Body piercing has become very popular in the past two decades, and it has been reported that approximately 50% of millennials receive at least one non-earlobe piercing [1
]. As most of these piercings are not performed in clinical settings, 17–46% of the piercings lead to complications [3
] including infection, metal allergy, bleeding, and tissue scarring [4
]. Among all these problems, local infection has been reported to be the most common type of piercing complication [6
]. Staphylococcus aureus
) and Streptococcus pyogenes
) (Lancefield Group A) are the most common pathogens causing these local infections [6
]. Particularly, as compared to the wounds stemming from the piercing of soft tissue, cartilage piercings (e.g., the auricle of the ear) have a greater predisposition to complications [9
]. These complications are due to the detachment of perichondrium and microfractures of the cartilage during piercing, which consequently leads to edema and bleeding into the cartilage [10
]. This trauma to the tissue ultimately results in a reduction of blood circulation to the relatively avascular cartilage tissue and increases the risk of infection.
Due to the avascular nature of cartilage, systemic antibiotic treatment through oral intake cannot be prescribed to patients with piercing infections in the auricle tissue, and the treatment for severe infections may require hospitalization and surgical intervention [11
]. It has been reported that a treatment delay greater than five days for cartilage infections can lead to severe outcomes, such as impaired hearing, ear deformation, and amputation of the auricle [10
]. There has been much effort to prevent infection resulting from wounds by administering a wide range of active agents to the wound locally: these agents include anti-infective [12
], anti-inflammatory [15
], and analgesic [18
] compounds. The common aftercare practice for preventing ear piercing infection is the regular application of antiseptics or antibiotics [18
] for a few weeks, which is not thoroughly effective as nearly 20% of all piercings lead to local infection [6
]. The median duration for the ear-piercing infection treatment using antibiotic therapy was reported as 16 days by Bellaud et al. [25
]. Therefore, it is imperative to develop alternative methods of prolonged and sustained piercing aftercare to prevent infection.
Many techniques have been used to reach sustained delivery of drugs, particularly antibiotics, in wound dressing applications. Although electrospinning of medicated nanofibers has been widely used recently to fabricate wound dressings [26
], the prolonged release of drugs from nanofibers remains a challenge. Yank et al. developed an electrospun wound dressing [29
]; however, 90% of the loaded antibiotic was released during the first 30 min of in vitro study. In another effort, electrospun drug loaded polylactic acid (PLA) scaffolds were used to achieve sustained drug release for wound dressing applications [30
]. However, the drug was released mainly in the first 72 h. Kim et al. enhanced the release of an antibiotic drug (cefoxitin sodium) out of electrospun PLGA scaffolds [31
]. The efficacy of the setup was proven against S. aureus
. However, the release of the drug reached up to one week with a rapid decrease in the zone of inhibition in the first two-hours for drug loaded scaffolds. Other fabrication methods have also faced similar challenges regarding the prolonged release. An emulsion solvent diffusion method was used to develop a drug loaded emugel microsponge to release mupirocin against S. aureus
]. However, the release was sustained only for 24 h. In another study, the spray drying technique was used to develop microparticles for controlling the delivery of mupirocin calcium [33
]. However, over 80% of the loaded drug was released during the first 72 h of the dissolution test. A different group reported a polyglyconate mesh dip coated in gentamicin loaded poly-lactic-co
-glycolic acid (PLGA) to develop a wound dressing [34
]. The inhibition of bacterial growth of the gentamicin loaded wound dressings maintained over two weeks. However, dip coating has limitations in terms of controlling the amount of the incorporated drug as well as thickness and geometry of the layers. Although great strides have been achieved in the field of active drug-eluting wound dressings, the release kinetics of drugs and bioactive compounds must be improved in terms of sustained and prolonged release to be used for piercing aftercare applications.
Three-dimensional (3D) printing has been widely used to fabricate patient-tailored drug-eluting systems to achieve arbitrary geometries and hence release profiles [35
]. However, the application of 3D printing is limited in drug-eluting wound dressings [39
]. Conventional high-temperature 3D printing techniques cannot be implemented as many of the active anti-infective compounds are heat- labile [44
]. Furthermore, although 3D printed wound dressings have shown improved drug-eluting characteristics, the release of the drugs has been limited to a few hours or days [40
]. Therefore, there is a need to develop alternative low-temperature 3D printing methods for the fabrication of constructs that provide a prolonged drug release for piercing infection prevention.
In this study, a drug-eluting bio-absorbable scaffold was developed and is intended to cover piercing studs. This “biopierce” will stay in human tissue following piercing for up to two weeks and to prevent the development of a wound infection. A novel, low-temperature 3D printing process was developed for fabrication of the PLGA biopierces, and the organic solvent (methyl ethyl ketone (MEK)) was successfully removed after printing without affecting the drug integrity (mupirocin). The amount of the release drug over time is quantified, and the efficacy of the printed biopierces against S. aureus is characterized through the measurement of the zone of inhibitions in bacterial tests. The release profile of varying grades (lactic to glycolic ratios) of PLGA and concentration of mupirocin is characterized over two weeks.
2. Materials and Methods
2.1. Preparation of Biomaterial Inks
PLGA, a bio-absorbable polymer suitable for wound dressing and drug delivery applications [34
], was selected as the polymeric matrix (PolySciTech, West Lafayette, IN, USA). Mupirocin, which is effective against S. aureus
and S. pyogenes
(Lancefield Group A), was selected as the antibacterial agent in this study [47
]. Mupirocin (AM26100, Biosynth Carbosynth, Compton, UK) was dissolved in MEK (Sigma Aldrich, St. Louis, MO, USA) and was stirred at 200 rpm at room temperature for one hour. Biomaterial inks were prepared by adding different concentrations of the mupirocin solution to PLGA (as shown in Table 1
). PLGA concentration in the biomaterial ink was set at 80% (w
) to maintain the optimum printable biomaterial ink concentration as described in our previous study [49
]. The biomaterial inks were left for overnight magnetic stirring at 200 rpm at room temperature to yield homogeneous biomaterial inks.
2.2. 3D Printing
A commercial 3D bioprinter (BioX, Cellink, Gothenburg, Sweden) with a pneumatic printhead (20340, Cellink, Gothenburg, Sweden) was used to print the biopierces as shown in Figure 1
. A 3-mL cartridge (CSC010311101, Cellink, Gothenburg, Sweden) was filled with the biomaterial ink and was left in an inverse position to remove air bubbles. An 8.00-mm hollow cylinder with 1.60 mm inner diameter and 2.00 mm outer diameter was designed using a computer-aided design (CAD) software (SolidWorks, Waltham, MA, USA). G-codes were generated with a slicer software (Slic3r, Repetier-Host, Willich, Germany) in such a way as to have only a single thread of the filament in each layer. Biopierces were printed with a 410 μm conical nozzle (NZ3220005001, Cellink, Gothenburg, Sweden) (Figure 1
a). A printed biopierce fitted on a piercing stud (R993-S, Studex, Gardena, CA, USA) is shown in Figure 1
b. The printing pressure was 550 kPa, and the printing speed was set at 1.00 mm/s to minimize any deformation of the printed construct while printing. A 10.00-mm length scaffold was printed for bacterial testing.
2.3. Post-Printing Requirements
Solvent removal: Proton nuclear magnetic resonance (1
H NMR) spectroscopy was used to confirm the complete removal of the solvent. Considering the vapor pressure of MEK at 20 °C (9.5 kPa), similar to our previous study [49
], the printed scaffolds were dried in a vacuum flask connected to a vacuum line of a fume hood for one week. A scaffold was placed in 1 mL of deuterium oxide (D2
O) 99.9% (Cambridge Isotope Laboratories Inc., Tewksbury, MA, USA) to leach any remaining solvent into D2
O overnight. The trace amount of MEK was detected by 1
H NMR spectroscopy (300 MHz, Bruker, MA, USA) with 16 scans setting [49
Drug integrity: The integrity of mupirocin after solvent removal and over the release duration was also investigated. Mupirocin contains structurally sensitive moieties, such as epoxide and ester, which upon hydrolysis could either alter or diminish its antibacterial properties. Liquid chromatography high-resolution mass spectrometry (LC-HRMS) was run for sample integrity analysis and was conducted as follows: Thermo Accela UHPLC Pump, Thermo Exactive HRMS fitted with an ESI source and Thermo PDA. A Kinetex core–shell 100 Å C18 column (2.1 × 50 mm, 1.7 μm, Phenomenex) was used with a mobile-phase flow rate of 0.5 mL/min and injection volume of 10 μL (all samples were prepared in CH3OH). The following elution method was used (A = H2O (0.1% formic acid), B = CH3CN (0.1% formic acid)): 5% B from 0.0 to 0.2 min, linear gradient from 5% B at 0.2 min to 99% B at 4.8 min, 99% B from 4.8 to 8.0 min, linear gradient from 99% B at 8.0 min to 5% B at 8.5 min, and 5% B from 8.5 to 10.0 min. The following HRMS parameters were used: positive ionization mode, mass resolution of 30,000, mass range of m/z 190 to 2000, spray voltage of 2.0 kV, the capillary temperature of 300 °C, S-lens RF voltage of 60.0%, maximum injection time of 10 ms, and 1 microscan. The system was controlled by Thermo Xcalibur software modules.
2.4. In Vitro Drug Release Study
Phosphate buffered saline (PBS) (VWRL0119, VWR, Radnor, PA, USA) was used as the dissolution medium (pH = 7.4). To appropriately simulate in vivo drug release conditions [40
], the scaffolds were incubated in 4 mL of PBS at 37 °C, and the medium was stirred at 200 rpm. To eliminate the effect of the medium removal on the drug release kinetics at each time point, the scaffolds were incubated 1 day, 2 days, 1 week, and 2 weeks prior to the tests. The scaffolds were taken out at the time points for the scaffold diffusion assay, and the dissolution medium was used in the in vitro drug release study and the disk diffusion assay.
The amount of released mupirocin from the scaffold over time was quantified via LC-HRMS. Each sample, in triplicate, was diluted by a half to ensure limit of detection was not reached prematurely. Peak areas were calculated from the extracted ion count of the released mupirocin, and a calibration curve was used to determine the total release. To develop the calibration curve, all parameters were maintained similar to the drug integrity study and the following concentrations were injected: 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, 75.00, and 100.00 µg/mL. The calibration curve was produced using the extracted ion count peak area for mupirocin.
2.5. In Vitro Mupirocin Efficacy
The European Committee on Antimicrobial Susceptibility Testing (EUCAST) reports the mupirocin minimum inhibitory concentration (MIC) of 1 mg/L for S. aureus
]. The efficacy of biopierces against S. aureus
was studied in terms of scaffold and disk diffusion assays by characterizing the zone of inhibition of the eluted mupirocin. S. aureus
25923™ (Oxoid, Nepean, ON, Canada) is the recommended quality control strain for disk diffusion testing following the Clinical and Laboratory Standards Institute (CLSI) standards [51
]. The isolate was grown from frozen stock and subcultured twice onto Columbia agar (Oxoid Columbia Blood Agar Base # CM0331) supplemented with 5% defibrinated sheep blood (SBA) (Quad Five Donor Sheep Blood Defibrinated # 610-500) before testing. Colonies were picked from 18 h growth on SBA using a cotton swab, re-suspended in tryptic soy broth (TSB) (BD Bacto Tryptic Soy Broth # 211825), and adjusted to 1.5 × 108
CFU/mL by visual comparison with a 0.5 McFarland standard. A bacterial lawn was inoculated on to cation-adjusted Mueller-Hinton agar plates (MHA) (BD BBL Mueller Hinton II Agar # 211438). All media were prepared by Atlantic Veterinary College (AVC) Central Services media preparation laboratory following manufacturers’ guidelines.
Scaffold diffusion assay: Printed scaffolds were placed onto inoculated MHA plates within 15 min of inoculation. The agar was sliced with sterile sharp tweezers and the scaffold embedded into the agar. MHA plates were incubated at 35 ± 2 °C in ambient air for 24 h. Images were taken of the agar plates and were processed by Fiji image processing software (ImageJ 1.53c, GNU General Public License, Bethesda, Rockville, MD, USA) [52
] to determine the zone of inhibition. The images were converted to 8-bit images, and the threshold was set between 151 and 170 to specify the zone of inhibition. The pixel size was 20 µm, and the uncertainty in the measurement of the zone of inhibition areas was 0.1%. To eliminate the effect of the size of the scaffolds, the area of the corrected zone of inhibition, simply called zone of inhibition, was calculated by deducting the scaffold area out of the zone of inhibition area. A drug-free scaffold was used as a negative control. All tests were implemented in triplicate and mean values and standard deviations were reported.
Disk diffusion assay: Sterile paper disks were moistened using 30 µL of the dissolution medium (2017-006, Whatman, Marlborough, UIK, GE Health Sciences, Chicago, IL, USA). The disks were dried at room temperature and were placed on MHA plates within 15 min of inoculation. MHA plates were incubated at 35 ± 2 °C in ambient air for 24 h [53
]. Images of agar plates were taken and were processed similar to the scaffold diffusion assay. The area of the corrected zone of inhibition, simply called zone of inhibition, was calculated by deducting the disk area out of the zone of inhibition area [34
]. A drug-free disk in the medium was used as a negative control, and a 200-µg mupirocin antimicrobial susceptibility disk (CT0523B, Oxoid, Hampton, VA, USA) was used as a positive control. All tests were implemented in triplicate and mean values and standard deviations were reported.
A novel drug-eluting bio-absorbable scaffold called biopierce, which is intended to cover piercing studs and prevent piercing infection, was introduced and characterized. In terms of the biomaterial ink composition, PLGA, mupirocin, and MEK were used as the biopolymer, antimicrobial agent, and solvent, respectively. A low-temperature 3D printing technique was used to print the biopierces, and 1H NMR spectroscopy was used after the vacuum drying of the scaffold to confirm the complete removal of MEK. Scaffolds in different PLGA composition and loaded mupirocin amounts were examined to characterize the release amounts of mupirocin. LC-HRMS was used to confirm the structural integrity of mupirocin and to quantify the amount of the released drug over time. In addition, the effective release of mupirocin against S. aureus, indicated as the zone of inhibition, was studied in the scaffold and the disk diffusion assays, respectively. The drug release of scaffolds decreased over time while the drug concentration in the dissolution medium increased. Increasing mupirocin concentration and lactic to glycolic ratio of PLGA increased the zone of inhibition in the scaffold and disk diffusion assays. PLGA 85:15 is recommended to be used as the polymeric matrix because of its superior drug release and printability characteristics.
The zone of inhibition was consistently maintained over 14 days that showed the efficacy of biopierces. The outcomes of this research can be used for tissue piercing applications as well as other wound dressing research. For further development of this research, incorporating analgesic and anti-proliferative agents is recommended. More importantly, the antimicrobial properties of mupirocin in the presence of other drugs must be investigated. Overall, this work is an exciting area of development that can lead to the next generation of drug-eluting wound dressings.