# Adhesion of Respiratory-Infection-Associated Microorganisms on Degradable Thermoplastic Composites

Authors: Teemu Tirri; Eva Söderling; Minna Malin; Matti Peltola; Jukka V. Seppälä; Timo O. Närhi
Journal: International Journal of Biomaterials
 (2009)

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## Abstract

The purpose of this study was to evaluate bacterial adhesion and early colonization on a composite consisting of bioactive glass (BAG) particles and copolymer ofε-caprolactone/D,L-lactide. Materials were incubated with suspensions of both type strains and clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa for 30 minutes (adhesion) and 4 hours (colonization). Clear differences exist in the microorganisms' ability to adhere on the experimental materials. However, the presence of BAG particles does not inhibit bacterial adhesion, but early colonization of the materials with P. aeruginosa was inhibited by the addition of 90–315 μm BAG particles.


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## Body

## 1. Introduction

Tissue integrating biomaterials can
be utilized in various applications in head and neck and craniomaxillofacial surgeries such as the sinus augmentations and obliterations, repair of fractured orbital floor, and occluding nasal septum perforations. Bioactive glass (BAG) plates and granules are
currently available for clinical use for these applications [1–5]. Good
biocompatibility and osteopromotive properties of bioactive glasses are well
known, which make them interesting materials for reconstructive surgery.
However, glass itself is a brittle material and it cannot be easily shaped
during the surgery. Composites of BAG and biodegradable polymers may offer
better handling properties without losing glasses bioactive function [6, 7].A composite of copolymer of poly(ε-caprolactone and D,L-lactide) and BAG (S53P4) was recently
developed. The composite degrades via hydrolysis and it forms hydroxyapatite
layer on its surface in simulated body conditions [8, 9]. The composite is
thermoplastic and it can be shaped during the surgery. Materials’
osteoconductive capacity has been proven in experimental animal study [7].
Thus, the composite of poly(ε-caprolactone
and D,L-lactide) and
BAG is a potential new material for surgical corrections of tissue defects in
craniofacial area.Biofilm forms usually on implant
surfaces within few hours when they are exposed to an environment where they
are continuously bathed in bacteria-containing fluids. Bacterial colonization
can either be beneficial or destructive for the implanted device [10].
Materials that enhance biofilm formation may cause chronic infections in the
implantation site. No data is available about early colonization of polymer
composites containing BAG. In principle, in the upper respiratory tract area, there is a risk to
bacterial colonization of implants. The present study aimed to evaluate the
adhesion and colonization reflecting early biofilm formation of clinically important head and
neck area pathogens Streptococcus pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa [11, 12] on a composite of copolymer
of poly(ε-caprolactone
and D,L-lactide) and BAG.


## 2. Materials and Methods

### 2.1. Composite Substrates

Bioactive thermoplastic composites were made by
incorporating 60 wt% of bioactive glass granules (BAG) (S53P4, Vivoxid Ltd.,
Turku, Finland) into the copolymer of poly(ε-caprolactone and D,L-lactide) through blending
[8]. The composition of the BAG by wt% was SiO2 53%, Na2O
23%, CaO 20%, and P2O5 4%, while the particle size was
90–315 μm (C90) and <45 μm (C45).
Copolymer without BAG was used as a control (C). Round test specimens
(surface area 78.5 mm2, thickness 2 mm) for bacterial cultivation were
punched out of the cast composite plates. Specimens were packed and sterilized
with γ-radiation
(dose 25 Gy, Gammaster, Waageningen, The Netherlands). A representative sample
of the specimens was characterized prior to the microbial cultivation by
measuring their surface roughness and wetting properties. Wetting performance
was determined by contact angle measurement.


### 2.2. Microorganisms

The following microorganisms were
used in the experiments:S. pneumoniae ATCC 49619, S. pneumoniae clinical isolate 1186; H. influenzae ATCC 49247, H. influenzae clinical isolate 1258; P.
aeruginosa ATCC 27853, P. aeruginosa clinical isolate 1218. The
clinical isolates were kind gifts from Dr. Erkki Eerola, Department of
Microbiology, University of Turku,
Finland.


### 2.3. Cultivation of the Microorganisms

For the adhesion experiments the
microorganisms were cultured in Brain Heart Infusion medium (BHI; Difco
Laboratories, Mich, USA).H. influenzae and P. aeruginosa were grown overnight at 37°C, producing log-phase cells with the inoculum used. For H. influenzae the medium was supplemented with β-nicotinamide
adenine dinucleotide (10 mg/L) and hemin (20 mg/L) according to Kuo et al. [13]. The fast-growing S.
pneumoniae was grown in BHI supplemented with heat-inactivated fetal calf serum (10% v/v; Promocell, Heidelberg,
Germany). It
was first grown overnight whereafter an inoculum was transferred to fresh
medium in the morning and the cells were cultured for 3–4 hours resulting in
log-phase cells. The cells were harvested by centrifugation, washed once with
saline (0.9% NaCl w/v) and suspended in saline to an optical density of appr.
0.25 at the wavelength 660 nm. The S.
pneumoniae cell suspension was prepared as described before but the optical
density of the suspension was adjusted to appr. 0.5. The cell densities were
chosen based on preliminary adhesion experiments showing in scanning electron
microscopy (SEM) even distribution of the cells on the material surfaces with
abundant space for surface colonization.


### 2.4. Adhesion Tests

The cell suspensions were
prepared for the adhesion experiments as described above. The adhesion
experiments were essentially performed as described earlier [14]. Each tested
disc was incubated with 1.5 mL of the cell suspension at room temperature for
30 minutes using gentle rolling (The Coulter Mixer, Luton, UK) in a 14 mL capped plastic
test tube with an inner diameter of 16 mm (Falcon, BD Biosciences, Bedford, Mass,
USA). The mixer was tilted in a 15-degree angle to ensure that the materials
were covered by the cell suspension at all times. After the rolling the
material was rinsed gently 3 times in 50 mL saline. The cells adhering to the two
flat surfaces of each disc were carefully scraped from the surfaces in 900 μL
of transport medium (Tryptic Soy Broth, Difco Laboratories), each side of the
disc in a separate vial. Three applicators dipped into fresh transport medium
before the procedure (Quick-Stick, Dentsolv AB,
Sweden) were
used to scrape the cells from one side on the disc, the brush ends of the
applicators were cut into the transport media. In preliminary experiments
increasing the number of scrapings did not increase the cell yield. Vortexing
removed efficiently the cells from the ends of the applicators. The experiments
were performed with 3–4 replicates and repeated at least once.For the enumeration of cells on the disc
surfaces as colony-forming units (CFU), the microbe samples collected from the
surfaces were thoroughly Vortexed and grown on agar plates after serial
dilutions of the samples.H. influenzae was grown on chocolate agar, S. pneumoniae and P. aeruginosa on blood agar
overnight at 37°C in air supplemented with 7% CO2.In some experiments, the materials
were subjected to fixation followed by SEM as described in what follows.


### 2.5. Early Surface Colonization

For testing early surface
colonization the materials were, after the 30 minutes rolling in the bacterial
suspensions described earlier, dipped in 50 mL saline to remove cells not
attached to the surfaces. The materials were then transferred to fresh growth media
described in Section2.3. The culturing was
performed using rolling in 1.5 mL of growth medium in the tube used in the
adhesion experiments at 37°C for 4
hours. The numbers of cells on the material surfaces were determined as
described in Section 2.4. The pHs of the growth media were
determined at the end of the incubation to ensure that the pH of the medium was
not affected by the material, the cells growing on the material surface or shed
to the medium.The early colonization experiments
were performed with 3–4 replicates. The experiments were repeated at least
once.


### 2.6. SEM

For the SEM examinations the samples
were fixed for 5 minutes (2% glutaraldehyde and 2% formaldehyde in
phosphate-buffered saline, pH 7.4), rinsed once in distilled water and dried in an ascending ethanol series: 50%
EtOH for 5 minutes, 70% EtOH for 10 minutes, two times 96% EtOH for 10 minutes, and absolute
EtOH for 5 minutes. Finally the specimens were sputter coated with gold and
examined with SEM (Model JSM 5500, JEOL Ltd., Tokyo, Japan).


### 2.7. Statistical Analysis

Statistical
analysis was done with StatView and Graphics Program (SAS institute, Cary, NC,
USA).
Differences in the mean numbers of the microbes (CFUs) harvested form the
experimental materials were tested with an ANOVA after logarithmic
transformation. Post Hoc comparisons were made with Fisher’s PLSD test after
theF test for equal means was found to be significant at P<.05 level.


## 2.1. Composite Substrates

Bioactive thermoplastic composites were made by
incorporating 60 wt% of bioactive glass granules (BAG) (S53P4, Vivoxid Ltd.,
Turku, Finland) into the copolymer of poly(ε-caprolactone and D,L-lactide) through blending
[8]. The composition of the BAG by wt% was SiO2 53%, Na2O
23%, CaO 20%, and P2O5 4%, while the particle size was
90–315 μm (C90) and <45 μm (C45).
Copolymer without BAG was used as a control (C). Round test specimens
(surface area 78.5 mm2, thickness 2 mm) for bacterial cultivation were
punched out of the cast composite plates. Specimens were packed and sterilized
with γ-radiation
(dose 25 Gy, Gammaster, Waageningen, The Netherlands). A representative sample
of the specimens was characterized prior to the microbial cultivation by
measuring their surface roughness and wetting properties. Wetting performance
was determined by contact angle measurement.


## 2.2. Microorganisms

The following microorganisms were
used in the experiments:S. pneumoniae ATCC 49619, S. pneumoniae clinical isolate 1186; H. influenzae ATCC 49247, H. influenzae clinical isolate 1258; P.
aeruginosa ATCC 27853, P. aeruginosa clinical isolate 1218. The
clinical isolates were kind gifts from Dr. Erkki Eerola, Department of
Microbiology, University of Turku,
Finland.


## 2.3. Cultivation of the Microorganisms

For the adhesion experiments the
microorganisms were cultured in Brain Heart Infusion medium (BHI; Difco
Laboratories, Mich, USA).H. influenzae and P. aeruginosa were grown overnight at 37°C, producing log-phase cells with the inoculum used. For H. influenzae the medium was supplemented with β-nicotinamide
adenine dinucleotide (10 mg/L) and hemin (20 mg/L) according to Kuo et al. [13]. The fast-growing S.
pneumoniae was grown in BHI supplemented with heat-inactivated fetal calf serum (10% v/v; Promocell, Heidelberg,
Germany). It
was first grown overnight whereafter an inoculum was transferred to fresh
medium in the morning and the cells were cultured for 3–4 hours resulting in
log-phase cells. The cells were harvested by centrifugation, washed once with
saline (0.9% NaCl w/v) and suspended in saline to an optical density of appr.
0.25 at the wavelength 660 nm. The S.
pneumoniae cell suspension was prepared as described before but the optical
density of the suspension was adjusted to appr. 0.5. The cell densities were
chosen based on preliminary adhesion experiments showing in scanning electron
microscopy (SEM) even distribution of the cells on the material surfaces with
abundant space for surface colonization.


## 2.4. Adhesion Tests

The cell suspensions were
prepared for the adhesion experiments as described above. The adhesion
experiments were essentially performed as described earlier [14]. Each tested
disc was incubated with 1.5 mL of the cell suspension at room temperature for
30 minutes using gentle rolling (The Coulter Mixer, Luton, UK) in a 14 mL capped plastic
test tube with an inner diameter of 16 mm (Falcon, BD Biosciences, Bedford, Mass,
USA). The mixer was tilted in a 15-degree angle to ensure that the materials
were covered by the cell suspension at all times. After the rolling the
material was rinsed gently 3 times in 50 mL saline. The cells adhering to the two
flat surfaces of each disc were carefully scraped from the surfaces in 900 μL
of transport medium (Tryptic Soy Broth, Difco Laboratories), each side of the
disc in a separate vial. Three applicators dipped into fresh transport medium
before the procedure (Quick-Stick, Dentsolv AB,
Sweden) were
used to scrape the cells from one side on the disc, the brush ends of the
applicators were cut into the transport media. In preliminary experiments
increasing the number of scrapings did not increase the cell yield. Vortexing
removed efficiently the cells from the ends of the applicators. The experiments
were performed with 3–4 replicates and repeated at least once.For the enumeration of cells on the disc
surfaces as colony-forming units (CFU), the microbe samples collected from the
surfaces were thoroughly Vortexed and grown on agar plates after serial
dilutions of the samples.H. influenzae was grown on chocolate agar, S. pneumoniae and P. aeruginosa on blood agar
overnight at 37°C in air supplemented with 7% CO2.In some experiments, the materials
were subjected to fixation followed by SEM as described in what follows.


## 2.5. Early Surface Colonization

For testing early surface
colonization the materials were, after the 30 minutes rolling in the bacterial
suspensions described earlier, dipped in 50 mL saline to remove cells not
attached to the surfaces. The materials were then transferred to fresh growth media
described in Section2.3. The culturing was
performed using rolling in 1.5 mL of growth medium in the tube used in the
adhesion experiments at 37°C for 4
hours. The numbers of cells on the material surfaces were determined as
described in Section 2.4. The pHs of the growth media were
determined at the end of the incubation to ensure that the pH of the medium was
not affected by the material, the cells growing on the material surface or shed
to the medium.The early colonization experiments
were performed with 3–4 replicates. The experiments were repeated at least
once.


## 2.6. SEM

For the SEM examinations the samples
were fixed for 5 minutes (2% glutaraldehyde and 2% formaldehyde in
phosphate-buffered saline, pH 7.4), rinsed once in distilled water and dried in an ascending ethanol series: 50%
EtOH for 5 minutes, 70% EtOH for 10 minutes, two times 96% EtOH for 10 minutes, and absolute
EtOH for 5 minutes. Finally the specimens were sputter coated with gold and
examined with SEM (Model JSM 5500, JEOL Ltd., Tokyo, Japan).


## 2.7. Statistical Analysis

Statistical
analysis was done with StatView and Graphics Program (SAS institute, Cary, NC,
USA).
Differences in the mean numbers of the microbes (CFUs) harvested form the
experimental materials were tested with an ANOVA after logarithmic
transformation. Post Hoc comparisons were made with Fisher’s PLSD test after
theF test for equal means was found to be significant at P<.05 level.


## 3. Results

Ra value
of the samples with small glass particles was lower than that of samples with
big glass particles (0.46±0.08 μm versus 0.51±0.11 μm). Contact angle of the samples
with small glass particles was also smaller than that with big glass particels
(46°±12° versus 68°±5°).Clear
differences were observed in bacterial adhesion and early stages of surface
colonization of all experimental materials when using the type strains.H.
influenzae (ATCC
49247, clinical isolate 1258) adhered
significantly better on the copolymer C when compared to P. aeruginosa (ATCC 27853, clinical isolate 1218; mean difference 0.944, P<.0001) and S. pneumoniae (ATCC 49619, clinical isolate 1186; mean difference 0.430, P<.05). Significant difference was also
noticed between S. pneumoniae and P. aeruginosa (mean difference 0.514,
P<.01).Adhesion ofP. aeruginosa was
significantly higher to C90 as compared to the C (mean difference 1.000;
P<.001). The adhesion was also higher to C90 as compared to C45 (mean
difference 0.756, P<.01). The adhesion of H. influenzae and S. pneumoniae did not differ between the tested materials
(Figure 1).Attachment of (lg CFU/mL)P. aeruginosa (ATCC 27853), H. influenzae (ATCC 49247), and S. pneumoniae (ATCC 49619) on the
experimental materials C, C45, and C90.
(a)(b)(c)Similar
results were obtained with the type strains and the clinical isolates of the
microorganisms in the adhesion experiments. Thus the surface colonization
experiments were performed with the type strains only.P. aeruginosa showed rapid surface colonization during the culturing and was superior to H. influenzae in this respect (Figures 1
and 2). S. pneumoniae was not able to
colonize the material surfaces in the experimental conditions used and the
number of viable cells harvested from the materials after the 4-hour culturing
actually decreased (P<.001).Early colonization (lg CFU/mL) byP.
aeruginosa (ATCC 27853) and H. influenzae (ATCC 49247) on the experimental materials C, C45,
and C90.
(a)(b)ForP. aeruginosa the differences observed in the cell colonization
evened up after the 4-hour culturing indicating that C90 inhibited surface
colonization (Figure 2): no differences among the materials were observed
although significantly more viable cells had adhered on C90 during the 30 minutes adhesion. H. influenzae showed surface colonization of all materials (Figure 2).
No colonization inhibition was observed, actually, slightly higher numbers of
cells were harvested from the C45 than the C discs after the 4-hour culturing.Presence of BAG in the material had
no effect on the final pH of the culture media. No differences among the
experimental materials were noticed in this respect. No visible turbidity
indicating growth in the medium was observed after the 4-hour culturing.


### 3.1. SEM

The SEM examinations verified the
results obtained by culturing the cells harvested from the material surfaces.
Roughness of the sample surfaces varied according to the size of BAG particles
incorporated into the copolymer matrix. The smoothest surface was noticed on the
copolymer surface C, while the material C90 had the roughest surface texture.
Microbes seemed to attach evenly on the materials’ surfaces, but no microbes
were detected directly on the surfaces of the exposed BAG granules as is shown
in Figure3 for P. aeruginosa after 4
hours of culturing. The rough surface of C45 and C90 appeared to favor
adhesion of P. aeruginosa but this
was not seen for H. influenzae and S. pneumoniae. The results of the SEM
analyses appeared to be in agreement with the quantitative CFU assessments of
the cells from the material surfaces.SEM image of theP. aeruginosa (ATCC 27853) attachment on the
composite (a) and on the C90 disc (b) after 30-minute immersion (white
arrows). Bioactive glass granules are visible on the surface of C90 material (black
arrows). Images are inverted into negatives to show the attached microbes.
(a)(b)


## 3.1. SEM

The SEM examinations verified the
results obtained by culturing the cells harvested from the material surfaces.
Roughness of the sample surfaces varied according to the size of BAG particles
incorporated into the copolymer matrix. The smoothest surface was noticed on the
copolymer surface C, while the material C90 had the roughest surface texture.
Microbes seemed to attach evenly on the materials’ surfaces, but no microbes
were detected directly on the surfaces of the exposed BAG granules as is shown
in Figure3 for P. aeruginosa after 4
hours of culturing. The rough surface of C45 and C90 appeared to favor
adhesion of P. aeruginosa but this
was not seen for H. influenzae and S. pneumoniae. The results of the SEM
analyses appeared to be in agreement with the quantitative CFU assessments of
the cells from the material surfaces.SEM image of theP. aeruginosa (ATCC 27853) attachment on the
composite (a) and on the C90 disc (b) after 30-minute immersion (white
arrows). Bioactive glass granules are visible on the surface of C90 material (black
arrows). Images are inverted into negatives to show the attached microbes.
(a)(b)


## 4. Discussion

Primary adhesion between
bacteria and implant devices is mediated by nonspecific interactions [10].
Before the adhesion, implant surface is conditioned by the adsorption of water
and proteins, which alters its original surface properties. Thus, in clinical
environment bacterial adhesion and subsequent biofilm development is strongly
determined by materials ability to adsorb fluids from their surroundings.In the present study all the
experimental materials consisted of poly(ε-caprolactone/D,L-lactide) or its composites
with BAG. Due to the polycaprolactone blocks the copolymer itself is
hydrophobic and has relatively poor wettability [8]. The presence of BAG
increases water adsorption, but the adsorption occurs mainly through the
interface of the BAG and copolymer matrix. As formed, all BAG granules are
immediately embedded in the copolymer matrix and even near the surface granules
are covered by a thin polymer film. The samples used in this study were prepared
by casting, which resulted in different topographies on the specimen surfaces.
Casting was chosen in order to achieve circumstances which resemble clinical
situation after material application. Due to the thermoplastic nature of the
materials, a thin polymer skin covers the embedded galss granules even after the
material is shaped into its desired form. Grinding and polishig would have
resulted in more uniform surface texture, but thermoplastic nature and softness
of the material prevented using this method.In this study the hydrophobic nature of
copolymer did not prevent the adhesion of microorganisms as all tested microbes
attached on the experimental materials.
Copolymer samples without BAG showed relatively smooth surface texture.
However, the presence of BAG particles induced irregularities on the sample
surfaces, which increased the surface area of all the samples containing BAG.The purpose of this work
was to study attachment ofH. influenzae,S. pneumoniae, andP. aeruginosa to the composites as well as net
accumulation of the bacteria to the materials during the 4-hour exposure to the
growth medium. The growth media did not show turbidity after the experiments,
thus cells shed from the material surfaces should not have interfered with the
experiments. In this study, we used scraping of the cells and not labelling of
them as earlier [14] since we wanted to collect both sides of the discoid
material specimens for separate assessment. This enabled also the assessment of
CFUs and SEM detection from the same specimen. The repeatability of the
scraping method was good as judged by the small standard deviations of the
replicates. Thus the differences in bacterial adhesion and early colonization
are related with the differences in the composition and surface topography of
the tested materials.In the previous studies BAG has shown to possess
direct antimicrobial properties againstS. mutans, Porphyromonas gingivalis, Actinobacillusactinomycetemcomitans, and Actinomyces naeslundii [4, 15, 16] and
even against Candida albicans [17]. In these studies a clear
antimicrobial effect has been noticed when BAG has been used in the particulate
form. In this form, BAG’s active surface area is large and it increases
significantly the pH of the aqueous environment on its surrounding. It is
likely that the main antimicrobial effects of BAG are related to its reactivity
and capability to increase the pH.In our study all the materials were in solid
discoid form. Although BAG particles are reactive and capable of changing pH in
the vicinity of the composite surface [18] it did not prevent bacterial
attachment in this study. The attachment of P.
aeruginosa was increased by the presence of large BAG granules, most probably
due to larger surface area, while the attachment of H. influenzae and S. pneumoniae was influenced by the chemical nature of the material. In a previous study H. influenzae and S. pneumoniae attached poorly on BAG plates [4, 19]. Embedding BAG
in the copolymer apparently hinders the surface reactions of the BAG particles
since only the granules close to the composite surface are exposed to
dissolution in the short-term immersion. Despite that, in our study the
presence of BAG appeared to inhibit colonization, reflecting early biofilm
formation, as the expected values based on bacterial attachment did not
increase. As expected, P. aeruginosa could colonize on the
materials, and also H. influenzae was
able to grow on the materials and stay attached to them, while S. pneumoniae did not colonize at all. The differences in the inhibiton of
colonization among the studied microorganisms is propably related with the
surface reactivity of the experimental materials. Prevention of P. aeruginosa colonization was especially
clear on substrates containing large BAG particles.Within the limitations of this study it can be
concluded that composites of poly(ε-caprolactone
and D,L-lactide) and BAG do not inhibit the adhesion of S. pneumoniae, H. influenzae or P. aeruginosa to the copolymer. However, the
presence of BAG seems to inhibit their colonization. This is especially clear for P. aeruginosa, which is known to colonize easily on surfaces. One of the major drawbacks in the use of
biomaterials is the occurrence of biomaterials centred infections [20] and,
thus, any method to prevent them is beneficial. The findings in the present
study suggest that in terms of
bacterial colonization the composite of poly(ε-caprolactone and D,L-lactide) and
BAG is a promising
material for medical devices in head and neck and craniomaxillofacial applications.



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*Source: 765813-2009-04-06.xml*