In Vitro Degradation and Physical Characterization of Antimicrobial Electrospun Scaffolds with Aligned Fibers

Meghan Wright (Institute of Biomaterials and Biomedical Engineering, University of Toronto)
Meilin Yang (Faculty of Dentistry, University of Toronto)
Paul Santerre (Institute of Biomaterials and Biomedical Engineering, Faculty of Dentistry, University of Toronto)

Introduction

Electrospun scaffolds may be used to create tissue engineered constructs for the regeneration of the gingival connective tissues that are destroyed during the progression of periodontal disease. However, the use of a synthetic material in the infectious oral environment has the potential to lead to a biomaterial-associated infection, while periodontal pathogens continue to delay or inhibit healing in periodontal tissue regeneration strategies despite systemic and topical administration of antibiotics and antiseptics [1]. Antibiotic incorporated directly into electrospun scaffolds impose challenges due to drug aggregation that can affect the physical properties of the scaffold and result in a burst release of antibiotic [2]. Polymer-based antimicrobial delivery systems have been explored as a means to deliver drug in a more sustained, controlled manner [3,4]. The objective of the current study was to incorporate an antimicrobial polymer containing ciprofloxacin into aligned electrospun nanofiber scaffolds and characterize the scaffolds’ properties. It is hypothesized that the antimicrobial polymer will promote a uniform distribution of drug throughout the fibers such that as the antimicrobial polymer and scaffold matrix degrade by hydrolysis, there will be a sustained release of antibiotic.

Materials and Methods

Scaffolds were made using a degradable polyurethane, synthesized according to previous methods with hexane diisocyanate:polycarbonate diol:butane diol in a molar ratio of 3:2:1 [5]. A proprietary antimicrobial polymer was incorporated via blend electrospinning at concentrations corresponding to 7 and 15wt% ciprofloxacin, or antimicrobial, with respect to the polyurethane. Scaffolds with 15wt% ciprofloxacin HCl (in non-polymeric form) were also fabricated. Polyurethane and antimicrobials were dissolved in hexafluoro-2-propanol and injected at a rate of 0.5 ml/h onto a cylindrical mandrel rotating at 1150 rpm (18 kV voltage difference). Fiber morphology and scaffold surface chemistry were investigated using scanning electron microscopy (SEM) and attenuated total reflectance Fourier transform IR spectroscopy (ATR-FTIR: top 5-10µm). Scaffold microstructure was investigated via differential scanning calorimetry. The distribution of ciprofloxacin in the fibers was imaged using confocal microscopy. Matrix degradation and antimicrobial release studies were carried out in a simple chemical hydrolysis model. Scaffolds were incubated in Dulbecco's PBS at 37°C for 7 days. Antimicrobial polymer and ciprofloxacin release were measured by high performance liquid chromatography with a photo-diode array detector.

Results

There was greater surface roughness visible on the fibers of scaffolds made with ciprofloxacin HCl (in non-polymeric form) when compared to scaffolds with antimicrobial polymer (SEM images not shown). The antimicrobial polymer and ciprofloxacin HCl alter intermolecular hydrogen bonding on the surface of the polyurethane scaffolds, as seen via ATR-FTIR (Fig. 1). Ciprofloxacin added in non-polymeric form significantly increased the ratio of hydrogen bonded to free carbonate carbonyls and decreased the ratio of hydrogen bonded to free urethane amines with respect to both the antimicrobial polymer scaffolds as well as scaffolds with no antimicrobial. The glass transition temperature of the scaffolds did not undergo any change (data not shown). Confocal microscopy revealed uniform fluorescence throughout the fibers of the 7 and 15wt% antimicrobial scaffolds (Fig. 2). Scaffolds with 15wt% ciprofloxacin HCl showed aggregated fluorescent ciprofloxacin in the fibers and outside the fibers in clumps. At 7 days, the scaffolds with 7wt% antimicrobial had a cumulative release of 7.37±0.87% of the total loaded antimicrobial polymer, while the 15wt% scaffolds had a cumulative release of 80.19±11.94% of the antimicrobial polymer (n= 3±SD) (Fig. 3). The scaffolds with 15wt% ciprofloxacin HCl released 96.14±9.83% of the loaded antimicrobial within 1 hour, while the scaffolds with 15wt% antimicrobial in polymeric form had only released 24.27±3.59%.

Discussion and Conclusion

Preliminary results reveal that the release of antimicrobial containing molecules is sensitive to the concentration loaded into the fibers, which is consistent with the findings of other researchers investigating drug-loaded nanofibers [6,7]. Cumulative release was approximately ten times greater at day 7 for the 15wt% antimicrobial scaffolds than the 7wt% scaffolds. It is hypothesized that following initial release, pores may have formed rapidly in the fibers of the high concentration scaffolds which accelerated further release. Additionally, an increase in the wettability of the scaffolds due to higher hydrophilic drug content may increase the penetration of water and result in a greater drug diffusion rate [8]. These hypotheses will be investigated in future work. The scaffolds with antimicrobial polymer and ciprofloxacin HCl loaded at the same relative concentration of antibiotic released similar amounts of antimicrobial containing molecules by day 7. However, the scaffolds with ciprofloxacin in non-polymeric form released the majority of the loaded drug 96.14±9.83% within the first hour. Confocal microscopy of the 15wt% ciprofloxacin HCl scaffolds revealed the presence of aggregated drug in the fibers and in non-fiber clumps, and roughness on the fibers of the 15wt% ciprofloxacin HCl scaffolds (SEM image not shown) is hypothesized to be due to aggregates of drug accumulating on the surface of the scaffolds. Drug aggregation would increase the rapid diffusion of antibiotic from the scaffolds, and may explain the fast burst release of drug from the 15wt% ciprofloxacin HCl scaffolds.

Figure 1. Ratio of hydrogen bonded to non-hydrogen bonded peak areas for characteristic peaks in the ATR-FTIR spectra. Data are the mean ± SD (n =3). *Represents significant difference between groups (p < 0.05).

Figure 2. Confocal images showing ciprofloxacin distribution in the scaffold fibers. A) 0wt% antimicrobial, B) 7wt% antimicrobial (polymeric form), C) 15wt% antimicrobial (polymeric form) and D) 15wt% ciprofloxacin HCl. Aggregated drug is seen in non-fiber clumps (white arrows). Scale bars = 50 µm.

Figure 3. Percent cumulative release of antimicrobial containing molecules from the 7wt% antimicrobial (polymeric form), 15wt% antimicrobial (polymeric form) scaffolds, and 15wt% ciprofloxacin HCl scaffolds. Data are the mean±SD (n=3). *Denotes a significant difference (p < 0.05).

Acknowledgements

Interface Biologics Inc. for consulting support on polymer characterization. NSERC Synergy grant 430828. NSERC CGS M. Milligan Scholarship.

References

[1] Bottino et al. Dental Materials. (2012) 28:7:703. [2] Toncheva et al. European Journal of Pharmaceutical Sciences. (2012) 47:4:642. [3] Yang, Santerre. Biomacromolecules. (2001) 2:134. [4] Woo, Mittelman, Santerre. Biomaterials (2000) 21:1235. [5] Tang et al. Journal of Biomedical Materials Research. (2001) 56:4516. [6] He et al. Journal of Biomedical Materials Research, Part A. (2009) 89:1:80. [7] Reise et al. Dental Materials. (2012) 28:2:179. [8] Weldon et al. Journal of Controlled Release.”¯(2012) 161:3:903.

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