ncentrations of medium are ap-
proximately 8.25 mg/ml in Group A, 4.125 mg/ml in
Group B, and 0 mg/ml in Group C, respectively. Trials
were conducted for each bacterial strain. As control,
same sample size of 200 μl each medium without bacte-
ria was used in determining the optical density (OD650)
of medium. To assess the presence or absence of growth,
each sample and each control were incubated using 96-
well cell plates (Becton Dickinson Labware, NJ, USA) at
37˚C shaking incubater. At 0, 4, 8, and 12 hours after
incubation starts, the optical density (OD650) was deter-
mined. To validate the identification of the bacteria and
to rule out the possibility of contamination, 10 colonies
for each bacterial strain were randomly chosen and ana-
lyzed via Gram staining.
2.3. Cellular Function Investigation
To determine the viable count of bacteria, total adenosine
triphosphate (ATP) was measured in E. coli and S.
aureus after 6 hours culture. For the determination of
total ATP, the BacTiter-Glo system (Promega) was used.
The BacTiter-Glo buffer was mixed with the lyophilized
BacTiter-Glo substrate and equilibrated at room tem-
perature. As control, 100 μl bacterial suspension (5.0 ×
105 cells) was added to 100 μl medium (total 200 μl)
without HA/CMC that is similar to medium of Group C
in bacterial growth inhibition assay (Group I). We pre-
pared 100 μl medium with well-dissolved a quarter HA/
CMC (Group II, approximately 175 mg HA), a half of
HA/CMC (Group III, approximately 350 mg HA), one
HA/CMC (Group IV, approximately 750 mg HA), and
twice HA/CMC (Group V, approximately 1500 mg HA).
So, HA concentrations of medium are approximately 0
mg/ml in Group I, 8.25 mg/ml in Group II, 16.5 mg/ml in
Group III, 33 mg/ml in Group IV, and 66 mg/ml in
Group V, respectively. Sample sizes (5.0 × 105 cells) of
each bacterial suspension were used in determining the
initial luminescence. Each sample was incubated using
96-well cell plates (Becton Dickinson Labware, NJ, USA)
for 6 hours at 37˚C shaking incubater. Trials were con-
ducted for each bacterial strain. At 0, 2, 4, and 6 hours
after incubation starts, each sample was mixed well and
100 μl bacterial suspension of each well was transferred
to white 96-well cell plates (Thermo Electron Corpora-
tion). An equal volume of the previously prepared Bac-
Titer-Glo reagent was added in each well. The lumines-
cence of the sample was measured with a multi-detection
microplate reader (Powerscan HT, Dainippon Pharma-
ceutical, Osaka, Japan) immediately after five minutes
incubation. A calibration curve with dilutions of pure
rATP (Promega, P1132) was measured before each ex-
periment. ATP value was evaluated in triplicate and the
average value was computed.
2.4. Statistics
Statistical analyses were performed using Statview 4.5
(Abacus Concepts, Berkeley, California). Results are
presented as the means ± SD. In bacterial growth inhibi-
tion assay, two-way repeated measures ANOVA was
used to compare the each two groups with respect to
bacterial growth over time. In the study of cellular func-
tion investigation assay, Spearman’s rank correlation test
was used to test the correlation between bacterial growth
and the concentration of HA/CMC for 0, 2, 4 and 6 hours.
A p value of <0.05 was considered significant.
Copyright © 2011 SciRes. SS
K. UCHIDA ET AL.
433
3. Results
3.1. Bacterial Growth Inhibition
Figure 1 shows the optical density (OD) of broth solu-
tions with S. aureus during 12 hours incubation among
three groups. OD curve gradually elevated and reached
to plateau at 6 hours. There was a significant difference
in OD between Group A and B (p < 0.0001 for HA/CMC
concentrations and p < 0.0001 for time × HA/CMC con-
centrations). Moreover, there was a significant difference
in OD between Group A and C (p = 0.0020 for HA/CMC
concentrations and p < 0.0001 for time × HA/CMC con-
centrations). However, there was no difference in OD
between Group B and C. (p = 0.0550 for HA/CMC con-
centrations and p = 0.5767 for time × HA/CMC concen-
trations). Figure 2 shows OD of broth solutions with E.
coli during 12 hours incubation among three groups. OD
curve gradually elevated and reached to plateau at 4
hours. There was a significant difference in OD between
Group A and B. (p < 0.0001 for HA/CMC concentrations
and p < 0.0001 for time × HA/CMC concentrations).
There was a significant difference in OD between Group
A and C (p = 0.0020 for HA/CMC concentrations and p
< 0.0001 for time × HA/CMC concentrations). Moreover,
there was a significant difference in OD between Group
B and C (p < 0.0001 for HA/CMC concentrations and p
< 0.0001 for time × HA/CMC concentrations).
3.2. Cellular Function Investigation
Figure 3 shows the serial change of mean ATP value of
S. aureus in all groups. The mean ATP production was
inhibited dose-dependently by HA/CMC and the differ-
ences in the value of ATP production are statistically
significant among 5 groups at 2, 4, and 6 hours after in-
cubation. Figure 4 shows the serial change of mean ATP
value of E. coli in all groups. At 6 hours after incubation,
the mean ATP production was inhibited dose-depen-
dently by HA/CMC and the differences in the value of
ATP production are statistically significant among 5
groups at 6 hours after incubation.
4. Discussion
Recently, as the usage of HA/CMC increases in ab-
dominal surgical scene, the relationship between HA/
CMC and postoperative infectious complications has
become the center of attention [9,11]. In the presence of
infection, fibrinolysis in the abdominal cavity is even
more depressed and intraabdominal adhesion becomes
more severe. After contaminated or dirty operations, ab-
scesses arising from contaminated fibrin are frequent
Figure 1. The optical density (OD) of broth solutions with S.
aureus during 12 hours incubation among three groups. HA
concentrations of medium are approximately 8.25 mg/ml in
Group A, 4.125 mg/ml in Group B, and 0 mg/ml in Group C,
respectively. There was a significant difference in OD be-
tween Group A and B (*p < 0.0001 for HA/CMC concen-
trations and p < 0.0001 for time × HA/CMC concentrations).
Moreover, there was a significant difference in OD between
Group A and C (†p = 0.0020 for HA/CMC concentrations
and p < 0.0001 for time × HA/CMC concentrations). How-
ever, there was no difference in OD between Group B and
C (‡p = 0.0550 for HA/CMC concentrations and p = 0.5767
for time × HA/CMC concentrations). Statistical analysis
was performed by Two-Way Repeated-Measures ANOVA.
Figure 2. The optical density (OD) of broth solutions with E.
coli during 12 hours incubation among three groupos. HA
concentrations of medium are approximately 8.25 mg/ml in
Group A, 4.125 mg/ml in Group B, and 0 mg/ml in Group C,
respectively. OD curve gradually elevated and reached to
plateau at 4 hours. There was a significant difference in OD
between Gr oup A and B (*p < 0.0001 for HA/CMC concen-
trations and p < 0.0001 for time × HA/CMC concentrations).
There was a significant difference in OD between Group A
and C (†p = 0.0020 for HA/CMC concentrations and p <
0.0001 for time × HA/CMC concentrations). Moreover,
there was a significant difference in OD between Group B
and C (‡p < 0.0001 for HA/CMC concentrations and p <
0.0001 for time × HA/CMC concentrations). Statistical
analysis was performed by Two-Way Repeated-Measures
ANOVA.
complications. In the treatment of infection development
in the abdominal cavity, elimination of the source of
contamination is the cornerstone of abdominal surgery.
Copyright © 2011 SciRes. SS
434 K. UCHIDA ET AL.
Figure 3. The serial change of mean ATP value of S. aureus
in all groups. HA concentrations of medium are approxi-
mately 0 mg/ml in Group I, 8.25 mg/ml in Group II, 16.5
mg/ml in Group III, 33 mg/ml in Group IV, and 66 mg/ml
in Group V, respectively. The mean ATP production was
inhibited dose-dependently by HA/CMC and the differ-
ences in the value of ATP production are statistically sig-
nificant among 5 groups at 2, 4, and 6 hours after incuba-
tion. *p = 0.0006, †p = 0.0001, ‡p = 0.0004 (Spearman’s
rank correlation test).
Figure 4. The serial change of mean ATP value of E. coli in
all groups. HA concentrations of medium are approxi-
mately 0 mg/ml in Group I, 8.25 mg/ml in Group II, 16.5
mg/ml in Group III, 33 mg/ml in Group IV, and 66 mg/ml
in Group V, respectively. At 6 hours after incubation, the
mean ATP production was inhibited dose-dependently by
HA/CMC and the differences in the value of ATP produc-
tion are statistically significant among 5 groups at 6 hours
after incubation. *p = 0.0002 (Spearman’s rank correlation
test).
Fibrin forms deposits that are a matrix for ingrowth of
fibrocollagenous tissue. Moreover, fibrin clots protect
bacteria against the immunological defences of the ab-
dominal cavity. Following this pathway, intraabdominal
fibrin may eventually lead to adhesion and/or abscess
formation. Persistence and reinforcement of fibrinolysis
is needed after surgery new fibrin deposits will occur.
Animal experiments have demonstrated that enhance-
ment of fibrinolysis reduces intraabdominal abscess for-
mation [19,20]. Several mechanisms may be involved in
the reduction of adhesions by HA/CMC, including me-
chanical separation of wound surfaces, improvement of
peritoneal healing and enhanced fibrinolysis. Reijnen et
al. [9] reviewed investigations into the pathophysiology
of intra-abdominal adhesions and abscess formation, and
showed that the potential of hyaluronan-based agents to
reduce intra-abdominal adhesions and abscess on ab-
dominal surgery and sepsis is a promising new concept.
Previous study at our institute demonstrated that HA/
CMC itself does not affect the functions of isolated hu-
man Polymorphonuclear neutrophils in in vitro experi-
mental models of inflammation and endotoxin-stimula-
tion [12]. In this study, we tried to determine whether
hyaluronan-based bioresorbable membrane HA/CMC
reduces the bacterial growth. In growth inhibition assay,
we investigated the effect of HA/CMC on both the loga-
rithmic growth phase and stationary phase. And we put
emphasis on the effect of HA/CMC itself on the total
number of bacteria. In this assay, the logarithmic growth
phase of S. aureus and E. coli continued for 6 hours at
least. In cellular function investigation, we put emphasis
on the association between the viablility of bacteria and
the concentration of HA/CMC. Therefore, we investi-
gated the effect of HA/CMC to the logarithmic growth
phase for 6 hours by measuring total ATP.
HA modulates the inflammatory response; it has free
radical scavenging and antioxidant properties [21], and
increases interferon γ levels during peritonitis, inhibits
inflammatory proteinases [22], and probably induces the
production of proinflammatory mediators such as inter-
leukin 1, interleukin 8 and TNF-α [23]. The role of HA
in the resistance of bacteria to opsonization and phgo-
cytic killing is not completely understood [24]. Carlson
et al. [5] suggested that the bacteriostatic effect of HA
may be due to the saturation of the bacterial hyaluronate
lyase by the excess HA, which prevents the bacteria from
maintaining elevated levels of tissue permeability and
penetrating the physical defenses of the host. This would
enhance the ability of the host’s immune system to
eradicate pathogens. HA molecules also form a random
network of chains that may act as a sieve preventing the
spread of the bacteria.
Pirnazar et al. [17] demonstrated the bacteriostatic ef-
fects of HA on selected oral and non-oral microorgan-
isms. They suggested that HA has no bactericidal effect,
however, the high concentrations (2.0 mg/ml) of the me-
dium (757 kD) molecular weight (MW) HA had the
greatest bacteriostatic effect on several bacterial strains
compared to the combinations with other concentrations
(0.5 or 1.0 mg/ml) and MW (141 kD or 1300 kD). HA in
HA/CMC and has been esterified with glucuronic acid
residues and is different from native HA in the human
body. Based on light-scattering measurements, the modi-
fied HA in comprises molecules of approximately 100
kD in MW [18]. HA/CMC is a synthetic product of HA
Copyright © 2011 SciRes. SS
K. UCHIDA ET AL.
435
and CMC in the ratio of two to one. A sheet of HA/CMC
(12.7 cm × 14.7 cm, 186.69 cm2) weighs about 1 g in-
cluding HA 0.7 g and CMC 0.3 g. A quarter sheet of HA/
CMC include approximately 175 mg HA. The concentra-
tion of HA formulation used in Group A was 3.5 mg/ml
and higher compared to Pirnazar’s study. This study
clarified the higher concentrations of HA/CMC intro-
duced, the greater bacteriostatic effects on S. aureus and
E. coli using bacterial growth inhibition and cellular func-
tion investigation. In conclusion, HA/CMC may have
dose-dependently bacteriostatic effects on S. aureus and
E. coli. Further investigations are needed about various
bacterial strains which could be present in the abdominal
cavity.
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