Study materials
Experimental animals
In the present experiment, a total of six healthy male minipigs raised under the same conditions for a certain period (approximately 35-40 kg, 24 months old, Prestige World Genetics, Pyeongtaek, Korea) were used as experimental animals.
Graft material
Autogenous tooth bone graft materials made from teeth extracted from the above minipigs were grafted on individual minipigs after placing implants (MS Implant Narrow Ridge 3.0×10 mm, OSSTEM).
Study method
Animal experiment
Each of the selected minipigs was pretreated with an intravascular injection (IV) of Atropin (Kwangmyung Pharmaceutical Ind. Co. Ltd., Seoul, Korea) and intramuscular injections of xylazine (Rompun, Bayer Korea Co. Seoul, Korea) and ketamin (Ketara, Yuhan Co., Seoul, Korea) and was put under anesthesia throughinhalation anesthesia using enflurane (Gerolan, Choongwae Pharmaceutical Co., Seoul, Korea). Infiltration anesthesia was conducted on the surgical site using lidocaine (2% lidocaine hydrochloride - epinephrine, 1.8 ml, Yuhan Co., Seoul, Korea) for anesthesia and hemostasis. Four molar teeth on the right side of the lower jar were extracted and processed into autogenous tooth bone. On day 10 after tooth extraction, a buccolingual valve was formed and lifted, 2 mm deep vertical bone defects were formed in the four extraction sockets, and four implants (MS Implant Narrow Ridge 3.0×10 mm, OSSTEM) were placed in the extraction sockets. The foremost implant was not grafted with the autogenous tooth bone so that it could be used as a control group and the remaining rear implants were grafted with the autogenous tooth bone as an experimental group. The grafted autogenous tooth bone was grafted in the form of a processed pineapple shape that surrounded the peri-implant vertical bone defects. (Figure 1) Thereafter, the valve was pulled into its place and sutured using an absorbable surgical suture (3/0 Vicryl). Immediately after the surgery, an antibiotic (Penicillin G Sodium, Woojin B&G, Korea, 1 ml/10 kg, 1 time/2 days) was intramuscularly injected and the minipig was made to take liquid food until macroscopic wound healing was completed to protect the surgical site.
Fabrication of autogenous tooth bone
Extracted teeth immersed in 70% ethyl alcohol were sent to a specialized processing institution (Korea Tooth Bank Co., Seoul, Korea), made into block shapes after attached soft tissues and foreign substances such as tartar were removed, put into a distilled water and hydrogen oxide solution, and cleaned with an ultrasonic cleaner to remove remaining foreign substances. The cleaned particles were dehydrated using ethyl alcohol and underwent a defatting process using an ethyl ether solution. The autogenous teeth that underwent these processes were made to undergo a lyophilization process, disinfected using ethylene oxide gas, packed and delivered to the laboratory, and used in graft processes.
Fabrication and analysis of tissue specimens
Before fabricating specimens for tissue observation from the experimental animals, a mixture of xylazine HCL (2.3 mg/kg, Bayer) and ketamine (5 mg/kg, Yuhan Corporation) was intravenously injected into the minipigs at four, eight, and 12 weeks after graft to put the minipigs under anesthesia. Then, KCL (2 mmol/1 g, Huons) was swiftly injected into the veins to administer euthanasia and bone fragments including implements and adjacent tissues were collected immediately after the sacrifice.
The tissues collected from two minipigs per each week of collection were used in histomorphometric analysis. The collected tissues were fixed in 70% alcohol for three days and then stained first by leaving them in a Villanueva staining solution for seven to10 days. For dehydration and bleaching, the tissues were left in each of 50, 70, 80, 95, and 100% alcohol solutions for four hours and then left in propylene oxide overnight for complete dehydration. Blocks were made using epoxy resin (Eponate, Ted Pella Inc., Redding, CA, USA) and hardened for three days in an incubator at 60°C. Thereafter, trimmed sections were made using an Accutom-50 (Struers Co., Copenhagen, Denmark) and the trimmed sections were ground using a micro-cutting and grinding system (EXAKT, Exakt Co., Norderstedt, Germany) to make 10-20 μm thick tissue slides.
Histomorphometric analysis
Images magnified by factors of 12.5 and 40 were obtained using an optical microscope (Axioscop, Carl Zeiss, Jena, Germany) and the images were reconstructed to photos of the entire tissues using the Photoshop program. From these photos, the ratio of newly formed mineralized bones (NBF, new bone formation) to the entire bone defect was calculated using an image analyzer (iMTechnology, Korea). The area of newly formed bones was regarded as the area to newly formed bone boundaries including mineralized bones, remaining bone graft materials, bone marrow, fibrous connective tissues, and newly formed blood vessels. In addition, the total length of bone defects in each implant was measured as well as the bone areas in contact with the implant to calculate the contact ratio between the bones and the implant. (Villanueva stain, original magnification ×12.5, magnified photo Villanueva stain, original magnification ×40).
As a statistical method, independent sample t-tests were conducted using SSPS ver.17.0 (SPSS, Chicago, IL, USA) and cases where the P value was smaller than 0.05 were regarded as being statistically significant.
Comparison and evaluation of peri-implant bone densities in panoramas
To compare and evaluate bone densities, panorama photos (DP-80-P/PM2002 EC Proline, Finland) were taken from the control group that was not treated at all after implant placement and the experimental group grafted with bone after implant placement at four weeks and 12 weeks after implant placement (Figure 2). The panorama photos, digitized as DICOM files, were converted into JPEG graphic files, and the JPEGs were stored. From the stored files, bone densities were measured in the area around each implant ranging from 2 mm in front of the implant boundary to 2 mm in the rear of the implant boundary and to 2 mm downward from the upper bone boundary considering the size of the autogenous tooth bone graft material using the average value of the 255 tonality grayscale using the gray-level histogram of the Adobe Photoshop CS3 program. The peri-implant bone densities were measured in panoramas using the Adobe Photoshop CS3 program.
As a statistical method, the non-parametric Wilcoxon test of the SPSS 12.0 program (SPSS Inc. Chicago, USA) was used, and the statistical significance level was set to P < 0.05.
Comparison and evaluation of peri-implant bone densities on CT
To compare and evaluate bone densities, CT (DCTP-110-p/AlphardVEGA-3030, Asahi Roentgen Ind. Co., Japan) imaging was conducted with the control group and the experimental group at 12 weeks after implant placement. Thereafter, the photos digitized as DICOM files were converted into JPEG graphic files and the JPEGs were stored. From the stored files, bone densities were measured in the area around each implant of the minipigs (ranging from 2 mm in the buccolingual direction to 2 mm downward from the lingual upper bone boundary) using the average value of the 255 tonality gray scale using the gray-level histogram of the Adobe Photoshop CS3 program. A measurement was conducted on the axial view. The non-parametric Mann–Whitney test of the SPSS 12.0 program (SPSS Inc. Chicago, USA) was used and the statistical significance level was set to P < 0.05.