Abstract
Objective
Our study was performed with the aim of preparing electrospun polylactide fibers with a combination of ampicillin (AMP) and metronidazole (MNZ) and investigating their drug release behavior and the antibacterial effect on Aggregatibacter actinomycetemcomitans and other oral pathogens.
Methods
AMP and MNZ were integrated as a combination in two separate fibers (dual fiber mats – DFW mix) of electrospun PLA fiber mats by means of multijet electrospinning and in a single fiber (single fiber mats – SFW mix). HPLC (high-performance liquid chromatography) was used to measure the released drug quantities. Agar diffusion tests were used to determine the antibacterial effect of the eluates on A. actinomycetemcomitans , Fusobacterium nucleatum , Porphyromonas gingivalis and Enterococcus faecalis . The neutral red test was made to examine the cytocompatibility of the eluates with human gingival fibroblasts (hGFs).
Results
The release of the active agents varied with the antibiotic concentrations initially used in the fiber mats, but also with the distribution of the active agents in one or two fibers. Of the total quantity of MNZ (AMP), the SFW mix fiber mats released >60% (>70%) within a span of 5 min, and 76% (71%) after 96 h. With these drug concentrations released by the fiber mats (≥5 m%), an antibacterial effect was achieved on A. actinomycetemcomitans and on all other species tested. Fiber mats and their eluates have no cytotoxic influence on human gingival fibroblasts (hGFs).
Significance
Electrospun AMP/MNZ-loaded polymer fibers are a potential drug delivery system for use in periodontal and endodontic infections.
1
Introduction
It is now a well established approach in periodontal therapy to apply adjuvant antibiotics for the suppression of periodontopathogenic species after mechanical removal of biofilms . To avoid undesirable systemic side effects on the antibiotics and to achieve a strong effect on the periodontal lesion, the use of electrospun drug delivery systems has recently been discussed in addition, in the context of local antibacterial periodontitis therapy .
Active substances currently available for local application include, among others, pharmaceutical gel and ointment formulations such as Ligosan ® Slow Release (Hereaus Kulzer, Hanau, Germany), Atridox ® (TOLMAR Inc., Fort Collins, United States) or Arestin ® (OraPharmaInc, Horsham, United States). Each of these systems contains an antibiotic, however their spectrum of activity only rarely includes the important periodontopathogenic species.
The suppression of the species Aggregatibacter actinomycetemcomitans must be rated as complicated . Several studies discuss the absent or distinctly reduced effect on A. actinomycetemcomitans if metronidazole or clindamycin is applied alone . Within the adjuvant systemic application of antibiotics against A. actinomycetemcomitans -associated periodontitis, therefore, use is made, besides metronidazole, of amoxicillin, as its spectrum of activity also comprises facultatively anaerobic bacterial species . For local application, however, no suitable form of application is known combining both antibiotics. Such a combined therapy could markedly increase the therapeutic effect of local application systems.
Due to similarly good antibacterial properties, it is possible to use ampicillin as an alternative to amoxicillin . Compared to amoxicillin, ampicillin is clearly more soluble, which makes it a candidate for local application in fiber mats. This antibiotic, also of the β-lactam group, is used clinically for treating bacterial Infections of the respiratory tract, the gastro-intestinal tract, the bile and urinary ducts, etc. .
In a previous study, we were able to establish electrospun metronidazole-loaded PLA fibers for periodontitis treatment with good antibacterial properties against Fusobacterium nucleatum and Porphyromonas gingivalis . However, in this former study, A. actinomycetemcomitans , the key pathogen of aggressive periodontitis, could not be suppressed sufficiently.
Meanwhile, the technique of electrospinning has been further optimized, so that a considerable number of synthetic polymers are suitable as matrix materials and can be used with combinations of active components such as antibiotics, hormones or growth factors . Especially electrospun micro- and nanomaterials, such as resorbable antibiotic-loaded polylactide (PLA) fiber mats, enable improved, targeted release of the active substances .
Especially with the aid of the advanced technique of multijet electrospinning used in this study, we tried for the first time to load PLA fiber mats with the combination of ampicillin (AMP) and metronidazole (MNZ) in particular to make possible the suppression of A. actinomycetemcomitans . We aimed to clarify whether the distribution of the antibiotics in the combination mentioned, in one or both of the separate fibers, influences the release of the drugs. In addition, we sought to rule out a cytotoxic influence of the new drug delivery systems on human gingival fibroblasts
2
Methods
2.1
Production of fiber mats
For in vitro tests we made three different fiber systems of poly( l -lactide-co- d / l -lactide) (70/30, Resomer LR 708, Boehringer Ingelheim, Germany): (i) single-fiber drug delivery system (SFW) with different concentrations of Na-Ampicillinat (AMP, d -(−)-α-aminobenzylpenicillin-sodium salt, A9518 Sigma–Aldrich, Germany); (ii) single-fiber drug delivery system mix (SFW mix) with metronidazole (MNZ, 2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethanol, FARGON GmbH & Co. KG Barsbüttel, Germany) and Na-Ampicillinat combined in a fiber; (iii) dual-fiber drug delivery system mix (DFW mix), in which two single fibers, each with one antibiotic, were spun simultaneously into one fiber mat.
Different concentrations of the antibiotics were added to the PLA, dissolved in acetone. The ampicillin SFW fiber mats were loaded with concentrations of 0.1, 0.5, 1, 5, 10, 20, 30 and 40 m%. The SFW mix fiber mats were loaded with the combination of ampicillin and metronidazole (20/20 m%). In the DFW mix, the active agents were integrated with proportions of 20 m% each per fiber. A fiber mat without antibiotic served as a negative control. Electrospinning was performed in an E-Spintronic apparatus (E. Huber GmbH, Gernlinden, Germany) ( Fig. 1 a ). The electrospinning procedure has been described previously . The morphology of the antibiotic-loaded PLA fiber mats was studied by scanning electron microscopy (SEM) (Supra 55VP; Zeiss, Oberkochen, Germany) ( Fig. 1 b).
2.2
Generation of aliquots
To ascertain the release kinetics and the cytocompatibility of the antibacterial agents, we prepared aliquots of the fiber mat versions in different eluants.
2.2.1
PBS aliquots
Four 2.00 mg (±0.02 mg) specimens of each SFW, SFW mix and DFW mix fiber mats were obtained (Genius ME 215 microbalance; Sartorius, Germany). They were placed into 24-well microplates and UV sterilized for 10 min, then topped with 2 ml PBS each (phosphate buffered saline, Invitrogen, Germany), and kept at 37 °C, 5% CO 2 for 28 days. Eluates were obtained at a fixed time pattern: on the first day after 5 min, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h; on days 2–7, and on days 14, 21 and 28. After eluates were taken, the batches were rinsed with 1 ml PBS and kept in fresh PBS. The eluates obtained were aliquoted and kept at −20 °C until they were used for the antibacterial (1 ml) and HPLC tests (1 ml).
2.2.2
DMEM aliquots
To make eluates for the cytotoxicity experiments, two 1.00 mg (±0.02 mg) specimens of each fiber mat were weighed. Eluates were obtained and the samples kept analogously to the PBS eluates, except that the eluant was DMEM (Dulbecco’s Modified Eagle Medium, Invitrogen, Germany) with 10% fetal bovine serum (FBS).
2.3
Drug release profiles
The quantities of ampicillin and metronidazole were determined by means of high-performance liquid chromatography (HPLC) using a Shimadzu system. This comprised a CTO-10AC column oven, a DGU-14A solvent degasser, an LC-10AT serial pump, an SPD-10A dual wavelength detector and an autosampler. Data were obtained via Shimadzu CLASS-VP v 5.0 software. UV detection at 254 and 320 nm was performed with the aid of isocratic elution (95:5, v/v) from bidistilled water and acetone nitrile at a flow rate of 1 ml/min (Fisher Scientific GmbH, Schwerte, Germany). Volumes of 10 μl were injected at room temperature. The buffer solutions were maintained 4 °C below the working temperature.
2.4
Antibacterial efficiency
2.4.1
Bacterial strains
Cultures of the following periodontopathogenic species were included in the investigations: A. actinomycetemcomitans (NCTC 9710/DSM 8324), F. nucleatum (ATCC 10953/DSM 20482), P. gingivalis (ATCC 33277/DSM 20709) and the endopathogenic species Enterococcus faecalis (DSMZ 20376). The species were anaerobically cultured at 37 °C for 24 h in a nutrient medium enriched with vitamin K (10 μg/ml) (Schaedler broth, Oxoid, Germany).
2.4.2
Agar diffusion assay
The antibacterial efficacy of the fiber mat eluates was evaluated by means of agar diffusion tests. 100 μl of the respective bacterial suspension (OD 546 0.1) was applied onto Schaedler agar plates (10% sheep’s blood, 1% vitamin K) with a spatula. 100 μl of the respective eluate was then applied into holes (dia. 8 mm) punched at the center of each agar surface. After an incubation time of 48 h (anaerobic, 37 °C), the diameters of the inhibition zones on the Schaedler agar plates were measured. Eight parallel measurements were made of each eluate.
2.5
Cytocompatibility
2.5.1
Cell cultures
Cytocompatibility was determined with human gingival fibroblasts (hGFs) from a primary culture (Ethics Commission Jena: 1881-10/06). The hGFs were cultured in DMEM (0.1% antibiotic antimycotic solution, 10% fetal bovine serum) at 37 °C and 5% CO 2 .
2.5.2
Neutral red uptake
To estimate the cytotoxic potential of the eluates, we used the neutral red uptake assay. The neutral red dye is taken up by the lysosomes and bound as a function of pH, so that it stains viable cells only. For this cytotoxicity test, hGFs (10,000 cells/well) were put into 96-well microplates and incubated each with 100 μl eluate or standard medium (control) for 48 h. Subsequently the viability of the exposed cells was analyzed on the basis of the dye uptake. The measurement was made after 4 h of incubation of the dye with the hGFs at 540 nm (Lambda Scan 200, BmT GmbH, Meerbusch-Osterath, Germany). Eight parallel measurements were made of each eluate.
2.5.3
Direct exposure of cells on fiber mats
To examine hGFs for their biocompatibility and adherence in direct contact with the antibiotic-loaded fiber mats, 2 cm 2 specimens of each mat were prepared and clamped into suitable sample holders (scaffold rings, Scaffdex Oy, Finland). The test batches were put into 24-well microplates and then sterilized with ethanol (70%, 5 min). The hGFs, previously rinsed with DMEM, were directly applied onto the fiber mat surfaces (about 20,000 cells/cm 2 ). The batches were topped with 2 ml DMEM (20% FBS) and incubated for 48 h at 37 °C and 5% CO 2 . Here, a combination dye of fluorescein diacetate (24 μl) for viable cells and ethidium bromide (22 μl) for non-viable cells, dissolved in PBS (4 ml), served as a marker. Viable and non-viable cells on the fiber mat surfaces were detected with a fluorescence microscope (Labophot-2, Nikon, Japan).
2.6
Statistics
The statistical evaluation of the antibacterial efficacy of the antibiotics-loaded PLA fiber mats was obtained by the computation of the pharmacokinetic quantity “AUC” (area under curve) as inhibition zones (mean overall effect) averaged over the entire observation time. Significant differences between the antibacterial efficacies of the fiber mat versions were determined by means of non-parametric tests (Mann–Whitney U test and Kruskal–Wallis test) (IBM SPSS Statistics Version 20).
2
Methods
2.1
Production of fiber mats
For in vitro tests we made three different fiber systems of poly( l -lactide-co- d / l -lactide) (70/30, Resomer LR 708, Boehringer Ingelheim, Germany): (i) single-fiber drug delivery system (SFW) with different concentrations of Na-Ampicillinat (AMP, d -(−)-α-aminobenzylpenicillin-sodium salt, A9518 Sigma–Aldrich, Germany); (ii) single-fiber drug delivery system mix (SFW mix) with metronidazole (MNZ, 2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethanol, FARGON GmbH & Co. KG Barsbüttel, Germany) and Na-Ampicillinat combined in a fiber; (iii) dual-fiber drug delivery system mix (DFW mix), in which two single fibers, each with one antibiotic, were spun simultaneously into one fiber mat.
Different concentrations of the antibiotics were added to the PLA, dissolved in acetone. The ampicillin SFW fiber mats were loaded with concentrations of 0.1, 0.5, 1, 5, 10, 20, 30 and 40 m%. The SFW mix fiber mats were loaded with the combination of ampicillin and metronidazole (20/20 m%). In the DFW mix, the active agents were integrated with proportions of 20 m% each per fiber. A fiber mat without antibiotic served as a negative control. Electrospinning was performed in an E-Spintronic apparatus (E. Huber GmbH, Gernlinden, Germany) ( Fig. 1 a ). The electrospinning procedure has been described previously . The morphology of the antibiotic-loaded PLA fiber mats was studied by scanning electron microscopy (SEM) (Supra 55VP; Zeiss, Oberkochen, Germany) ( Fig. 1 b).
2.2
Generation of aliquots
To ascertain the release kinetics and the cytocompatibility of the antibacterial agents, we prepared aliquots of the fiber mat versions in different eluants.
2.2.1
PBS aliquots
Four 2.00 mg (±0.02 mg) specimens of each SFW, SFW mix and DFW mix fiber mats were obtained (Genius ME 215 microbalance; Sartorius, Germany). They were placed into 24-well microplates and UV sterilized for 10 min, then topped with 2 ml PBS each (phosphate buffered saline, Invitrogen, Germany), and kept at 37 °C, 5% CO 2 for 28 days. Eluates were obtained at a fixed time pattern: on the first day after 5 min, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h; on days 2–7, and on days 14, 21 and 28. After eluates were taken, the batches were rinsed with 1 ml PBS and kept in fresh PBS. The eluates obtained were aliquoted and kept at −20 °C until they were used for the antibacterial (1 ml) and HPLC tests (1 ml).
2.2.2
DMEM aliquots
To make eluates for the cytotoxicity experiments, two 1.00 mg (±0.02 mg) specimens of each fiber mat were weighed. Eluates were obtained and the samples kept analogously to the PBS eluates, except that the eluant was DMEM (Dulbecco’s Modified Eagle Medium, Invitrogen, Germany) with 10% fetal bovine serum (FBS).
2.3
Drug release profiles
The quantities of ampicillin and metronidazole were determined by means of high-performance liquid chromatography (HPLC) using a Shimadzu system. This comprised a CTO-10AC column oven, a DGU-14A solvent degasser, an LC-10AT serial pump, an SPD-10A dual wavelength detector and an autosampler. Data were obtained via Shimadzu CLASS-VP v 5.0 software. UV detection at 254 and 320 nm was performed with the aid of isocratic elution (95:5, v/v) from bidistilled water and acetone nitrile at a flow rate of 1 ml/min (Fisher Scientific GmbH, Schwerte, Germany). Volumes of 10 μl were injected at room temperature. The buffer solutions were maintained 4 °C below the working temperature.
2.4
Antibacterial efficiency
2.4.1
Bacterial strains
Cultures of the following periodontopathogenic species were included in the investigations: A. actinomycetemcomitans (NCTC 9710/DSM 8324), F. nucleatum (ATCC 10953/DSM 20482), P. gingivalis (ATCC 33277/DSM 20709) and the endopathogenic species Enterococcus faecalis (DSMZ 20376). The species were anaerobically cultured at 37 °C for 24 h in a nutrient medium enriched with vitamin K (10 μg/ml) (Schaedler broth, Oxoid, Germany).
2.4.2
Agar diffusion assay
The antibacterial efficacy of the fiber mat eluates was evaluated by means of agar diffusion tests. 100 μl of the respective bacterial suspension (OD 546 0.1) was applied onto Schaedler agar plates (10% sheep’s blood, 1% vitamin K) with a spatula. 100 μl of the respective eluate was then applied into holes (dia. 8 mm) punched at the center of each agar surface. After an incubation time of 48 h (anaerobic, 37 °C), the diameters of the inhibition zones on the Schaedler agar plates were measured. Eight parallel measurements were made of each eluate.
2.5
Cytocompatibility
2.5.1
Cell cultures
Cytocompatibility was determined with human gingival fibroblasts (hGFs) from a primary culture (Ethics Commission Jena: 1881-10/06). The hGFs were cultured in DMEM (0.1% antibiotic antimycotic solution, 10% fetal bovine serum) at 37 °C and 5% CO 2 .
2.5.2
Neutral red uptake
To estimate the cytotoxic potential of the eluates, we used the neutral red uptake assay. The neutral red dye is taken up by the lysosomes and bound as a function of pH, so that it stains viable cells only. For this cytotoxicity test, hGFs (10,000 cells/well) were put into 96-well microplates and incubated each with 100 μl eluate or standard medium (control) for 48 h. Subsequently the viability of the exposed cells was analyzed on the basis of the dye uptake. The measurement was made after 4 h of incubation of the dye with the hGFs at 540 nm (Lambda Scan 200, BmT GmbH, Meerbusch-Osterath, Germany). Eight parallel measurements were made of each eluate.
2.5.3
Direct exposure of cells on fiber mats
To examine hGFs for their biocompatibility and adherence in direct contact with the antibiotic-loaded fiber mats, 2 cm 2 specimens of each mat were prepared and clamped into suitable sample holders (scaffold rings, Scaffdex Oy, Finland). The test batches were put into 24-well microplates and then sterilized with ethanol (70%, 5 min). The hGFs, previously rinsed with DMEM, were directly applied onto the fiber mat surfaces (about 20,000 cells/cm 2 ). The batches were topped with 2 ml DMEM (20% FBS) and incubated for 48 h at 37 °C and 5% CO 2 . Here, a combination dye of fluorescein diacetate (24 μl) for viable cells and ethidium bromide (22 μl) for non-viable cells, dissolved in PBS (4 ml), served as a marker. Viable and non-viable cells on the fiber mat surfaces were detected with a fluorescence microscope (Labophot-2, Nikon, Japan).
2.6
Statistics
The statistical evaluation of the antibacterial efficacy of the antibiotics-loaded PLA fiber mats was obtained by the computation of the pharmacokinetic quantity “AUC” (area under curve) as inhibition zones (mean overall effect) averaged over the entire observation time. Significant differences between the antibacterial efficacies of the fiber mat versions were determined by means of non-parametric tests (Mann–Whitney U test and Kruskal–Wallis test) (IBM SPSS Statistics Version 20).
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