Abstract
Objectives
We aimed to achieve detailed biomaterials characterization of a drug delivery system for local periodontitis treatment based on electrospun metronidazole-loaded resorbable polylactide (PLA) fibers.
Methods
PLA fibers loaded with 0.1–40% (w/w) MNA were electrospun and were characterized by SEM and DSC. HPLC techniques were used to analyze the release profiles of metronidazole (MNA) from these fibers. The antibacterial efficacy was determined by measuring inhibition zones of drug-containing aliquots from the same electrospun fiber mats in an agar diffusion test. Three pathogenic periodontal bacterial strains: Fusobacterium nucleatum , Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were studied. Cytotoxicity testing was performed with human gingival fibroblasts by: (i) counting viable cells via live/dead staining methods and (ii) by exposing cells directly onto the surface of MNA-loaded fibers.
Results
MNA concentration influenced fiber diameters and thus w/w surface areas: diameter being minimal and area maximal at 20% MNA. HPLC showed that these 20% MNA fibers had the fastest initial MNA release. From the third day, MNA release was slower and nearly linear with time. All fiber mats released 32–48% of their total drug content within the first 7 days. Aliquots of media taken from the fiber mats inhibited the growth of all three bacterial strains. MNA released up to the 28th day from fiber mats containing 40% MNA significantly decreased the viability of F. nucleatum and P. gingivalis and up to the 2nd day also for the resistant A. actinomycetemcomitans. All of the investigated fibers and aliquots showed excellent cytocompatibility.
Significance
This study shows that MNA-loaded electrospun fiber mats represent an interesting class of resorbable drug delivery systems. Sustained drug release properties and cytocompatibility suggest their potential clinical applicability for the treatment of periodontal diseases.
1
Introduction
For treatment of periodontal disease, there is a need for an optimal local drug delivery system since the widespread systemic administration of antibiotics might cause undesired side effects or favor the development of resistances.
The use of antibacterial biomaterials becomes increasingly important in medical and dental science. Especially in the field of conservative dentistry, the elimination of bacteria and plaque is foundational for effective treatment . For instance, the conventional treatment of periodontitis by scaling and root planning is advantageously accompanied by the adjuvant administration of antibiotics . Antibacterial drug compounds can be applied by systemic or local administration. Compared to systemic drug delivery the local administration of drugs in periodontology is considered to be more effective, since the pathogen-specific drug can be placed directly in the periodontal pocket achieving effective concentrations. In addition the risk of undesired side effects caused by high systemic doses or resistance development can be reduced . For effective elimination of pathogenic bacteria, the antibiotic agent has to be available in the periodontal pocket in adequate concentrations for a sufficiently long period of time. It is therefore necessary to use local delivery systems that control the release of their agents and guarantee lasting drug concentrations in the pocket in spite of high sulcular fluid rates.
Non-resorbable drug carrier systems such as tetracycline-loaded fibers are placed from seven up to ten days in the periodontal sulcus. In this period concentrations up to 1300 μg/ml in the sulcus fluid can be maintained. However, the insertion of such non-resorbable fibers is time consuming and when their removal is required this incurs the risk of tissue damage.
Many resorbable drug delivery systems were developed during recent decades, such as drug loaded hydroxypropylcellulose films , which were first described by Noguchi et al. (1984), or drug carrying gels such as Elyzol ® (Dumex GmbH, Bad Vilbel, Germany) dental gel, based on melted glycerol mono-oleate . However, also for these systems, the periodontal milieu often poses the major problem that the required period of drug exposure (7–10 days) cannot be achieved . Also in the field of periodontal surgery – as in the transplantation of a mucous membrane – resorbability of the scaffold material is important to avoid inflammatory effects and surgical removal.
Therefore the aim of this study was to investigate a resorbable drug reservoir, which releases essential amounts of its ingredients within an adequate period of time. A possible drug delivery system based on electrospinning of polylactide was developed whereby mats of electrospun fibers containing the antibiotic metronidazole (MNA) were generated having a large surface area per volume ratio. Fiber mats incorporating different proportions of MNA (from 0.1 to 40.0%, w/w) were created to investigate release characteristics and determine the concentrations necessary for effective antibacterial action when placed in an appropriate host environment.
2
Materials and methods
2.1
Electrospinning
Poly( l -lactide-co- d / l -lactide) 70/30 (Resomer LR 708, Boehringer Ingelheim, Germany) was used for electrospinning ( Fig. 1 ). A weight-average molecular weight of 1.5 × 10 6 g mol −1 was determined for the polymer by gel permeation chromatography using CHCl 3 as solvent and polystyrene as external standard. All solvents used for electrospinning purposes were of HPLC grade (Sigma–Aldrich, Germany). Micronized 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol (Metronidazole, MNA, Ph Eur 6.0 specification) was purchased from FARGON GmbH & Co. KG (Barsbüttel, Germany). Tetrahydrofuran, chloroform, dichloromethane, and acetone were tested for their suitability to dissolve poly( l -lactide-co- d / l -lactide) (PLA) and MNA for its subsequent use for electrospinning. Acetone was chosen because of its good solubilizing of both PLA and MNA, its low boiling point and its established use in dental adhesive applications. We determined that a 3–5% (w/w) polymer solution, depending on the MNA content, was necessary to spin the copolymer under the conditions described below, to obtain similar fiber diameters. A homogeneous solution was prepared by slow stirring of appropriate amounts of PLA and MNA in acetone at room temperature for 3 h using a magnetic stirrer at 250 rpm. The obtained clear and viscous solutions were transferred directly into a 5 ml plastic syringe. The PLA/MNA mixture was then deployed in the electrospinning process using a custom designed electrospinning apparatus. This incorporated an adjustable high-voltage power supply (ESV-100; Ingenieurbüro G. Fuhrmann, Leverkusen, Germany) and an infusion pump (LA-100, Landgraf Laborsysteme, Germany). The syringe was connected by a 35 cm PTFE tube to a stainless-steel straight-end hollow needle (0.4 mm) under conditions adapted from those that have been previously described . A mirrored glass surface (20 cm × 20 cm; glass thickness 2 mm) was used as the electrically grounded plate to collect the drug loaded fibrous mat. The needle was connected to the ESV-100 DC power supply adjusted to 20 kV. The syringe was mounted vertically against the collector and the sample solution was fed to the nozzle at a constant flow rate of 1.5 ml h −1 . The distance between the needle tip and the mirror was defined at 16–18.5 cm. The formation of smooth fibers with diameters ranging from 0.6 to 1.2 μm was observed. Diameters were determined by software analysis of microscopic images (Image-Pro Plus 5.0, Media Cybernetics Inc., Silver Spring, MD, USA) and scanning electron microscopy (SEM). The applied amounts of PLA, MNA and solvent, together with parameters of the electrospinning process and mean diameters of the resulting fibers are shown in Table 1 . The overall area of the obtained electrospun fiber matrices was approximately 25 cm 2 , and the thickness ranged between 70 and 100 μm.
| PLA (mg) | MNA (mg) | MNA conc. (w/w) % | Acetone (g) | PLA conc. (w/w) % | Target distance (cm) | Relative humidity (%) | Temp. (°C) | Mean fiber diameter (μm) |
|---|---|---|---|---|---|---|---|---|
| 200 | 0.2 | 0.1 | 3.81 | 5.3 | 16 | 29 | 25.9 | 1.20 |
| 203 | 1.1 | 0.5 | 3.84 | 5.3 | 16 | 29 | 26.5 | 1.06 |
| 198 | 2.0 | 1.0 | 3.81 | 5.2 | 16 | 29 | 26.4 | 1.15 |
| 190 | 10.1 | 5.0 | 3.81 | 5.0 | 16 | 29 | 26.9 | 0.89 |
| 85 | 9.5 | 10 | 3.06 | 2.8 | 18.5 | 30 | 25.9 | 0.74 |
| 101 | 25.0 | 20 | 3.03 | 3.3 | 18.5 | 32 | 21.6 | 0.64 |
| 132 | 57.0 | 30 | 2.98 | 4.4 | 18.5 | 30 | 23.5 | 0.98 |
| 133 | 89.0 | 40 | 2.94 | 4.5 | 18.5 | 32 | 22.1 | 1.06 |
2.2
Material characterization
2.2.1
Scanning electron microscopy (SEM)
The drug-loaded fiber morphology was examined by field-emission scanning electron microscopy (SEM) (Supra 55VP; Zeiss, Oberkochen, Germany). For SEM, a Si wafer was used as substrate for the fibers and Au was sputtered on the specimens to ensure sufficient electrical conductivity. The images were taken using an InLens-detector with 5 keV excitation energy. The aim of this microscopic analysis was to determine whether the MNA concentration influenced the fiber structure or diameter.
2.2.2
Energy dispersive X-ray spectroscopy (EDX)
The SEM was equipped with an EDX-system (Quantax with Si(Li)-detector, Bruker, Berlin, Germany). The fiber mats were coated with evaporated carbon. For the measurements, an excitation energy of 2 keV was used. Spectra were taken from the crossed fiber regions of the mat to get a larger excitation volume for EDX.
2.2.3
Differential scanning calorimetry (DSC)
These experiments were performed on dry fiber mats in a Pyris 1 apparatus (PerkinElmer). The samples were subjected to a heating scan from 25 °C up to 200 °C at 10 °C/min. After cooling down to 25 °C at a rate of 10 °C/min, another heating run was performed from that temperature up to 200 °C at 10 °C/min. The first scans are those analyzed here.
2.3
Generation of aliquots
2.3.1
PBS aliquots
To evaluate the release of MNA from fibers placed in liquid media and its antibacterial efficiency, aliquots were produced for HPLC analysis and agar diffusion tests. Due to the varying fiber density, the weight of the individual fiber mats containing different percentages of MNA (0%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, w/w) had to be standardized to 2 mg (±100 μg). Therefore rectangular parts of appropriate sizes (4–100 mm 2 ) were cut of each mat with a scalpel and weighed with a microscale (Genius ME215S; Satorius, Germany). Afterwards these weighted parts from the fiber mats were placed into 24-well plates and exposed to 2 ml of PBS (phosphate buffered saline/Invitrogen, Germany). Aliquots for study were removed on successive days (1–7) and weeks (day 14, 21 and 28) for HPLC analysis and agar diffusion tests. After each aliquot removal the fiber mats were rinsed with 1 ml of PBS and wetted with new liquid medium. For the investigation of initial MNA release characteristics, another set of aliquots was produced. Therefore MNA loaded fiber mats (1%, 10%, 20% and 40%, w/w) of 2 mg were rinsed with 500 μl PBS from each side.
2.3.2
DMEM aliquots
For cytotoxicity tests (MTT-test, Neutral Red uptake) using human gingival fibroblasts, aliquots from fiber mats with different MNA concentrations (0.1–40.0%, w/w) were generated as described above for PBS aliquots. Instead of PBS, DMEM (Dulbecco’s Modified Eagle Medium; Invitrogen, Germany) with 10% of fetal calf serum was used as aliquot medium. The plates were cultivated at 37 °C with 5% CO 2 .
All aliquots were stored in Eppendorf tubes at −20 °C until they were used for further tests.
2.4
Drug release profiles
2.4.1
HPLC
Determination of MNA concentrations in the test aliquots was carried out by high-performance liquid chromatography (HPLC). The HPLC system consisted of a Shimadzu system with a column oven CTO-10AC, a series pump LC-10AT, a solvent degasser DGU-14A, an autosampler, a dual wavelength detector SPD-10A and Shimadzu CLASS-VP v 5.0 software. A LiChrosorb R18 column, 5 μm, 25 mm × 4.0 mm (Göhler HPLC-Analysentechnik, Chemnitz, Germany) was used. The separation was carried out under isocratic elution with double distilled water/acetonitrile (95:5, v/v) at a flow rate of 1 ml min −1 with UV detection at 254 and 320 nm which was applied for analysis. Acetonitrile was HPLC grade and purchased from Fisher Scientific (Fisher Scientific GmbH, Schwerte, Germany). The column temperature was ambient and an injection volume of 10 μl was used. The buffer solutions of drug eluting experiments were stored at 4 °C until those were used.
2.5
Antibacterial efficacy
2.5.1
Bacterial strains
For this study three pathogenic periodontal bacterial strains were used: Fusobacterium nucleatum (ATCC 10953/DSM 20482), Aggregatibacter actinomycetemcomitans (NCTC 9710/DSM 8324) and Porphyromonas gingivalis (ATCC 33277/DSM 20709). For the following test procedures, suspension cultures of these bacterial strains were cultivated in nutrient solution (Oxoid, Germany) that was enriched with vitamin K for 24 h at 37 °C under anaerobic conditions. After centrifugation of the bacterial strains they were rinsed twice with PBS and in physiological saline resuspended to an optical density of 0.1 at 640 nm, that equals a bacterial density of 10 8 bacteria/ml.
2.5.2
Agar diffusion assay
This method of testing was prepared to evaluate the antibacterial efficiency of the test aliquots. 100 μl of each bacterial suspension (optical density: 0.1) was spread onto a Petri dish with Schaedler agar (Oxoid, Germany) enriched with 1% of vitamin K and 10% of sheep blood. Holes of 8 mm in diameter were punched-out of the agar and filled with 100 μl of each test aliquot. Aliquots from fibers without MNA were uses as negative control. After incubation time of 48 h at 37 °C the diameters of the inhibition zones were measured. Six specimens were tested for each aliquot.
2.6
Statistical analysis of antibacterial action
The diameters of affected areas can vary with concentration and time for each of the three bacterial species investigated. This dependence was analyzed statistically, as follows. A two-way analysis of variance (ANOVA) was conducted using SPSS statistical software (IBM; version 19), by means of univariate analysis in a general linear model, to determine F -ratios for concentration and time, and their interaction, for each species separately. This was followed by application of the post hoc Scheffé test to determine homogenous subsets (alpha = 0.05) for both concentration and time.
2.7
Cytocompatibility
2.7.1
Cell cultures
For cytotoxicity tests human gingival fibroblasts (HGFs) were used (Ethics Commission Jena: 1881-10/06). These cells were cultivated in DMEM with 10% of fetal calf serum and 0.1% of AAS (antibiotic antimycotic solution) at 37 °C with 5% of CO 2 . To investigate the cytotoxicity of the fiber mats the neutral red uptake test and the MTT-test were used.
2.7.2
Neutral red uptake test
Here, HGFs were pipetted into 96-well microplates with a cell density of approximately 10,000 cells per well and covered with 100 μl of the test aliquot (prepared as described above). After two days of incubation time (37 °C, 5% CO 2 ) the viability of the cells was analyzed by measuring their neutral red (Sigma–Aldrich, Germany) uptake using a microplate reader (Lambda Scan 200, BmT GmbH, Meerbusch-Osterath, Germany) with wavelength of 540 nm.
2.7.3
MTT-test
To evaluate the influence of the test aliquots on the viability of the HGFs, the cells were treated with the water-soluble dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) (Roche, Germany). Due to the glycolysis equivalent transformation of the colorant MTT, the viability of the cells could be analyzed by measuring the optical density.
For both cytotoxicity tests DMEM was used as negative control substance. Positive control tests already existed with HGFs that were performed previously at the University of Jena, as already published .
2.7.4
Direct exposure of cells on MNA-loaded fiber mats
Fiber mats containing different percentages of MNA (0%, 1.0%, 5.0%, 10.0%, 20.0% and 40.0%, w/w) were clamped into CellCrown (Scaffdex Oy, Finland) cell culture inserts and sterilized with ethanol (70%, v/v). After rinsing with 2 ml of PBS, a cell suspension (10,000 cells/cm 2 ) was applied onto each fiber mat that was then covered with 2 ml of DMEM medium. After the cultivation period of 48 h the fiber mats were taken out the cell culture inserts and after rinsing with PBS stained with a live-dead colorant (12 ml of PBS + 12 μl of fluorescein diacetate (vital) + 16 μl ethidium bromide [EtBr] (avital)) and evaluated microscopically.
2
Materials and methods
2.1
Electrospinning
Poly( l -lactide-co- d / l -lactide) 70/30 (Resomer LR 708, Boehringer Ingelheim, Germany) was used for electrospinning ( Fig. 1 ). A weight-average molecular weight of 1.5 × 10 6 g mol −1 was determined for the polymer by gel permeation chromatography using CHCl 3 as solvent and polystyrene as external standard. All solvents used for electrospinning purposes were of HPLC grade (Sigma–Aldrich, Germany). Micronized 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol (Metronidazole, MNA, Ph Eur 6.0 specification) was purchased from FARGON GmbH & Co. KG (Barsbüttel, Germany). Tetrahydrofuran, chloroform, dichloromethane, and acetone were tested for their suitability to dissolve poly( l -lactide-co- d / l -lactide) (PLA) and MNA for its subsequent use for electrospinning. Acetone was chosen because of its good solubilizing of both PLA and MNA, its low boiling point and its established use in dental adhesive applications. We determined that a 3–5% (w/w) polymer solution, depending on the MNA content, was necessary to spin the copolymer under the conditions described below, to obtain similar fiber diameters. A homogeneous solution was prepared by slow stirring of appropriate amounts of PLA and MNA in acetone at room temperature for 3 h using a magnetic stirrer at 250 rpm. The obtained clear and viscous solutions were transferred directly into a 5 ml plastic syringe. The PLA/MNA mixture was then deployed in the electrospinning process using a custom designed electrospinning apparatus. This incorporated an adjustable high-voltage power supply (ESV-100; Ingenieurbüro G. Fuhrmann, Leverkusen, Germany) and an infusion pump (LA-100, Landgraf Laborsysteme, Germany). The syringe was connected by a 35 cm PTFE tube to a stainless-steel straight-end hollow needle (0.4 mm) under conditions adapted from those that have been previously described . A mirrored glass surface (20 cm × 20 cm; glass thickness 2 mm) was used as the electrically grounded plate to collect the drug loaded fibrous mat. The needle was connected to the ESV-100 DC power supply adjusted to 20 kV. The syringe was mounted vertically against the collector and the sample solution was fed to the nozzle at a constant flow rate of 1.5 ml h −1 . The distance between the needle tip and the mirror was defined at 16–18.5 cm. The formation of smooth fibers with diameters ranging from 0.6 to 1.2 μm was observed. Diameters were determined by software analysis of microscopic images (Image-Pro Plus 5.0, Media Cybernetics Inc., Silver Spring, MD, USA) and scanning electron microscopy (SEM). The applied amounts of PLA, MNA and solvent, together with parameters of the electrospinning process and mean diameters of the resulting fibers are shown in Table 1 . The overall area of the obtained electrospun fiber matrices was approximately 25 cm 2 , and the thickness ranged between 70 and 100 μm.
