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segmented polyurethane blends as coating materials for urinary catheters: in vitro bacterial adhesion and encrustation behavior
Biomaterials 23 (2002) 3991–4000
Assessment of PEO/PTMO multiblock copolymer/segmented polyurethane blends as coating materials for urinary catheters: in vitro bacterial adhesion and encrustation behavior Jae Hyung Parka, Yong Woo Chob, Ick Chan Kwonb, Seo Young Jeongb, You Han Baea,* a
Department of Materials Science and Engineering, Center for Biomaterials and Biotechnology, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, South Korea b Biomedical Research Center, Korea Institute of Science and Technology, Cheongryang, Seoul 136-791, South Korea Received 27 September 2001; accepted 4 April 2002
Abstract The effective long-term use of indwelling urinary catheters has often been hindered by catheter-associated infection and encrustation. In this study, the suitability of poly(ethylene oxide) (PEO)-based multiblock copolymer/segmented polyurethane (SPU) blends as coating materials for the commercial urinary catheters was assessed by measuring swellability, bacterial adhesion, and encrustation behavior. When exposed to PBS (pH 7.4), the blends absorbed a significant amount of water, which was proportional to the copolymer content. It was demonstrated from bacterial adhesion tests that compared to bare SPU, the blend surfaces could significantly reduce the adhesion of E. coli, P. mirabilis, and S. epidermidis; the number of adherent bacteria correlated with the amount of copolymer additive, indicating that the swellability of the blends affected bacterial adhesion. Of the bacteria studied, the greatest effect of the copolymer additive was observed in S. epidermidis adhesion, in which there was an 85% decrease compared to bare SPU with a small amount of copolymer additive as low as 5% based on a dried blend. By using an artificial bladder model, allowing the catheter to be blocked by encrustation, it was revealed that the blend surfaces could effectively resist encrustation. The duration of patency was extended up to 2073.1 h on the blend surface containing 10% of the copolymer additive, whereas the silicone-coated catheter, a control, required the least time for blockage, 7.873.1 h. The superior characteristics of the blends compared to other surfaces might be attributed to their PEO-rich surfaces, produced by the migration of PEO phase in the copolymer chain of the blends in an aqueous environment, and provide promising potential as a coating material on the urinary catheter for long-term catheterization. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Poly(ethylene oxide); Multiblock copolymer; Polyurethane; Bacterial adhesion; Encrustation; Urinary catheter
1. Introduction While the urinary catheter is one of the most commonly used medical devices in urology, catheterization has been the most frequent cause of nosocomial infections. Of the two million nosocomial infections, occurring annually in the United States, 40% involve the urinary tract [1–3] and more than 60% of these are related to an indwelling urinary catheter [4]. The bacteria, related to catheter-associated urinary tract infections (CAUTIs), may gain access to the bladder by the extraluminal or intraluminal ascendance on the *Corresponding author. Tel.: +82-62-970-2305; fax:+82-62-9702304. E-mail address: [email protected] (Y.H. Bae).
urinary catheters. Among various bacilli, Esherichia coli (E. coli) alone accounts for nearly 13% of CAUTIs [5]. Gram-positive cocci, which are responsible for approximately 34% of CAUTIs, including enterococci and coagulase negative staphylococci are also in abundance, inducing in significant inflammatory responses. Since these CAUTIs can be a source of nosocomial bacteremia [6] and be associated with an increase in mortality [7], many researchers have sought to develop urinary catheters that inhibit bacterial ascendance [8–10]. Encrustation of the urinary catheter has been another common complication, combined with CAUTIs, in the treatment of patients who should undergo long-term indwelling bladder catheterization [11,12]. It is believed that encrustation is associated with the adhesion and colonization of urease-producing organisms such as
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 1 4 4 - 8
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Proteus mirabilis (P. mirabilis) [13,14] and leads to the deposition of rigid crystals, primarily composed of magnesium ammonium phosphate and calcium phosphate, onto the catheter lumen surfaces [15,16]. These crystalline deposits may produce pain in the patient due to the occurrence of mechanical trauma in urethral mucosa upon catheter withdrawal. In addition, the accumulation of deposits contributes to the blockage of the catheter lumen, thereby resulting in retention of urine, ascending infection of the urinary tract, and increased discomfort for the patient. Due to the nature of the soft and slippery surfaces when hydrated, hydrogels may be beneficial if used as a coating material for urinary catheter. Surfaces coated with hydrogels are less likely to attract bacterial adhesion [9,10], damage the urethral mucosa [17,18], or cause discomfort to the patient. However, it is not fully understood whether hydrogel-coated surfaces are also effective in resisting encrustation or not. For example, Talja et al. [11] found that a hydrogel-coated catheter was less prone to encrustation compared to full silicone or siliconized catheters. A similar encrustation trend on various indwelling double-J stents was also observed by Cormio et al. [19]. In other groups, significant differences were not observed between hydrogel-coated catheters and silicone [20,21] or latex catheters [14]. Moreover, the possibility of higher encrustation on hydrogel surfaces than on other surfaces has recently been suggested [22,23]. In a previous report, our group developed a novel type of hydrogels (multiblock copolymers) based on poly(ethylene oxide) (PEO) and poly(polytetramethylene oxide) (PTMO) [24]. They exhibited outstanding resistance to protein adsorption and platelet adhesion, which was dependant on the PEO block length and
polymer topology. In addition, the promising potential of the copolymer (PEO2kPTMO) as an additive was demonstrated with the surface modification of commercial segmented polyurethane (SPU) [25]. The results showed that the high molecular weight and compatible chemical structure of the copolymer with SPU made the surfaces PEO rich and stable in an aqueous environment, resulting in greater resistance to protein adsorption and platelet adhesion, without significant deterioration in physical properties, compared to bare SPU. Herein, we aimed to evaluate the copolymer/SPU blends as coating materials for the surface modification of urinary catheters, suffering from CAUTIs and encrustation. By using five different blends varying in copolymer content, bacterial adhesion to and encrustation on blend surfaces were assessed.
2. Materials and methods 2.1. Test materials A multiblock copolymer, PEO2kPTMO, was prepared by polycondensation reaction between hydrophilic PEO (Mn ¼ 2000; Aldrich) and hydrophobic PTMO (Mn ¼ 2000; Aldrich) in the presence of hexamethylene diisocyante as reported previously [24]. A SPU, Pellethanes 2363-80AE (Dow chemical), was used as a control after precipitating SPU solution (10 wt% in dimethyl acetamide) in methanol, followed by drying for two days at 501C in a vacuum. As shown in Fig. 1, the copolymer contains an equimolar amount of PEO and PTMO, which forms physically crosslinked hydrogel in an aqueous environment, while a SPU consists of PTMO as a soft segment and 4,40 -methylenediphenylene
Pellethane ® (SPU)
O O
H
H
N
N
CH2
x
H CH2
O
O
4
H
N
O
O
N CH2 O
O
Hard segment
Soft segment
PEO2kPTMO (multiblock copolymer) H N
O O
x
CH2
O
Hydrophilic block
H N 6
O O O
yO
O N
CH2
H
6N
H
z
Hydrophobic block
Fig. 1. Chemical structure of SPU (Pellethanes) and copolymer additive (PEO2kPTMO).
y
z
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diisocyanate connected with 1,4-butane diol as a hard segment, which forms phase-separated thermoplastic elastomer. 2.2. Film preparation A SPU was dissolved in dimethyl acetamide to give a 10 w/v% solution and the various amounts of the copolymer at 5, 10, 20 and 30 wt% (based on dried blends) were added. The homogeneous polymer solutions were poured into clean petri dishes and the films were prepared by evaporating the solvent at 501C for 2 days in a vacuum. The thickness of films prepared was adjusted to 200 mm by the amounts of the poured polymer solution and they were cut into circular shape (12 mm in diameter) for further experiments such as for the swelling property and bacterial adhesion. The SPU films containing the copolymer additive were designated as a Pell-X ; where X means the weight percent of the copolymer based on the dried blend. 2.3. Catheter coating A urinary catheter, silicone-coated two-way foley catheter (16 Fr, Rusch, USA), was used after being treated with ethanol for 30 min to remove any additives or low-molecular weight components. To prepare blend solutions for catheter coating, desired amounts of SPU and copolymer additive were dissolved in tetrahydrofurane (THF, HPLC grade, J.T. Baker) with a concentration of 1 w/v%. The catheters were then coated with blend solutions by dipping, which was repeated five times at 10-min intervals to coat catheters evenly. The blend-coated catheters were placed in a clean bench for 1 day at room temperature, followed by being dried for 1 day in a vacuum to remove any residual solvent. 2.4. Swelling behavior The swelling kinetic of the blends was examined by measuring the water capacity as a function of time. The dried films (Wpolymer ) were immersed in PBS (pH 7.4), which was pre-warmed to 371C, and the weights of the swollen polymers (Wswollen ) were measured in the desired periods after removal of the excess surface water by patting the samples with filter paper. The water capacity was then calculated using the following equation: Water capacity; Wc ð%Þ ¼ ðWswollen Wpolymer Þ 100=Wpolymer :
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Collection for Type Cultures and used for the observation of initial bacterial adhesion to the blend surfaces and encrustation test. The day before the experiment, each of the species which had been maintained on a nutrient agar plate was grown for 24 h at 371C in a nutrient broth (Difco Chemical) solution. The bacterial inoculum was then washed twice by centrifuging in PBS (pH 7.4). After being resuspended in PBS, the cell density was adjusted to 1 108/ml by dilution using a PBS. These resulting inoculums were directly used for the bacterial adhesion tests. Of the species studied, P. mirabilis inoculum was further used for encrustation test since it is known to be urease-producing bacteria accelerating the deposition of rigid crystals onto the catheter lumen surfaces. 2.6. Bacterial adhesion test Before the bacterial adhesion experiment, the polymer films were exposed to a 40 W UV irradiation using a mercury-vapor UV lamp (Sangwoo Inc., Korea) in a clean bench, where a distance between films and lamp was 60 cm. After exposing to UV light for 2 h, the films were hydrated for 1 day in PBS, immersed into a bacterial suspension (10 ml), and incubated for 2 h at 371C. After incubation, the films were gently rinsed three times with 10 ml sterile PBS to remove weakly adhered bacteria. The resulting films were vortexed and sonicated for 2 min, respectively, with 10 ml of PBS containing 0.1% Tween-80 to detach bacteria adhering to the surfaces, as suggested by Flemming et al. [26]. The number of adherent bacteria was quantitatively determined by colony counts and expressed as the number of colony-forming units (CFU) per square centimeter. 2.7. Encrustation tests 2.7.1. Artificial urine The composition of the artificial urine, shown in Table 1, was similar to that described by Tunney et al. [27] except for the use of nutrient broth and P. mirabilis instead of urease. Four solutions were separately prepared to prevent bacterial growth and brushite (CaHPO4 2H2O) formation. Before the experiments, 500 ml each of compounds A and B were homogeneously mixed and compound C (50 ml) was added to supply minimal nutrients to P. mirabilis, urease-producing bacterium, during the encrustation tests [14]. After being filter-sterilized using a cellulose acetate membrane (0.2 mm, Corning, USA), the artificial urine was applied for further experiments within 2 h.
2.5. Bacterial strains and culture conditions E. coli (ATCC No. 11775), P. mirabilis (ATCC No. 25993), and Staphylococcus epidermidis (S. epidermidis, ATCC No. 12228) were obtained from the Korean
2.7.2. Artificial bladder model and intraluminal encrustation A model for the evaluation of encrustation on the catheter surface, mimicking the human bladder, was
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Table 1 Composition of artificial urine used in this studya Compound A (g/l)
Compound B (g/l)
Compound C (g/l)
Compound D
Magnesium chloride hexahydrate (3.6) Potassium dihydrogen orthophosphate (7.6) Urea (16)
Calcium chloride hexahydrate (5.3) Chicken ovalbumin (2)
Nutrient broth (8)
Suspension of P. mirabilis (1 108 cell/ml in PBS)
a
Compounds A, B, and C were dissolved in distilled water.
composed of a cylindrical glass flask (5 cm in diameter and 10 cm in height) similar to that designed by Stickler et al. [8], a peristaltic pump, and a drainage bag (Fig. 2). The catheters coated with SPU and blends were inserted into the autoclaved flask and inflated using a polystyrene syringe to fix the position like a catheterizaion in the human body. With a peristaltic pump, the sterile urine was supplied to the flask at a rate of 0.7 ml/min, which made 30 ml of residual volume below the level of the catheter eyelet. The artificial urine in the flask was then inoculated by adding 300 ml of compound D to achieve a cell density of B1 106/ml. This artificial bladder model was maintained for 1 h to acclimate P. mirabilis to a new environment and to stimulate contamination for the initiation of encrustation. The supply of sterile urine was then switched on and monitored until the artificial urine became impassable through the catheter due to intraluminal blockage by encrustation. Data regarding lumen patency were statistically analyzed using a Student’s unpaired t test with a 95% confidence level (po0:05) and were reported as mean7standard deviation.
0.7 ml/min
Peristaltic pump
Bacterial inoculum
Water (37 oC)
Encrustation
Sterile urine
2.8. Scanning electron microscopy To evaluate the surface topologies of the catheters before encrustation test, the catheter surfaces were observed by using a scanning electron microscopy (SEM, JSM-5800, JEOL) after being gold-coated with a sputter coater (SPI-MODULEt, SPI sppl). With an identical method, the surfaces of the polymer films, prepared in Section 2.2, were observed before the experiments such as bacterial adhesion and swelling behavior tests. Encrustation on the catheter surfaces, retrieved immediately after blockage from the model and then gently rinsed in distilled water to remove threads of albumin [22], was observed using a low-vacuum SEM (Hitachi, S-2460N) after being gold-sputtered with a ion sputter (Hitachi, E1010) as suggested by Morris et al. [28].
3. Results The swelling kinetics for the blends at 371C in PBS (pH 7.4) are shown in Fig. 3. All the blends demon-
Drainage bag Fig. 2. Artificial bladder model for encrustation study.
strated time-dependant water absorption with initial rapid swelling followed by sluggish equilibrium, which was mainly ascribed to the presence of a hydrophilic PEO block in the copolymer additive. The water capacity was proportional to the copolymer content in the blends, indicating that the increase in the copolymer content enhanced the hydrophilicity of the blends [25]. The results also showed that the equilibrium swelling of the blends, in which the Wc values ranged from 3.05 (Pell-5) to 17.54 (Pell-30), was attained within 2 h. The polymer films, observed by SEM, exhibited uniform and smooth surfaces without considerable surface irregularities, allowing this study to evaluate the effect of surface chemistry on bacterial adhesion. To
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Attached cells (X102 CFU/cm2)
20
Water capacity (%)
15
10
5
300
200
100
150
200
250
Time (min)
(a)
Pe ll 30
100
Pe ll 5
50
SP U
0
Pe ll10 Pe ll20
0 0
300
200
100
Pe
ll 30
20 llPe
10 llPe
5 Pe Pe
ll-
SP
(b)
SP U
U
0
14000
Attached cells (X102 CFU/cm2)
12000 10000 8000 6000 4000 2000
ll 30 Pe
20 llPe
10 llPe
(c)
5
0
ll-
observe the effects of the copolymer additive on the adhesion of bacteria, relevant to infection and encrustation during catheterization, bare SPU and the blends were treated with inoculums containing three different species. All bacteria including E. coli, S. epidermidis, and P. mirabilis were found to adhere to the bare SPU surfaces much greater than to the blend surfaces (Fig. 4), a small amount of copolymer additive as low as 5% significantly improved the resistance to bacterial adhesion (e.g., the number of adherent E. coli to bare SPU was 275731 102 CFU/cm2 but that to Pell-5 was 83713 102 CFU/cm2, respectively). On all the polymeric surfaces, the adhesion numbers of S. epidermidis were consistently higher than those of E. coli and P. mirabilis, where the differences were often larger than a factor of 10. It was also revealed that the number of adherent bacteria decreased gradually with increasing the copolymer content in the blends, which thus resulted in the lowest bacterial adhesion to Pell-30 containing the highest amount of copolymer additive among the blends studied. The greatest effect of the copolymer additive was observed in S. epidermidis adhesion, while the smallest effect was in P. mirabilis, e.g., the numbers of adherent S. epidermidis and P. mirabilis to Pell-5 were 88.7% and 33.5% lower than those adhering to bare SPU, respectively. After being coated with SPU or the blends, the surface topologies of the urinary catheters were observed using a SEM. Fig. 5 shows the representative pictures for each of the outer surfaces before and after catheter coating. All the surfaces had different topologies, in which the uncoated catheters showed a large quantity of surface irregularities with deep cracks, pits and fissures. A catheter coated with SPU showed a
Attached cells (X102 CFU/cm2)
Fig. 3. Swelling behavior of copolymer/SPU blends as a function of time: (J) Pell-5; (&) Pell-10; (W) Pell-20; (X) Pell-30.
Fig. 4. Bacterial adhesion to copolymer/SPU blend surfaces: (a) E. coli, (b) P. mirabilis, (c) S. epidermidis. The error bar is for standard deviation (n ¼ 5).
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Fig. 5. Scanning electron micrographs of urinary catheter surfaces coated with SPU and copolymer/SPU blends.
30 †‡
25
Lumen patency (hours)
†‡
†‡
20 †‡
15
10
5
Pe ll 20 Pe ll 30
ll 10 Pe
ll5 Pe
SP U
lic
on e
0
Si
smoother surface with the occasional presence of a small size of pits (0.1–0.5 mm). The blend-coated catheter surfaces, however, were relatively even and much smoother without significant cracks or pits although they showed rippled characteristics, indicating that the surface roughness of the uncoated catheters could decrease remarkably via coating with the blends. The artificial bladder model, which contained artificial urine inoculated with P. mirabilis, allowed the catheters to be blocked, primarily attributed to the encrustation proceeded by the deposition of magnesium ammonium phosphate and calcium phosphate onto the catheter lumen surfaces [27]. As shown in Fig. 6, none of the catheters were capable of resisting encrustation by P. mirabilis. A catheter coated with silicone, a control sample, required the least time for blockage (7.872.8 h), which was prolonged by coating with SPU (11.372.0 h) but without statistically significant difference (p > 0:05). It was noteworthy that the duration of lumen patency could be further extended by coating the catheters with the blends, e.g., the time for blockage significantly increased to 14.871.2 h by Pell-5, approximately double the period compared to that for a silicone-coated catheter. The maximal duration of lumen patency could be achieved when coated with Pell-10 (20.373.1 h), which showed a comparable period to those for Pells-20 and 30. The cross-sections and intraluminal surfaces were observed using SEM along the length of the catheters blocked by encrustation. Similar results for all the catheters studied were observed and the representative pictures are shown in Fig. 7 for the silicone-coated catheter. The results imply that the encrustation was dependent on the position along the catheter length, where the most extensive encrustation was observed in the region just below the eyehole. The quantity of crystal
Fig. 6. Duration of lumen patency of urinary catheters coated with SPU and copolymer/SPU blends (The error bar is for standard deviation, n ¼ 3). w, z, located above the bars, indicate significantly different values at the 95% confidence level, po0.05, when compared to silicone and SPU, respectively.
deposits, composed of struvite and hydroxyapatite, gradually reduced as the distance from the catheter tip increased, which was also observed by Morris et al. [28].
4. Discussion The use of indwelling urinary catheters involving prolonged contact with urine has been limited due to
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Fig. 7. Low-vacuum scanning electron micrographs of cross-sections of silicone-coated urinary catheters retrieved after blockage in the bladder model: (a) 1 cm, (b) 5 cm, (c) 10 cm from the catheter tip, (d) and (e) are intraluminal surfaces at 5 and 10 cm from catheter tip, respectively.
associated problems such as bacterial adhesion [9,10] and encrustation [11,12], resulting in biomaterial-centered infection [4,5] and urethral stricture [12], respectively. In this study, we explored bacterial adhesion to and encrustation behavior on the multiblock copolymer/ SPU blend-coated urinary catheter surfaces to evaluate their suitability for long-term catheterization. The multiblock copolymer was composed of hydrophilic PEO and hydrophobic PTMO blocks, forming physically crosslinked hydrogel via preserving a threedimensional structure in an aqueous environment [24]. When this copolymer was blended with SPU, a small amount as low as 5% based on the dried blend was able to significantly improve surface hydrophilicity and reduce protein adsorption and platelet adhesion without considerable deterioration in physical properties [25]. This was due to the achievement of stable PEO-rich surfaces, attributed to the high molecular weight and compatible structure of the copolymer with SPU. Along this line of research, by simple and convenient dipping method using a volatile polymer solution in THF, the copolymer/SPU blends were coated on the catheters to allow the surfaces to be PEO rich. It is known that the time taken to reach equilibrium swelling in distilled water for chemically crosslinked hydrogel is at least 10 h [29–31] and its swelling is known to be mediated by the hydration of a polymer chain, followed by the diffusion of water molecules to the gel matrix [32]. The swelling kinetic and degree of equilibrium swelling for chemically crosslinked hydrogel are determined by the hydrophilicity of the polymer, the amount and nature of the cross-linking agent, and the degree of dilution prior to polymerization. In the case of
physically crosslinked hydrogels, which often maintain their physical stability by aggregates of hydrophobic blocks associated with hydrophobic interaction, equilibrium swelling can be achieved within 4 h, depending on the type of hydrophobic block, due to the rapid response of a hydrophilic block such as PEO [33,34]. The copolymer itself, used in this study, also reached equilibrium swelling (Wc ¼ 114) within 20 min, where the flexible and amorphous hydrophobic block, PTMO, allows the PEO chain to be hydrated rapidly in an aqueous environment. The retarded period for equilibrium swelling in the blends, which was approximately 2 h, seemed to result from the restricted mobility of the PEO chain, attributed to the compatibility of the copolymer with the SPU matrix [25]. The low water capacity of the blends, which was approximately half of the expected value judging from the copolymer content, indicated that the hydration of the copolymer was inhibited by the intra or intermolecular interactions with the SPU chain such as van der Waals force and hydrogen bonding (Fig. 3). Bacterial adhesion to biomaterial surfaces is believed to be based both on physicochemical interactions (phase one) and on molecular and cellular interactions (phase two) between bacteria and material surfaces [35]. The latter is the process that bacteria adhere firmly to the biomaterial surface by the bridging function of the polymers releasing from or existing on a bacterial surface including capsules, fimbriae, and slime. Before phase two, the bacteria may attach themselves to the biomaterial surface via shot- or long-range interactions (phase one) such as electrostatic attraction, van der Waals force, hydrophobic interaction, or hydrogen
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bonding [36]. Of the physicochemical interactions, hydrophobic adsorption appears to be the most important since most bacterial surfaces contain some degree of hydrophobic moiety [10,37]. The lower adhesions of all the bacteria to the blend surfaces, compared to bare SPU, also suggest that hydrophilic surfaces can effectively resist bacterial adhesion (Fig. 4). The gradual decrease in bacterial adhesion, proportional to the copolymer content, can be more evidence of the correlation between hydrophilicity and bacterial adhesion since the water capacity, related to the hydrophilicity, increased with the copolymer content, as discussed above. These effects were also observed by Cook et al. [38], who synthesized hydrogels varying in water content from 33 to 69 wt% and demonstrated that P. aeruginosa adhered less to hydrogels with higher water contents. However, it should be considered that the bacterial adhesion depends not only on the degree of water absorption but also on the functional groups exposed on the biomaterial surfaces [35]. Among the bacteria studied, E. coli and S. epidermidis were representative uropathogens, relevant to the catheter-associated infections [39], while P. mirabilis was the predominant microorganism observed in encrusted catheter biofilms [13]. It is known that urease-producing species, including Proteus vulgaris, P. mirabilis, and Providencia rettgeri, elevate the pH of the urine through hydrolysis of the urea to produce ammonia [14,40]. In the resulting alkaline environment, magnesium and calcium ions readily precipitate and deposit on the catheter surfaces in the form of struvite (NH4MgPO4 6H2O) and poorly crystalline hydroxyapatite (Ca10(PO4)6(OH)2) [15,16]. Recently, the ability of P. mirabilis to swarm over catheters has also been demonstrated by Stickler et al. [41], which may enhance the adhesiveness on and allow the migration over the catheter surfaces because the swarmer cells secrete slime and move rapidly over surfaces [42]. Therefore, it can be suggested that the low adherence of P. mirabilis to the blend surfaces provides the potential to delay the onset of encrustation since it is an event subsequent to bacterial adhesion and colonization. There has been controversy with respect to the effectiveness of hydrogel-coated urinary catheters on resistance to encrustation [11,14,20–23]. Even though various factors affect these inconsistent results, the different surface chemistries of hydrogels employed in each study should play a critical role since encrustation is a phenomenon occurring on the catheter surface. The surface morphologies of the catheters attributed to the methods of manufacture could be another possible reason [43,44]. In this study, the simple dip-coating method provided smooth surface of the blend on the catheters (Fig. 5). This fact allows this study to evaluate the effects on surface chemistry and hydrophilicity. As shown in Fig. 6, a small amount of copolymer content as
low as 5% could increase the time for blockage, which could be further extended by 10% copolymer content. This implies that PEO-rich surfaces, produced due to the migration of the PEO phase in the copolymer chain of the blends when exposed to artificial urine, can effectively resist encrustation. Superior resistance to encrustation of PEO surface was also demonstrated by Gorman et al. [44] who evaluated comparative encrustation on Aquavenes (PEO/SPU composite hydrogel), SPU, and silicone. It is also interesting to note that the addition of a large amount of the copolymer (>10%) cannot increase the duration of lumen patency any more, indicating that hydrophilicity is not always a critical factor for encrustation. For long-term catheterization, urinary catheters should not vary significantly in terms of surface hydrophilicity and mechanical properties to preserve the repellency of bacterial adhesion and prevent catheter fracture, respectively. In a previous report [25], we demonstrated that the mechanical properties of the blends maintained up to 10% addition of the copolymer compared to those of SPU even though surface hydrophilicity was significantly improved. The blends also showed minimal extraction of the copolymer additive when exposed to a buffer solution over 2 months; o1% weight loss of the dried polymer occurred, indicating the creation of permanent PEOrich surfaces. These results also suggest that SPU/ copolymer blends are suitable as coating materials for the catheters. As an overview, urinary catheters coated with blends, especially containing 10% of copolymer additive (Pell-10), might be promising for long-term catheterization, based on better resistance to the bacterial adhesion and encrustation in vitro compared to SPU and silicone-coated catheters.
5. Conclusion The SPU/copolymer blends were assessed for application as coating materials onto the urinary catheters. The blend films adsorbed a significant amount of water, which was proportional to copolymer content. From the bacterial adhesion tests, it was demonstrated that the blend surfaces effectively suppressed the adhesion of E. coli, S. epidermidis, and P. mirabilis. The catheter surfaces coated with blends were much smoother and relatively even without significant surface irregularities, compared to silicone- and SPU-coated catheters. It was also observed that the catheters coated with blends showed good resistance to encrustation, which was maximal when the copolymer content was higher than 10%. These in vitro results of the urinary catheter coated with blends provide promising potential for longterm catheterization without serious infection and encrustation.
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Acknowledgements This study was supported by Grant No. HMP-98-G2-034 from the Ministry of Health and Welfare, Korea.
References [1] Jarvis WR. Selected aspects of the socioeconomic impact of nosocomial infection: morbidity, mortality, cost, and prevention. Infect Control Hosp Epidemiol 1996;17:552–7. [2] Stamm WE. Catheter-associated urinary tract infections: epidemiology, pathogenesis, and prevention. Am J Med 1991;91(Suppl 3B):65S–71S. [3] Bronsema DA, Adams JR, Pallares R, Wenzel RP. Secular trends in rates and etiology of nosocomial urinary tract infections at a university hospital. J Urol 1993;150:414–6. [4] Vincent JL, Bihari DJ, Suter PM. The prevalence of nosocomial infection in intensive care units in Europe: results of the European prevalence of infection in intensive care (EPIC) study. J Am Med Assoc 1995;274:639–44. [5] Tambyah PA, Halvorson KT, Marki DG. A prospective study of pathogenesis of catheter associated urinary tract infections. Mayo Clin Proc 1999;74:131–6. [6] Maki DG. Nosocomial bacteremia: an epidemiologic overview. Am J Med 1981;70:719–32. [7] Platt R, Polk BF, Murdock B, Rosner B. Mortality associated with nosocomial urinary tract infection. N Engl J Med 1982;307:637–42. [8] Stickler DJ, Morris NS, Williams TJ. An assessment of the ability of a silver-releasing device to prevent bacterial contamination of urethral catheter drainage systems. Br J Urol 1996;78:579–88. [9] Gabriel MM, Mayo M, May LL, Simmons RB, Ahearn DG. In vitro evaluation of the efficacy of a silver-coated catheter. Curr Microbiol 1996;33:1–5. [10] Roberts JA, Fussell EN, Kaack MB. Bacterial adherence to urethral catheters. J Urol 1990;144:264–9. [11] Talja M, Korpela A, J.arvi K. Comparison of urethral reaction to full silicone, hydrogel-coated and siliconised latex catheters. Br J Urol 1990;66:652–7. [12] Denstedt JD, Wollin TA, Reid G. Biomaterials used in urology: current issues of biocompatibility, infection, and encrustation. J Endourol 1998;12:493–500. [13] Stickler DJ, Ganderton L, King J. Proteus mirabilis biofilms and the encrustation of urethral catheters. Urol Res 1993;21:407–11. [14] Sarangapani S, Cavedon K, Gage D. An improved model for bacterial encrustation studies. J Biomed Mater Res 1995;29: 1185–91. [15] Hedelin H, Eddeland A, Larsson L, Pettersson S, Ohman S. The composition of catheter encrustations, including the effects of allopurinol treatment. Br J Urol 1984;56:250–4. [16] Cox AJ, Hukins DWL. Morphology of mineral deposits on encrusted urinary catheters investigated by scanning electron microscopy. J Urol 1989;142:1347–50. [17] Nickel JC, Olson ME, Costerton JW. In vivo coefficient of kinetic friction: study of urinary catheter biocompatibility. Urology 1987;29:501–3. [18] Nacey JN, Delahunt B. Toxicity study of first and second generation hydrogel-coated latex urinary catheters. Br J Urol 1991;67:314–6. [19] Cormio L, Talja M, Koivusalo A, Makisalo H, Wolff H, Ruutu M. Biocompatibility of various indwelling double-J stents. J Urol 1995;153:494–6.
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[20] Cox AJ, Millington RS, Hukins DWL, Sutton TM. Resistance of catheters coated with a modified hydrogel to encrustation during an in vitro test. Urol Res 1989;17:353–6. [21] Cox AJ, Hukins DWL, Sutton TM. Comparison of in vitro encrustation on silicone and hydrogel-coated latex catheters. Br J Urol 1988;61:156–61. [22] Tunney MM, Keane PF, Jones DS, Gorman SP. Comparative assessment of ureteral stent biomaterial encrustation. Biomaterials 1996;17:1541–6. [23] Desgrandchamps F, Moulinier F, Daudon M, Teillac P, Le Duc A. An in vitro comparison of urease-induced encrustation JJ stents in human urine. Br J Urol 1997;79:24–7. [24] Park JH, Bae YH. Hydrogels based on poly(ethylene oxide) and poly(tetramethylene oxide) or poly(dimethyl siloxane): synthesis, characterization, in vitro protein adsorption and platelet adhesion. Biomaterials 2002;23:1797–808. [25] Park JH, Bae YH. Physicochemical properties and in vitro biocompatibility of PEO/PTMO multiblock copolymer/ segmented polyurethane blends. J Biomater Sci Polym Edn, in press. [26] Flemming RG, Capelli CC, Cooper SL, Proctor RA. Bacterial colonization of funtionalized polyurethanes. Biomaterials 2000;21:273–81. [27] Tunney MM, Bonner MC, Keane PF, Gorman SP. Development of a model for assessment of biomaterial encrustation in the upper urinary tract. Biomaterials 1996;17:1025–9. [28] Morris NS, Stickler DJ. Encrustation of indwelling urethral catheters by proteus mirabilis biofilms growing in human urine. J Hosp Infect 1998;39:227–34. [29] Iza M, Stoianovici G, Viora L, Grossiord JL, Couarraze G. Hydrogels of poly(ethylene glycol): mechanical characterization and release of a model drug. J Control Rel 1998;52:41–51. [30] Kaneko Y, Sakai K, Kikuchi A, Yoshida R, Sakurai Y, Okano T. Influence of freely mobile grafted chain length on dynamic properties of comb-type grafted poly(N-isopropylacrylamide) hydrogels. Macromolecules 1995;28:7717–23. [31] Karada&g E, Saraydin D, C . O. In vitro swelling - etinkaya S, Guven studies and preliminary biocompatibility evaluation of acrylamide-based hydrogels. Biomaterials 1996;17:67–70. [32] Kost J, Langer R. Equilibrium swollen hydrogels in controlled release applications. In: Peppas NA, editor. Hydrogels in medicine and pharmacy: properties and applications, vol. 3. Boca Raton, FL: CRC Press, 1987. p. 95–108. [33] Choi YK, Bae YH, Kim SW. Star-shaped poly(ether-ester) block copolymers: synthesis, characterization, and their physical properties. Macromolecules 1998;31:8766–74. [34] Bae YH, Huh KM, Kim Y, Park K. Biodegradable amphiphilic multiblock copolymers and their implications for biomedical applications. J Control Rel 2000;64:3–13. [35] An YH, Fredman RJ. Concise review of mechanisms of bacterial adhesion to biomaterial surfaces. J Biomed Mater Res 1998;43:338–48. [36] Gristina AG. Biomaterial-centered infection: microbial adhesion versus tissue integration. Science 1987;237:1588–95. [37] Ong Y, Razatos A, Georgiou G, Sharma MM. Adhesion forces between E. coli bacteria and biomaterial surfaces. Langmuir 1999;15:2719–25. [38] Cook AD, Sagers RD, Pitt WG. Bacterial adhesion to poly(HEMA)-based hydrogels. J Biomed Mater Res 1993;27: 119–26. [39] Hawthorn L, Reid G. The effect of protein and urine on uropathogen adhesion to polymer substrata. J Biomed Mater Res 1990;24:1325–32. [40] Stickler D, Morris N, Moreno M, Sabbuba N. Studies on the formation of crystalline bacterial biofilms on urethral catheters. Eur J Clin Microbiol 1998;17:649–52.
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[41] Stickler D, Hughes G. Ability of Proteus mirabilis to swarm over urethral catheters. Eur J Clin Microbiol Infect Dis 1999;18:206–8. [42] Rahman MM, Guard-Petter J, Asokan K, Hughes C, Carlson RW. The structure of the colony migration factor from pathogenic Proteus mirabilis. A capsular polysaccharide that facilitates swarming. J Biol Chem 1999;274:22993–8.
[43] Cox AJ. Comparison of catheter surface morphologies. Br J Urol 1990;65:55–60. [44] Gorman SP, Tunney MM, Keane PF, Van Bladel K, Bley B. Characterization and assessment of a novel poly(ethylene oxide)/ polyurethane composite hydrogel (Aquavenes) as a ureteral stent biomaterial. J Biomed Mater Res 1998;39:642–9.
Assessment of PEO/PTMO multiblock copolymer/segmented polyurethane blends as coating materials for urinary catheters: in vitro bacterial adhesion and encrustation behavior Jae Hyung Parka, Yong Woo Chob, Ick Chan Kwonb, Seo Young Jeongb, You Han Baea,* a
Department of Materials Science and Engineering, Center for Biomaterials and Biotechnology, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, South Korea b Biomedical Research Center, Korea Institute of Science and Technology, Cheongryang, Seoul 136-791, South Korea Received 27 September 2001; accepted 4 April 2002
Abstract The effective long-term use of indwelling urinary catheters has often been hindered by catheter-associated infection and encrustation. In this study, the suitability of poly(ethylene oxide) (PEO)-based multiblock copolymer/segmented polyurethane (SPU) blends as coating materials for the commercial urinary catheters was assessed by measuring swellability, bacterial adhesion, and encrustation behavior. When exposed to PBS (pH 7.4), the blends absorbed a significant amount of water, which was proportional to the copolymer content. It was demonstrated from bacterial adhesion tests that compared to bare SPU, the blend surfaces could significantly reduce the adhesion of E. coli, P. mirabilis, and S. epidermidis; the number of adherent bacteria correlated with the amount of copolymer additive, indicating that the swellability of the blends affected bacterial adhesion. Of the bacteria studied, the greatest effect of the copolymer additive was observed in S. epidermidis adhesion, in which there was an 85% decrease compared to bare SPU with a small amount of copolymer additive as low as 5% based on a dried blend. By using an artificial bladder model, allowing the catheter to be blocked by encrustation, it was revealed that the blend surfaces could effectively resist encrustation. The duration of patency was extended up to 2073.1 h on the blend surface containing 10% of the copolymer additive, whereas the silicone-coated catheter, a control, required the least time for blockage, 7.873.1 h. The superior characteristics of the blends compared to other surfaces might be attributed to their PEO-rich surfaces, produced by the migration of PEO phase in the copolymer chain of the blends in an aqueous environment, and provide promising potential as a coating material on the urinary catheter for long-term catheterization. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Poly(ethylene oxide); Multiblock copolymer; Polyurethane; Bacterial adhesion; Encrustation; Urinary catheter
1. Introduction While the urinary catheter is one of the most commonly used medical devices in urology, catheterization has been the most frequent cause of nosocomial infections. Of the two million nosocomial infections, occurring annually in the United States, 40% involve the urinary tract [1–3] and more than 60% of these are related to an indwelling urinary catheter [4]. The bacteria, related to catheter-associated urinary tract infections (CAUTIs), may gain access to the bladder by the extraluminal or intraluminal ascendance on the *Corresponding author. Tel.: +82-62-970-2305; fax:+82-62-9702304. E-mail address: [email protected] (Y.H. Bae).
urinary catheters. Among various bacilli, Esherichia coli (E. coli) alone accounts for nearly 13% of CAUTIs [5]. Gram-positive cocci, which are responsible for approximately 34% of CAUTIs, including enterococci and coagulase negative staphylococci are also in abundance, inducing in significant inflammatory responses. Since these CAUTIs can be a source of nosocomial bacteremia [6] and be associated with an increase in mortality [7], many researchers have sought to develop urinary catheters that inhibit bacterial ascendance [8–10]. Encrustation of the urinary catheter has been another common complication, combined with CAUTIs, in the treatment of patients who should undergo long-term indwelling bladder catheterization [11,12]. It is believed that encrustation is associated with the adhesion and colonization of urease-producing organisms such as
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 1 4 4 - 8
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Proteus mirabilis (P. mirabilis) [13,14] and leads to the deposition of rigid crystals, primarily composed of magnesium ammonium phosphate and calcium phosphate, onto the catheter lumen surfaces [15,16]. These crystalline deposits may produce pain in the patient due to the occurrence of mechanical trauma in urethral mucosa upon catheter withdrawal. In addition, the accumulation of deposits contributes to the blockage of the catheter lumen, thereby resulting in retention of urine, ascending infection of the urinary tract, and increased discomfort for the patient. Due to the nature of the soft and slippery surfaces when hydrated, hydrogels may be beneficial if used as a coating material for urinary catheter. Surfaces coated with hydrogels are less likely to attract bacterial adhesion [9,10], damage the urethral mucosa [17,18], or cause discomfort to the patient. However, it is not fully understood whether hydrogel-coated surfaces are also effective in resisting encrustation or not. For example, Talja et al. [11] found that a hydrogel-coated catheter was less prone to encrustation compared to full silicone or siliconized catheters. A similar encrustation trend on various indwelling double-J stents was also observed by Cormio et al. [19]. In other groups, significant differences were not observed between hydrogel-coated catheters and silicone [20,21] or latex catheters [14]. Moreover, the possibility of higher encrustation on hydrogel surfaces than on other surfaces has recently been suggested [22,23]. In a previous report, our group developed a novel type of hydrogels (multiblock copolymers) based on poly(ethylene oxide) (PEO) and poly(polytetramethylene oxide) (PTMO) [24]. They exhibited outstanding resistance to protein adsorption and platelet adhesion, which was dependant on the PEO block length and
polymer topology. In addition, the promising potential of the copolymer (PEO2kPTMO) as an additive was demonstrated with the surface modification of commercial segmented polyurethane (SPU) [25]. The results showed that the high molecular weight and compatible chemical structure of the copolymer with SPU made the surfaces PEO rich and stable in an aqueous environment, resulting in greater resistance to protein adsorption and platelet adhesion, without significant deterioration in physical properties, compared to bare SPU. Herein, we aimed to evaluate the copolymer/SPU blends as coating materials for the surface modification of urinary catheters, suffering from CAUTIs and encrustation. By using five different blends varying in copolymer content, bacterial adhesion to and encrustation on blend surfaces were assessed.
2. Materials and methods 2.1. Test materials A multiblock copolymer, PEO2kPTMO, was prepared by polycondensation reaction between hydrophilic PEO (Mn ¼ 2000; Aldrich) and hydrophobic PTMO (Mn ¼ 2000; Aldrich) in the presence of hexamethylene diisocyante as reported previously [24]. A SPU, Pellethanes 2363-80AE (Dow chemical), was used as a control after precipitating SPU solution (10 wt% in dimethyl acetamide) in methanol, followed by drying for two days at 501C in a vacuum. As shown in Fig. 1, the copolymer contains an equimolar amount of PEO and PTMO, which forms physically crosslinked hydrogel in an aqueous environment, while a SPU consists of PTMO as a soft segment and 4,40 -methylenediphenylene
Pellethane ® (SPU)
O O
H
H
N
N
CH2
x
H CH2
O
O
4
H
N
O
O
N CH2 O
O
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Soft segment
PEO2kPTMO (multiblock copolymer) H N
O O
x
CH2
O
Hydrophilic block
H N 6
O O O
yO
O N
CH2
H
6N
H
z
Hydrophobic block
Fig. 1. Chemical structure of SPU (Pellethanes) and copolymer additive (PEO2kPTMO).
y
z
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diisocyanate connected with 1,4-butane diol as a hard segment, which forms phase-separated thermoplastic elastomer. 2.2. Film preparation A SPU was dissolved in dimethyl acetamide to give a 10 w/v% solution and the various amounts of the copolymer at 5, 10, 20 and 30 wt% (based on dried blends) were added. The homogeneous polymer solutions were poured into clean petri dishes and the films were prepared by evaporating the solvent at 501C for 2 days in a vacuum. The thickness of films prepared was adjusted to 200 mm by the amounts of the poured polymer solution and they were cut into circular shape (12 mm in diameter) for further experiments such as for the swelling property and bacterial adhesion. The SPU films containing the copolymer additive were designated as a Pell-X ; where X means the weight percent of the copolymer based on the dried blend. 2.3. Catheter coating A urinary catheter, silicone-coated two-way foley catheter (16 Fr, Rusch, USA), was used after being treated with ethanol for 30 min to remove any additives or low-molecular weight components. To prepare blend solutions for catheter coating, desired amounts of SPU and copolymer additive were dissolved in tetrahydrofurane (THF, HPLC grade, J.T. Baker) with a concentration of 1 w/v%. The catheters were then coated with blend solutions by dipping, which was repeated five times at 10-min intervals to coat catheters evenly. The blend-coated catheters were placed in a clean bench for 1 day at room temperature, followed by being dried for 1 day in a vacuum to remove any residual solvent. 2.4. Swelling behavior The swelling kinetic of the blends was examined by measuring the water capacity as a function of time. The dried films (Wpolymer ) were immersed in PBS (pH 7.4), which was pre-warmed to 371C, and the weights of the swollen polymers (Wswollen ) were measured in the desired periods after removal of the excess surface water by patting the samples with filter paper. The water capacity was then calculated using the following equation: Water capacity; Wc ð%Þ ¼ ðWswollen Wpolymer Þ 100=Wpolymer :
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Collection for Type Cultures and used for the observation of initial bacterial adhesion to the blend surfaces and encrustation test. The day before the experiment, each of the species which had been maintained on a nutrient agar plate was grown for 24 h at 371C in a nutrient broth (Difco Chemical) solution. The bacterial inoculum was then washed twice by centrifuging in PBS (pH 7.4). After being resuspended in PBS, the cell density was adjusted to 1 108/ml by dilution using a PBS. These resulting inoculums were directly used for the bacterial adhesion tests. Of the species studied, P. mirabilis inoculum was further used for encrustation test since it is known to be urease-producing bacteria accelerating the deposition of rigid crystals onto the catheter lumen surfaces. 2.6. Bacterial adhesion test Before the bacterial adhesion experiment, the polymer films were exposed to a 40 W UV irradiation using a mercury-vapor UV lamp (Sangwoo Inc., Korea) in a clean bench, where a distance between films and lamp was 60 cm. After exposing to UV light for 2 h, the films were hydrated for 1 day in PBS, immersed into a bacterial suspension (10 ml), and incubated for 2 h at 371C. After incubation, the films were gently rinsed three times with 10 ml sterile PBS to remove weakly adhered bacteria. The resulting films were vortexed and sonicated for 2 min, respectively, with 10 ml of PBS containing 0.1% Tween-80 to detach bacteria adhering to the surfaces, as suggested by Flemming et al. [26]. The number of adherent bacteria was quantitatively determined by colony counts and expressed as the number of colony-forming units (CFU) per square centimeter. 2.7. Encrustation tests 2.7.1. Artificial urine The composition of the artificial urine, shown in Table 1, was similar to that described by Tunney et al. [27] except for the use of nutrient broth and P. mirabilis instead of urease. Four solutions were separately prepared to prevent bacterial growth and brushite (CaHPO4 2H2O) formation. Before the experiments, 500 ml each of compounds A and B were homogeneously mixed and compound C (50 ml) was added to supply minimal nutrients to P. mirabilis, urease-producing bacterium, during the encrustation tests [14]. After being filter-sterilized using a cellulose acetate membrane (0.2 mm, Corning, USA), the artificial urine was applied for further experiments within 2 h.
2.5. Bacterial strains and culture conditions E. coli (ATCC No. 11775), P. mirabilis (ATCC No. 25993), and Staphylococcus epidermidis (S. epidermidis, ATCC No. 12228) were obtained from the Korean
2.7.2. Artificial bladder model and intraluminal encrustation A model for the evaluation of encrustation on the catheter surface, mimicking the human bladder, was
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Table 1 Composition of artificial urine used in this studya Compound A (g/l)
Compound B (g/l)
Compound C (g/l)
Compound D
Magnesium chloride hexahydrate (3.6) Potassium dihydrogen orthophosphate (7.6) Urea (16)
Calcium chloride hexahydrate (5.3) Chicken ovalbumin (2)
Nutrient broth (8)
Suspension of P. mirabilis (1 108 cell/ml in PBS)
a
Compounds A, B, and C were dissolved in distilled water.
composed of a cylindrical glass flask (5 cm in diameter and 10 cm in height) similar to that designed by Stickler et al. [8], a peristaltic pump, and a drainage bag (Fig. 2). The catheters coated with SPU and blends were inserted into the autoclaved flask and inflated using a polystyrene syringe to fix the position like a catheterizaion in the human body. With a peristaltic pump, the sterile urine was supplied to the flask at a rate of 0.7 ml/min, which made 30 ml of residual volume below the level of the catheter eyelet. The artificial urine in the flask was then inoculated by adding 300 ml of compound D to achieve a cell density of B1 106/ml. This artificial bladder model was maintained for 1 h to acclimate P. mirabilis to a new environment and to stimulate contamination for the initiation of encrustation. The supply of sterile urine was then switched on and monitored until the artificial urine became impassable through the catheter due to intraluminal blockage by encrustation. Data regarding lumen patency were statistically analyzed using a Student’s unpaired t test with a 95% confidence level (po0:05) and were reported as mean7standard deviation.
0.7 ml/min
Peristaltic pump
Bacterial inoculum
Water (37 oC)
Encrustation
Sterile urine
2.8. Scanning electron microscopy To evaluate the surface topologies of the catheters before encrustation test, the catheter surfaces were observed by using a scanning electron microscopy (SEM, JSM-5800, JEOL) after being gold-coated with a sputter coater (SPI-MODULEt, SPI sppl). With an identical method, the surfaces of the polymer films, prepared in Section 2.2, were observed before the experiments such as bacterial adhesion and swelling behavior tests. Encrustation on the catheter surfaces, retrieved immediately after blockage from the model and then gently rinsed in distilled water to remove threads of albumin [22], was observed using a low-vacuum SEM (Hitachi, S-2460N) after being gold-sputtered with a ion sputter (Hitachi, E1010) as suggested by Morris et al. [28].
3. Results The swelling kinetics for the blends at 371C in PBS (pH 7.4) are shown in Fig. 3. All the blends demon-
Drainage bag Fig. 2. Artificial bladder model for encrustation study.
strated time-dependant water absorption with initial rapid swelling followed by sluggish equilibrium, which was mainly ascribed to the presence of a hydrophilic PEO block in the copolymer additive. The water capacity was proportional to the copolymer content in the blends, indicating that the increase in the copolymer content enhanced the hydrophilicity of the blends [25]. The results also showed that the equilibrium swelling of the blends, in which the Wc values ranged from 3.05 (Pell-5) to 17.54 (Pell-30), was attained within 2 h. The polymer films, observed by SEM, exhibited uniform and smooth surfaces without considerable surface irregularities, allowing this study to evaluate the effect of surface chemistry on bacterial adhesion. To
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Attached cells (X102 CFU/cm2)
20
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10
5
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0 0
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ll-
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U
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12000 10000 8000 6000 4000 2000
ll 30 Pe
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0
ll-
observe the effects of the copolymer additive on the adhesion of bacteria, relevant to infection and encrustation during catheterization, bare SPU and the blends were treated with inoculums containing three different species. All bacteria including E. coli, S. epidermidis, and P. mirabilis were found to adhere to the bare SPU surfaces much greater than to the blend surfaces (Fig. 4), a small amount of copolymer additive as low as 5% significantly improved the resistance to bacterial adhesion (e.g., the number of adherent E. coli to bare SPU was 275731 102 CFU/cm2 but that to Pell-5 was 83713 102 CFU/cm2, respectively). On all the polymeric surfaces, the adhesion numbers of S. epidermidis were consistently higher than those of E. coli and P. mirabilis, where the differences were often larger than a factor of 10. It was also revealed that the number of adherent bacteria decreased gradually with increasing the copolymer content in the blends, which thus resulted in the lowest bacterial adhesion to Pell-30 containing the highest amount of copolymer additive among the blends studied. The greatest effect of the copolymer additive was observed in S. epidermidis adhesion, while the smallest effect was in P. mirabilis, e.g., the numbers of adherent S. epidermidis and P. mirabilis to Pell-5 were 88.7% and 33.5% lower than those adhering to bare SPU, respectively. After being coated with SPU or the blends, the surface topologies of the urinary catheters were observed using a SEM. Fig. 5 shows the representative pictures for each of the outer surfaces before and after catheter coating. All the surfaces had different topologies, in which the uncoated catheters showed a large quantity of surface irregularities with deep cracks, pits and fissures. A catheter coated with SPU showed a
Attached cells (X102 CFU/cm2)
Fig. 3. Swelling behavior of copolymer/SPU blends as a function of time: (J) Pell-5; (&) Pell-10; (W) Pell-20; (X) Pell-30.
Fig. 4. Bacterial adhesion to copolymer/SPU blend surfaces: (a) E. coli, (b) P. mirabilis, (c) S. epidermidis. The error bar is for standard deviation (n ¼ 5).
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Fig. 5. Scanning electron micrographs of urinary catheter surfaces coated with SPU and copolymer/SPU blends.
30 †‡
25
Lumen patency (hours)
†‡
†‡
20 †‡
15
10
5
Pe ll 20 Pe ll 30
ll 10 Pe
ll5 Pe
SP U
lic
on e
0
Si
smoother surface with the occasional presence of a small size of pits (0.1–0.5 mm). The blend-coated catheter surfaces, however, were relatively even and much smoother without significant cracks or pits although they showed rippled characteristics, indicating that the surface roughness of the uncoated catheters could decrease remarkably via coating with the blends. The artificial bladder model, which contained artificial urine inoculated with P. mirabilis, allowed the catheters to be blocked, primarily attributed to the encrustation proceeded by the deposition of magnesium ammonium phosphate and calcium phosphate onto the catheter lumen surfaces [27]. As shown in Fig. 6, none of the catheters were capable of resisting encrustation by P. mirabilis. A catheter coated with silicone, a control sample, required the least time for blockage (7.872.8 h), which was prolonged by coating with SPU (11.372.0 h) but without statistically significant difference (p > 0:05). It was noteworthy that the duration of lumen patency could be further extended by coating the catheters with the blends, e.g., the time for blockage significantly increased to 14.871.2 h by Pell-5, approximately double the period compared to that for a silicone-coated catheter. The maximal duration of lumen patency could be achieved when coated with Pell-10 (20.373.1 h), which showed a comparable period to those for Pells-20 and 30. The cross-sections and intraluminal surfaces were observed using SEM along the length of the catheters blocked by encrustation. Similar results for all the catheters studied were observed and the representative pictures are shown in Fig. 7 for the silicone-coated catheter. The results imply that the encrustation was dependent on the position along the catheter length, where the most extensive encrustation was observed in the region just below the eyehole. The quantity of crystal
Fig. 6. Duration of lumen patency of urinary catheters coated with SPU and copolymer/SPU blends (The error bar is for standard deviation, n ¼ 3). w, z, located above the bars, indicate significantly different values at the 95% confidence level, po0.05, when compared to silicone and SPU, respectively.
deposits, composed of struvite and hydroxyapatite, gradually reduced as the distance from the catheter tip increased, which was also observed by Morris et al. [28].
4. Discussion The use of indwelling urinary catheters involving prolonged contact with urine has been limited due to
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Fig. 7. Low-vacuum scanning electron micrographs of cross-sections of silicone-coated urinary catheters retrieved after blockage in the bladder model: (a) 1 cm, (b) 5 cm, (c) 10 cm from the catheter tip, (d) and (e) are intraluminal surfaces at 5 and 10 cm from catheter tip, respectively.
associated problems such as bacterial adhesion [9,10] and encrustation [11,12], resulting in biomaterial-centered infection [4,5] and urethral stricture [12], respectively. In this study, we explored bacterial adhesion to and encrustation behavior on the multiblock copolymer/ SPU blend-coated urinary catheter surfaces to evaluate their suitability for long-term catheterization. The multiblock copolymer was composed of hydrophilic PEO and hydrophobic PTMO blocks, forming physically crosslinked hydrogel via preserving a threedimensional structure in an aqueous environment [24]. When this copolymer was blended with SPU, a small amount as low as 5% based on the dried blend was able to significantly improve surface hydrophilicity and reduce protein adsorption and platelet adhesion without considerable deterioration in physical properties [25]. This was due to the achievement of stable PEO-rich surfaces, attributed to the high molecular weight and compatible structure of the copolymer with SPU. Along this line of research, by simple and convenient dipping method using a volatile polymer solution in THF, the copolymer/SPU blends were coated on the catheters to allow the surfaces to be PEO rich. It is known that the time taken to reach equilibrium swelling in distilled water for chemically crosslinked hydrogel is at least 10 h [29–31] and its swelling is known to be mediated by the hydration of a polymer chain, followed by the diffusion of water molecules to the gel matrix [32]. The swelling kinetic and degree of equilibrium swelling for chemically crosslinked hydrogel are determined by the hydrophilicity of the polymer, the amount and nature of the cross-linking agent, and the degree of dilution prior to polymerization. In the case of
physically crosslinked hydrogels, which often maintain their physical stability by aggregates of hydrophobic blocks associated with hydrophobic interaction, equilibrium swelling can be achieved within 4 h, depending on the type of hydrophobic block, due to the rapid response of a hydrophilic block such as PEO [33,34]. The copolymer itself, used in this study, also reached equilibrium swelling (Wc ¼ 114) within 20 min, where the flexible and amorphous hydrophobic block, PTMO, allows the PEO chain to be hydrated rapidly in an aqueous environment. The retarded period for equilibrium swelling in the blends, which was approximately 2 h, seemed to result from the restricted mobility of the PEO chain, attributed to the compatibility of the copolymer with the SPU matrix [25]. The low water capacity of the blends, which was approximately half of the expected value judging from the copolymer content, indicated that the hydration of the copolymer was inhibited by the intra or intermolecular interactions with the SPU chain such as van der Waals force and hydrogen bonding (Fig. 3). Bacterial adhesion to biomaterial surfaces is believed to be based both on physicochemical interactions (phase one) and on molecular and cellular interactions (phase two) between bacteria and material surfaces [35]. The latter is the process that bacteria adhere firmly to the biomaterial surface by the bridging function of the polymers releasing from or existing on a bacterial surface including capsules, fimbriae, and slime. Before phase two, the bacteria may attach themselves to the biomaterial surface via shot- or long-range interactions (phase one) such as electrostatic attraction, van der Waals force, hydrophobic interaction, or hydrogen
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bonding [36]. Of the physicochemical interactions, hydrophobic adsorption appears to be the most important since most bacterial surfaces contain some degree of hydrophobic moiety [10,37]. The lower adhesions of all the bacteria to the blend surfaces, compared to bare SPU, also suggest that hydrophilic surfaces can effectively resist bacterial adhesion (Fig. 4). The gradual decrease in bacterial adhesion, proportional to the copolymer content, can be more evidence of the correlation between hydrophilicity and bacterial adhesion since the water capacity, related to the hydrophilicity, increased with the copolymer content, as discussed above. These effects were also observed by Cook et al. [38], who synthesized hydrogels varying in water content from 33 to 69 wt% and demonstrated that P. aeruginosa adhered less to hydrogels with higher water contents. However, it should be considered that the bacterial adhesion depends not only on the degree of water absorption but also on the functional groups exposed on the biomaterial surfaces [35]. Among the bacteria studied, E. coli and S. epidermidis were representative uropathogens, relevant to the catheter-associated infections [39], while P. mirabilis was the predominant microorganism observed in encrusted catheter biofilms [13]. It is known that urease-producing species, including Proteus vulgaris, P. mirabilis, and Providencia rettgeri, elevate the pH of the urine through hydrolysis of the urea to produce ammonia [14,40]. In the resulting alkaline environment, magnesium and calcium ions readily precipitate and deposit on the catheter surfaces in the form of struvite (NH4MgPO4 6H2O) and poorly crystalline hydroxyapatite (Ca10(PO4)6(OH)2) [15,16]. Recently, the ability of P. mirabilis to swarm over catheters has also been demonstrated by Stickler et al. [41], which may enhance the adhesiveness on and allow the migration over the catheter surfaces because the swarmer cells secrete slime and move rapidly over surfaces [42]. Therefore, it can be suggested that the low adherence of P. mirabilis to the blend surfaces provides the potential to delay the onset of encrustation since it is an event subsequent to bacterial adhesion and colonization. There has been controversy with respect to the effectiveness of hydrogel-coated urinary catheters on resistance to encrustation [11,14,20–23]. Even though various factors affect these inconsistent results, the different surface chemistries of hydrogels employed in each study should play a critical role since encrustation is a phenomenon occurring on the catheter surface. The surface morphologies of the catheters attributed to the methods of manufacture could be another possible reason [43,44]. In this study, the simple dip-coating method provided smooth surface of the blend on the catheters (Fig. 5). This fact allows this study to evaluate the effects on surface chemistry and hydrophilicity. As shown in Fig. 6, a small amount of copolymer content as
low as 5% could increase the time for blockage, which could be further extended by 10% copolymer content. This implies that PEO-rich surfaces, produced due to the migration of the PEO phase in the copolymer chain of the blends when exposed to artificial urine, can effectively resist encrustation. Superior resistance to encrustation of PEO surface was also demonstrated by Gorman et al. [44] who evaluated comparative encrustation on Aquavenes (PEO/SPU composite hydrogel), SPU, and silicone. It is also interesting to note that the addition of a large amount of the copolymer (>10%) cannot increase the duration of lumen patency any more, indicating that hydrophilicity is not always a critical factor for encrustation. For long-term catheterization, urinary catheters should not vary significantly in terms of surface hydrophilicity and mechanical properties to preserve the repellency of bacterial adhesion and prevent catheter fracture, respectively. In a previous report [25], we demonstrated that the mechanical properties of the blends maintained up to 10% addition of the copolymer compared to those of SPU even though surface hydrophilicity was significantly improved. The blends also showed minimal extraction of the copolymer additive when exposed to a buffer solution over 2 months; o1% weight loss of the dried polymer occurred, indicating the creation of permanent PEOrich surfaces. These results also suggest that SPU/ copolymer blends are suitable as coating materials for the catheters. As an overview, urinary catheters coated with blends, especially containing 10% of copolymer additive (Pell-10), might be promising for long-term catheterization, based on better resistance to the bacterial adhesion and encrustation in vitro compared to SPU and silicone-coated catheters.
5. Conclusion The SPU/copolymer blends were assessed for application as coating materials onto the urinary catheters. The blend films adsorbed a significant amount of water, which was proportional to copolymer content. From the bacterial adhesion tests, it was demonstrated that the blend surfaces effectively suppressed the adhesion of E. coli, S. epidermidis, and P. mirabilis. The catheter surfaces coated with blends were much smoother and relatively even without significant surface irregularities, compared to silicone- and SPU-coated catheters. It was also observed that the catheters coated with blends showed good resistance to encrustation, which was maximal when the copolymer content was higher than 10%. These in vitro results of the urinary catheter coated with blends provide promising potential for longterm catheterization without serious infection and encrustation.
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Acknowledgements This study was supported by Grant No. HMP-98-G2-034 from the Ministry of Health and Welfare, Korea.
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