This experiment protocol was approved by the Ethics Committee on Animal Experimentation of the Institutional Animal Care and Use Committee, Cronex (Hwasung, South Korea) (CRONEX-IACUC 201801002). This study conformed the Arrive guidelines.
The subjects of this study were three mongrels (10 months old, male, average 30 kg). The mongrels were selected for the study because their bone size and dentistry were able to accommodate human dental implants and allow for the use of mechanical force on implants.Orti, Bousquet  The subject were housed in separate cages with regular washing system, air conditioning, and lighting. The health and oral hygiene were checked and maintained daily.
Each mongrel underwent extraction of all mandibular premolars. After three months, four implants—two at each hemimandible, were placed in each of the mongrels. The three mongrels were each designated as control, normal dose PTH (PTH-1; TeriboneTM 56.6 µg, Asahi Kasei Corporation, Tokyo, Japan), high dose PTH (PTH-2) groups. The study groups were then further subdivided into non-surgery and surgery (corticotomy) subgroups (Fig. 1; Control_N, Control_S, PTH-1_N, PTH-1_S, PTH-2_N, PTH-2_S).
The subjects were anesthetized with 1 mL/10 kg of intravenously administered tiletamine hydrochloride and zolazepam hydrochloride (1:1) mixture (Zoletil 50®, Virbac, Carros, France) and xylazine hydrochloride (Rumpun®, Bayer, Leverkusen, Germany). The mongrel was put to sleep via inhalation anesthesia for implant placement and antibiotics (Biotril, Komi, Siheung, South Korea) were administered for three days following surgery.
All mandibular premolars were extracted, and the implants were placed three months later. Two implants were placed in each hemimandible and they were spaced approximately 2.5 cm apart for a total of 4 implants placed in each dog. Implant TS lll SA 3.5 × 10 mm (Osstem, Seoul, South Korea) was used, and its size was selected based on mongrel bone width and nerve location.
Miniscrews (1.4 × 6 mm, Jeil Medical Co., Seoul, South Korea) were placed 5 mm away from the distal implant as a reference point. Osseointegration of all implants were confirmed using x-ray and resonance frequency analyzer (Ostell Mentor, Ostell, Gothenburg, Sweden) and ISQ values of all implants were over 70. Customized abutment and metal crowns were fabricated for Nickel Titanium (NiTi) closed coil spring engagement. The maxillary first and second premolars were further extracted to avoid unnecessary occlusal loading.
Surgical intervention—Corticotomy around implant
Corticotomy was performed twice—once before prosthetic force loading and once 12 weeks after force loading. A flap incision was made to reveal the bone, then using a piezo-ultrasonic instrument (VarioSurg 3, Nakanishi.Inc, Tokyo, Japan), and corticotomy was performed vertically in the mesial and distal position 3 mm away from the implant and horizontally 3 mm below the implant position. Cortical bone around the implant was additionally perforated 20 times to induce active blood flow.
Non-precious metal crowns were delivered and heavy NiTi closed coil spring (Tomy International, Tokyo, Japan) was loaded one week post first corticotomy. A force equivalent of 500 g was applied on the implants. (Fig. 1)
Application of pharmacologic agents & Implant movement evaluation
Pharmacologic agent was injected locally around the implant for 20 weeks. The control group was injected with normal saline. One vial of teriparatide acetate (56.6㎍) was diluted with 1 ml saline and injected in the normal dose PTH group. Two vials of teriparatide acetate were injected in the high dose PTH group.
X-rays and impression of the study quadrants of each mongrel were taken every two weeks to measure implant movement. The impressions at 0,1,9,12,14,16 weeks were scanned with a 3D dental scanner (Medit i500, Medit, Seoul, South Korea) and the scanned images at each time point were each superimposed with its week 0 image using the program Rapidform (Inus Technology, Seoul, South Korea). Using miniscrews as the reference point, serial superimposition of the scanned images at all different time points determined the mobility of the implants.
Fluorescence staining for histologic analysis
New bones were labeled with three intramuscularly injected fluorochromes: 30 mg/kg of alizarin red (Sigma, St. Louis, USA), 10 mg/kg of calcein green (Sigma, St, Louis, USA), and 30 mg/kg of oxytetracycline yellow (Fluka, Shanghai, China). Fluorochromes were administered at 4 weeks, 6 weeks, and 55 days post second corticotomy.
After sacrificing the mongrels (20 weeks after agent injection), tissue and block bones (including implants) were removed from the mandibles. The specimen were fixed in 4% paraformaldehyde (Duksan Chemicals Co. Ltd, Gyeonggi-do, Korea) for 48 hours. After washing, they were assessed with micro computed tomography (µCT, SkyScan1173 Ver 1.6, Bruker-CT, Kontich, Belgium). The sample was imaged with pixel size of 29.83 µm. The voltage and current intensity of the images were 130 kV and 60 µA, respectively. Eight hundred images were obtained with 500 ms exposure time, 1 mm aluminum filter, and 30 µm 2240 × 2240 pixels. Nrecon Ver 126.96.36.199 (Skysan, Aartselaar, Belgium) was used to reconstruct cross-section images from the micro CT slices.
Dataviewer and CTAn Imaging Software (Bruker micro CT, Kontich, Belgium) were used for microarchitectural analysis of the specimen. The images of each implants were cropped along the implant axis, from the area of bone-implant contact under the metal crown to 1 cm below the implant using Dataviewer. Sagittal view images were used to observe the mesial and distal areas around the implants. The regions of interest (ROIs) were determined as the 10 mm x 10 mm square area, 3 mm from the bottom of the implant. The percentage of bone volume to tissue volume (BV/TV%), trabecular pattern factor (Tb.Pf, /1 mm), trabecular number (Tb. N, /1 mm), and trabecular thickness (Tb. Th, mm) were analyzed.
Specimens were dehydrated in increasing concentration of ethanol and embedded in a mixture of ethanol and Technovit 7200 resin (Heraeus Kulzer, Wehrheimm, Germany) with an increasing ratio of resin. Following resin infiltration, the samples were hardened in an UV embedding system (KULZER EXAKT 520, Norderstedt, Germany) for a day. The undecalcified specimens were cut with an EXAKT diamond cutting system (EXAKT 300 CP, Norderstedt, Germany) and the tissue and bone were attached to a glass slide with an adhesive system. The width of the tissue section was adjusted to 40 ± 5 µm using a grinding system (EXAKT 400CS, KULZER, Norderstedt, Germany). Fluorescence images were obtained with a Pannoramic 250 Flash lll system (Histech, Budapest, Hungary). The specimens were stained with Masson Goldner Trichrome and photographed with a Pannoramic 250 Flash lll system.
Bone implant contact (BIC) was assessed with a case viewer program (3DHISTECH Ltd., Budapest, Hungary). The implant threads (a total of 12 threads) were divided into top, middle and bottom and tension and pressure side.[2, 19] The Implant surface was divided into three regions at low magnification (15x). Tension and pressure side of implant in bone contacted surface was classified. Tension and pressure side existed in the opposite side of implant bottom because mechanical force influenced on metal crown and force was delivered to bottom of implant by center of rotation and center of resistance. BIC was calculated as the average of tension and pressure side with measurement at a higher magnification (200x). Adjusted BIC (Adj. BIC) was the average bone implant contact in tension and pressure side, except of thread exposure region.
In the fluorescence slide photographs, the outlines of the labeled bones were traced, and the distance between the inter-labeled outlines was measured. The mean distance between five points of inter-labeled outlines in 12 different regions was calculated. The mean distance between the labels was divided by the time between the injection of the labels to yield the mineral apposition rate (MAR, µm/day). The bone formation rate(BFR, µm3/ µm2/day) was determined from the formula BFR = MAR*(BS/MS) (MS; mineralizing surface, BS; bone surface). The mineralizing surface and the bone surface were calculated with Image Pro Premier (Media Cybernetics Inc., Washington Street, USA).