Anisotropic swelling and mechanical behavior of composite bacterial cellulose–poly(acrylamide or acrylamide–sodium acrylate) hydrogels

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Research paper

Anisotropic swelling and mechanical behavior of composite bacterial cellulose–poly(acrylamide or acrylamide–sodium acrylate) hydrogels A.L. Buyanov a,∗ , I.V. Gofman a , L.G. Revel’skaya a , A.K. Khripunov a , A.A. Tkachenko b a Institute of Macromolecular Compounds, Russian Academy of Sciences, 31, Bolshoi pr., 199004, St. Petersburg, Russia b St. Petersburg State University, Universitetskaya nab., 7-9, 199034 St. Petersburg, Russia

A R T I C L E

I N F O

A B S T R A C T

Article history:

Bacterial cellulose–polyacrylamide (BC–PAAm) composite hydrogels are prepared by

Received 23 September 2008

synthesis of PAAm networks inside the BC matrices. The behavior of these gels and of

Received in revised form

the ionic ones obtained via partial hydrolysis of BC–PAAm gels is studied under swelling

16 June 2009

and compressive deformation conditions. The dependences of the hydrogels’ properties

Accepted 17 June 2009

on the BC matrix preparation conditions, gel synthesis conditions and the BC content in

Published online 23 June 2009

the hydrogel compositions are studied. Two types of BC gel pellicle are used in the hydrogel synthesis, namely matrix pellicles subjected to pre-pressing (samples of series A) and those not subjected to any mechanical actions before synthesis (series B samples) containing about 99% water. The effect of anisotropic swelling of type A hydrogels is detected. The type B specimens swell isotropically. Both types of hydrogel exhibit substantial anisotropy of their mechanical properties, apparent in different shapes of compression stress–strain curves of samples cut out from the gel plates in various directions. Composite hydrogels show superb mechanical properties, including compression strength up to 10 MPa and the ability to withstand long-term cyclic stresses (up to 2000–6000 cycles) without substantial reduction of mechanical properties. c 2009 Elsevier Ltd. All rights reserved.

1.

Introduction

Hydrogels are three-dimensional crosslinked structures formed by hydrophilic polymers. Hydrogels of ionic type can substantially change their volume as a result of pretty modest variations of the outside ambient such as pH, ionic strength of the solution, and electric current. These effects make such hydrogels promising materials for chemo–mechanical ∗ Corresponding author. Tel.: +7 904 6100516; fax: +7 8123286869. E-mail address: [email protected] (A.L. Buyanov). c 2009 Elsevier Ltd. All rights reserved. 1751-6161/$ - see front matter doi:10.1016/j.jmbbm.2009.06.001

systems, hydrogel actuators, and so on (Calvert, 2004, 2009; Kaneko et al., 2002). Another promising field of hydrogels’ use is biomaterials — implants for medical applications (Calvert, 2009; Kopecek, 2007; Seal et al., 2001). For example, these materials are already used as artificial cartilages to substitute the injured natural ones. The richest information concerning the gels’ properties and their practical use as artificial cartilages was

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collected for hydrogels based on poly(vinyl alcohol) (Grant et al., 2006; Oka, 2001; Stammen et al., 2001). The broad use of different hydrogel types is hindered by the insufficiently high level of their mechanical properties. For example, the PAAm hydrogels, characterized by good biological compatibility (Bosch et al., 2000; Novaes and Berg, 2003), demonstrate relatively low values of elastic modulus and mechanical strength (Baselga et al., 1987; Buyanov et al., 1992). One of possible methods of material design with improved functional properties is the synthesis of compositions with the structure of interpenetrating polymer networks. By using this method we have developed hydrogel membranes consisting of cellulose–PAAm and cellulose–polyacrylic acid (PAA) compounds. At the expense of the high rigidity of the cellulose carcass, these hydrogels possess high mechanical strength and stiffness, and at the same time demonstrate all valuable properties inherent to PAAm and PAA (Buyanov et al., 1998, 2001). In recent years, a special type of cellulose, namely bacterial cellulose (BC), has been used for the synthesis of various types of hydrogel compositions which also include xyloglucan, pectin (Astley et al., 2001), or gelatin (Nakayama et al., 2004). BC is the subject of intensive studies and is treated now as a promising biomaterial for medical applications (Czaja et al., 2007; Svensson et al., 2005). In our works (Buyanov et al., 2005; Gofman et al., 2006, 2008), we have used BC in the synthesis of BC–PAAm composite hydrogels which were shown to have mechanical properties close to those of natural cartilages of different localizations. For some of these types of hydrogel, pronounced anisotropic behavior was detected both at swelling and at deformation (Buyanov et al., 2005). This behavior differs significantly from that of mono-component PAAm hydrogels, which demonstrate isotropic swelling and mechanical behavior. The results of in vitro and in vivo experiments carried out on animals (these hydrogels were implanted into rat and dog body tissues for three months) have shown a high level of biocompatibility of these types of hydrogel composition (Gofman et al., 2006). The present work aims at a careful study of the peculiarities of anisotropic behavior of BC–PAAm hydrogel compositions depending on the preparation method of the BC matrices. It was also important to study the mechanical behavior of these composite hydrogels in the conditions of long-term fatigue cyclic compression tests, because this mode of loading will take place in practical use of such materials. Such data can hardly be found in the literature. As a rule only the modulus of elasticity and strength values, measured during monotonic deformation of the gels studied, can be found in the previous articles, or only the Young’s modulus values are reported. The latter data are insufficient for predicting the behavior of hydrogel materials in different practical applications. In particular, such information is valuable to evaluate possible hydrogel use as artificial joint cartilages because natural ones are subjected to the action of intense long-term cyclic mechanical loads (Huang et al., 2005; Oka, 2001; Stammen et al., 2001; Wong and Carter, 2003).

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2.

Materials and methods

2.1.

Starting materials

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Acrylamide (Aldrich Chemicals) was recrystallized twice from benzene. All other reagents of analytical grade were used as received.

2.2. Synthesis of BC and preparation of BC pellicles for the synthesis of composite hydrogels BC, or so-called acetobacter xylinum, was grown by using VKM880 strain in water solutions containing 2 wt% of glucose, 0.3 wt% of yeast extract and 2 wt% of ethanol at 30 ◦ C for 14–21 days in cylindrical glass vessels, as described in detail in Khripunov and Tkachenko (1998). The BC was subsequently washed in water solutions of potassium hydroxide at ∼100 ◦ C and then in water at room temperature. The resulting BC samples were gel-like pellicles up to 25 mm thick, containing about 99 wt% water. These BC pellicles are materials of very low elasticity in the direction perpendicular to their surface, and they have very poor water retention — the liberation of water from the pellicles takes place even under low mechanical loading. For the synthesis of composite hydrogels the BC pellicles were used in one of two states: partially dehydrated by pressing with a manually operated press or in the original non-pressed state. After pressing, the pellicles look like wet cotton cloth. The water content in the pellicles was determined by weighing several (∼5) samples, taken from various regions of the pellicle, while hydrated and when dehydrated, by averaging the test results, and dividing the difference in those weights by the hydrated weight. Samples were dehydrated in an oven at 160–170 ◦ C for 5 h. The gravimetric control of the pressing process has shown that in the conditions of moderated loading, produced by the press, which we have used in our work, the water content in pressed BC pellicles is almost independent of the applied loading. It was 86 ± 2 wt%. To decrease the residual water content in pressed pellicles, a powerful press should be used, for example, a hydraulic one.

2.3.

Synthesis of composite hydrogels

Samples of BC–PAAm hydrogels were prepared using the technique of composite hydrogel material synthesis that we have developed previously (Buyanov et al., 1998, 2001). BC plates with two different water contents (∼99 or ∼86 wt%) were used as matrices for hydrogel synthesis. It should be emphasized that for the synthesis of composite hydrogels, conducted in concentrated acrylamide solutions, the use of pressed BC matrices with moderated water content is more convenient. These matrices swell easily in the reaction solutions and the BC content in the resulting hydrogels can be varied to some extent by changing the swelling duration and, as a result, the amount of acrylamide monomer which has diffused inside the BC layer. This amount was controlled gravimetrically by weighing the pellicles in the initial state and after swelling in the reaction solutions. According to our previous work

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Table 1 – Synthesis conditions, composition and properties of composite BC–PAAm (1A–6A, and 1B samples) and BC–PAAm–PAA(Na+ ) (1A-H, 4A-H, and 1B-H samples) hydrogels. Sample 1A⊥ 1Ak 2A⊥ a 2Ak a 3A⊥ 3Ak 4A⊥ 5A⊥ 6A⊥ 1B⊥ 1Bk 1A-H⊥ b 1A-Hk b 4A-H⊥ b 6A-H⊥ b 6A-Hk b PAAm

[M0 ] (wt%)

BC content (wt%)

Q (g/g)

55

4.5

5.06

55

4.5

4.93

55

8.0

5.01

55 30 10

14.2 9.2 25.0

5.30 6.82 11.2

55

2.0

3.43

55

4.5

8.22

55

14.2

10.7

10

25.0

20.9

55

0

10.8

E⊥ /Ek

E|10%–15% (MPa) 4.67 0.90 4.84 1.06 8.01 0.65 8.84 2.34 4.0 7.39 3.85 4.46 2.48 7.19 2.64 0.24 0.0088

4.9 4.6 12.3

1.92 1.80

11.0

E|25%–30% (MPa) 7.19 3.09 7.74 3.18 22.2 1.89 19.5 13.3 16.2 8.55 5.41 6.43 3.39 8.93 9.49 1.11 0.0145

E⊥ /Ek

2.3 2.4 11.7

1.58 1.90

8.55

σmax (MPa)

ε|σmax (%)

2.6 9.9 2.3 9.0 3.1 9.9 5.6 3.3 3.9 8.52 8.88 2.26 2.24 2.31 2.05 4.4 1.16

49 85 43 80 30 85 43 43 42 75 75 32 53 32 39 59 80

a The MBA concentration was 7 × 10−3 M; for all other samples it was 1.4 × 10−3 M. b Hydrolyzed ionic hydrogels were swollen and tested in 0.5 M NaCl water solution.

(Buyanov et al., 1992), practically complete (∼99%) acrylamide conversion into PAAm is ensured in our reaction conditions. Consequently, we can determine the PAAm content in the hydrogel composition from the monomer amount absorbed by the BC matrix during the swelling process. The BC contents in compositions were also determined gravimetrically, based on the weight of dry BC involved in the synthesis. By using pressed matrices containing 14 wt% of BC, a series of hydrogel samples designated as series A was prepared. Due to the variation of swelling duration of the matrices in the reaction solutions (0.5–16 h), hydrogel samples with BC content in the BC–PAAm compositions varying from 4 to 14 wt% (Table 1) were obtained at constant initial monomer concentration ([M0 ]) equal to 55 wt% (8.26 M). Synthesis of hydrogels with greater BC content was found to be impossible under these conditions. Therefore, to increase the BC content in hydrogels the initial monomer concentration was reduced to 30 or even to 10 wt% (4.37 and 1.42 M, respectively). At [M0 ] = 10 wt%, samples with BC content in BC–PAAm composition as high as 25 wt% were synthesized (Table 1). N,N0 -methylene-bis-acrylamide (MBA) was used as the crosslinking agent at a concentration of 1.4 × 10−3 M (in some cases this concentration was 5 times higher, i.e. 7 × 10−3 M). To initiate free-radical polymerization, cobalt (III) acetate was proposed in our previous work (Buyanov et al., 1992). To obtain the hydrogel compositions under study it was used at a concentration of 0.5 × 10−3 M. After impregnation in the reaction solutions, the swollen BC matrices were placed between two glass plates for 3 h to conduct the polymerization of acrylamide at ∼30 ◦ C. BC–PAAm composite hydrogels of series B were synthesized by immersing non-pressed matrices containing about 1 wt% of BC into a large amount of aqueous reaction solutions containing 55 wt% of acrylamide for 16 h. In this case, the BC matrices were shaped in the form of cylinders and the polymerization was conducted in cylindrical glass vessels of

corresponding diameter. All other synthesis conditions coincided with those used for the synthesis of hydrogels of series A. Because the BC content in non-pressed matrices is not high (∼1 wt%), its content in the polymer compositions was about 2 wt%. Despite this low BC content in composite hydrogels, their properties differ strongly from those of the PAAm hydrogels synthesized in similar conditions, as shown in Table 1. When the synthesis was over, the composite hydrogels were placed in distilled water for several days to remove low molecular weight components and to let the gels swell. To prepare ionic hydrogels the method of alkaline hydrolysis of amide groups was used. For this purpose, BC–PAAm hydrogels were placed for 48 h in 0.5 M NaOH solution at 23 ◦ C, then in water. Under these conditions the PAAm chains converted into copolymer of PAAm with polyacrylate of alkali metal (in our case sodium). These BC–PAAm–PAA(Na+ ) hydrogels (where PAA(Na+ ) is sodium polyacrylate) are indexed with H in Table 1. The extent of hydrolysis calculated using the data of elemental analysis was 0.4–0.5. The BC content in these gels was the same as in the original BC–PAAm hydrogel. The degree of equilibrium hydrogel swelling was determined by using the equation Q = (Ws − Wd )/Wd , where Ws and Wd are the weights of swollen and dry polymer compositions (BC–PAAm or BC–PAAm–PAA(Na+ )), respectively.

2.4.

Mechanical tests

Cylindrically shaped specimens (5–10 mm diameter and 3–10 mm height) for compression tests (single-shot compression and cycling) were cut from the hydrogel sheets in two directions: the cylinder axis oriented in the direction parallel or perpendicular to the horizontal surfaces of the hydrogel samples (Fig. 1). These surfaces correspond to the top and bottom

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Table 2 – Anisotropic swelling behavior of the hydrogel samples of series A.

Fig. 1 – BC–PAAm hydrogel plates from which the samples were cut out in two directions. a: The direction of cutting is perpendicular to the top and bottom surfaces of the BC matrix; b: the direction of cutting is parallel to these surfaces; c: typical BC–PAAm hydrogel specimen for mechanical tests (this specimen was cut out in the direction perpendicular to the surfaces of the BC matrix).

Fig. 2 – Unconfined compression test of BC–PAAm hydrogel composition.

surfaces of the BC matrix at which the BC growth begins and stops during its cultivation. Stainless steel cylindrical moulds were used to cut these specimens. Special care was taken to obtain cylinders with the flat ends parallel to each other and to ensure the perpendicularity of these flat ends to the cylinder vertical axis. The mechanical tests were carried out with a UTS 10 mechanical test system (Germany) in unconfined compression between two impermeable platens (Fig. 2). The deformation speed was usually 1 mm/min for single compression tests and 10 mm/min for tests in the cycling mode. To avoid the damage of the test equipment the maximal value of the specimens’ deformation in the single compression tests did not exceed 80%–85% (depending upon the initial height of the specimen). Long-term cycling tests of the gel samples were carried out in water medium to prevent the drying of the gel. The data reported for each type of hydrogel is the average of results obtained with five specimens. Compression

Sample Swelling medium

Hydrogel plate dimensions, relative units l1 l2 h

Q (g/g)

4A 5A 4A 5A 4A 5A 4A-H 5A-H 4A-H 5A-H 4A-H 5A-H

1.00 1.00 0.977 0.970 0.968 0.960 1.03 1.06 1.03 1.07 1.05 1.18

5.29 6.82 0.98 1.50 0.27 0.35 10.7 13.4 12.6 15.0 16.7 21.1

H2 O H2 O 50% EtOH 50% EtOH 90% EtOH 90% EtOH 0.50 M NaCl 0.50 M NaCl 0.17 M NaCl 0.17 M NaCl 0.0001 M NaCl 0.0001 M NaCl

1.00 1.00 0.971 0.980 0.970 0.965 1.02 1.02 1.02 1.06 1.04 1.09

1.00 1.00 0.479 0.411 0.355 0.284 2.03 1.83 2.00 2.08 2.56 2.75

moduli and ultimate stresses of materials under study were calculated using the initial cross-sections of uncompressed specimens.

3.

Results

3.1.

Swelling behavior of composite hydrogels

It was found that, for the series A hydrogel plate-like samples, only an increase in their thickness takes place during the swelling process. The other sample dimensions (length and width of plates) remain approximately the same. It was nearly impossible to get quantitative data on the increase of linear dimensions of the samples during swelling (in the beginning of this process the swelling speed is higher at the peripheral regions of the gel plates, which causes some deformation of these plates). This is why only data on equilibrium swelling of the samples in water–ethanol mixtures of various concentrations are presented in Table 2. The dimensions of the samples are expressed in relative units — as ratios of the dimensions of samples, swollen in equilibrium conditions in water–ethanol mixtures, to those of the same samples, swollen in water. The degree of equilibrium swelling Q decreases (deswelling) with the increase of ethanol concentration in the mixtures because ethanol is the precipitant for PAAm. As can be seen from Table 2, the thickness of samples (h) decreases by approximately a factor of three during deswelling (the ethanol concentration increases from 0% to 90%) while the other dimensions (l1 and l2 ) decrease by no more than 4%. To investigate the influence of the polyelectrolyte effect on the properties of the composite hydrogels, ionic carboxylate groups were introduced into them via basic hydrolysis of amide groups. Such a procedure carried out in soft conditions does not cause any destruction of polymer network chains and, thus, their topology does not change. Similarly to other ionic hydrogels, the degree of equilibrium swelling exhibits strong dependence on the concentration of low molecular weight electrolyte. The electrolyte (NaCl) concentration in these experiments was reduced gradually from 0.5 M to 10−4 M, because the most significant change of the equilibrium swelling degree was usually observed in this range of

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Table 3 – Isotropic swelling behavior of the hydrogel samples of series B. Sample

Swelling medium

Hydrogel cylinder Q dimensions, relative units (g/g) d h

1B 1B 1B 1B-H 1B-H 1B-H 1B-H

H2 O 50% EtOH 90% EtOH 0.17 M NaCl 0.01 M NaCl 0.0001 M NaCl 0.17 M NaCl

1.00 0.875 0.697 1.344 2.469 3.094 1.938

1.00 0.857 0.644 1.286 2.143 2.643 1.785

3.43 2.02 0.846 9.77 49.2 87.7 25.0

concentrations (Buyanov et al., 1992). The data in Table 2 show that the value of Q of ionic hydrogels of series A is maximal in 10−4 M solution of NaCl and is approximately thrice the Q value of the nonionic analogues. This is the result of the Donnan effect and the effect of repulsion of the charges fixed on polymer chains (Flory, 1953). The ionic hydrogels in this series also demonstrate anisotropic swelling. The thickness of the corresponding hydrogel plates is 2.6–2.8 times larger than that in nonionic gels (Table 2). In contrast to the anisotropic behavior of series A samples, the nonionic BC–PAAm samples of series B exhibit isotropic behavior on swelling in water–ethanol mixtures. The variations of the diameter (d) and height (h) of cylindrical samples are approximately equal (Table 3). The ionic hydrogels of series B also swell isotropically if the polyelectrolyte effect is substantially suppressed (down to NaCl concentration 1 wt%). The decrease of NaCl concentration to 0.01 M and further to 10−4 M leads to the increase in swelling values that are more substantial than those in samples of series A, and some anisotropy of the swelling process was registered. At the same time some qualitative changes of these hydrogels’ properties were observed, namely the sharp loss of elasticity and hardness. Additionally, the hydrogels changed visually from being milky-white into being semi-transparent. It is obvious that the BC matrix in this case does not hold the pressure produced by the polyelectrolyte swelling of PAAm–PAA(Na+ ) network and that matrix fragmentation takes place. As has already been noted, the BC content in series B hydrogels is low (∼2 wt%). The degree of swelling of series B hydrogels swollen sequentially in 10−4 M NaCl solution and then in more concentrated NaCl solution did not reach the same level as in a one-step cycle of swelling in concentrated NaCl solution. The data summarized in Table 3 show that a single-step swelling in 1% NaCl results in the Q value of 9.8 g/g, whereas the same hydrogel, subjected sequentially to strong swelling in dilute NaCl solution ([NaCl] = 10−4 M) with Q value up to 88 g/g and then in 1% NaCl solution, demonstrates a resulting swelling approximately twice as high (25 g/g) as after a single-step swelling. This effect was not observed in series A hydrogels: the cycles of swelling–deswelling were completely reversible for them.

3.2.

Mechanical behavior of composite hydrogels

Compression stress–strain curves for specimens of series A and B are presented in Fig. 3(a) and (b), respectively. The essentially different shapes of the stress–strain curves for

Fig. 3 – Anisotropic mechanical behavior of BC–PAAm composite hydrogels of series A (a) and B (b), containing 4.5 (a) and 2.0 (b) wt% of BC (in BC–PAAm composition). 1: compression in the direction parallel to the top and bottom surfaces of the BC matrix; 2: compression in the perpendicular direction.

samples cut out in various directions are evidenced, and it should be stressed that this difference is more pronounced while analyzing the specimens of series A. The resilience of specimens cut in the direction perpendicular to the top and bottom surfaces of the BC matrix is substantially higher than that of the specimens cut in the parallel direction. In other words the well defined anisotropy of mechanical properties of such hydrogels was seen. To characterize the stiffness of such hydrogel compositions, the usual procedure of the compressional modulus calculation as the initial slope of the stress–strain curve cannot be used. The reason behind this is the pronounced nonlinearity of the stress–deformation dependence: the dσ/dε value, where σ stands for stress and ε for deformation, demonstrates the progressive rise in a broad range of deformations. The modulus calculation using the Mooney–Rivlin equation gives similar results. For this reason we have selected two regions of the compression curves to characterize the hydrogel stiffness, namely these from 10% to 15% and from 25% to 30% of compressive deformation. The mean slope values of the compression curves at these two regions were used as the measure of the hydrogels’ elasticity (E10%–15% and E25%–30% , respectively).

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Fig. 4 – Schematic diagram of crack formation during hydrogel compression.

The following values are listed in Table 1, characterizing the gel compositions under study: – E10%–15% and E25%–30% , – the compressive stresses at maximal compression (at specimen failure or at 80%–85% compression) σmax , – the deformation values ε|σmax corresponding to σmax . The data from specimens cut in the two directions (parallel and perpendicular to the surface of the BC matrix) are denoted by “k” and “⊥” subscripts, respectively. The E⊥ values in series A hydrogels are 5–12 times higher than the Ek values (in the compression range of 10%–15%). For the specimens of series B the anisotropy is much weaker: the E⊥ value is only ∼2 times higher than Ek in the same deformation range. The presence of a maximum is ubiquitous for the stress– compression curves obtained from specimens cut in the direction perpendicular to the surface of the BC matrix (Fig. 3). The gradual fall of the slope of such curves registered in natural cartilage specimens was treated in Kerin et al. (1998) as one of the symptoms of the material destruction and as a criterion for mechanical failure. Our compression tests show that the deformation of series A specimens up to the region of the stress maximum leads to the formation of micro-cracks. As a rule these were radial cracks: their formation took place at the border of the specimen’s faces (Fig. 4). Macroscopic failure of the specimens was not observed just at this moment due to the high elasticity of the PAAm network, and the samples kept their cylindrical shape, but these cracks grow along with the further compression of the sample until its fragmentation (Fig. 3(a), curve 2). Specimens cut in the direction parallel to the surface of the BC matrix exhibit a monotonic rise. No symptoms of the specimen’s cracking were detected up to the deformation levels of 80%. The specimens of series B, cut in the direction perpendicular to the surface of the BC matrix, are more elastic compared to those of series A. No cracks were detected visually while deforming these specimens beyond the local stress maximum (up to 80%). The dependence of the compression behavior of series A specimens cut in the direction perpendicular to the surface of the BC matrix upon the BC concentration in these hydrogel

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Fig. 5 – Stress–strain curves in compression of series A specimens with different BC concentrations, cut in the direction perpendicular to the surface of the BC matrix: the BC concentration in the polymer composition is 0 (1), 4.0 (2), 8.0 (3) and 14 (4) wt%.

compositions is presented in Fig. 5. The increase of BC content in the polymer composition from 4 to 14 wt% results in the increase of both the modulus and strength values by a factor of two (Table 1) and to the significant variations of the shape of the stress–strain curves. The hydrogel compositions with elevated BC content demonstrate the tendency to a shift of the stress maximum toward the depressed deformations. However, for the material containing 14% of BC in the BC–PAAm composition this maximum is situated at a deformation that is substantially more than 30%. In other words, the destruction of these hydrogels begins at a deformation substantially more than the maximal deformation of the joint’s cartilage in a human body (Stammen et al., 2001). The monocomponent PAAm hydrogel is substantially less rigid than BC–PAAm compositions (Table 1, Fig. 5). The mechanical properties of the specimens cut in the direction parallel to the surface of the BC matrix demonstrate less pronounced dependence upon the BC content in the hydrogel composition (Table 1). Moreover, some decrease of the hydrogels’ compressive modulus takes place along with the increase of the BC content. The mechanical properties of the similar gel specimen containing 14 wt% of BC were not measured since the hydrogel layer was too thin to prepare such a specimen. The increase of the BC concentration in BC–PAAm composition up to 25 wt%, ensured by decreasing the acrylamide monomer concentration at the stage of PAAm synthesis to 10 wt%, did not lead to an increase of the hydrogel stiffness. But in this case we should take into account that the degree of equilibrium swelling of this specimen is about twice higher than the Q value of hydrogels synthesized at [M0 ] = 55 wt% (Table 1). This result is in agreement with our previous data, obtained for hydrogels of PAAm, crosslinked by the macromolecular polyfunctional agents, such as allyl ethers of water-soluble cellulose derivatives (Buyanov et al., 1990). Leveling down the initial monomer concentration in hydrogel synthesis leads to a reduction of the crosslink density and, as a result, to an increase of the degree of equilibrium swelling and to a reduction of the modulus. To carry out a more precise analysis of the experimental data we should

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Fig. 6 – Cyclic compression–unloading curves of BC–PAAm hydrogel samples of series A, cut in the direction perpendicular to the surface of the BC matrix (BC content in polymer composition = 8.0 wt%). 1: 1-st cycle; 2: 700-th cycle; 3: 2000-th cycle; 4: cycle after 2000 cycles + one day’s relaxation. For convenient data presentation each consecutive cyclic curve was shifted by 5% against the previous one along the deformation axis.

take into account the dependence of the modulus of elasticity on the degree of equilibrium swelling. For PAAm and PAAm–PAA(Na+ ) hydrogels synthesized using macromolecular polyfunctional crosslinkers these dependences can be expressed via a scaling-type equation: E = A(1/Q)m , where m is 2.63 for nonionic hydrogels and 2.41 for ionic ones swollen in 1 M NaCl solution (Buyanov et al., 1992). For hydrogel compositions BC–PAAm these data are so far unavailable. Hydrolyzed BC–PAAm–PAA(Na+ ) hydrogels also exhibit anisotropy of mechanical properties. Their degree of equilibrium swelling substantially exceeds that of the original BC–PAAm hydrogels (Table 1). The extent of this anisotropy depends upon the synthesis conditions and hydrogel composition. All hydrogels studied in our work (except composition 2A) were synthesized at constant and moderate concentration of the crosslinking agent. We have previously used the same range of crosslinking agent concentration for the synthesis of PAAm gels (Buyanov et al., 1992). It should be emphasized that the concentration of crosslinking agent can be varied broadly enough without any marked variations of characteristics of synthesized materials. Table 1 shows that the increase of the crosslinking agent concentration by a factor of five does not result in any substantial change of hydrogel properties (compositions 1A and 2A). This result can be explained by the fact that the degree of equilibrium swelling of mono-component PAAm hydrogels, synthesized even at the relatively high concentrations of crosslinking agent, is substantially higher than that of the hydrogel compositions studied in this work. The crosslink centers of the PAAm network in BC–PAAm hydrogels are practically unused and the extent of the degree of equilibrium swelling is restricted by the BC matrix.

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Fig. 7 – Cyclic compression–unloading curves of BC–PAAm hydrogel samples of series A, cut in the direction parallel to the surface of the BC matrix (BC content in polymer composition = 8.0 wt%). 1: 1-st cycle; 2: 2000-th cycle.

The variations of the mechanical properties of the hydrogels of series A under the action of cyclic compressive stresses were tested by cycling up to 30% compression. It has already been pointed out that 30% compression is less than the critical point at which the onset of mechanical failure of these hydrogels takes place. Fig. 6 represents a set of cyclic stress–strain diagrams registered at different stages of fatigue tests of a series A hydrogel specimen (compressions up to 30% with subsequent decompressions without any relaxation periods in the unloaded state between cycles) along with an additional curve which demonstrates the behavior of the specimen subjected previously to 2000 compression cycles in the conditions described above, and then to relaxation in an unloaded state for 24 h. For convenient data presentation each consecutive cyclic curve was shifted by 5% along the deformation axis. All curves presented in Fig. 6 demonstrate a definite hysteresis, but after being subjected to 2000 compression cycles no significant irreversible changes of the specimen’s appearance and behavior were detected. The maximum stress during the cycle slightly reduces after 2000 compression cycles but the one day relaxation period of the specimen leads to a partial restoration of this value. The cyclic compression curves for the specimens cut in the direction parallel to the surface of the BC matrix are presented at Fig. 7. For these specimens only very modest hysteresis can be seen, and they are more resistant to the action of cyclic compression stresses as compared to the specimens cut in the perpendicular direction. In fact after 2000 compression cycles some increase of the maximum load at cycle still takes place. The specimens of series B cut in the perpendicular direction were subjected to fatigue tests up to 6000 cycles because their behavior in the cyclic test conditions was more stable than that of the specimens of series A: after 2000 compression cycles an increase of the maximal load at cycle was registered (Fig. 8). As can be seen in this figure, during further fatigue tests up to 4000–6000 cycles some depression of the maximal load at cycle takes place, but the value of this depression is not large at all.

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Fig. 8 – Cyclic compression–unloading curves of BC–PAAm hydrogel samples of series B, cut in the direction perpendicular to the surface of the BC matrix (BC content in polymer composition = 2.0 wt%). 1: 1-st cycle; 2: 2000-th cycle; 3: 4000-th cycle; 4: 6000-th cycle.

Since all cyclic compression tests were carried out at rather high loading–unloading speed (10 mm/min), while the compression speed for all single compression test was 10 times less (1 mm/min), it was important to clarify the action of the deformation speed on the character of the mechanical behavior of the hydrogels. Fig. 9 demonstrates that the 10 times increase of the deformation speed does not cause any significant variations of cyclic stress–strain curve shape.

4.

Discussion

As we have already stressed above, the monocomponent PAAm hydrogels demonstrate isotropic swelling behavior. Their mechanical properties also do not depend on the direction (vertical or horizontal) in which we have cut the specimen from the hydrogel plate. In other words, these properties do not depend on the direction in which we deform the specimen. The anisotropic properties of BC–PAAm hydrogel compositions originate in the specific features of the BC matrix structure and in the peculiarities of matrix preparation method. The comparison of the series A and B specimens’ swelling (Tables 2 and 3) shows that the anisotropic behavior of series A samples originates in the pre-pressing of BC matrices before the synthesis: the reason for this effect is the specific method of matrix preparation. The matrices subjected to pressing “remember” their initial equilibrium swollen state because of their three-dimensional network structure. In bi-component hydrogel compositions these matrices let the PAAm chains swell easily in the direction of the matrix pre-compression (in the direction perpendicular to the surface of the BC matrix). The swelling of PAAm network in the horizontal plane (in the direction parallel to the surface of the BC matrix) is restricted by the resilience of the BC network. On another hand, the BC matrices, which were not subjected to the prepressing, ensure the isotropic swelling of the PAAm chains in all directions (specimens of series B), because in this case the strain ability of the BC network is equal in all these directions. Another situation was found while analyzing the mechanical properties of the hydrogels under study. The pronounced

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Fig. 9 – Two sets of cyclic compression–unloading curves of a series A specimen cut in the direction perpendicular to the surface of the BC matrix (BC content in polymer composition = 2.0 wt%). The deformation speed was 1: 1 mm/min; 2: 10 mm/min.

anisotropy of mechanical properties was found in specimens of both A and B series. The moduli of specimens of both series cut in the direction perpendicular to the surface of the BC matrix are substantially higher than those of the specimens cut in the parallel direction. Hence this effect cannot be ascribed only to the peculiarities of the specimen preparation process. This reason can only partially affect the anisotropy of the mechanical properties of series A hydrogels. In fact, the anisotropy of the mechanical behavior is more pronounced in this series of hydrogels than in series B. This difference originates obviously in the anisotropic swelling of the former gels that causes the elongation of PAAm chains along the axis perpendicular to the surface of the plate. This effect may lead to the increase in the hydrogel’s elastic modulus in the direction of macro-chain elongation. But only the specific features of the structure of the gels under study can be put forward as the principal reason for the anisotropy of BC–PAAm hydrogels’ mechanical behavior. This anisotropy can be explained by the different structure of the BC matrix, observed in two directions: parallel and normal to its surface. In the direction perpendicular to the surface, the reinforcing action of the BC matrix upon the PAAm network is larger than in the parallel direction. Only this reason can explain the anisotropy of mechanical behavior of series B hydrogels. BC has a unique three-dimensional structure in the swollen state, formed by the micro-fibrillar tapes and stabilized by H-bonds (Czaja et al., 2007). According to the data of Haigh et al. (1973) these micro-fibrils may form some local structures, such as bands, parts of spherolites, or threadlike structures. A high level of structural organization of swollen BC was shown in Thompson et al. (1988) using an SEM method. Some tunnel-like lacunae were detected in the crosssections of the BC gel sample, normal to the surface of the pellicle. These tunnels of the effective transversal dimension at about 7 µm are characterized by a regular (with the same intervals) arrangement between micro-fibril bundles. All these tunnels and bundles are oriented in only one direction: along the axis perpendicular to the surface of the BC pellicle. These

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Fig. 10 – Scheme of tunnel-like structural organization of swollen BC, according to Thompson et al. (1988). a: tunnel-like lacunae detected in the cross-sections of a BC gel sample normal to the surface of the pellicle; b: tunnel “sidewalls”, formed by the oriented cellulose fibrils.

tunnels were also detected at the horizontal cross-sections of the BC layer, but in substantially lower concentration. The tunnels detected at these horizontal cross-sections have no preferential orientation — they are oriented in different directions. These tunnels are formed by oriented spiral-like cellulose fibrils. Thompson supposed that they were formed by Acetobacter bacteria as the passageways along their movement during cellulose formation. The BC structure reported in this paper is rather unusual. As was pointed out in Ross et al. (1991), the morphology observed by Thompson et al. (1988) may indicate the existence of a well organized structure in BC that was not previously suspected. Our data concerning anisotropic mechanical behavior of BC–PAAm hydrogels are in good agreement with such tunnel structure of the BC matrix, in which the tunnels are oriented predominantly along the direction normal to its surface. The scheme of this structure is given in Fig. 10. The sizable free volume, formed by these tunnels, would be filled by the polyacrylamide network during the synthesis of the hydrogel composition. This network has a depressed stiffness. As a result, the compression of this composite material along the direction parallel to the surface of the BC matrix will result predominantly in the compression of these comparatively soft hydrogel regions, and so the effective compression modulus in this direction will be substantially less than that measured in the perpendicular direction. The tunnels’ “sidewalls”, formed by the oriented cellulose fibrils, can produce a substantial reinforcement of PAAm network just in the direction perpendicular to the BC matrix surface. Exactly this type of mechanical behavior was detected in our experiments. On the other hand, the tunnel-like spiral structure of BC, reported by Thompson et al. (1988), is in agreement with the anisotropic swelling behavior of BC–PAAm hydrogels. Pressing of the BC pellicles at the initial stage of the hydrogel synthesis causes the compression and shrinkage of these spirals. The swelling of BC–PAAm composite hydrogels leads to their

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reshaping, and to the restoration of initial shapes of these spirals. We do not pretend to the close accordance of the results obtained with namely this type of the structural organization of BC. The structure of BC can depend on the type of microorganism strain used for its synthesis (Czaja et al., 2007). Moreover, a different structure was detected in different regions of the BC pellicles. The structure of a gel-film’s layer near the surface differs from that of the deep layers (Czaja et al., 2007). All these structural features can affect the resulting structure of the BC-PAAm composite hydrogel. To make more precise conclusions we should obtain additional information concerning the structure of the hydrogel compositions under consideration and of the initial BC matrices. We plan to carry out this work in the next stage of our investigations. We should emphasize that a high level of mechanical properties of BC–PAAm hydrogel compositions is ensured. They are very close to the properties of natural cartilage. In fact, according to the data of Kerin et al. (1998), the value of compressive modulus of a human’s joint cartilage increases from 1.9 to 14 MPa along with the increase in the compression level. Mechanical loads, which act on natural cartilage in a human’s body, can attain a level as high as 5 MPa (Covert et al., 2003). It can be seen (Table 1) that some BC–PAAm hydrogels satisfy this feature well. The broad variations of the modulus of these compositions can be ensured by changing the synthesis conditions, and the stiffness values of some compositions obtained in our work are very close to that of natural cartilages. The further improvement of the mechanical properties of composite BC–PAAm hydrogels can be ensured both by chemical modification of the cellulose matrix, and by the introduction of nanoparticles (for example montmorillonite) into this polymer composition (Gofman et al., 2008). By using these approaches we obtained an increase of the hydrogel’s compressive strength up to 16 MPa. The hydrolyzed BC–PAAm–PAA(Na+ ) hydrogels demonstrate a depressed level of mechanical properties. But these gels have a very valuable feature. Their behavior is sensitive to the variations of such conditions as pH, ionic strength and composition of the solution surrounding the gel specimen, and to the action of an electric field. The modest variations of these parameters can cause substantial changes of the volume of these gels. In our work we have not studied systematically the actions of these factors on the hydrolyzed gel’s behavior, and the actions of pH value and of electric current were not examined at all. But there are a lot of already published data concerning these effects in different systems with chemical compositions close to that of our hydrogels (Calvert, 2004; Kaneko et al., 2002). When the ionic forms of hydrogel compositions are used as gel actuators, the anisotropy of their properties can be a very advantageous feature, especially the anisotropic swelling effect. In fact, if we use the hydrogel actuator to produce the displacement of some parts of the device along one axis, then the gel’s swelling along the perpendicular axis is undesirable. In such mechanisms the use of a gel which swells anisotropically is more effective than the employment of gels demonstrating isotropic swelling.

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5.

Conclusions

The synthesis of PAAm networks inside BC matrices leads to the formation of high strength composite BC–PAAm hydrogels which can show anisotropic behavior on both swelling and deformation. The swelling can be isotropic or anisotropic depending on the method of BC matrix preparation. The anisotropy of mechanical properties is detected in both series A and B hydrogels. This effect can be explained only by the formation of ordered regions in the BC structure. The anisotropic behavior encountered in our work can be ascribed to the existence of the tunnel-like oriented structure of BC reported by Thompson et al. (1988). But to clarify the origins of these effects completely work should be carried out to study the structural features of both the BC used in our experiments and of composite hydrogels synthesized based on this BC. REFERENCES

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