Urethral in situ biocompatibility of new drug-eluting biodegradable stents: an experimental study in the rabbit

Authors


Andres Kotsar, Tampere University Hospital, Department of Urology, Teiskontie 35 Tampere 33521, Finland.
e-mail: andres.kotsar@uta.fi

Abstract

OBJECTIVE

To assess the effect of drug-eluting properties on the degradation process and the biocompatibility of biodegradable drug-eluting urethral stents.

MATERIALS AND METHODS

Braided biodegradable 80 L/20D-PLGA (copolymer of polylactide and polyglycolide) stents with drug-eluting properties were used as the test material. The drugs analysed were indomethacin, dexamethasone and ciprofloxacine. 80 L/20D-PLGA stents without a drug coating served as controls. In all, 16 male rabbits were used and divided into four groups. The stents were inserted under general anaesthesia into the posterior urethra. After 1 month, the rabbits were killed and the urethra removed for histological and optic microscopy analyses.

RESULTS

Control stents and the dexamethasone-eluting stents degraded totally during the follow-up period. Conversely, in both indomethacin- and ciprofloxacine-eluting stent groups, the degradation process was significantly delayed and they induced an increase in epithelial hyperplasia. Histological analysis showed that all the stents induced eosinophilia, but there were no significant differences in the intensity of acute or chronic inflammatory reactions and fibrosis.

CONCLUSIONS

A drug-eluting capacity can be added to biodegradable stents. The addition of a drug influences the biodegradation time of PLGA urethral stents. Further studies are needed, to find the proper concentrations and releasing profiles of the drugs to achieve the desired bioactivity and biocompatibility properties.

Abbreviations
PLGA

copolymer of polylactide and polyglycolide acid

PLA

polylactide

PGA

polyglycolide.

INTRODUCTION

Internal urethrotomy is the primary treatment for urethral strictures, but 46–76% recur within 2 years and their treatment involves an even greater risk of further recurrences [1]. The essential problem is how to prevent the edges of the cut stricture from adhering together and the scar from shrinking [1]. Many patients do not accept the option of repeated self-catheterization to maintain a satisfactory urethral lumen after internal urethrotomy [2]. To solve the problem of recurrence, permanent self-expanding metallic stents [3], temporarily placed biocompatible metallic stents [4] and polyurethane temporary stents [5] have been introduced for the treatment of urethral strictures. However, problems with epithelial hypertrophic proliferation, stent encrustation and difficulties with removal have been associated with the permanent and temporary endoscopically placed urethral stents used to date [2].

A biodegradable self-reinforced poly-l-lactic acid stent with helical spiral configuration was developed to avoid these problems [1,6,7]. However, its clinical use was limited by the risk of a sudden breakdown of the helical spiral configuration of the stent, leading to transient obstruction of the lower urinary tract and migration of the stent from its position [8,9]. To overcome these problems a new tubular mesh configuration for the biodegradable stent was recently developed by our group [10]. However, animal studies showed that the new configuration did not affect in itself the amount of epithelial hyperplasia and reactive inflammation caused by tissue trauma. This gave impetus to the development of a drug-eluting biodegradable urethral stent [11].

Drug-eluting stents have already been used for several years in interventional radiology of coronary arteries and intracranial arteries. The development of drug-eluting stents is one of the major revolutions in coronary artery disease, as the re-stenosis rate has been significantly reduced in comparison with bare metal stents [12]. Elective intracranial stenting with drug-eluting stents appears similarly to be feasible and safe [13]. The ideal drug for use in the prevention of urethral stricture recurrence should have an antiproliferative effect as well effectively inhibiting the inflammatory response induced in the tissue by the internal urethrotomy. The potential drugs of choice were estimated to be anti-inflammatory or anti-microbial. We chose the most cost-effective alternative in each drug group, and the in vitro drug-release profiles of selected drugs were tested first.

The aim of this pilot study was to evaluate the effects of indomethacin, dexamethasone and ciprofloxacine on the biodegradation and biocompatibility of braided PLGA [a copolymer of polylactide (PLA) and polyglycolide (PGA)], stents in the rabbit posterior urethra, and thus to predict whether these drug-eluting stents could be a subject for further research to prevent the repeated recurrence of urethral strictures in the future.

MATERIALS AND METHODS

The biodegradable braided 80 L/20D-PLGA stents were designed and manufactured at the Institute of Biomaterials, Tampere University of Technology, Finland. The PLA/PGA polymer ratio in the material was 80:20; the material was obtained from PurAc Biochem b.v., Groeningen, the Netherlands. The stent filament was first melt-spun using a single screw extruder (Extrudex, Mühlacker, Germany), then the internal orientation of the filament fibrils was achieved by drawing in a three-step process, obtaining a final filament diameter of 0.3 mm. The stents were manufactured by braiding 16 melt-spun 80 L/20D-PLGA monofilaments on a metallic mandrel (diameter 6 mm) in a one-over-one pattern using a 32 carrier braiding machine (Pick Master). The structure was stabilized by thermal treatment and the stents were cut from the braid to a length of 30 mm.

The stents were coated by immersion in a solution containing rasemic 50 L/50D-PLA and one of the three drugs, indomethacin, dexamethasone or ciprofloxacin. Stents coated with pure 50 L/50D-PLA solution served as controls. Acetone was used as a solvent in the coating solutions. The stents were dipped into the solution on a mandrel, withdrawn and air-dried, and the procedure repeated until a sufficiently thick coating was achieved (Table 1). Finally, the stents were vacuum-dried to evaporate the acetone and sterilized using γ-irradiation at a dose of 25 kGy at 42 °C.

Table 1.  Drug variables of the stent coatings
Stent coatingMean (sd) drug load in stent, mgDrug in coating, weight %
50 L/50D-PLA +:
 Indomethacin2.2 (0.3)32
 Dexamethasone2.2 (0.5)29
 Ciprofloxacin1.1 (0.1)25

All animal protocols were reviewed and approved by the Institutional Committee for Animal Research and by the Western-Finland Provincial Government. The investigation conformed to the Guide for Care and Use of Laboratory Animals published by the USA National Institute of Health. In all, 16 male New Zealand White rabbits were used and divided into four groups. The rabbits were anaesthetized with medetomidine hydrochloride 0.3 mL/kg i.m. (Domitor® 1 mg/mL, Orion Pharma, Finland) and ketamine hydrochloride 0.3 mL/kg i.m. (Ketalar® 50 mg/mL, Pfizer, USA). All rabbits before surgery received a single dose of orofloxacin 5 mg/kg s.c. (Baytril vet.® 100 mg/mL, Bayer, Germany) for antibacterial prophylaxis.

The full length of the rabbit urethra was dilated with a Hegar probe to a diameter of 6 mm and the stents inserted into the prostatic urethra with a specially designed delivery system consisting of a simple metallic tube and piston. The outer diameter of the insertion device was 5 mm. The stent was packed into the tube immediately before insertion and released with the piston into the prostatic urethra immediately proximal to the external sphincter. Localization was then assessed endoscopically using a 13 F paediatric cystoscope.

The rabbits, four in each group, were killed after 1 month using medetomidine hydrochloride 0.3 mL/kg i.m. and ketamine hydrochloride 0.3 mL/kg i.m. as sedation, and an overdose of pentobarbital sodium i.v. (Mebunat® 60 mg/mL, Orion Pharma, Finland). The urethra surrounding the stent was dissected from the rabbits en bloc. Representative samples, cross-sections and sections taken perpendicular to the wall of the urethra were taken for histological analysis. The tissue blocks were fixed in 10% formalin and embedded in paraffin, and sections cut and stained with haematoxylin and eosin following routine techniques.

The biodegradation process in the stent and the development of epithelial hyperplasia (polyposis) were evaluated by optic microscopic analyses. The amount of polyposis was scored semiquantitatively in a range of 0–3. All histological analyses were performed ‘blinded’ by an experienced pathologist (P.M.). The biological response variables assessed and recorded included: eosoniphilia, acute inflammatory changes (polymorphonuclear leucocytes), chronic inflammatory changes (lymphocytes, plasma cells) and the amount of fibrosis. Tissue reactions in the variables analysed were scored semiquantitatively on a scale of 0–3 as ‘no’, ‘mild’, ‘moderate’ and ‘marked reaction’.

RESULTS

One of the control stents with pure 50 L/50D-PLA coating was located in the seminal vesicle and was omitted from the analyses. In the optic microscopy analysis all the other three control stents had degraded at 1 month, as had three of the four dexamethasone-eluting stents. In the indomethacin- and ciprofloxacine-eluting stent groups, the tubular mesh configuration of the stent was still unbroken in three of the four rabbits (Fig. 1) (Table 2). The polyposis was equal in the pure PLA-coated and the dexamethasone-eluting stents (Fig. 2), whereas the indomethacin- and ciprofloxacine-eluting stents induced slightly more polyposis.

Figure 1.

Epithelial polyposis and indomethacin-eluting stent structure at 1 month.

Table 2.  Optic microscopy analyses of tissue reactions induced by indomethacin, dexamethasone or ciprofloxacin blended and unblended (control) 50 L/50D-PLA-coated 80 L/20D-PLGA stents at 1 month. The mean values for polyposis (range 0–3) and the degradation process of the stents
Stent coatingMean polyposis values (range 0–3)Degradation, n/N
Indomethacin1.3Configuration unbroken 3/4
Dexamethasone0.3Degraded 3/4
Ciprofloxacine1.0Configuration unbroken 3/4
Control0.3Degraded 3/4
Figure 2.

Epithelial polyposis induced by dexamethasone-eluting urethral stent at 1 month.

Upon histological analysis of the tissue reactions, all three different drug-eluting stent groups induced slightly more eosinophilia than the controls. However, there were no significant differences in the intensity of acute or chronic inflammatory reactions and fibrosis (Table 3).

Table 3.  Mean values of histological tissue reactions induced by the 80 L/20D-PLGA stents at 1 month, scoring semiquantitatively, range 0–3
Stent coatingMean value (range 0–3) of histological tissue reactions
Acute inflammationChronic inflammationFibrosisEosinophilia
Indomethacin0.30.50.52.0
Dexamethasone0.300.31.3
Ciprofloxacine0.50.50.32.0
Control00.30.30.5

DISCUSSION

After surgical treatment of urethral stricture, catheterization or stenting is useful in supporting the repair and achieving urinary drainage. Previous work on rabbits has shown that re-epithelization occurs within days of superficial mucosal injury, while 7–10 days are necessary for a strong mucosal bridge [14]. Healing characteristics are affected by factors such as vascularization and the presence of inflammatory agents with or without infection. Among other factors urine may pose such an inflammatory risk and lead to unpredictable scar formation. The use of drugs eluting from urethral stents could offer a means of overcoming these risks of scar recurrence. In the present study, three different drugs were selected to represent anti-inflammatory and anti-microbial agents added to the stents for local drug delivery, the aim being to test their biocompatibility as well as to assess their affect on the stent degradation process.

In assessing the degradation process of the stents and epithelial polyposis we used optical microscopy analyses. All pure PLA-coated stents (one omitted from analyses due to incorrect location) were degraded at 1 month after placement, as well as in three of the four dexamethasone-eluting stents. In the indomethacin- and ciprofloxacine-eluting groups, the tubular mesh configuration of the stent was still unbroken in three of the four rabbits. It is thus clear that the drugs affected the stent degradation time in vivo. We speculate that slowing down of the degradation process of the indomethacin- and ciprofloxacine-eluting stents could be explained by the hydrophobic nature of these drugs. Dexamethasone is more hydrophilic than the other drugs tested. The hydrophobicity of these two drugs may prevent absorption of water into the stent. Water access between orientated PGA or PLA filaments inside the stent is an essential step in biodegradation.

Although intraurethral stents are generally well tolerated, epithelial overgrowth follows placement of the device and fibrosis of this overgrowth is the most common cause of stricture formation. The amount of polyposis in the present study was equal in the pure 50 L/50D-PLA-coated stents and the dexamethasone-eluting stents, whereas the indomethacin- and ciprofloxacine-eluting stents induced slightly more polyposis. Dexamethasone might thus be thought to be the drug of choice for further research. However, the reason for the slightly increased polyposis in the other drug groups could also be the concentration chosen in the present study. Even more important in the indomethacin- and ciprofloxacine-eluting stent groups, the stent structure was still unbroken at 1 month after placement, causing mechanical irritation and thus more polyposis.

Histological analyses showed that the drug-eluting stents induced slightly more eosinophilia than the controls. This is a normal finding considering that the time of this analysis was quite early after stent placement, and we can therefore presume that the eosinophilia would decrease or disappear with time.

The significance of eosinophilia in biocompatibility testing is also controversial. From the standpoint of biocompatibility it was important that there were no significant differences in the amount of inflammatory reaction or fibrosis between the test groups. We may consider that all the materials used proved to be highly biocompatible and potential candidates for further investigation. It is important to note that in the present study the test was carried out on uninjured posterior urethra, whereas the effect of these bioactive stents in a strictured urethra could be different.

The focus of further research is to establish whether it is possible to affect the re-stricture formation process with these drugs; the key issue being the appropriate concentration and release of the drug. There is also a need to test other drugs with antiproliferative activity.

The present results showed that all the tested materials were highly biocompatible and that the drug-eluting property can be safely added to biodegradable stents. In the aim to reduce epithelial hyperplasia, tests with different drug concentrations are ongoing. The drugs affected the biodegradation time of braided 80 L/20D-PLGA urethral stents in vivo. To ascertain the appropriate concentrations and releasing profiles of the drugs further studies are needed to establish desired bioactivity and biocompatibility properties.

CONFLICT OF INTEREST

None declared. Source of funding: the National Technology Agency of Finland (TEKES) and the Medical Research Fund of Tampere University Hospital.

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