Viscoelasticity of Biomaterials - American Chemical Society

Chapter 14

Effect of Water on the M a i n Chain Motion of Polysaccharide Hydrogels H . Yoshida , T. Hatakeyama , and Hyoe Hatakeyama Downloaded by STANFORD UNIV GREEN LIBR on October 12, 2012 | http://pubs.acs.org Publication Date: May 8, 1992 | doi: 10.1021/bk-1992-0489.ch014

1

2

3

Department of Industrial Chemistry, Tokyo Metropolitan University, Minami-ohsawa, Hachiohji-shi, Tokyo 192-03, Japan Research Institute for Polymers and Textiles and Industrial Products Research Institute, Tsukuba, Ibaraki 305, Japan 1

2

3

The main chain motion of hyaluronic acid, xanthan and pullulan with various water contents ranging from 0 to 3 (grams of water per gram of dry polysaccharide) was investigated by differential scanning calorimetry ( D S C ) . Water in the polysaccharide system was classified into three types: non-freezing, freezing bound, and free wa ter. Hydrogel derived from polysaccharide, hyaluronic acid, and xanthan contained a large amount of freezing bound water. The water-polysaccharide systems formed the glassy state by cooling from 300°Κ to 150°K at the rate of 10°K/min. The glass transition temperature of the system was influenced by non-freezing water and freezing bound water. A part of the freezing bound wa ter in hydrogels formed amorphous ice. As a result of cooperative motion of the polysaccharide, the glass tran sition of these systems absorbed non-freezing water and the amorphous ice. Hydrogels have become increasingly important in industrial and medical fields, such as food processing, delivery systems, manufacture of artificial biological tissues, civil engineering, etc. The phase transition of gels i n duced by thermal and electrical stimulations, and also the gelation mech anism, have attracted much attention in both the theoretical and applied fields (1-4). Biological gels show better mechanical properties than syn thetic gels, since biological gels are constituted of rigid molecules such as proteins which have helix coil conformation, or polysaccharides which con sist of glucan or glucosamino glycan having /?-glycoside linkages. Because these rigid biomolecules show high glass transition temperatures in the dry state, it is difficult to observe the main chain motions below those ther mal decomposition temperatures. Not only for synthetic polymers but also 0097-6156/92AM89-0217$06.00/0 © 1992 American Chemical Society

In Viscoelasticity of Biomaterials; Glasser, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by STANFORD UNIV GREEN LIBR on October 12, 2012 | http://pubs.acs.org Publication Date: May 8, 1992 | doi: 10.1021/bk-1992-0489.ch014

218

VISCOELASTICITY OF BIOMATERIALS

biopolymers, the main chain motion affects their mechanical and physical properties and also their functionality. It is well known that the main chain motion of hydrophilic polymers depends on the interaction between water and polymers. In order to i n vestigate the physical properties and functionality of hydrogels, it is i m portant to consider the molecular motion of polysaccharide and water in hydrogels. We have reported the structural change of water in the waterpolyelectrolyte system (5), the water-polysaccharide systems (6,7), and hydrogels (8-11) using differential scanning calorimetry (DSC) and nuclear magnetic resonance spectrometry ( N M R ) . Most of the water-polyelectrolyte systems formed the liquid crystalline state at high concentrations due to the electrostatic interaction among the hydrated ions (5-7, 11). From D S C and N M R studies, water molecules interacting with polysaccharide molecules played an important role in forming the liquid crystalline state i n these systems. Several kinds of water-polysaccharide systems form both a liquid crystalline phase and a hydrogel. It is expected that the higher structures, such as liquid crystals or junction zones of hydrogels, influence the main chain motion of polysaccharides. In this study, three kinds of polysaccharides were used to investigate the effect of water on the main chain motion of polysaccharides. Hyaluronic acid and xanthan form hydrogel; xanthan also forms the liquid crystal phase. The water-pullulan system forms neither liquid crystal nor hydrogel. The effect of higher structures on the molecular motion of polysaccharides and water was also discussed. Experimental Materials. Three kinds of polysaccharides—hyaluronic acid, xanthan and pullulan—were used in this study. Hyaluronic acid and pullulan were obtained from Wako Pure Chemical Industries, L t d . , Tokyo, Japan. Xanthan used was Kelzan, supplied by Kelco Co., L t d . Hyaluronic acid and xanthan were purified and neutralized as described elsewhere (9). The sodium content per tetraoligosaccharide unit of hyaluronic acid and xanthan was 2.5 and 2.6 mole/mole, respectively. Apparatus. A Seiko differential scanning calorimeter Model D S C 200 connected to a Seiko Thermal Analysis System Model S S C 5000 was used. The scanning rate was 10°K/min. The sample weight used was 2-5 mg, depending on water content. The sample was weighed and dissolved i n distilled water in a D S C aluminum sample vessel. After the sample had completely dissolved, the water was slowly evaporated in order to obtain a sample with the predetermined water content ( W ) . After sealing the D S C sample vessel, all samples were kept at room temperature for a few days in order to homogenize them. The W was defined as follows: c

c

Wc(g/g) =

amount of water/g dry sample weight/g


(1)

14. Y O S H I D A E T A L .

Main Chain Motion of Polysaccharide Hydrogels 219

The dry sample weight was determined by the weight after heating to 460° Κ at which the endothermic peak due to the evaporation of water disappeared (12).


Results and Discussion Type of Water in Hydrogels. The state of water in various polymers, includ ing polymers in aqueous solutions, have been studied using thermal, dielectrical and spectroscopic analysis (13-16). From the value of their diffusion coefficients observed by N M R , water molecules i n aqueous protein solutions were classified into three categories (15). Water molecules interacting with biomolecules are called trapped water, solid water, bound water, interfacial water, etc. In previous papers (5-12, 17-19), water molecules which showed first-order phase transition, such as crystallization and melting, were called freezing water, and those molecules which never crystallized even at 130° Κ were called non-freezing water. In the case of hydrophilic polymers insol uble in water, freezing water was classified into two types: free water and bound water (17). These two types of water were defined by the crystalliza tion temperature on cooling at a given rate. A t a cooling rate of 10°K/min, free water crystallized at 255°K and bound water at around 230°K. These values are independent of W (18,19). Three types of water in hydrogels of the water-polysaccharide system were found by D S C (20). Hydrogels and the water-polysaccharide system contain much freezing bound water, compared with other hydrophilic poly mers. As the crystallization temperature of bound water in these systems changed continuously from 230°K to 250°K on cooling, it is difficult to classify freezing water into bound water and free water only by a D S C cooling curve (20). Figure 1 shows the schematic D S C curves for three types of water—non-freezing water, freezing bound water, and free water— undergoing cooling and heating at 10°K/min. Free water showed a sharp exothermic peak due to crystallization on cooling and a sharp endothermic peak due to melting on heating. The starting temperature of cryst allization on cooling ( T ) of free water in hy drogels was observed at temperatures between 254 and 256°K, depending on its thermal histories. The starting temperature of melting ( T ) of free water was close to 273°K, and the melting peak temperature ( T ) was the same as that of pure water. The values of T - and T of free water scarcely depended on W and the thermal history of the system. O n the other hand, freezing bound water shows a broad exothermic peak which can be attributed to the slow rate of crystallization and lower T on cool ing. From the isothermal crystallization analysis of free and freezing bound water, the crystallization rate of freezing bound water was 10 times slower than that of free water (21). For freezing bound water, T - and T were much lower than those of free water. Both temperatures, T - and T of freezing bound water, depended on W and the thermal history of the sys tem. Non-freezing water showed no first-order phase transition either on cooling or heating, but showed glass transition. From the melting enthalpy of water, the freezing water content ( W / ) c

c

m t

m

m t

m

c

c

m t

m

m i

c


m

220

VISCOELASTICITY O F BIOMATERIALS

which contained both freezing bound and free water was calculated as shown in the following equation: amount of freezing water/g

rrr / / \ /

(

g

/

g

)

~

dry sample weight/g

(

.

(

}

B y the subtraction of W / from W , the non-freezing water content ( W / ) was also calculated. The changes i n W / for the water-polysaccharide systems are shown in Figure 2. Only non-freezing water existed i n the W range below 0.5 for all polysaccharide systems. W i t h increasing W , W / approached a constant value which depended on the type of polysaccharide. From the constant value of W / observed i n the W range above 0.5, the number of moles of non-freezing water which interacted strongly with ionic groups or polar groups of polysaccharides was calculated. Sodium ions in the water-xanthan and the water-hyaluronic acid systems accompanied about 7 moles of non-freezing water in the hydration shell. In the W range above 0.5, the hyaluronic acid system showed the minimum value of W / . It may be appropriate to consider that the minimum value of W / was caused by the overlap of the hydration shell (22). In the water-pullunan system, each hydroxy group of pullulan bonded to one water molecule as non-freezing water. Figure 3 shows the D S C heating curves of polysaccharides having the same W (ca. 0.6). A l l polysaccharide systems contained both nonfreezing and bound water. The D S C curves of hyaluronic acid show glass transition temperature (T^), an exothermic peak temperature due to coldcrystallization of water ( T ) , and an endothermic peak temperature ( T ) due to the melting of water. In addition to T^, T and T , the xanthan system shows the transition from the liquid crystalline state to the isotropic liquid state (11). T * shows the peak temperature of this transition. The heat of liquid crystalline transition of the xanthan system was larger than that of the other lyotropic liquid crystals (23). The liquid crystalline state of the xanthan system differs from the cholesteric type liquid crystal which was observed i n the case of the hydroxypropyl cellulose system (24,25). The D S C curve of the water-pullulan system shows T^, T and T as seen i n Figure 3. The exotherm started immediately after T^ i n the D S C heating curve of the pullulan system. Despite the same W , the values of T^, T and T are different in samples. The T values suggested that each polysaccharide system had different molecular mobility of main chain even at the same W . A s can be seen i n Figure 2, each polysaccharide system had different amounts of non-freezing water and freezing bound water. The lower T suggested that the water-hyaluronic acid and the water-xanthan system contained a large amount of freezing bound water even at the same w . c

n

n

c


c

n

n

c

c

n

n

c

c c

m

c c

m

c c

m

c

c c

g

m

c

m

c

Glass Transition of the Water-Poly saccharide Systems. Figure 4 shows the W dependence on T^ of the water-hyaluronic acid system. The T^'s were observed for the sample which was prepared by cooling from 300° Κ to 150°K at 10°K/min. In the low W range, T decreased rapidly to the c

c

g


14.


YOSHIDA ET AL.


Je

free water

freezing bound water

non-freezing water Figure 1. Schematic D S C cooling and heating curves of non-freezing, freez ing bound and free water i n hydrogel. T is the starting temperature of crystallization on cooling, T - and T are the starting temperature and the peak temperature of melting on heating. c

m t

m

1.0k-

A

Β 0.5

ί-

jf-

c

•J 0.5

15

1.0 Wc

/

20

g.g"

1

Figure 2. Relationship between non-freezing water content ( W / ) and water content ( W ) for the water-hyaluronic acid system ( A ) , the waterxanthan system (B), and the water-pullulan system ( C ) . n

c



222


7 /

Κ

Figure 3. D S C heating curves of the water-hyaluronic acid system (A), the water-xanthan system (B), and the water-pullulan system (C) having the same W . W was about 0.6. T^, T , T , and T * are the glass transition temperature, the cold crystallization temperature of amorphous ice, melting temperature of regular ice, and transition temperature from the liquid crystalline state to the isotropic liquid state, respectively. c

c

c c

m




minimum value with increasing W . After passing the minimum value, increased gradually and approached the constant value with increasing W . Not only the water-hyaluronic acid system, but also other waterpolysaccharide systems, showed a similar W dependency on T^. Each polysaccharide showed a different value of W at which the minimum was observed. In order to discuss the effect of water on T^, the W range was divided into four regions as shown i n Figure 4. Each region was defined in terms of type of water in the system. c

c

c

c


c

Molecular Motion in Region I. In region I, the system which contained only non-freezing water showed glass transition only i n the heating D S C curve. In the completely dry state, glass transition of all polysaccharides was not observed because overlapped with their decomposition tem perature. For all polysaccharide systems, decreased markedly with the addition of water due to the plasticizing effect. The main chain motion of the polysaccharide was restricted by a large number of hydrogen bonds in the dry state. When the system absorbed a small amount of water, some water molecules broke the hydrogen bonds i n the same way as the hydrogen bonds of hydrophilic polymers were broken (19). Molecular Motion in Regions II and III. In regions II and III, the system contained non-freezing water and freezing bound water. When the system was cooled at 10°K/min, the crystallization of bound water was observed for the system in region H I , but not for the system in region II. In region II, T^ still decreased gradually with increasing W . After reaching the minimum value, T^ increased and approached a constant value with increasing W i n region III. In regions II and III, the exothermic peak between T^ and T was observed in the heating D S C curve. The exothermic peak was influenced by annealing at temperatures between T^ and T . Figure 5 shows the schematic D S C curves of the water-polysaccharide system with W corre sponding to regions II and III. The sample, which was prepared by cooling at 10°K/min from 300°Κ to 150°K, was used as the original sample. This original sample was heated to the end temperature of the exothermic peak, and then quenched to 130°K. The sample having this thermal history was used as the annealed sample. The heating D S C curve of the annealed sample does not show the exothermic peak. The value of T^ shifted to the higher temperature and the heat capacity difference between the glassy and liquid states at T^ ( A C p ) decreased by annealing. This fact suggested that the disordered part, which contributed to glass transition of the original sample, changed to the ordered part, which no longer contributed to glass transition. The freezing bound water of the system in region II did not crystallize on cooling. However, the T of ice was observed in the heating D S C curve. This fact suggests that the freezing bound water crystallized on heating at around the exothermic peak and then melted at T . Therefore, we assume that the freezing bound water formed amorphous ice during cooling and the exothermic peak observed i n the heating D S C curve was c

c

m

m

c

m

m



224


Figure 4. Relationship between glass transition temperature (T^) and water content ( W ) for the water-hyaluronic acid system. Samples were prepared by cooling at 10°K/min from 300°Κ to 150°K. The W range is divided into four regions which were defined in terms of type of water in the system. c

c


14.

Y O S H I D A ET A L


Ο Χ LU


ORIGINAL

S.

ANNEALED

S.

O "O C LU

150

200 T

250 /

300

K

Figure 5. Schematic D S C curves of the water-polysaccharide system having different thermal history. The original sample was prepared by cooling at 10°K/min from 300°Κ to 150°K. T^ and A C p are glass transition temper ature and heat capacity difference between glassy state and liquid state at T^, respectively.


226

VISCOELASTICrTY OF BIOMATERIALS

due to the crystallization of amorphous ice. From the melting enthalpy of ice and the area of the exothermic peak, the amount of amorphous ice which crystallized at T ( W 2 ) was estimated. The value of Δ Ο ρ of amorphous ice ( A C p ) was calculated using the following equation: c c

2

ΔΟρ

=

(ΔΟ

Ρ ο

-ΔΟ

Ρ α

)

(3)

where Δ Ο ρ and Δ Ο ρ were Δ Ο ρ of the original and the annealed sample, respectively. The calculated values of Δ 0 ρ 2 are shown in Figure 6 as a function of W . For all polysaccharide systems, Δ 0 ρ 2 decreased with increasing W and approached the value of amorphous ice prepared from pure wa ter (26). This fact suggests that freezing bound water formed amorphous ice when the system was cooled at 10°K/min and the molecular motion of amorphous ice contributed to the glass transition of the original sample in regions II and III. As can be seen in Figure 6, the formation of amor phous ice occurred in a wide W range in the hyaluronic acid and xanthan systems while the formation of amorphous ice occurred in a narrow W range in the pullulan system which did not form hydrogel readily. This fact indicated that the hydrogel contained a large amount of bound water and that part of the bound water changed easily to amorphous ice even if the system was cooled slowly. A t a given W , the hyaluronic acid system showed the largest value of Δ 0 ρ 2 , and the value of Δ 0 ρ 2 of the xanthan and pullulan systems decreased as shown in Figure 6. The large value of Δ 0 ρ 2 suggests that the structure of amorphous ice in the system adopts a more random arrangement than the system which showed the small Δ 0 ρ 2 values. Recently, it was reported that glass transition of amorphous ice i n hydrogel prepared from poly(2-hydroxyethyl methacrylate) ( P H E M A ) was observed at 136° Κ by dynamic mechanical analysis (27). As the reported T^ is close to that of pure water which is obtained by calorimetric measure ments (26,28), amorphous ice in P H E M A hydrogel scarcely interacts with P H E M A in contrast to the water-polysaccharide systems. In order to explain the molecular mechanism of W dependence on T in regions II and III, the following hypothesis was presented. A s the system in regions II and III contained almost the same amount of nonfreezing water, it was assumed that the change of T occurred as the result of the plasticizing effect of amorphous ice on the main chain motion of polysaccharide absorbed non-freezing water. Actually, the minimum value of T observed between regions II and III seemed to depend on amount of freezing bound water. The T^ change in the polymer-plasticizer system is well described by the Gordon-Taylor equation as shown below. 0


2

α

c

c

c

c

c

c

g

g

g

frAC rji φιΑΟρ Pl

+ faACp r + ψ ΔΟρ a

χ

2

#a

(4)

2

where φι, Δ Ο ρ ι , and Τ χ are weight fraction, Δ Ο ρ and Τ^ of i-th compo nent, respectively. The amount of amorphous ice (w ) which is calculated 9

2



14. Y O S H I D A E T A L

from the area of the exothermic peak gives the weight fraction of amor phous ice for each W in regions II and III. The values of T ^ i and Δ Ο ρ ι are obtained as the experimental values of the sample with W 0.5, which were 185.7°K and 0.321 J / g K for the hyaluronic acid system, 198°K and 0.345 J / g K for the xanthan system. The relationship between T^ and φι of the original samples i n regions II and III for hyaluronic acid and xanthan systems are shown in Figure 7. The solid lines show the calculated val ues using Equation 4 with 135°K and 1.94 J / g K as the values of T i and ΔΟρ2, respectively (26). The experimental values correspond well with the calculated values. The result shows that the T^ of polysaccharide-absorbed non-freezing water decreased with increasing amounts of amorphous ice in region II. This fact suggests that the glass transition observed in the original sample was a result of the cooperative molecular motion of the polysaccharide and the amorphous ice in regions II and III. c

c


9

Molecular Motion in Regions III and IV. It is well known that T^ depends not only on the amount of plasticizer but also the degree of crystallinity. In region III, a part of the freezing bound water was crystallized to the regular ice on cooling. The system in region I V contained free water in addition to non-freezing water and freezing bound water. A l l of the free water in the system formed regular ice at around 250°K on cooling at 10°K/min, since the crystallization rate of free water was much faster than the cooling rate (21). In order to explain the molecular motion of the system in regions III and I V , it is necessary to estimate the amount of regular ice in the system. The deviation of experimental value from the calculated value shown in Figure 7 was probably caused by the presence of regular ice which formed in the cooling process. From the heat of crystallization during cooling at 10°K/min, the regular ice content ( W ) was obtained using the following equation: l c e

___ W

i

, M

. ν &

)

amount of ice formed during cooling/g = dry sample weight/g

t

λ ( 5 )

Figure 8 shows the relationship between T^ and W , of the original samples in regions III and I V for the water-hyaluronic acid system. The relation curve consists of two lines having different gradients corresponding to re gions III and I V . This fact indicates that the increase of T^ in region III was due to the increase of the amount of regular ice which formed during cool ing at 10°K/min. The system in region III was plasticized by amorphous ice. However, the main chain motion of the polysaccharide was restricted by the presence of the regular ice. When the system contained free water, the freezing bound water did not form the amorphous ice and the system showed a constant T . The constant T^ depended on the amount of nonfreezing water irrespective of types of polysaccharide, because all of the freezing water crystallized on cooling in the system W corresponding to region I V . When the system contained a certain amount of regular ice at high W , the glass transition was difficult to detect. It was hard to deter mine the T^ of the water-hyaluronic acid system in the W range above 3. c e

g

c

c

c



228


0.6

0.8

1.0 V\fc

/

1.2 g.g'

1.4

1

Figure 6. Relationship between heat capacity difference between glassy state and liquid state of amorphous ice (ΔΟρ2) and W for the waterhyalurem (A), the water-xanthan system (B), and the water-pullulan sys tem (C). c

200

T50" 0

« 0.05

0

1 0.1

I 0.15

2

Figure 7. Relationship between and amorphous ice content (^2) for the water-hyaluronic acid (A) and the water-xanthan (B) systems. The solid lines show the calculated value using Equation 4.





200

180h

Wjce Figure 8. Relationship between hyaluronic acid system.

0.8

0.6

OA

0.2

0

.-1

/

g-g

and ice content (W e) for the waterlC

Conclusions The influence of water on the main chain motion of polysaccharides was discussed in four regions (I, II, III and IV) depending on W . In the completely dry state, the main chain motion of polysaccharides did not occur below the decomposition temperature. B y sorption of water, glass transition of polysaccharides appeared and T^ decreased rapidly with increasing W . The sorbed water existed as non-freezing water in region I. The decrease of T^ of hydrogel was observed even if freezing water existed in the system in region II. In this region, freezing bound water formed amorphous ice by cooling at 10°K/min. The main chain motion of the polysaccharideabsorbed non-freezing water was plasticized by the amorphous ice in region II. W i t h increasing W , T^ increased after passing the minimum value due to the presence of regular ice which formed on cooling in region III. In regions II and III, the glass transition of the system appeared as the result of the cooperative motion of polysaccharide, non-freezing water, and amorphous ice. In region I V where free water existed, the T of the system approached a constant value irrespective of the type of polysaccharide and was hard to observe with increasing W . c

c

c

g

c

Literature Cited 1. Djabourov, M. Contemp. Phys. 1988, 29, 273-297. 2. A m i y a , T . ; Tanaka, T. Macromolecules 1987, 20, 1162-1164. 3. Andrade, J. D . , E d . Hydrogels for Medical and Related Applications; A C S Symp. Ser. 31; Washington, D C : American Chemical Society, 1976.



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December 16,

1991


Viscoelasticity of Biomaterials - American Chemical Society

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