Poly(vinyl alcohol) Hydrogels Prepared under Different Annealing

Chapter 20

Poly(vinyl alcohol) Hydrogels Prepared under Different Annealing Conditions and Their Interactions with Blood Components 1

Downloaded by GRIFFITH UNIV on September 4, 2017 | http://pubs.acs.org Publication Date: November 30, 1993 | doi: 10.1021/bk-1994-0540.ch020

Keiji Fujimoto , Masao Minato, and Yoshito Ikada Research Center for Biomedical Engineering, Kyoto University, 53 Kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606, Japan Poly(vinyl alcohol) (PVA) hydrogels obtained by annealing in the presence of glycerol exhibited a remarkable decrease in the water solubility and water content because of an increase in the crystallinity. The physical properties of the hydrogels were greatly dependent on the glycerol concentration in the PVA films during annealing. The hydrogels showed reduced protein adsorption in vitro and reduced platelet adhesion both in vitro and ex vivo, compared with that obtained without the addition of glycerol. Characterization of the surface of PVA hydrogels by contact angle and zeta potential suggested that the hydrated PVA chains existed at the surface region. Probably, as the surface coverage with the tethered PVA chains was increased by the addition of glycerol, the blood components were much more protected from direct contact with the material surface because of its steric hindrance effect. In previous papers(i-J), we have reported that a specially prepared poly(vinyl alcohol) (PVA) film is very low in protein adsorption and platelet adhesion compared with conventional PVA and other polymers. When PVA is used for practical applications as non aqueous solutions, physical or chemical crosslinking or acetalization is introduced to PVA to make it insoluble in water. Acetalization with formaldehyde and physical crosslinking by heat treatment (annealing) are the most common among the insolubilization methods(^). However, for the minimum protein adsorption and platelet adhesion, these physical and chemical modifications have to be avoided because significant interactions with biological components are observed for such PVA, similar to the conventional polymers.

Current address: Faculty of Applied Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223, Japan

0097-6156/94/0540-0228$06.00/0 © 1994 American Chemical Society

Shalaby et al.; Polymers of Biological and Biomedical Significance ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


20. FUJIMOTO ET AL.

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Polyvinyl alcohol) Hydrogels

The purpose of the present work is to twofold: (1) to clarify why only mild heat treatment of PVA can produce a surface which greatly prevents protein adsorption and platelet adhesion and, (2) to find the optimal annealing condition for production of PVA hydrogels with low water content. In previous studies, we could not obtain high strength PVA hydrogels with low water content because, at that time, a very mild annealing condition (heat treatment at low temperature for a short time ) was chosen without paying attention to the mechanical properties. In the present study, we added glycerol to PVA films to be annealed, since Lee and Hitomi reported about 50 years ago that heat treatment of PVA in the presence of glycerol enhanced crystallization of PVA ( Lee,S.;Hitomi,K. presented at the 2nd meeting of Japanese Association for Synthetic Fiber Research, Oct. 4, 1943, Kyoto, Japan.). To our knowledge, no detailed studies on the effects of glycerol on the PVA crystallization have been reported since then. We describe here the effects of glycerol concentration and annealing temperature on the water content, crystallinity, protein adsorption, and platelet adhesion for PVA hydrogels obtained under different annealing conditions. Experimental Preparation of PVA Films. Commercial PVA with a viscosity- average degree of polymerization of 1,700 and a saponification degree of 99.5 mol% was employed in this study. Glycerol was purchased from Wako Pure Chemical Inc, Osaka, Japan. Both were used as received. Distilled water was added to the PVA powder in a Pyrex flask to prepare a PVA concentration of 10 wt%. Glycerol was added to the mixture by 10-40 wt% of PVA and placed in an autoclave at 110°C for 30 min to completely dissolve PVA. The resulting PVA aqueous solution containing glycerol was cast on Pyrex Petri dishes and dried at room temperature for a week in a clean room. The PVA films obtained with 80-120 /zm thickness were annealed at temperatures from 70 to 15Q°C for 10 min, followed by slow cooling, unless otherwise noted. After heat treatment, the PVA films were immersed in methanol to remove glycerol at 37°C for 48 hr and then air-dried. Determination of the Sol Fraction and Water Content. The dried PVA films were immersed in water at 37 °C for 72 hr to determine the sol fraction and the water content of the insoluble PVA hydrogel. They were calculated from equations (1) and (2): Sol fraction = [(W -W )/W ] χ 100

(1)

Water content = [(W -W )/W ] x 100

(2)

F

D

8

F

D

S

where W is the weight of the dry PVA film after glycerol removal, W is the weight of the insoluble hydrogel after drying in air, and W is the weight of the hydrogel swollen in water at 37°C to equilibrium. F

D

s

Crystallinity Determination. The crystallinity of dried PVA hydrogels was evaluated by the density gradient tube method using CCl -toluene mixtures at 37°C (5). The crystallinity χ, defined as the weight ratio of 4


230

POLYMERS OF BIOLOGICAL AND BIOMEDICAL SIGNIFICANCE

crystalline PVA to the total PVA, was calculated from equation (3); 1/d = * / d

c

+ (l-*)/d

(3)

A

where d is the density of the dried PVA hydrogel, d is the density of 100 % amorphous PVA, and d is the density of 100 % crystalline PVA. d and d are assumed to be 1.27 and 1.34 g c m , respectively (6). A

c

A

-3


c

Contact Angle Measurement. Dynamic contact angle measurement was performed on the dried hydrogels at 25°C by the Wilhelmy plate technique using equipment manufactured by Shimadzu Inc. (Automated System for Dynamic Contact Angle Measurement, ST-1S type) ( 7). Water used for the measurement was purified by de-ionization after double distillation. Five measurements on different parts of the film were averaged. The movement rate of the water vessel into which the gel specimen was immersed was kept at 0.3 mm sec" . 1

Zeta Potential Measurement. Streaming potentials of the hydrogel surface were measured at and pH 7.4 using the cell method (6). The electric potentials created by streaming an electrolyte solution were recorded on an X-Y recorder. The electrolyte solution was prepared from NaOH and HC1 aqueous solutions to have an ionic strength of 6xl0~ . Five measurements were averaged, and zeta potentials were calculated from the average streaming potentials using the Helmholtz- Smoluchowski equation. 3

Protein Adsorption. Bovine y -globulin (IgG, crystallized), purchased from Sigma Chemical Co., was used as received. N a I for protein labeling was purchased from Daiichi Pure Chemical Co. Ltd., Tokyo, Japan and the Chloramine-T method was employed for labeling (5). Unbound I was removed from the protein solution by applying to an anionic exchange resin (Dowex 1-X8, Dow Chemicals, USA). The aqueous solution of labeled IgG was stored at 4TJ and used for adsorption study within 12 hours after labeling. Protein adsorption was carried out under static conditions at a protein concentration of 1 mg m l , adjusted with phosphate buffered solution (PBS, pH7.4), at 37 °Chj immersing five pieces of hydrogel film of 1 cm χ 1 cm in 2 ml of protein solution for each experiment. After a 3 hr adsorption, without taking them out of the protein the films were first rinsed with PBS solution and then blotted with tissue paper. Radioactivity of the protein-adsorbed films was measured with a gamma-ray scintillation counter. Five readings were performed on the different pieces cut from the same large film. 125

1 Z 5

- 1

Platelet Adhesion. Venous blood from healthy human volunteers, who did not receive any agents which affect platelet function, was collected with a vacuum syringe containing 5 % citric acid. The blood was centrifuged at 800 rpm for 10 min at 25^, and the platelet-rich plasma (PRP) was withdrawn with a polyethylene pipette and placed in clean vials at room temperature. A portion of PRP was diluted by adding PBS and centrifuged at 1800 rpm for 10 min to prepare platelet pellets. The residue of the blood was further centrifuged at 3,000 rpm for 10 min to obtain platelet-poor plasma (PPP). The platelet count was determined with Coulter Counter (Type 4) and adjusted to have 150,000 platelets in 1


20. FUJIMOTO ET AL.

Poly (vinyl alcohol) Hydrogels

231

3

mm . Plasma-free platelet solution (washed platelet, WP) and PRP were prepared by adding the platelet pellets to PBS and PPP, respectively. 0.6 ml of WP or PRP was placed on each of the hydrogel films of 1.8 cm in a vial and allowed to stand for 1 hr at 37 °C. Then, the films were vigorously washed with PBS and put into 2 ml of 0.1 M phosphate buffer (PB) containing 0.5 % Triton-X100 to lyse the adhered platelets. Lactic acid dehydrogenase (LDH) activity of the lysate was determined with an enzymatic method to count the adhered platelets with the use of a calibration curve of platelet counts(Tamada,Y., Kyoto University, Doctor Thesis, 1989.). To confirm the reproducibility, the platelet adhesion experiment was repeated five times for the same film. A Silastic film, donated by Dow Corning Cor., Ltd., Midland, USA, was used as a reference material. Downloaded by GRIFFITH UNIV on September 4, 2017 | http://pubs.acs.org Publication Date: November 30, 1993 | doi: 10.1021/bk-1994-0540.ch020

2

Ex vivo Arterio-venous Shunt Experiment. A plasticized poly(vinyl chloride) (PVC) tube, provided by Japan Medical Supply, Co., Ltd., Hiroshima, Japan, was coated with a thin layer of PVA containing 40 wt% glycerol, and heat treatment of this layer was done at 70 °C for 30 min followed by glycerol removal with methanol. Rabbits weighing 2-3 kg were anesthetized and their carotid artery and jugular vein were exposed surgically to make the arterio-venous (A-V) shunt. In the case of venous(V)-venous(V) shunt model, the shunting between the right and the left vein was performed (1Û). The PVA-coated PVC tube was connected to the shunt circuit with the help of a Vessel tip made of fluorine polymer (Quinton Instrument Co., Seattle, WA). After removal of the tube from the shunt, the blood-contacting surface of the tube was rinsed with 100 ml PBS at 100 cmHg and fixed with glutaraldehyde for scanning electron microscopy (SEM). Results Bulk Properties of PVA Hydrogels. Figure 1 shows the effect of annealing of PVA films containing different concentrations of glycerol on the sol fraction. In the absence of glycerol, a considerable sol fraction was observed even by heat treatment at 110 °Cfor 30 min, whereas annealing at low temperatures such as 30°C could yield PVA gel fractions when glycerol was added. The annealing temperature required for formation of the same amount of gel became lower as the addition of glycerol increased. The water content of PVA gel fractions also decreased with the annealing temperature as shown in Figure 2. Addition of glycerol led to an abrupt decrease in water content when the weight percentage of glycerol added to PVA was above 20 %. A similar tendency was found for the crystallinity of PVA gel. The result is given in Figure 3. Clearly, the crystallinity of the PVA gel film gradually increased as the annealing temperature was higher, regardless of the amount of glycerol added. Glycerol addition always promoted the crystallization of PVA. Figure 3 shows that crystallinity changed discontinuously around Tg of PVA when glycerol was absent, although this phenomenon was not seen in the presence of glycerol probably because of the lowering of Tg. Figure 4 shows the relationship between the water content and the crystallinity of PVA hydrogel. As is seen, the effect of glycerol addition on the water content-crystallinity relationship is not remarkable, but it is apparent that the water content drastically changed in the region of low crystallinity between 0.2 and 0.3.


232

POLYMERS OF BIOLOGICAL AND BIOMEDICAL SIGNIFICANCE 80


Φ

30

50

70

90

110

130 150

Annealing temperature / ° C

Fig.l Effect of annealing temperature on the sol fraction of PVA films. · ; 0 %, O; 10 %, O; 20 %, Δ; 30 %, • ; 40 % (weight percent of glycerol to PVA).


Fig.2 Effect of annealing temperature on the water content of PVA hydrogels. · ; 0 %, Ο; 10 %, O i 20 %, Δ ; 30 %, • ; 40 % (weight percent of glycerol to PVA).



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233


0.11 50

•

ι

ι

ι

70

90

110

130

• I 150 170


Fig.3 Effect of annealing temperature on the crystallinity of dried hydrogels. · ; 0 %, Ο; 10 %, Ο; 20 %, Δ ; 30 %, • ; 40 % (weight percent of glycerol to PVA). 0.6

ι

1

j

1

r

30

40

50

60

70

80

Water content / %

Fig.4 Relation between the water content and the crystallinity of PVA hydrogels, φ ; 0 %, Ο; 10 %, Ο; 20 %, Δ; 30 %, • ; 40 % (weight percent of glycerol to PVA).


234


Table I Water Contact Angles and Zeta Potentials of PVA Annealed in the Presence of Glycerol by 40 wt% of PVA Annealing Temp.

Contact Angle (deg.)

(X) 90 110 130 150 Original*

5

29±2.5 35±3.1 37±1.8 42±3.1 36±1.7

14±1.5 16±1.2 18±0.8 18±0.6 18±1.1

Zeta Potential (mV) -0.8±0.1 -0.8 ± 0 . 1 -1.5±0.5 -2.1±0.3 -5.7±0.6


a) annealed at 150 °C for 30 min without glycerol

Surface Properties of PVA Hydrogels. Table I shows contact angles and zeta potentials of PVA hydrogels obtained by annealing in the presence of glycerol, together with those obtained in the absence of glycerol. The surface measurements were carried out for the PVA hydrogels in the water-swollen state. It is seen that the receding contact angles ( θ ) were v e r y low in contrast to the advancing contact angles ( θ ) , i r r e spective of the annealing temperature. Both the contact angles θ and θ slightly increased with the annealing temperature. It is also apparent from Table I that the zeta potentials are virtually zero or negative for all samples. Seemingly, annealing of PVA shifted the zeta potentials to a little higher negative values. r

a

r

a

In vitro Protein Adsorption. The results of IgG adsorption onto PVA hydrogels annealed at various temperatures are shown in Figures 5 and 6 as a function of crystallinity and water content of the PVA hydrogels, respectively. The amount of protein adsorbed per unit surface area, as well as platelets adhered, was calculated under the assumption that the surface of the PVA hydrogels was entirely smooth. As seen from Figure 5, IgG adsorption drastically increased as crystallinity increased in the a b sence of glycerol during annealing; however, it exhibited a slow increase over a wide range of crystallinity when annealed in the presence of glycerol. Protein adsorption leveled off at crystallinity above 0.4 when glycerol was added by 40 % of PVA. Furthermore, the result in Figure 6 reveals that adsorption was remarkably reduced with decreased water content for the hydrogels annealed in the absence of glycerol; whereas, reduced protein adsorption was maintained over a relatively wide range of water content when PVA films were annealed in the presence of glycerol. In vitro Platelet Adhesion. Figures 7 and 8 show the time course of platelet adhesion onto Silastic and PVA hydrogels when WP and PRP were used, respectively. PVA hydrogels were prepared under two different conditions: by heat treatment at 150T) in the absence of glycerol and by heat treatment at 70 °C in the presence of glycerol by 40 wt% of PVA. The number of adhered platelets was expressed as a percentage of adhered p l a t e l e t s onto the r e f e r e n c e m a t e r i a l

(Silastic film).

As can be seen i n


20. FUJIMOTO ET AL.

235



0.25

0.05 I 0.2

,

,

ι

0.3

ι 0.4

.

1 0.5

.

1 0.6

Crystallinity

Fig.5 Dependence of IgG adsorption on the crystallinity of PVA hydrogels. · ; 0 %, Ο; 20 %, • ; 40 % (weight percent of glycerol to PVA). 0.22

0.081 30

ι

ι

ι

1

40

50

60

70

1 80

Water content / %

Fig.6 Dependence of IgG adsorption on the water content of PVA hydrogels. · ; 0 %, Ο; 20 %, • ; 40 % (weight percent of glycerol to PVA).



236


0

20 40 Incubation time /min

60

Fig.7 Effect of incubation time on the platelet adhesion to PVA hydrogels and Silastic in WP. O ; Silastic, · ; 0 % (150°C), • ; 40 % (70 °C) (weight percent of glycerol to PVA).

0

20

40

60

Incubation time /min Fig.8 Effect of incubation time on the platelet adhesion to PVA hydrogels and Silastic in PRP. O; Silastic, · ; 0 % (150T:), • ; 40 % (70°C) (weight percent of glycerol to PVA).


20. FUJIMOTO ET AL.

237


Figure 7, platelet adhesion onto Silastic film gradually increased with the incubation time, but platelet adhesion onto PVA hydrogels always r e mained at low levels. Compared with platelet adhesion in WP, adhesion of platelets from PRP onto the PVA annealed in the absence of glycerol significantly increased with the incubation time as seen in Figure 8. Influences of the crystallinity and water content of PVA hydrogels on platelet adhesion in WP for 60 min are shown in Figures 9 and 10, respectively. The PVA hydrogel prepared by heat treatment at 150 °C in the a b sence of glycerol was selected as a reference material, to which 6.5 x 10 platelets were adhered per mm . As can be seen in Figure 9, the profile of the relation between the crystallinity and the number of platelets adhered is similar to that observed for IgG adsorption. A similar profile is also recognized in Figure 10, where platelet adhesion was found to be greatly suppressed over the wide water content, when PVA was annealed in the presence of glycerol. 3


2

Ex vivo Platelet Adhesion. An ex vivo experiment for platelet adhesion was carried out after coating the surface of a PVC tube with PVA hydrogels. Figure 11 shows SEM photographs of the surface of the PVA hydrogel exposed to blood in the A - V shunt system for different periods of time, together with those of PVC. Platelets adhered to the virgin PVC surface gradually increased in number with the shunting time, whereas platelet deposition was not observed on the surface of PVA-coated PVC even 30 min after exposure to blood. Figure 12 shows SEM photographs of virgin PVC and PVA-coated PVC surfaces 15 min after blood exposure in the V - V shunt system where thrombus was formed more quickly than in the A - V shunt. The surface of PVC was covered with a tightly deposited platelet layer containing red blood cells in fibrin-like network, but v e r y few adherent red blood cells could be observed on the surface of PVA-coated PVC.

Discussion PVA molecules have one hydroxy group for each of the - C H - C H repeating units. This simple chemical structure allows PVA to readily crystallize to yield high strength fiber and film which are currently used in industry. Once the crystallites are destroyed, PVA becomes soluble in water even at room temperature. Because of these unique properties, several researchers have attempted to utilize PVA for biomedical materials. For instance, Peppas et al. (11) and Noguchi et al. (12) tried to use PVA as articular cartilage . Merril et al. (13) and Sefton (14-17) studied the interaction of PVA with blood components. Tomita et al. (13) investigated the possibility of using the PVA fiber as suture material. All of these PVA samples seem to have received severe heat treatment to enhance the mechanical properties, probably resulting in significant interactions with blood components. As shown in Figures 1 to 3, annealing in the presence of glycerol gave rise to the enhanced gel fraction and the lowered water content. This must have resulted from promotion of PVA crystallization by glycerol. It is likely that glycerol molecules present in the PVA matrix served as plasticizer to help the Brownian motion of the PVA segments for reorientation. Glycerol is known to become a solvent of PVA when the 2



238


1

0 0.1

«

«

ι

ι

I

0.2

0.3

0.4

0.5

0.6

Crystallinity Fig.9 Dependence of platelet adhesion on the crystallinity of PVA hydrogels in WP. · ; 0 %, O ; 20 %, • ; 40 % (weight percent of glycerol to PVA).

0 I 20

I

I

I

I

I

30

40

50

60

70

Water content / % Fig. 10 Dependence of platelet adhesion on the water content of PVA hydrogels in WP. · ; 0 %, O ; 20 %, • ; 40 % (weight percent of glycerol to PVA).



20. FUJIMOTO ET AL.


Fig. 11 S E M photographs of PVA hydrogels and PVC surfaces after exposure to blood at an A - V shunt system for different times.

Fig. 12 S E M photographs of PVA hydrogels and PVC surfaces 15 min after exposure to blood at a V - V shunt system.


239


240


(c) Medium water content with small crystallites

(d) Medium water content with large crystallites

Fig. 13 Assumed models of interfacial structure of a PVA hydrogels of different water contents in contact with water.



20. FUJIMOTO ET AL.


241

temperature is higher than 100 °C. It is v e r y reasonable that the promoted crystallization of PVA brought about the increase in gel content and the decrease in water content of PVA gel even at low annealing temperatures. Since the protein adsorption and platelet adhesion are the surface-associated phenomena, it is obvious that glycerol had also altered the surface structure of PVA hydrogel. Unfortunately, v e r y few analytical methods are currently available for characterizing the surface structure of hydrogels in aqueous environments. One of them is the zeta potential measurement of the surface. Contact angles against water may provide information on the surface structure in contact with water. Based on the results obtained by these measurements, it has previously been concluded that there exists some semi-soluble PVA chains or loops extruding from the material surface into the outer aqueous phase (1). This is called a diffused surface (2). The results in Table I seem to support the diffuse structure model, since almost zero zeta potential means that the slipping plane is shifted toward the outer aqueous phase by the tethered, semi-soluble PVA chains. They should also interfere with protein adsorption to the surface through the steric hindrance effect. The surface structure of hydrogels with microcrystallites is schematically represented in Figure 13. It is highly possible that the number density and size of the microcrystallites are different among the materials even if their crystallinity is similar with each other. It seems probable that larger microcrystallites are formed with the increased addition of glycerol because of its promotion effect on PVA crystallization. At the same time, wider amorphous regions should be formed with the increasing glycerol concentration, leading to a more complete covering of the surface with many semi-soluble PVA chains, when compared at a similar crystallinity. The glycerol effect is compared in the surface model of Figure 13(c) and (d). If the diffuse surface can prevent the protein adsorption, platelet adhesion would be also reduced because it is well known that platelet adhesion is promoted by adsorbed proteins such as IgG and fibrinogen. Figures 5 and 6 give evidence for this assumption. In conclusion, it may be summarized that the annealing of PVA film in the presence of plenty of glycerol makes it possible to produce a diffuse layer on the PVA surface without any chemical modification. In addition, the resulting PVA hydrogels have low water contents such as 30 %. Finally, it should be stressed that blood interactions of PVA are greatly influenced by the history of the PVA sample preparation. In the future, we need to accumulate more information on the phase structure of PVA hydrogel, especially in the surface region, although it is v e r y difficult because of its highly hydrated structure.

REFERENCES 1. Ikada,Y.;Iwata,H.;Horii,F.;Matsunaga,T.;Suzuki,M.;Taki,W.;Yamagata,S.; Yonekawa,Y.;Handa,H. J. Biomed. Mater. Res. 1981, 15, 697. 2. Ikada,Y. Adv. Polym. Sci. 1984, 57, 103. 3. Ikada,Y.;Suzuki,M.;Tamada,Y. In Polymers as Biomaterials; Shalaby,S.W., Ed.; Plenum Press, N.Y., 1984, pp 51. 4. Polyvinyl Alcohol; Finch,C.A., Ed.; John Wiley & Sons Ltd., 1973.



242


5. Boyer,R.F.;Spencer,R.S.;Wiley,R.M. J. Polym. Sci. 1946, 1, 249. 6. Sakurada,I.;Nukushina,Y.;Sone,Y. Polymer Chem. (in Japanese), 1955, 12, 506. 7. Uyama,Y.;Inoue,H.;Ito,K.;Kishida,A.;Ikada,Y. J. Colloid. Inter. Sci. 1991, 41, 275. 8. van Wagenen R.V.;Andrade,J.D. J. Colloid. Inter. Sci. 1980, 76, 305. 9. Methods in Immunology; a laboratory text for instruction and research; Garvey,J.S.;Cremer,N.E.;Sussdorf,D.H., Eds., W.A. Benjamin Inc., 1977. 10. Fujimoto,K.;Minato,M.;Tadokoro,H.;Ikada,Y. J. Biomed. Mater. Res. 1993, 27, 335. 11. Peppas,N.A.;Merrill,E.W. J. Polym. Sci., Polym. Chem. Ed. 1976, 14, 441. 12. Noguchi,T.;Yamamura,T.;Oka,M.;Kumar,P.;Kotoura,Y.;Hyon,S.-H.; Ikada,Y. J. Appl. Biomaterials, 1991, 2, 101. 13. Merril,E.W.;Salzman,E.W.;Wong,P.S.L.;Ashford,T.D.;Brown,A.H.; Austen,W.G. Physiol. 1970, 29, 723. 14. Goosen,M.F.A.;Sefton,M.V. J. Biomed. Mater. Res. 1983, 17, 359. 15. Cholakis,C.H.;Sefton,M.V. J. Biomed. Mater. Res. 1989, 23, 399. 16. Cholakis,C.H.;Zingg,W.;Sefton,M.V. J. Biomed. Mater. Res. 1989, 23, 417. 17. Smith,B.A.H.;Sefton,M.V. J. Biomed. Mater. Res. 1993, 27, 89. 18. Tomita,N.;Tamai,S.;Shimaya,M.;Mii,Y.;Ikeuchi,K.;Ikada,Y. Biomed. Mater. Eng. 1992, 2, 71. Received May 25, 1993


Poly(vinyl alcohol) Hydrogels Prepared under Different Annealing

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