J Mater Sci: Mater Med (2008) 19:3335–3343
`DOI 10.1007/s10856-008-3476-4
`
`Physical properties of crosslinked hyaluronic acid hydrogels
`
`Maurice N. Collins Æ Colin Birkinshaw
`
`Received: 15 March 2008 / Accepted: 15 May 2008 / Published online: 5 June 2008
`Ó Springer Science+Business Media, LLC 2008
`
`Abstract
`In order to improve the mechanical properties
`and control the degradation rate of hyaluronic acid (HA) an
`investigation of the structural and mechanical properties of
`the hydrogels crosslinked using divinyl sulfone (DVS),
`glutaraldehyde (GTA) and freeze-thawing, or autocross-
`linking has been carried out. The thermal and mechanical
`properties of the gels were characterised by differential
`scanning calorimetry (DSC), dynamic mechanical thermal
`analysis (DMTA) and compression tests. The solution
`degradation products of each system have been analysed
`using size exclusion chromatography (SEC) and the
`Zimm–Stockmayer theory applied. Autocrosslinked gels
`swell the most quickly, whereas the GTA crosslinked gels
`swell most slowly. The stability of the autocrosslinked
`gels improves with a reduction in solution pH, but is still
`poor. GTA and DVS crosslinked gels are robust and elastic
`when water swollen, with glass transition values around
`20°C. SEC results show that the water soluble degradation
`products of the gels show a reduction in the radius of
`gyration at any particular molecular weight and this is
`interpreted as indicating increased hydrophobicity arising
`from chemical modification.
`
`1 Introduction
`
`Hydrogels based on hyaluronic acid (HA) or hyaluron have
`gained attention as possible cell transplantation vehicles for
`the regeneration of a variety of tissues. The mechanical and
`
`M. N. Collins (&) C. Birkinshaw
`Department of Materials Science and Technology,
`University of Limerick, Limerick, Ireland
`e-mail: maurice.collins@ul.ie
`
`physical properties of the gel depend on the interaction of
`the constituent polymer with water and generally as the
`specific amount of absorbed water increases, the gels per-
`meability to oxygen and its strength decreases [1]. A high
`equilibrium swelling promotes nutrient diffusion into the
`gel and cellular waste removal out of the gel, while the
`insolubility provides the structural integrity necessary for
`tissue growth. The structural integrity of hyaluron hydro-
`gels is determined by the crosslinks formed by chemical
`bonds and by physical interactions. These gels are gener-
`ally biodegradable, processed easily, can be delivered in a
`minimally invasive manner but must have mechanical and
`structural properties similar to tissues and the extra cellular
`matrix (ECM) due to their similarity to the body’s own
`highly hydrated composition. It is of great interest to create
`hydrogels with controlled mechanical properties for bio-
`medical
`applications
`and
`these must
`possess
`the
`mechanical strength and flexibility sufficient to withstand
`compressive forces from the surrounding tissues in vivo
`without deformation or collapse [2].
`In addition,
`the
`mechanical properties of materials to which cells adhere
`can profoundly affect the function of the cells [3].
`Hyaluronic acid a glycosaminoglycan (GAG) is a par-
`ticularly attractive hydrogel material
`for biomedical
`applications [4]. GAG’s have a multifunctional role in
`wound healing. The early presence of a HA rich matrix
`enhances infiltration of migratory cells into the injured
`tissue and also creates an environment that promotes both
`cell motility and prolifereation [5]. HA also acts as a sig-
`naling molecule for cell migration and proliferation [6] and
`the degradation products modulate the inflammatory
`response and stimulate angiogenesis. For these reasons
`HA is an attractive building block for new biocompati-
`ble and biodegradable polymers with possible appli-
`cations in drug delivery [7], tissue engineering [8–10], and
`
`123
`
`ALL 2071
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
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`

`3336
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`J Mater Sci: Mater Med (2008) 19:3335–3343
`
`visco-supplementation [11, 12]. As a polysaccharide of the
`extra cellular matrix (ECM), it plays a multi-task role,
`having many structural, rheological, physiological and
`biological functions in the body. It is a linear and anionic
`polymer consisting of two modified sugars, glucuronic acid
`and N-acetylglucosamine, with the molecular structure:
`[D glucuronic acid (1-b-3) N-acetyl-D-glucosamine (1-b-4)]n.
`It has a high capacity for lubrication, and water sorption
`and retention allowing application in ophthalmic surgery
`[13]. HA also plays a critical role as a signaling molecule
`in cell motility [14], cell differentiation [15], wound
`healing [16, 17]. However, poor mechanical properties and
`rapid degradation in solution limit broader ranges of clin-
`ical application and emphasise the need for some property
`improvement.
`To improve the mechanical properties and control the
`degradation rate, HA can be chemically modified or
`crosslinked. Crosslinking is the most common modification
`of hyaluronan to form a hydrogel and a number of mech-
`anisms have been reported in the literature [18–21]. The
`functional groups which are mainly responsible for cross-
`linking of HA molecules are the hydroxyl and carboxyl
`groups. Hydroxyl groups may be crosslinked via an ether
`linkage and carboxyl groups via an ester linkage. If desired
`the HA may be chemically modified prior to crosslinking to
`form other chemically reactive groups. Thus for example
`HA may be treated with acid or base such that it will
`undergo at least partial deacetalisation, resulting in the
`presence of free amino groups which can then be cross-
`linked via an amide (–C(O)– NH–); imino (–N=CH–) or
`secondary amine (–NH–CH–) bond. An imino linkage can
`be converted into an amine linkage in the presence of a
`reducing agent.
`The fabrication of novel HA-based materials has been
`described previously [21, 22]. Here, we report a systematic
`investigation of the structural and mechanical, properties of
`the hydrogels focusing on homogeneous reactions using
`divinyl sulfone (DVS), glutaraldehyde (GTA) and auto-
`crosslinked samples, it having been demonstrated that such
`hydrogels remain insoluble for periods of time ranging
`from a few hours to several days. GTA is believed to form
`either a hemiacetal or an ether link with HA under acidic
`conditions. With DVS the crosslinking occurs via the
`hydroxyl groups forming an ether bond. The proposed
`reaction schemes have been reported in a previous publi-
`cation [21]. ‘‘Autocrosslinking’’ was also carried out and
`the mechanism here is postulated to occur through the
`development of extensive and semi-permanent secondary
`bonding between HA molecules. Okamoto et al.
`[23]
`maintain that during the freezing period at a low pH the
`electrostatic repulsive forces between the HA molecules
`are suppressed so the HA molecules are packed closely
`together to facilitate the formation of a gel. For such a gel
`
`123
`
`to be stable at higher temperatures than the temperature of
`formation, it must be presumed that either a crystallisation
`process occurs, or at least a process analogous to crystal-
`lisation in which the relationship between the gelation
`temperature and the gel melt temperature is similar to that
`between Tc and Tm in conventional melt processed
`polymers.
`The present study examines the relationships between
`crosslink density and mechanical properties and degrada-
`tion rate and mechanism of HA hydrogels. The relationship
`between crosslink density and solvent swelling is described
`by the well known Flory–Rhener equation which demon-
`strates that
`increasing crosslinker effectiveness will be
`shown by a reduced volumetric swelling. For this work the
`swelling ratio (SR) was calculated via the equation:
`Swelling ratio ¼ Ws
`Wd
`
`ð1Þ
`
`where Ws is the weight of the sample at equilibrium at each
`temperature and Wd is the weight of the dried sample.
`All the hydrogels were mechanically characterised by
`dynamic mechanical thermal analysis (DMTA) and ther-
`mally by differential scanning calorimetry (DSC). The
`hydrolytic degradation products of the gels were investi-
`gated using the Zimm–Stockmayer
`theory using size
`exclusion chromatography (SEC). HA is a linear molecule
`prior to crosslinking and if the water soluble degradation
`fragments are examined, their architecture and in particular
`their degree of branching, will give some indication of the
`original structure and the mechanism of breakdown.
`
`2 Materials and methods
`
`2.1 Materials
`
`The sodium salt of HA with an average molecular weight
`of 2.06 9 106 was supplied by Clear Solutions (New York,
`NY) as dry powder. This material is prepared in high yield
`from streptococcus bacteria by fermenting the bacteria
`under anaerobic conditions in CO2 enriched growth med-
`ium [24]. GTA, and DVS were purchased from Lancaster
`(UK).
`
`2.2 Hydrogel preparation
`
`The crosslinking procedure has been outlined previously
`[25]. Briefly, in order to obtain a crosslinked HA gel with
`DVS, HA was dissolved in dilute alkaline solution (4 wt%)
`and DVS was added dropwise. Generally, one hour was
`enough for completion of the crosslinking reaction. All gels
`were optically clear, with a smooth surface. For cross-
`linking to proceed with GTA the aqueous HA mixture
`
`

`

`J Mater Sci: Mater Med (2008) 19:3335–3343
`
`3337
`
`
`
`(
` 1
`2
`
`g ¼ 6
`BW
`
`randomly branched polymer,
`trifunctional
`Stockmayer equation is:
`2 þ BW
`BW
`
`
`
`"
`
`the Zimm–
`
`#
`
`) ð
`
`4Þ
`
`1=2
`
`In
`
`ð
`ð
`
`2 þ BW
`2 þ BW
`
`Þ1=2þB1=2
`Þ1=2B1=2
`
`W
`
`W
`
` 1
`
`where Bw is the weight average number of branches per
`molecule of a polydisperse sample.
`The branching frequency is calculated as follows:
`k ¼ R Bn
`Mw
`
`ð5Þ
`
`where R is the repeat unit, Mw is the molecular weight.
`A repeat unit of 37800 was used for these calculations, this
`value is equivalent to 100 HA units [26–28].
`
`2.5 Characterisation of hydrogels
`
`Differential scanning calorimetry analysis was carried out
`using a TA Instruments DSC 10 differential scanning cal-
`orimeter. The samples were hermetically sealed within the
`DSC pans. The thermal analysis profiles were of water
`swollen samples and the temperature was increased from
`ambient to 300°C at a rate of 10°C /min under 60 cc/min of
`nitrogen gas flow. Samples were tested in triplicate to
`ensure reproducibility. The DMTA of the materials was
`carried out with a Polymer Laboratories DMTA MK-1
`apparatus, operating in the parallel plate mode. The scans
`were performed on samples maintained under room con-
`ditions, at a frequency of 1 Hz, temperature range of -30
`to 100°C, and a heating rate of 4°C min-1. Multi-frequency
`tests were carried out on the DVS crosslinked gels.
`The more robust HA gels crosslinked by GTA were
`compression tested and Fig. 1 shows a typical sample used
`
`Fig. 1 A typical hydrogel samples tested in compression
`
`123
`
`should be acidic and varying amounts of crosslinker were
`used. This crosslinker produced the most mechanically
`robust gels and they were also easily moulded into any
`shapes. All gels were washed to remove any unreacted
`crosslinker. With autocrosslinking HA aqueous (1 wt%)
`solutions were prepared. The pH of the solution was
`adjusted to the required value using 1 M HCl and was then
`placed in a freezer at -20°C for 2.5 days and then thawed
`at 25°C.
`
`2.3 Swelling measurements
`
`The swelling ratio (SR) of a hydrogel was measured after it
`was swollen to a desired state at 25°C. It was carefully
`taken out from the solution, wiped with a filter paper for
`the removal of the free water on the surface, and then
`weighed. SR (g/g) of a sample was calculated according to
`Eq. 1. All measurements were made in triplicate for each
`sample.
`
`2.4 Analysis of degradation
`
`A degrading crosslinked hydrophilic polymer can be
`expected to generate large soluble fragments and so
`characterisation of these is a useful indicative parameter
`of the mechanism and original structure. The soluble
`degradation products can be compared with the original
`polymer using SEC and this was done using Viscotek
`Model 270 chromatograph with a GMPWXL mixed bed
`column at 25°C. Solvent flow was maintained at 0.7 ml/min
`through the column. The model 270 system consisted of a
`dual detector, a viscometer and a light scattering detector
`(LALS and RALS). Both detectors are then connected
`with a refractive index detector (RI 2000). HA is a ran-
`dom coil molecule, so it has an a value between 0.5 and
`1.0, or greater is possible for extended rod-like molecules.
`The g0 branching index is calculated as the ratio of
`sample [g]B value against its linear reference [g]L value of
`same molecular weight:
`
`ð2Þ
`
`g0 ¼ g½ ŠB
`g½ ŠL
`To relate the viscosity branching index g with g0, an
`assumption has to be made, i.e. g and g0 have a relationship
`as shown in Eq. 3.
`ð3Þ
`g0 ¼ ge
`where e is the shape factor which is taken to be 0.75
`assuming random branching [26].
`The relation between the number of branches per mol-
`ecule and the branching ratio depends on the branching
`functionality and the polydispersity of the sample of
`branched molecules. Assuming the degraded gel
`is a
`
`

`

`3338
`
`J Mater Sci: Mater Med (2008) 19:3335–3343
`
`0
`
`100
`
`200
`
`300
`400
`500
`600
`Swelling time (hrs)
`
`700
`
`800
`
`900
`
`0123456789
`
`Swelling Ratio (g/g)
`
`Fig. 3 Longer term behaviour of DVS gel swollen at 37°C in
`distilled water (HA:DVS mole ratio 1:2)
`
`2
`
`7
`pH
`
`9
`
`2.5
`
`2
`
`1.5
`
`1
`
`0.5
`
`0
`
`Swelling Ratio (g/g)
`
`Fig. 4 Autocrosslinked Hyaluronic acid after 60 mins swelling at
`25°C in different pH levels
`
`suggests that GTA is the most effective crosslinker with the
`autocrosslinking technique the least effective. Both of the
`covalently crosslinked gels are at their equilibrium swell-
`ing ratio after 6 h in distilled water and are stable beyond
`24 h whereas the autocrosslinked samples have dispersed
`into solution before this time is reached. The effect of
`reducing pH is in accordance with the proposals of
`Okamoto et al. [28] that maximum chain interaction can be
`obtained at low pH, but it is apparent from the results
`presented here that even at 25°C the forces of solvation
`greatly exceed the inter-molecular binding forces. The
`GTA crosslinked samples are the least swollen and are in
`turn the most dimensionally stable of the gels; observations
`which are consistent with a high crosslink density. During
`swelling of these gels, they initially turned white in colour,
`and then a colourless swelling front moved inwards, sep-
`arating the highly swollen surface from the less swollen
`core of the gel and gradually the entire gel turned colour-
`less and was swollen evenly. This colour phenomenon is
`presumed to be due to molecular rearrangement as the
`water diffuses through the material. Between 24 and 48 h
`
`for this. A compression rig was set up on an Instron 4302
`machine using 100 N load cell with a crosshead speed of
`5 mm/min. Samples were compressed to 50% of their
`initial thickness and the swollen modulus, Ge, of each
`sample was calculated using Eq. 6 since uniaxial com-
`
`pression measurements were carried out on the hydrogels
`between two parallel plates [29].
`F=A ¼ Ge k k2
`
`ð6Þ
`
`
`
`where F is the force, A is the original cross sectional area of
`the swollen hydrogel, and k = l/lo where l and lo are the
`lengths of the gel after and before compression, respec-
`tively. Plotting F/A versus k - k-2 resulted in a straight
`line with a slope of Ge, which was the modulus of elasticity
`of the swollen hydrogel.
`Cyclical compressive testing between a compressive
`strain of 0.2 and 0.4 at a crosshead speed of 1.25 mm/min
`was used to evaluate sample resilience and hysteresis. All
`tests were performed at room temperature.
`
`3 Results and discussion
`
`3.1 Swelling studies
`
`Figure 2 shows the short term swelling behaviour of the
`DVS, GTA and pH 1.5 autocrosslinked gels at 25°C in
`distilled water and Fig. 3 shows the longer term behaviour
`of
`the DVS crosslinked gels. Figure 4 compares the
`swelling behaviour of three autocrosslinked gels prepared
`at pH values of 2.0, 7.0 and 9.0.
`The speed at which the gels swell to their equilibrium
`water content is presumed to be a function of crosslink
`density and hydrophobicity of the system, and comparison
`of the results for the three different crosslinking methods
`
`DVS
`GTA
`Autocrosslinked
`
`0.5
`
`1
`
`3
`2
`Swelling Time (hrs)
`
`6
`
`24
`
`10
`
`0123456789
`
`Swelling Ratio (g/g)
`
`Fig. 2 Swelling ratio at 25°C in distilled water. Hyaluronic acid is
`crosslinked at 1:2 mole ratio with DVS and GTA
`
`123
`
`

`

`J Mater Sci: Mater Med (2008) 19:3335–3343
`
`3339
`
`Table 1 DSC data for hyaluronic acid hydrogels prior to swelling
`
`Sample
`
`Endothermic
`peak (°C)
`
`Degradation temperature
`(°C)
`
`Autocrosslinked
`
`DVS
`
`GTA
`
`78.2
`
`119.1
`
`91.1
`
`1st peak
`
`2nd peak
`
`225.5
`
`222.9
`
`215.5
`
`234.3
`
`231.5
`
`228.2
`
`Log Elastic Modulus (pa)
`
`012345678
`
`0.8
`
`0.7
`
`0.6
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`0
`
`Tan (σ)
`
`-20
`
`-10
`
`0
`
`20
`10
`30
`Temperature (oC)
`
`40
`
`50
`
`60
`
`Fig. 6 Autocrosslinked sample after 2 h swollen in distilled water
`(scanning rate 4°C min-1; frequency 1 Hz)
`
`the gels began to undergo additional swelling and this is
`presumed to indicate degradation.
`
`3.2 Mechanical and thermal characterisation
`of the hydrogels
`
`Figure 5 shows the DSC thermograms for the covalently
`crosslinked and autocrosslinked and slightly swollen
`hydrogels and Table 1 details the important peak temper-
`atures. The two chemically crosslinked gels produced
`thermograms which were similar in shape and all of the
`samples showed the presence of a broad endothermic peak
`around 100°C, which is associated with the loss of moisture
`remaining after the initial drying procedure. The relatively
`smaller endotherm obtained with the autocrosslinked
`material may reflect lower water retention through the
`drying period. Significant sharp exothermic peaks were
`also observed for each sample at higher temperatures and
`crosslinking changes the relative magnitude of these two
`exothermic peaks. It is thought that the first of these peaks
`represents conversion into a less-ordered state, and the
`second represents thermal degradation.
`Figures 6–8 show the DMTA responses of the gels
`investigated and Table 2 summarises some of the impor-
`tant results. As expected the samples show a reduction in
`elastic modulus with increased water content, with the
`height of the tan d peak (the a transition) being reduced and
`the peak broadened in the more swollen samples. In all
`three figures the glass transition is apparent at approxi-
`mately 20–25°C. Increased water content slightly lowers
`
`Fig. 5 DSC thermograms of
`the crosslinked hydrogels (a)
`DVS (b) GTA (c)
`autocrosslinked
`
`(a)
`
`(b)
`
`Exothermic
`
`(c)
`
`Exothermic
`
`50
`
`100
`
`200
`150
`Temperature oC
`
`250
`
`300
`
`50
`
`100
`
`150
`200
`Temperature oC
`
`250
`
`300
`
`50
`
`100
`
`200
`150
`Temperature oC
`
`250
`
`300
`
`123
`
`

`

`3340
`
`J Mater Sci: Mater Med (2008) 19:3335–3343
`
`Divinyl sulfone crosslinked materials were selected for
`frequency sweeps and Figs. 9 and 10 show results obtained
`using frequencies of 0.1, 1.0 and 10 Hz. As expected, the
`storage modulus increases with increasing frequency, but
`above the glass transition the effects are small. In the glass
`transition region, the effect of the measurement frequency
`is pronounced between 0.1 and 1 Hz but less so between 1
`and 10 Hz. At 37°C when the modulus reaches a rubbery
`plateau, the modulus increases by 18% across the fre-
`quency range. The measurement frequency effect is more
`uniform across the frequency range above the Tg and the
`shift to higher apparent stiffness with increasing frequency
`correlates with the increase in Tg as indicated by the tan d
`peak. The small peak on the storage modulus directly
`preceding the drop associated with the Tg is thought to be
`due to molecular rearrangement as the material relieves
`stresses frozen in by the processing method [30].
`Figure 11 shows the results of cyclic loading on a GTA
`crosslinked gel. A maximum stress of 20.3 KPa was
`required to induce an initial compressive strain of 0.2. The
`first compressive cycle results in a permanent deformation
`
`10 Hz
`1 Hz
`0.1 Hz
`
`012345678
`
`Log Elastic Modulus (pa)
`
`-20
`
`0
`
`20
`Temperature (oC)
`
`40
`
`60
`
`Fig. 9 Multiple frequency analysis of DVS hydrogels
`
`10 Hz
`1 Hz
`0.1 Hz
`
`0
`
`20
`Temperature (oC)
`
`40
`
`60
`
`1.8
`
`1.6
`
`1.4
`
`1.2
`
`1
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0
`-20
`
`Tan (δ)
`
`Fig. 10 Multiple frequency analysis of DVS hydrogels
`
`Fig. 7 GTA crosslinked sample after 2 h swollen in distilled water
`(scanning rate 4°C min-1; frequency 1 Hz)
`
`Log Elastic Modulus (pa)
`
`012345678
`
`0
`
`20
`Temperature (oC)
`
`40
`
`60
`
`1.2
`
`1
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0
`
`-20
`
`Tan (δ)
`
`Fig. 8 DVS crosslinked sample after 2 h swollen in distilled water
`(scanning rate 4°C min-1; frequency 1 Hz)
`
`Table 2 Summary of the transition temperatures and 37°C moduli of
`the partially swollen hydrogels
`
`Swelling
`ratio (g/g)
`
`Tg (°C) Tan (d)
`
`Storage modulus
`@ 37°C (Kpa)
`
`Autocrosslinked
`
`DVS
`
`GTA
`
`2.2
`
`2.5
`
`1.9
`
`24.6
`
`23.5
`
`20.8
`
`0.78
`
`1.12
`
`0.62
`
`2.20
`
`0.55
`
`4.18
`
`the glass transition temperature of the hydrogels, however
`the GTA crosslinked materials have a lower than expected
`Tg and this may arise from the presence of unreacted
`crosslinker which is having a plasticising effect. Since the
`materials are crosslinked the elastic modulus in shear does
`not decline to near zero values and they still exhibit useful
`load bearing characteristics above their glass transitions
`and at 37°C.
`
`123
`
`

`

`J Mater Sci: Mater Med (2008) 19:3335–3343
`
`3341
`
`incompressible elastic material, is shown Figure 12. The
`elastic modulus was calculated from the slope and found to
`be 24.4 KPa for a 1:2 mole ratio crosslinked gel, which
`compared very favourably with elastic modulii of similar
`glycosaminoglycans (GAGs) measured by Kirker et al.
`[31].
`
`3.3 Analysis of degradation
`
`To characterise the degradation products samples were
`taken from the PBS solution surrounding the degrading gel,
`analysed with SEC and compared with a reference linear
`HA of similar original molecular weight. As an example of
`typical results Figs. 13 and 14 show the relationships
`between intrinsic viscosity and molecular weight and
`retention volume and molecular weight for the autocross-
`linked sample. From the Mark Houwink equation, the plot
`of the Log IV and Log M should be linear, however in
`Fig. 13 the slope is not constant for the degraded sample
`which suggests a branched structure and Fig. 14 shows that
`the degraded sample has a higher molecular weight than
`the reference sample at the same elution volume, but the
`difference is decreased as the molecular weight decreases.
`This indicates that
`the polymeric degradation products
`possess a smaller radius of gyration for a given molecular
`weight, and this behaviour is usually considered to be
`indicative of short chain branching. Applying the Zimm–
`Stockmayer theory gives the branching results shown in
`Table 3, however these results are amenable to a number of
`interpretations. The polymer molecule in its original sol-
`vated form assumes a rod like conformation and any
`crosslinking process is likely to increase the hydropho-
`bicity of the polymer and therefore reduce the radius of
`gyration and increase the molecular density of the degra-
`dation
`products. Additionally with
`the
`chemically
`crosslinked materials the large molar excess of reactant,
`
`0
`
`5
`
`10
`15
`Strain (% Compressed)
`
`20
`
`25
`
`20
`
`15
`
`10
`
`5
`
`0
`
`Stress (Kpa)
`
`Fig. 11 Cyclic loading of swollen GTA swollen @ 23°C (1:2) mole
`ratio swelling ratio 9.2
`
`Stress (Kpa)
`
`25
`
`20
`
`15
`
`10
`
`5
`
`0
`
`a
`y = -24.4x
`
`b
`y = -14.3x
`
`-0.8
`
`-0.7
`
`-0.6
`
`-0.5
`
`-0.3
`
`-0.2
`
`-0.1
`
`0
`
`-0.4
`(λ - λ -2)
`
`Fig. 12 Engineering stress versus strain curve for GTA gels swollen
`@ 23°C for 24 h (a) HA: GTA (1:2) mole ratio and (b) HA: GTA
`(1:1) mole ratio
`
`of around 10% with subsequent cycles showing greater
`relative recovery. Although significant hysteresis is evident
`the overall elasticity of the gel is apparent. The engineering
`stress versus strain curve, obtained by treating the gel as an
`
`2005-10-28_19;59;22_LMW_01.vdt - Branching Data View
`
`0.974
`0.900
`
`0.800
`
`0.700
`
`0.600
`
`0.500
`
`0.400
`
`0.300
`
`0.200
`
`0.123
`
`Log Intrinsic Viscosity
`
`Fig. 13 Intrinsic viscosity
`versus molecular weight plot for
`autocrosslinked gel degradation
`products (lower). The linear
`sample is upper
`
`4.983
`
`5.100
`
`5.200
`Log Molecular Weight
`
`5.300
`
`5.371
`
`123
`
`

`

`3342
`
`J Mater Sci: Mater Med (2008) 19:3335–3343
`
`Fig. 14 Log of molecular
`weight versus retention volume.
`The reference sample (lower)
`has a lower molecular weight
`than the degraded sample at the
`same elution volume
`
`Table 3 Zimm–Stockmayer analysis of degraded samples
`
`Apparent
`branches/
`molecule
`
`–
`
`129
`
`164
`
`330
`
`Apparent
`branching
`frequency
`
`–
`
`36.8
`
`51.1
`
`71.3
`
`Linear reference
`
`Autocrosslinked
`
`DVS
`
`GTA
`
`needed to achieve acceptable crosslink densities, suggests
`that many crosslinker molecules may be covalently bound
`to the polymer, but not
`forming effective crosslinks.
`However such a scenario cannot be presumed with the
`autocrosslinked material where although some chain deg-
`radation may be occurring, it must be presumed that the
`principal process is dissolution, and the reduction in the
`radius of gyration at any given molecular weight must be
`presumed to come from changes in chain conformation. It
`is therefore considered that
`the output of the Zimm–
`Stockmayer approach is best considered to reflect changes
`in the hydrophobility of the polymer following the cross-
`linking processes. In that sense the apparent branching
`values correlate well with the efficiencies of the different
`crosslinking techniques used.
`
`4 Conclusions
`
`Comparing autocrosslinking with the use of covalent
`crosslinkers, then clearly autocrosslinking is attractive in
`that it avoids the need for reactants which may present
`problems of residues, and should lead to gels with the same
`tissue response as natural HA. However the stability of the
`resulting gels is poor, even when prepared at low pH. If
`autocrosslinking functions through crystallisation, or at
`least
`through the development of semi-ordered quasi-
`crystal structures, then the use of a very slow cooling rate
`
`123
`
`may be beneficial in giving longer times for the polymer
`molecules to diffuse into the appropriate conformations
`relative to each other, however the results presented here
`for the technique are not encouraging.
`Gels produced using GTA and divnyl sulphone show
`much higher stability having useful lifetimes of several
`days. Once swollen these gels are dimensionally stable, are
`mechanically resilient and have 37°C modulus values
`comparable with soft tissue.
`Considering the gel degradation products the size
`exclusion results show a reduction in the radius of gyration
`at any particular molecular weight and this can be inter-
`preted as indicating short chain branching or increased
`hydrophobicity with the latter explanation being consid-
`ered the most likely. The inverse correlation between speed
`of swelling of the gel in water and the molecular density
`measured using size exclusion chromatograpy supports this
`interpretation.
`
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`