The Difference Between Stabilization and Crosslinking

The Difference Between Stabilization and Crosslinking
Katarina Edsman, Åke Öhrlund, Cecilia Sturesson, Lars Nord, Anne Helander Kenne and Jacques Näsström
Q-Med AB, Seminariegatan 21, SE-752 28 Uppsala, Sweden.
Objective
Conclusions
Conclusions
The objective of this study was to investigate if there are measurable differences between hyaluronic acid
(HA) dermal fillers manufactured using the NASHA™ process and products using other crosslinking
processes.
Introduction
Most hyaluronic acid (HA) dermal fillers are chemically modified with crosslinkers to improve the
mechanical properties and the duration in vivo. The crosslinking can be made in many different ways.
The most common method is traditional crosslinking where most of the gel strength is achieved by
introducing chemical (synthetic) crosslinks. Alternatively there is stabilization where a small amount of
synthetic crosslinks stabilize the natural crosslinks (entanglements), see Figure 1.
Entanglement
of HA chains
(natural crosslinks)
Synthetic crosslink
a
+
HA
Reagent
b
shows the differences between the HA-dermal fillers manufactured
with the NASHA™ process and products manufactured with other
crosslinking processes.
• A high modification efficiency (MoE) was obtained for Restylane
products manufactured using the NASHA™ process, which
preserves entanglements that contribute to the gel strength.
• NASHA™ technology is unique since it combines a strong gel and a
minimal modification of the HA.
Results
+
Reagent
• The modification efficiency (MoE) is a measurable quantity that
HA
c
Figure 1. a) Synthetic and natural crosslinks (entanglements). b) Traditional crosslinking processes result
in a gel with numerous chemical crosslinks and few natural entanglements. c) Stabilization gives a low
number of chemical crosslinks and a large number of natural entanglements.
The modification efficiency (MoE) is shown in Figure 2. All the examined HA-fillers have MoE values
below one, whereas the Restylane products have MoE values over four. The NASHA stabilization process
used for manufacturing the Restylane products results in a very high gel strength for each modification
compared to the other HA-fillers. It seems like the NASHA™ process is unique in preserving
entanglements when compared to the other processes used for manufacturing the HA-fillers examined
in this study.
A similar result can be seen in Figure 3 using another measure of the gel strength, G*. The Restylane
products have a higher gel strength and a lower degree of modification compared to the other HA-fillers
in this study.
Degree of Modification (MoD)
The degree of modification (MoD) is defined as the ratio of moles (number of molecules) of linked
crosslinker to moles of HA-disaccharides. To preserve biocompatibility of the HA molecules the modification
should preferably be kept at a minimum.
Crosslinker
HA-disaccharide
Gel strength
The strength of a gel is dependent on the total number of crosslinks (both chemical crosslinks and natural
entanglements). The stronger the gel the better is the lifting capacity.
Figure 2. The modification efficiency (MoE) for different HA-fillers.
In this study we have used two measures of gel strength, Cmin and G*:
Cmin is the concentration of HA in the fully swollen gel in an excess of solvent. Stronger gels swell less and
have a higher value for Cmin.
15
14
G* is the complex modulus measured by rheology. It is a measure of the total resistance to deformation.
Stronger gels have a higher resistance to deformation and a higher value for G*.
13
Modification efficiency
2
5
The modification efficiency (MoE) is a measure of the gel strength achieved for each introduced
crosslinker molecule. MoE can be calculated from the gel strength, Cmin (in mg/ml) and the degree of
modification, MoD (in %).
MoE=
Cmin
MoD
9
4
The strength of
the gel
Degree of
modification
Materials
12
3
1
8
10
11
Figure 3. The gel strength measured as the total resistance to deformation, G* plotted against the degree
of modification (MoD). Sample numbering as in Table 1.
Methods
Table 1. Investigated HA-filler products
#
Produkt
Lot No
Manufacturer
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Teosyal™ Deep Lines
Teosyal™ Ultra Deep
Esthelis BasicTM
Fortelis ExtraTM
Stylage MTM
Stylage LTM
Stylage XLTM
Juvéderm® Ultra 2
Juvéderm® Ultra 3
Juvéderm® Ultra 4
Princess Filler
Renofill Perfectly Volume
Restylane Touch™
Restylane®
Restylane PerlaneTM
TS27-083502B
TSU-082903B
311103/3
510063/2
EMB090061
ELB083301
EXC083231
X24L506559
X30L509376, X30L504649
S30L482804
903015/2, 903006/2, 903008/1
410065/2
10254
10368
10312
Teoxane, Switzerland
Teoxane, Switzerland
Anteis, Switzerland
Anteis, Switzerland
Laboratories Vivacy, France
Laboratories Vivacy, France
Laboratories Vivacy, France
Allergan, France
Allergan, France
Allergan, France
Croma Pharma GmbH, Austria
Laboratories Renophase, France
Q-Med AB, Sweden
Q-Med AB, Sweden
Q-Med AB, Sweden
Determination of MoD
Gel filler samples were washed by 0.9% NaCl and filtered on a 0.22 micrometer filter. The washed gel samples were
completely enzyme digested with Chondroitinase AC in deuterated water and 1H NMR spectra were recorded at 400
MHz from the resulting digests. The degree of modification was determined by integrating the signal from the N-acetyl
group in HA and one signal from the BDDE crosslinker. The ratio between the integrals for these two signals (crosslinker/
HA NAc) gives the degree of modification after correction for the number of protons responsible for each signal.
Determination of Cmin
Cmin is calculated as the concentration of HA in the product multiplied by the gel content and divided by the swelling
factor. The gel content is the fraction of HA that cannot pass through a 0.22 micrometer filter when filtering a diluted
suspension of the product. The swelling factor is calculated as V/Vo where Vo is the initial volume of the gel and V is the
volume of the fully swollen gel in 0.9% NaCl.
Determination of G*
The viscoelastic properties were measured on an Anton Paar MCR 301 rheometer using the parallel plate measuring
system using a gap of 1mm. G* was measured in a frequency sweep within the linear viscoelastic range determined by a
strain sweep and evaluated at 0.1Hz.
8th Anti-aging Medicine World Congress (AMWC) Monte-Carlo, Monaco - April 8-9-10, 2010