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Biomaterials 25 (2004) 795–801
ARTICLE IN PRESS
*Correspondin
4994.
E-mail addres1Present addr
University of Ca
Cathays Park, C
0142-9612/$ - see
doi:10.1016/S014
Molecular interactions in collagen and chitosan blends
A. Sionkowskaa, M. Wisniewskia, J. Skopinskaa, C.J. Kennedyb,1, T.J. Wessb,*,1
aFaculty of Chemistry, N. Copernicus University, 87-100 Torun, PolandbDepartment of Biological Science, Centre for Extracellular Matrix Biology, University of Stirling, Stirling FK9 4LA, UK
Received 12 May 2003; accepted 14 July 2003
Abstract
Molecular interactions between collagen and chitosan (CC) have the potential to produce biocomposites with novel properties.
We have characterised the molecular interactions in CC complexes by viscometry, wide angle X-ray scattering and Fourier
transform infrared spectroscopy. It was found that CC are miscible at the molecular level and exhibit interactions between the
components; X-ray diffraction of CC blends indicate that the collagen helix structure is lost in CC films with increasing chitosan
content. Non-linear viscometic behaviour with decreasing chitosan content is interpreted as evidence of a third structural phase
formed as a complex of CC. The blending of collagen with chitosan gives the possibility of producing new bespoke materials for
potential biomedical applications.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Collagen; Chitosan; FTIR; XRD; Structure; Viscometry; Gelatin
1. Introduction
Collagen and chitin are amongst the most abundantpolymers in life. They both have intrinsic properties thatprovide a strong but manipulable scaffolding structurein many multi-cellular organisms. Collagen and chitosan(CC) (a more soluble derivative of chitin) do not existtogether as blends in nature, but the specific propertiesof each may be used to produce man made blends thatconfer unique structural and mechanical properties. Theuses of relatively low cost, low pollution biomaterialswith specific properties has great potential for thedevelopment of a new generation of prosthetic implants.One of the most promising features of chitosan is itsexcellent ability to be processed into porous structuresfor use in cell transplantation and tissue regeneration. Anumber of researchers have examined the tissueresponse to various chitosan-based implants [1–3]. Ingeneral, these materials have been found to evoke aminimal foreign reaction. In most cases, no major
g author. Tel.: +44-1786-46-7775; fax:+44-1786-46-
s: [email protected] (T.J. Wess).
ess: Department of Optometry and Vision Sciences,
rdiff, Redwood Building, King Edward VII Avenue,
ardiff, CF10 3NB Wales, UK.
front matter r 2003 Elsevier Ltd. All rights reserved.
2-9612(03)00595-7
fibrous encapsulation has been observed [4–6]. Medicaland pharmaceutical applications of chitosan include itsuse in bandages, sponges, membranes, artificial skin,contact lenses, control release drugs, bone diseasetreatment and surgical sutures [7,8].An important aspect of the properties of a blend is the
miscibility of its components. Miscibility in polymerblends is assigned to specific interactions betweenpolymeric components, which usually give rise to anegative free energy of mixing in spite of the highmolecular weight of polymers. The most commoninteractions in the blends are: hydrogen bonding, ionicand dipole, p-electrons and charge-transfer complexes.Most polymer blends are immiscible with each other dueto the absence of specific interactions, however mixturesof collagen with synthetic and natural polymers are ofincreasing interest to scientists and technologists [9,10].Blends of CC have been used for design of polymericscaffolds for the in vitro culture of human epidermoidcarcinoma cells [11], as membrane for controlled release[12–14], and as implant fibres [15,16]. The influence ofchitosan on physicochemical and biochemical propertiesof collagen has been studied previously [17–20]. Ithas been shown that chitosan can modify the proper-ties of collagen when the biological or mechanicalproperties are considered. Moreover, the formation of
ARTICLE IN PRESSA. Sionkowska et al. / Biomaterials 25 (2004) 795–801796
polyanion–polycation complexes between CC wasobserved [19]. From the foregoing, further physicaland structural characterisation of the CC is required todevelop novel biomaterial properties that allow a moreextensive characterisation of the effect of miscibility atthe molecular level. In the study presented here we haveused a variety of complementary biophysical character-isation methods to produce a better insight into CCinteractions.
0
2
4
6
8
10
12
0.29 0.87 1.45 2.03 2.61 3.19 3.79 4.36
nm-1
Inte
nsi
ty
Fig. 1. Wide angle X-ray diffraction linear profiles of collagen (dark
blue), chitosan (green) and a blend (80:20 collagen:chitosan; pink). The
collagen profile displays clear peaks at 0.84 and 3.5 nm�1, which
correspond to the helix–helix interactions of collagen and the turn of
the collagen helix respectively. Chitosan displays a peak at 1.3 nm�1
which is characteristic of hydrated chitosan. The blend only displays
the peak at 2.1 nm�1 which is an amorphous ring present in all
samples.
2. Materials and methods
Collagen was obtained in our laboratory from tailtendons of young albino rats. Briefly, tendons wereexcised and washed in distilled water, and blended in aWaring blender in 0.5m acetic acid, samples were thenspun at 10,000 rpm in a Sorvall centrifuge and thesoluble fraction decanted and lyophilised. Chitosan(360,000 molecular weight) was obtained from Fluka,Switzerland. Polymeric blends were prepared by mixingof suitable volumes of CC in 0.5m acetic acid such thata series of 11 solutions were produced in duplicatecontaining collagen:chitosan blends with a final con-centration of 1 g per litre. Solutions were produced ofblends ranging from 100% chitosan to 100% collagen at10% weight contribution intervals.Polymer films were obtained by casting solutions onto
glass plate or CaF2 spectrophotometric windows. Aftersolvent evaporation, the samples were dried in vacuumat room temperature.Viscosity measurements of collagen, chitosan and
collagen/chitosan blend solutions were performed at20�C in 0.5M acetic acid pH 2.7, using a quartzUbbelohde viscometer. For calculation of miscibility theprocedures as described by Pingping were used [21].IR spectra were obtained using a spectrophotometer
Mattson Genesis II (USA). All spectra were recorded byabsorption mode at 4 cm�1 intervals and 16-timesscanning. Films of a sufficient thickness (typically50 mm) were used to ensure that absorbance valuesobtained in the optimal linear range of the spectro-photometer-up to 1AU in the wavelength range of3200–1000 cm�1 wavenumbers.For wide angle X-ray scattering (WAXS) measure-
ments, the thin film samples were loaded into a samplechamber of the NanoSTAR (Bruker AXS, Karlsruhe)X-ray facility at the University of Stirling. The datacollection procedure used followed that described indetail by Wess et al. [22]. Scattering profiles were takenover 3 h exposures using a sample to detector distance of4.5 cm. Collected data was corrected for camera distor-tions, a background image was subtracted, and imageswere analysed using in-house software. The two-dimen-sional detector output was converted into one-dimen-sional profiles in preparation for analysis.
Principal components analysis (PCA) was employedfor detailed examination of the data. The mathematicalbasis for PCA has been well documented [23–30]. PCArotates the original data into a new set of axes, such thatthe first few axes reflect most of the variations within thedata. By plotting the data on these axes, majorunderlying structures may be spotted automatically.PCA generates basis functions that explains the natureof the variance in the data. There are as many basisfunctions as there are initial variables, and they aresorted in decreasing order of importance, as dictated bytheir associated eigenvalues.
3. Results and discussion
The analytical techniques used to characterise theinteractions of the CC blends are described below.
3.1. X-ray diffraction
Wide angle X-ray diffraction of CC displays char-acteristic X-ray diffraction pattern, in the samplesconsidered here, much of the scattered intensity ispresent in all samples as a broad isotropic amorphouspeak at a spacing of around 0.5 nm, features at around0.85 nm indicative of the 020 reflection from hydratedchitosan were also observable as were the 1.2 nmequatorial spacing from the collagen helix–helix inter-actions as well as the 0.286 nm axial periodicity which isindicative of one turn of the collagen helix. Blending ofCC caused the immediate attenuation of the signalindicative of the collagen helix character, this isdemonstrated in Fig. 1 where the diffraction intensityplots of 100% collagen, 90% collagen:10% chitosan andthe 100% chitosan samples are shown. The plots reveal
ARTICLE IN PRESS
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
100 90 80 70 60 50 40 30 20 10
Percentage of Collagen in Blend
Co
effi
cien
tto
Bas
isF
un
ctio
n2
Fig. 3. Coefficients of basis function 2. As can be observed, at high
collagen content the coefficients are positive. At high chitosan levels
the coefficients are negative. In between there appears to be a plateau
region where the coefficients are approximately zero, indicating that
there is no native collagen or chitosan present in the sample.
A. Sionkowska et al. / Biomaterials 25 (2004) 795–801 797
that low levels of chitosan can alter the diffractionsignature from the collagen helix.
3.2. PCA of WAXS data
PCA of the high angle X-ray diffraction data was ableto distinguish clearly between the collagen, the chitosanand the blends. Basis function 2 gave the clearestdistinction in this regard. Fig. 2 shows the profile ofbasis function 2. The positive peaks in this functioncorrespond to peaks at 1.2 and 0.286 nm molecularspacings, which are characteristic of the collagenintermolecular spacing and the turn of the collagenhelix, respectively. Also prominent is the large diffusepeak at around 5 (A which appears in both CCdiffraction profiles.As the 1.2 and 0.286 nm peaks appear positive in the
basis function profile, samples that display these features(i.e. collagen features) will have positive coefficients tobasis function 2. A peak corresponding to spacings at0.85 nm is shown as negative in the basis functionprofile. This peak is characteristic of the presence ofhydrated chitosan [31]. Samples that display this featurewill have negative coefficients to basis function 2. Fig. 3shows the coefficients for basis function 2 from thespectrum of CC blends, from pure collagen to purechitosan. This figure shows that at 100% collagen, and ablend of 90:10 collagen:chitosan, the coefficients arepositive, indicating the presence of native collagen.From the blend 20:80 collagen:chitosan to 100%chitosan, the coefficients appear negative indicating thepresence of native chitosan.The blends from 80:20 to 20:80 collagen:chitosan
show no correlation to basis function 2, with coefficientvalues of approximately zero. This indicates that thereare no diffraction features from either collagen or
-0.04
-0.03
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.29 1.06 1.84 2.61 3.38 4.17 4.94
nm-1
Basis func. 2 profile
Fig. 2. Basis function 2 from PCA of the WAXS data. Positive peaks
occur at 0.84 and 3.5 nm�1; a negative peak appears at 1.2 nm�1. The
positive peaks can be attributed to the presence of collagen features in
the diffraction profiles. The negative peak can be attributed to the
presence of chitosan in the samples.
chitosan in these blends, the major contributor beingthe amorphous ring. Examination of the diffractionprofiles of these samples confirms that the peaks at 1.2,0.85 and 0.286 nm are not present, indicating that thereis no native collagen or chitosan in these samples.
3.3. Viscosimetry
According to the classical Huggins equation [21], usingthe data obtained here, the concentration-dependentspecific viscosity, Zsp can be obtained. The changes inintrinsic viscosity of blends with collagen content isshown in Fig. 4, where the viscosity of blends is seen to begreater than that of either collagen or chitosanalone. Polymer mixtures might exhibit positive ornegative deviations from the defined ideal behaviourbecause of the existence or absence of interaction. The
0
0.5
1
1.5
2
2.5
3
3.5
0 20 40 60 80 100
Percentage of Chitosan in Blend
gll
Fig. 4. Concentration dependence of intrinsic viscosity. The limiting
viscosity number (gll) is a measure of the viscosity of the samples. As
can be observed, the blends (’) display a higher viscosity that either
the collagen (E) or the chitosan (m). This indicates that a third
component exists in the blends that can be accounted for by
interactions between the individual components generating a new,
viscous substance.
ARTICLE IN PRESS
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
100015002000250030003500
Wavenumber [cm-1]
Ab
sorb
ance
Fig. 5. FTIR spectra of collagen (red) and chitosan (black). There is a
great deal of difference between the two samples in terms of peak
intensity and position. In particular, the collagen spectrum displays a
larger amide I peak (1662 cm�1), smaller amide II peak (1567 cm�1)
and greater amide III peak (1240 cm�1) than the chitosan spectrum.
Chitosan displays greater absorbance than collagen in the range of
2000–4000 cm�1, with characteristic bands at 3352, 2932 and
2890 cm�1, and intense peaks at 1563 and 1414 cm�1.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
100015002000250030003500
Wavenumber [cm-1]
Ab
sorb
ance
Fig. 6. FTIR spectra of CC blend (80% collagen:20% chitosan; dark
blue) with theoretical spectra (aqua) and difference profile (pink). In
these spectra, it is clear to see that the experimental data displays a
greater intensity than the theoretical data, particularly in the region of
1000–1700 and 2300–3700 cm�1.
A. Sionkowska et al. / Biomaterials 25 (2004) 795–801798
polymer–polymer miscibility can be determined by aparameter Db this describes the deviation of viscosityfrom that expected by proportions of the individualcomponents signify. The intrinsic viscosity of the blendsare higher than either of the individual polymers, andaccording to Pingping [21] the parameter being Db > 0;indicates polymer miscibility.
3.4. Fourier transform infrared spectroscopy
The Fourier transform infrared spectroscopy (FTIR)spectra of chitosan [11] depict characteristic absorptionbands at 3352, 2932 and 2890 cm�1, which represent the–OH, –CH2 and –CH3 aliphatic groups, and bands at1563 and 1414 cm�1 which represent the NH-groupbending vibration and vibrations of –OH group of theprimary alcoholic group, respectively. The amino grouphas a characteristic absorption band in the region of3400–3500 cm�1, which is masked by the broad absorp-tion band from the –OH group. The shoulders at1635 cm�1 represents the C=O groups and suggestschitosan is a partially deacetylated product.Collagen displays bands at 1658, 1554 and 1240 cm�1,
which are characteristic of the amide I, II and III bandsof collagen [32,33]. The amide I absorption arisespredominantly from protein amide C=O stretchingvibrations, the amide II absorption is made up of amideN–H bending vibrations and C–N stretching vibrations(60% and 40% contribution to the peak, respectively);the amide III peak is complex, consisting of componentsfrom C–N stretching and N–H in plane bending fromamide linkages, as well as absorptions arising fromwagging vibrations from CH2 groups from the glycinebackbone and proline side-chains. The main amide IIIpeak is observed at 1240 cm�1, with smaller peaks seenat 1204 and 1283 cm�1. FTIR spectra corresponding toCC are shown in Fig. 5.In the miscible blends of CC, the changes in FTIR
spectra are judged by comparing the appropriatelyscaled addition of CC in a theoretical blend to that inthe experimental blend and the production of differenceprofiles. Examples of these can be seen in Fig. 6. Themost obvious deviations between theoretical and experi-mental composites are in the modes of vibrationsassigned to amide groups. The peaks corresponding tothe amide I peak displays a change as the level ofcollagen is reduced relative to the level of chitosan in asample. As the level of collagen in the sample decreases,the amide I peak decreases also, until it is present only asa small shoulder to the amide II peak. The amide II peakremains relatively consistent with varying levels of CC.The intensity of the peak shows a general increase as thelevel of chitosan is increased in the sample relative to thelevel of collagen. When there is no collagen left in asample (e.g. 100% chitosan) the amide II peaks displaysa shift from 1554 to 1564 cm�1. The peaks correspond-
ing to the amide III peak of collagen display a loss ofintensity with increasing content of chitosan anddecreasing collagen content. The peak at 1283 cm�1
persists until the relative amounts of CC are equivalentin the sample. The peaks at 1204 and 1240 cm�1 persistin the samples until 30% of the sample material iscollagen. Increasing the level of chitosan in the samplebeyond 70% brings about a loss of these two peaks.
3.5. PCA of FT-IR data
PCA was able to define a trend in the data from 100%collagen to 100% chitosan. Fig. 7 shows the basisfunctions of PCA carried out on the FTIR data. Basisfunction one shows positive peaks at 1664 (with ashoulder at 1637), 1283, 1240 and 1204 cm�1, which arerepresentative of the amide I and III peaks of collagen.The basis function also shows small negative peaks at1417 and 1583 cm�1. These peaks are due to the shifting
ARTICLE IN PRESS
-0.15-0.1
-0.050
0.050.1
0.150.2
0.25
999
1211
1423
1635
1847
2060
2272
2484
2696
2908
3120
Wavenumber [cm-1]
Fig. 7. Basis functions 1(dark blue), 2(pink) and 3(green) from PCA of
FT-IR spectra of the collagen/chitosan blends. The greatest level of
variance in the samples is observed in the region of 1000–1700 cm�1.
The positive peaks on basis function 1 can be accounted for by the
presence of the amide I and III peaks.
-0.4
-0.2
0
0.2
0.4
0.6
0 30 60 100
Percentage of Chitosan in Blend
Co
effi
cien
t
Fig. 8. Coefficients of the blends to basis function 1. As the level of
collagen decreases and the level of chitosan increases in the blends,
there is a clear trend present; the coefficients change from positive to
negative. The reason for this can be extrapolated from basis function 1
and the data. Basis function 1 shows intense positive peaks at the
amide I and III peaks. As the level of collagen decreases in the samples,
so do the intensities of these peaks. Thus, the coefficients decrease also.
A. Sionkowska et al. / Biomaterials 25 (2004) 795–801 799
of peaks from 1457 to 1413 cm�1 and 1554 to 1654 cm�1
from collagen to chitosan, which give rise to greaterintensities in these areas. Fig. 8 shows the trend of thechange in coefficients from collagen to chitosan throughthe blends. When collagen is present as the majorconstituent of the blend, the samples are positivelycorrelated to basis function 1; when chitosan is themajor constituent of the blend the samples arenegatively correlated to the basis function 1.
3.6. PCA of FT-IR difference maps
Difference maps were obtained by subtracting experi-mental FT-IR profiles from the theoretical profiles. Thetraces of the maps display a range of variations betweenthe real and theoretical data. In all cases the generaltrend of the difference profiles is a positive one,indicating that real data displays a greater level of
absorbance than theoretical data; however, the differ-ences are specific and vary from blend to blend. PCAwas employed to examine in greater detail the variationsin the samples.PCA was able to discern a number of peaks in the
samples that constitute a great deal of the changes thatare occurring in the sample. The trend for thecoefficients of the basis functions are of interest. Basisfunction 1 shows no trend except for one sample (50:50collagen:chitosan). Basis function 2 showed a generalincrease in coefficients, from negative to positive, as therelative contribution of chitosan to collagen increased.Basis function 2 is of interest as its main features were
positive peaks at 1552 and 1452 cm�1. These peaks areconsistent with the positions of the amide II peak and ashoulder peak that was observed in experimental but nottheoretical data that corresponds to N–H bending. Thispeak is only present when chitosan make up more than50% of the blend, as is a peak at 1612 cm�1, whichrepresents exocyclic C=C bonds.
4. Discussion
Previous research by Taraval and Domard [17,19] andDomard and Taraval [18] indicated that CC interactionsare polyelectrolytic, and from interpretation of datapostulated the presence of a polyanion/polycationcomplex and a competing collagen gelation process.They also proposed a physical separation of collagenmicrogels encapsulated by the complex, and thatcomplexes contain denatured collagen. Their FTIRevidence also points to the presence of a novel hydrogenbonding in the CC interactions and they concluded thatthe collagen in the complexes is denatured. A recentstudy of CC scaffolds [11] also indicated the viability ofusing CC miscible blends as scaffolds in bioprostheseshowever this study utilised glutaraldehyde linked blendsthat may stabilse the collagen helix and cannot becompared directly with evidence presented here. Theconclusion of the physical characterisation made by theShanmugasundaram study concluded that CC blendsboth retained the characteristics of the parent species,however the crosslinking of complexes with glutaralde-hyde, means that a direct comparison with resultspresented here cannot be made. It is possible that thepresence of glutaraldehyde is responsible for themaintenance of the collagen helical structure andthe tri-phasic system of collagen–gelatin–chitosan isminimised.The data presented here indicate that the CC blend is
miscible and alters the molecular properties of thecomponents. Of particular note is the deviation of theviscosimetric measurements toward a more viscoussolution of the blends. This indicates a specific interac-tion between CC that cannot be accounted for by a
ARTICLE IN PRESSA. Sionkowska et al. / Biomaterials 25 (2004) 795–801800
simple biphasic mixture. Our hypothesis is that a thirdcomponent is responsible for the enchanced viscosity ofblends with an optimum at around 50% collagen and50% chitosan (by weight). Since both components inisolation have a lower viscosity, the presence of a thirdcomponent induced by the action of chitosan oncollagen can account for this behaviour. Since gelatinhas a higher intrinsic viscosity than collagen, it points toa more disordered form of collagen in the complex.In collagen the –OH groups of hydroxyproline are
involved in hydrogen bonds between chains, whileinteractions between other side groups are thought tobe important in formation of fibrils from a number ofindividual molecules. These side groups are capable offorming hydrogen bonds with –OH and NH2 groups inchitosan. Moreover, the end group –COOH and NH2 incollagen-may also form hydrogen bonds with –OH and–NH2 groups from chitosan, as chitosan possesses largenumbers of –OH groups. The long chain of chitosan canwind around the collagen triple helix; the entanglementof two different macromolecules (CC) may form acomplex, which have much higher viscosity than singlecomponents.Additionally, CC may be bonded ionically. These
molecules are capable of forming complexes withoppositely charged ionic polymers, particularly thecationic polysaccharide chitisan and anionic –COOHgroup in collagen. These interactions may form the basicof a new materials approach based on the blends of CC.Wide angle X-ray diffraction data also indicates the
loss of the collagen helical character in the blends as wellat the loss of any collagen–collagen interactions that areusually conspicuous in the dry phase. From the X-raydiffraction data, it is attractive to speculate thatmiscibility induces a deviation of the collagen helixstructure to a more disordered phase, similar to gelatin.Production of such a third phase in the blend would beconsistent with increased viscosity and a loss of helicaldiffraction peaks. The presence of gelatin in X-raydiffraction is somewhat of a ‘negative proof’, it isusually characterised by the absence of the characteristichelical features and the relative increase of scattering at5 (A [34]. Examination of the PCA of the X-raydiffraction from pure collagen, chitosan and blendsshows that the fractional contribution of component 2(Fig. 2) has a relationship with the blend composition (apositive correlation as a function of collagen content),however there is also evidence of a plateau regionindicating that a low percentage contribution ofchitosan to the blend can significantly reduce the X-ray diffraction contribution from helical collagen.Although PCA functions are often not directly inter-pretable, it is possible to see that this function containsnegative and positive peaks that correspond to theprincipal diffraction features of CC films. The firstprincipal component has a linear relationship to the
blend composition, but contribution of this componentto the 100% collagen film diffraction profile issignificantly different. This also adds to the evidencethat blend formation produces a strong deviation from aformal collagen structure and substantiates the evidencefrom Taravel and Domard [17,19] and Domard andTaravel [18].The evidence of FTIR measurements also points to
the formation of new resonances that cannot beaccounted for in a simple mixture. The indications arethat a new composite is being formed, as shown by thecomparison of theoretical composite FTIR spectra andspectra from experimental blends. If there was no trendin the data, and no difference between the calculatedand observed results, then it would be a fair assumptionthat there is no interaction between the constituents ofthe blend, and that the spectra generated are simplyfrom the CC existing together in the blend but notinteracting. However, PCA was able to determine anumber of areas in the spectra that were different, anddisplayed at what respective levels of CC thesedifferences became apparent. This indicates that theCC are interacting with each other, forming a newcomplex. In addition, if the collagen was denatured andexisted as gelatin along side the chitosan, a shift in peakintensity from 1560 to 1530 cm�1 would be expected[35–37]. This is not observed, indicating that a newstructure between the CC is formed.
5. Conclusion
Our overall conclusion is that collagen chitosanblends are miscible and interact at the molecular level,new hydrogen bonding networks appear to alter thecollagen helical character and therefore the overallphysical parameters of the blend. Our explanation ofthe changes in viscosity is through a triphasic systemwhere the CC blend contains a third ‘gelatin’ like phase.The presence of a collagen substrate without collagenhelical characteristics may be beneficial for biomaterialdesign. By careful alterations in the composition ofblends and conditions of blend formation, it may bepossible in the future to alter the levels of collagen orderin the complex and thus alter the resorbtive, hydrationand biomechanical properties.
Acknowledgements
Financial support from NATO GRANT 978595 theScientific Research Committee (KBN, Poland, grant no.3 P05A 06922) and the SHEFC JREI fund is gratefullyacknowledged.
ARTICLE IN PRESSA. Sionkowska et al. / Biomaterials 25 (2004) 795–801 801
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