Evaluation of the biocompatibility of a coating material for an implantable bladder volume sensor

Abstract

As the applications for implantable medical devices have increased, the need for biocompatible packaging materials has become important. Recently, we reported an implantable sensor for real‐time monitoring of the changes in bladder volume, which necessitated finding a safe coating material for use in bladder tissue. At present, materials like polyethylene glycol (PEG), polydimethylsiloxane (PDMS) and parylene‐C are used in biomedical devices or as coating materials, owing to their excellent safety in various medical fields. However, few studies have assessed their safety in bladder tissue, therefore, we evaluated the biocompatibility of PEG, PDMS and parylene‐C in the bladder. All three materials turned out to be safe in in vitro tests of live/dead staining and cell viability. In vivo tests with hematoxylin and eosin and immunofluorescence staining with MAC387 showed no persistent inflammation. Therefore, we consider that the three materials are biocompatible in bladder tissue. Despite this safety, however, PEG has biodegradable characteristics and thus is not suitable for use as packaging. We suggest that PDMS and parylene‐C can be used as safe coating materials for the implantable bladder volume sensor reported previously.

Introduction

The development of implantable medical devices has led to improvement in the treatment of patients with chronic diseases. For example, an implantable medical device such as a pacemaker can control arrhythmia that does not respond to medical treatment. Such devices can improve the quality of life. Therefore, great efforts have been made to invent implantable medical devices in various medical fields including urology.

Neurogenic bladder is one of the voiding dysfunctions caused by disorders of the nervous system, such as cerebral attack or hemorrhage, spinal cord injury, and diabetes mellitus. In these patients, monitoring of intravesical pressure is important for protecting renal function, therefore, some studies have reported the use of implantable devices for monitoring intravesical pressure or changes of the bladder in animals [[1], [2], [3]].

Before an implantable device can be used clinically, not only efficacy but also safety should be evaluated. Generally, materials like polyethylene glycol (PEG) and polydimethylsiloxane (PDMS) are used to coat the implantable device safely [[4], [5]]. In addition, parylene‐C has recently been suggested as another safe coating material [6].

We previously have reported on the usefulness of a bladder volume sensor in small animals [3]. For this sensor to be used in clinical applications, appropriate, safe packaging materials for the bladder volume sensor are necessary. Materials such as PEG, PDMS and parylene‐C can be used to coat the implantable device: however, few studies have evaluated their safety as coating materials in bladder tissue.

Accordingly, in the present study, we evaluated the safety of PEG, PDMS and parylene‐C in rabbit bladder.

Materials and methods

Study design

The research was conducted in accordance with the National Institutes of Health guidelines on care and use of laboratory animals, and was approved by the Catholic Animal Ethics Committee (Catholic University Medical College 2010‐0115‐03).

Preparation of coating materials

We investigated the biocompatibility of three coating materials: PEG, PDMS and parylene‐C. A coin‐shaped piece of aluminum was coated with PEG, PDMS or parylene‐C. Coin‐shaped pieces of aluminum without packaging were used as controls (n = 3).

Coating with PEG, PDMS or parylene‐C

PEG solution was prepared by using 99% (by wt%) PEG (molecular weight 258; Aldrich) and 1% (by wt%) hydrophilic 2‐hydroxy‐2‐methylpropiophenone photoinitiator (Aldrich) [7]. The aluminum coin was then immersed in the PEG solution (molecular weight 258; Sigma‐Aldrich, St. Louis, USA) and exposed to 250–400 nm ultraviolet light for seconds.

The aluminum coin was immersed in PDMS solution (Sylgard 184; Dow Corning, Seoul, Korea; silicone elastomer: curing agent = 10: 1), and heated at 80 °C for 2 hours.

Parylene‐C was vaporized at 1 barometric pressure and 150 °C. Even at 0.5 vaporized pressure and 690 °C, the heat degradation reaction was maintained, and at that time, parylene‐C was converted to the monomer para‐xylene. It was polymerized to poly‐para‐xylene at 0.1 barometric pressure and 35 °C, and this was used to coat the aluminum coin. The coating reaction was carried out with the Nuricell Parylene coating system (NPCR‐400; Nuricell, Seoul, Korea), as described previously [8].

Implantation of coated aluminum coins

Twelve New Zealand male rabbits (2.5–3 kg) were used. The rabbits were assigned to three experimental groups of three animals each: PEG, PDMS and parylene‐C. The rabbits were anesthetized by the intramuscular injection of 10 mg/kg xylazine (Bayer Korea, Seoul, Korea) and 25 mg/kg ketamine (Yoo Han Yang Hang, Seoul, Korea). The bladder was then exposed by midline incision, and the aluminum coins coated with PEG, PDMS, or parylene‐C were attached to the external wall of the bladder by use of 4‐0 polydioxanone (Fig. 1). Sham operation was performed in the control group (n = 3) in the same manner.

Figure 1.

Coin‐shaped pieces of aluminum coated with polyethylene glycol, polydimethylsiloxane or parylene‐C were attached to the external bladder wall.

In vitro cytotoxicity test

Live/dead staining

To study the cytotoxicity of the packaging materials, NIH3T3 fibroblasts were used as the model cell. NIH3T3 cells were expanded in T‐175 flasks supplemented with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Packaging materials on 1‐cm discs were fitted into a six‐well tissue culture plate. To evaluate cell viability, NIH3T3 cells were seeded at a density of 10⁵ cells/mL on the surface of the packaging materials. After incubation for a predetermined time, the medium was removed and the packaging materials were washed twice in phosphate‐buffered saline (PBS). Live and dead cells were assessed by use of the LIVE/DEAD Viability/Cytotoxicity Assay Kit (Molecular Probes, Carlsbad, CA, USA). Cultured packaging materials were incubated in assay reagents containing 2 μM calcein AM (labeled live cells) and 4 μM ethidium homodimer‐1 (labeled dead cells). These packaging materials were incubated in a covered dish to prevent drying of the samples for about 45 minutes at 37 °C. The live and dead cell images were taken with an LSM 510 confocal microscope (LSM510 META, Carl Zeiss, Germany).

Cell viability

The cytotoxicity test of the prepared packaging materials was determined by cell counting with trypan blue. In the dynamic condition, an extract of the test sample (PEG, PDMS or parylene‐C) was made by immersing the test sample into DMEM containing 100 U/mL penicillin, 100 g/mL streptomycin, and 10% fetal bovine serum in an incubator at 37 °C in a humidified atmosphere of 5% CO₂ for 24 hours. First, NIH3T3 fibroblasts (10⁶/well) were plated in six‐well plates and incubated for 24 hours. Then, the medium was aspirated and the plates were rinsed twice with PBS. The extract of the test sample was added to each well, and the plates were incubated for 24 hours. The cells detached by trypsin/EDTA were stained with 500 μL 4% trypan blue (Gibco, Carlsbad, CA, USA). The living cells were subsequently counted in a hemocytometer.

In vivo cytotoxicity tests

Hematoxylin and eosin staining

Rabbits were sacrificed after 1, 2 and 4 weeks, and tissues attached to the aluminum coated with PDMS, PEG or parylene‐C were collected, and fixed in 10% neutral formalin for 1 day. For the preparation of the fixed tissues as samples for light microscopy, the tissues were dehydrated with alcohol, embedded in paraffin, sectioned in 5‐μm sections with a microtome, and stained with hematoxylin and eosin (H&E). Histological images of the implantation site were obtained by use of a light microscope at 40× magnification. Neovascularization was observed at 400× magnification. The degree of neovascularization was assessed in a semiquantitative way by using a modified histological scale [9] (Table 1).

Table 1.

Histological scoring scale.

Score	Inflammation	Collagen organization	Vascularity
0	None	Disorganized	None (No vessel/hpf)
1	Minimal–mild	Slightly organized	Minimal (1–3 vessels/hpf)
2	Moderate	Moderately organized	Moderate (4–10 vessel/hpf)
3	Severe	Well organized

Hpf = high‐power field.

Immunofluorescence staining for macrophages

Rabbits were sacrificed and the bladder attached with PEG, PDMA, or parylene‐C – coated aluminum coin of each rabbit was excised after 1, 2 and 4 weeks and then washed with ethanol for 5 minutes. The slides were added to 0.01% sodium citrate (pH 6), boiled in a microwave oven, and cooled for 20 minutes at room temperature. To inhibit nonspecific reactions during staining, the slides were reacted with a blocking solution (1.5% normal goat serum, 1.5% normal horse serum, 1% bovine serum albumin, 0.1% Triton X‐100 in PBS) at room temperature for 1 hour, and were reacted with the primary antibody MAC387 (Abcam, Cambridge, UK) at room temperature for 2 hours. The slides were then washed with PBS three times, and reacted at room temperature for 1 hour with the secondary antibody (Alexa Fluor 568 goat anti‐mouse IgG; Invitrogen, Carlsbad, CA, USA) diluted to 1:500 with the blocking solution. The slides were then washed with PBS three times, and mounted with 4′,6‐diamidino‐2‐phenylindole, and images were obtained with a fluorescence microscope.

Data analysis

The data were analyzed statistically by using one‐way analysis of variance and were expressed as the mean ± standard deviation. The data of each group were compared by using the Newman–Keuls' multiple comparison test. Significance was set at p < 0.05.

Results

In vitro cytotoxicity

As shown by the results of the live/dead staining, the expression of live cells in the PEG, PDMS and parylene‐C groups was similar to that in the controls, and dead cells were rarely observed. Cell viability was >80% in each of the three groups, which was the same as for the controls (Fig. 2).

Figure 2.

In vitro cytotoxicity test. (A) LIVE/DEAD staining. Green: live cell; red: dead cell (scale bar: 50 μm). (B) Cell viability. PEG = polyethylene glycol; PDMS = polydimethylsiloxane.

In vivo cytotoxicity

Histological findings

We observed that the aluminum coins coated with PEG, PDMS or parylene‐C were implanted well in the bladder. H&E staining at 1 week after implantation showed the presence of inflammatory cells resulting from the early inflammatory response (Fig. 3). The formation of new blood vessels was noted in the PEG, PDMS and parylene‐C groups compared with the controls after 1 week. The degree of neovascularization, however, was mild in all three groups (Fig. 4).

Figure 3.

Histological findings at 1 week after implantation. Arrow: implantation site and inflammatory cells, (hematoxylin and eosin, 40×). PEG = polyethylene glycol; PDMS = polydimethylsiloxane.

Figure 4.

(A) Neovascularization at 1 week after implantation. Arrow: formation of new blood vessels near the implantation site (hematoxylin and eosin, 40×). (B) Number of new blood vessels. *p < 0.05 compared with normal tissue. PEG = polyethylene glycol; PDMS = polydimethylsiloxane.

Immunofluorescence findings

Immunofluorescence staining with MAC387 showed mild expression of macrophages in the implantation site in the PEG, PDMS and parylene‐C groups compared with the controls after 1 week. A decrease in expression of macrophages was observed after 2 weeks. At 4 weeks, macrophages were rarely expressed in all three groups (Fig. 5).

Figure 5.

(A) Immunofluorescence staining with MAC387. Red: macrophage; blue: cell nucleus (4′,6‐diamidino‐2‐phenylindole staining), 100×. Arrow: macrophage near the implantation site. (B) Number of MAC387‐positive cells on each packaging material. *p < 0.01. PEG = polyethylene glycol; PDMS = polydimethylsiloxane.

Discussion

The results of the present study showed that PEG, PDMS and parylene‐C seemed to be safe for use as coating materials in bladder tissue. Therefore, these materials could be used to package a pressure or volume sensor in the bladder.

Neurogenic bladder is a voiding dysfunction caused by disorders in the central or peripheral nervous system, such as cerebrospinal disease or injury, diabetes mellitus, or complications after radical pelvic surgery [[10], [11]]. According to the characteristics of the voiding status of the neurogenic bladder, it can be divided into hyperreflexic and underactive or acontractile neurogenic bladder. Of these two types of neurogenic bladder, it is important to monitor changes in bladder volume in patients with underactive or acontractile neurogenic bladder [12]. Usually, these patients cannot recognize distension over their normal bladder capacity. As a result, normal voiding is not able to occur and, moreover, repetition of inappropriate voiding causes deterioration of renal function. We have previously reported development of a real‐time implantable bladder volume sensor that was attached to both sides of the outside wall of the bladder [3]. Undoubtedly, safety is a significant factor in the design of an implantable medical device. Many studies on the safety of PEG, PDMS and parylene‐C as packaging materials in various organs have been done, and the safety of these materials has been demonstrated [[13], [14], [15], [16]]. However, evaluation of safety has rarely been done in the bladder.

According to the present in vitro results with live and dead staining, almost all of the cells in the PEG, PDMS and parylene‐C groups appeared as live cells. Moreover, all of the three materials showed excellent viability by cell culture. This finding is similar to previous research on the safety of these three materials [[17], [18], [19]].

After implantation of a medical device, an inflammatory reaction occurs against the foreign body. In the early phase, the biomaterial‐mediated inflammatory response is modulated by histamine‐mediated macrophage recruitment and adhesion to the implant surface [20]. Next, mononuclear cells such as monocytes and lymphocytes are present at the implant site in the chronic inflammation phase. If the implant device is biocompatible, early resolution of the acute and chronic inflammation responses occurs. The persistence of inflammatory responses for longer than 3 weeks usually indicates an infection [[20], [21]].

In the present study, inflammatory cells were observed only in the area adjacent to the implants, and these changes were not significantly different from the controls, as shown by H&E staining after 1 week. In addition, we noted only mild neovascularization in the area adjacent to the implants coated with PEG, PDMS or parylene‐C, by H&E staining. The formation of new blood vessels was not observed after 4 weeks. These findings suggest that PEG, PDMS and parylene‐C are safe as coating materials in bladder tissue.

According to the results of the immunofluorescent staining with MAC387, a mild increase in the expression of macrophages was noted in the rabbit bladder tissues that were in contact with the coated implants after 1 week. These changes were not significantly different from those in the controls. After 2 weeks, expression of macrophages seemed to decrease, and macrophages were rarely observed after 4 weeks. As previously mentioned, macrophages play an important role in the inflammatory reaction against foreign bodies [[20], [21]]. The decrease in the expression of macrophages after 4 weeks suggests that PEG, PDMS and parylene‐C were not the cause of infection and, therefore, all three materials seem to be biocompatible.

The inflammatory response did not persist in the rabbit bladder tissue in contact with PEG, PDMS or parylene‐C beyond 3 weeks. For these reasons, PEG, PDMS and parylene‐C may all be appropriate packaging materials for the implantable bladder volume sensor that we have reported previously [3].

Among the three materials we used in our study, PEG is not supposed to be suitable as a packaging material because it has biodegradable characteristics [[22], [23]]. Nevertheless, we chose PEG as one of our study materials because of its excellent biocompatibility compared with other polymers. PEG has cationic and hydrophilic characteristics, therefore, it inhibits early and late macrophage adhesion and then inhibits lymphocyte proliferation for longer times [[24], [25], [26]]. Therefore, PEG is considered to have good biomaterial surface chemistry. In the present study, the inflammatory responses of PDMS and parylene‐C were similar to those for PEG; therefore, we conclude that PDMS and parylene‐C may show the same safety profile as PEG in rabbit bladder tissue. Meanwhile, there are some differences between PDMS and parylene‐C. For example, PDMS shows more elasticity than parylene‐C does. In contrast, parylene‐C may have better characteristics with regard to the coating process. Therefore, it is necessary to consider which material is appropriate according to the shape and characteristics of the various implantable sensors in the bladder.

A limitation of the present study was that we evaluated only the short‐term safety of PDMS and parylene‐C coating materials in the bladder tissue. Thus, more studies about the long‐tem safety are necessary to determine the ideal packaging materials for clinical application.

In conclusion, we suggest that PDMS and parylene‐C can be used as biocompatible packaging materials for the implantable bladder volume sensor. For clinical application, further study of the long‐term safety of PDMS and parylene‐C appears to be necessary.

Acknowledgments

This work was supported by the Converging Research Center Program through the Ministry of Education, Science and Technology (2010K001094) and the Healthy Medical Treatment Research and Development Program of the Ministry of Health & Welfare (No. A090481).