Brazilian Green Propolis Carried in Lipid-Based Nanostructures: A Potent Adjuvant Therapy to Non-Surgical Periodontal Treatment in the Management of Experimental Periodontitis

Abstract

Objective: This study aimed to evaluate the effects of local use of Brazilian Green Propolis (BGP), either as an ethanolic extract (the most common formulation) or incorporated into lipid-based nanostructures, as an adjuvant therapy for non-surgical periodontal treatment in managing experimental periodontitis (EP) in ovariectomized rats. Methods: Fifty-six female Wistar rats underwent bilateral ovariectomies. After 10 weeks, a cotton ligature was placed around the lower first molar and remained in place for two weeks to induce EP. The ligature was removed, and the rats were randomly assigned in the groups NLT (n = 14), SRP (n = 14), SRP-BGPee (n = 14), and SRP-BGPlns (n = 14). In the NLT group, no local treatment was performed. The SRP group received scaling and root planing (SRP), along with irrigation using a physiological saline solution. The SRP-BGPee group underwent SRP and irrigation with ethanolic extract of BGP. The SRP-BGPlns group underwent SRP and irrigation with BGP-loaded lipid nanostructure (BGPlns). Each group received one SRP session followed by four irrigation sessions with the specified solutions, which were conducted immediately after SRP and subsequently after 2, 4, and 6 days. Euthanasia was performed at 7 and 28 days following the removal of the ligatures. The hemimandibles were processed for the following analyses: microtomographic analysis; histological analysis; histometric analysis of the percentage of bone tissue in the furcation region (PBT); and immunohistochemical analysis for tartrate-resistant acid phosphatase activity (TRAP), transforming growth factor beta 1 (TGFβ1), and osteocalcin (OCN). Results: The SRP-BGPlns group demonstrated superior periodontal tissue repair, reduced alveolar bone loss, fewer TRAP-positive cells (at 7 days), and higher levels of immunolabeling for TGFβ1 (at both 7 and 28 days) and OCN (at 28 days) compared to the other experimental groups. Conclusions: The irrigation with BGP is an effective adjuvant therapy for non-surgical periodontal treatment in managing EP in ovariectomized rats. Its application in lipid-based nanostructures proved to be more effective than the ethanolic extract form.

Keywords: green propolis; lipid-based nanostructures; periodontitis; rat; scaling and root planing.

Figure 1

Study design. (a) Scheme depicting the procedures performed over time in NLT, SPR, SPR-BGPee, and SRP-BGPlns groups. (b) Clinical aspect after the removal of the ligature, which was kept for two weeks. (c) SRP procedure. (d) Local irrigation with ethanolic extract of BGP after SRP. (e) Local irrigation with BGPlns after SRP.

Figure 2

BGP-loaded lipid nanostructure (BGPlns). (a) Schematic illustration of BGPlns. (b) Table containing the major phytochemical compounds identified in the BGP extract by HPLC-DAD (λ = 275 nm). (c) Electron micrograph showing the appearance of BGPlns after 90 days of preparation (storage at 25 °C). Scale bars: 200 nm.

Figure 3

Microtomographic appearance of the lower first molar at 28 days. (a,b) Graphs showing the mean linear distance from the cementoenamel junction to the alveolar bone crest (CEJ-ABC) (a) and Bone Volume/Total Volume (BV/TV) in the furcation region at 28 days. Statistical tests: Analysis of Variance (ANOVA) and Tukey post hoc test. (c–j) Microtomographic representative images of NLT (c,g), SRP (d,h), SRP-BGPee (e,i), and SRP-BGPlns (f,j) groups. Symbols: †, statistically significant difference compared to the NLT group; ‡, statistically significant difference compared to the SPR group; ¶, statistically significant difference compared to the SPR-BGPee group. Scale bars: 1 mm.

Figure 4

Bone tissue in the furcation region of the lower first molar. (a) Graph showing the percentage of bone tissue in the furcation region (PBT) in the different experimental groups. Statistical tests: Analysis of Variance (ANOVA) and Tukey post hoc test. (b–e) Photomicrographs showing the histological aspect of the furcation region of the lower first molar in the NLT (b), SPR (c), SPR-BGPee (d), and SRP-BGPlns (e) groups at 7 days. Abbreviations and symbols: *, inflammatory infiltrate; ct, connective tissue; ab, alveolar bone; †, statistically significant difference compared to the NLT group in the same period; ‡, statistically significant difference compared to the SPR group in the same period; ¶, statistically significant difference compared to the SPR-BGPee group in the same period. Staining: Hematoxylin and Eosin (H&E). Original magnification: (b–e): 200×. Scale bars: (b–e): 100 μm.

Figure 5

TRAP immunolabeling in the furcation region of the lower first molar. (a) Graph showing the number of TRAP-positive cells in the different experimental groups. Statistical tests: Analysis of Variance (ANOVA) and Tukey post hoc test. (b–e) Photomicrographs showing the immunolabeling pattern for TRAP in the furcation region of the lower first molar in the NLT (b), SPR (c), SPR-BGPee (d), and SRP-BGPlns (e) groups at 7 days. Abbreviations and symbols: ab, alveolar bone; white arrows, TRAP-positive cells; †, statistically significant difference compared to the NLT group in the same period; ‡, statistically significant difference compared to the SPR group in the same period; ¶, statistically significant difference compared to the SPR-BGPee group in the same period; §, statistically significant difference compared to 7 days in the same group. Original magnification: (b–e): 400×. Scale bars: (b–e): 30 μm.

Figure 6

TGFβ1 immunolabeling in the furcation region of the mandibular first molar. (a) Graph showing the density of immunolabeling for TGFβ1 in the different experimental groups. Statistical tests: Analysis of Variance (ANOVA) and Tukey post hoc test. (b–e) Photomicrographs showing the immunolabeling pattern for TGFβ1 in the furcation region of the mandibular first molar in the NLT (b), SPR (c), SPR-BGPee (d), and SRP-BGPlns (e) groups at 7 days. Abbreviations and symbols: ct, connective tissue; ab, alveolar bone; †, statistically significant difference in relation to the NLT group in the same period; ‡, statistically significant difference compared to the SPR group in the same period; ¶, statistically significant difference compared to the SPR-BGPee group in the same period; §, statistically significant difference compared to 7 days in the same group. Original magnification: (b–e): 400×. Scale bars: (b–e): 30 μm.

Figure 7

OCN immunolabeling in the furcation region of the lower first molar. (a) Graph showing the density of immunolabeling of OCN in the different experimental groups. Statistical tests: Analysis of Variance (ANOVA) and Tukey post hoc test. (b–e) Photomicrographs showing the pattern of immunolabeling for OCN in the furcation region of the lower first molar in the NLT (b), SPR (c), SPR-BGPee (d), and SRP-BGPlns (e) groups at 28 days. Abbreviations and symbols: ab, alveolar bone; †, statistically significant difference compared to the NLT group in the same period; ‡, statistically significant difference compared to the SPR group in the same period; ¶, statistically significant difference compared to the SPR-BGPee group in the same period; §, statistically significant difference compared to 7 days in the same group. Original magnification: (b–e): 400×. Scale bars: (b–e): 50 μm.