Preparation of the scaffold
Zirconia powder (50 μm, 3 mol YTZP, E grade, Tosoh, Japan) was mixed with 50 wt.% resin beads (50 μm polymethyl methacrylate powder) added to create microscopic pore sizes. Thirty weight percent coarse sodium chloride particles (500–700 μm) were added to the mix to create large interconnected pores. The powder was mixed in a rotating cylinder for 24 h to insure homogenous powder distribution. A binder material (1 wt.% polyvinyl glycol) was added to the mix, and the powder was isostatically pressed into cylinders (40 mm in diameter and 80 mm in length) to create the required dimensions of the CAD/CAM milling blocks. The blocks were partially sintered at 1250 ○C for 30 min then soaked in deionizer water for 24 h to dissolve the salt particles.
Jaw defect and scaffold design
The ethics committee of Alexandria University approved the working protocol used in this study (STDF reintegration grant 489) according to the university code of conduct regarding using animals in scientific studies. After approval of the ethics committee on publishing parts of the obtained data, 2-year-old healthy male Beagle dogs (weighing 10–12 kg) were generally anesthetized by administration of subcutaneous injection of atropine (0.05 mg/kg; Kwangmyung Pharmaceutical, Seoul, Korea) and an intravenous injection of a mixed xylazine (Rompun, Bayer Korea, Seoul, Korea) and zoletil (Virbac, Carros, France) and maintained by inhalation anesthesia (Gerolan, Choongwae Pharmaceutical, Seoul, Korea). Surgical flap was reflected to expose the premolar-molar region of the lower jaw with minimum amount of trauma, afterwards the involved teeth were removed and a surgical block 2 cm long × 2 cm deep was cut using a surgical guide to demarcate the wound boundaries. Finally, the surgical flap was repositioned and sutured and the dogs received an antibiotic (20 mg/kg of cefazoline sodium, intramuscularly; Yuhan, Seoul, Korea) for 3 days, given soft diet, and the surgical site was sprayed with topical 0.2% chlorhexidine solution. After 3-month healing time, three-dimensional images of the boney defect was performed using cone beam CT radiographic imaging (I cat, Imaging science international, Hatfield, PA). The images were transferred to an open access CAD/CAM software (CAMworks, Geometric Americas INC, Scottsdale, AZ) and the design of the required zirconia scaffold was reconstructed to accurately fit the modeled boney defect putting in account the expected sintering shrinkage of the material (25 vol.% shrinkage). The scaffold was designed to restore normal contour of the resected ridge. Five axes dry milling unit (DWX-51D 5, Roland, Parkway, Irvin, Cal) was used to mill the prepared blocks into the required shape and the scaffold was sintered at 1350 ○C for 4 h.
Enriching with nano-hydroxyapatite
Nano-hydroxyapatite particles were prepared using sol gel chemical precipitation method. The sol was thermally aged at low temperature at 50 °C for 2 h. Upon drying the sol particles agglomerated into a dry gel through van der Waals forces composed of 10–14-nm particles. A crystalline apatite is achieved after sintering at 450 °C resulting in a gained structure of 25–55 nm in diameter. Twenty-five weight percent suspension of nano-hydroxyapatite particles were added to 80% ethyl alcohol and stirred to achieve a homogenous suspension, and the right scaffold of each dog was immersed in the prepared suspension for 15 min under vacuum to insure adequate filling of all pores. Scaffolds were dried at 120 °C for 180 min and the process was repeated two times. Finally, the coated scaffolds were heated at 900 °C for 30 min to ensure proper drying of the particles without changing the chemistry of the particles or the supporting scaffold.
Characterization of the prepared scaffolds
Mercury porosimetery was performed for evaluate pore size and distribution and to measure the total porosity percent of the scaffolds. Pore sizer (Porosimeter, Micromeritics 9320, USA) was used for testing the produced porosity on the nanoscale covering pore diameter in range from 360 to 0.006 μm.
Energy dispersive X-ray analysis (EDX) (INCA Penta FETX3, OXFORD Instruments, Model 6583, England) and X-ray diffraction analysis (XRD) (PANalytical, X Pert PRO, The Netherlands) with Cu target (λ = 1.54 Å), 45 kV, 40 mA, and 2Ɵ (10°–80°) were used to analyze elemental surface composition and crystal structure of the scaffolds. Density of the prepared scaffolds was compared to theoretical density of fully sintered zirconia.
Twelve weeks after healing of the resected ridges, the animals were exposed to the second stage surgery where the created defect size was exposed using the same procedures described previously and each scaffold was seated in its final position. Resorbable collagen membrane (Biomend, Zimmer Inc, CA, USA) was used to cover the exposed surface of the scaffold and soft tissue was gently expanded and sutured to secure proper wound closure using resorbable suture material (Vicryl Rapide 5; Ethicon Inc., Somerville, NY). To increase primary retention, the scaffolds were fixed using resorbable polylactic acid fixation screws (Rapidsorb, Deput Synthes, PA, USA).
Six weeks after insertion of the scaffolds, the animals were given an over dose of an anesthetic injection and section blocks were obtained by cutting the mandible maintaining 10 mm of sound bone around the scaffolds. Cut sections were immediately fixed in 4% buffered formaldehyde and dehydrated in graded ethanol solutions using a dehydration system under agitation and vacuum, and the specimens were then defatted in xylene solution. Finally, the specimens were embedded in transparent chemically polymerized methyl methacrylate resin (methyl methacrylate 99%, Sigma-Aldrich, Steinheim, Germany). After polymerization, the specimens were cut along the long axis of the scaffolds using a diamond-coated disc rotating in a micro-sectioning system (Micracut 150 precision cutter, Metkon, Bursa, Turkey) followed by polishing using 800 grit silicon carbide paper. One hundred-micrometer-thick cut sections were stained using Stevenel’s blue and van Gieson picro-fuchsin. Histomorphometric analysis was performed using digital images obtained using a light stereomicroscope (Olympus BX 61, Hamburg, Germany) equipped with a high-resolution digital camera (E330, Olympus, Imaging Corp, Beijing, China).
Measurements were made by first calculating the pore volume on the images using digital tracing option of the software (white pores on the images), and the amount of new bone formation (mineralized tissue stained red) was calculated as a percent of the total pore volume (Olympus CellM & CellR, version 3.3, Olympus Soft Imaging Solutions).
Examiner reliability was cross checked by re-evaluating randomly selected digital images by another expert examiner. The recorded correlation coefficient ranged from 0.83 to 0.92, indicating high reliability for all measured parameters. The data obtained were expressed as mean and standard deviation values and were analyzed using Student’s t test (SPSS 15.0, SPSS, Chicago, IL).