Bone Regeneration of Hydroxyapatite with Granular Form or Porous Scaffold in Canine Alveolar Sockets

Materials and Methods

Experimental animals. Six adult (18-22 months old) male beagle dogs, weighing between 8.5 and 9.5 kg, were included in this study. The protocol for the animal experiment was approved by the Institutional Animal Care and Use Committee of Chungbuk National University (no. 677-14-01). All dogs were kept in individual cages throughout the experimental period. Water and food were supplied ad libitum during the experimental period. The alveolar extraction sites were divided randomly and a split-mouth design was established in three groups: control group: extracted socket only, four alveolar sockets, gHA group: filled with granulated HA, four alveolar sockets, and pHA group: filled with porous HA scaffold, four alveolar sockets.

Preparation of gHA form and pHA scaffold. A gHA slurry was made by mixing 3 g nano-HA powders (OssGen Co., Daegu, Korea) and binders (2% high molecular weight polyvinyl alcohol, 1% carboxymethylcellulose). The gHA slurry was dripped in liquid nitrogen using a micropipette to make a granular structure. After lyophilization, gHA granules were sintered at 1,230°C for 3 h. The Particle size of gHA was 500 μm to 2 mm.

pHA scaffold was fabricated using a polymeric template-coating technique, as described elsewhere (13). A polyurethane sponge (60 pores per inch) was coated with nano-alumina powders (OssGen Co., Daegu, Korea) in distilled water-based slurry. Binders (3% high molecular weight polyvinyl alcohol, 3% carboxymethylcellulose, 5% ammonium polyacrylate dispersant, and 7% N,N-dimethylformamide drying agent) were added to the slurry mixture to improve sintering and stabilize the scaffold structure. Coated sponges were dried overnight at room temperature before sintering at 1,500°C for 3 h. Final mandible molar root-like shape of pHA scaffold dimensions were 4 mm in diameter, and 10 mm in length (Figure 1). The properties of the scaffold produced were observed using a stereoscope (Fisher Micromaster; Fisher Scientific, Fremont, CA, USA) and scanning electron microscope (SEM; EVO 40; ZEISS, Dublin, CA, USA).

Each type of HA was placed in a 24-well petri-dish using plastic forceps. Two milliliters of 70% ethanol was poured on the well. After 1 h, the ethanol was removed using suction. HA was washed three times with phosphate-buffered saline (pH 7.4), soaked in fetal bovine serum-free medium, then left overnight under ultraviolet light. HA was washed three times with fresh medium. After removing the water from the HA, it was implanted.

Surgical procedures. Surgical procedures were performed under sterile conditions and general anesthesia. The left and right mandibular first molars were extracted without injury using the closed extraction technique. The graft site was closed with 4-0 single interrupted absorbable sutures (Surgifit, Ailee Co., Busan, Korea) with gingival flap technique. Enough tissue was elevated for the flap to be placed over the socket without spontaneous retraction. Commercial dog feed soaked in water was fed to minimize the damage of surgical area during the entire experimental period. Antibiotics and analgesics were administered in the postoperative period.

Evaluation of CT and micro-CT. CT images were taken sequentially at baseline, four, and eight weeks post-implantation to assess the new bone formation and mineralization. CT images were obtained by a single-slice spiral CT (Hi Speed CT/e, GE Medical Co., USA). CT exposures were performed at 120 kVp (150 mA), with 1 mm thickness and 512×512 voxel matrix.

At 8 weeks post-implantation, the harvested mandible was scanned using a micro-CT. A Skyscan Desktop Micro-CT 1172 (Skyscan; Aartselaar, Belgium), with a source voltage of 60 kV, a current of 167 μA, resolution 26.7 μm was used to acquire x-ray radiographs. The specimens were attached to a stage that rotated 180° with images acquired every 0.6°. After scanning, cross-sectional slices were reconstructed and each scan result was reconstructed using the 0.008-0.031 threshold values to distinguish bone and air. The parameters of bone mass and microarchitecture were evaluated using the micro-CT built-in software, including BMD, bone volume, tissue volume, bone surface, percent bone volume, bone-specific surface, bone surface density, trabecular thickness, trabecular number, trabecular separation and interception surface.

Fluorescent bone labeling and preparation of specimens for fluorescence microscopy. To label the mineralized tissue and assess the time course of new bone formation and mineralization, tissue was conducted by a polychrome sequential fluorescent labeling method. Oxytetracycline (25 mg/kg; Green Cross, Yongin, Gyeonggi, Korea) and calcein (20 mg/kg, Sigma-Aldrich Co., St Louis, MO, USA) were intravenously injected at four and six weeks after the implantation. Alizarin red S (30 mg/kg, Sigma-Aldrich Co.) was intraperitoneally injected at 8 weeks after the implantation.

The formalin-fixed samples from the sacrificed alveolar sockets in each beagle were dehydrated in a graded series of ethanol, then immersed and infiltrated using Technovit 7200VLC (Technovit; Hatfield, PA, USA) before light catalysis for 24 h. A macrocutting and grinding system (Exakt 310CP series; Exakt Apparatebau, Norderstedt, Germany) was used to produce undecalcified cut and ground sections. A final thickness of approximately 50 μm of each section was produced for fluorescence microscopy. Labeling of new bone formation was evaluated with confocal laser scanning microscope. Active bone formation was evaluated relative to the presence or absence, intensity, and width of the fluorochrome markers.

Statistical analysis. Statistical analyses were performed using SPSS statistical software package version 19.0.1.1. (IBM SPSS Statistics for Windows, Version 19.0; IBM Corp., Armonk, NY, USA). Data are presented as the mean±standard deviation (SD). Normality and homogeneity of the data were confirmed before analysis of variance (ANOVA). Differences among the experimental groups were assessed by one-way ANOVA followed by Duncan's multiple range tests. Null hypotheses of no difference were rejected if p-values were less than 0.05.