Photodynamic Therapy with Natural Photosensitizers in the Management of Periodontal Disease Induced in Rats

Abstract

This study aims to investigate the effect of new natural photosensitizers (PS) (based on oregano essential oil, curcuma extract, and arnica oil) through in vitro cytotoxicity and biological tests in rat-induced periodontal disease, treated with photodynamic therapy (aPDT). The cytotoxicity of PS was performed on human dental pulp mesenchymal stem cells (dMSCs) and human keratinocyte (HaCaT) cell lines. Periodontal disease was induced by ligation of the first mandibular molar of 25 rats, which were divided into 5 groups: control group, periodontitis group, Curcuma and aPDT-treated group, oregano and aPDT-treated group, and aPDT group. The animals were euthanized after 4 weeks of study. Computed tomography imaging has been used to evaluate alveolar bone loss. Hematological and histological evaluation showed a greater magnitude of the inflammatory response and severe destruction of the periodontal ligaments in the untreated group.. For the group with the induced periodontitis and treated with natural photosensitizers, the aPDT improved the results; this therapy could be an important adjuvant treatment. The obtained results of these preliminary studies encourage us to continue the research of periodontitis treated with natural photosensitizers activated by photodynamic therapy.

Keywords: computed tomography; cytotoxicity; gels; photodynamic therapy; photosensitizers.

Figure 1

Cell viability of photosensitizers on human dental pulp mesenchymal stem cells (dMSCs) and human keratinocyte cell lines (HaCaT) compared with untreated control. Each bar represents mean and standard deviation (n = 3).

Figure 2

Comparative analysis of CT images of experimental groups on the first day of treatment: (A) positive control group; (B) aPDT + curcumin group; (C) aPDT + oregano group; (D) laser therapy group. The arrow indicates the first molar on the left hemimandible where periodontal disease was induced.

Figure 3

Comparative analysis of CT images of experimental groups on 21st day of treatment: (A) positive control group; (B) aPDT + curcumin group; (C) aPDT + oregano group; (D) laser therapy group. The arrow indicates the first molar on the left hemimandibula where periodontal disease was induced.

Figure 4

Control group: (A) histopathological images from the dental crown and partially from the dental root area (hematoxylin-eosin); (B,C) higher resolution, 50 µm and 20 µm, of the marked zone with red.

Figure 5

Periodontisis group: (A,B) the superficial area of the inflammatory outbreak covered by tissue and fodder debris; (C) detail of the dental support device, with moderate-segmental osteoclastic resorption of the alveolar bone and suppurated periodontitis; (hematoxylin–eosin).

Figure 6

Laser group: (A) hyperplasia and marked hyperkeratosis of the gingival epithelium (hematoxylin–eosin); (B) high resolution, 50 µm, of the marked zone with red from image A; (C) numerous neutrophils with rare mononuclear and reactive fibroblasts.

Figure 7

GC group: (A) hyperplasia and marked hyperkeratosis of the gingival epithelium. The superficial gingival area presents a focal ulcer (minimal), covered by a serocellular crust mixed with cellular debris and forage (the area demarcated by the rectangle); (B) higher resolution, 50 µm, of the marked zone with red; (C) detail of inflammatory granulation tissue, with the abundance of neutrophils (viable and degenerate) in combination with rare mononuclear and reactive fibroblasts.

Figure 8

GO group: (A,B) hyperplasia and marked hyperkeratosis of the gingival epithelium, and partial replacement of the dental ligament with partially oriented fibro-vascularized connective tissue; (C) high resolution, 20 µm, of the marked zone with red from image B.

Figure 9

Means and standard deviations for all groups and variables without outliers: (a) WBC; (b) LYM; (c) MID; (d) GRA; (e) RBC; (f) HGB; (g) MVC; (h) MCH; (i) MCHC; (j) PLT. (Physiological values: WBC: 4–12 × 10⁹/L, LYM: 2–14.1 × 10⁹/L, MID: 0–0.98 × 10⁹/L, GRA: 0.1–5.4 × 10⁹/L, RBC: 9–15 × 10¹²/L, HGB 90–150 mg/dL, HCT: 24–45%, PLT: 250–750 × 10⁹/L).

Figure 10

(A). The treatment regimen followed in the periodontal disease induced in rats; (B) (a,b) different stages during aPDT procedure; (c) oregano and curcuma-based gels used in the study.