Histomorphometric and microbiological assessment of photodynamic therapy as an adjuvant treatment for periodontitis: a short-term evaluation of inflammatory periodontal conditions and bacterial reduction in a rat model

Abstract

Objective: The aim of this study was to investigate the short-term effects of photodynamic therapy (PDT) in periodontal tissue when it is used as an adjuvant treatment for periodontitis.

Background data: PDT has been used as an adjuvant in the combat of local infections, such as periodontitis, and combines a photosensitizer (PS) with a light source to induce reactive oxygen species (ROS) and kill microbial cells.

Methods: Fifty healthy male rats were used in this study. Periodontitis was induced by placing a cotton ligature around the upper left second molar in a subgingival position. Posterior maxillas were removed and histologically prepared with hematoxylin & eosin (H&E) staining techniques. PDT was performed with a diode laser (λ=660 nm) with an output power of 100 mW. Methylene blue aqueous solution (100 μM) was used as the PS while control group used phosphate buffered saline (PBS). Collagen organization, inflammatory infiltrate, and bone loss were evaluated. Bacterial samples were collected before and immediately after treatment to determine bacterial reduction.

Results: The experimental group that was treated with PDT presented better periodontal healing, as measured by collagen organization, inflammatory infiltrate, and bone loss. Significant bacterial reduction was achieved following treatment with or without PDT compared to control, with a higher microbial reduction observed in the PDT group.

Conclusions: PDT used as an adjuvant treatment showed effective short-term control of periodontitis infection.

FIG. 1.

Induction of periodontitis on day −7. After 7 days, all treatments were performed. The animals were euthanized after 8 h, 24 h, 48 h, and 7 days.

FIG. 2.

Induction of periodontitis on day −15. After 15 days, all treatments were performed and microbiological analyses were subsequently performed.

FIG. 3.

(A) An example of a ligature surrounding the second upper molar. (B) An example of the scaling and root planing therapy (C) Methylene blue photosensitizer staining the periodontal tissue. (D) The laser irradiation of the inflamed tissue.

FIG. 4.

(A) Healthy periodontal tissue with fewer inflammatory cells and organized connective tissue. (B) Loss of the interproximal architecture as well as destruction of the periodontal tissue before treatment. (C) Reorganization of the tissue architecture (SRP, 48 h after treatment). (D) Group SRP+PDT shows better tissue organization at the same time point (compare C and D).

FIG. 5.

The data are means of bone loss and standard deviation. The Y axis represents the percentage of bone loss of the two groups evaluated in this study. The control shows the bone level after periodontitis induction and before treatment. Differences between the groups were not observed at 8, 24, and 48 h after treatment. After 7 days, the SRP+PDT group presented a statistically significant gain in bone level compared to the control and SRP groups (p<0.05). The SPR group showed additional bone loss compared to the control group.

FIG. 6.

Representative amplicons of A. actinomycetemcomitans reference DNA (left) and clinical samples (right). PCR generated amplicons in the electrophoresis gel (1.5%). DNA from A. actinomycetemcomitans HK 1651 strain as positive control (+) and clinical samples (lane 1, before treatment; lane 2, after treatment) are characterized by a specific amplicon of 547 bp.

FIG. 7.

The mean recovery of (A) microaerophile and (B) anaerobic bacteria from periodontal tissue before and after treatment. Error bars show the standard deviation.