RESI

LIENT H

YALURONIC ACIDTM

PATENTED TECHNOLO

GY

RHA

REFERENCES

1- Sall I., Férard G., "Comparison of the sensitivity of 11 crosslinked hyaluronic acid gels to bovine testis hyaluronidase", Polymer Degradation and Stability 2007;92:915-919. 2-

Jones D., Tezel A., Borell M., "In Vitro Resistance to Degradation of Hyaluronic Acid Dermal Fillers by Ovine Testicular Hyaluronidase", Dermatol Surg 2010;36:804-809. 3- Rzany

B., Becker-Wegerich P., Bachmann F., Erdmann R., Wollina U., "Hyaluronidase in the correction of hyaluronic acid-based fillers: a review and a recommendation for use", Journal

of Cosmetic Dermatology 2009;8:317-323. 4- Borrell M., Leslie D., Tezel A., “Lift capabilities of hyaluronic acid fillers”, J Cosmet Laser Ther. 2011; 13:21-27. 5- Bourdon F., Charton

E., Meunier S., "Lift Capabilities evaluation of Hyaluronic Acid fillers", Poster presentation AMWC Monaco 2012.

T H E B E S T O F H Y A L U R O N I C A C I D

RE

F: 5

0019

6/00

www.teoxane.com

In Vitro Model for the Assessment of the Lasting-Capabilities of Hyaluronic Acid-based Dermal Fillers

The mostcomplete rangewith or withoutlidocaine

In Vitro Model for the Assessment of the Lasting-Capabilitiesof Hyaluronic Acid-based Dermal Fillers François Bourdon, Emeline Burton, Stéphane Meunier PhD*

Teoxane SA, Les Charmilles, Rue de Lyon 105, CH-1203 GENEVA

CONCLUSION■ The lasting capabilities are essential for a dermal filler to ensure an optimal and regular aesthetic result throughout the life time of the implant. A higher resistance to degradation enables to maintain the mechanical properties of the implant over time, and thus to offer better Lasting Capabilities.

■ The measurement of the decrease of G’ after incubation of the gel with HAase is an intuitive method that leads to the direct analysis of the dermal filler resistance to degradation. This is an in vitro and not an in vivo study, which means that the real longevity needs to be confirmed with clinical results.

■ We demonstrated in this in vitro study a correlation between the gel’s resistance to flattening,i.e. its cohesivity, and the gel’s resistance to HAase. Thus, the cohesivity of the gels (measured by their resistance to compression) 4, 5 appears to be an essential factor in the dermal filler lasting capabilities.

■ Teosyal® PureSense Deep Lines and Ultra Deep are the most cohesive gels among the dermal fillers presently tested. They offer in vitro a better resistance to HAase degradation, and thus better mechanical performances over time. These fillers are intended for the filling of deep wrinkles and the restoring of the face volumes. For such indications, these Teosyal® fillers show optimal lasting capabilities, in order to ensure longer aesthetic results.

Juvéderm®Ultra 3

(Allergan)

X30L571190

Juvéderm®Ultra 4

(Allergan)

S30L652347

Restylane® L(Q-Med)

10040

Perlane® L(Q-Med)

10060

Teosyal®PureSenseDeep Lines

(Teoxane SA)

TS27L-112701

Teosyal®PureSenseUltra Deep

(Teoxane SA)

TSUL-112603

Cohesivity is a mechanical property quite different from G’ and recently introduced to define the resistance of the gel to compression. It has been established to be an essential and complementary parameter to G‘ for the description of the mechanical characteristics of a dermal filler 4, 5.

The results on figure 2 demonstrate that there is no correlation between the G’ value levels of the gels before degradation, and their resistance to the depolymerizing action of HAase.

On the contrary, we can observe a good correlation between the resistance to HAase of the gels, and their resistance to compression, i.e. cohesivity (figure 3): the gels with lower cohesive properties show lower capacity to resist to Haase. We can presume that a weaker structure of the gel may facilitate the diffusion and the action of degrading agents such as HAase.

In this way, it appears that the capacity of a dermal filler to resist to HAase - and thus its lasting capability - is closely related to its cohesivity property (or mechanical resistance to flattening).

At last, it was also verified, using the same protocol, that the most resistant filler from this dataset(i.e. Teosyal® PureSense Ultra Deep) was totally digested (the measured value of G’ was zero) with a low “therapeutic dose” of HAase (set up at 30U/g of gel), thus confirming the legitimacy of such treatment to destroy the implant.

We represent hereinafter the resistance to HAase as a function of the initial G' value (figure 2), and as a function of the cohesivity of the tested products (figure 3):OBJECTIVE

This in vitro study evaluates the sensitivity to bovine hyaluronidase (HAase) of 6 commercially available hyaluronic acid (HA) – based dermal fillers. A correlation has been established between the gel’s resistance to HAase, and the gel’s resistance to compression, i.e. its cohesivity. Results obtained are good indicators to assess the best candidates for long-lasting capabilities products.

INTRODUCTION HA-based dermal fillers are non-permanent implants that will degrade in vivo via complexmechanisms including free radicals and HAase. The resistance of the gels against such degradation could be linked to their intrinsic mechanical properties. However, there is no study so far that demons-trates this relationship. Besides, the methods described in the literature to characterize the in vitro sensitivi-ties of HA gels to HAase are generally non-direct, and consist in assaying the fragments of HA released upon HAase action 1,2. We describe here a simple and intuitive test, based on the effect of the degradation on the gel’s mechanicals properties. This new method resides in measuring the decrease of the gel G’ (elastic modulus measured in oscillatory rheology) after incubation with a precise dose of HAase during a limited time. The decrease of G’ can be linked to the decline of the lift capabilities, when the gel is being degraded in vivo. It is to be noticed that the dose of HAase used for the present test is about 50 times lower than the dose possibly used for a therapeutic action 3, i.e. treatment of overcorrection or rare complications. With such “therapeutic doses”, all the dermal fillers of this study are totally degraded.

MATERIAL AND METHODSThe fillers were obtained from commercial sources. A same batch of product was used for all the tests before expiry date.

■ HAase degradation100µL of a solution of HAase (from bovine testicular, type IV-S, Sigma), prepared at 16U/mL in H2O, is added to 3g of the tested HA gel filler. Thus the global activity of HAase is 0,5U/g of gel, which represents globally 1,5U HAase in each sample. The resulting mixture is homogenized and incubated 24 hours at 37°C, then immediately cooled at 25°C for rheology measurement.

■ Rheology: Dynamic oscillatory testEach sample is tested before and after incubation with HAase, with the following protocol: measurements at 25°C and 1Hz ω frequency, with amplitude sweep φ corresponding to an applied deformation strain from 1 to 1400Pa, using a Thermo Haake RS3000 rheometer with a 35mm / 1° Titanium cone-plate geometry. The resulting stress response is measured: G' and δ are recorded at low strain (τ = 5Pa), i.e. almost at rest.

■ Compression test2.5 g of gel are placed between the 2 plates of 35mm plane-plane geometry, using a Thermo Haake RS3000 rheometer. The rheometer is set to a normal force mode: the upper plate is put in contact with the gel and is lowered toward the bottom plate, thus compressing the gel. The course is stopped when a 70% compression rate is reached. The resulting normal force is measured and integrated during the experiment, from 0 to 70% compression rate, which leads to the resistance to compression (N.s.).

RESULTS AND DISCUSSIONFigure 1 shows the G’ values measured for the HA gel fillers before and after incubation with HAase.

Elastic modulus G' is a measure of the energy which is stored by the gel and returned when the gel is submitted to small deformations in oscillatory rheology: it represents the hardness of the gel at rest. The G’ values of the HA gel fillers tested are quite different. But in all cases, the incubation with HAase results in a decrease of the G’ values, which demonstrates the degradation of the gels due to the enzyme (figure 1). Thus, we assess the resistance to HAase as the % of the initial G' remaining after degradation. The fillers usually described as “particulate gels” (Restylane® L and Perlane® L) show the highest G’ values in this dataset, but we observe a significant degradation of these fillers after incubation with Haase (respectively 25 and 39% of the initial G’ value remained after degrada-tion). In comparison, the decrease of G’ after incubation is lower for the Juvéderm® products (56 and 69% of initial G' remained), and far lower for Teosyal® products (81 and 89% of initial G’ remained).

Teosyal® PureSenseUltra Deep

Teosyal® PureSenseDeep Lines

20

40

60

80

100

Res

ista

nce

to H

Aas

e (%

)

Resistance to compression, or "Cohesivity" (N.s.)

00 5 10 15 20 25 30 35 40

Juvéderm® Ultra 4

Juvéderm® Ultra 3

Perlane® L

Restylane® L

Restylane® L

LONG-LASTING CAPABILITIES

0

50

100

150

200

250

300

350

69%

Juvéderm®

Ultra 3

56%

Teosyal®PureSenseDeep Lines

81%

Perlane® L

39%

Teosyal®PureSenseUltra Deep

Restylane® L

25%

G' (

Pa)

Initial Gel Gel after degradation by HAase

Juvéderm®

Ultra 4

REFERENCES

1- Sall I., Férard G., "Comparison of the sensitivity of 11 crosslinked hyaluronic acid gels to bovine testis hyaluronidase",

Polymer Degradation and Stability 2007;92:915-919. 2- Jones D., Tezel A., Borell M., "In Vitro Resistance to Degradation of

Hyaluronic Acid Dermal Fillers by Ovine Testicular Hyaluronidase", Dermatol Surg 2010;36:804-809. 3- Rzany B., Becker-

Wegerich P., Bachmann F., Erdmann R., Wollina U., "Hyaluronidase in the correction of hyaluronic acid-based fillers: a

review and a recommendation for use", Journal of Cosmetic Dermatology 2009;8:317-323. 4- Borrell M., Leslie D., Tezel A.,

“Lift capabilities of hyaluronic acid fillers”, J Cosmet Laser Ther. 2011; 13:21-27. 5- Bourdon F., Charton E., Meunier S.,

"Lift Capabilities evaluation of Hyaluronic Acid fillers", Poster presentation AMWC Monaco 2012.

* Corresponding author:s.meunier@teoxane.com

www.teoxane.com

Figure 1: G' values measured before and after incubation with HAase

89%

Resistance to HAase = % of remaining G' after HAase degradation

Figure 3: Resistance of HAase as a function of the cohesivity

Teosyal® PureSenseUltra Deep

Teosyal® PureSenseDeep Lines

20

40

60

80

100

Res

ista

nce

to H

Aas

e (%

)

Elastic Modulus G' (Pa)

00 100 150 200 250 300 350

Juvéderm® Ultra 4

Juvéderm® Ultra 3

Perlane® L

Figure 2: Resistance to HAase as a function of the initial G' value