Antimicrobial properties of drug-containing electrospun scaffolds

Abstract

Endodontic treatment of the infected immature tooth has undergone a dramatic change. Conventional endodontic treatment can control infection, but root development usually remains impaired. A novel regenerative endodontic procedure, the revascularization method, can now control the infection and enable such teeth to continue root development. This is done by creating a fibrin-matrix scaffold in the antibiotic treated root canal space (RCS). Dental stem cells and growth factors have been able to continue root development in such an environment. The fibrin-matrix scaffold is dependent on the induction of a blood clot into the RCS, and this cannot always be predictably induced. PDS is a biocompatible material that can be electrospun to provide a matrix for cells and growth factors and perhaps improve on the blood clot induced fibrin scaffold by incorporating metronidazole as an adjuvant antimicrobial. A metronidazole containing electrospun PDS scaffold was examined in vitro using a turbidimetric test, the modified direct contact test. This scaffold significantly inhibited growth of an anaerobic primary endodontic pathogen Porphyromonas gingivalis. This scaffold may improve the treatment of the infected immature tooth by providing a designed matrix for root regeneration while serving simultaneously as an antibiotic drug delivery device to disinfect the RCS. The aim of this study is to evaluate in vitro the property of a synthetic scaffold to function as an antibacterial drug delivery device. PDS*II (polydioxanone) suture was obtained from Ethicon, INC. (Somerville, NJ) and was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP (Sigma Aldrich). Three different scaffolds were electrospun onto an aluminum foil background; (1) control scaffold with no antibiotic incorporated, (2) scaffold with 5.0-wt % metronidazole incorporated, and (3) 25-wt % metronidazole incorporated. All scaffolds were cut using a 4-mm diameter biopsy punch under aseptic conditions and removed from foil, control scaffold (n = 64), scaffold containing 5.0-wt % metronidazole (n = 32), and scaffold containing 25-wt % metronidazole (n=32). Experimental scaffolds were placed in a 96- well sterile flat bottom microtiter plate. Porphyromonas gingivalis a known primary endodontic pathogen was grown in 5 ml of BHI + YE with 0.25 μl of vitamin K with incubation at 37°C under anaerobic conditions for 48 hours. Microplates were sterilized before inoculation with Pg with 400 μl of 70-percent EtOH for a minimum of 30 minutes then pipetted out. After sterilization the microwells were washed with 400 μl of sterile water and pipetted out. Group 1 (negative control) microwells (n = 8) contained control scaffold and 190 μl of broth only. Group 2 (positive control) microwells (n = 8) contained 190 μl of broth and Pg only. Group 3 microwells (n = 8) contained control scaffold, 190 μl of broth, and 10 μl of Pg inoculum. Group 4 microwells (n = 8) contained scaffold with 5 wt % metronidazole, 190 μl of broth, and 10 μl of Pg inoculum. Group 5 microwells (n = 8) contained scaffold with 25 wt % metronidazole, 190 μl of broth, and 10 μl of Pg inoculum. Group 6 contained 190 μl of uninoculated broth for spectrophotometer calibration. Sterile microplate lids were used to isolate microwells from the surrounding environment. Microplates were incubated at 37°C under anaerobic conditions for 48 hours. After 48 hours the microplates were read by using an endpoint reading in the spectrophotometer. This was repeated four times. Comparisons among the groups for differences in optical density as a measure of bacterial growth were made using mixed-model ANOVA, with a fixed effect for group and a random effect for experimental run. Pair-wise group comparisons were performed using Tukey's multiple comparisons procedure to control the overall significance level at 5 percent. The analyses were performed using the ranks of the data. Broth had significantly lower OD than all other groups (p < 0.0001). Broth+Pg and Broth+Pg+Scaffold had significantly higher OD than 5-wt % Metro (p < 0.0001) and 25-wt % Metro (p < 0.0001), but Broth+Pg and Broth+Pg+Scaffold were not significantly different from each other (p = 0.97). 5-wt % Metro and 25-wt % Metro were not significantly different from each other (p = 0.24). From the results of our study, we concluded that the 5.0-wt % and 25-wt % metronidazole containing scaffolds significantly inhibited bacterial growth and could be effectively utilized for the endodontic regeneration procedure.