HYALURONAN
`Volume 1 - Chemical, Biochemical and Biological Aspects
`
`Editors:
`JOHN F. KENNEDY BSe, PhD, OSe, EurChem CChem FRSc, CBiol FIBiol, FCIWEM, FCMI, FIFST
`Director of Birmingham Carbohydrate and Protein Technology Group,
`SchoolofChemical Sciences,The University ofBinningham, BirminghamB15 2IT, England,UK,
`Director of Chembiotech Ltd,
`University of Birmingham Research Park, Birmingham B15 2SQ, England, UK,
`Director of Inovamed Ltd,
`Chembiotech Laboratories, University of Birmingham Research Park, Vincent Drive,
`Birmingham B15 2SQ, England, UK, and
`Professor of Applied Chemistry,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LUI 2AW, Wales, UK
`
`GLYN O. PHILLIPS BSe, PhD, OSe, HODOSe, HODLIB, CChem FRSC
`Chairman of Research Transfer Ltd,
`Newtech Innovation Centre,
`Professorial Fellow,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LUI 2AW, Wales, UK, and
`Professor of Chemistry,
`The University of Salford, England, UK
`
`PETER A. WILLIAMS BSe, PhD, CChem FRSC
`Director of the Centre for Water Soluble Polymers,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`LUI 2AW, Wales, UK,
`Director of the Centre for Advanced and Renewable Materials at
`Institute and University of Wales, Bangor,
`The North East Wales Institute of Higher Education, P1as Coch, Mold Road, Wrexham, Clwyd,
`LUI 2AW, Wales, UK
`Professor of Polymer and Colloid Chemistry,
`The North East Wales Institute of Higher Education, Plas Coch, Mold Road, Wrexham, Clwyd,
`. LL11 2AW, Wales, UK
`
`the North East Wales
`
`Guest Editor:
`VINCE C. HASCALL PhD
`Co-Direetor of the Orthopaedic Surgery Musculoskeletal Research Center,
`Department of Biomedical Engineering ND-20, Lerner Research Institute. Cleveland Clinic
`Foundation, Cleveland, Ohio 44195, USA
`Adjunct Professor
`Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA
`Adjunct Professor
`Department of Biochemistry, Rush Presbyterian S1. Lukes Medical Center, Chicago, Illinois,
`60612 USA
`
`WOODHEAD PUBLISHING LIMITED
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`ALL 2029
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`Published by Woodhead Publishing Ltd, Abington Hall, Abington,
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`www.woodhead-publishing.com
`
`First published 2002
`
`© 2002, Woodhead Publishing Ltd
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`

`

`RHEOLOGY OFHYALURONAN PRODUCTS
`
`Ove Wik, Bengt Agerup and Hege Bothner Wik
`
`Q-Med AB. Seminariegatan 21. S-752 28 Uppsala, Sweden
`
`ABSTRACT
`
`Various modified (stabilized or cross-linked) hyaluronan products used for tissue
`augmentation were examined by rheometry. Five products - Restylane Fine Lines,
`Restylane, Perlane, Hylaform and Dermalive - exhibited typical gel-like behaviour to
`varying degree after examination of the viscoelastic response as a function of frequency.
`This suggests that all products contain hyaluronan with permanent linkages between
`polysaccharide chains. One product (Rofilan) claimed to be a 'Hylangel' containing
`'cross-linked' hyaluronan at a concentration of 20 mg/ml exhibited a behaviour typical
`of hyaluronan solutions. The results demonstrate that
`this product contains free
`hyaluronan chains with a molecular weight of 2 million.
`
`KEYWORDS
`
`Rheology, viscoelasticity, cross-link, tissue augmentation.
`
`INTRODUCTION
`
`Hyaluronan is intimately linked with rheology. The remarkable viscous and elastic
`behaviour of hyaluronan solutions in general and of body fluids such as synovial fluid
`in particular has been studied for decades. After the pioneering work by Balazs 1-2,
`modem rheometers were utilised for the subsequent development of hyaluronan
`products for use in e.g. ophthalmology and joint disorders. Fittingly, the multifaceted
`use ofhyaluronan in medicine has been described by Balazs as "viscosurgery" 3.
`In recent years hyaluronan has been modified by means of various types and varying
`degree of cross-linking 4-6. As a consequence of these modifications, products with quite
`different
`rheological behaviour have been marketed. We have performed basic
`rheological
`studies on some commercial products containing modified, gel-like
`hyaluronan derivatives, and report data on the viscous and elastic properties as a
`function of frequency.
`
`MATERIALS AND METHODS
`
`Samples
`
`The following samples of modified hyaluronan products used in tissue augmentation
`were used. Lot numbers are given in parenthesis. Restylane Fine Lines (6083),
`Restylane (5922) and Perlane (6106) were obtained from Q-Med AB, Uppsala, Sweden.
`Hylaform (A709) was obtained from Biomatrix Inc., Ridgefield NJ, USA. Dermalive
`(VR26060) was obtained from Dermatech, Paris, France. Rofilan (4991) was obtained
`from Rofil Medical Nederland B.V., Breda, Netherlands.
`The products manufactured by Q-Med AB (Restylane Fine Lines, Restylane and
`Perlane) contain non-animal stabilised hyaluronan (NASHA) at a concentration of 20
`
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`

`

`202
`
`Rheological behaviourofhyaluronan
`
`mg/m!. These products are specifically designed for tissue augmentation in different
`layers of the skin. Information on the other products was obtained from the packaging
`inserts.
`Hylaform contains Rylan B at a concentration of5.5 mg/m!.
`Dermalive is a suspension of non resorbable fragments of acrylic hydrogel and a
`solution of slightly cross-linked hyaluronan. The product contain 200 mg/ml of acrylic
`fragments and 14.4 mg/ml ofhyaluronan.
`Rofilan is stated to be a 'Hylangel' containing 'cross-linked' hyaluronan at a
`concentration of 20 mg/m!.
`
`Rheological characterisation
`
`Bohlin VOR Rheometer System (Bohlin Reologi AB, Sjobo, Sweden) with the
`Windows compatible Millenium software was used for rheological characterisation. The
`samples were more or less gel-like and therefore all samples were studied in the
`oscillation mode by recording the response to varying frequency and strain. All
`experiments were performed at 25°C. When sufficient amount of sample (about 3 m!)
`was available the cup and bob measurement system C14 was used. Otherwise the cone
`and plate system CP5/30 (diameter 30 mm, cone angle 5°) was used. Precautions were
`taken to exclude possible effects of the formation of a dry hyaluronan film on surface
`layers. The strain-dependent response was recorded to ascertain determination of the
`viscoelastic response within the linear region.
`
`RESULTS AND DISCUSSION
`
`The viscoelastic response is shown in Fig. 1 where the elastic modulus (0') and
`viscous modulus (0") are plotted as function of frequency. All data were recorded at
`low enough strain to ascertain recordings in the linear region.
`
`Elastic modulus, G' (Pa) - - -
`Viscous modulus, G" (Pa) ••• _•••
`100 0 , . . . . . . - - - - - - - - - - - - - - - ,
`
`Elastic modulus, G' (Pa)
`Viscous modulus, G" (Pa) ••••••.
`1000 . , . . . . . . - - - - - - - - - - - - - - - ,
`
`100
`
`10
`
`• :0:: : ~: ~ : ~ : : e:=0 =:0:: : .-"
`.~.
`
`••
`
`100L__- - , - - -........-4I~~~~
`
`10
`
`• Reslyl.ne Fine Lines
`• Reslyl.ne
`o Perlane
`
`• Hyl.form
`o Derm.llve
`... RoliI.n
`
`.001
`
`.1
`.01
`Frequency (Hz)
`
`1
`
`10
`
`.001
`
`.01
`
`.1
`Frequency (Hz)
`
`1
`
`10
`
`Figure 1.
`
`The elastic and viscous moduli as a function of frequency
`plotted on log-log scale.
`
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`

`

`Rheology ofhyaluronan products
`
`203
`
`The classical fashion of describing the viscoelastic properties shown in Figure 1 is,
`though proper from a rheological point of view, somewhat complicated when discussing
`the rheological properties with the end user of products. Therefore, EA Balazs
`introduced a simple, yet very illustrious way to present and describe the viscoelastic
`properties of hyaluronate solutions and products.
`In most instances the interesting aspect of the viscoelastic properties as shown in
`Figure 1 is the proportion between elasticity and viscosity. A simple relationship
`describing the viscoelastic properties is obtained by calculation the percentage elasticity
`(Elasticity, % in graphs below) as follows:
`
`G'
`Elastic modulus· 100
`Elasticity ('Yo) = Elastic modulus + Viscous modulus = G' + G" • 100
`
`The same information is, of course, also obtained from the frequency dependence of
`the phase angle. However, the response for a viscoelastic sample changing from viscous
`to elastic gives a change in the phase angle from 90° to 0°, whereas the introduced
`parameter Elasticity (%) changes from 0 to 100. These data are plotted in Fig. 2
`demonstrating that most products are predominantly elastic at all frequencies, whereas
`Restylane Fine Lines change behaviour in a somewhat complicated fashion and Rofilan
`is viscous at low frequencies and elastic at high frequencies.
`From the viscoelastic response the dynamic viscosity - 11' - may be calculated. The
`frequency dependence of the dynamic viscosity coincides with the shear rate
`dependence of the shear viscosity according to the Cox-Merz rule (Fig. 3). The results
`demonstrate a gel-like behaviour with a continuously increasing viscosity even at low
`
`Elasticity ("!o)
`100 r - - - - - - - - - - - - - - - - ,
`
`Dynamic viscosity, Il' (Pa-s]
`
`80
`
`60
`
`40
`
`20
`
`10000
`
`o Perlane
`• Restylane
`• Restylane Fine Lines
`o Oermalive
`• Hylaform
`.... Rofilan
`
`• Hylaform
`• Restylane
`o Perlane
`o Dermallve
`• Restylane Fine Lines
`....Rofilan
`
`10
`
`.01
`
`1
`.1
`Frequency (Hz)
`
`10
`
`.01
`
`.1
`Frequency (Hz)
`
`1
`
`10
`
`Figure 2. The percentage elasticity (see
`formula in text) as a function
`of frequency plotted on lin-log
`scale
`
`Figure 3. The dynamic viscosity as a
`function of frequency plotted
`on log-log scale
`
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`

`

`204
`
`Rheological behaviourofhyaluronan
`
`frequencies for all samples except Rofilan. For the latter product a Newtonian, constant
`zero shear viscosity at low frequencies was recorded. The zero shear viscosity of
`hyaluronan is dependent on the concentration and molecular weight
`7. Using the
`published formulas Rofilan was found to contain hyaluronan with a molecular weight of
`2 million using the zero shear viscosity obtained (80 000 Pa • s) and the concentration
`(20 mg/m1) stated.
`
`CONCLUSIONS
`
`The viscoelastic data obtained demonstrated that most products exhibit a gel or gel(cid:173)
`like behaviour to varying degree with an almost constant response independent on
`frequency. A slight variation in response was observed for Hylaform. Restylane Fine
`Lines showed a mixed behaviour changing from gel-like to solution-like indicating that
`the product
`is a pseudo-gel with a complicated mixture of solution- and gel-like
`response. Rofilan changed behaviour from predominantly viscous at low frequencies to
`elastic at high frequencies typical of a solution containing non-modified, separate
`hyaluronan molecules with molecular weight 2 million.
`
`REFERENCES
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`6.
`
`7.
`
`D.A. Gibbs, E.W. Merrill, K.A. Smith & E.A. Balazs, The rheology of
`hyaluronic acid, Biopolymers, 1968,6,777-791.
`E.A. Balazs & D.A. Gibbs, D.A. The rheological properties and biological
`In Chemistry and Molecular Biology of the
`function of hyaluronic acid,
`Intercellular Matrix, E.A. Balazs (ed.), Academic Press, London and New York,
`1970, pp. 1241-1254.
`E.A. Balazs & J.L. Denlinger, Clinical uses of hya1uronan, CIBA Foundation
`Symposium, 1989, 143,265-275.
`E.A. Balazs et aI., Chemically modified hyaluronic acid preparation and method
`of recovery thereof from animal tissues, U.S. Patent No 4,713,448, 1987.
`B. Agerup, Polysaccharide gel composition, PCT/SE/96/00684, 1996.
`G.D. Prestwich et ai, Chemical modification of hyaluronic acid for drug delivery,
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`Applications of Hyaluronan and its Derivatives, T.e. Laurent (ed.) Portland
`Press, London and Miami, 1998, pp. 43-65.
`H. Bothner, Rheological studies of sodium hyaluronate in pharmaceutical
`preparations, Thesis, Uppsala University, 1991.
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`