collagen in the concentrated phase of the anisotropic collagen gel with the critical concentration for exhibiting liquid crystalline phases in collagen solutions reported in previous studies.18−21,44 Let us estimate the concentration of collagen in the concentrated phase of the anisotropic collagen from 3D CLSM image. A relationship between the volume of collagen before and after anisotropic gelation is given by

V ϕi0 = V1ϕ1 + V2 ϕ2 (7) where V, V1, V2, ϕi0, ϕ1, and ϕ2 are, respectively, the entire volume of 3D CLSM image, the volumes of concentrated and diluted phases, the volume fractions of collagen in the initial collagen solution, the concentrated phase, and the diluted phase. The density of the concentration of collagen fibrils in the concentrated phase is much higher than that in the diluted phase. Thus, we assume the volume fraction of collagen in the concentrated phase is much larger than that in the diluted phase (ϕ1 ≫ ϕ2). Under this assumption, the relationship between volume fractions of collagen before and after the anisotropic gelation is given by

V ϕi0 ≈ V1ϕ1

Figure 5. SALS patterns for incident beam direction A measured at different distances from the top of the gel (d). The anisotropic collagen gel for measuring the SALS patterns was prepared using the chamber A and the phosphate solution with pH of 7.1 and ionic strength of 256 mM at room temperature (25 °C) for 24 h. The arrow heads indicate the scattering maximum on the meridian of the SALS patterns. The contrasts of SALS patterns are adjusted to make the scattering maximum on the meridian more visible.

broadened toward the azimuth direction. The SALS patterns measured for the incident beam direction B are shown in Figure 6A−D. A blurred scattering streak was observed in regions with smaller scattering angles. In contrast to the SALS patterns for the incident beam direction A, there was no scattering maximum in the SALS pattern for the incident beam direction B. The direction of the scattering streak in the SALS patterns for the incident beam direction A was independent of d. By contrast, the direction of the blurred scattering streak in the SALS patterns for the incident beam direction B was strongly dependent on the position on the gel. The scattering maximum was not observed in the SALS pattern for the incident beam direction B when measuring at different sample-to-screen distances (data not shown). From the results of SALS measurements and macroscopic observations (Figure 3A,D), we concluded that the scattering streak observed in the smaller-angle region is due to the macroscopic morphologies of the anisotropic collagen gel: the turbid stripes and transparent layer in the cross-section A and the turbid network and transparent pores in the cross-section B. The scattering maximum in the SALS patterns for the incident beam direction A can be attributed either to a semiperiodic structure where linear scattering elements align perpendicular to the growth direction of the gel45,46 or to the formation of extremely monodisperse aggregates that become suspended in the gel in an oriented array. 47 If there were extremely monodisperse aggregates in the gel, the scattering maximum should be observed in the SALS patterns for the incident beam direction B. However, the SALS patterns for the incident beam direction B show neither the semiperiodic structure nor the extremely monodisperse fibrils in the cross-section B. Therefore, the anisotropic collagen gel probably has the semiperiodic

(8)

From eq 8 and the density of collagen (=1.4 g/mL), the concentration of collagen in the concentrated phase is estimated to be 8.1 mg/mL. The concentration of collagen in the concentrated phase is much lower than the critical concentrations for exhibiting liquid crystalline phases in collagen solutions reported by Marie Madeleine Giraud-Guille et al.18−21,44 The result shows that the formation of ordered structure of the anisotropic collagen gel is not attributable to a simple increase in collagen concentration due to the phase separation of collagen solution. We can observe a significant number of collagen fibrils aligned parallel to an interface between the concentrated phase and the diluted phase. The result was consistent with the birefringence pattern shown in Figure 3E, suggesting that the orientation of collagen fibrils mainly occurs at the interface region between the concentrated phase and the diluted phase. Therefore, the phase separation of collagen solution probably plays a key role in a formation of ordered structure of the anisotropic collagen gel. 3.1.3. Small Angle Light Scattering of Anisotropic Collagen Gel. Typical SALS patterns measured for the incident beam direction A as a function of d are shown in Figure 5A−D. In the equatorial direction, a sharp streak was observed, irrespective of d. Moreover, a scattering maximum was observed on the meridian (it is indicated by white arrows in Figure 5A−D). With increasing d, the scattering maximum shifted toward the regions with smaller scattering angles and 33


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Figure 6. (A−D) SALS patterns for incident beam direction B measured at the different positions (P1−P4) on the anisotropic collagen gel prepared using the chamber B. (E) Schematic illustration of the incidence positions of laser beam. The anisotropic collagen gel for measuring the SALS patterns was prepared using the chamber B and the phosphate solution with pH of 7.1 and ionic strength of 256 mM at room temperature (25 °C) for 1 h.

Figure 7. (A−B) Semilog plots for the SALS profiles of the collagen anisotropic gel measured at different distances from the top of the gel (d). (C) The positional dependence of the period of semiperiodic structure (ξ).

structure in the cross-section A. As shown in Figure 4B, the collagen fibrils are aligned parallel to the interface between the concentrated and diluted phases. In other words, the collagen fibrils are aligned perpendicular to the growth direction of the gel. Therefore, the semiperiodic structure consisted of linear

scattering elements could be attributable to the collagen fibrils. The 1D SALS profiles obtained by sectors averaging the 2D SALS patterns for incident beam direction A are shown in Figure 7A,B. In the equatorial direction (Figure 7A), the 34


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Figure 8. (A−D) Azimuth angle dependence of the scattering intensity at the peak position (qc) measured as a function of d. Ibase is the scattering intensity of the baseline of the azimuth angle dependences. (E) The positional dependence of the order parameter of the collagen anisotropic gel (S(d)). (F) The positional dependence of the birefringence of the anisotropic collagen gel.

scattering intensity in higher q regions increased with increasing d. In the meridional direction (Figure 7B), however, a broad peak was observed in the larger q regions. With increasing d, the position of the broad peak (qc) shifted toward the smaller q regions, and the peak intensity increased. Because the scattering maximum shifts toward the smaller q regions and broadens toward the azimuth direction, it overlaps the scattering streak in the equatorial direction. Therefore, the increase of the scattering intensity in the higher q regions for the equatorial profile is attributable mainly to the changes in the position and shape of the scattering maximum on the meridian. As discussed below, the results show that the degree of orientation is dependent on the position on the gel. The peak shift toward the smaller q regions shows that the period of semiperiodic structure increases with increasing d. The period of semiperiodic structure (ξ) can be estimated using Bragg’s relation (ξ = 2π/qc). The positional dependence of ξ is shown in Figure 7C; ξ increased with increasing d. The result suggests that a packing-density of the collagen fibrils in the anisotropic collagen gel decreases with increasing d. The azimuth angle (ϕ) dependences of the normalized scattering intensity measured at qc (I(qc,ϕ)/Ibase) are shown in Figure 8A−D, where Ibase is the scattering intensity of the baseline of the azimuth angle dependences. The scattering peak was observed approximately around the azimuth angle of 90° with respect to the equatorial direction. The peak width increased with increasing d. In particular, at d = 7 mm, the scattering intensity at qc was not dependent on the azimuth

angle. The results suggest that the anisotropy decreases with increasing d. The anisotropy of the sample can be quantified using Herrmann’s orientation function S,48 given by the following equation:

3⟨cos2 ϕ⟩ − 1 (9) 2 where ⟨cos2 ϕ⟩ can be calculated from the azimuth angle dependence of I(qc,ϕ)/Ibase (Figure 8A−D) using the following integral: S=

ϕ

⟨cos2 ϕ⟩ =

∫ϕ 2 I(ϕ, qc)cos2 ϕsin ϕdϕ 1

ϕ

∫ϕ 2 I(ϕ, qc)sin ϕdϕ 1

(10)

where ϕ1 and ϕ2 are the limits of integration (15° < ϕ < 165°). S assumes a value of 1 for the system with complete orientation of the scattering elements parallel to the growth direction of the gel layer and −1/2 for the system with complete orientation of the scattering elements perpendicular to the growth direction of the gel layer. If S is 0, the system has an isotropic structure. Figure 8E shows the positional dependence of the order parameter (S) of the anisotropic collagen gel. The value of S is negative, suggesting that the collagen fibrils orient perpendicular to the growth direction of the gel layer. The result is consistent with the CLSM image in the cross-section B of 35


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Figure 9. (A−D) Time evolution of the thickness of the collagen gel layer observed under natural light. (E) The anisotropic gel formation process expressed by the function (ỹ(x̃(t)̃ )) against the scaled time (t)̃ .

formation rate of 3D network structure of collagen gel is much higher than the growth rate of collagen gel layer, the growth process of the collagen gel layer is limited by the diffusion of ions. The growth rate of collagen gel layer (K) determined from the slope of ỹ(x̃(t)̃ ) versus t ̃ was 2.3 × 10−5 cm2/s. Because the rate of the diffusion is dependent on the concentration gradient of ions, the value of K can be controlled by the concentration of ions in the phosphate solution. 3.2.2. Kinetics of Phase Separation. To investigate the dynamics of phase separation during the anisotropic gelation, the time course of the radius of the transparent pore (rpore) observed in Figure 3D was measured using the phase-contrast microscopy. The time course of rpore was shown in Figure 10.

the anisotropic collagen gel (see Figure 4(B)). Although the birefringence was obviously observed, the value of S shows that the collagen gel is almost isotropic. The small negative value of S is due to the coexistence of the anisotropic and isotropic phases in the anisotropic collagen gel. Since the SALS patterns reflect the ensemble average of structures in a scattering volume, the value of S might be underestimated. S decreased with increasing d. The result shows that the orientation of the anisotropic collagen gel decreased with increasing d, and the anisotropic collagen gel had an orientation gradient. The positional dependence of the birefringence (Δn) of the anisotropic collagen gel is shown in Figure 8F. Δn decreased with increasing d, indicating that the anisotropic collagen gel has an orientation gradient. As discussed above, the packingdensity of the collagen fibrils decreases with increasing d. Therefore, the orientation gradient of the anisotropic collagen gel could be attributed to the positional dependence of the packing density of the collagen fibrils. 3.2. Kinetics of Gelation and Phase Separation During Anisotropic Gelation of Collagen. 3.2.1. Kinetics of Gelation. To investigate the kinetics of the anisotropic gelation, the time course of the thickness of the gel layer was measured. Typical photographs for measuring the time course of the thickness of the collagen gel layer (x(t)) are shown in Figure 9A−D. In the phosphate solution, the interface between the collagen solution and the phosphate solution was immediately gelled. The frontline of the gel layer was cylindrically symmetrical and moved to the center of the cover glass during the anisotropic gelation. The gelation was complete after 65 min. To elucidate the kinetics of the gelation of collagen, the time course of the thickness of the collagen gel layer was analyzed using the “moving boundary picture”.34 Figure 9E shows a scaled-time course of the universal function describing the dynamics of gelation (ỹ(x̃(t)̃ )). The scaled time course of (ỹ(x̃(t)̃ )) was proportional to t ̃ during the entire process, suggesting that the growth process of the collagen gel layer is limited by the diffusion of ions in the phosphate solutions into the collagen solution.34 During the anisotropic gelation, the ions, such as Na+, K+, HPO42−, and H2PO4−, in the phosphate solution diffuse to the collagen solution, whereas H+ and Cl− in the collagen solution diffuse to the phosphate solution. The diffusion of ions neutralizes the pH of collagen solution. At a temperature of 20 °C, the collagen solution is immediately gelled by neutralizing the pH of collagen solution. Since the

Figure 10. Time course of the average radius of the transparent pore (rpore) observed in Figure 3D during the anisotropic gelation process.

At the initial stage, rpore was constant. At the intermediate stage, on the other hand, the time course of rpore can be expressed by a power law with an exponent of 1/3. The features are universally observed in the initial stage and intermediate stage of spinodal decomposition.37−39 Thus, the result suggests that the dynamics of phase separation during the anisotropic gelation are the spinodal decomposition. The spinodal decomposition of collagen solution could be induced by the pH and the ionic strength quench of the collagen solution due to the inflow of ions from the phosphate solution into the collagen solution and the outflow of hydrochloric acid from the collagen solution. Finally, the growth of rpore stopped at late stage. As shown in Figure 4B, the 3D network structures of the collagen gel are formed both in the concentrated and the diluted phases. 36


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3.3. Effects of pH, Ionic Strength, and Temperature on Changes in State of the Collagen Solution. The gelation of collagen solutions is due to the fibrillogenesis of collagen molecules. The fibrillogenesis is induced by neutralizing the pH and increasing the temperature of collagen solutions, resulting in a collagen fibril. The kinetics of collagen fibrillogenesis consist of two stages: a nucleation process and the subsequent growth of nuclei.35,54,55 The rate of fibrillogenesis increases with increasing pH and temperature, whereas it decreases with increasing ionic strength.54,55 The fibrillogenesis is followed by the aggregation of the collagen fibrils.35 The aggregation of collagen fibrils results in the 3D network structure of collagen gel. Thus, the formation rate of 3D network structure of collagen gel increases with increasing the pH and temperature, whereas it decreases with increasing the ionic strength. As discussed above, the final structure of anisotropic collagen gel is mainly determined by the kinetic processes: phase separation and gelation. Therefore, preparation conditions to produce the anisotropic collagen gel could be strongly dependent on the pH, ionic strength, and temperature. To find the preparation conditions to produce the anisotropic collagen gel, we examined changes in state of the collagen solutions added dropwise to phosphate solutions with various pHs, ionic strengths, and temperatures. The changes in state of the collagen solutions added dropwise to phosphate solutions are summarized in Figure 11A−D. An anisotropic collagen gel was observed above a threshold pH around 6.5. The threshold pH slightly increased with increasing temperature and ionic strength. By contrast, an isotropic collagen gel was obtained by dropping the collagen solution into the phosphate solutions below the threshold pH. The collagen solution was completely mixed with the phosphate solutions with high ionic strengths under low-temperature conditions. Under high temperature and neutral pH conditions, because the rates of gelation are fast, the formation of a 3D network structure of collagen gel occurred before the collagen solution was mixed with the phosphate solution. Therefore, the gel membrane was immediately formed at the interface between the collagen solution and phosphate solution. The gel membrane formed by the initial insolubilization reaction plays a role of dialysis membrane. The collagen gel network grew from the gel membrane to the center of the collagen solution drop. Finally, we obtained the collagen gel beads. Under low temperature and high ionic strength conditions, the rates of gelation are slow. Therefore, the collagen solution drop was completely mixed with the phosphate solutions because the collagen molecules diffused into the phosphate solution before forming the 3D network structure of collagen gel. Thus, the boundary between gel and sol phases observed in Figure 11 is determined kinetically rather than thermodynamically. The anisotropic collagen gel bead was formed by dropping the collagen solution into the phosphate solutions above the threshold pH. The pH and ionic strength of the phosphate solution affect the net surface charges of the collagen molecules,56,57 and the net surface charge affects the electrostatic interaction between the molecules. Under low pH conditions, the electrostatic interaction between collagen molecules is repulsive force. Therefore, the collagen molecules are homogeneously dissolved in the 1 mM HCl solution with pH of 3.0. Under neutral pH conditions, the electrostatic repulsion is weakened by neutralization of cationic functional groups and dissociation of anionic fuctional groups. The temperature affects the water-mediated hydrogen-bonding

The development of the 3D network structure inhibits a diffusion of collagen molecules from diluted phase to concentrated phase and a coalescence of the diluted regions. Thus, the collagen solution does not separate into macroscopic two phases completely, that is, the microphase-separated structure formed during the anisotropic gelation of the collagen solution was pinned by the formation of 3D network structure. The time at which the phase-separated structure is pinned by the gelation (tp) is an important parameter for determining the final hierarchical structure of the anisotropic collagen gel. If tp is long, the diameter of the pores will grow larger. If tp is short, the diameter of the pores will grow smaller. Therefore, the final hierarchical structures of the anisotropic collagen gels vary with tp. The growth process of pores to the growth direction of the gel is shown in Movie 1 uploaded as Supporting Information. We observed convection flows in the vicinity of the sol−gel front. In Movie 1, we can observe that the aggregates flow along the streamline of the convection flow. A similar convection flow has been observed in formation process of capillary tubes in alginate gels by Kohler et al.49−51 They attributed the existence of periodical array of capillary tubes in the alginate gels to the convection flow. However, this is not the case for the present system. The pore fusion as observed for alginate gel was not observed in the entire process for forming anisotropic collagen gel including the process for formation of the primary membrane. Instead, we observed that the tubular pores grew and the diameters increased. The nuclei of capillaries were not observed during the gelation of collagen. Therefore, the process of formation of tubular pores in the gelation of collagen is much different from that of alginate, and it cannot be explained by the theory of Kohler for the present system. On the other hand, in Movie 1, the growth of several tubular pores stopped during the anisotropic gelation. Thus, the number of pores decreases with increasing d. The diameters of tubular pores when stopped were smaller than those of growing tubular pores. The result indicates that the pores with larger sizes absorb the pores with smaller sizes. It could be explained by the “Ostwald ripening”, which was observed in the intermediate stage of the spinodal decomposition. The classical spinodal descriptions refer to a given location within the material with a morphology progressively growing coarser, and such a simple picture cannot be directly applied to the present case of gelation from the contact surface. However, it is natural to assume that the power law characterizing the spinodal decomposition should hold on the interface between the gel layer and the solution in the present system. In this context the experimentally observed 1/3 power in the logarithmic plot of the radius of the pore against time suggests the spinodal decomposition. To verify the mechanism perfectly requires further experiments and a theoretical support, which is ongoing and published in the near future. During the anisotropic gelation, the pH of inner sol layer could decrease with time, because the hydrochloric acid diffuses from the inner sol layer to the outer phosphate solution. Therefore, the depth of pH quench changes with the distances from the top of the gel (d; for the chamber geometry) or the perimeter of the cover glass (x; for the circular geometry). The quench depth is “distance” from the critical points in the phase diagram, which affects the compositions in concentrated and diluted phases formed by the phase separation and the dynamics of phase separation.52,53 Therefore, the change in quench depth of pH during the anisotropic gelation influences the final structure of the anisotropic collagen gel. 37


Biomacromolecules

Article

Figure 11. Changes in state of the collagen solutions added dropwise to the phosphate solutions with various pHs, ionic strengths, and temperatures. Circles, squares, and triangles denote anisotropic collagen gel, isotropic collagen gel, and isotropic solution, respectively.

between collagen molecules. 58,59 The changes in the interactions could be important for the morphologies of the fibrils, such as diameter and length of fibrils. The morphologies of collagen fibrils would be expected to affect a microscopic structure of the collagen gel. As mentioned above, the phase separation of collagen solution probably plays a key role in formation of the ordered structure of the anisotropic collagen gel. The changes in the interactions might be also important for the phase separation of collagen solution. The temperature affects various parameters related to the formation of the anisotropic collagen gel. The formation rate of 3D network structure of collagen gel increases with increasing the temperature.35 On the other hand, the pH and the ionic strength of the phosphate solutions are dependent on the temperature. The formation rate of 3D network structure of collagen gel is also dependent on the pH and the ionic strength. Furthermore, the diffusion coefficients of ions in the phosphate solutions increase with increasing the temperature. The growth process of the gel layer during the anisotropic gelation of collagen is limited by the diffusion of ions in the phosphate solution to the collagen solution. Thus, the temperature mainly affects the kinetics of gelation of collagen solution. The formation of 3D network structure pins the micro phase-separated structure formed during the anisotropic gelation of collagen. Thus, effects of temperature on the kinetics of gelation and phase separation of collagen solution are important for controlling the hierarchical structure of the anisotropic collagen gel and will be investigated in future works.

4. CONCLUSION We investigated the hierarchical structure and the formation mechanism of the anisotropic collagen gel prepared by means of dialysis-induced anisotropic gelation. During the anisotropic gelation, the phase separation and gelation of the collagen solution occur simultaneously. By observing the time course of the size of the dilute region (transparent region), the dynamics of phase separation during the anisotropic gelation appear to be limited by a spinodal decomposition-like process. The phase separation results in the characteristic macroscopic morphologies, the turbid stripes and transparent layer for the crosssection A and the turbid network and transparent pores for the cross-section B, and can induce the isotropic−anisotropic phase transition in the collagen solution. The temporal macroscopic morphology and anisotropic structure could be pinned by the formation of a 3D gel network consisting of collagen fibrils. The SALS patterns suggested that the collagen fibrils align perpendicular to the growth direction of the collagen gel. The order parameter (S) and birefringence (Δn) of the anisotropic collagen gel decreased with increasing distance from the top of the gel (d). It shows that the anisotropic collagen gel had gradient properties. The dynamics of the anisotropic gelation of collagen were limited by the diffusion of ions in the phosphate solutions. The anisotropic gelation of collagen was induced by the dialysis of the collagen solutions into phosphate solutions with pH values above a threshold. The threshold pH values are dependent on the temperature and ionic strength of the phosphate solution. We suggested that the hierarchical structure and properties of anisotropic collagen gel can be 38


Biomacromolecules

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controlled by regulating the kinetics of phase separation and gelation. Because the anisotropic collagen gel has both anisotropic properties and gradient properties, it could be utilized as a novel scaffold for tissue engineering.

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AUTHOR INFORMATION

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REFERENCES

S Supporting Information * The movie of the growth process of pores to the growth direction of the gel. This movie was recorded by using a phasecontrast microscope (CKX41, Olympus, Tokyo, Japan) equipped with a digital camera (CAMEDIA C-5050 ZOOM, Olympus) at room temperature. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author *E-mail: [email protected]. Fax: +81-11-706-4493.

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Studies on the Formation Mechanism and the Structure of the

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