Abstract
Background
Antibiotic beads are used to treat local bacterial infections by delivering high drug concentrations to infected tissue.
Objectives
This study examined the elution characteristics of metronidazole from metronidazole-calcium sulfate (MCa) and metronidazole-calcium-potassium sulfate (MCaK) beads over 20 days and the antibacterial efficacy of the beads after storage.
Methods
The MCa and MCaK beads were prepared by mixing 250 mg of metronidazole and 10 g of calcium sulfate hemihydrate with water and a 3% potassium sulfate solution, respectively. The beads were placed in phosphate-buffered saline for the elution study. The metronidazole eluents were determined using high-performance liquid chromatography. The microstructures were examined by scanning electron microscopy (SEM), and the antimicrobial activity was evaluated by a microbioassay.
Results
For the 20-day study, the total amount of metronidazole released was greater in the MCa beads than in the MCaK beads by 6.61 ± 0.48 mg (89.11% ± 3.04%) and 4.65 ± 0.36 mg (73.11% ± 4.38%), respectively. The amounts of eluted drugs from the MCa and MCaK beads were higher than the minimum inhibitory concentration at 0.5 µg/mL against anaerobic bacteria at both 20 days and 14 days. SEM showed that calcium crystals on the outer surface had dissolved after elution, and thinner calcium crystals were prominent in the MCaK beads. The MCa and MCaK beads exhibited antibacterial activity after setting, followed by storage at room temperature or 4°C for 21 days.
Conclusions
The MCa beads could release more drug than the MCaK beads, but all eluted metronidazole amounts were effective in controlling bacterial infections. Both metronidazole beads could be stored at ambient temperature or in a refrigerator.
Keywords: Anaerobic bacteria, antibacterial agent, drug carrier, drug release, infection
INTRODUCTION
Antibiotic beads have been used to prevent or treat local bacterial infections, particularly orthopedic infections, and have been developed continuously for effective treatment [1]. Antibiotic beads have been used in veterinary medicine to treat various infections, such as orthopedic and periodontal infections, in various animals [2,3]. The antibiotic beads delivered a high concentration of antibiotic locally that could control infections effectively without systemic toxicity [4].
The materials used for antibiotic beads can be classified into two groups: absorbable and non-absorbable composites. Calcium phosphate, calcium sulfate (Ca), and chitosan are absorbable materials, whereas polymethylmethacrylate (PMMA) and hydroxyapatite ceramic are non-absorbable materials. Ca is an inorganic, absorbable, osteoconductive substance used as a bone substitute to fill bone defects [5]. Antibiotic-impregnated Ca beads have been used to control bacterial infection locally because they released high levels of antibiotics locally and continuously [6,7]. Various antibiotics have been used to prepare antibiotic beads, such as amikacin, penicillin, and vancomycin [8,9].
Various methods have been used to prepare antibiotic beads for subsequent drug release. Combined antibiotic Ca beads had increased the antibacterial properties [9]. In addition, the setting reaction of Ca was accelerated by adding inorganic salts, such as potassium sulfate (K2SO4), potassium chloride (KCl), and sodium chloride (NaCl) [5]. These inorganic salts increased the density of the calcium crystals, which affected drug elution from the Ca beads [6]. A potassium sulfate solution mixed with Ca instead of sterile water improved drug elution [10,11]. The antibiotic elution profiles were related to the microscopic structures of the antibiotic beads observed using scanning electron microscopy (SEM). The enhanced porosity of the Ca beads increased ceftazidime elution [12].
Metronidazole is an antimicrobial agent used to treat anaerobic bacteria and protozoa. In this study, metronidazole was selected to address local bacterial infections in animals caused by anaerobic bacteria. Numerous studies have shown that anaerobic bacteria can cause serious infections in veterinary medicine, such as odontogenic abscesses in guinea pigs [13], osteomyelitis in dogs and cats [14,15], and osteomyelitis and myositis in red kangaroos [16]. The available information on anaerobic bacteria infections may not reflect the complete situation because of the difficulty in isolating anaerobic bacteria. Nevertheless, metronidazole is an effective antimicrobial agent for controlling anaerobic bacteria [17]. Ramos et al. [18] studied metronidazole-impregnated PMMA beads as a release drug for 21 days. On the other hand, the metronidazole elution profile of Ca beads has not been investigated.
This study examined the metronidazole elution profiles from Ca beads: metronidazole-calcium sulfate (MCa) and metronidazole-calcium-potassium sulfate (MCaK) beads for 20 days. The characteristics of the calcium beads were investigated using SEM. In addition, the antibacterial efficacy was studied of metronidazole–calcium beads under different storage conditions (room temperature and 4°C) for 21 days.
MATERIALS AND METHODS
Preparation of Ca beads
The beads were prepared from Ca hemihydrates (Sigma-Aldrich, USA) and metronidazole (Dr. Ehrenstorfer GMbH, Germany). The Ca beads were prepared by mixing 10 g of Ca hemihydrate and 4.2 mL of water [19]. The calcium-potassium sulfate (CaK) beads were prepared by mixing 10 g of Ca hemihydrate and 6.5 mL of 3% potassium sulfate. The calcium mixtures were set in molds at room temperature. The Ca and CaK beads were used as the controls in the SEM study.
MCa beads were prepared by mixing 250 mg of metronidazole and 10 g of Ca hemihydrate with 4.6 mL of sterile water. MCaK beads were prepared by mixing 250 mg of metronidazole, 10 g of Ca hemihydrate, and 6.5 mL of 3% potassium sulfate solution. The metronidazole mixtures were poured into molds. After the calcium beads had hardened at room temperature, they were kept in a dry container at 4°C and used the next day.
Metronidazole elution studies for 20 days
An elution method was used to determine the metronidazole released from the different Ca beads. The dissolution medium was a phosphate buffer saline solution (0.1 M phosphate-buffered saline [PBS], pH 7.4). Sixteen beads were sampled separately from both the MCa and MCaK groups and weighed. Two beads were placed in tubes containing 1.5 mL of PBS, and tubes were incubated at 37°C for 24 h. The PBS was removed, and 1.5 mL of fresh PBS was added daily for 20 days. All eluted mediums were kept at −20°C until analysis.
Metronidazole determination
The metronidazole concentrations were determined from the eluted medium based on high-performance liquid chromatography (HPLC, Agilent 1260; Agilent Technologies, USA) using a slight modification of the methodology reported by Ramos et al. [18]. Each sample was passed through a 13 mm diameter filter with a 0.22 µm pore size. Each 15 µL sample was injected into a 3.9 × 150 mm C18 column (Nova-Pak®; Waters, USA) with a 4 µm particle size. The mobile phase was a mixture of 30% methanol and 70% PBS (0.01 M, pH 3.0) and was delivered at a flow rate of 0.6 mL/min, with a diode array detector at 313 nm. The lowest limit of detection of metronidazole was 0.1 µg/mL. Dimetridazole was used as the internal standard.
Microstructure of various Ca beads after elution
All four types of calcium beads (Ca, CaK, MCa, and MCaK) were immersed in PBS as an elution study. The PBS was removed and replaced with fresh PBS daily. The micromorphology of all calcium beads, including the control non-immersed beads, was examined by SEM after 1, 5, 7, and 20 days of elution. The beads were sputter-coated with platinum before the cross-sectional and surface images were obtained using SEM (SU8020; Hitachi, Japan).
Antibacterial activity of metronidazole beads after storage
The MCa and MCaK beads were kept in separate containers for 20 days at room temperature (27°C) and 4°C. The antibacterial activity was assessed using an agar-well diffusion microbiological assay using a methodology reported elsewhere [20]. Briefly, Bacillus subtilis (ATCC 6633) was seeded into Mueller Hinton agar as an indicator organism. Agar wells, 5 mm in diameter, were made using a cork borer. The beads were placed into each well, and the agar plates were incubated at 37°C for at least 18 h. Each bead type was tested in triplicate. The zones of inhibition were measured for each bead based on the different temperatures on days 0, 7, 14, and 21 and compared for antibacterial activity.
Statistical analysis
The results are reported as mean ± SD values. All statistical analyses were performed using the SigmaPlot software (Systat Software, USA). The differences between the eluted drugs from the MCa and MCaK beads were compared using a Student’s t-test or a Mann-Whitney U test where appropriate. The differences in bead shape, weight, and antibacterial activity were investigated based on an analysis of variance (ANOVA), followed by a Tukey multiple comparison test. Statistical significance was tested at p < 0.05.
RESULTS
Calcium bead preparation
Four types of calcium beads were formed in cylindrical shapes with an average diameter and height of 5 mm and 5.3–5.6 mm, respectively. The weights of the Ca, CaK, MCa, and MCaK beads ranged from 0.13g to 0.15 g (Table 1), with the weights of the Ca and MCa beads (0.142 ± 0.003 g and 0.152 ± 0.008 g, respectively) being significantly different (p < 0.001). In contrast, the weights of the CaK and MCaK beads (0.127 ± 0.002 g and 0.130 ± 0.005 g, respectively) were similar (p = 0.44). The heights and weights of the MCa and MCaK beads were significantly different (p = 0.04).
Table 1. Size and weight of the four types of calcium beads.
| Bead types | Diameter (mm) | Height (mm) | Weight (g) |
|---|---|---|---|
| Ca | 4.940 ± 0.0652 | 5.264 ± 0.074a | 0.142 ± 0.003a |
| CaK | 5.006 ± 0.110 | 5.574 ± 0.126b | 0.127 ± 0.002b |
| MCa | 5.061 ± 0.192 | 5.470 ± 0.136b | 0.152 ± 0.008c |
| MCaK | 5.000 ± 0.109 | 5.280 ± 0.176a | 0.130 ± 0.005b |
The values are shown as mean ± SD (n = 16).
Ca, calcium sulfate; CaK, calcium-potassium sulfate; MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate.
Different lowercase superscripts (a, b, and c) within the column indicate significant differences using multiple comparison procedures with a Tukey test, p < 0.05.
Table 2 lists the weights of the MCa and MCaK beads before and after the elution experiment for 20 days. The mass losses of the MCa and MCaK beads were 3.15% ± 1.25% and 10.38% ± 3.43%, respectively.
Table 2. Weights of metronidazole-calcium beads before and after the elution and percentage mass loss after the elution test.
| Bead type | Weight before elution (g) | Weight after elution (g) | Mass loss (%) |
|---|---|---|---|
| MCa | 0.152 ± 0.008 | 0.147 ± 0.006 | 3.15 ± 1.25 |
| MCaK | 0.130 ± 0.005 | 0.117 ± 0.007 | 10.38 ± 3.43 |
The values are shown as mean ± SD (n = 16).
MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate.
Concentration of eluted metronidazole for 20 days
The release of metronidazole from the MCa and MCaK beads was quantified by HPLC. The release behavior of the two beads was comparable, with a large release on the first day and a decline over 20 days. On the first day, the concentrations of the drug released from the MCa and MCaK beads were 1467.95 ± 219.74 µg/mL and 1751.48 ± 220.94 µg/mL, respectively (Table 3), with the MCaK beads releasing significantly more metronidazole than the MCa beads (p = 0.02). The concentrations of metronidazole eluted daily from the MCa and MCaK beads were significantly different except on day 14 (p = 0.08). On the first day, the metronidazole released from the MCa and MCaK beads was 29.58 ± 3.1% and 41.32 ± 4.52%, respectively. Compared to the amounts released at 20 days, the amounts of metronidazole released from the MCa and MCaK beads on the first day were 33.17% and 56.43%, respectively. On day 2, the quantity of metronidazole released from the MCa beads was significantly higher than that from the MCaK beads (612.74 ± 124.26 µg/mL and 490.34 ± 49.53 µg/mL, respectively) (p = 0.02). From day 4, the cumulative amount of metronidazole released from the MCa beads was significantly greater than from the MCaK beads (5.27 ± 0.89 mg and 4.31 ± 0.39 mg, respectively) (p < 0.001).
Table 3. Daily and cumulative released metronidazole from MCa beads and MCaK beads for 20 days.
| Day | MCa beads | MCaK beads | ||||
|---|---|---|---|---|---|---|
| Eluted metronidazole (µg/mL) | Cumulative metronidazole (mg) | Cumulative metronidazole (%) | Eluted metronidazole (µg/mL) | Cumulative metronidazole (mg) | Cumulative metronidazole (%) | |
| 1 | 1,467.95 ± 219.74 | 2.2 ± 0.33 | 29.58 ± 3.1 | 1,751.48 ± 220.94a | 2.63 ± 0.33a | 41.32 ± 4.52a |
| 2 | 612.74 ± 124.26 | 3.12 ± 0.49 | 41.9 ± 4.7 | 490.34 ± 49.53a | 3.36 ± 0.33 | 52.92 ± 4.45a |
| 3 | 738.81 ± 147.88 | 4.23 ± 0.71 | 56.76 ± 6.91 | 564.65 ± 67.36a | 4.21 ± 0.36 | 66.22 ± 4.13a |
| 4 | 692.14 ± 129.79 | 5.27 ± 0.89 | 70.69 ± 8.72 | 64.75 ± 77.81a | 4.31 ± 0.39a | 67.76 ± 4.85 |
| 5 | 454.9 ± 72.07 | 5.95 ± 0.91 | 79.9 ± 8.68 | 120.73 ± 35.26a | 4.49 ± 0.35a | 70.64 ± 4.44a |
| 6 | 181.24 ± 74.99 | 6.22 ± 0.81 | 83.64 ± 7.17 | 52.86 ± 14.7a | 4.57 ± 0.34a | 71.89 ± 4.27a |
| 7 | 88.14 ± 54.74 | 6.35 ± 0.73 | 85.48 ± 6.05 | 24.05 ± 7.75a | 4.6 ± 0.34a | 72.46 ± 4.25a |
| 8 | 53.95 ± 48.19 | 6.43 ± 0.66 | 86.61 ± 5.14 | 10.41 ± 2.8b | 4.62 ± 0.34a | 72.71 ± 4.27a |
| 9 | 31.08 ± 30.66 | 6.48 ± 0.62 | 87.27 ± 4.59 | 6.45 ± 2.99b | 4.63 ± 0.35a | 72.86 ± 4.28a |
| 10 | 21.88 ± 23.66 | 6.51 ± 0.59 | 87.73 ± 4.21 | 3.78 ± 2.63b | 4.63 ± 0.35a | 72.95 ± 4.3a |
| 11 | 17.38 ± 19.65 | 6.54 ± 0.56 | 88.1 ± 3.89 | 2.5 ± 3.07b | 4.64 ± 0.35a | 73 ± 4.32a |
| 12 | 12.22 ± 15.01 | 6.56 ± 0.54 | 88.36 ± 3.65 | 1.48 ± 2.02b | 4.64 ± 0.35a | 73.04 ± 4.34a |
| 13 | 8.78 ± 11.19 | 6.57 ± 0.52 | 88.55 ± 3.47 | 0.96 ± 1.27b | 4.64 ± 0.35a | 73.06 ± 4.35a |
| 14 | 3.56 ± 4.21 | 6.58 ± 0.52 | 88.63 ± 3.42 | 0.66 ± 0.97 | 4.64 ± 0.35a | 73.08 ± 4.36a |
| 15 | 5.36 ± 6.49 | 6.59 ± 0.51 | 88.74 ± 3.33 | 0.46 ± 0.74b | 4.64 ± 0.35a | 73.09 ± 4.36a |
| 16 | 4.26 ± 4.96 | 6.59 ± 0.5 | 88.83 ± 3.26 | 0.3 ± 0.47b | 4.64 ± 0.35a | 73.09 ± 4.37a |
| 17 | 3.62 ± 4.62 | 6.6 ± 0.5 | 88.91 ± 3.2 | 0.26 ± 0.37b | 4.64 ± 0.36a | 73.1 ± 4.37a |
| 18 | 3.26 ± 4.3 | 6.6 ± 0.49 | 88.98 ± 3.14 | 0.24 ± 0.37b | 4.64 ± 0.36a | 73.1 ± 4.37a |
| 19 | 3.12 ± 4.29 | 6.61 ± 0.48 | 89.05 ± 3.09 | 0.22 ± 0.3b | 4.64 ± 0.36a | 73.11 ± 4.38a |
| 20 | 2.99 ± 4.23 | 6.61 ± 0.48 | 89.11 ± 3.04 | 0.22 ± 0.27b | 4.65 ± 0.36a | 73.11 ± 4.38a |
The values are shown as mean ± SD (n = 8).
MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate.
aSignificant difference in the means between MCa and MCaK bead using Student’s t-test at p < 0.05.
bSignificant difference in the medians between MCa and MCaK bead using Mann–Whitney rank sum test at p < 0.05.
Approximately 90% of the cumulative metronidazole was released from the MCa and MCaK beads on days 5 and 3, respectively, showing that the MCaK beads discharged their contents more quickly. The percentages of cumulative metronidazole released from both beads were significantly different (p < 0.05), except on day 4 (p = 0.42). The amount of metronidazole released for 20 days was greater for the MCa beads than the MCaK beads by 6.61 ± 0.48 mg and 4.65 ± 0.36 mg, respectively. The percentage of drug release was also higher for the MCa beads (89.11% ± 3.04%) than the MCaK beads (73.11% ± 4.38%) (Supplementary Fig. 1). The MCa and MCaK beads released metronidazole at levels greater than 0.5 µg/mL for 20 days and 14 days, respectively (Fig. 1), which was the minimum inhibitory concentration of metronidazole against anaerobic bacteria isolated from dogs with periodontal disease [21].
Fig. 1. Concentration of eluted metronidazole from MCa and MCaK beads during the 20-day experimental period (n = 8).
MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate; MIC, minimum inhibitory concentration.
Microstructure of various Ca beads after elution
SEM images were taken to compare the cross-section and surface characteristics of the different calcium beads. The microstructure of the beads was identical based on calcium crystals presented as long needle-like shapes that were tightly packed together to form a compact structure. The internal structures of the beads had comparable porosity. The calcium crystals on the outer surfaces of the CaK and MCaK beads were sparser than those of Ca and MCa (Fig. 2).
Fig. 2. Scanning electron microscopy images showing the microstructure of calcium beads: Ca (A, B), CaK (C, D), MCa (E, F), and McaK (G, H) on day 0 (before elution), where subfigures A, C, E, and G show outer surface view and subfigures B, D, F, and H show cross-sectional view. Scale bars = 50 µm (A–H).
Ca, calcium sulfate; CaK, calcium-potassium sulfate, MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate.
After one day of immersion in PBS, the outer surfaces of all bead types changed (Fig. 3). The thinning of the calcium crystals indicated the dissolution of calcium on the outer surface of the crystals, while the interior structure of the beads remained unchanged (Supplementary Fig. 2). The outer modifications of the CaK and MCaK beads differed from those of the Ca and MCa beads, which was consistent with the results of the MCaK beads releasing more metronidazole than the MCa beads on day 1.
Fig. 3. Scanning electron microscopy images showing surface view of calcium beads: Ca (A, B), CaK (C, D), MCa (E, F), and McaK (G, H) 1 day and five days after elution, where subfigures A, C, E, and G show beads one day after elution and subfigures B, D, F, and H show beads five days after elution. Scale bars = 50 µm (A–H).
Ca, calcium sulfate; CaK, calcium-potassium sulfate, MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate.
The outer surface of the beads was significantly thinner after immersion for 5, 7, and 21 days, particularly for the CaK beads (Figs. 3 and 4). The calcium crystals of the CaK and MCaK beads were more fragile and prone to splitting than the Ca beads. The beads not submerged in PBS appeared to have an unaltered interior microstructure (Supplementary Figs. 2 and 3).
Fig. 4. Scanning electron microscopy images showing the surface view of calcium beads: Ca (A, B), CaK (C, D), MCa (E, F), and McaK (G, H) at seven days and 20 days after elution, where subfigures A, C, E, and G show beads seven days after elution and subfigures B, D, F, and H show beads 20 days after elution. Scale bars = 50 µm (A–H).
Ca, calcium sulfate; CaK, calcium-potassium sulfate, MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate.
After immersion for 21 days, the outer surface of all beads (particularly the MCa and MCaK beads) was significantly thinner (Fig. 4). There were no discernible alterations to the inside microstructure of the calcium crystals (Supplementary Fig. 3).
Antibacterial activity of metronidazole beads after storage
The MCa and MCaK beads exhibited antibacterial activity on days 0, 7, 14, and 21 of the experimental period, with a noticeable inhibition zone against B. subtilis around the bead samples. There was no inhibitory zone for either the Ca or the CaK beads in the absence of metronidazole (Fig. 5). The antibacterial activity of the MCa and MCaK beads was maintained at room temperature and 4°C for 21 days, and there were no significant differences (ANOVA, p = 0.113) (Table 4).
Fig. 5. Metronidazole beads showing antibacterial activity with the zone of inhibition against B. subtilis after preparation (day 0).
Ca, calcium sulfate; CaK, calcium-potassium sulfate; MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate.
Table 4. Antibacterial activity of MCa and MCaK beads stored at room temperature and 4°C on days 0, 7, 14, and 21 was determined using an agar diffusion test.
| Day | Inhibition zone (mm) | |||
|---|---|---|---|---|
| Storage at room temperature | Storage at 4°C | |||
| MCa | MCaK | MCa | MCaK | |
| 0 | 28.783 ± 1.177 | 28.323 ± 0.650 | - | - |
| 7 | 29.857 ± 0.789 | 30.363 ± 0.652 | 29.710 ± 1.018 | 29.090 ± 0.249 |
| 14 | 29.213 ± 1.133 | 29.310 ± 0.802 | 29.190 ± 0.208 | 29.253 ± 1.242 |
| 21 | 29.687 ± 0.794 | 30.510 ± 1.279 | 29.830 ± 0.632 | 28.170 ± 1.068 |
The values (mean ± SD) (n = 3) with no significant difference among all treatments using ANOVA, p = 0.113.
MCa, metronidazole-calcium sulfate; MCaK, metronidazole-calcium-potassium sulfate.
DISCUSSION
Throughout the 20 days of the drug release study, the MCa and MCaK beads released metronidazole continuously. High metronidazole levels were eluted from the beads on the first day, after which the metronidazole levels decreased gradually. The amounts of metronidazole released from the MCa and MCaK beads were above the MIC50 and MIC90 values against anaerobic Porphyromonas spp. and Prevotella spp. at 0.125 ppm and 0.5 ppm over 20 and 14 days, respectively. These bacteria were isolated from dogs with periodontal disease [21]. Approximately 54% of the aerobic and anaerobic periodontal bacteria isolated from the rabbit abscesses were susceptible to metronidazole (MIC < 1 µg/mL) [22]. Therefore, the amounts of metronidazole eluted from the MCa and MCaK beads in the present study were greater than 1 µg/mL for 20 and 12 days, respectively. In addition, the high concentration of eluted antibiotics was beneficial for preventing and treating biofilm and resistant bacteria [23,24].
Metronidazole is an effective concentration-dependent antimicrobial drug when its concentration exceeds the MIC by at least 10-fold [25]. In the present study, the release level of the drug was 10 times (equal to 5 µg/mL) the MIC of 0.5 µg/mL for the MCa and MCaK beads for 15 and 9 days, respectively. Consequently, a substantial metronidazole release of MCa and MCaK could effectively treat certain infections. Biofilms are a major obstacle preventing the drug from destroying the infection, with the administration of systemic antibiotics resulting in inadequate drug levels at the site of infection or causing systemic toxicity. In addition, local-high drug levels can eradicate biofilm-forming organisms [26,27,28,29]. The choice of antibiotic used in the preparation of antibiotic beads should consider the impact of the drug susceptibility on pathogens. In addition, bacteriostatic antibiotics should be avoided in osteomyelitis treatment [30]. The use of antibiotic beads, in conjunction with surgical intervention for bone infections in canine and feline cases, has produced predominantly favorable outcomes. Frequently, the occurrence of reinfection after applying the antibiotic beads can be attributed to the selection of a drug that is not susceptible to the causative agent [3].
Drug release from the MCa and MCaK beads corresponded physically with the SEM images of both bead types. The calcium needle crystals were farther apart on the outer surface of the CaK and MCaK beads than on the Ca and MCa beads. When immersed in the PBS solution, the outer surface of the calcium crystals became gradually thinner, indicating that the release of calcium beads occurred more at the periphery and that the MCaK beads discharged metronidazole more rapidly than the MCa beads due to the loosened calcium crystals. The internal structure of the beads showed no obvious changes. The drug release from the bead was assumed to be biphasic, where the drug is released from the bead surface and cracks. The drug was released from inside through the interconnected pores [31]. The metronidazole drug contained in the structure of the outer calcium crystals was dissolved in the PBS solution used to soak the beads. Therefore, the initial drug release rate was elevated. Subsequently, the drug within the structure of the beads became available through the interconnected pores. In the present study, the weight of the MCa and MCaK beads soaked in PBS solution decreased by 3%–10%, and the Ca crystals on the surface of the beads were notably thinner. A hydrophilic drug can be released easily from the calcium bead structure [32], and metronidazole beads could be eluted readily from the calcium beads. Furthermore, calcium crystals disintegrate or dissolve in solution and the body [1,33,34]. Therefore, this calcium dissolution is beneficial for minimizing bacterial colonization and biofilm formation on the beads.
Parker et al. [10] reported that Ca beads prepared with a 3% potassium sulfate solution released more daptomycin than Ca without potassium sulfate, which was different from the results from the present study. They reported that antibiotics added to the material affected the calcium crystal structure of the antibiotic beads, resulting in variable drug release abilities [35,36]. In contrast, Parker et al. [10] pre-mixed the Ca in the daptomycin-Ca pellet with water or potassium sulfate solution, which differed from the present study. When the solid powder and liquid were combined first, as reported by Parker et al. [10], the mixture could not be molded because daptomycin prevented the transformation of Ca hemihydrate into Ca dihydrate or gypsum [37].
Combining the powder or solution form of antibiotics (such as amikacin, cefazolin, clindamycin, gentamicin, tobramycin, and vancomycin) with Ca powder produces antibiotic Ca beads. Some antibiotics, including ciprofloxacin, cefepime, ceftriaxone, and daptomycin, are prepared differently by mixing Ca powder into water or solution until a paste is formed and the antibiotic powders are added [38].
The MCa beads were heavier than the MCaK beads. On the other hand, after immersion in PBS solution, the weight of MCa beads decreased approximately three times less than for the MCaK beads. This may have been due to the use of a potassium sulfate solution instead of water, in which the potassium sulfate solution accelerated the crystallization of Ca [10,39]. In the present study, SEM suggested that the MCa beads had a denser crystal structure than the MCaK beads. Therefore, the metronidazole on the outside dissolved more readily in the PBS solution. The MCaK crystals were more dispersed than the MCa beads. Therefore, the MCaK beads contained less drug than the MCa beads. Furthermore, the calcium crystals had a distinct structure on the different antibiotic Ca beads [36]. Therefore, the components used to produce the antibiotic beads can influence the structure and drug release rate.
In the present study, there was no difference between the levels of antibacterial activity of the MCa and MCaK beads stored for 21 days at either room temperature or at 4°C. This result was consistent with studies involving vancomycin and tobramycin Ca beads, which retained their antimicrobial properties after at least 120 days of storage at room temperature and ambient humidity [40]. In addition, cefazolin-calcium beads maintained their antibacterial activity for at least six months when stored at either room temperature or 4°C [19].
Ramos et al. [18] examined the release of metronidazole from PMMA beads prepared using a metronidazole-to-PMMA ratio of 1:40, with a metronidazole percentage of 2.17%, which was comparable to the Ca concentration of 2.43% in the present study. The elution of metronidazole from the PMMA beads was characterized by the high initial release followed by the gradual decline in the amount of metronidazole released, which was 1,093 µg over 21 days (10 PMMA spheres, 6.4 mm diameter), representing 3.53% of the drug released from the beads. In comparison, in the present study, the amounts of metronidazole released over the 20 days from the MCa and MCaK beads were 6,610 µg and 4,650 µg, respectively, representing 89.11% and 73.11% of the drug present in the beads, respectively. These studies agreed with other investigations that showed that the antibiotic release from PMMA beads was less than that from Ca beads [19]. The preparation of metronidazole with PMMA revealed prolonged polymerization during preparation [18]. The present study was the first to investigate metronidazole release from Ca beads, with MCa beads having the potential to prevent and treat topical infections because of their simplicity of preparation and high drug release rate.
In conclusion, the metronidazole–calcium beads showed a sustained high drug-release efficacy throughout the 20-day study. The volume of fluid and fluid exchange in the infected area may differ from the results of this in vitro study. Therefore, MCa beads could control or prevent localized infections where the amount of released metronidazole is greater than the MIC for most anaerobes. Furthermore, it could be used in conjunction with systemic antibiotic administration in severe infections. The beads can be stored at ambient temperature or in the refrigerator for at least 21 days and retain effective antibacterial activity. The MCa beads were more stable in solution and could discharge more drug than the MCaK beads. Therefore, using a potassium sulfate solution as a catalyst in producing MCa beads may be unnecessary. Further development involving the clinical application of metronidazole beads will be essential for conducting in vivo investigations.
Footnotes
Funding: This research was supported by grants from the Kasetsart Veterinary Development Funds, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.
Conflict of Interest: The authors declare no conflicts of interest.
- Conceptualization: Udomkusonsri P.
- Formal analysis: Ithisariyanont B, Poapolathep S.
- Funding acquisition: Udomkusonsri P.
- Methodology: Ithisariyanont B, Poapolathep S, Poapolathep A.
- Writing - original draft: Ithisariyanont B.
- Writing - review & editing: Ithisariyanont B, Udomkusonsri P.
SUPPLEMENTARY MATERIALS
Cumulative percentage of metronidazole eluted from MCa beads and MCaK beads during the 20-day experiment. Values are mean ± SD (n = 8).
Scanning electron microscopy images showing cross-sectional views of calcium beads: Ca (A, B), CaK (C, D), MCa (E, F), and MCaK (G, H) on day 1 and five days after elution, where subfigures A, C, E, and G show beads on day one after elution and subfigures B, D, F, and H show beads five days after elution. Scale bars = 50 µm (A–H).
Scanning electron microscopy images showing the cross-sectional view of calcium beads: Ca (A, B), CaK (C, D), MCa (E, F), and MCaK (G, H) on seven days and 20 days after elution, where subfigures A, C, E, and G show beads seven days after elution and subfigures B, D, F, and H show beads 20 days after elution. Scale bars = 50 µm (A–H).
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Associated Data
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Supplementary Materials
Cumulative percentage of metronidazole eluted from MCa beads and MCaK beads during the 20-day experiment. Values are mean ± SD (n = 8).
Scanning electron microscopy images showing cross-sectional views of calcium beads: Ca (A, B), CaK (C, D), MCa (E, F), and MCaK (G, H) on day 1 and five days after elution, where subfigures A, C, E, and G show beads on day one after elution and subfigures B, D, F, and H show beads five days after elution. Scale bars = 50 µm (A–H).
Scanning electron microscopy images showing the cross-sectional view of calcium beads: Ca (A, B), CaK (C, D), MCa (E, F), and MCaK (G, H) on seven days and 20 days after elution, where subfigures A, C, E, and G show beads seven days after elution and subfigures B, D, F, and H show beads 20 days after elution. Scale bars = 50 µm (A–H).