Viscoelasticity of Biomaterials - American Chemical Society

Chapter 2

Physicochemical Properties of Polysaccharides in Relation to Their Molecular Structure Downloaded by STANFORD UNIV GREEN LIBR on June 18, 2012 | http://pubs.acs.org Publication Date: May 8, 1992 | doi: 10.1021/bk-1992-0489.ch002

Marguerite Rinaudo Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique, B.P. 53X, Grenoble, 38041 France Several relationships between the chemical structure of some bacterial polysaccharides and their physical properties i n aqueous solutions are reviewed. These native microbial polysaccharides adopt an ordered conformation in dilute solution which controls the stiffness of the molecule and thereby the viscoelastic behavior of their solutions. Two types of gelation observed with polysaccharides are described. Especially characteristic are cooperative interactions in thermoreversible gels that are influenced by the thermodynamic behavior of the solutions. This review concerns some native polysaccharides produced by plants, animals, and microorganisms. Even though modified polysaccharides are very important for industrial applications, this class of polymers will not be considered here because chemical modifications usually induce heterogeneities in chemical structure that result in the disappearance of some characteristic such as stereoregularity. The polysaccharide cellulose is one of the most important natural polymers; it is used as an insoluble material in the textile and paper industries. Different reviews have been published on its structure, biosynthesis and physical properties (1-6). It will not be covered here. Starch is also of great importance; in some conditions, it is soluble in an aqueous medium and forms gels by rétrogradation. A large bibliography exists concerning starch, starch derivatives, and the morphology of starch granules (7-10); this compound will also not be considered in the following. Among the most important sources of polysaccharides is also the crab shell or other Crustacea from which chitin is extracted (11-14). Alkaline deacetylation of chitin produces chitosan, which becomes soluble in acid; this polysaccharide has a specific chelating property related to the - N H 2 group which 0097-6156/92/0489-0024$06.00/0 © 1992 American Chemical Society

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

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Physicochemical Properties of Polysaccharides

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is unusual i n natural polysaccharides. In the fully deacetylated form, it is poly[/?(l —+ 4)-2-amino-2-deoxy-D-glucose] which becomes an important and interesting polymer for many applications. Most of the water-soluble native polysaccharides are used as thickening polymers mainly for food applications but also for other industrial purposes (15). This review will be devoted i n large part to the microbial water-soluble polysaccharides. The aim will be to show how the chemical structure (monomeric unit, osidic linkage, ionic site, side groups, etc.) controls the physical properties in solution and during gelation. Solution Properties Xanthan Gum. The most important bacterial polysaccharide produced on large scale is the xanthan gum excreted by Xanthomonas campestris (Figure l a ) . M a n y papers have been published on this polysaccharide, but there are still some questions under discussion. The thermodynamic behavior (i.e., activity coefficients of counterions) suggests that xanthan is a single chain molecule which adopts a helical conformation under certain conditions (excess of external salt, low tem perature) (16,17). Xanthan undergoes a reversible conformational transi tion (helix coil) as demonstrated by optical rotation-temperature ex periments. A characteristic temperature, T , taken at half the transi tion, is assumed to characterize the conformational transition. As for other biopolymers, a linear dependence relates log Q r to ( 1 / T ) for monovalent counterions where Qp is the total ion concentration taking into account the external salt concentration C and the osmotic free counterions Φ 0 ( C is the concentration of polymer ionic sites expressed in equivalents/liter, and Φ is the osmotic coefficient directly related to the charge parameter of the ionic polymer). The change in viscosity from helical conformation to coil conformation when temperature increases is not important due to the intrinsic stiffness of the β(1 —• 4)D-glucose chain and the presence of the three sugar units as a side chain. In dilute solution, in the helical conformation, xanthan behaves as a worm-like chain characterized by an intrinsic persistence length around 350Â for the preheated molecule (18); this value was determined using Yamakawa-Fujii and Odijk (19) treatments. This great stiffness of the xanthan molecule justifies the high viscosity for a given molecular weight and the small dependence of viscosity on the ionic strength; a large solvent draining effect exists even in infinite ionic strength in monovalent electrolyte (20,21). This insensitivity to an excess of salt was appreciated for tertiary oil recovery compared to the usual flexible ionic synthetic polymers. In direct relation to the stiffness of the molecule, in concentrated solutions, cholesteric liquid crystals are formed (22); the same type of structure was also obtained with succinoglycan (23) and scleroglucan (24). The most important physical properties of xanthan and related polysaccharides are the Theological properties in dilute and semi-dilute solutions. This point has been much investigated in the literature. Neverm

m

s


Ρ

p

26

VISCOEXASTICITY O F BIOMATERIALS

K

4]-B-D-Glcp[l-^]-B-D-Glcp-[l-^]-Q-0-Glcp-[i^4]-B-0-Glcp-Ci3

6-D-Manp-[l->4]-B-D-GlcAp-[l->2]-a-D-Manp-6-0Ac


β-D-Manp-[ 4 \

(a)

'

1-^4]-3-D-GlcAp-[1^2]-a-D-Manp-6-0Ac

6

C / \ HC COOH 3

p4]-S-D-Glcp-[1^4]-B-D-Glcp-[l-3]-fl-0-Galp-[l-^4]-B-0-G1cp-[l-^ 6

t 1 B-0-Glcp-[M]-B-0-Glcp-[l-3]-Q-0-Glcp-[l-6]-S-D-Glcp

Λ

HC 3

1 COOH

Ô CH C0 2

(b)

CH C00H 2

+3]-B-D-Glcp-[l+3]-B-D-Glcp-[M]-B-D-G1cp-[> (oL β-D-Glcp 1

t 6 +3]-B-D-Glcp-[l+3]-B-D-Glcp-[M]-B-D-Glcp-[l(J)L Figure 1. Repeating unit structure of some microbial polysaccharides, (a) xanthan; (b) succinoglycan (29); (c) curdlan (30); (d) scleroglucan (35).


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theless, many of the results were obtained on commercial samples i n which microgels and aggregates preexist due mainly to post-fermentation treatments. This allowed some authors to draw conclusions about the gel-like properties of xanthan solutions (25). In fact, xanthan directly isolated from the broth behaves as a single chain solution as recently discussed (26). In the dilute regime (i.e., polymer concentration lower than the overlap concentration, C * ~ [rç]" , where [η] is the intrinsic viscosity), the viscosity in the Newtonian regime is directly related to the polymer concentration C and the molecular weight M or the intrinsic viscosity following the relation: Downloaded by STANFORD UNIV GREEN LIBR on June 18, 2012 | http://pubs.acs.org Publication Date: May 8, 1992 | doi: 10.1021/bk-1992-0489.ch002

1

in which [η] = KM*. In helical conformation, in direct connection with the stiffness of the chain, the parameter a equals 1.14 in 0.1 M N a C l (27), and it approaches 1 for infinite ionic concentration (18). Such high values for the a coeffi cient are still not justified in the literature; they seem to correspond to the original behavior of worm-like chains in which the exponent a increases with the persistence length in the range of intermediate molecular weights even when the persistence length is considered in infinite salt concentration thereby eliminating the electrostatic contribution (see Yamakawa and Fujii treatment, ref. 19a, Figure 1). The semi-dilute regime appears over C * ; in this range, the nonNewtonian behavior (i.e., decrease of the viscosity as a function of the shear rate) becomes more and more important when the polymer concentration and molecular weight increase. A second critical concentration, C * * , corresponds to a linear variation of log η with C[rç] in the range of C**[?/] ~ 8-10; over C * * , the viscosity follows the relation: 9ρ

This dependence looks like that of synthetic polymers in the melt (28). Over C * * , the viscoelastic properties of the fluid become important and a yield stress ( r ) rapidly appears when the polymer concentration increases. The low values of C[rj] corresponding to the appearance of r seem to be related to the stiffness of the xanthan molecule. This may be related to the conditions under which the screening length, ξ, in semi-dilute solution is of the same order as the persistence length (20). In the same range of polymer concentration, the free diffusion of the molecules becomes nearly independent of the polymer concentration (20). The viscoelastic behavior of xanthan solutions has recently been ex amined in flow and dynamic experiments (26). To conclude, xanthan gum behaves as a worm-like chain with a large local stiffness in its helical conformation. Some essential differences be tween xanthan gum and synthetic flexible polymers become apparent. The influence of the chemical structure (i.e., content of pyruvate or acetyl substituents depending on strain type and/or on post-fermentation treatment) on the main physical properties i n solution seems to be negligible, but it r

r


28

VISCOELASTICITY OF BIOMATERIALS


may be important for polymer-polymer interactive processes (i.e., adsorp tion on solids, etc.). Other Microbial Polysaccharides. More recently, the behavior of a succinoglycan excreted by Pseudomonas sp. N C I B 11592 and produced by Shell Company has been examined. The main chain is a β(ΐ —• 4)/?(l —• 3) glycanic chain with a four sugar unit side chain attached to each fourth unit of the main chain (Figure l b ) . The stiffness of this backbone is much lower than that of xanthan. In the disordered conformation, the viscosity is very low; by contrast, in the helical conformation induced by excess salt, the stiffness becomes greater than that of xanthan (29). The role of the interaction of the side chain with the backbone is essential for stabilizing the helical conformation, and this seems of prime importance for the co ordination of the conformational transitions (30,31). In direct relation, a large isotopic effect is demonstrated when the melting temperature T is determined in H2O vs. D 0 , and this indicates the role of H bonds i n the helical conformation (23). m

2

β(1 —» 3) Glucans. Curdlan is a polysaccharide produced by Alkaligenes faecalis var. myxogenes 10 C 3 and specially investigated by Harada in Japan (32). This β(1 3) linear glucan is a gel-forming polymer (33); it is necessary to heat the aqueous dispersion of curdlan to dissolve it (Figure le). A gel is formed upon cooling, and this process is thermally reversible. Curdlan forms firm gels with some syneresis; the mechanism of gelation was investigated by Fulton and Atkins (34). Scleroglucan has the same backbone structure as curdlan; it is a β(1 —• 3) glucan with a β(1 —• 6) glucose as side chain on every third glucose unit of the main chain. Scleroglucan is produced by Sclerotium rolfsii. A very regular structure has been revealed by C N M R (35). It was demonstrated that schizophyllan (same structure as scleroglucan) produced by Schizophyllum commune forms a stiff triple helix with a persistence length of 200 n m (36). It is a neutral polymer soluble in water but with some aggregation. Solubility increases in N a O H (0.01N), and this produces good solutions (37). The triple helix of curdlan (38) (whose near-insolubility is reflected by the formation of strong and opaque gels) is rendered soluble by substitution of every third glucosyl sidechain, and this inhibits packing without affecting the local helical structure (36). A n irreversible conformational transition was demonstrated in D M S O and 0.2 M N a O H from the stiff triple helix conformation of scleroglucan to a single chain coil with a very low viscosity. Thermal melting was also observed in water around 135°C (36b). In the ordered conformation, the molecule is very stiff, and it forms liquid crystals (24). The presence of the side group is essential for increasing the solubility of the polymer and decreasing the interchain interactions compared with curdlan. A phase transition is observed around 8°C leading to the formation of a gel. This behavior is due to interchain interactions with a large isotopic effect. This effect demonstrates the interaction between the side group and the main chain involving the surrounding water molecules (39,40). 1 3



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Gellan-Like Polysaccharides. Gellan is a polysaccharide excreted by the bacteria Pseudomonas elodea. In the literature, it has been referred to as S-60 or PS60. It is produced by Kelco (USA) as a gel-forming polymer. The chemical structure of the native polymer was examined by different groups (41) (Figure 2). The substituents reduce the ability to gel, but they are readily hydrolyzed i n alkaline conditions. The linear polymer thus obtained is called Gelrite (Kelco trade name) (Figure 3a), and this produces firm gels under certain thermodynamic conditions (i.e., excess of external salt, low temperature, etc.). The gels are capable of displacing agarose in some applications (42). In the solid state, Chandrasekaran et al. established a double helical structure in which the helices are parallel (43). In dilute aqueous solution, gellan exists as a coil at high temperature, and forms a double helix when the temperature decreases or when an excess external salt is present. In the dilute regime, i n the ordered conformation, gellan behaves as a worm-like chain with a persistence length of 72 n m . In the disordered conformation the length was determined to be 6.1 nm (42). Gellan is a weakly charged polyelectrolyte even in double helix struc ture (charge parameter λ = 0.75). The activity of counterions in dilute solution confirms the double helical conformation which is more stable i n the presence of divalent counterions (44). Even with its low charge parameter, this polyelectrolyte displays a high ionic selectivity in the double helix form. W i t h monovalent counterions, this ionic selectivity is in the order: L i > Na+ > K > T M A + ( T M A = tetramethyl-ammonium ion). This selectivity also controls the degree of aggregation and the gel-forming ability (45). Welan is a new polysaccharide produced by Alcaligenes A T C C 31555 and referred to as S-130 or Biozan, the trade name of Kelco. This polymer has the same backbone as that of gellan, but since it is branched (Figure 3b), it is a non-gelling polymer. It is advocated as a thickening and suspending agent just as rhamsan (S194—produced by Alcalignes A T C C 31961) (Fig ure 3c) or the polysaccharide S-657 excreted by Xanthomonas A T C C 53159 (Figure 3d) (46). For welan, the following relation was found +

[η] = 3.37 χ 1 0 " M 7

1 4 1

+

+

([η] in dl/g)

between the intrinsic viscosity [η] and the molecular weight, M , i n 0.1 M N a C l solution. The high exponent (a = 1.41) seems to indicate a very stiff conformation (47). Brant et ai seem to rule out a double helix confor mation. Based on thermodynamic arguments, it was recently proven that welan forms a very stable double helix structure (48). The physical properties of these three polysaccharides with comb-like structure were also investigated by Crescenzi et al. (49) and Brant (50) so as to discuss the role of the comb-like structure on the physical properties. A s pointed out by Robinson (51), no conformational transition was observed in welan for different ionic strengths, and no detectable high resolution N M R


30


L-GIyceric 1 1 2 H3)-/3-i>Glc/;-( 1 ^ 4 ) - ^ 6 Î


A c

0.5

Figure 2. Repeating unit structure of the native gellan (39a). ( a ) - 3)-/3-D-Glcp.(1 --4)-^D-GlcpA-(1 -4)-/3-D.GIcp-(1 -4)-a-L;Rhap-(1(fc>) -3)-jS-D.GIcp-(1 -4)-/3-D.GIcpA-(1 -4)-/3.D-Glcp-(1 - 4)-o-LRhap-(1 3 :

î

1 β - L - Rhap or α - L-Manp

- 3).j3-D.GIcp-(1 - 4)-/3-D.GicpA.(1 - 4)-0-D-GIcp-(1 - 4)-a-L-Rhap-(1 -

1

or-D-GIcp ~" 6

1

/3-D-Gicp -^3)-)3-D-Glc/7-(1^4)-)3-D-GlcpA-(1^4).^D-Glcp-(l-^4)-a-L-Rha/?-(l-* 3 ( C a > Sr, corresponding to decreasing stability of the gel. For both polymers, no gel is ever formed i n the presence of M g . Applications of alginate are developed i n different industrial fields and especially for cell immobilization (67). The mechanical properties of these gels were investigated by Bouffar-Roupe; i n this work, the role of the guluronic block on the elastic modulus was clearly demonstrated (68). In the absence of divalent counterions, alginates have a moderate stiffness (per sistence length around 100Â) (69), depending on their chemical structure (68). 2 +


34



Conclusion In this study, the main results obtained on some polysaccharides were reviewed. The water-soluble polymers were chosen to demonstrate the role of chemical structure on their physical properties i n aqueous solution. The microbial polysaccharides form a series of new polysaccharides with a large variety of physical properties, and they hold great interest for investigating the fundamental structure-properties relationship. Xanthan and succinoglycan can be considered as single-chain polymers adopting a helical conformation i n the presence of an excess of neutral salts. Their stiffness is great, and they behave as worm-like chains with persistence lengths of about 400À. The /?(1 —» 3) glucans form triple helical structures, such as curdlan, a well-known gel-forming polysaccharide. When side groups (β(1 —• 6) glucose) exist, as i n scleroglucan, the polysaccharide becomes soluble i n water, and then it is a good thickening polymer. It forms a gel i n water only at temperatures below 8°C. The gellan-like polysaccharides are produced by bacteria. Gellan is a gelling polymer, but the comb-like polysaccharides (welan, rhamsan, S-657) having the same backbone also form a double helical structure but without gelation. Many analogies exist among all these polysaccharides and are related to the local stiffness of the molecules, the low ionic content sensitivity, and large draining effect were explained. Finally, some information concerning the gelling polysaccharides are recalled for the two series of systems usually recognized: the thermore versible systems and the divalent crosslinked gels. In light of various data obtained on these polysaccharides, i t appears that general conclusions relating their chemical structure to their physical properties can now be drawn. Some points are still under discussion and will be investigated i n the near future. Literature Cited 1. Ott, E.; Spurlin, H. M . ; Grafflin, M . W . In High Polymers; Interscience Publishers, 1963; Vol. V , Parts I, II, III. 2. Bikales, Ν. M . ; Segal, L. In High Polymers; Interscience Publishers, 1971; Vol. V , Parts IV, V . 3. Mark, H. F . ; Bikales, N. M.; Overberger, C. G . ; Menges, G . ; Kroschwitz, J . I., Eds. In Encyclopedia of Polymer Science and Engineering; John Wiley: New York, 1985; Vol. 3, pp 60-270. 4. Pigman, W.; Horton, D.; Herp, Α., Eds. In The Carbohydrates; Aca demic: New York, 1970; Vol. II, Chs. 34-36. 5. Marchessault, R. H.; Sundarajan, P. R. In Polysaccharides; Aspinall, G. O., Ed.; Academic: New York, 1983; Vol. 2, p 11-95. 6. Arthur, J . C., Ed. Cellulose Chemistry and Technology; ACS Sym posium Series No. 48; American Chemical Society: Washington, D C , 1983.



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7. Guilbot, Α.; Mercier, C. In The Polysaccharides; Aspinall, G . O., Ed.; Academic: New York, 1985; Vol. 3, pp 209-282. 8. Greenwood, C. T . In The Carbohydrates; Pigman, W.; Horton, D.; Herp, Α., Eds.; Academic: New York, 1970; Vol. IIB, pp 471-513. 9. Imberty, A . Thesis, Grenoble, 1988. 10. Whistler, R. L.; BeMiller, J . N.; Paschall, E . F., Eds. Starch: Chemistry and Technology; Academic: New York, 1984. 11. Muzzarelli, R. A . A . Chitin; Pergamon Press, 1977. 12. Domard, Α.; Rinaudo, M . Int. J. Biol.Macromol.1983, 5, 49-51. 13. Batista, I.; Roberts, G . A . F . Makromol. Chem. 1990, 191, 429-434. 14. Rinaudo, M . ; Domard, A . In Chitin and Chitosan; Skjak-Braek, G . ; Anthonsen, T . ; Sandford, P., Eds.; Elsevier, 1989; pp 71-86. 15. Blanshard, J . M . V . ; Mitchell, J . R. Polysaccharides in Food; Butterworths, 1979. 16. Rinaudo, M . ; Milas, M . Biopolymers 1978, 17, 2663-2678. 17. Rinaudo, M . ; Milas, M . Carbohydr. Res. 1979, 76, 186-196. 18. Tinland, B. Thesis, Grenoble, 1988. 19a. Yamakawa, H.; Fujii, M .Macromolecules1974, 7, 128-135. 19b. T . Odijk. Biopolymers1979,18,3111-3113. 20. Tinland, B.; Maret, G.; Rinaudo, M . Macromolecules 1990, 23, 596602. 21. Tinland, B.; Rinaudo, M . Macromolecules 1989, 22, 1863-1865. 22. Maret, G . ; Milas, M . ; Rinaudo, M . Polymer Bull. 1981, 4, 291-297. 23. Gravanis, G . Thesis, Grenoble, 1985. 24. Kojima, T . ; Itou, T . ; Teramoto, A . Polymer J. 1987, 19, 1225-1229. 25. Milas, M . ; Rinaudo, M . ; Knipper, M . ; Schuppiser, J . L. Macromolecules, 1990, 23, 2506-2511. 26a. Milas, M . ; Rinaudo, M . ; Tinland, B. Polym. Bull. 1985, 14, 157-164. 26b. Kulicke, W . M . ; Kniewske, R. Rheo. Acta 1984, 23, 75-83. 27. Rinaudo, M . ; Milas, M . in Industrial Polysaccharides: Genetic Engi neering, Structure/Property Relations, and Applications; Yalpani, M . , Ed.; Elsevier, 1987; pp 217-228. 28. Gravanis, G . ; Milas, M . ; Rinaudo, M . ; Tinland, B. Carbohydr. Polym. 1987, 160, 259-265. 29. Gravanis, G . ; Milas, M . ; Rinaudo, M . ; Sturman, A . J. C. Int. J. Mol. Biol., 1990, 12, 195-200; 201-206. 30. Harada, T . ; Masada, K.; Fujimori, K.; Maeda, I. Agr. Biochem. 1966, 30, 196. 31. Kasai, N.; Harada, T . In Fiber Diffraction Methods; French, A . D.; Gardner, K. C. H., Eds.; ACS Symposium Series No. 141; American Chemical Society: Washington, D C , 1980; pp 363-383. 32. Fulton, W . S.; Atkins, E . D. T . In Fiber Diffraction Methods; French, A . D.; Gardner, K. C. H., Eds.; ACS Symposium Series No. 141; American Chemical Society: Washington, D C , 1980; pp 384-410. 33. Deslandes, Y . ; Marchessault, R. H.; Sarko, A . Macromolecules 1980, 13, 1466-1471.



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34a. Yanaki, T . ; Norisuye, T . ; Fujita, H. Macromolecules 1980, 13, 14621466. 34b. Yanaki, T . Thesis, Osaka, Japan, 1984. 35. Rinaudo, M . ; Vincendon, M . Carbohydr. Polym. 1982, 2, 135-144. 36. Bo, S.; Milas, M . ; Rinaudo, M . Int. J. Biol. Macromol. 1987, 9, 153157. 37. Itou, T . ; Teramoto, Α.; Matsuo, T . ; Suga, H. Carbohydr. Res. 1987, 160, 243-257. 38. Itou, T.; Teramoto, Α.; Matsuo, T . ; Suga, H. Macromolecules1986,19, 1234-1240. 39a. Kuo, M . ; Mort, A . J.; Dell, A . Carbohydr. Res. 1986, 156, 173-187. 39b. Jansson, B.; Lindberg, B.; Sandford, P. A . Carbohydr. Res. 1983, 124, 135-139. 39c. O'Neill, Ν. Α.; Selvendran, R. R.; Morris, V . J. Carbohydr. Res. 1983, 124, 123-133. 39d. Chowhury, Τ. Α.; Lindberg, B.; Lindquist, U.; Baird, J . Carbohydr. Res. 1987, 164, 117-122. 40. Kang, K. S.; Velder, G . T . ; Cottrell, I. W . Progr. in Ind. Microbiol. 1983, 18, 231-253. 41a. Chandrasekaran, R.; Millane, R. P.; Arnott, S.; Atkins, E . D. T . Car bohydr. Res. 1988, 175, 1-15. 41b. Attwool, P. T . ; Atkins, E . D. T . ; Miles, J.; Morris, V . J . Carbohydr. Res. 1986, 148, C1-C4. 42a. Shi, X . Thesis, Grenoble, 1990. 42b. Milas, M.; Rinaudo, M . ; Shi, X . Polym. Prepr. 1988, 29, 629-630. 43. Milas, M . ; Shi, X . ; Rinaudo, M . Biopolymers 1990, 30, 451-464. 44. Urbani, R.; Brant, D. A . Carbohydr. Res. 1989, 11, 169-191. 45. Campana, S.; Andrade, C.; Milas, M . ; Rinaudo, M . Int. J. Biol. Macro mol. 1990, 12, 379-384. 46. Crescenzi, V . ; Dentini, M.; Dea, I. C. M . Carbohydr. Res. 1987, 160, 283-302. 47. Crescenzi, V . ; Dentini, M . ; Coviello, T . ; Rizzo, R. Carbohydr. Res. 1986, 149, 425-432. 48. Talashek, Τ. Α.; Brant, D. A . Carbohydr. Res. 1987, 160, 303-316. 49. Robinson, G . ; Manning, C. E . ; Morris, E . R.; Dea, I. C. M . In Gums and Stabilizers for Industry; Phillips, G . O.; Wedlock, D. J.; Williams, P. Α., Eds.; IRL Press, 1988; Vol. 4, pp 173-181. 50a. Jansson, P. E . ; Lindberg, B.; Widmaim, G.; Sandford, P. A. Carbohydr. Res. 1985, 139, 217-223. 50b. Jansson, P. E . ; Lindberg, B.; Lindberg, J . Maekawa, E . ; Sandford, P. A. Carbohydr. Res. 1986, 156, 157-163. 51. Frank, F . C.; Keller, A . Polymer Comm. 1988, 29, 186-189. 52. Hikmet, R. M . ; Callister, S.; Keller, A . Polymer 1988, 29, 1378-1388. 53. Rinaudo, M . ; Karimian, Α.; Milas, M . Biopolymers 1979, 29, 13781388. 54. Rinaudo, M . ; Landry, S. Polymer Bull. 1987, 17, 563-565. 55. Rochas, C.; Rinaudo, M . Biopolymers 1984, 23, 735-745.


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1990, 66. 67. 68. 69.

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