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JOURNAL OF MATERIALSSCIENCE: MATERIALS N MEDICINE 5(1994) 872-876Blends of hyaluronic acid derivatives withethylene vinyl alcohol copolymers as potentialbiornaterialsM . S E G G I A N I , L. L A Z Z E R I , P . G I U S T I , N . B A R B A N I , L. L E L L IDip artim ent o di Ingegneria C himica Chim ica Industriale e Scienza dei Materiali Universita diPisa Via Diotis alvi 2 1-56126 Pisa ItalyM . P A L L AC.N.R. Centro Studi Processi Ionici Via Diotis alvi 2 1-56126 Pisa ItalyM . T A V E R IFidia Research Via Ponte della Fabbrica 3/ 4 3503 1 Aba no Terme Ita ly

Films were prepared by solution casting from blends of hyaluronic acid derivatives andethylen e-vinyl alcoho l copolymers. A chem ico-physical and biological characterization wascarried out on these bioa rtificial m aterials made of synth etic and biological polymers. Themorphological and chemical properties of the films were investigated by scanning electronmicroscopy and differential scanning calorimetry. The transport properties of these films weretested in liquid systems to evaluate their possible use in dialysis and/or haemodialysis. Thebioco mp atibi l i ty wa s investigated by a haemoco mpa tibi l ity test based on the contact activationof plasma prekallikrein. No particular interaction between the two components was observed.The results of the permeation tests were compared with those obtained using commercialproducts such as Cuprophane and poly(acrylonitrile) membranes. These tests indicate that thepermeability of the blends decreases as the content of the synthetic polymer increases. Thegood haemocompatibility of these materials suggests their possible use as biomaterials.

1 IntroductionIn the last few years great attention has been devotedto the preparation and the characterization of poly-meric blends in order to obtain new materials withdifferent properties from those of the two originalpolymeric components, and variable as a function ofthe composition.Blends of natural and synthetic polymers are alsothe object of great interest in the biomaterial field forpossible biomedical applications. Natural polymers(biopolymers) show good biocompatibility, but poormechanical properties and often high productioncosts. On the other hand, synthetic polymers have, ingeneral, much better physical-mechanical propertiesand low production costs, but their biocompatibility isoften inadequate. Thus biopolymer/synthetic polymerblends were prepared to obtain new bioartificial [1]polymeric materials that could combine the goodmechanical properties of the synthetic componentwith the biocompatibility of the natural polymer.Bioartificial materials have been prepared using asbiopolymers, hyaluronic acid (HA) and its derivatives(HYAFF), and as synthetic components, commerciallyavailable polymers such as ethylene-vinyl alcohol co-polymers (tradename, Clarene). These copolymersare already in use in the biomedical field owing to theirhydrophilic properties and high biocompatibility.87

Since hyaluronic acid is hydrosoluble, the HA-basedfilms redissolve when in contact with biological fluids.Therefore water-insoluble derivatives of HA, obtainedby esterifying the carboxylic groups of HA with benzylalcohol [2], have been used in this work.

This paper details the preparation and chemico-physical characterization of benzyl hyaluronic acidesters (HYAFF)/Clarene blends.The transport properties of HYAFF/Clarene filmsobtained by casting were also studied to evaluate theirpossible use in haemodialysis. Haemocompatibilitytests were carried out on both the pure polymericcomponents and the bioartificial materials.

2 Experim ental procedures2.1. MaterialsBenzyl hyaluronic acid esters with degree of esterific-ation of 75% (HYAFF1 lp75) and 100% (HYAFF11)were supplied by FAB S.p.A, Italy.Clarene with different ethylene content: Clarene L6 (low ethylene content: 29 mol %) Clarenee P10 (moderate ethylene content:

36 tool %) Clarene R20 (high ethylene content: 40 mol %)were supplied by Solvay & C.i.e., Italy.

0957-4530 1994 Chapman Hal l

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2 2 Films preparationFor each type of HYAFF and for each type of Clarene,a 1 (w/v) solut ion in dimethylsulfoxide DMSO) wasprepared. The formulated solutions were mixed at80 C to obtain blends having the following composi-tion in polymer weight ratio: 20/80, 50/50, 80/20 (thefirst number refers to HYAFF content). The blendsobtained were as follows:

HYAFF11/Clarene L6 (20/80, 50/50, 80/20)HYAFF11/Clarene P10 (20/80, 50/50, 80/20)HYAFF11/Clarene R20 (20/80, 50/50, 80/20)HYAFF1 lp75/Clarene L6 (20/80, 50/50, 80/20)HYAFF1 lp75/C larene P10 (20/80, 50/50, 80/20)HYAFFllp75/Clarene R20 (20/80, 50/50, 80/20)

After stirring, films were obtained by solution castingat 60C. HYAFF and Clarene homopolymers filmwere also prepared by the same method. The filmswere stored in distilled water until use. The thicknessof the water-swollen membranes was measured using amicrometer with an accuracy of + 1 I-tin. The averagethickness of the wet films was about 20 ~m.

3 Appar atus and techniqu es3 1 Thermal analysisDifferential scanning calorimetry (DSC) measure-ments were performed with a Perkin Elmer DSC-7, ina N 2 atmosphere with a heating rate of 10 C/min inthe range 30 to 190 C. The thermal properties of theblends and homopolymers were analysed in one hea-ting scan. The calorimetric melting temperature, Tm,and the fusion heat, AHf, of Clarene were determinedfrom the maximum and the area of the melting peak,respectively. Within the explored temperature rangethe biopolymer does not show any relevant thermaltransition.

diffusion cell as follows [4]:PAJs -- h (Cd - Cr)

where P is the solute permeability, A is the membranearea, h is the swollen membrane thickness, Ce and C rare the solute concentrations in donor and receptorchambers of the diffusion cell, respectively. The aboveequation has been used to calculate the solute per-meability, P, provided that the concentration gra-dient, the flux under steady-state conditions and theswollen membrane thickness are known.

3 4 Haemocompatibility analysisThe activation of human plasma prekallikrein (PKK)to kallikrein (KK), induced by the contact of bloodwith foreign materials, is a useful in vitro haemo-compatibility test [5]. The activation of PK K to KK isdetermined by the proteolytic reaction between KKand the chromogenic substrate H-H-Pro-Arg-pNa (S-2302 Kabi Diagnostica). The test was carried out onthe following materials: borosilicate glass (as a high-activation reference material), silicone (as a low-activa-tion reference material), ethylene-vinyl alcohol copol-ymers (Clarene L6, Clarene P10, Clarene R20), benzylhyaluronic acid esters (HYAFF11 and HYAFF1 lp75)and HYAFF/Clarene blends.

3 2 Morphological analysisThe surfaces and the cross-sections of the films wereanalysed with a Jeot T 300 scanning electron micro-scope (SEM). The films were freeze-fractured in liquidN z and sputter-coated with gold before SEM analysis.

3 3 Permeability measurementsPermeability measurements were performed at 37 Cusing solutes with different molecular weight, includ-ing sodium chloride NaC1 (MW 58 D), vitamin B12(MW 1355D) and bovine albumin (MW 69000D).The experimental apparatus used for solute permeabil-ity measurements is described in details elsewhere [3].It essentially consists of a stirred diffusion cell made upof two chambers separated by the membrane to betested. The solute concentration on the permeate sideof the membrane gradually increased until it becameconstant (i.e. until s teady-sta te conditions were ob-tained). When the steady state is reached, the flux ofsolute across the membrane, Js, can be written in termsof solute concentrations in the two chambers of the Figure SEM images of HYAFFllp75/Clarene L6 80/20 film(a) surface and (b) cross-section.

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4 Resul ts4 1 Thermal characterizationThe Clarene thermal transit ion varied with the com-positi on o f the blen ds a nd with the degree of esterific-a t ion of HYA FF. The H YA FF homopolymer f i lmsshowed no significant transition in the explored tem-peratu re i ange (30 190C). The blends show ed novariation s with regard to the melting point, Tm, of thecrystall ine polymeric compone nt, while the enthalpy offusion AHf decreased with increasing HY AF F content.Ma ybe this is due to morphological effects [6 -8] thatreduce the crystallinity degree of the synthetic poly-mer.

4.2. SEM characterizationSEM images showed structures gradually changingfrom homog eneous (Fig. la , b) to heterogeneous, withevident phase separa tion (Fig. 2a, b) depe nden t on thecontent of Clarene. H omo gene ous structures, asshown in Fig. 1, refer to blends with higher values ofA H f ; heterog eneou s structures, as sh own in Fig. 2,refer instead to blends with lower values of AHfindicating poor miscibil i ty between the two compon-ents in the blend.

4 3 P e r m e a b i l i t y m e a s u r e m e n t sThe permeabili ty coeff icients of HYAFF/Clarenemembranes are shown in Fig. 3, and Fig. 4 for NaC1,

vit. B12 and bovine albumin. The permeability values,P (cm /s), are normalized to a thickness o f 20 gm. Theresults are compared with those obtained using com-mercial products such as Cuprophane and polyacryl-onitrile (AN69) membranes. These results show thatpure HYAF F1 lp75 and HYA FF11 m embranes have

t~4-

Ea. 2

AN 69

Cuprophane

0 0(a) 20 4 0 60 80 100HYAFF11 content (wt %)

10

f 8x

26

2.0

4b 1.5x

E~ 1.0

~ 0 . 511_

(b)2.50.

AN 69

Cuprophane

20 40 60 80 100HYAFF11 content (wt %)

Figure SEM images of HYAFFllp75/Clarene L6 20/80 f ilm(a) surface and (b) cross-section.874

2.25-

E0.5

E~, 0.2513

AN 69

Cuprophane

20 z~0 60 80 100(c) HYAFF11 conte nt (wt %)Figure3 Permeability of HYAFFll/Clarene blend membranesversus percentage weight of HYAFFll in the blend, for (a) NaC1,(b) vit. B12 and (c) bovine albumin. (e H YA FF ll/ Cl are ne L6, HYA FF11/Clarene P10; HYAF F11/Clarene R20).

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7- 600

O 0(a)

, ./7 *4 0 doHYAFF1 p75 co ntent (wt %)

S

x 5-E '~ 4~ 3 -55B2-

AN 69/C u p r o p h a n e

100

1.2

m-- 1,0 -bX.~ 0.8-

v

0.6~ 0 . 4 -Ed~

D 0.2 A N 69C u p r o p h a n e

0 T 720 do 8 o lOO(b) HYAFF11 con ten t (wt %)

~ ' 4O

X

E~2d~Eo - 1

0 0c)

AN 69

C u p r o p h a n e. . . . .20 40 60 80HYAFF11 con tent (w t %) 100

Figure 4 P e r m e a b il i ty o f H Y A F F 1 l p 7 5 / C l a r e n e b l e nd m e m b r a n e sv e rsu s p e rc e n t a g e we i g h t o f HY AF Fl t p 7 5 i n th e b l en d , fo r(a) NaC1, (b ) v i t. B12 and (c) bovine a lbum in . (eH Y A F F l l p 7 5 / C l a r e n e L 6 , H Y A F F l l p 7 5 / C l a r e n e P 1 0, H Y A F F 1 l p 7 5 / C l a r e n e R 20 ).

good permeability while pure Clarene membraneshave poor permeability to all tested substances (Figs. 3and 4). Consequently, the permeability of blends de-creases drastically with increasing Clarene content,and this is due to well-known barrier properties ofethylene-vinyl alcohol copolymers.

500.~ 400D- - 3 0 0