Hyperbaric oxygen therapy efficacy on mandibular defect regeneration in rats with diabetes mellitus: an animal study

Animals were kept on 12-h fasting before inducing T1DM. A single (50 mg/kg) intraperitoneal injection of streptozotocin (STZ) (Sigma-Aldrich, St. Louis, MO, USA) was used, dissolved in 0.1 M citrate buffer (4.5 pH) immediately prior to the injection. Blood glucose level analysis was performed to confirm DM after 72 h from the administration of STZ, then on a weekly basis for the duration of the experiment, using OneTouch digital glucometer (LifeScan, Milpitas, CA, USA) and obtaining blood samples from the animals tail vein. Diabetes was regarded as fasting blood glucose levels above 250 mg/dL [3].

The procedure was done under general anesthesia via intramuscular injection of (7 mg/kg) xylazine 2% (XylaJect, Adwia Co, 10th of Ramadan city, Egypt) and (80 mg/kg) ketamine 10% (Ketamine Alfasan, El Yoser Co, Cairo, Egypt) [26]. Using an aseptic technique, a 15 mm linear incision was made along the line of the posterior right mandibular basal bone. Afterward, a full-thickness mucoperiosteal flap was reflected and a round 4 mm-diameter critical-sized osseous defect was prepared, using a sterile trephine bur (Medesy, JDentalCare, Cairo, Egypt) [3, 27]. After irrigation with sterile saline, the osseous defect was filled with β-TCP bone graft (Nano Gate, Nasr city, Cairo, Egypt). Then, the flap was repositioned and stitched with resorbable sutures (Vicryl, Ethicon, Somerville, NJ, USA) [28]. The operative sites were checked daily, and the rats were monitored for any symptoms. Postoperatively and for 3 days, antibiotic and analgesic were given in the form of 30 mg/kg cefazolin sodium (ALPAC-AL Wady Pharmaceutical, Helwan, Egypt) and 5 mg/kg Carprofen (Adwia Co, 10th of Ramadan city, Egypt) [26].

Starting 24 h postoperatively, Group B was subjected to HBOT using a mono-place chamber. Animals were placed in custom-made plastic boxes with oxygen administration tubes [22]. They were subjected to 90-min HBO sessions at 2.4 ATA, five consecutive days per week (from Saturday to Wednesday) for 3 weeks [22, 29]. To prevent barotrauma and discomfort, pressurization and depressurization processes took place over 10 min each. The untreated control animals in group A were kept in the same room outside the hyperbaric chamber.

Euthanasia was done after 3 weeks of therapy via the injection of an overdose of barbiturates intravenously (Nembutal, pentobarbital sodium, Akorn Pharmaceuticals Co.) [30]. The mandibles were dissected out and cleaned of soft tissues. The experimental segments with the osseous defects were used for histological evaluation, immunohistochemical and histomorphometric analysis.

Specimens were labeled and then fixed instantly in 10% neutral-buffered formalin. Washing, decalcification by 8% tri-chloroacetic acid, dehydration with increasing grades of alcohol, clearing with xylene, then infiltration and embedding in paraffin wax blocks were carried out. Serial tissue sections, 5-μm thick, were cut sagittally through the center of the defect. Staining with Hematoxylin & eosin (H&E) and Gomori trichrome was performed for the general examination of bone regeneration and the evaluation of collagen formation, respectively. Sections were examined by a light microscope (OPTIKA microscopes B-290 series, Ponteranica, Italy) by two different investigators for qualitative histological evaluation.

Morphometric analysis was performed on H&E-stained sections using the ImageJ software (ImageJ 1.53k, NIH, Bethesda, MD, USA) to calculate the percentage of the newly formed bone surface area within the defects. Three sections were obtained from each sample to be used for quantification by two blinded investigators. Then one image was taken from each section under the same magnification power (X40), via a light microscope (OPTIKA Microscopes B-290 series, Ponteranica, Italy) with an attached camera (OPTIKA Microscopes C-B10, Ponteranica, Italy) and Optika Proview software (OPTIKA Microscopes, version 3.7, Ponteranica, Italy). In each digital image taken, the surface area of a selected region of interest (ROI) was measured and recorded as the total surface area. The area that the marrow spaces and other tissue spaces occupied was also measured. Afterward, the surface area occupied solely by bone was obtained by subtracting these two measurements, and its percentage from the total area was calculated. The mean of the measurements from the three images was determined to be used for statistical analysis.

For quantitative assessment of intraosseous microvessel density (MVD), immunostaining against the endothelial progenitor cell marker (CD34) was performed using monoclonal anti-CD34 antibodies (Thermo Scientific, Fremont, CA, USA) [31].

Surveying of sections was initially done at (×100) magnification, followed by a randomized selection of five microscopic regions with the densest concentration of vessels (vascular hot spots) per section. In each image, a standardized counting frame was placed over these areas, and the microvessel number was counted under (×400) magnification using the ImageJ software (ImageJ 1.53k, NIH, Bethesda, MD, USA). A single microvessel was considered as any positively immuno-stained endothelial cells or endothelial cell clusters, clearly separated from the connective tissue elements or adjacent microvessels. This was done by two different observers to avoid bias. The mean value of microvessel number per mm² was used to calculate the MVD based on an established protocol by Weidner et al. [32].

The obtained quantitative data were subjected to statistical analysis using Student’s t-test and expressed as mean values and standard deviations (SD) with a statistical significance level set at 5% (p < 0.05). IBM SPSS software package version 20.0 (Armonk, NY: IBM Corp.) was utilized to perform the analysis.

Microscopic examination showed the newly formed bone originating from the borders of the defect and extending a short distance toward the defect center in the form of irregular trabeculae in association with fibrous connective tissue of low density and limited vascularity. A discontinuous layer of osteoblasts lined the immature bone trabeculae which contained many large, entrapped osteocytes. A line of demarcation was observed in some areas of the defect boundary between the native and newly formed bone denoting their fusion (Figs. 1, 2).

A striking picture of active bone formation could be observed occupying most of the defect regions. Together with segments of bone with primary osteon formation, areas of new trabecular bone were seen in the form of intercommunicating trabeculae of better organization than what was observed in group A. The newly formed bone was lined by a continuous layer of voluminous active osteoblasts and had osteocytes that appeared more organized than those of the control group. High vascularity of the bone marrow and the regeneration front was observed. An outstanding osteointegration of the new bone with the native bone was noted in most of the sections examined (Figs. 1, 2).

Comparing the study group and the control group, there was a statistically significant difference (p < 0.001) regarding the percentage of newly formed bone surface area with mean values of (36.26 ± 4.16%) and (15.55 ± 1.91%), respectively. The percentages of the new bone surface area are demonstrated in Table 1.

The MVD exhibited a higher value in group B (96.87 ± 7.12/mm²), treated with HBO, in comparison with group A (73.96 ± 7.12 /mm²), with a statistically significant difference between the two groups (p = 0.004). The mean values of MVD in the defects are demonstrated in Table 2.