Effects of Experimental Agents Containing Tannic Acid or Chitosan on the Bacterial Biofilm Formation in Situ

Abstract

Chitosan and tannic acid are known for their antibacterial properties. In the present in-situ study, their antibacterial and anti-adherent effects on biofilm formation on enamel were investigated. Six subjects carried upper jaw splints with bovine enamel specimens, allowing in-situ biofilm formation. During the two-day trial, subjects rinsed with experimental solutions that contained either chitosan, tannic acid (pH = 2.5), tannic acid (pH = 7) or hydrochloric acid. Water served as the negative and chlorhexidine as the positive control. Rinsing occurred four or five times following two different rinsing protocols to investigate both the immediate and long-lasting effects. After 48 h of intraoral exposure, the dental plaque was stained with LIVE/DEAD^® BacLight, and fluorescence micrographs were evaluated by using the software ImageJ. The results were verified by scanning electron microscopy. Rinsing with chitosan resulted in little immediate antibacterial and anti-adherent effects but failed to show any long-lasting effect, while rinsing with tannic acid resulted in strong immediate and long-lasting effects. Except for a slightly lower antibacterial effect, the neutral solution of tannic acid was as good as the acidic solution. Hydrochloric acid showed neither an antibacterial nor an anti-adherent effect on dental biofilm formation. Experimental solutions containing tannic acid are promising anti-biofilm agents, irrespective of the pH values of the solutions. Chitosan, on the other hand, was not able to prevent biofilm formation.

Keywords: biofilm; chitosan; tannic acid.

Figure 1

Specimens fixed to splints. Four specimens were fixed with silicone impression material to individual upper jaw splints. After the two-day trial, specimens were examined with fluorescence and scanning electron microscope, each with two specimens.

Figure 2

Rinsing sequences every subject has to go through. In each round, one test substance was applied according to one rinsing protocol.

Figure 3

Coverage of specimens with bacteria in (%). The height of the bars corresponds to the mean value, and the line applied corresponds to ± the standard deviation. During two days of biofilm formation on bovine enamel specimens in situ, subjects rinsed 4 or 5 times with different experimental solutions. By using two different rinsing protocols, the immediate and long-lasting effects were tested. For the ex-vivo examination with a fluorescence microscope, the biofilm was stained with LIVE/DEAD^® BacLight™. Friedmann test: p < 0.05. Mouthwashes that differ significantly from water are marked with a for the first and b for the second protocol. HCl = hydrochloric acid, TA 2.5 = tannic acid (pH = 2.5), TA 7 = tannic acid (pH = 7) and CHX = chlorhexidine.

Figure 4

Representative LIVE/DEAD^® BacLight™ images. During two days of biofilm formation on bovine enamel specimens in situ, subjects rinsed 4 or 5 times with different experimental solutions. By using two different rinsing protocols, the immediate and long-lasting effects were tested. For the ex-vivo examination with a fluorescence microscope, the biofilm was stained with LIVE/DEAD^® BacLight™. Living bacteria are fluorescent green, and dead bacteria are fluorescent red. HCl = hydrochloric acid, TA 2.5 = tannic acid (pH = 2.5), TA 7 = tannic acid (pH = 7) and CHX = chlorhexidine.

Figure 5

Viability of bacteria in (%). The height of the bars corresponds to the mean value, and the line applied corresponds to ± the standard deviation. During two days of biofilm formation on bovine enamel specimens in situ, the subjects rinsed 4 or 5 times with different experimental solutions. By using two different rinsing protocols, the immediate and long-lasting effects were tested. For the ex-vivo examination with a fluorescence microscope, the biofilm was stained with LIVE/DEAD^® BacLight™. Friedmann test: p < 0.05. Mouthwashes that differ significantly from water are marked with a for the first and b for the second protocol. Wilcoxon Test: p < 0.05. Significant differences between both rinsing protocols are marked with c. HCl = hydrochloric acid, TA 2.5 = tannic acid (pH = 2.5), TA 7 = tannic acid (pH = 7) and CHX = chlorhexidine.

Figure 6

Representative scanning electron micrographs of specimens in 1000-fold magnification. Bovine enamel specimens were attached to upper jaw splints that were carried by subjects (n = 6) for 48 h. In the first protocol, rinsing occurred 5 times and, in the second protocol, 4 times with different experimental solutions. Micrographs show specimens covered by a biofilm that consists of the pellicle (1) and bacteria (2). HCl = hydrochloric acid, TA 2.5 = tannic acid (pH = 2.5), TA 7 = tannic acid (pH = 7) and CHX = chlorhexidine.

Figure 7

Representative scanning electron micrographs of specimens in 20,000-fold magnification. Bovine enamel specimens were attached to upper jaw splints that were carried by subjects (n = 6) for 48 h. In the first protocol, rinsing occurred 5 times and, in the second protocol, 4 times with different experimental solutions. Micrographs show specimens covered by a biofilm that consists of the pellicle (1) and bacteria (2). HCl = hydrochloric acid, TA 2.5 = tannic acid (pH = 2.5), TA 7 = tannic acid (pH = 7) and CHX = chlorhexidine.