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May 2016 600 Volume 15 • Issue 5
Copyright © 2016 ORIGINAL ARTICLES Journal of Drugs in Dermatology
SPECIAL TOPIC

Effect of Different Crosslinking Technologies on Hyaluronic

Acid Behavior: A Visual and Microscopic Study of Seven
Hyaluronic Acid Gels
Patrick Micheels MD,a Didier Sarazin MD,b Christian Tran MD,c and Denis Salomon MDd
a
Private Practice, Geneva, Switzerland
Laboratoire Viollier, Geneva, Switzerland
b

c
Department of Dermatology, HCU, Geneva, Switzerland
d
CIDGE International Dermatology Clinic, Geneva, Switzerland

ABSTRACT
Background: The mechanical, rheological, and pharmacological properties of hyaluronic acid (HA) gels differ by their proprietary
crosslinking technologies.
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Objective: To examine the different properties of a range of HA gels using simple and easily reproducible laboratory tests to better
understand their suitability for particular indications.

Penalties Apply
Methods and materials: Hyaluronic acid gels produced by one of 7 different crosslinking technologies were subjected to tests for
cohesivity, resistance to stretch, and microscopic examination. These 7 gels were: non-animal stabilized HA (NASHA® [Restylane®]), 3D
Matrix (Surgiderm® 24 XP), cohesive polydensified matrix (CPM® [Belotero® Balance]), interpenetrating network-like (IPN-like [Stylage®
M]), Vycross® (Juvéderm Volbella®), optimal balance technology (OBT® [Emervel Classic]), and resilient HA (RHA® [Teosyal Global Action]).
Results: Cohesivity varied for the 7 gels, with NASHA being the least cohesive and CPM the most cohesive. The remaining gels could
be described as partially cohesive. The resistance to stretch test confirmed the cohesivity findings, with CPM having the greatest resis-
tance. Light microscopy of the 7 gels revealed HA particles of varying size and distribution. CPM was the only gel to have no particles
visible at a microscopic level.
Conclusion: Hyaluronic acid gels are produced with a range of different crosslinking technologies. Simple laboratory tests show how
these can influence a gel’s behavior, and can help physicians select the optimal product for a specific treatment indication.
Versions of this paper have been previously published in French and in Dutch in the Belgian journal Dermatologie Actualité. Micheels P,
Sarazin D, Tran C, Salomon D. Un gel d’acide hyaluronique est-il semblable à son concurrent? Derm-Actu. 2015;14:38-43.

J Drugs Dermatol. 2016;15(5):600-606.

INTRODUCTION

S
ince their introduction in Europe in 1996, crosslinked hyal- The raw material in the production of HA gels for aesthetic use
uronic acid (HA) gels have progressively replaced bovine consists of pharmacological grade HA chains or HA powder of
collagen as the preferred treatment for filling lines and the same purity, but with different molecular weights, which
folds,1 and account for the vast majority of non-invasive aes- may vary from 600 kDa to more than 2,500 kDa. The final prod-
thetic procedures used in daily practice. ucts differ in terms of their HA concentration and method of
crosslinking. Crosslinking methods may be either chemical or
In its native form, the chemical structure of HA is identical physical, but in the field of aesthetic medicine the crosslinking
across different species. This feature, along with its unique agent that is used to stabilize the majority of HA-based dermal
viscoelastic and physicochemical properties, has led to the de- fillers currently on the market is 1,4-butanediol diglycidyl ether
velopment of numerous HA-based medical devices. However, (BDDE). The stability, biodegradability, and toxicity profile of
due to the short half-life of endogenous HA, chemical modifica- BDDE put it ahead of other crosslinking agents such as divinyl
tions are required to obtain long-lasting gels.2 This is achieved sulfone.5 It should be noted that “natural” crosslinks in the form
by a crosslinking process, which changes the 3-dimensional of Van der Waals forces are also found in all HA preparations
structure of the HA chains and results in the formation of either developed for aesthetic use.
HA microspheres “pearls” or a jelly. While the risk of immu-
nogenicity to HA-derived products is generally low, the altered The basic crosslinking process takes place in 2 steps and is the
structure of the 3-dimensional HA gels may result in them be- same for many currently used HA products that use BDDE as the
ing recognized as foreign by the dermis.3,4 crosslinking agent: (1) dissolution in an alkaline medium and
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May 2016 • Volume 15 • Issue 5

linearization of the HA, and (2) addition of crosslinking agent TABLE 1.

under temperature control. However, crosslinking techniques Definitions Used to Describe the Properties of Gels Produced
differ from one manufacturer to another, and gels vary in the With Different Crosslinking Technologies
final amount of crosslinked HA they contain. These differences Water-soluble polymer crosslinked via
modify the behavior of the gels so that injection techniques and (Hydro)gel
chemical or physical bonds.
depths have to be adapted for the HA gel used. The terms used
A monophasic gel consists of a single phase
to describe the properties of the different gels are defined in
Monophasic and is usually used to describe a gel looking
Table 1.
non-particulate (cohesive).

At least 7 different types of crosslinking technology are used in A biphasic gel traditionally describes a
the production of current HA gels. All of these gels are available particulate gel, which consists of a phase of
Biphasic
with lidocaine, which is introduced during the crosslinking pro- semi-solid crosslinked hyaluronic acid particles
cess by the manufacturers. suspended in a liquid phase.
Cohesion represents the internal forces that unite a
1. Non-Animal Stabilized Hyaluronic Acid (NASHA ) ® solid or liquid particles. A gel is said to be cohesive
In this technique developed by Bengt Agerup MD, the addi-
tion of a small amount of BDDE introduces minute amounts of Do Not Copy
Cohesivity/
if it conserves its unity, its cohesivity or cohesion,
when placed into an aqueous solution (characteristic

Penalties Apply
crosslinks between the polysaccharide chains, resulting in the of monophasic gels) at a low dilution, for instance
cohesion
formation of an entangled matrix.6 The degree of crosslinking 1:3, without agitation. In contrast, a gel is said to
in the original matrix is estimated to be around 10% to 15% be non-cohesive if it is unable to conserve its unity,
and between 1% to 2% in the final product.7 It is hypothesized its cohesion, once placed into an aqueous solution
that the slightly viscous matrix thus obtained is dried and then (characteristic of biphasic gels).
sieved or passed through cleaver filters of different diameters A gel is described as monodensified if it
to produce gel particle sizes adapted to the clinical indications Monodensified consists of a single homogeneous crosslinking
of the final product. This process creates solid HA “pearls,” grade/density zone inside the gel itself.
which are then suspended in a non-crosslinked vector such as
A gel is described as polydensified if it consists
NaCl 0.9% in phosphate buffer (phosphate buffered saline) or
Polydensified of several crosslinking grades/density zones
a non-crosslinked HA gel. The number and size of the pearls
inside the gel itself.
varies depending on the gel indication. The current study used
Restylane® (Q-Med, Uppsala, Sweden), a gel with an average 4. Optimal Balance Technology (OBT®)
pearl diameter of 250 μm (100,000 pearls/mL).8 This technology is used to produce the Emervel® range of HA
gels (Q-Med, Uppsala, Sweden). These have the same HA con-
2. 3D Matrix centration (20 mg/mL) but, unlike the Restylane products that
3D Matrix represents an advancement of Hylacross® tech- differ only in their particle sizes, Emervel products differ in their
nology, but unlike Hylacross is not yet US Food and Drug degrees of crosslinking as well as gel calibration, depending
Administration (FDA) approved (personal communica- on their indication. Thicker or thinner fillers are obtained by
tion, Dr. P. Lebreton, Allergan). Surgiderm® products varying gel calibration, and firmer or softer fillers by varying
(Allergan-Corneal Industry, Pringy, France) are formulated crosslinking.
with 3D Matrix and contain a high ratio of high molecular
weight HA to lower molecular weight molecules. In a single- 5. Cohesive Polydensified Matrix (CPM®)
step crosslinking process, the high and low molecular weight Cohesive polydensified matrix (CPM®) technology is used for the
molecules are mixed. A greater number of BDDE molecules Belotero® range of products (Anteis S.A., Geneva, Switzerland,
are attached by both ends or extremities, resulting in more a wholly owned subsidiary of Merz Pharmaceuticals GmbH)
efficient crosslinking. and is based on a dynamic double crosslinking. In addition to
the classic crosslinking process, 2 additional steps are added:
3. Vycross® the addition of a new amount of HA followed by a continua-
This uses the same crosslinking technique as 3D Matrix, but tion of the crosslinking process. This produces a monophasic
the proportion of high to low molecular weight HA is re- polydensified gel that combines high levels of crosslinked HA
versed, with Vycross® containing a higher proportion of low with lighter levels of crosslinked HA in a cohesive matrix.9
molecular weight HA. It therefore contains less HA (lower HA
concentration) compared with 3D Matrix. Juvéderm Voluma® 6. Resilient Hyaluronic Acid (RHA®)
is so far the only product using this technology to have This is the crosslinking technology used in the Teosyal® (Teo-
received FDA approval. xane Laboratories, Geneva, Switzerland) range of gels. The
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Journal of Drugs in Dermatology P. Micheels, D. Sarazin, C. Tran, D. Salomon
May 2016 • Volume 15 • Issue 5

technology produces gels with long HA chains stabilized by Cohesivity Test

natural and chemical crosslinks. Only a small amount of BDDE When conducted in private practice, 0.6 mL of saline solution
is used to create the gels, which differ in their degree of cross- (NaCl 0.9%) was combined with 2 drops of a coloring agent (Eco-
linking (1.9%-4.0%) as well as their HA concentration. line® no.548 Talens® blue violine from the Royal Talens Society).
To this was added 0.2 mL of the HA gel to be tested by simple
7. Interpenetrating Network-Like (IPN-Like®) pressure on the syringe plunger to avoid any change in the vis-
The Stylage® range of gels (Laboratoires VIVACY®, Archamps, coelastic properties of each gel. No other distortion or stress was
France) use several individual crosslinked matrices, which un- applied. Finally, 2 drops of ethanol 70% were added and the re-
dergo an interpenetrating network-like (IPN-like®) process to cipient gently rotated. Photos were taken before and after the
achieve a monophasic gel, resulting in an increased density of addition of the ethanol. Products were measured precisely us-
crosslinking. The product also contains mannitol, which claims to ing Omnican® syringes (Braun, Switzerland). The same test was
protect the gel to a certain extent from the effects of free radicals. conducted in a private laboratory by coloring 40 mL of saline
serum with Ecoline 548. The investigators then placed 0.9 mL of
In this paper we report on simple and easily reproducible tests this colored saline solution in a Petri dish and added 0.3 mL HA
that can be conducted in the laboratory to allow us to better un- gel. Tests were performed a minimum of 3 times for each gel. The
derstand the properties of HA gels produced by the 7 different
types of crosslinking technology. Do Not Copy
different gels were observed visually and under a microscope
between slides to see if they remained as long, cohesive strands

Penalties Apply
or disintegrated into multiple stands or smaller particles.
"Hyaluronic acid gels produced by one
Resistance to Stretch Test
of 7 different crosslinking technologies We placed 0.2 mL of each gel on a Petri dish. The gels were then
were subjected to tests for cohesivity, pinched with an Adson’s plier to draw them out. A photo was taken
of the gel at maximum stretch and the length noted using a measur-
resistance to stretch, and microscopic ing tape.The test was performed a minimum of 3 times for each gel.
examination."
Equipment
Each laboratory had its own camera, and photographic images
METHODS were taken with the following cameras: Nikon(R) digital camera
Tested Gels D 40 X, lens AF Micro Nikkor 60 mm; Sony® Cyber-shot; Nikon
Between 2006 and 2014, we tested HA gels available on the DXM1200F; and Olympus SC100. Microscopic examinations
Swiss market manufactured by one of the 7 different crosslink- were performed with a Leica® MS5 and a Zeiss Axiokop 40.
ing technologies: NASHA (Restylane), 3D Matrix (Surgiderm
24 XP), CPM (Belotero Balance), IPN-like (Stylage M), Vycross RESULTS
(Juvéderm Volbella), OBT (Emervel Classic), and RHA (Teosyal Microscopic Examination
Global Action). All of the gels were available with lidocaine, in- For the 4 HA gels available for testing in 2011-- NASHA, CPM,
troduced during the crosslinking process by the manufacturers. 3D Matrix, IPN-like -- a difference in viscosity was noted when
The tests were conducted on the gels as they became available, preparing the slides for examination, particularly when spread-
with the last tests conducted in 2014 on Vycross, OBT, and RHA. ing the gels, with NASHA being remarkable for having the least
All gels were marketed for aesthetic indications (filling lines or viscosity. In addition, when rinsing with double distilled water, a
creating volume). The tests were conducted in private practice large amount of the NASHA gel was washed away. This was not
as well as in private and university laboratories observed with the other gels. The most viscous gel was the IPN-
like and the most adherent was CPM. 3D Matrix had an adherence
Microscopic Examination between NASHA and IPN-like. Gels produced with the most recent
For microscopic examination, 0.1 mL of each gel was placed crosslinking technologies (Vycross, OBT, and RHA) were tested in
on a glass slide and spread as for a hematological examina- 2014. Of these, RHA had the greatest viscosity and resistance to
tion. The gel’s resistance to spreading was noted as a simple spreading, but was poorly adherent to the glass slide. Vycross and
estimate of their viscosity. The gels were then colored with tolu- OBT were similar in having an important viscosity and resistance
idine blue at 1 of 2 concentrations (depending on the laboratory to spreading, but less so than RHA. During rinsing, the adherence
where the tests were realized): 0.1% and 0.069% for 30 seconds of Vycross and OBT was also similar and greater than that of OBT.
to 60 seconds before being rinsed twice with double distilled
water. Adhesion to the slide during rinsing was examined. The Observation of the gels, with or without added lidocaine, un-
slide was then covered and placed under the microscope for der a light microscope revealed some significant differences in
examination of the gel’s structure. structure (Figures 1 and 2).
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Journal of Drugs in Dermatology P. Micheels, D. Sarazin, C. Tran, D. Salomon
May 2016 • Volume 15 • Issue 5

FIGURE 1. Appearance of hyaluronic acid gels (NASHA®, CPM® and FIGURE 2. Appearance of hyaluronic acid (HA) gels (Vycross®, OBT®,
3D-Matrix) under the light microscope. The top row images were tak- and RHA®) under the light microscope. The top row images show a
en at HCU, Geneva (toluidine blue, original magnification x12.5). The macroscopic view of the HA gels Vycross, OBT®, and RHA colored
bottom row images were taken at the Laboratory of Histopathology, with toluidine blue. The images below show the appearance of the
Viollier, Geneva (toluidine blue, original magnification x25). same gels under the light microscope (original magnification x25).

Do Not Copy
NASHA
Penalties Apply
Hyaluronic acid particles were clearly emphasized and balloon-
shaped rather than a round pearl. The structure of the gel was
clearly non-cohesive and biphasic.

CPM
The gel had a very specific structure appearing as a continuous
network complex, with some areas of the gel having greater
staining and appearing more dense than others.

3D Matrix and IPN-Like

These gels had similar structures that were totally different
from NASHA or CPM. Compared with CPM, they appeared
lighter, less dense, and with less continuous networks.

RHA
The gel appeared as large grains of compressed particles with a
nice spreading. The gel resembled Vycross, but with larger par-
ticles. There was no real complex continuous network and the
gel could be described as non-cohesive or partially cohesive.

Vycross
When spread on the microscope slide, the gel appeared as fine
grains, finer than RHA and OBT. With magnification, the gel
appeared as many particles compressed closely together and
could be described as particulated, similar to NASHA. Vycross
could be described as a non-cohesive or partially cohesive gel.

OBT
On spreading, the gel appeared as fine grains, but not as fine as
Vycross. On magnification, the gel appeared as a more or less
continuous network comprising particles of different sizes with
an appearance similar to IPN-Like. OBT was also classed as a
non-cohesive or partially cohesive gel.
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May 2016 • Volume 15 • Issue 5

FIGURE 3. Cohesivity test. Investigators placed 0.9 mL of colored TABLE 2.

saline solution in a Petri dish and added 0.3 mL hyaluronic acid gel. Behavior of Hyaluronic Acid Gels Produced by Different
Once placed in the solution, the NASHA® gel disintegrated into Crosslinking Technologies After Contact With Saline Solution
multiple very small particles, indicating it was non-cohesive. Only Crosslinking
CPM® was truly cohesive, remaining as a continuous long strand. Technology
Cohesivity Test Observation
The other gels split into multiple strands, indicating that they were
partially cohesive. Disintegration once in contact with saline
solution. Microscopic particles, “pearls,” of gel
NASHA®
visible. Particles are even palpable when the pure
gel is massaged between thumb and forefinger.
Gel disintegrates into multiple strands or
3D Matrix “sausages” after several seconds. Addition of
ethanol increases the process.
IPN-like® Gel disintegrates like 3D Matrix®.
Gel remains perfectly cohesive with or without
the addition of lidocaine. It remains as a single,

Do Not Copy
CPM ®
long strand “continuous sausage,” even after the
addition of ethanol.

Penalties Apply
Vycross® Gel disintegrates as for 3D Matrix®.
RHA ®
Gel disintegrates as for 3D Matrix®.
OBT® Gel disintegrates as for 3D Matrix®.

University of Geneva, Department of Dermatology, in 2008 on

the 3 FDA-approved gels (Figure 3).10 The results illustrate the
cohesivity of the different gels, with NASHA being the least co-
hesive and CPM the most cohesive. The results were the same
for all gels, whether or not they contained added lidocaine.

Resistance to Stretch Test

For all the HA gels with the exception of CPM, it was not pos-
sible to draw out the gel to a distance greater than 1 cm to
2 cm without the gel breaking (Figure 4, Table 3). This was the
case whether lidocaine had been added by the manufacturer or
not; addition of liquid lidocaine to an HA gel may modify a product’s
cohesivity and change its viscoelastic properties. The CPM gel
could be drawn to a distance of 3.5 cm to 5 cm without break-
ing.

DISCUSSION
A simple set of tests that can be performed in private practice
or in a laboratory reveal large differences in the behavior of
currently available HA gels manufactured using different cross-
linking technologies. Crosslinking is required to slow down
the degradation of endogenous HA, but is also harnessed to
change the rheological properties of HA gels with consequenc-
es on the effectiveness of a product for a particular indication.
Cohesivity Test
Tests performed in private practice showed that the NASHA gel Cohesivity is used to assess the ability of a filler to resist de-
dispersed immediately after contact with saline solution (Fig- formation and maintain product integrity and, along with the
ure 3, Table 2). The addition of ethanol increased the dispersion. elastic modulus (G prime) of a gel, is an important determi-
CPM gel remained completely intact followed in descending nant of the lift capability of a filler. Cohesivity of gels can be
order by Vycross, OBT, RHA, 3D Matrix, IPN-like, and finally NA- measured quantitatively by the amount of pressure required to
SHA. The same results were observed in tests performed at the compress them between 2 plates. In a qualitative measure of
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May 2016 • Volume 15 • Issue 5

FIGURE 4. Resistance to stretch test results. The length of stretch was measured against a metric scale (visible in the background of the lower images).

Do Not Copy
Penalties Apply

cohesivity, we observed the dispersion of the gels after mixing large pools. These patterns are consistent between patients and
with a classic colored saline solution. Some of the gels dis- therefore predictable.8
persed completely, others partially, and one remained totally
cohesive. Products with high cohesivity such as CPM remain as The ability of CPM to distribute homogenously across the tar-
long continuous strands when mixed. In contrast, the non-co- geted area and into the surrounding tissues is due to the fact
hesive gels are dispersed. A further measure of cohesivity was that it contains variable zones of crosslinking density, with ar-
provided by the resistance to stretch test. The results supported eas of higher crosslinking density (harder) interspersed with
the findings above, with the CPM gel demonstrating the great- areas of lower crosslinking density (softer).12 This creates a gel
est resistance to stretching (3.5 cm-5 cm), while the remaining that retains its integrity on injection and has high resistance to
gels could not be stretched for distances greater than 2 cm.
TABLE 3.
Although simple, these easily reproducible laboratory tests can
Behavior of Hyaluronic Acid Gels Produced by Different
help us understand how the different HA gels integrate with
Crosslinking Technologies in Resistance to Stress Test
the collagen and elastin fibers of the dermis. Biopsies of hu-
Crosslinking Maximum Distance Gel can be Drawn (cms)
man skin after injection have shown that the different HA gels
Technology (Minimum of 3 Tests)
have a predictable histologic behavior, which differs by their
type of crosslinking.8,11 CPM, the only monophasic polydensi- NASHA® ≤ 1.0
fied gel, demonstrates homogenous staining and penetrates 3D Matrix ≤ 1.5
all the dermis in a diffuse and evenly distributed manner. Bi- IPN-like ®
≤ 2.0
phasic products such as NASHA appear as large pools of HA
CPM ®
3.5–5.0
distributed as clumps or beads of material in the lower portion
Vycross® ≤ 1.0
of the dermis, with the upper and mid reticular dermis being
free of material. Monophasic monodensified products such RHA ®
≤ 0.5
as 3D Matrix show HA material throughout the dermis, but in OBT ®
≤ 1.5
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May 2016 • Volume 15 • Issue 5

deformation, for example in areas of high facial movement. The

product’s low viscosity also means that it is easily injected, with REFERENCES
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3. Micheels P. Human anti-hyaluronic acid antibodies: is it possible? Dermatol
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low molecular weight (short chain) HA with a higher proportion with the use of chemically-modified animal and nonanimal source hyaluronic
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ness) and medium cohesivity, making it suitable for volumizing tanediol diglycidyl ether-crosslinked hyaluronic acid dermal fillers. Dermatol
Surg. 2013;39(12):1758-1766.
and subcutaneous or supraperiosteal injection. 6. Edsman K, Nord LI, Ohrlund A, Lärkner H, Kenne AH. Gel properties of hyal-
uronic acid dermal fillers. Dermatol Surg. 2012; 38(7 pt 2):1170-1179.
7. Cours à l’Hôpital Tarnier-Cochin, Paris- DU des injectables-Université Paris
Light microscopy confirmed the particulate nature of each V 2009.
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the microspheres has previously been shown to be a factor in Première étude complète des caractéristiques physico-chimiques et essais
cliniques. J Méd Esth Chir Derm. 2005;32:11-20.

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foreign-body reactions, with granulomatous reactions occur- 10. Micheels P, Besse ST, Sarazin D, Grand Vincent A, Portnova N, Diana MS.
ring less frequently after implantation of microspheres with Quantifying depth of injection of hyaluronic acid in the dermis: data from clin-
ical, laboratory, and ultrasound settings. J Drugs Dermatol. 2016;15(4):483-
smooth surfaces.13 Irregular and sharp-edged particles may 490.
also induce more severe granulomatous reactions. 11. Taufig AZ, Szöke A, Kühnel W. A new strategy to detect intradermal reac-
tions after injection of resorbable dermal fillers. J Ästhetische Chirurgie.
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CONCLUSION 12. Micheels P, Besse S, Flynn TC, Sarazin D, Elbaz Y. Superficial dermal injection
With the wide choice of HA gels available on the market, it is of hyaluronic acid soft tissue fillers: comparative ultrasound study. Dermatol
Surg. 2012;38(7 pt 2):1162-1169.
not always easy to select the best filler for a specific purpose. 13. Lemperle G, Gauthier-Hazan N, Wolters M, Eisemann-Klein M, Zimmermann
Despite beginning with the same starting material, HA fillers U, Duffy DM. Foreign body granulomas after all injectable dermal fillers: part
1. Possible causes. Plast Reconstr Surg. 2009;123(6):1842-1863.
are produced with a range of different crosslinking technolo-
gies. With a few simple and easily reproducible tests, we have AUTHOR CORRESPONDENCE
shown how these can influence a gel’s behavior and con-
sequently require an adaptation of injection technique and Patrick Micheels MD
probably depth of injection.10 E-mail:................……..................................... [email protected]

There is no single HA gel for all indications, each treatment

indication requiring a targeted product. Knowledge of the rheo-
logical properties of a gel, combined with proper selection of
injection technique and patients individual anatomy, (eg, skin
thickness and restrictions regarding nerves and blood vessels),
will help physicians select the right product to achieve optimal
cosmetic outcomes.

ACKNOWLEDGMENTS
We would like to thank the Laboratory of Histopathology,
Viollier, Geneva, Switzerland, for its precious and gracious col-
laboration, and for the use of its facilities. We would also like to
thank the manufacturers of the products used for their answers
to our questions. The authors wish to acknowledge the con-
tribution of Jenny Grice for assistance with translation of the
French text and for helping to finalize this manuscript. Editorial
assistance was funded by Merz Pharmaceuticals GmbH.

DISCLOSURES
The authors have no financial disclosure related to the present
study. Medical writing was funded by Merz Pharmaceuticals GmbH.
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