Animals
Forty-eight albino rats of the Wistar strain (age 6 months, mean weight 476±0.5 kg) obtained from a certified breeder were used in the study. All animals were housed in appropriately dimensioned cages under standard conditions of temperature in a light-controlled environment and were provided water and special diet ad libitum. The study protocol was approved by the appropriate local authority (Regierungspräsidium Darmstadt, Germany).
The present reporting followed the ARRIVE Guidelines [16].
Study design and surgical procedures
Following the extraction of both maxillary first molars, smooth-surfaced titanium mini-implants (Ustomed® Micro-Screws, Cross, ⌀ 1,2 mm, shortened to 2–3 mm) were inserted at respective sites and left to heal for 6 weeks. Subsequently, the animals had randomly received the following commonly applied antiresorptive/antiangiogenic medications, including n=8 animals each: (1) amino-bisphosphonate (Zoledronate 5mg/kg intravenous, Ribometa® 4mg/5ml, Hikma Pharma, Gräfelfing, Germany) (Zo), (2) RANKL inhibitor (Denosumab 60mg/kg subcutaneous, Prolia®, Amgen, Munich, Germany) (De), (3) antiangiogenic medication (Bevacizumab 5mg/kg intravenous, Avastin® 400 mg/16 ml, Roche Pharma, Grenzach-Wyhlen, Germany) (Be), (4) Zo+Be, (5) De+Be, or (6) no medication serving as control group (Co). Drug administration was repeated at 12 weeks. Subsequently, peri-implantitis lesions were induced by an established and validated procedure [17]. This included an intraperitoneal booster lipopolysaccharide (Lipopolysaccharide Escherichia coli O111:B4, EMD Millipore, Merck, Darmstadt, Germany) injection along with daily topical injections in the peri-implant sulcus at each implant site for 3 days. Subsequently, miniature polyester ligatures (6-0) were placed in a submarginal position around both implants in each animal for 4 weeks [18]. In brief, ligatures were forced into a position directly apical of the mucosal margin. Subsequently, a “pocket” was created to facilitate the establishment of a submucosal microflora. This was followed by a progression period of 12 weeks (Fig. 1a).
Anesthesia protocol
For each surgical intervention, the animals were anesthetized by intraperiotoneal injection of 7.5 mg/kg ketamine (Ketanest®, Pfizer Pharma GmbH, Karlsruhe, Germany) and 5 mg/kg xylazine (Rompun®, Bayer HealthCare, Leverkusen, Germany). For postoperative analgesia, 4.5 mg/kg carprofene was administered subcutaneously immediately after surgery, as well as 1, 2, and 3 days postoperatively.
Histological and immunohistochemical processing
The animals were euthanized with an overdose of pentobarbitone at 100 mg/kg. The pharyngeal space was dissected from intraorally and included the mucosal and submucosal tissue compartments (Fig. 1b).
The samples were decalcified using ultrasound supported water bath and Ethylenediaminetetraacetic (EDTA) for 1 week prior to processing and embedding in paraffin. Two sections of each block were cut in the horizontal plane with the micrometer set at 3–4 μm. One section was stained with hematoxylin and eosin (HE). The second section was used for immunohistochemical staining for cluster of differentiation 68 (CD68). After dewaxing in xylene, rehydration by a graded alcohol series, the samples were incubated in antigen retrieval solution (citrate buffer, pH=6) for 20 min in 96°C. Next, the sections were stained using a Lab VisionTM Autostainer 360-2D instrument (ThermoFisher Scientific, Germany) as described previously [19]. A primary mouse anti-rat monoclonal antibody was used to stain CD68 (dilution 1:1000, Bio-rad, MCA341R USA) for 30 min. Sections were washed with TBS buffer and positively stained cells were visualized using UltraVision™ Quanto Detection system HRP DAB (ThermoFisher Scientific, Germany). For negative controls, the primary antibody was replaced with non-immune serum.
Histological and histomorphometrical analysis
Digital images (original magnification x 200; BX53, Olympus, Hamburg, Germany) were obtained from each specimen and evaluated using a software program (cellSens, Olympus).
In all sections, the inner and outer confines of the lateral pharyngeal and retropharyngeal connective tissue was delineated and defined as total surface area (TA). Within TA, the cross-sectional surface area and respective percentage of infiltrated connective tissue (ICT), as evidenced by the presence of CD68-positive cells, were assessed. Moreover, at 5 randomly selected regions of interest within TA but outside the demarcated ICTs, the total number of CD68-positive cells was counted (Fig. 1b).
All measurements were performed by one previously calibrated examiner. Calibration was accepted when repeated measurements of n=5 different sections were similar at >95% level.
Adequately preserved, intact pharyngeal tissue sections allowing a reproducible depiction of TA could be obtained from a total of twenty-five animals (Zo: n=4; De: n=4; Be: n=4; Zo+Be: n=3; De+Be: n=5; Co: n=5). The sections prepared from the remaining animals revealed an incomplete TA and were therefore not considered for the present analysis.
Statistical analysis
The statistical analysis of the data sets was performed using a commercially available software program (IBM SPSS Statistics 27.0, IBM Corp., Armonk, NY, USA).
