Chapter 8
`
`Biomedical Applications of Hyaluronic Acid
`
`Samuel J. Falcone, David Palmeri, and Richard A. Berg
`
`FzioMed, Inc., 231 Bonetti Drive, San Luis Obispo, CA 93401
`
`Hyaluronic acid (HA), also named hyaluronan, is a high
`molecular weight polysaccharide
`found
`in the body
`in
`pericellular matrices, various bodily fluids, and in specialized
`tissues such as the vitreous humor of the eye and cartilage.
`Hyaluronic acid possesses both biological activities and
`physical properties that add to the uniqueness of this
`ubiquitous polysaccharide. The uniqueness of HA and its
`importance both biologically and physically apparently
`accounts
`for
`its
`identical structure when synthesized
`in
`bacteria, birds, and mammals. Because of this property, HA
`has been purified from chickens or bacteria for use as a
`biomaterial in medical devices in humans or other mammals.
`Hyaluronic acid is unique because of its viscoelastic and
`hydrodynamic properties, its assembly into extracellular and
`pericellular matrices, and its effects on cell signaling. Its use as
`a biomaterial has been driven largely by its physical properties
`and viscoelastic behavior. The biological properties of HA and
`its fragments have
`largely been ignored in its use as a
`biomaterial. As HA is more widely used and studied, these
`properties are becoming increasingly apparent and important
`in its use in medical devices.
`
`© 2006 American Chemical Society
`
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`
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`156
`
`Hyaluronic acid (HA) is a component of the extracellular matrix and is a
`ubiquitous substance found abundantly in nature. Many types of cells synthesize
`HA; it interacts with other constituents (proteins) of the extracellular matrix
`including the cell surface to create the supportive and protective structure around
`the cells. HA has also been shown to provide extracellular signals to cells related
`to locomotion and gene expression. It is a constituent of all body fluids and
`tissues, and it is found in higher concentrations in the vitreous humor of the eye,
`hyaline cartilage, and the synovial fluid. It was initially isolated from the vitreous
`of the eye.
`Structurally, it is a polydisaccharide containing D-glucuronic acid and D-N-
`acetylglucosamine with repeating β(1-3), β(1-4) saccharide linkages. Three
`isozymes of hyaluronan synthases have been identified in bacterial and animal
`cells (for review, see reference(/)). These enzymes produce HA of variable
`molecular weights depending on the tissue. It is a most unusual polymer in that
`the same polysaccharide is produced in bacteria, birds, and mammals although
`the molecular weights may differ depending on its source. The highest molecular
`weight HA is found in cartilage. Hyaluronic acid being an extracellular and
`pericellular polymer is catabolized by receptor-mediated endocytosis and
`lysosomal hydrolysis in various tissues (2) or after transport to lymph nodes (3).
`Hyaluronidases are broadly distributed enzymes involved in tissue invasion and
`remodeling, (4) and are found in plasma (5).
`Of special interest is that HA can be degraded by and fragmented to smaller
`oligosacharides via reactive oxygen (6) such as that produced by inflammatory
`cells, supporting a role for HA fragments in wound healing and inflammation (7)
`(for review of HA homeostasis in the body see reference (/)).
`Hyaluronic acid has been purified in quantity from cartilage and bacteria
`owing to the relative abundance of HA in those materials. Once HA was purified
`its rheological properties as a viscoelastic polymer became apparent. The
`physical properties of HA point to its pivotal role in the extracellular and
`pericellular matrix in tissues. Hyaluronic acid, in solution, is viscoelastic and the
`rheological properties depend on concentration,
`ionic strength, pH, and
`molecular weight (8, 9). These rheological properties have been exploited for
`use in some biomedical applications. Solutions of high molecular weight HA are
`viscous, cohesive, lubricious, and hydrophilic. These properties have found use
`as viscoelastic adjuncts in ophthalmic surgery, viscosupplementation
`in the
`synovium of arthritic joints, and as covalently bound lubricious hydrophilic
`coatings for medical devices, e.g., stents, catheters {10,11). For medical
`applications that require extended residence time in situ, HA can also be
`crosslinked by a variety of chemical methods. Cross-linking HA dramatically
`increases the elastic component of the overall modulus of HA causing the
`polymer gel to behave more like an elastic solid and less like a viscous fluid in
`response to deformation. Crosslinked HA products have found use as surgical
`adhesion barriers, synovial viscosupplements, and more recently as materials for
`augmentation of the dermis (12-14).
`This review will focus on the biological and physical properties of HA that
`have been successfully exploited for its use as a biomaterial.
`
` Marchessault et al.; Polysaccharides for Drug Delivery and Pharmaceutical Applications
`
`ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
`
`

`

`Biological Properties of Hyaluronic Acid
`
`157
`
`Hyaluronic acid is found both in the extracellular matrix between cells in
`tissues and associated with cell surfaces, in the pericellular matrix. Furthermore,
`HA is not an inert material. It is recognized specifically by several proteins in
`the extracellular matrix that give rise to important biological functions (75).
`The first two proteins discovered to interact with HA are the link protein
`and the protein aggrecan found in the extracellular matrix of cartilage (16). The
`association of these two proteins with HA contributes to the formation of
`proteoglycan aggregates in cartilage, which are responsible for the bulk elastic
`and hydrodynamic properties of cartilage. A second type of interaction is with
`receptor proteins found on cell surfaces (for review see (17)). Two of these
`proteins are CD 44, a receptor found on many cell types (18) and R H A M M, a
`receptor for hyaluronan-mediated motility (19).
`CD 44 is a transmembrane receptor that connects the pericellular matrix on
`the outside of cells with cytoskeletal proteins (17). The close association of HA
`with the cell pericellular matrix suggests an explanation for the role of CD44 in
`leukocyte migration, neoplasia, and in wound healing (17). In order for cells to
`migrate in tissues they must be capable of disrupting connections to the
`extracellular matrix. As cells migrate they are exposed to HA that is present in
`the extracellular matrix. A protein that is also implicated in cell mobility is
`R H A MM which is present in several intracellular compartments and is also
`exported to the cell surface where it interacts with HA (17). R H A MM was
`shown to be involved in fibroblast locomotion (20) and cell mobility (for review,
`see reference (17) ).
`Since HA is found in pericellular matrix it is not surprising that cell
`migration, mobility and wound healing may be influenced by HA. Of interest is
`the finding that fragments of HA, presumably resulting from its degradation, are
`more active in interaction with these cell surface receptors than native intact
`leading to a role for fragments of HA in acute (22) and chronic
`HA(21)
`inflammation (7). HA fragments can induce genes coding for inflammatory
`mediators (22, 23). These observations suggests that HA fragments, resulting
`from degradation of high molecular weight H A, are capable of cell signaling
`through
`interaction of receptor-mediated control pathways,
`followed by
`alterations in ceil mobility and gene expression. Support for this pathway is that
`fragments of HA are capable of inducing cytokine expression in macrophages
`(24).
`The finding that HA interacts specifically with proteins in the body serving
`both structural and cell signaling functions points to a potentially complex
`response by tissue when HA is injected into the body in the form of a medical
`device. For example, as HA is degraded it may have effects on leukocyte
`mobility and therefore inflammation and wound healing.
`
` Marchessault et al.; Polysaccharides for Drug Delivery and Pharmaceutical Applications
`
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`
`

`

`158
`
`Physical Properties of Hyaluronic Acid
`
`HA Structure in Solution and Rheology
`
`0
`
`The structure of uncrosslinked HA in dilute and concentrated solution has
`been extensively studied (for a review see reference (25)). The original work
`envisioned that uncrosslinked HA is a high molecular weight unbranched
`polysaccharide that behaves as a stiffened random coil in solution. The molecule
`occupies a large hydrated volume and shows solute-solute interactions at
`unusually low concentration. Since HA is a polyelectrolyte, the solution
`properties are greatly affected by ionic strength (25). The conformation of HA in
`solution has been studied at neutral pH and at physiological concentration,
`using nuclear magnetic resonance and circular dichroism. These studies support
`a model incorporating dynamically formed and broken hydrogen bonds that
`contribute to the semi-flexibility of the polymer chain (26).
`Hyaluronic acid that is not crosslinked is water soluble, rapidly resorbed, and
`has a short residence time in situ that limits its utility for use in biomedical
`applications. HA in solution is subject to degradation via ultrasound, UV
`irradiation, thermal, and free radicals (25). Uncrosslinked HA in solution
`behaves as a pseudoplastic shear thinning fluid and the zero shear viscosity ( η
`)
`correlates to the solution concentration multiplied by the molecular weight (27).
`Figure 1 describes the relationship of the log η
` to the log of the (concentration
`*molecular weight) for a series of HA solutions prepared from HA of three
`molecular weights, 1800 kg mol'1, 680 kg mol"1, and 350 kg mol"1, at several
`concentrations, between 10-90mg/ml,
`in phosphate buffered saline. Also
`included in Figure 1 are seven commercial preparations of uncrosslinked HA
`used as medical devices. The data for these HA medical products are included
`in Table 1. The plot of the log of the solution η
` vs. the log of the solution
`(concentration*MW) is linear. This means that the viscosity of an HA solution
`can be controlled by adjusting polymer molecular weight and/or the solution
`concentration.
`The data in Figure 1 also imply that the properties of two uncrosslinked HA
`solutions of a given viscosity can be quite different. Consider two HA solutions
`both with a η
` of —170 Pas. Solution A is a 16mg/ml solution of HA 1800 kg
`mol^and solution Β is a 70mg/ml solution of HA 350 kg mol"1. The η* at low
`frequency, 0.0628 rad/s, low deformation rates, or low shear is 172 Pas for both
`materials. The viscoelastic properties of these two solutions are quite different
`and this is shown in Figure 2. This Figure describes the complex viscosity (η*)
`and the storage viscosity (η"), the elastic component of the complex viscosity vs.
`frequency for HA solutions A and B. At low frequency both solutions have
`
`0
`
`0
`
`0
`
` Marchessault et al.; Polysaccharides for Drug Delivery and Pharmaceutical Applications
`
`ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
`
`

`

`159
`
`the same η *; as the frequency is increased, the η* of the high molecular weight
`HA solution decreases more rapidly than the η* of lower molecular weight HA.
`At high frequency, the η* of the concentrated low molecular weight HA is
`greater than 10 times higher than the dilute solution of high molecular weight
`HA. The data in Figure 2 also show that the elastic component of the η *, η", at
`low frequency for the solution of high molecular weight HA is much higher than
`that of low molecular weight HA. At low deformation rates, the high molecular
`weight HA solution with an entangled or aggregated chain structure responds
`elastically to deformation. Under these conditions the concentrated
`low
`molecular weight solution responds in a primarily viscous manner. The high
`molecular weight HA solution, up to a frequency of ~ 1 rad/s, is more elastic
`than the solution of low molecular weight H A. Both materials have the same
`viscosity at low deformation rates, but solution A is a dilute entangled cohesive
`solution ideally suited for bulk removal during eye surgery, while solution Β is a
`concentrated non-cohesive tissue adhesive material possibly better suited for
`tissue coating and lubrication medical applications. The higher viscosity and
`elasticity of low molecular weight HA at high frequency also indicate that it may
`be a better tissue coating and lubricating material under conditions of high
`deformation rates.
`
`Cohesive Properties of Hyaluronic Acid: Cohesion-Dispersion Index (CDI)
`
`The cohesive nature of HA is of prime importance to its use as a viscoelastic
`adjunct in eye surgery (28). The more cohesive the material is the more likely it
`is to be readily removed from the anterior chamber of the eye during cataract
`surgery. Cohesion is a result of intermolecular entanglement of the HA polymer
`chains that imparts a bolus-like behavior to the viscoelastic solution. A
`quantitative dynamic aspiration method that measures
`the cohesion of
`viscoelastic agents has been developed (29). The principle of the technique is to
`measure the amount of sample aspirated with increasing vacuum applied to the
`sample; vacuum levels of 127, 254, 381, 533, and 711 mm of Hg were used. At
`each vacuum level, the weight of material aspirated through a 0.5mm pipette tip
`in two seconds is measured. The data are plotted as the percent aspirated vs.
`vacuum level and the slope of the steepest portion of the curve is determined.
`The cohesive-dispersive index (CDI) is equal to the slope at steepest portion of
`the curve. Materials that are more cohesive are removed in bulk at a lower
`vacuum than materials of lesser cohesiveness. Table I list the CDI results for a
`series of HA viscoelastic agents that have been used in eye surgery. From these
`data it was concluded that the cohesive nature of HA increases with increasing
`molecular weight (29). The study was extended to include the effect of
`concentration and molecular weight on the cohesive properties of uncrosslinked
`HA (27). It was found that the CDI correlates very well to solution concentration
`
` Marchessault et al.; Polysaccharides for Drug Delivery and Pharmaceutical Applications
`
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`

`

`160
`
`10000η
`
`1000
`
`looi
`
`10
`
`1 1
`
`0.1
`1000
`
`• HA products
`
`Ο Reported HA solution Data
`
`Ο
`Ο
`
`οο
`ο ο
`
`Ο
`
`HA Products
`Amvisc
`Healon
`Provisc
`Healon G V
`Orthovisc
`Hyalgan
`Hylartil
`
`10000
`C(mg/ml)*MW (kg mol-1)
`
`10000C
`
`) of uncrosslinked HA materials as a
`Figure 1. The zero shear viscosity (η
`function ofMWand concentration. Open circles are data from Falcone et
`al. (27) and filled diamonds are data from Ρ oyer et al. (29)
`
`0
`
`1000
`
`îoo 1
`
`c3
`
`10
`
`Ρ"
`
`0.1
`
`0.01
`
`•·«:::
`
`aRlfl200oo°oA.
`αοα°ο5ρΐΐοοοοο5·,
`
`π
`
`• HA 1800 kg mol1 η*
`• HA 1800 kg mol'1 η"
`• HA 350 kg mol1 η*
`Ο HA 350 kg mol1 η"
`
`0.1
`
`1
`
`10
`
`100
`
`1000
`
`Frequency rad/s
`
`Figure 2. The complex modulus (η*) and storage modulus (η") vs. frequency for
`two HA solutions of different molecular weight and concentration; 1.6% HA
`1800 kg mot1 and 7% HA 350 kg moT1 in phosphate buffered saline.
`
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`
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`

`

`161
`
`0
`
`of HA, and both sets of data are plotted in Figure 3. The results of this study also
`confirmed that the cohesiveness of HA increases with increasing molecular
`weight and that the cohesive nature of HA correlates to the complex viscosity at
`high frequency.
`, but
`The cohesive properties of HA polymeric solutions are a function of η
`the zero shear viscosity does not accurately predict the cohesive behavior of the
`solution. The cohesive properties of uncrosslinked HA preparations were found
`to be proportional to the dynamic rheological properties of the HA polymer
`solutions. The cohesive properties, CDI, of the HA solutions were found to
`correlate well to the dynamic viscosity at high frequency, 62.8 rad/s. Figure 4
`describes the relationship of the log of the CDI to the log of the dynamic
`viscosity, η' for a series of HA solutions prepared from HA of three molecular
`weights, 1800 kg mol'1, 680 kg mol'1, and 350 kg mol"1, at several
`concentrations, between 10-90 mg/mL, in phosphate buffered saline. Also
`included in Figure 4 are three commercial preparations of uncrosslinked HA
`used as medical devices: Healon, Pro vise, and Viscoat. For both series, the CDI
`correlates well to the η' at high frequency. For this data, the CDI decreases as
`the η' increases and the cohesive nature of uncrosslinked HA correlates to the
`high frequency viscous component of the viscoelastic material. High molecular
`weight HA has less viscosity and stiffness and elastic response at high
`deformation rates. These rheological properties produce a more cohesive
`entangled structure that rapidly and easily flows through a small orifice, thus
`yielding a high CDI. The data presented here reveal that the CDI correlates well
`with the dynamic viscosity at high frequency in dynamic viscoelastic testing.
`From the rheological properties of high molecular weight HA, it appears to
`be ideally suited for a wide variety of medical applications. Under conditions of
`low shear, the applications include viscoelastic gels for eye surgery in the
`anterior chamber of the eye or as a temporary replacement for the vitreous
`humor where a gel-like material is required. Their use in ocular surgery is
`improved by their high viscosity at low shear rates in the anterior chamber of the
`eye, whereas their low high shear viscosity facilitates their injection into and
`removal from the eye through a small-bore needle.
`Studies of the synovial joint have indicated that HA is responsible for the
`viscoelasticity of synovial fluid (28). Analysis of the synovial fluid of an
`osteoarthritic joint has shown that, compared to a healthy joint, the molecular
`weight and viscosity of the HA are reduced (30, 31). Hyaluronic acid would be
`expected to function well as a shock absorber under low shear in the resting
`knee. However, owing to the shear thinning nature of high molecular weight HA
`and its cohesiveness, high molecular weight HA would be expected to be a less
`effective lubricant than low molecular weight HA. This observation may account
`for some of the discrepancy in results of various formulations of HA in the
`treatment of osteoarthritis, OA (32, 33) where it is not clear that the function of
`HA in reducing pain in joints is due to a lubricating effect, a shock absorber
`effect, or a biological effect.
`
` Marchessault et al.; Polysaccharides for Drug Delivery and Pharmaceutical Applications
`
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`

`

`162
`
`οι
`1.0
`
`10.0
`C (mg/ml)
`
`100.0
`
`Figure 3. The cohesiveness of uncrosslinked HA vs. the solution concentration.
`This data is taken form two independent studies. Filled squares, data from Table
`I. Open circles, data from Falcone et al (27)
`
`•
`
`I
`
`0.1
`
`0.01
`
`0.1
`
`10
`
`100
`
`Dynamic Viscosity @ 62.8 rad/s η* (Pas)
`Figure 4. The log of the CDI vs. the log of the dynamic viscosity, η ', for
`uncrosslinked HA solutions and products. Filled squares, data from Falcone et
`al. (27)
`
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`

`163
`
`Crosslinked Hyaluronic Acid
`
`Hyaluronic acid has been chemically covalently crosslinked to provide HA
`derivatives that effectively have an increased molecular weight, improved
`residence time in the body, and provide different viscoelastic properties than
`uncrosslinked HA (//, 33) (see references in Table II). Typically, crosslinking
`of HA is done by adding the crosslinker to HA in solution to make a solid gel
`and the crosslinked gel is dried and broken into particles and screened for
`different sizes. These crosslinked particles are suspended in aqueous solution to
`form the products listed in Table I. The molecular weight and material
`properties now depend on crosslink density and particle size (34). The gel
`suspension of crosslinked HA also has different rheological properties than
`uncrosslinked HA.
`In dilute solution, the modulus at low frequency of
`crosslinked HA is higher that uncrosslinked HA and this indicates that a network
`structure forms for the crosslinked materials. At higher concentrations, when the
`entangled network has formed, the steady shear viscosity or elastic modulus at
`high frequency for crosslinked HA is not higher than that of uncrosslinked HA
`(33). The increase in molecular weight of crosslinked HA increases elastic
`component of the viscoelastic properties at low frequency which are generally
`thought to improve efficacy of HA containing formulations for a given medical
`indication, e.g., viscosupplementation for treatment of arthritis and dermal skin
`augmentation (30, 34-36). The covalently crosslinked HA products are gel-like
`in structure whereas uncrosslinked HA forms dilute to concentrated polymer
`solutions depending on the molecular weight and concentration of HA (33).
`Figure 5 describes the effect of frequency on the elastic and viscous modulus for
`two uncrosslinked and two crosslinked HA products. The uncrosslinked HA
`products, Healon and Provisc, both exhibit a transition from a predominately
`viscous regime to a predominately elastic regime at ~ 0.5 rad/sec and ~ 25Pa.
`This behavior is typical of an entangled high molecular weight HA concentrated
`solution (27). These modulus data support data in Table I indicating that Healon
`and Provisc are of the same solution concentration and same approximate
`molecular weight. The modulus data for the crosslinked products Restylane and
`Hylaform in Figure 5 support a gel-like structure. For these materials, the elastic
`modulus is considerably higher than the loss modulus across the entire frequency
`range. It is also noteworthy that the elastic modulus for Restylane is -10 higher
`than that of Hylaform and the loss modulus is >10 higher over the entire
`frequency range tested.
`Table II list the cross-linkers that are used for each of the HA crosslinked
`products. Hylaform, Hylaform Plus, and Synvisc are crosslinked with divinyl
`sulfone (DVS). The DVS crosslinked HA materials can be prepared with
`varying amounts of cross-link density in the HA matrix by changing the mole
`ratio of D VS to HA in the crosslinking reaction. The cross-linking reaction is
`conducted as a single phase in acidic or basic medium and addition of DVS to
`HA at high pH forms an insoluble gel matrix. After neutralization, the insoluble
`
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`
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`

`

`164
`
`Table I. Products derived from HA and their properties
`
`Product
`
`C(mg//ml) MW(kg mot1)
`
`(Ρα-ϊ)
`
`η
`
`0
`
`% Elasticity®
`6.2 rad/s
`
`Amvisc Plus
`
`Healon
`
`Healon GV
`Healon 5
`
`Proviso
`
`Viscoat*
`
`Orthovisc
`
`Hyalgan
`
`Hylartil (Healon)
`
`Artzal
`
`Synvisc**
`
`Restylane**
`
`Perlane**
`
`Hylaform**
`
`Hylaform Plus**
`Juvederm 24**
`Juvederm 24 HV**
`Juvederm 30**
`
`Hyalobarrier**
`
`Intergel**
`
`Eye viscoelastic adjuncts
`100
`2000
`300
`1900
`3000
`5000
`7000
`4000
`256
`2300
`72
`>500*
`
`16
`10
`14
`23
`10
`40*
`
`Injectables for Osteoarthritis
`160
`1300
`.06
`700
`
`15
`10
`
`8
`
`20
`20
`4.5-6.0
`4.5-6.0
`24
`24
`24
`
`1400
`
`6000
`Skin Augmentation
`23320
`7396
`14970
`10270
`1436
`6857
`3462
`
`3000
`3000
`3000
`
`Anti adhesion products
`652
`284
`
`40
`5
`
`69
`
`69
`46
`
`56
`
`67
`27
`78
`
`82
`84
`90
`95
`65
`75
`71
`
`65
`59
`
`* HA + chondroitin sulfate: HA of low molecular weight (500 kg mol" l<) at 40mg/ml.
`** Crosslinked HA product
`CDI values for eye viscoelastic adjuncts taken from Poyer et al. (29)
`% elasticity @ 6.2 rad/s taken from Balazs (30)
`
`CDI
`
`21.4
`31.2
`72.3
`
`46
`3.4
`
`16.7
`
`12.4
`
`2.7
`38
`
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`
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`

`

`1000
`
`ι—ι 100
`eu
`q
`
`b
`
`Provisc crossover
`
`165
`
`Restylane _
`
`G' = f(co)
`G" = f (ω)
`
`G' = f(<o)
`G" = f (ω)
`
`G' = f((o)
`G" = f (ω)
`
`G' = f(co)
`G" = f (ω)
`
`Healon
`—
`—
`Hylaform
`•
`•
`ProVisc
`Φ
`" °~
`
`η π π Π
`
`•
`
`0.01
`
`0.10
`
`1.00
`10.00
`Frequency [rad/s]
`
`100.00
`
`1000.00
`
`Figure 5. 7%e e/ort/c (G 7 and loss (G ") modulus vs. frequency for two
`crosslinked, Restylane and Hylaform, and two uncrosslinked, Healon and
`Provisc, HA products.
`
`Table II. Cross-linking Methods for HA
`
`Product
`Intergel
`Hyalobarrier
`Orquest HA
`Restylane
`Perlane
`Hylaform
`Hylaform Plus
`Juvederm 24
`Juvederm 24HV
`Juvederm 30
`Synvisc
`N/A
`
`Cross-linker
`
`Ferric iron
`Internal esterification
`Oxidation
`1,4-butanediol diglycidylether
`1,4-butanediol diglycidylether
`Vinyl sulfone
`Vinyl sulfone
`1,4-butanediol diglycidylether
`1,4-butanediol diglycidylether
`1,4-butanediol diglycidylether
`Vinyl sulfone
`Multifunctional succinimidyl esters of
`polyethylene glycol
`
`Reference
`US 5,532,221
`US 5,676,964
`US 6,303,585
`US 5,827,937
`US 5,827,937
`US 4,500,676
`US 4,500,676
`US 6,685963
`US 6,685963
`US 6,685963
`US 4,500,676
`US 5,470,911
`
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`
`

`

`166
`
`composition is isolated and dried, and the dehydrated powder can be completely
`re-hydrated in saline solution. The crosslink density controls the rheological
`properties and the heat resistance of the gel matrix. Varying the mole ratio of
`DVS to HA and the percent solids in solution can control the viscosity of the
`resultant Hylan from 2-185 Pas before sterilization. More importantly, changing
`the cross-link density can also control the viscosity after terminal sterilization.
`Using a DVS:HA mole ratio of 0.6-1, virtually no decrease in viscosity is seen
`after steam sterilization of the formulation.
`Restylane is a biopolymer gel consisting of hyaluronan from Streptococcus
`equi or S. zooepidemicus that is crosslinked with the di-functional di-glycidyl
`ether of 1,4 butane diol. The crosslinking reaction can be carried out in basic
`medium to promote the formation of ether linkages as the cross-links. The cross(cid:173)
`link density is kept low, <1.0%, which after neutralization and evaporation
`affords a biocompatible viscoelastic gel. This formulation is reported to have
`optimal viscoelastic properties for augmenting the dermis (36). Perlane is also
`crosslinked with the di-glycidyl ether of 1,4 butane diol (see Table II).
`Juvederm 24, 24HV, and 30 are all crosslinked insoluble networks of HA
`for dermal augmentation. These materials are covalently crosslinked with
`butanediol diglycidyl ether by a proprietary process that allows a high solids
`formulation of crosslinked HA that still has good injectability. The difference
`between these materials is the crosslink density and the size of the crosslinked
`particles in the viscoelastic gel.
`Hyalobarrier is an anti-adhesion gel consisting of autocrosslinked HA. In this
`case, the crosslinking chemistry is an inter- and intramolecular esterification
`promoted by several reagents. Using this technique, it was envisioned that the in
`situ residence time of the HA formulation could be increased without introducing
`toxic crosslinking agents that could be leached out of the crosslinked polymer
`matrix (12).
`
`HA Products and % Elasticity
`
`Several studies concerning HA products have referred to a rheological
`property called the percent elasticity (30, 35). Percent elasticity is calculated as
`(G')*100/(G'+G") and is reported as the proportion of elasticity in an HA
`formulation. The higher the percent elasticity the more viscoelastic the HA
`formulation. These studies have also suggested that the efficacy of a HA
`preparation is related to the viscoelasticity of the formulation. The efficacy of
`both viscosupplementation and dermal augmentation are reportedly improved as
`the percent elasticity of the formulation increases. This has been reported for
`HA products that are uncrosslinked and crosslinked. The percent elasticity vs.
`frequency for several HA preparations used in augmenting the dermis are shown
`in Figure 6. The percent elasticity for all of these formulations is relatively
`
` Marchessault et al.; Polysaccharides for Drug Delivery and Pharmaceutical Applications
`
`ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
`
`

`

`167
`
`• Hylaform
`Ο Hylaform Plus
`° Restylane
`
`• Perlane
`A Juvederm 24
`Δ Juvederm 24HV
`• Juvederm 30
`
`^ • • • • •
`
`Ππ
`
`A
`
`À
`
`AA
`
`A
`
`A
`
`A
`
`A
`
`0.1
`
`1
`
`10
`
`100
`
`1000
`
`Frequency (rad/s)
`
`Figure 6. The change in percent elasticity with frequency for several HA
`products currently indicated for augmentation of the dermis
`
` Marchessault et al.; Polysaccharides for Drug Delivery and Pharmaceutical Applications
`
`ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
`
`

`

`168
`
`0
`
`constant over the entire frequency range tested. The Juvederm products have the
`lowest percent elasticity and the Hylaform products have the highest elasticity
`with Restylane and Perlane in between. For the crosslinked HA products, which
`are used for augmentation of the dermis, the relationship of the percent elasticity
`of the formulation to efficacy of the formulation is still in question.
` for several HA
`The percent elasticity at a frequency of 1 Hz and the η
`products are listed in Table I. The percent elasticity at 1 Hz is plotted against the
`η
` for all of these HA products in Figure 7. The data are presented for all
`products in Table I. that have both percent elasticity® 6.2 rad/s and η
` values
`listed. For all these products, including both uncrosslinked and crosslinked H A,
`the percent elasticity correlates in a logarithmic manner to the η
`.
`
`0
`
`0
`
`0
`
`Biomedical Uses of Hyaluronic Acid
`
`Hyaluronic acid has a number of physical properties that vary depending on
`molecular weight, concentration and whether it is uncrosslinked, crosslinked, or
`formed into particles. Table HI lists some of the properties that are expressed by
`various forms of HA. Each application depends on a specific combination of
`physical and biological properties.
`
`HA in Ophthalmology
`
`In vitro experiments have
`Hyaluronic acid is contained in ocular fluid.
`shown that HA provides a protective effect on damaged animal endothelium or
`human corneal endothelium after intraocular lens implantation (57). HA was
`introduced into ophthalmic surgery to take advantage of its physical property of
`high viscosity, and its presence in the vitreous body of the eye. For viscoelastic
`adjuncts used in eye surgery, high concentrations, 10-70 mg/ml of high
`molecular weight H A, 2000-5000 kg mol'1 have been used. The notable
`exception is Viscoat, which is a mixture of relatively low molecular weight HA,
`< 500 kg mol"1 and chondroitin sulfate. These viscoelastic formulations are
`uncrosslinked and characterized as being highly entangled concentrated HA
`polymer solutions with η
` of -100-7000 Pas. These materials are formulated to
`be highly cohesive so that they can be injected and removed entirely as a bolus
`before and after eye surgery (29).
`
`0
`
`Rheumatoid or Osteoarthritic Tissues
`
`In the normal undamaged joint, hyaluronic acid pervades the surface layer
`of the articular tissues and diffuses into the synovial space to lubricate the joint
`at low deformation rates such as resting or walking. Hyaluronic acid is reported
`
` Marchessault et al.; Polysaccharides for Drug Delivery and Pharmaceutical Applications
`
`ACS Symposium Series; American Chemical Society: Washington, DC, 2006.
`
`

`

`169
`
`Ο
`
`5000
`
`10000
`
`15000
`
`20000
`
`25000
`
`Tlo(Pas)
`
`Figure 7. Plotted here is the percent elastic