Experimental animals and groups
After being fed adaptively for 1 week, 25 female SD rats at the age of 5 weeks old were randomly divided into five groups (N = 5/groups) as follows: (1) NaCl group, NaCl group with no ligation; (2) NaCl + ligation group, rats with ligature-induced periodontitis alone; (3) hBD3 + ligation group, rats with ligature-induced periodontitis that were treated with hBD3; (4) AuNPs + ligation group, rats with ligature-induced periodontitis that were treated with AuNPs; and (5) AuNPs-hBD3 + ligation group, rats with ligature-induced periodontitis that were treated with AuNPs combined with hBD3. Reagents and configurations.
hBD3 was commercially available from Peprotech (Rocky Hill, NJ, USA), and AuNPs with a diameter of 45 nm were synthesized by adopting a chemical reduction method. The concentrations of AuNPs and hBD3 were used according to our published data [13, 14].
Rat ligature-induced experimental periodontitis model
The rats were anesthetized with pentobarbital sodium (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at 50 mg/kg. The silk threads were soaked in medium containing P. gingivalis for 2 h ahead of time and were then used to ligate the bilateral maxillary second molars of the SD rats. The control group was treated with no ligation, and the ligated groups were treated with sterile silk thread. According to the groups, 100 μL of 5 μg/mL hBD3, 10 μM AuNPs, or 0.9% NaCl solution was injected into the mesial, central, and distal sides of the maxillary second molars; this process was repeated every 3 days. At the study endpoint, when the rats were 7 weeks old, they were euthanized, blood was collected from retro-orbital, and the maxillary bone, gums, and other tissues were collected and fixed in a 4% neutral paraformaldehyde solution.
Micro-CT scanning
The maxillary samples of the SD rats were soaked in a 4% paraformaldehyde solution and then were scanned by micro-CT with a Skyscan 1176 scanner (Bruker, Karlsruhe, Germany). The scanning layer thickness was 18 μm, the X-ray exposure time was 404 ms, the tube voltage was 70 kV, and the tube current was 353 μA. After scanning, 3D volume rendering technology from CTVox software was used to convert the 2D CT tomography images into 3D images proportionally and to reconstruct the images to measure the bone mineral density (BMD), bone volume (BV), and tissue volume (TV).
Detection of serum inflammatory factors
After the rats were anesthetized, the hair around the eyes was cut off, the eyeball was protruding out of the socket, a fine-walled Pasteur pipette (o.d. of 1–2 mm) was inserted into the corner of the eye socket underneath the eyeball, the tip was directed at a 45-degree angle toward the middle of the eye socket. Gentle downward pressure was applied and then released until the vein was broken and blood was visualized entering the pipette. When amounts of blood began filling the pipette, the Eppendorf tube was used to collect blood. After standing at room temperature for 2 h at 2000 × g and centrifuging at 4 °C for 10 min, the serum was transferred to a new centrifuge tube. ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to detect the concentrations of TNF-α, IL-6, and IFN-γ.
Histological and immunohistochemical analysis
The maxillary samples were soaked in 10% ethylenediaminetetraacetic acid (EDTA) for 4 weeks, dehydrated with an ethanol gradient, and embedded in paraffin. The sections of 5 μm thickness were obtained from sagittal aspects of the second molars. The sections were selected including the mesial and distal roots of the second molar and their root bifurcation. The sections were started to retain from the simultaneous appearance of the roots and root bifurcation of the second molars, until the roots and root bifurcation of the second molars disappeared. Subsequently H&E and Masson staining, as well as the specific tartrate-resistant acid phosphatase (TRAP) staining, was used to evaluate bone resorption and osteoclast activity in the tissue (H&E, Masson, and TRAP staining kits were all obtained from Servicebio, Wuhan, China). The number of osteoclasts per square millimeter around the alveolar bone surface was counted and analyzed on the basis of the TARP staining images. Alkaline phosphatase (ALP) (antibody obtained from Huabio, Hangzhou, China, #ET1601-21, rabbit pAb), osteoprotegerin (OPG) (antibody obtained from Huabio, #EM1701-98, rabbit pAb), and receptor activator of NF-κB ligand (RANKL) (antibody obtained from Servicebio, Wuhan, China, #gb11235, rabbit pAb) expression levels in the periodontal tissues around maxillary second molars were measured by the immunohistochemical method. Stained sections were scanned using CaseViewer software (3Dhistech, Budapest, Hungary). Then, the quantitative analyses of ALP, OPG, and RANKL expression were performed with ImageJ software.
Statistical analysis
In our study, all statistical computations were performed using GraphPad Prism 6.0 software, and the experimental data of each group are expressed as the mean ± standard deviation (SD). The statistical significance was analyzed using one-way analysis of variance (ANOVA) followed by the Tukey post hoc test. When the P value < 0.05, the difference was considered statistically significant.