All methods in this animal experiment were performed in conformity with the principles of the 3R (Replacement, Reduction, and Refinement) and two major laws in Korea which are Animal Protection Act established by the Ministry of Agriculture Food and Rural Affairs, and the Laboratory Animal Act established by the Ministry of Food and Drug Safety. The animal experiment was evaluated and authorized by the Institutional Animal Care and Use Committee of Seoul National University (IACUC; approval no. SNU-210115-–1) and performed in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The study comprised four 1-year-old male beagle dogs, weighing approximately 10–12 kg. The manuscript was written in compliance with the ARRIVE guidelines. The timeline of this study is presented in Fig. 1.

Clinical and radiographic photograph of single- and double-root 3D-printed implant. All protective caps were removed 2 weeks after implant placement for plaque control and implant stability measurements. There were no clinical signs of peri-implant inflammation, including redness, spontaneous bleeding, swelling, or ulceration. The clinical and radiographic photos were taken at the time of implant placement and 2, 4, 6, 8, 10, 12 weeks after implant placement.

Fabrication of 3D-printed implants

The fabrication process of the 3D-printed implants was conducted according to a previous study¹⁶. In brief, CT datasets of the mandible were obtained using a CT scanner (GE, Boston, USA) and were imported into 3D reconstruction software (Materialise, Leuven, Belgium) via Digital Imaging and Communications in Medicine format. Both sides of the mandibular third and fourth premolars were virtually extracted and isolated as a stereolithography (STL) file with the software. The STL file was transferred into software (Materialise) to fabricate 3D single-root implants at the distal root of the third premolar area and double-root implants at the fourth premolar area (Fig. 1) with a direct metal laser sintering machine using Ti–6Al–4V powder through Dentium Build Processor 1.4.7 (Dentium, Seoul, Korea) powered by KETI Slicing Engine. The single-root implant and mesial root of the double-root implants were manufactured to be 2 mm longer than the corresponding teeth with a groove to obtain primary stability. The implants were marked with numbers and letters in the upper area to denote animals and locations. The root dimensions of the 3D-printed implants were different for each tooth, but the abutment was manufactured with a constant size. Following large-grit sandblasting and acid-etching (SLA) surface treatment according to a previous study, the 3D-printed implants were sterilized using gamma-ray irradiation, which emits short wavelength light from a cobalt-60 (60Co) radioactive isotope. A surgical guide and drills to perform osteotomies at the mesial roots of the planned sites were fabricated through digital light processing (DLP) 3D-printer (Dental 3DPrinter-P, Dentium, Seoul, Korea) using material Surgical Guide (DG-1). The protective cap was fabricated with polymer with a thickness of 1 mm to minimize loading on the implant (DG-1, Hephzibah, Inchon, Korea).

Immediate placement of 3D-printed implants

The animals were anesthetized by a veterinarian using intravenous injections of tiletamine/zolazepam (5 mg/kg, Virbac, Carros, France), xylazine (2.3 mg/kg, Bayer Korea, Ansan, Korea), and 0.05 mg/kg atropine sulfate for the surgery. Complementary local anesthesia was injected at the mandibular third and fourth premolar area with 2% lidocaine HCl with epinephrine (1:1,000,000, Huons, Seongnam, Korea). The third and fourth premolars were hemisectioned with a diamond fissure bur in the buccolingual direction of the teeth and atraumatically extracted with elevator and forceps without flap reflection. The apical portion of the extraction socket was prepared using a 2.3 mm drill with a motor-driven handpiece (EXPERTsurg LUX, KaVo, Warthausen, Germany) to be 2 mm longer than the corresponding root for single-root 3D-printed implant, and mesial root for double-root 3D-printed implant. The 3D-printed implant heads were directly tapped using a surgical mallet. The protective cap was attached to the adjacent teeth using resin-modified glass ionomer cement (GC FujiCEM2, Tokyo, Japan).

Postoperative care

An antibiotic (cefazoline, 20 mg/kg, Chongkundang Pharm., Seoul, Korea) and analgesic (tramadol hydrochloride, 5 mg/kg, Samsung Pharm., Hwaseong, Korea) were intravenously injected after surgery to relieve postoperative pain and inflammation. For 3 days after the surgery, antibiotics and analgesics were administered by mixing with the animals’ diet. To prevent any mechanical pressure that might hinder wound healing, a soft diet was provided for a month. The surgical sites were inspected every 2 weeks and rinsed with 0.12% chlorhexidine gluconate solution (Hexamedine, Bukwang Pharm., Seoul, Korea).

Implant stability measurements

Based on a previous study^11,16,18, damping capacity analysis (Anycheck, Neobiotech, Seoul, Korea) was performed at implant placement and at every two weeks following until 12 weeks to measure implant stability. Measurements were taken five times from the buccal side of each implant, and the average value was considered representative.

Marginal bone changes

The marginal bone level was measured with periapical radiographs taken at implant placement, 6 weeks and 12 weeks followed by implant placement. The measurement was performed at the mesial and distal sites of each implant and middle sites in the case of double-root implants. Mesial and distal marginal bone loss at 6 weeks and 12 weeks were each compared between the two 3D-printed implant groups. The marginal bone loss of the double-root 3D-printed implants at 6 and 12 weeks was compared among mesial, middle, and distal sites.

Micro-CT analyses

Animals were sacrificed 12 weeks after implant placement with potassium chloride (75 mg/kg, Jeil Pharm., Daegu, Korea). The block biopsy from each experimental site was harvested for micro-CT and histological preparation. The scan was performed at an energy of 60 kV, intensity of 167 μA, and resolution of 13.3 μm using a 0.5-mm aluminum filter and a 3-dimensional micro-CT machine (SkyScan 1172, SkyScan, Aartselaar, Belgium). The data were reconstructed with the manufacturer’s software (DataViewer 1.5.2.4 64-bit version, Bruker micro-CT, Skyscan, Kontich, Belgium) and quantitatively analyzed with CTAn (Bruker-CT, Kontich, Belgium). Based on a previous study¹⁶, the volume of interest (VOI) was set to a 190-μm circular band stretching 60–2250 μm from the implant surface of each root, limiting 1 mm to 4 mm above the fixture apex.

Histological preparation

After 1 week in a fixative solution containing 10% neutral formalin buffer, the tissue sections were dehydrated in a series of ethanol solutions. Subsequently, the samples were embedded in methacrylate (Technovit 7200, Heraeus Kulzer, Hanau, Germany). The central mesiodistal sections were prepared and polished to approximately 45 ± 5 μm and stained with Goldner trichrome.

Histological and histomorphometric analyses

Histological slides were stored as digital images after scanning with Panoramic 250 Flash III (3DHISTECH, Budapest, Hungary). The region of interest (ROI) was selected from 1 to 4 mm above the fixture apex using a computer-aided slide image analysis program (CaseViewer 2.2; 3DHISTECH Ltd., Budapest, Hungary). As described in a previous study¹⁶, bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO) were measured from each 3D printed implant.

Statistical analysis

A sample size calculation was not performed due to the pilot nature of the study. All data of the two types of 3D-printed implants are presented as the means ± SDs. Two-way ANOVA (implant type and time period) was conducted, and Sidak’s multiple comparisons test was performed for implant stability and marginal bone changes. An unpaired t test was conducted in the micro-CT analysis. Due to the lack of normality test passes, the Mann‒Whitney test was performed for BIC and BAFO.