Five healthy male miniature pigs weighing approximately 40 kg and with ages of 24 months or so (Prestige World Genetics, Pyeongtaek, Korea) were used as animal subjects. In accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines, every experiment was conducted in the operating room within the laboratory, and all procedures for selection, care, and surgical treatments of animal subjects (lab animals) were implemented after obtaining IACUC's approval (Medikinetics-IACUC: 100407-001). Any and all surgical procedures and techniques were performed and applied under general anesthesia.

Atropin (Kwangmyung Pharmaceutical Ind. Co. Ltd., Seoul, Korea) was intravenously administered; xylazine (Rompun; Bayer Korea Co., Seoul, Korea) and ketamin 30 mg (Ketara; Yuhan Co., Seoul, Korea) were intramuscularly injected to induce general anesthesia; endotracheal intubation was then performed using Miller sized-4 laryngoscope and standard straight tube with inner diameter of 5.5 mm and cuff (Portex, Kent, UK); general anesthesia was maintained with 1% euflurane (Gerolan; Choongwae Pharmaceutical Co., Seoul, Korea). To prevent possible infections, epocelin (40 mg/kg body weight; Dong-A Pharm, Seoul, Korea) was intra muscularly administered both preoperatively and postoperatively; to protect the surgical site, soft food was fed to the animals until the completion of wound healing. Miniature pigs were placed in the ventral recumbency position, and then their heads were fixed followed by shaving. After disinfecting the surgical site with betadine, the site was isolated with sterile drapes. For local anesthesia, infiltration anesthesia was performed with lidocaine (2% lidocaine HCl and epinephrine, 1.8 mL; Yuhan Co., Seoul, Korea). Afterward, the left and right mandibular premolars and molars were extracted and delivered to the Korea Tooth Bank (Seoul, Korea) for processing into powdery bone graft material. After about 8 weeks, the maxillary sinus was exposed through the lateral approach, and maxillary sinus floor elevation was carried out using the outfracture osteotomy sinus graft technique.(Fig. 1) An autogenous tooth bone graft was placed on the right side for the experiment group, and hydroxyapatite (Bone Gros; BioAlpha Inc., Seongnam, Korea), on the left side for the control group. Initially included in experimental group, an animal was excluded from the experimental group because the right maxillary sinus membrane got perforated, indicating failure of maxillary sinus bone grafting. In both experimental group and control group, a bone window was prepared again in the grafted site followed by interrupted suture.

The mandibular premolars of the minipigs were extracted on both sides and prepared 8 weeks before the graft procedure. Immediately after extraction, the teeth were trimmed and cleaned of all extraneous material and sterilized in 70% alcohol. The tooth was cut in the cementoenamel junction to divide it into crown and root. Using the crown dentin block, the pulp tissue was totally removed by the retrograde technique. The teeth were crushed into powders measuring 400-800 µm under a dried and cold environment (2℃). After being washed with sterile saline at 2℃ for one hour, the teeth were immersed in 0.6N HCl (hydrochloric acid solution) at 2℃ for 30 minutes for demineralization. Afterward, the specimens were washed in distilled water for 1 hour under constant agitation to remove the acid completely. They were defatted in chloroform-methanol for 30 minutes under the same conditions as those of demineralization. After defatting, the specimens were then washed in distilled water for 3 hours under constant agitation to remove the reagents completely. Freeze-drying and sterilization were done under appropriate conditions, with the specimens stocked for graft until 8 weeks before graft.

The laboratory animals were sacrificed 12 weeks after grafting, and specimens were harvested in blocks from the grafted site along with the surrounding normal bone tissue on the floor of the maxillary sinuses. The prepared specimens were cleanly washed with normal saline, and then kept in formalin solution and sent to an independent laboratory without delay to request slide preparation and analysis. The specimens were demineralized with formic acid, and paraffin blocks were made in a routine manner. For each sample, paraffin blocks were cut into 4 µm sections, followed by hematoxylin-eosin staining and histopathological examination under an optical microscope (Olympus BX-51; Olympus Co., Tokyo, Japan). Histomorphometric analyses were performed by observing the respective slides at 100× magnification under an optical microscope. Specifically, after acquiring digital images from a digital camera mounted on the microscope, image analyzing software program (Kappa Image Base-metreo; Opto-Electronics, Gleichen, Germany) was used to determine and analyze the area of newly formed mineralized bone (NB%) in the entire lesion as seen in the acquired images.

The NB% of each graft material was statistically analyzed using the Mann Whitney U-test at 95% confidence level Using the IBM SPSS Statistics (version 19.0; IBM Co., Armonk, NY, USA).

During this study, miniature pigs remained healthy, and outcomes of the treatment in their grafted sites were good. No major complication such as infection, edema, or exposed bone graft material was observed in any group.

Typical histological findings for the experimental group are presented in Figs. 2-4. Fig. 2 shows the experimental specimens under magnification of ×100. Grafted tooth-derived graft materials (asterisks) were evident, showing multiple linear streaks as a dentinal tubular structure. New bone (arrowheads) was growing at the periphery of the tooth-derived materials in Fig. 2. Other specimen under magnification of ×400 showed bone remodeling activity as evidenced by the multinucleated giant cells of osteoclastic activity at the periphery of the graft material with multiple linear streaks (arrowhead in Fig. 3). Another experimental specimen under magnification of ×100 is shown in Fig. 4. Encroaching on the tooth-derived graft material (asterisk), new bone formation (arrowhead) was identified, showing bone remodeling as evidenced by the incremental lines. In Fig. 5, synthetic hydroxyapatite (asterisk) was partially evident in the histological section of the control group. New bone was also found at the periphery of the grafted hydroxyapatite (arrowheads) but not in the inner portion of the graft space (Fig. 5).

The area of new bone formation (B), total examined area (T), and ratio of new bone formation (B/T) were presented in Table 1. The area was measured in µm² for the area of new bone and total area, and the ratio of new bone formation was calculated in unit %. Although there was no statistical significant difference between the two groups because of the small number of experimental animals (P>0.05), the mean values and standard deviation of the ratio of new bone formation were calculated as 57.19%±11.16% for the experimental group and 34.07%±13.09% for the control group as summarized in Table 2.