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Effects of hyaluronic acid on the rheological properties of zinc carboxylate gels
Materials Science and Engineering C 24 (2004) 703 – 707 www.elsevier.com/locate/msec
Effects of hyaluronic acid on the rheological properties of zinc carboxylate gels Norifumi Azumaa,*, Toshiyuki Ikomab,c, Akiyoshi Osakaa, Junzo Tanakab,c a b
Biomaterial Laboratory, Faculty of Engineering, Okayama University, Tsushima, Okayama 700-8530, Japan Biomaterials Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan c CREST, Japan Science and Technology Corporation, Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan Received 10 June 2004; received in revised form 8 July 2004; accepted 11 August 2004 Available online 29 September 2004
Abstract Hyaluronic acid (HyA) and zinc carboxylate gels were synthesized by using L-malic acid (MA) and NaOH solutions. Obtained gels (HyA/Zn/MA gels) had a characteristic with transparency of 12.9 to 48.2% at a wavelength of 500 nm, pH values ranging from 6.8 to 7.1. The polymerization reaction and the formation of network structure were completed within 12 h. Rheological measurements indicated that the storage (GV) moduli were higher than the loss (GU) moduli in all specimens and the elastic moduli were greater than 10,000 Pa, which is higher than gels cross-linked with covalent bonds. Moreover, gel strength values (S calculated from the complex (G* ) moduli at 1.0 Hz) were higher than other chemically cross-linked gels. The gel strength increased at zinc concentrations up to 0.45 mol/l, however, decreased at a zinc concentration of 0.5 mol/l with an increasing HyA concentration. The rheological properties of HyA/Zn/MA gels strongly depended on zinc and HyA concentrations. D 2004 Elsevier B.V. All rights reserved. Keywords: Gel; Zinc; Malic acid; Rheology; Hyaluronic acid
1. Introduction Hyaluronic acid (HyA) is a major component of the extracellular matrix in the viscoelastic tissues and of the synovial fluid in joints [1]. It has unique rheological properties that are unparalleled by any other natural or synthetic polymers, and it has been used as a surgical aid, especially for the replacement of the natural vitreous humor in ophthalmic procedures [2]. The carboxyl and hydroxyl groups of HyA has been shown to coordinate with metal ions such as Fe(III), Cu(II), Zn(II), Ca(II), Ag(I), Cd(II) and Pb(II) [3–8]. Particularly, Fe(III) and Cu(II) ions with HyA form gels. The rheological properties of HyA gels crosslinked with Fe(III) ions can be enhanced dramatically depending on a Fe(III) ion concentration [9].
* Corresponding author. Tel.: +81 298 51 3354; fax: +81 298 51 8291. E-mail address: [email protected] (N. Azuma). 0928-4931/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2004.08.015
Rheological properties of the gels are obtained by examining the gel network and cross-linking density [10– 14]. The structure of the network and the cross-linking density are important factors to determining the rheological characteristics of the gels. There are two general parameters to describe the rheological properties: the dynamic moduli ( GV, storage modulus; GU, loss modulus) and gel strength (S) determined by oscillatory shear experiments [13,15]. The dynamic moduli show the elastic and viscous characters of the gels [17–20], and the gel strength is a material parameter, depending on the cross-linking density [21–23]. The gel strength is determined with the complex moduli ( G*(x)) if the gels show the linear viscoelastic properties [24–28]. The complex moduli are given the information about both of the elastic and viscous properties of the gels. In our previous study, we established a new method to produce zinc carboxylate gels based on L-malic acid (MA) with using a NaOH solution. MA is one of carboxylate in
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the citric acid cycle of metabolic system. The a-hydroxyl carboxyl acids, such as malic acid, have been used as ligands for precursor metal complexes to synthesize metal oxide [29,30]. Zinc carboxylate gels based citric acid with ammonium gels had described as a precursor to obtain zinc oxide [31]. The gels containing zinc ions have been widely used for biomaterials, such as ointment, since zinc has an anti-inflammation property [32]. However, zinc carboxylate gels based on malic acid gels have not been described previously. In this study, we synthesized zinc-malic acid gels containing HyA. The effect of a HyA concentration on the rheological properties of gels was characterized by using the dynamic moduli and the gel strength values obtained from oscillatory rheological measurements.
vessel for 24 h. Before starting any measurement, the HyA/Zn/MA gels were rested for one minute, allowing the stress induced during sample loading to be released. All measurements were conducted at room temperature (298F0.5 K). The extent of the linear viscoelastic region was determined by performing a stress sweep under the conditions that the shear stress was changed from 0.1 to 1000 Pa and the frequency was fixed at 1.0 Hz. In the linear viscoelastic regions, the GV, GU and tangent-delta values were collected from oscillatory measurement at various HyA concentrations. In order to obtain the gel strength values, the dynamic frequency sweep was conducted by applying a constant shear stress of 1.0 Pa at a frequency range between 0.1 and 10 Hz. The gel strength was calculated from the frequency sweep at various pH values and HyA concentrations.
2. Experiments
2.3. Time dependence on polymerization process
2.1. Synthesis and characterization of HyA/Zn/MA gels
The polymerization process after adding 12.0 ml of a NaOH solution to 10.0 ml of HyA/Zn/MA solutions (HyA concentration of 1.5 wt.%) was evaluated by measuring the change of viscoelastic properties against still standing time. The GV, GU, and tangent-delta and gel strength values were collected from stress-strain measurement at 1.0 Pa and 1.0 Hz.
L-malic acid (MA, 97.0% of purity), zinc chloride (ZnCl2, 98.0% of purity) and a 1.0 mol/l sodium hydroxide solution (NaOH, 96.0% of purity) were purchased from Wako Pure Chem., Japan. Zinc and MA solutions with different molar ratios and lower pH values than 1.0 were prepared as the starting solutions as follows. MA was dissolved to 0.5 mol/l in distilled water and then ZnCl2 was added to the solution up to zinc concentrations of 0.4, 0.45 and 0.5 mol/l at room temperature. HyA powder (hyaluronic sodium, MW; 720 kDa, Seikagaku Kogyo, Japan) was added to 10 ml of the starting solutions up to concentrations of 0.1–1.5 wt.% (10–150 mg). Twelve milliliters of a NaOH solution adjusted to 1.0 mol/l were dropped into 10.0 ml of HyA/Zn/MA solutions to increase the pH values while stirring. The HyA/Zn/MA solutions were further stirred for 5 min at the desired pH values and then stood for 24 h at room temperature. The optical transmittance of the specimens obtained was measured using a HITACHI U-2000 double beam spectroscope at 500 nm of wavelength. The samples were put into a PMMA cell with a path length of 10 mm. Distilled water was used as a reference sample. The measurements were conducted three times at room temperature. 2.2. Rheological measurements Viscoelastic properties were investigated with the viscoelastic meter RheoStress RS1 (HAAKE, Germany), equipped with plate-plate tools of 20 mm in diameter and one mm in gap length. After stirring of HyA/Zn/MA solutions for 5 min, the solutions were run into a silicone sheet bored through 20 mm in diameter and 1.5 mm in depth. The samples were carefully pinched with polypropylene and glass boards, extra samples were flew out, and then the samples were held in a constant humidity
2.4. Theory of rheology The viscoelastic properties of the cross-linking system of the sol through a transition to the gel state were monitored by measuring the oscillatory shear moduli: Z t r ðt Þ ¼ S ðt tVÞn c˙ ðt VÞdt V ð1Þ l
where r is the shear stress; dc , lb t Vbt, is the rate of deformation of the sample at a gel point; t is the present time; S is the gel strength, depending on cross-density and molecular chain flexibility; n is the relaxation exponent. It has been shown that the share relaxation modulus G(t) is characterized by a power-law in time at a gel point [25–28,33]: Gðt Þ ¼ St n
k0 btbl
ð2Þ
An oscillatory test at fixed frequency, x, is qualitatively equivalent to a transient experiment at time t=1/x; therefore, if linear viscoelasticity is held, it may be assumed that Eq. (2) transforms into GðxÞ ¼ Sxn
f or 0bxb1=k0
ð3Þ
The GV modulus provides information about the elasticity or the energy stored in the material during deformation, whereas the GU modulus demonstrating the viscous character or the energy dissipates as heat. The ratio
N. Azuma et al. / Materials Science and Engineering C 24 (2004) 703–707
between the viscous and elastic moduli is expressed by the loss tangent: tand ¼ GW=GV
ð4Þ
where d is the phase angle. The gel strength is obtained from the complex shear ( G* in Pa) moduli as follows. The combined viscous and elastic behavior is given by the absolute value of the complex shear module G* as function of the frequency x in Hz. ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi r
G4ð-Þ ¼
GVðxÞÞ2 þ GWð-ÞÞ2
ð5Þ
The experimental data of all frequency sweep tests were correlated by the following power law: r ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi G4ð-Þ ¼
GVðxÞÞ2 þ GWð-ÞÞ2 ¼ Sx1=z
ð6Þ
where S is the gel strength and z is the coordination number which represents the number of flow units interacting with one another to give the observed flow response of the materials. The correlation between G* and x was obtained by the following linear regression analysis: logG4 ¼ z1 logx þ logS
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which indicates that HyA/Zn/MA gels have an elastic property rather than a viscous property. The elastic modulus values of HyA/Zn/MA gels were greater than 1000 Pa, notably over 10000 Pa at a zinc concentration of 0.5 mol/l. Furthermore, the dynamic moduli of the gels at a zinc concentration of 0.5 mol/l were one order magnitude higher than those of the gels at a zinc concentration of 0.4 mol/l. We thus conclude that the rheological properties of the gels strongly depend on the zinc concentration. These values of HyA/Zn/MA gels obtained in the present study are greater than the previously reported values of other polymer gels such as poly (L-lactide) (PLLA) and poly (ethylene oxide) (PEO) (500–1000 Pa) [36], and SnO2 colloidal gels (G V; 0.1–1 kPa, GU; 0.1–0.2 kPa) [37]. HyA gels as a biomaterial with a kPa range of GV attract widespread attention since many native tissues have moduli in this range although most show nonlinear response [36]. For example, human nasal cartilage (234F27 kPa) [38], bovine articular cartilage (990F50 kPa) [38], pig thoracic aorta (43.2F15 kPa) [39], right lobe of human liver (270F10 kPa) [39], canine kidney cortex and medulla (~10 kPa) [40], and nuleus pulposus and eye lens (~1 kPa) [41] have moduli in this range.
ð7Þ
The slope is the reciprocal of z and the intercept at x=1.0 Hz is log S [25–28].
3. Results and discussions Fig. 1 shows transparency (Fig. 1a) and opaque (Fig. 1b) HyA/Zn/MA gels which were obtained at a zinc concentration of 0.4 mol/l and a HyA concentration of 1.5 wt.%. Transmittance values of 12.9–48.2% were obtained from HyA/Zn/MA gels at all HyA concentrations and a pH range of 6.8–7.1. The polymerization reaction did not occur at lower pH values (ca pH 6.0), the specimens had high transmittance (ca. 98.0%) and zinc-malate-trihydrate crystals were precipitated. Zinc malate trihydrate (Zn(C4H4O5)24H2O) are tetragonal crystal which is easily precipitated and grown by slow evaporation of an zinc and malic acid aqueous solution [34,35]. From these results, the dissociation of carboxyl groups of MA (the dissociation constant pK 2 of 5.05) was strongly correlated with the polymerization process. At higher pH range (ca pH7.6), opaque HyA/Zn/MA gels were obtained. By increasing a pH value, viscous solutions with the precipitations of small fragments were obtained, and then zinc hydroxide precipitations were formed. The higher pH values probably induced the polymerization/chelation of zinc ions with the MA molecules and the formation of zinc hydroxide. Fig. 2 shows the dynamic moduli of the HyA/Zn/MA gels with 1.5 wt.% HyA at various zinc concentrations against a frequency range between 0.1 and 10 Hz. The GV moduli were higher than the GU moduli in all specimens,
Fig. 1. Photo images of HyA/Zn/MA gels used in rheological experiments; (a) the transparent gel prepared by an addition of 12.0 ml of a NaOH solution at a pH value of 7.1, and (b) the opaque gel prepared by an addition of 13.0 ml of a NaOH solution at a pH value of 7.6. A HyA concentration was 1.5 wt.%, and a zinc concentration was 0.4 mol/l.
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Fig. 2. A change of dynamic moduli of the HyA/Zn/MA gels with a frequency range between 0.1 and 10 Hz at zinc concentrations of 0.4, 0.45 and 0.5 mol/ and a HyA concentration of 1.5 wt.%. The open marks indicate GV values, and the filled marks indicate square GU values. The circle marks indicate HyA/Zn/MA gels at a zinc concentration of 0.5 mol/l, and the square and triangle marks indicate HyA/Zn/MA gels at zinc concentrations of 0.45 and 0.4 mo/l.
Fig. 3. Gel strength values of HyA/Zn/MA gels against a HyA concentration. The circle marks indicate the values at a zinc concentration of 0.5 mol/l, and the square and triangle marks show 0.45 and 0.4 mol/l gels.
Fig. 4. Changes of dynamic moduli and tangent delta values of HyA/Zn/ MA gels against the time evaluation at 298 K. A Zinc concentration was 0.4 mol/l, and a HyA concentration was 1.5 wt.%. The circle marks indicate the G Vvalues, and the square and triangle marks indicate the GU and tangentdelta values, respectively.
The dynamic moduli are linear and parallel in a typical frequency range. The linearity of these values suggests that HyA/Zn/MA gels behave like a critical gel having a threedimensional gel network [24]. Gel strength values of HyA/ Zn/MA gels were calculated from the complex moduli with Eq. (5)–(7). Fig. 3 shows the gel strength of HyA/Zn/MA gels against HyA concentrations. At a HyA concentration of
Fig. 5. Changes of gel strength values of HyA/Zn/MA gels against the time evaluation at 298 K. A Zinc concentration was 0.4 mol/l, and a HyA concentration was 1.5 wt.%.
N. Azuma et al. / Materials Science and Engineering C 24 (2004) 703–707
0 wt.%, the gel strength values were 1.73F0.24, 7.36F1.28 and 118.47F30.15 kPa at zinc concentrations of 0.4, 0.45 and 0.5 mol/l, respectively. The gel strength strongly depended on the zinc concentration. Furthermore, at a HyA concentration of 1.5 wt.%, the gel strength values were 7.62F1.39, 7.36F1.28 and 66.76F6.36 kPa at zinc concentrations of 0.4, 0.45 and 0.5 mol/l, respectively. An increase of a HyA concentration up to a zinc concentration of 0.45 mol/l caused an increase of the gel strength. It is thus indicated that HyA molecules interact with zinc and MA molecules, and the gel network of HyA/Zn/MA gels becomes firmer than gels without HyA. However, the strength of the gels decreased at a zinc concentration of 0.5 mol/l. This means that when an excess amount of zinc is added to HyA/Zn/MA starting solutions, an interaction of HyA molecules with zinc and MA molecules is blocked. The gel strength values of HyA/Zn/MA gels were higher than other critical gels such as an ethyl(hydroxyethyl)cellulose (EHEC)/cetyltriammonium bromide gel (0.11 kPa), an EHEC/sodium dodecyl sulfate gel (0.11 kPa) [22], polycaprolactone gels (0.09–19 kPa) [16] and PVA gels (0.01–0.9 kPa) [23]. Therefore, we conclude that HyA/Zn/MA gels are stronger than any other critical gels. Figs. 4 and 5 show a change of the dynamic moduli, and tangent delta and gel strength values of HyA/Zn/MA gels at a zinc concentration of 0.4 mol/l and a HyA concentration of 1.5 wt.% against time evolution. The dynamic moduli and gel strength values increased with an increase of time, and reached equilibrium after 12 hours. On the contrary, tangent-delta values of the gels decreased with reaction time, and reached equilibrium after 12 h. These results indicate that the polymerization reaction and formation of the network structure are completed within 12 h after an addition of a NaOH solution.
4. Conclusion HyA/Zn/MA gels prepared in this study are classified into a metal carboxylate gel with a three-dimensional network formed through an inorganic polymerization process. The HyA/Zn/MA gels showed linear viscoelastic properties, and, therefore, the gel strength values could be calculated from the complex moduli. The rheological measurements showed that an increase of the network connectivity was dependent on HyA and zinc concentrations. An increase of a HyA concentration caused an increase of the gel strength. It is thus indicated that HyA molecules interact with Zinc and MA molecules each, which results in the formation of a firmer gel network than gels without HyA. However, an addition of an excess amount of zinc to the original HyA/Zn/MA solutions blocked an interaction of HyA molecules with zinc and MA molecules.
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Effects of hyaluronic acid on the rheological properties of zinc carboxylate gels Norifumi Azumaa,*, Toshiyuki Ikomab,c, Akiyoshi Osakaa, Junzo Tanakab,c a b
Biomaterial Laboratory, Faculty of Engineering, Okayama University, Tsushima, Okayama 700-8530, Japan Biomaterials Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan c CREST, Japan Science and Technology Corporation, Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan Received 10 June 2004; received in revised form 8 July 2004; accepted 11 August 2004 Available online 29 September 2004
Abstract Hyaluronic acid (HyA) and zinc carboxylate gels were synthesized by using L-malic acid (MA) and NaOH solutions. Obtained gels (HyA/Zn/MA gels) had a characteristic with transparency of 12.9 to 48.2% at a wavelength of 500 nm, pH values ranging from 6.8 to 7.1. The polymerization reaction and the formation of network structure were completed within 12 h. Rheological measurements indicated that the storage (GV) moduli were higher than the loss (GU) moduli in all specimens and the elastic moduli were greater than 10,000 Pa, which is higher than gels cross-linked with covalent bonds. Moreover, gel strength values (S calculated from the complex (G* ) moduli at 1.0 Hz) were higher than other chemically cross-linked gels. The gel strength increased at zinc concentrations up to 0.45 mol/l, however, decreased at a zinc concentration of 0.5 mol/l with an increasing HyA concentration. The rheological properties of HyA/Zn/MA gels strongly depended on zinc and HyA concentrations. D 2004 Elsevier B.V. All rights reserved. Keywords: Gel; Zinc; Malic acid; Rheology; Hyaluronic acid
1. Introduction Hyaluronic acid (HyA) is a major component of the extracellular matrix in the viscoelastic tissues and of the synovial fluid in joints [1]. It has unique rheological properties that are unparalleled by any other natural or synthetic polymers, and it has been used as a surgical aid, especially for the replacement of the natural vitreous humor in ophthalmic procedures [2]. The carboxyl and hydroxyl groups of HyA has been shown to coordinate with metal ions such as Fe(III), Cu(II), Zn(II), Ca(II), Ag(I), Cd(II) and Pb(II) [3–8]. Particularly, Fe(III) and Cu(II) ions with HyA form gels. The rheological properties of HyA gels crosslinked with Fe(III) ions can be enhanced dramatically depending on a Fe(III) ion concentration [9].
* Corresponding author. Tel.: +81 298 51 3354; fax: +81 298 51 8291. E-mail address: [email protected] (N. Azuma). 0928-4931/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2004.08.015
Rheological properties of the gels are obtained by examining the gel network and cross-linking density [10– 14]. The structure of the network and the cross-linking density are important factors to determining the rheological characteristics of the gels. There are two general parameters to describe the rheological properties: the dynamic moduli ( GV, storage modulus; GU, loss modulus) and gel strength (S) determined by oscillatory shear experiments [13,15]. The dynamic moduli show the elastic and viscous characters of the gels [17–20], and the gel strength is a material parameter, depending on the cross-linking density [21–23]. The gel strength is determined with the complex moduli ( G*(x)) if the gels show the linear viscoelastic properties [24–28]. The complex moduli are given the information about both of the elastic and viscous properties of the gels. In our previous study, we established a new method to produce zinc carboxylate gels based on L-malic acid (MA) with using a NaOH solution. MA is one of carboxylate in
704
N. Azuma et al. / Materials Science and Engineering C 24 (2004) 703–707
the citric acid cycle of metabolic system. The a-hydroxyl carboxyl acids, such as malic acid, have been used as ligands for precursor metal complexes to synthesize metal oxide [29,30]. Zinc carboxylate gels based citric acid with ammonium gels had described as a precursor to obtain zinc oxide [31]. The gels containing zinc ions have been widely used for biomaterials, such as ointment, since zinc has an anti-inflammation property [32]. However, zinc carboxylate gels based on malic acid gels have not been described previously. In this study, we synthesized zinc-malic acid gels containing HyA. The effect of a HyA concentration on the rheological properties of gels was characterized by using the dynamic moduli and the gel strength values obtained from oscillatory rheological measurements.
vessel for 24 h. Before starting any measurement, the HyA/Zn/MA gels were rested for one minute, allowing the stress induced during sample loading to be released. All measurements were conducted at room temperature (298F0.5 K). The extent of the linear viscoelastic region was determined by performing a stress sweep under the conditions that the shear stress was changed from 0.1 to 1000 Pa and the frequency was fixed at 1.0 Hz. In the linear viscoelastic regions, the GV, GU and tangent-delta values were collected from oscillatory measurement at various HyA concentrations. In order to obtain the gel strength values, the dynamic frequency sweep was conducted by applying a constant shear stress of 1.0 Pa at a frequency range between 0.1 and 10 Hz. The gel strength was calculated from the frequency sweep at various pH values and HyA concentrations.
2. Experiments
2.3. Time dependence on polymerization process
2.1. Synthesis and characterization of HyA/Zn/MA gels
The polymerization process after adding 12.0 ml of a NaOH solution to 10.0 ml of HyA/Zn/MA solutions (HyA concentration of 1.5 wt.%) was evaluated by measuring the change of viscoelastic properties against still standing time. The GV, GU, and tangent-delta and gel strength values were collected from stress-strain measurement at 1.0 Pa and 1.0 Hz.
L-malic acid (MA, 97.0% of purity), zinc chloride (ZnCl2, 98.0% of purity) and a 1.0 mol/l sodium hydroxide solution (NaOH, 96.0% of purity) were purchased from Wako Pure Chem., Japan. Zinc and MA solutions with different molar ratios and lower pH values than 1.0 were prepared as the starting solutions as follows. MA was dissolved to 0.5 mol/l in distilled water and then ZnCl2 was added to the solution up to zinc concentrations of 0.4, 0.45 and 0.5 mol/l at room temperature. HyA powder (hyaluronic sodium, MW; 720 kDa, Seikagaku Kogyo, Japan) was added to 10 ml of the starting solutions up to concentrations of 0.1–1.5 wt.% (10–150 mg). Twelve milliliters of a NaOH solution adjusted to 1.0 mol/l were dropped into 10.0 ml of HyA/Zn/MA solutions to increase the pH values while stirring. The HyA/Zn/MA solutions were further stirred for 5 min at the desired pH values and then stood for 24 h at room temperature. The optical transmittance of the specimens obtained was measured using a HITACHI U-2000 double beam spectroscope at 500 nm of wavelength. The samples were put into a PMMA cell with a path length of 10 mm. Distilled water was used as a reference sample. The measurements were conducted three times at room temperature. 2.2. Rheological measurements Viscoelastic properties were investigated with the viscoelastic meter RheoStress RS1 (HAAKE, Germany), equipped with plate-plate tools of 20 mm in diameter and one mm in gap length. After stirring of HyA/Zn/MA solutions for 5 min, the solutions were run into a silicone sheet bored through 20 mm in diameter and 1.5 mm in depth. The samples were carefully pinched with polypropylene and glass boards, extra samples were flew out, and then the samples were held in a constant humidity
2.4. Theory of rheology The viscoelastic properties of the cross-linking system of the sol through a transition to the gel state were monitored by measuring the oscillatory shear moduli: Z t r ðt Þ ¼ S ðt tVÞn c˙ ðt VÞdt V ð1Þ l
where r is the shear stress; dc , lb t Vbt, is the rate of deformation of the sample at a gel point; t is the present time; S is the gel strength, depending on cross-density and molecular chain flexibility; n is the relaxation exponent. It has been shown that the share relaxation modulus G(t) is characterized by a power-law in time at a gel point [25–28,33]: Gðt Þ ¼ St n
k0 btbl
ð2Þ
An oscillatory test at fixed frequency, x, is qualitatively equivalent to a transient experiment at time t=1/x; therefore, if linear viscoelasticity is held, it may be assumed that Eq. (2) transforms into GðxÞ ¼ Sxn
f or 0bxb1=k0
ð3Þ
The GV modulus provides information about the elasticity or the energy stored in the material during deformation, whereas the GU modulus demonstrating the viscous character or the energy dissipates as heat. The ratio
N. Azuma et al. / Materials Science and Engineering C 24 (2004) 703–707
between the viscous and elastic moduli is expressed by the loss tangent: tand ¼ GW=GV
ð4Þ
where d is the phase angle. The gel strength is obtained from the complex shear ( G* in Pa) moduli as follows. The combined viscous and elastic behavior is given by the absolute value of the complex shear module G* as function of the frequency x in Hz. ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi r
G4ð-Þ ¼
GVðxÞÞ2 þ GWð-ÞÞ2
ð5Þ
The experimental data of all frequency sweep tests were correlated by the following power law: r ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi G4ð-Þ ¼
GVðxÞÞ2 þ GWð-ÞÞ2 ¼ Sx1=z
ð6Þ
where S is the gel strength and z is the coordination number which represents the number of flow units interacting with one another to give the observed flow response of the materials. The correlation between G* and x was obtained by the following linear regression analysis: logG4 ¼ z1 logx þ logS
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which indicates that HyA/Zn/MA gels have an elastic property rather than a viscous property. The elastic modulus values of HyA/Zn/MA gels were greater than 1000 Pa, notably over 10000 Pa at a zinc concentration of 0.5 mol/l. Furthermore, the dynamic moduli of the gels at a zinc concentration of 0.5 mol/l were one order magnitude higher than those of the gels at a zinc concentration of 0.4 mol/l. We thus conclude that the rheological properties of the gels strongly depend on the zinc concentration. These values of HyA/Zn/MA gels obtained in the present study are greater than the previously reported values of other polymer gels such as poly (L-lactide) (PLLA) and poly (ethylene oxide) (PEO) (500–1000 Pa) [36], and SnO2 colloidal gels (G V; 0.1–1 kPa, GU; 0.1–0.2 kPa) [37]. HyA gels as a biomaterial with a kPa range of GV attract widespread attention since many native tissues have moduli in this range although most show nonlinear response [36]. For example, human nasal cartilage (234F27 kPa) [38], bovine articular cartilage (990F50 kPa) [38], pig thoracic aorta (43.2F15 kPa) [39], right lobe of human liver (270F10 kPa) [39], canine kidney cortex and medulla (~10 kPa) [40], and nuleus pulposus and eye lens (~1 kPa) [41] have moduli in this range.
ð7Þ
The slope is the reciprocal of z and the intercept at x=1.0 Hz is log S [25–28].
3. Results and discussions Fig. 1 shows transparency (Fig. 1a) and opaque (Fig. 1b) HyA/Zn/MA gels which were obtained at a zinc concentration of 0.4 mol/l and a HyA concentration of 1.5 wt.%. Transmittance values of 12.9–48.2% were obtained from HyA/Zn/MA gels at all HyA concentrations and a pH range of 6.8–7.1. The polymerization reaction did not occur at lower pH values (ca pH 6.0), the specimens had high transmittance (ca. 98.0%) and zinc-malate-trihydrate crystals were precipitated. Zinc malate trihydrate (Zn(C4H4O5)24H2O) are tetragonal crystal which is easily precipitated and grown by slow evaporation of an zinc and malic acid aqueous solution [34,35]. From these results, the dissociation of carboxyl groups of MA (the dissociation constant pK 2 of 5.05) was strongly correlated with the polymerization process. At higher pH range (ca pH7.6), opaque HyA/Zn/MA gels were obtained. By increasing a pH value, viscous solutions with the precipitations of small fragments were obtained, and then zinc hydroxide precipitations were formed. The higher pH values probably induced the polymerization/chelation of zinc ions with the MA molecules and the formation of zinc hydroxide. Fig. 2 shows the dynamic moduli of the HyA/Zn/MA gels with 1.5 wt.% HyA at various zinc concentrations against a frequency range between 0.1 and 10 Hz. The GV moduli were higher than the GU moduli in all specimens,
Fig. 1. Photo images of HyA/Zn/MA gels used in rheological experiments; (a) the transparent gel prepared by an addition of 12.0 ml of a NaOH solution at a pH value of 7.1, and (b) the opaque gel prepared by an addition of 13.0 ml of a NaOH solution at a pH value of 7.6. A HyA concentration was 1.5 wt.%, and a zinc concentration was 0.4 mol/l.
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N. Azuma et al. / Materials Science and Engineering C 24 (2004) 703–707
Fig. 2. A change of dynamic moduli of the HyA/Zn/MA gels with a frequency range between 0.1 and 10 Hz at zinc concentrations of 0.4, 0.45 and 0.5 mol/ and a HyA concentration of 1.5 wt.%. The open marks indicate GV values, and the filled marks indicate square GU values. The circle marks indicate HyA/Zn/MA gels at a zinc concentration of 0.5 mol/l, and the square and triangle marks indicate HyA/Zn/MA gels at zinc concentrations of 0.45 and 0.4 mo/l.
Fig. 3. Gel strength values of HyA/Zn/MA gels against a HyA concentration. The circle marks indicate the values at a zinc concentration of 0.5 mol/l, and the square and triangle marks show 0.45 and 0.4 mol/l gels.
Fig. 4. Changes of dynamic moduli and tangent delta values of HyA/Zn/ MA gels against the time evaluation at 298 K. A Zinc concentration was 0.4 mol/l, and a HyA concentration was 1.5 wt.%. The circle marks indicate the G Vvalues, and the square and triangle marks indicate the GU and tangentdelta values, respectively.
The dynamic moduli are linear and parallel in a typical frequency range. The linearity of these values suggests that HyA/Zn/MA gels behave like a critical gel having a threedimensional gel network [24]. Gel strength values of HyA/ Zn/MA gels were calculated from the complex moduli with Eq. (5)–(7). Fig. 3 shows the gel strength of HyA/Zn/MA gels against HyA concentrations. At a HyA concentration of
Fig. 5. Changes of gel strength values of HyA/Zn/MA gels against the time evaluation at 298 K. A Zinc concentration was 0.4 mol/l, and a HyA concentration was 1.5 wt.%.
N. Azuma et al. / Materials Science and Engineering C 24 (2004) 703–707
0 wt.%, the gel strength values were 1.73F0.24, 7.36F1.28 and 118.47F30.15 kPa at zinc concentrations of 0.4, 0.45 and 0.5 mol/l, respectively. The gel strength strongly depended on the zinc concentration. Furthermore, at a HyA concentration of 1.5 wt.%, the gel strength values were 7.62F1.39, 7.36F1.28 and 66.76F6.36 kPa at zinc concentrations of 0.4, 0.45 and 0.5 mol/l, respectively. An increase of a HyA concentration up to a zinc concentration of 0.45 mol/l caused an increase of the gel strength. It is thus indicated that HyA molecules interact with zinc and MA molecules, and the gel network of HyA/Zn/MA gels becomes firmer than gels without HyA. However, the strength of the gels decreased at a zinc concentration of 0.5 mol/l. This means that when an excess amount of zinc is added to HyA/Zn/MA starting solutions, an interaction of HyA molecules with zinc and MA molecules is blocked. The gel strength values of HyA/Zn/MA gels were higher than other critical gels such as an ethyl(hydroxyethyl)cellulose (EHEC)/cetyltriammonium bromide gel (0.11 kPa), an EHEC/sodium dodecyl sulfate gel (0.11 kPa) [22], polycaprolactone gels (0.09–19 kPa) [16] and PVA gels (0.01–0.9 kPa) [23]. Therefore, we conclude that HyA/Zn/MA gels are stronger than any other critical gels. Figs. 4 and 5 show a change of the dynamic moduli, and tangent delta and gel strength values of HyA/Zn/MA gels at a zinc concentration of 0.4 mol/l and a HyA concentration of 1.5 wt.% against time evolution. The dynamic moduli and gel strength values increased with an increase of time, and reached equilibrium after 12 hours. On the contrary, tangent-delta values of the gels decreased with reaction time, and reached equilibrium after 12 h. These results indicate that the polymerization reaction and formation of the network structure are completed within 12 h after an addition of a NaOH solution.
4. Conclusion HyA/Zn/MA gels prepared in this study are classified into a metal carboxylate gel with a three-dimensional network formed through an inorganic polymerization process. The HyA/Zn/MA gels showed linear viscoelastic properties, and, therefore, the gel strength values could be calculated from the complex moduli. The rheological measurements showed that an increase of the network connectivity was dependent on HyA and zinc concentrations. An increase of a HyA concentration caused an increase of the gel strength. It is thus indicated that HyA molecules interact with Zinc and MA molecules each, which results in the formation of a firmer gel network than gels without HyA. However, an addition of an excess amount of zinc to the original HyA/Zn/MA solutions blocked an interaction of HyA molecules with zinc and MA molecules.
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