In vivo killing of Porphyromonas gingivalis by toluidine blue-mediated photosensitization in an animal model

Abstract

Porphyromonas gingivalis is one of the major causative organisms of periodontitis and has been shown to be susceptible to toluidine blue-mediated photosensitization in vitro. The aims of the present study were to determine whether this technique could be used to kill the organism in the oral cavities of rats and whether this would result in a reduction in the alveolar bone loss characteristic of periodontitis. The maxillary molars of rats were inoculated with P. gingivalis and exposed to up to 48 J of 630-nm laser light in the presence of toluidine blue. The number of surviving bacteria was then determined, and the periodontal structures were examined for evidence of any damage. When toluidine blue was used together with laser light there was a significant reduction in the number of viable P. gingivalis organisms. No viable bacteria could be detected when 1 mg of toluidine blue per ml was used in conjunction with all light doses used. On histological examination, no adverse effect of photosensitization on the adjacent tissues was observed. In a further group of animals, after time was allowed for the disease to develop in controls, the rats were killed and the level of maxillary molar alveolar bone was assessed. The bone loss in the animals treated with light and toluidine blue was found to be significantly less than that in the control groups. The results of this study show that toluidine blue-mediated lethal photosensitization of P. gingivalis is possible in vivo and that this results in decreased bone loss. These findings suggest that photodynamic therapy may be useful as an alternative approach for the antimicrobial treatment of periodontitis.

FIG. 1.

Effect of toluidine blue-mediated photosensitization on the viability of P. gingivalis on the gingival margins of the maxillary molars of rats. A range of light doses (6 to 48 J) and a range of toluidine blue concentrations (0.01 to 1.0 mg/ml) were used. Control animals were not administered toluidine blue and were not irradiated with laser light. In the case of animals administered 1.0 mg of toluidine blue per ml, no viable bacteria were detected with any of the light doses used.

FIG. 2.

Numbers of viable P. gingivalis organisms from samples taken from the palatal (open bars) and buccal (black bars) gingival margins of maxillary molars after photosensitization. Light energy doses of between 6 and 48 J were used in combination with toluidine blue concentrations ranging from 0.01 to 1.0 mg/ml. In the case of animals administered 1.0 mg of toluidine blue per ml, no viable bacteria were detected in either of the two sites with any of the light doses used.

FIG. 3.

Histological sections of the periodontal structures of rats photosensitized with 1.0 mg of toluidine blue per ml plus 48 J of laser light (a and b) and control animals which were not irradiated and not treated with toluidine blue (c and d). The sections show normal gingival epithelium (arrow) and sulcular epithelium (arrowhead) abutting the teeth (asterisk) (for clarity, the structures are labeled only on panel b). There is no evidence of ulceration or inflammation.

FIG. 4.

Fluorescence microscopy images showing the biodistribution of toluidine blue administered topically to the rat gingiva. (a, c, e, and g) Fluorescence images after application of toluidine blue at 1.0, 0.1, and 0.01 mg/ml and no toluidine blue, respectively. (b, d, f, and h) Corresponding sections stained with hematoxylin-eosin. Scale for each panel, 880 by 550 μm.

FIG. 4.

Fluorescence microscopy images showing the biodistribution of toluidine blue administered topically to the rat gingiva. (a, c, e, and g) Fluorescence images after application of toluidine blue at 1.0, 0.1, and 0.01 mg/ml and no toluidine blue, respectively. (b, d, f, and h) Corresponding sections stained with hematoxylin-eosin. Scale for each panel, 880 by 550 μm.

FIG. 5.

Representative photographs showing the alveolar bone supporting the maxillary molars of rats administered toluidine blue (0.1 mg/ml) and irradiated with 48 J of laser light (a) and control animals which were not irradiated and not treated with toluidine blue (b).

FIG. 6.

Representative radiographs showing the alveolar bone of the maxillary molars of rats administered toluidine blue (0.1 mg/ml) and irradiated with 48 J of laser light (a) and control animals which were not irradiated and not treated with toluidine blue (b).

FIG. 7.

Resorption of alveolar bone of the maxillary molars assessed by morphometric measurement. The bone loss was regarded as the distance between the CEJ and the alveolar bone crest. Animals in the test group were treated with various concentrations of toluidine blue and irradiated with 48 J of laser light (T+L+). Control groups consisted of rats which were not irradiated with laser light and which were not administered toluidine blue (T−L−), rats which were not irradiated but which were administered toluidine blue (T+L−), and rats which were not administered toluidine blue but which were irradiated with 48 J of laser light (T−L+).

FIG. 8.

Resorption of alveolar bone of the maxillary molars assessed by radiographic measurement. The bone loss was regarded as the distance between the deepest bony defect and cusp tips. Animals in the test group were treated with various concentrations of toluidine blue and irradiated with 48 J of laser light (T+L+). Control groups consisted of rats which were not irradiated with laser light and which were not administered toluidine blue (T−L−), rats which were not irradiated but which were administered toluidine blue (T+L−)and rats which were not administered toluidine blue but which were irradiated with 48 J of laser light (T−L+).