Vol.2, No.7, 764-768 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.27096
Pyrogenicity of hyaluronic acid hydrogel cross-linked
by divinyl sulfone for soft tissue augmentation
Jin-Tae Kim1, Jae-Ha Choi1, Deuk-Yong Lee2*
1Department of Advanced Materials Engineering, Chungbuk National University, Cheongju, Korea
2Department of Materials Engineering, Daelim University College, Anyang, Korea; *Corresponding Author:
Received 3 March 2010; revised 23 April 2010; accepted 29 April 2010.
ABSTRACT
Hyaluronic acid hydrogels (HAHs) were synthe-
sized by immersing the micro-beads in phos-
phate buffered saline solution to assess short-
term biocompatibility of the gels by means of
the rabbit pyrogen test and the bacterial en-
dotoxin test. The rise in body temperature of 3
male New Zealand white rabbits weighing about
2~3 kg (12~16 weeks old) following intravenous
injection of the test article (10 mL/kg) was mo-
nitored at 30 min intervals in 3 h to examine the
pyrogenicity. No rabbits showed an individual
rise in temperature of 0.5oC or more above its
respective control temperature. The tempera-
ture rises of the rabbits after injection were
0.12oC, 0.13oC, and 0.18oC, respectively, sugge-
sting that HAH meets the requirements for the
absence of pyrogens. The bacterial endotoxin
test revealed that the concentration of endoto-
xins required to cause the lysate to clot under
standard conditions was < 0.125 EU/mL. Com-
paring the HAHs that was synthesized in this
experiment to the ones approved by FDA, the
amount of < 0.125 EU/mL endotoxins is rela-
tively safe and effective. The test solution did
not contain any interfering factors under the ex-
perimental conditions used. It is conceivable
that the HAHs are likely to be suitable injectable
dermal filler for facial soft tissue augmentation
due to the absence of pyrogens.
Keywords: Hyaluronic Acid Hydrogel; Injectable
Dermal Filler; Pryogenicity; Endotoxin; Interfering
Factor; Soft Tissue Augmentation
1. INTRODUCTION
Hydrogels as injection augmentation of facial soft tissue
have attracted immediate attention due to their regenera-
tion properties of various tissues, mechanical properties,
softness, oxygen permeability, similarities the body’s
own highly hydrated composition and excellent bio-
compatibilities [1-8]. They demonstrated their efficacy
in correcting aesthetic defects such as congenital or hy-
povolumetries, nasolabial furrows, forehead, glabella
wrinkles, cheekbone, chin hypovolumetry and lip aug-
mentation. They are known to be highly swollen and
insoluble networks that can be used to entrap cells. It is
noted that higher equilibrium swelling promotes nutrient
diffusion into the gel and cellular waste removal out of
the gel, while the insolubility provides the structural
integrity necessary for tissue growth [1,2].
Among natural polymers, such as collagen, gelatin,
fibrin, alginic acid, chitosan and hyaluronic acid (HA),
crosslink-stabilized HA is highly acknowledged as a
naturally derived injectable filler due to its longevity of
correction, a reduced risk of immunogenicity and hy-
persensitivity, and its controllable mechanical and deg-
radation properties [1,7]. HA is a linear polysaccharide
formed from disaccharide units containing N-acetyl-D-
glucosamine and glucuronic acid [2,8]. HA molecule is
stabilized to produce cross-linked gel suitable for soft-
tissue implantation, resulting in improving its resistance
to enzymatic degradation within the dermis without
compromising its biocompatibility.
HA hydrogels cross-linked by divinyl sulfone (HAHs)
were prepared by immersing the micro-beads in phos-
phate buffered saline solution (NaH2PO4) [9]. HAHs for
soft tissue augmentation are reported to be biologically
inert and non-allergic and do not require allergy testing
before implantation. Prior to the evaluation of long-term
toxicity (genotoxicity) and carcinogenicity after the in-
jection because they have been used within the dermis
for several months, it is necessary to examine adverse
and allergic reactions of the HAH, such as short-term
pyrogenicity [5]. Although the rabbit pyrogen test has
played a key role to control pyrogenicity of the drugs for
long time, the test has limitations due to insufficient ac-
curacy. The bacterial endotoxin test, which is based on

J.-T. Kim et al. / Natural Science 2 (2010) 764-768
765
765
highly sensitive clotting of Limulus Amoebocyte Lysate
(LAL) by endotoxin, has been applied in place of the
pyrogen test. However, the LAL test has also limitations
in detection of such in vivo synergistic effect of endo-
toxin and the drugs [10]. The pyrogenicity of the HAH is
investigated to assess the short-term biocompatibility by
means of the rabbit pyrogen test and the LAL test
(gel-clot method) [10,11].
2. METHODS
2.1. Materials
HA solutions of 4.0 wt% concentration were prepared by
dissolving a 3.2 g of sodium hyaluronate (Mw = 1 ⅹ 106
Da, Shiseido Co., Japan) in 8 mL of 0.05 mol/L NaOH at
room temperature. A pH in the range of 12 to 14 was
achieved by adding 0.4 mL of 10 mol/L NaOH to the HA
solution. Then, the HA solution was placed in a solution
hopper attached to the Masterflex L/S tubing pump
(Cole Parmer, USA) and fed into a syringe equipped
with a 14-gage metal needle at a flow rate of 0.2 mL/min.
Micro-beads with diameters of 0.2 to 0.3 mm were fab-
ricated by supplying compressed air with a pressure of
34.475 Pa along the HA solution nozzle. The nozzle was
enclosed by a delivery tube with a diameter of 6 mm [9].
Micro-beads were collected into a solution mixture of
0.4 mL of divinyl sulfone (≥ 98%, Sigma and Aldrich,
Germany) and 40 mL of 2-methyl-1-propanol (99%,
Aldrich), followed by a stirring process (140~160 rpm)
for 24 h at room temperature. Then, the cross-linked
micro-beads were immersed in ethanol for 0.5 h to clean
the beads by removing impurities such as divinyl sulfone
and 2-methyl-1-propanol. After 3-time cleaning in etha-
nol, micro-beads were dried at 60oC in vacuum (20 torr).
The as-dried micro-beads were immersed in 80 mL of
phosphate buffered saline solution (NaH2PO4) for 2 h to
obtain HAHs, as shown in Figure 1. The HAHs were
then provided in a 1 mL sterilized syringe with luer-lok
type capped.
2.2. Rabbit Pyrogen Test
Male New Zealand white rabbits weighing about 2~3 kg
(12~16 weeks old) were employed for pyrogenicity. The
material extract was prepared in the static conditions in
sterile physiological saline solution (1 g in 50 mL) for 72
h at 37oC, followed by centrifuging for 10 min at 3000
rpm and filtering (Whatman, Grade 4, England). Not
more than 30 min prior to the injection of the test dose,
the control temperature of each rabbit was determined.
The control temperature is the base for the determination
of any temperature increase resulting from the injection
of a test solution. Rabbits, whose control temperatures
did vary by more than 1oC from each other, were ex-
Figure 1. Photographs of (a) the micro-beads immersed in
phosphate buffered saline solution and (b) hyaluronic acid
hydrogels.
cluded. In addition, any rabbits having a temperature
exceeding 39.8oC were also excluded. The test involved
measuring the rise in body temperature of rabbits fol-
lowing intravenous injection of the test article and was
designed for products that can be tolerated by the test
rabbit in a dose not exceed 10 mL/kg injected intrave-
nously, within a period of not more than 10 min. Injec-
tion was performed after warming the test solution to a
temperature of 37 ± 2oC.
After the injection, the body temperature is monitored
at 30 min intervals in 3 h. If no rabbit shows an individ-
ual rise in temperature of 0.5oC or more above its re-
spective control temperature, the product meets the re-
quirements for the absence of pyrogens. If any rabbit
shows an individual temperature rise of 0.5oC or more,
continue the test using 5 other rabbits. If not more than 3
of 8 rabbits show individual rises in temperature of
0.5oC or more and if the sum of 8 individual maximum

J.-T. Kim et al. / Natural Science 2 (2010) 764-768
766
temperature rises does not exceed 3.3oC, the material
under examination meets the requirements for the ab-
sence of pyrogens.
2.3. In Vitro Endotoxin Test
The pyrogens that almost invariably contaminate par-
enteral pharmaceuticals are bacterial endotoxins (lipo-
polysaccharides, LPS)-cell wall material from gram ne-
gative bacteria [10]. The LAL test detects only LPS,
which caused extracellular coagulation of the blood of
the horseshoe crab, Limulus polyphemus. The bacterial
endotoxin test, which is based on highly sensitive clot-
ting of LAL by endotoxin, has been applied in place of
the pyrogen test for testing parenteral human drugs [10,
11]. The gel-clot testing was performed to detect bacte-
rial endotoxin. The gel-clot method is based upon the
reaction between bacterial endotoxin and a single test
LAL (CAMBREX, Lot No. GL1403).
All glassware was depyrogenated for 4 h at 180oC and
the test was carried out in clean-bench to avoid the en-
dotoxin contamination. The material extract was pre-
pared in the static conditions in LAL reagent water (BIO
Whittaker, 1 g in 40 mL) for 1 h at 37oC. The concentra-
tion of endotoxins required to cause the lysate to clot
under standard conditions is the labeled lysate sensitivity
(), expressed IU/mL. Endotoxin is expressed in Inter-
national Unit (IU). One IU of endotoxin is equal to one
Endotoxin Unit (EU). Standard solutions of at least four
concentrations equivalent to 2, 1, 0.5, and 0.25
were prepared by diluting the standard endotoxin stock
solution with LAL reagent water. A volume of the lysate
solution was mixed with an equal volume of one of the
standard solutions. The mixture was incubated for 60 ± 2
min at 37 ± 1oC. Following 1 h of incubation at 37°C,
the test tube was examined by 180o inversion for the
presence of a stable solid clot. A clotted incubation mix-
ture is considered to be a positive result. A result is
negative if an intact gel is not formed. The test is not
valid unless the lowest concentration of the standard
solutions shows a negative result in all replicate tests.
The end-point is the last positive result in the series of
decreasing concentrations of endotoxin. The mean value
of the logarithms of the end-point concentrations and
then the antilogarithm of the mean value is calculated.
The geometric mean end-point concentration is the
measured sensitivity of the lysate solution. If this is not
less than 0.5 and not more than 2, the labeled sensitivity
is confirmed and is used in the tests performed with this
lysate. Solutions, as shown in Table 1 (A, B, C, and D),
are prepared to confirm the labeled lysate sensitivity.
The test is not valid unless both replicates of two posi-
tive control solutions B and C are positive and those of
the negative control solution D are negative.
3. RESULTS AND DISCUSSION
The rise in body temperature of 3 Male New Zealand
white rabbits following intravenous injection of the test
article (10 mL/kg) was monitored at 30 min intervals in
3 h to examine the pyrogenicity. It is found that the in-
crease in body weight was normal, as summarized in
Table 2. No mortality and abnormal clinical signs were
detected. No rabbits showed an individual rise in tem-
perature of 0.5oC or more above its respective control
temperature, as shown in Figure 2. The temperature
rises of the rabbits after injection were 0.12oC, 0.13oC,
and 0.18oC, respectively, suggesting that the HAH meets
the requirements for the absence of pyrogens.
The gel-clot technique allows detection or quantifica-
tion of endotoxins and is based on clotting of the lysate
in the presence of endotoxins [11]. The concentration of
endotoxins required to cause the lysate to clot under
standard conditions is the labeled lysate sensitivity. To
ensure both the precision and validity of the test, the
Table 1. Solutions for the gel-clot test.
solution endotoxin concentration/solution to
which endotoxin is added replicates
A None/sample solution 2
B 2/sample solution 2
C 2/LAL reagent water 2
D none/ LAL reagent water 2
Table 2. Body weight changes.
Rabbits Receipt day (g) Injection day (g)
1
2
3
2268.6
2161.6
1957.4
2377.8
2276.0
2091.1
Mean
SD
2129.2
158.1
2248.3
145.3
0.0 0.5 1.0 1.5 2.0 2.5 3.0
38.0
38.5
39.0
39.5
40.0
rabbit 1
rabbit 2
rabbit 3
Body temperature (oC)
Time (h)
Figure 2. The variation in body temperature of 3 Male
New Zealand white rabbits after intravenous injection
of the test article. Note that the body temperature is
monitored at 30 min intervals in 3 h.

J.-T. Kim et al. / Natural Science 2 (2010) 764-768
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767
labeled lysate sensitivity and the test for interfering fac-
tors were performed by using solutions for the gel-clot
test as summarized in Table 1. The end-point is the last
positive results in the series of decreasing concentrations
of endotoxin. The geometric mean end-point concentra-
tion is the measured sensitivity of the lysate solution.
The mean value of the logarithms of the end-point con-
centrations is equivalent to the antilogarithm of the mean
value, (Antilog Mean = 0.12 EU/mL). The antilogarithm
of the mean value is calculated to be −0.921. If the geo-
metric mean end-point concentration of 0.12 EU/mL is
not less than 0.5(0.06) and not more than 2,
the labeled sensitivity is confirmed and is used in the
tests performed with this lysate, as shown in Table 3.
For the lysate to clot under standard conditions, < 0.125
EU/mL was found to be the required concentration of
endotoxins. The material extract was prepared in the
static conditions in LAL reagent water for 1 h at 37oC
[11]. After considering the dilution factor, the final con-
centration of endotoxin was determined to be < 0.005
EU/mg. According to FDA’s “Summary of Safety and
Effectiveness Data,” endotoxin concentrations are 0.5
EU/mL, 20 EU/syringe, 0.08 EU/mL for the three ap-
proved injectable dermal HAH fillers in market, respec-
tively [12-14]. Comparing the HAHs that was synthe-
sized in this experiment to the ones approved by FDA,
the amount of < 0.125 EU/mL endotoxins is relatively
safe and effective [12-14].
The test for interfering factors is repeated when any
changes are made to the experimental conditions that are
likely to influence the result of the test. The test is not
valid unless all replicates of solutions A and D show no
reaction and the result of solution C confirms the labeled
lysate sensitivity. If the sensitivity of the lysate deter-
mined with solution B is not less than 0.5 and not greater
than 2, the test solution does not contain interfering fac-
tors under the experimental conditions used. Otherwise,
the solution interferes with the test. Test results, as listed
in Table 4, implied free of interfering factors because
solutions A and D showed reaction and the lysate sensi-
tivity with solution B was within the experimental range
(0.5~2) [11].
Table 3. Test for confirmation of the labeled lysate sensitivity.
Endotoxin dilution (EU/mL)
No.
of
time 0.24 0.12 0.06 0.03 LAL RW*
(2) (1) (0.5) (0.25)
End-
point
(EU
/mL)
1
2
3
4
+ + - - -
+ + - - -
+ + - - -
+ + - - -
0.12
0.12
0.12
0.12
+: positive, -: negative, RW: reagent water.
Table 4. Test for interfering factors.
Endotoxin dilution
(EU/mL)
Solution
No.
of
time 0.24 0.12 0.06 0.03
(2) (1) (0.5) (0.25)
End-point
(EU/mL)
B
1
2
3
4
+ + - -
+ + - -
+ + - -
+ + - -
0.12
0.12
0.12
0.12
C 1
2
+ + - -
+ + - -
0.12
0.12
*solution A: all positive, solution D: all negative.
4. CONCLUSIONS
Short-term biocompatibility of the HAHs prepared by
immersing the micro-beads in phosphate buffered saline
solution was evaluated by examining the rabbit pyrogen
test and the bacterial endotoxin test. No rabbits showed
an individual rise in temperature of 0.5oC or more above
its respective control temperature after intravenous in-
jection of the test article (10 mL/kg) to 3 male New Zea-
land white rabbits. The temperature rises of the rabbits
after injection were 0.12oC, 0.13oC, and 0.18oC, respec-
tively, suggesting that the HAH meets the requirements
for the absence of pyrogens. The concentration of en-
dotoxins required to cause the lysate to clot under stan-
dard conditions was determined to be < 0.125 EU/mL.
Comparing the HAHs that was synthesized in this study
to the injectable dermal fillers approved by FDA (0.5
EU/mL), the amount of < 0.125 EU/mL endotoxins is
relatively safe and effective. The test solution was free
of interfering factors under the experimental conditions
used. It is suggested that the HAHs are likely to be suit-
able filler for facial soft tissue augmentation due to the
absence of pyrogens.
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