Polymer Degradation and Stability 92 (2007) 915e919

www.elsevier.com/locate/polydegstab

Comparison of the sensitivity of 11 crosslinked hyaluronic acid

gels to bovine testis hyaluronidase
Ibrahima Sall*, Georges Férard
Laboratoire de Biochimie Appliquée, Faculté de Pharmacie, Université Louis Pasteur, 74 Route du Rhin, BP 60024, F-67401 Illkirch Cedex, France
Received 12 October 2006; accepted 15 November 2006
Available online 27 January 2007

Abstract

Crosslinked hyaluronan gels are used in various applications where their stability is a prerequisite. The sensitivity of such gels to hyaluron-
idase can be determined as an index of stability by several approaches: chromatography, electrophoresis, and viscometry. We describe here a test
based on the colorimetric determination of the N-acetyl-D-glucosamine released by hyaluronidase in standardized conditions. The sensitivities to
bovine testicular hyaluronidase of 11 different gels used to fill skin wrinkles (Restylane; Perlane; Juvéderm 18, 24, 24HV, 30, and 30HV;
Surgiderm 18, 24XP, 30, and 30XP) were compared.
The method was reproducible, easy to perform, not time-consuming and allowed us to demonstrate that the sensitivity to testicular hyaluron-
idase was dependent on the degree of crosslinking of the gels and also on their monophasic/biphasic nature. Under our conditions, Surgiderm 30,
24XP and 30XP were the most resistant gels.
We propose to retain the hyaluronidase test to predict the in situ stability of a crosslinked gel used to fill skin wrinkles.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Crosslinked hyaluronan gels; Stability; Sensitivity to hyaluronidase; N-Acetyl-glucosamine reducing end assay

1. Introduction a major role in the organization and integrity of the extracel-

lular matrix, thereby participating in the preservation of the
Hyaluronic acid or hyaluronan (HA) [1e3] is a high-molec- form and in the spatial arrangement of tissue components.
ular mass linear, anionic polysaccharide without branching Unlike collagen, it is present in an identical form in all animal
side-chains, composed of 2000e25 000 disaccharide units and bacterial species with the largest amount in the skin. HA is
formed by glucuronic acid and N-acetyl-D-glucosamine involved in numerous biological and physiological functions
(NAG), which are linked by b(1,4)-glycosidic bond, and can such as cell motility, cell matrix adhesion, cell proliferation,
reach 105e107 Da in molecular mass. This polymer represents water homeostasy of tissues, or joint lubrication [6]. The HA
a class of ubiquitous molecules [glycosaminoglycans (GAG)], molecules can absorb a large volume of water which expands
the only one not linked to a core protein, non-synthesized in the in the extracellular space, hydrates tissues and finally main-
Golgy apparatus, and the only non-sulfated GAG [4]. In tains the moisture of the skin [7,8]. Degradation in the body
the human body, HA is a major component of the extracellular can occur in any of the three ways: attack by free radicals, en-
matrix, the skin, the synovial fluid, loose connective tissues, zymes (hyaluronidases), or thermal [9]. Natural HA provides
umbilical cord, vitreous body of the eye and the cartilage a biological material with high viscoelastic and rheological
[5]. It exhibits a wide range of biological functions and plays properties which, in addition to its non-immunogenicity, its
biocompatibility, and its total biodegradability, makes it suit-
able for various medical applications such as dermatology, sur-
* Corresponding author. Fax: þ33 390 24 42 86. gery and wound healing, embryo implantation and drug
E-mail address: [email protected] (I. Sall). delivery [10]. Hyaluronidases are endo-glucosidases that can

0141-3910/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymdegradstab.2006.11.020
916 I. Sall, G. Férard / Polymer Degradation and Stability 92 (2007) 915e919

break down GAG such as HA, chondroitin, or chondroitins 4- 2. Materials and methods
and 6-sulfate [11]. These enzymes fall into three classes,
according to their hydrolysis mechanism. Testicular or 2.1. Chemicals and instruments
venom-type hyalurono-glucosaminidases (EC 3.2.1.35) break
the b(1,4) link [12]; they are vertebrate-type endo-b-acetyl- We tested the sensitivity of the following 11 crosslinked
hexosaminidases generating tetrasaccharides as predominant gels to hyaluronidase: Restylane (lot 7345-1), Perlane (lot
end-products. They lack substrate specificity, because in 6380-1) are obtained from Q-Med (Uppsala, Sweden); Juvé-
addition to HA, they can hydrolyze chondroitin sulfate and derm 18 (lot IN180410), Juvéderm 24 (lot IN240450), Juvé-
dermatan sulfate but at a much slower rate. Various enzymes derm 24HV (lot HV240441), Juvéderm 30 (lot IN3004126),
of the mammalian group are involved in the pathophysiology Juvéderm 30HV (lot HV300427), Surgiderm 18 (lot SG182
of many disorders like cancers, osteoarthritis, mucopolysac- 10620), Surgiderm 24XP (lot XP24210626), Surgiderm 30
charidosis, liver, and skin diseases [13,14]. Hyalurono-glucu- (lot SG30210622), and Surgiderm 30XP (lot XP30210624)
ronidases (EC 3.2.1.36) which are produced by salivary are from Cornéal (Pringy, France). Restylane and Perlane
glands of leeches, parasites and crustaceans break the b(1,3) gels are biphasic in constitution which means they are com-
link [15]; they are endo-b-glucuronidases generating tetra- posed of distinct swollen particles in a continuous homoge-
and hexasaccharides. This group is specific mainly for HA. neous phase; Restylane has 100 000 particles per ml and
Hyaluronate-lyases (EC 4.2.99.1) are the third class of en- Perlane has 10 000 particles per ml of gel. The other tested
zymes found mainly in bacteria; they can degrade HA and gels (Surgiderm and Juvéderm families) are all monophasic
to some extent chondroitin and dermatan sulfate. They are and homogeneous without distinct particles. The Surgiderm
operated by a b-elimination reaction yielding essentially gels have the same features as those of Juvéderm family,
disaccharides. except that they are produced with another more efficient
In order to increase the biomechanical properties and the crosslinking process. All these gels are used as dermal fillers
resistance to enzymatic break down of natural HA containing in the treatment of aging skin and for soft tissue augmentation:
preparations (one-third of the human body’s HA is turned facial lines, wrinkles, and lips.
over each day by lymphatics and the liver), various methods The amount of crosslinking agent in the tested gels, noted
have been developed for chemical modification or crosslink- as a number of (þ) is also variable: (þ) for Restylane and Per-
ing of native HA by covalent links, resulting in larger and lane; (þþ) for Juvéderm 18, Surgiderm 18 and Juvéderm 24;
most stable derivatives that retain HA biocompatibility and (þþþ) for Juvéderm 30, Juvéderm 24HV, Surgiderm 30, and
biodegradability in vivo [16]. Crosslinking HA hydrogels re- Surgiderm 24XP; (þþþþ) for Juvéderm 30HV and Surgi-
sult in significant increase in tissue residence time, slow deg- derm 30XP.
radation, and the same bioresorbability. They are significantly All the tested gels are made of crosslinked non-animal HA
more stable than their non-crosslinked counterparts, because from a streptococcus bacterial fermentation source, swollen in
of the formation of a dense network by intermolecular bonds phosphate buffer at a defined concentration. The crosslinking
or bridges, that is much less susceptible to degradation by process (also named stabilization by QMED) uses 1,4-butane-
tissue enzymes such as the hyaluronidases [17,18], oxygen- diol diglycidylether as the crosslinker.
derived free radicals [19] or the temperature [20]. This makes Hyaluronidase from bovine testis was purchased from Sigma
modified HA derivatives obtained by bacterial cultures suit- Chemical Co. (St Louis, USA; ref H3506, 608 USP Units/mg).
able to be employed safely in most various medical and All other chemicals were of the highest purity available.
biological applications [21], and also without any risk of aller- The absorbances were measured, using an Uvikon 922
gic reactions. Crosslinked HA gels have multiple uses, such as (Kontron, Milano, Italy) dual-beam spectrophotometer.
aesthetic surgery (wrinkle smoothing), gynaecology, or drug
transport [22]. 2.2. Preparation of gel pellets
A wide number of techniques have been reported in the lit-
erature to follow-up in vitro the degradation rate of crosslinked The gel syringes were stored in sealed plastic bags at a tem-
HA hydrogels under the action of testicular hyaluronidase perature of þ4  C until use. They were brought to laboratory
[5,23,24]: change in viscosity, in water content, in molecular room temperature for 1 h before use.
mass by chromatography, gel plate assay in a Petri dish, or col- Gel samples’ weights between 0.1 and 0.5 g were placed in
orimetric assays for the liberated glucuronic acid. All these the bottom of stoppered glass tubes. These tubes were then
methods are complex, time-consuming or they need sophisti- centrifuged for 5 min at 1000 g in a Beckman Allegro bench
cated and expensive instruments. top refrigerated centrifuge equipped with a swinging bucket
The aim of this study was to develop a heterogeneous phase rotor. At the end of the centrifugation, thin pellets firmly at-
approach based on the measurement of the NAG release by an tached at the bottom of the tubes were obtained.
improved colorimetric method to evaluate the sensitivities of
different crosslinked HA hydrogels to the action of beef testis 2.3. Hyaluronidase sensitivity test
hyaluronidase. The test was then applied to 11 different com-
mercially available gels, differing in their crosslinking degree A solution of hyaluronidase was prepared as required at
and their monophasic/biphasic nature. a concentration of 10 mg/ml (6080 U/ml) in an isotonic
 I. Sall, G. Férard / Polymer Degradation and Stability 92 (2007) 915e919 917

phosphateeNaCl buffer (10e155 mmol/l) at a pH of 7.4. To sufficient to obtain the maximum hydrolysis rate (correspond-
measure the degradation power of the hyaluronidase on the ing to enzyme substrate saturation) for the added hyaluroni-
gels, a heterogeneous method with no prior dilution of the dase activity. Moreover, with such gel amount, only thin
gel was used. The glass tubes containing the gel pellets and pellets were formed at the bottom of the tubes after centrifu-
the hyaluronidase solution were pre-incubated separately for gation. Therefore, to compare the sensitivities of the 11 gels
10 min at 37  C. Then 100 ml of the enzyme solution was in- to hyaluronidase, a fixed test sample consisting of 0.3 g for
troduced gently, without stirring, onto the surface of the gels. each of the gels was used.
After incubation for 120 min at 37  C, the enzymatic reaction
was stopped by the addition of 0.1 ml of a potassium tetrabo-
3.2. Sensitivities of the 11 gels to hyaluronidase
rate solution (0.8 mol/l, at a pH of 9.1), followed by immediate
stirring on a vortex mixer and heating for 3 min at 100  C. The
Under our conditions, (a pellet consisting of 0.3 g of gel,
tubes were then cooled at room temperature and the released
enzyme volume of 100 ml, pH 7.4, temperature 37  C, incuba-
NAG was assayed.
tion time of 120 min), we compared the absorbance values at
585 nm that corresponded to the amounts of the released NAG
2.4. Assay of the released NAG due to the effect of the hyaluronidase on the 11 gels (three
samples for each gel). The mean absorbance values obtained
We used Ehrlich’s reagent, in accordance with the method at 585 nm varied according to the gel with decreasing values
described by Reissig et al. [25]. To each tube we added 3 ml from Restylane to Juvéderm 24HV (Fig. 2).
of freshly prepared reagent. The contents of the tubes were The absorbance values were related to the initial HA
mixed by vigorous stirring on a vortex mixer. The tubes content of the gels and standardized, taking Restylane as the
were then stoppered and incubated for 20 min at 37  C, which reference gel, for calculating relative rates of degradation
allowed the development of a violet colour. The tubes were (Table 1). These relative rates also decreased from Restylane
then centrifuged at 1000 g for 15 min to remove at the same to Juvéderm 24HV.
time the turbidity in the reaction medium and also the gel frag- In another set of experiments, we compared the absorbance
ments that were still present. After confirming previously that values at 585 nm for four Surgiderm gels to that of Restylane
gels incubated without addition of enzyme did not produce (Fig. 3). Surgiderm 30, 24XP and 30XP were the most resis-
any violet colour with Ehrlich’s reagent, the reagent blanks tant in this second group of gels.
were prepared with only the phosphate buffer and the colour
reagent.
4. Discussion

3. Results In this study, we first determined the amount of a Juvéderm

gel required to reach the maximum rate of hydrolysis pro-
3.1. Variation of the amount of NAG released as duced by a fixed activity of hyaluronidase from bovine testis,
a function of gel amount
0,30
Results which were obtained with increasing weights of Ju-
Abs (585 nm) per mg of initial amount of NaHA

véderm 30 crosslinked gel (Fig. 1), showed that the NAG

absorbance curve at 585 nm as a function of gel amount was 0,25
hyperbolic, tending toward a plateau from 0.3 g of gel and ex-
hibiting MichaeliseMenten kinetics. Between 0.3 and 0.5 g of 0,20
gel, the increase in absorbance reached hardly 3e4%. This
means that the test sample containing 0.3 g of gel was
0,15

1.5
0,10
Absorbance (585 nm)

1.0 0,05

0,00
0.5 e
an ne 18 24 30 HV HV
tyl rla v. v. v. 24 30
s Pe Ju Ju Ju . .
Re Juv Juv
Gel wt = 0.3 g; Vol hyaluronidase = 100 µl; Conc hyaluronidase sample = 6080 U/ml;
0.0 Temp= 37 º C; pH = 7.4; Time = 120 min.
0.0 0.1 0.2 0.3 0.4 0.5 0.6 Each point is the mean of three measurements ± 2 SD
Weight of gel per tube (g)
Fig. 2. Comparison of the sensitivity to hyaluronidase for Juvéderm gels vs
Fig. 1. Effect of Juvéderm 30 gel weight on the amount of NAG released. Restylane/Perlane gels.
918 I. Sall, G. Férard / Polymer Degradation and Stability 92 (2007) 915e919

Table 1
Comparison of the HA hydrolysis rates for the seven gels by hyaluronidase
Gel NaHA content (mg/g) Weight (g) NaHA (mg) Absorbance (585 nm) Absorbance per Relative rate
g of NaHA of degradationa
Restylane 20.0 0.300 6.00 1.185  0.328 197.50  54 100
Perlane 20 0.300 6.00 1.165  0.175 194.17  29 98.3
Juvéderm 18 17.8 0.300 5.34 0.952  0.172 178.28  32 90.3
Juvéderm 30 22.1 0.300 6.63 0.756  0.215 114.03  32 57.7
Juvéderm 24 23.3 0.300 6.99 0.792  0.172 113.30  24 57.4
Juvéderm 30HV 22.1 0.300 6.63 0.690  0.109 104.07  16 52.7
Juvéderm 24HV 22.8 0.300 6.84 0.624  0.078 91.23  11 46.2
The indicated values correspond to the mean of three measurements  2 SD for each gel.
a
Expressed in percent by taking Restylane as the reference gel.

which falls in the same mammalian group as the skin enzyme As previously pointed by Takahashi et al. [28], it was neces-
[26], with significant homology between them. This source of sary to centrifuge the final medium reaction prior to absor-
enzyme was retained because of its frequent use for the study bance measurement in order to remove turbidity and small
of action of mammalian enzyme on HA [27]; its mechanism of gel fragments which were still present. Moreover, it was pos-
action is similar to that of human hyaluronidases as from skin, sible to determine the degradation rate of HA hydrogels on
and also for its easy commercial availability in pure form. The several series with different samples. In our hands, the hyal-
determination of the optimum gel amount then allowed us to uronidase test was reproducible enough to show marked differ-
compare the sensitivities of 11 crosslinked HA gels, from dif- ences in the sensitivity to the enzyme between the 11 gels
ferent sources or grades, to hyaluronidase. tested here. Furthermore, in contrast to tests using chromatog-
The variation curve of the amount of NAG produced as raphy, electrophoresis or viscometry, the present colorimetric
a function of the Juvéderm 30 sample weight, showed a maxi- test is direct in the sense that it permits us to determine the
mum rate with 0.3 g sample. After initial centrifugation of the number of b(1,4) links hydrolyzed by hyaluronidase.
gel tubes, this sample amount finally yielded thin pellets that For the heterogeneous phase measurement assay, we have
were fairly close to the in situ operating conditions. The het- retained conditions (solid surface erosion effect, temperature
erogeneous phase results, i.e. with no prior dilution of the 37  C, and neutral pH 7.4) very similar to the in vivo condi-
gels, also appeared to be more reproducible than the results tions on the skin. The obtained results indicated that the sen-
obtained in a homogeneous phase with prior dilution, that is sitivity of the gels to the effect of the enzyme decreased as
after gels were diluted in a buffer solution (data not shown). a function of their amount of crosslinker. Restylane was the
The heterogeneous enzyme test was easy and rapid to per- most sensitive, while Surgiderm 30, 24XP and 30XP gels
form with only a small amount of gel, and required neither were the most resistant. The sensitivities of Juvéderm 18, 24
specialized reagents nor a particular measuring instrument. and 30 and Surgiderm 18 were intermediate. This difference
in sensitivity among the 11 gels may be due to more limited
access by the enzyme to its HA substrate for Juvéderm
0,30 24XP and 30XP that are more highly crosslinked than Resty-
lane/Perlane, and more efficiently crosslinked than Juvéderm
Abs (585 nm) per mg of initial

0,25 24HV and 30HV. Similarly, among the group of five studied
Juvéderm gels, the Juvéderm 18 (the least crosslinked gel) dis-
amount of NaHA

0,20 played the greatest sensitivity to the effect of the enzyme

(Table 2). In general the viscosity of tested gels increases
0,15 with their concentration, but also with their reticulation de-
gree; the latter being in the following increasing order: Juvé-
0,10 derm 18 and Surgiderm 18 < Surgiderm 30 and Juvéderm
30 < Surgiderm 24XP and Juvéderm 24HV < Surgiderm
0,05 30XP and Juvéderm 30HV. The consequence for the most
heavily crosslinked gels is a greater stability in comparison
0,00 with the other non-crosslinked gels as observed with another
e 18 30
lan XP XP analytical approach [29,30].
ty rm rm 24 30
es e e m m
R r gid r gid id er id er Gels such as Restylane and Perlane displayed greater sensi-
Su Su rg rg
Su Su tivity to the effect of the enzyme among all of the 11 studied
Gel wt = 0.3 g; Vol hyaluronidase = 100 µl; Conc hyaluronidase sample = 6080 U/ml;
Temp= 37 ºC; pH = 7.4; Time = 120 min. gels. Their biphasic nature can also explain their greatest sen-
Each point is the mean of three measurements ± 2 SD
sitivity toward hyaluronidase: the distinct particles offer
Fig. 3. Comparison of the sensitivity to hyaluronidase for Surgiderm gels vs a greater surface to the attack of the enzyme than monophasic
Restylane gel. gels like Juvéderm or Surgiderm gels. In addition, we observed
 I. Sall, G. Férard / Polymer Degradation and Stability 92 (2007) 915e919 919

Table 2
Comparison of the HA hydrolysis rates for five gels by hyaluronidase
Gel NaHA content (mg/g) Weight (g) NaHA (mg) Absorbance (585 nm) Absorbance per Relative rate
g of NaHA of degradationa
Restylane 20.0 0.300 6.00 1.391  0.056 231.83  9.3 100
Surgiderm 18 17.8 0.300 5.34 0.737  0.010 138.01  1.9 59.5
Surgiderm 30XP 24.6 0.300 7.38 0.747  0.012 101.22  1.6 43.7
Surgiderm 24XP 25.5 0.300 7.65 0.759  0.079 99.21  10.3 42.8
Surgiderm 30 24.3 0.300 7.29 0.707  0.102 96.98  14 41.8
The indicated values correspond to the mean of three measurements  2 SD for each gel.
a
Expressed in percent by taking Restylane as the reference gel.

with the naked eye a much more rapid liquefaction of these [13] Frost GI, Csóka TB, Stern R. The hyaluronidases: a chemical, biological
two gels after the addition of hyaluronidase to the incubation and clinical overview. Trends Glycosci Glycotechnol 1996;8:
419e34.
tubes. Meanwhile, the Juvéderm 30XP gel required more time [14] Jedrzejas M. Structural and functional comparison of polysaccharide
before starting to liquefy under the action of the enzyme. It degrading enzymes. Crit Rev Biochem Mol Biol 2000;35:221e51.
should be later possible to compare the heterogeneous phase [15] Sutherland IW. Polysaccharide lyases. FEMS Microbiol Rev 1995;16:
sensitivity results with viscosity measurements: measurement 323e47.
of elasticity change as a function of time. [16] Balazs EA. In: Laurent TC, editor. The chemistry, biology and medical
application of hyaluronan and its derivatives. London: Portland Press;
The calculation of the relative rates referred to the initial 1998. p. 185.
HA content of the gels indicates that Restylane and Perlane [17] Yui N, Nihira J, Okano T, Sakurai Y. Regulated release of drug micro-
are the most sensitive gels, followed closely by Juvéderm spheres from inflammation responsive degradable matrices of cross-
18. The least sensitive of the 11 studied gels were clearly linked hyaluronic acid. J Controlled Release 1993;25:133e43.
the Juvéderm 24XP and the Juvéderm 30XP. [18] Yui N, Okano T, Sakurai Y. Inflammation responsive degradation of
cross-linked hyaluronic acid gels. Regulated release of drug micro-
This study also demonstrates that all tested Juvéderm and spheres from inflammation responsive degradable matrices of cross-
Surgiderm gel implants compared to Restylane are indeed linked hyaluronic acid. J Controlled Release 1992;22:105e16.
less degradable by hyaluronidase that means better stability [19] McNeil JP, Wiebkin OW, Betts WH, Cleland LE. Depolymerization
and longer half-life. On the other hand Surgiderm gels have products of hyaluronic acid after exposure to oxygen-derived free
also better stability than Juvéderm gels. radicals. Ann Rheum Dis 1985;44:780e9.
[20] Bothner H, Waaler T, Wik O. Limiting viscosity number and weight of
hyaluronate samples produced by heat denaturation. Int J Biol Macromol
1988;10:287e91.
References [21] Volpi N. Therapeutic applications of glycosaminoglycans. Curr Med
Chem 2006;13:1799e810.
[1] Meyer K, Palmer JW. The polysaccharide in the vitreous humour. J Biol [22] Luo Y, Kirker KR, Prestwitch GD. Cross-linked hyaluronic acid hydrogel
Chem 1934;107:629e34. films: new biomaterials for drug delivery. J Controlled Release 2000;69:
[2] Laurent TC, Fraser J. Hyaluronan. FASEB J 1992;6:2397e404. 169e84.
[3] Toole BP. Hyaluronan. In: Iozzo RV, editor. Proteoglycans. New York: [23] Vercruysse KP, Marecak DM, Marecek JF, Prestwich GD. Synthesis and
Marcel Dekker; 2000. p. 61. in vitro degradation of new polyvalent hydrazide cross-linked hydrogels
[4] Laurent TC, Laurent UB, Fraser JR. The structure and function of of hyaluronic acid. Bioconjugate Chem 1997;8:686e94.
hyaluronan: an overview. Immunol Cell Biol 1996;74:A1e7. [24] He D, Zhou A, Wei W, Nie L, Yao S. A new study of the degradation of
[5] Prestwitch GD, Marecak DM, Marecek JF, Vercruysse KP, Ziebel MR. hyaluronic acid by hyaluronidase using quartz crystal impedance tech-
Controlled chemical modification of hyaluronic acid: synthesis, applica- nique. Talanta 2001;53:1021e9.
tions, and biodegradation of hydrazide derivatives. J Controlled Release [25] Reissig JL, Strominger JL, Leloir LF. A modified colorimetric method
1998;53:93e103. for the estimation of N-acetylamino sugars. J Biol Chem 1955;217:
[6] Hirano K, Sakai S, Ishikawa T, Avci FY, Linhardt RJ. Preparation of 959e96.
methyl ester of hyaluronan and its enzymatic degradation. Carbohydr [26] Cashman DC, Laryea JU, Weissman B. The hyaluronidase of rat skin.
Res 2005;340:2297e304. Acta Physiol Scand 1988;134:405e11.
[7] Comper WD, Laurent TC. Physiological functions of connective tissue [27] Asteriou T, Deschrevel B, Delpech B, Bertrand P, Bultelle F, Merai C,
polysaccharides. Physiol Rev 1978;58:255e315. et al. An improved assay for the N-acetyl-D-glucosamine reducing ends
[8] Stern R. Hyaluronan catabolism: a new metabolic pathway. Eur J Cell of polysaccharides in the presence of proteins. Anal Biochem
Biol 2004;83:317e25. 2001;293:53e9.
[9] Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribution, [28] Takahashi T, Ikegami-Kawai M, Okuda R, Suzuki K. A fluorimetric Mor-
functions and turnover. J Intern Med 1997;242:27e33. ganeElson assay method for hyaluronidase activity. Anal Biochem
[10] Brown MB, Jones SA. Hyaluronic acid: a unique topical vehicle for the 2003;322:257e63.
localized delivery of drugs in the skin. J Eur Acad Dermatol Venerol [29] Zhong SP, Campoccia D, Doherty PJ, Williams RL, Benedetti L,
2004;19:308e18. Williams DF. Biodegradation of hyaluronic acid derivatives by hyaluron-
[11] Kreil G. Hyaluronidases e a group of neglected enzymes. Protein Sci idase. Biomaterials 1994;15:359e65.
1995;4:1666e9. [30] Li P, Dehazya P. In vitro degradation of cross-linked hyaluronic acid
[12] Meyer K. Hyaluronidases. In: Boyer PD, editor. The enzymes. New matrices by hyaluronidase: effect of post polymerization cross-linking.
York: Academic Press; 1971. p. 307. Technical Report 04: Clear Solutions Biotech, Inc., NY, USA; 2002.