Bacteriostatic Effects of Hyaluronan-Based Bioresorbable Membrane

doi:10.4236/ss.2011.29094

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Surgical Science, 2011, 2, 431-436

doi:10.4236/ss.2011.29094 Published Online November 2011 (http://www.SciRP.org/journal/ss)

Bacteriostatic Effects of Hyalurona n - Bas ed

Bioresorbable Membrane

Keiichi Uchida, Kohei Otake, Mikihiro Inoue, Yuhki Koike, Kohei Matsushita,

Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Masato Kusunoki

Department of Gastrointestinal and Pediatric Surgery, Graduate School of Medicine,

Mie University, Tsu, Mie, Japan

E-mail: [email protected]

Received June 14, 2011; revised September 16, 2011; accepted October 31, 2011

Abstract

Purpose: The purpose of this study is to determine the presence of bacteriostatic effects of hyaluronan-based

bioresorbable membrane (HA/CMC) on selected major bacterial strains in digestive organs. Methods: We

firstly evaluated the growth inhibition effect of HA/CMC for E. coli and S. aureus by determining the optical

density (OD)650 in the incubation medium. At second, to determine the viable counts of bacteria, total adeno-

sine triphosphate (ATP) were measured with five groups; several concentrations of HA/CMC and control.

Results: OD curve gradually elevated and reached to plateau at 4 hours in E. coli. and 6 hours in S. aureus.

After reaching plateau, the growth inhibition of both strains was statistically significantly correlated to the

concentrations of HA/CMC. The ATP productions had statistically significant differences at 6 hours after

incubation and inhibited in dose-dependence of a well-dissolved HA/CMC. Conclusion: HA/CMC may

have dose-dependently bacteriostatic effects on S. aureus and E. coli.

Keywords: HA/CMC, Bacteriostatic, Hyaluronan, Adhesion, Surgery

1. Introduction

Clinical effectiveness and safety of hyaluronan-based

bioresorbable membrane (HA/CMC) on preventing in-

traperitoneal adhesions has been widely accepted in ab-

dominal and pelvic surgical use [1-4]. Recently, a few

cases with postoperative intraperitoneal infection and

inflammation after HA/CMC placement have been re-

ported [5,6]. Though the relationship between HA/CMC

and such morbidities was not clear, several clinical and

animal studies demonstrated that HA/CMC neither re-

sulted in the propagation of infection, nor affected ab-

scess formation, intraperitoneal septic condition and sys-

temic inflammatory response adversely [7-10].

A previous study at our institute [11] demonstrated

that HA/CMC did not aggravate an inflammatory cyto-

kine response or produce extensive intra-abdominal ad-

hesions, even in patients with intraperitoneal septic com-

plications and rat models with bacterial peritonitis in-

duced by cecal ligation and puncture. In experimental

basic study, K. Otake et al. [12] revealed HA/CMC itself

does not affect the functions of isolated human Poly-

morphonuclear neutrophils in in vitro experimental mo-

dels of inflammation and endotoxin-stimulation. Al-

though our results showed that HA/CMC can be safely

used even in contaminated conditions, further basal ex-

periments about HA/CMC against infectious pathogens

are needed.

HA/CMC is a bioresorbable membrane made of sodium

hyaluronate (Hyaruronic acid, HA) and another polyan-

ionic poly saccharide, carboxymethylcellulose (CMC).

These components are chemically modified to reduce

water solubility and provide longer in vivo residence

time than the native polymers. Preclinical evaluation

demonstrated that HA/CMC is nontoxic, nonmutagenic,

nonimmunogenic, nonirritating and nonpyrogenic [13].

HA is a connective tissue glycosaminoglycan that has a

number of embryonic and wound healing properties,

including the facilitation of cell migration and differen-

tiation during tissue formation and repair [14,15]. Previ-

ous studies suggested that HA has great bacteriostatic

effects on the common orthopedic and periodontal bacte-

rial pathogens [16,17]. The purpose of this study is to

determine the presence of any bacteriostatic or bacteri-

cidal effects of HA/CMC on selected microorganisms

such as Staphylococcus aureus (S. aureus) and Escherichia

432 K. UCHIDA ET AL.

coli (E. coli).

2. Materials and Methods

2.1. Bacterial Preparations

The two bacterial strains, S. aureus (ATCC27217) and E.

coli (ATCC25922) were purchased from the ATCC

(American Type Culture Collection). S. aureus and E.

coli were cultured in Dulbecco’s modified Eagle’s media

(D-MEM) (Invitrogen Co., Tokyo, Japan) for 10 h at

37˚C. The culture medium was centrifuged at 1700 × g

for 10 min at room temperature to pellet the S. aureus or

E. coli, and resuspended in sterile normal saline. A 100

μL quantity of the suspension was serially diluted with

sterile saline, applied on 1.5% Agar-B (Qbiogene, Inc.)

and 0.8% Difco Nutrient Broth (Difco Laboratories,

USA.) plate, and incubated for 18 h to determine the

bacterial concentration. The remainder was stored at

–80˚C until use. Just before use, the bacterial suspension

was diluted in medium to achieve a final concentration of

5.0 × 106 colony-forming units (cfu)/mL. The study was

approved by the local ethics committee.

2.2. Bacterial Growth Inhibition

Seprafilm® (HA/CMC, Genzyme Corporation, Japan) is

a synthetic product of HA and CMC in the ratio of two to

one. A sheet of Seprafilm® (12.7 cm × 14.7 cm, 186.69

cm2) weighs about 1 g including HA 0.7 g and CMC 0.3

g. A quarter sheet of HA/CMC include approximately

175 mg HA [18]. The incubation medium of Group A

was 10 ml D-MEM with a well-dissolved quarter sheet

of HA/CMC (approximately 175 mg HA). The incuba-

tion medium of Group B was 10 ml D-MEM with a well-

dissolved one-eighth sheet of HA/CMC (approximately

87.5 mg). The incubation medium of Group C was only

D-MEM without HA/CMC. 100 μl bacterial suspension

(5.0 × 105 cells) was added to 100 μl each group medium

(total 200 μl). So, HA concentrations 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.

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.

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

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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