Animals
Six healthy male beagle dogs, aged 18–20 months (weighed 8.5-9.5kg) were used and this study was approved by the institutional review committee of Shanghai Jiao Tong University School of Medicine (No. HKDL [2017]368). The study was conducted in accordance with ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines for preclinical animal studies and the Guidelines laid down by the National Institute of Health (NIH) in the USA regarding the care and use of animals for experimental procedures. The animals were adapted to a 12-h light/12-h dark cycle for 1 weeks before the surgery. The dogs had free access to food and water in the entire experiment.
Surgical management
The experimental study was performed in two surgical phases. All the animals were anesthetized with 1.25% Sodium pentobarbital (0.4 ml/kg, IV) in the surgery. Buccal dehiscence type defects were created in the first phase. Six beagle dogs received dehiscence defects on the distal root of the mandibular 3rd, and 4th premolars (P3-P4) bilaterally in each animal (n=4 defects per animal) with a total of 24 defects created under general anesthesia. Briefly, a full thickness mucoperiosteal flap was reflected, and standardized buccal dehiscence-type defects (6 mm in height from the cemento-enamel junction and 5 mm and 2mm in width at the top and bottom, respectively) in each side of the lower jaw were created as described previously. 15,16 Root surfaces were denuded of periodontal ligament using a curette and then a mucoperiosteal flap was repositioned (Fig.S1, Supporting Information). After 1-month healing period, this model could resemble buccal dehiscence defects confirmed by cone beam computed tomography (CBCT) (Fig.1A-C& Fig.S2, Supporting Information). Each group included 2 animals. Randomization was carried out by a research assistant and computer-generated random codes were sealed in opaque envelopes. Then a total of 12 sides (n=2 sides of the lower jaw per animal ) were randomly assigned into the following treatment groups, including 4 sides each: Group A: blood clot in an untreated defect; Group B: deproteinized bovine bone mineral with granule size of 0.25mm-1mm and a volume of 0.25ml per defect (DBBM, Bio-Oss, Geistlich Biomateirals AG, Wolhuser, Switzerland) was applied to bone regeneration, covered with a collagen barrier; Group C: DBBM covered with the periosteum.17 In the second surgery, a horizontal incision was first performed at the mucogingival junction from mesial P3 to distal P4 without vertical releasing incisions for the group A. The mucoperiosteal flap was reflected coronally till near the gingival margin and then repositioned and sutured, only leaving blood clot in the defect area (Fig. S3, Supporting Information). Untreated defects served as the control group. For the group B, the operative approach was the same as group A, and Bio-Gide membrane were placed over the defect which was filled with DBBM (Fig.1D, S4, Supporting Information). For the group C, care was taken not to cut the periosteum when a horizontal incision was performed according to our previous clinical report.18 A partial full-thickness flap was elevated apically with sharp dissection so as to ensure the operation remaining on the periosteal surface. Then the periosteum was incised 5mm below the initial incision and reflected coronally so that the periosteum dimension was similar to that of the collagen membrane (Fig. 1E). The periosteum was repositioned and sutured after bone grafting with DBBM (Fig. 1F). Finally, the mucosal flap was repositioned and securely sutured (Fig. S5, Supporting Information). Penicillin (30000 u/kg) was administered every day for 1 week and all animals were fed soft food with a plaque control regimen during the whole period of the experiment.
Sequential fluorescent labeling
To evaluate the new bone formation and mineralization with time, the animals were injected intramuscularly with tetracycline (25 mg/kg, TE; Sigma, USA), alizarin red (30 mg/kg, AL; Sigma) and calcein (20 mg/kg, CA; Sigma) at postoperative 3, 6 and 9 weeks, respectively. The outcomes were analyzed as the reported methods 8. Half of the samples were fixed for nondecalcified sectioning, dehydrated using ascending concentration of alcohols from 75%-100%, infiltrated and embedded along the buccolingual plane in methyl methacrylate (MMA). Three serial buccolingual sections, with a thickness of 150 µm, were taken along the longitudinal direction of the teeth by a diamond-coated internal-hole saw microtome (Leica SP 1600, Milan, Italy), Each section was grounded and polished to a final thickness of 50-70 µm for fluorescent labeling observation a confocal laser scanning microscope (Leisa TCS, Germany). Sequential fluorochrome labeling for the newly formed mineralized bone was calculated according to the previous report.19 The number of pixels labeled with yellow (TE), red (AL), and green (CA) in each image was measured as a percentage of the mineralization area, respectively, using an image analysis system (Image-Pro Plus software, Media Cybernetic, USA).
CBCT and Micro-CT evaluation
CBCT (Imaging Sciences International, Hatfield, PA, USA) was taken immediately and 1 month after operation for dehiscence creation in this study. Vertical alveolar bone loss (VABL) was defined as the distance between the crest of the alveolar bone and the cement-enamel junction in the long axis direction of distal root of P3 or P4 along the largest buccal-lingual section, which reflected effective periodontal bone support around the teeth.
To observe the internal structure of bone, micro-CT was performed for all animals that were sacrificed at 3 months post implantation. Blocks in the lower premolar regions, including bone, teeth and surrounding soft tissue, were harvested and fixed in 4% paraformaldehyde (PFA). The fixed samples were scanned using an animal Micro-CT scanner (mCT-80, Scanco Medical, Switzerland) to observe the newly formed bone at the buccal defects. The parameters of the micro-CT were set at 70 kV, 114 mA, 700 ms of integration time, a resolution of 2048 × 2048 pixels and an isotropic voxel size of 18 μm. The volume of interest was selected as the dehiscence defect and extended for a total of 500 slices. Bone volume to total volume ratio (BV/TV), trabecular number (Tb. N.) and bone mineral density (BMD) were analyzed. 20 Because micro-CT can be cut freely and view from different perspectives, VABL was measured as the above method mentioned.
Histological and histomorphometric observations
After fluorescence microscopy, the undecalcified sections were stained with Van Gieson’s picro-fuchsin. The percentages of newly formed bone and the residual bone substitutes were quantified at low magnification from 3 randomly selected sections from each specimen using image analysis system (Image-Pro-Plus, Media Cybernetic, USA). The other half of the blocks was decalcified with 10% of ethylenediaminetetraacetic acid (EDTA-2Na, pH= 7.4) at 37℃ for 9 months and dehydrated in ethyl alcohol with gradually increasing concentrations from 70% to 100%. After embedding in paraffin, serial sagittal cross sections were made to a thickness of 4-5 µm. Three slices were chosen from each specimen. The first slice was from the long axis of distal part of teeth in a buccolingual direction and the rest were obtained 0.5 mm mesial and distal from the initial section. Sections were stained with hematoxylin and eosin (H-E) and images were captured by a light microscope for the observation of bone regeneration.
Immunohistochemistry
For the detection of the bone remodeling proteins expression in the defect area, immunohistochemical analysis was performed using antibodies for osteopontin (OPN), and osteocalcin (OCN). The sections were treated with 3% H2O2 in methanol for 30 min and blocked endogenous peroxidase. And then these sections were incubated in Tris-buffered saline (TBS). Primary antibodies contained OPN (Rabbit; 1:100 dilution; Novus Biologicals, USA) and OCN (Rat; 1:100 dilution; Abcam, USA) were applied to the sections at 4℃ overnight. After 3 times washes with PBS, the slices were incubated for 30 min with biotiny-conjugated secondary antibody anti-rabbit or anti-rat IgG (Boster Bio Co. Ltd., Shanghai, China), and incubated with a preformed streptavidin biotin complex for 30 min. Staining was carried out by 3-3’-diaminobenzidine substrate (DAKO, Cambridge, UK), and the specimens were then counterstained with hematoxylin.
Protein expression levels were assessed by the mean optical density (MOD) [16]. Both the area and the integrated optical density (IOD) of positive stains were quantified with Image J software. MOD was calculated as follows: MOD= IOD/ observed area. 21,22 The mean value was calculated and used as the final value.
Statistical analysis
SPSS v.17.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. All the data are presented as mean ± standard deviation (SD). The differences among groups A, B and C were analyzed by analysis of variance. According to the data distribution and equal variance assumption test, Student-Newman-Keuls (SNK) post hoc or Friedman's test followed by Wilcoxon test for multiple comparisons were performed. All comparisons were conducted at the 0.05 level of significance.