Commercially pure, grade 4 titanium discs (Dentium Co., Seoul, Korea) with 10 mm diameter and 2 mm thickness were divided into three groups as follows; machined-surface discs (A), sandblasted, large-grit, acid-etched (SLA)-treated discs (B), SLA-treated and microgroove (width × depth, 200 µm × 100 µm; distance between microgrooves, 50 µm)-formed discs (C). All samples were washed in an ultrasonic bath by using distilled water and stored in 100% ethanol at room temperature.

Surface roughness was estimated using laser scanning confocal microscope (Zeiss LSM 5 Pascal, Carl Zeiss Inc., Oberkochen, Germany) with imaging software (Zeiss LSM Image Examiner, Carl Zeiss Inc.). The multi-argon laser emits light with a wavelength of 633 nm. This allows the calculation of the arithmetic mean of surface roughness from a mean plane in the sampling area (900×900×350 µm). To evaluate surface wettability, water contact angle was measured by sessile drop method at room temperature. A video camera with an image analyzer (Phoenix 300, Surface Electro Optics, Seoul, Korea) visualized the shape of the drop and provided the contact angle. Three probe liquids of different polarities were used: 1-bromonaphthalene, formamide, and deionized water. The volume of liquid drops was controlled using an instrument with a computerized interface and pictures of the drops were taken as soon as they landed on the surfaces using a video camera. Right and left contact angles of each drop were automatically averaged to give one contact angle per drop. Then, the surface free energy was calculated using three contact angle values from three different probe liquids according to the van Oss model. Each experiment was repeated 5 times for 3 specimens of each experimental group. The surface energy was calculated using the Owens-Wendt equation [6].

Human gingival fibroblasts (hGFs), purchased from ScienCell (Carlsbad, CA, USA), and murine osteoblastic MC3T3-E1 cells were seeded at 1×10⁴ cells/well in a 24-well plate. After 24 hours cell culture, the cells were washed with PBS and fixed using 4% paraformaldehyde solution. The fixed cells were permeabilized with buffered 0.3% Triton X-100 at room temperature. The samples were blocked with 1% bovine serum albumin (BSA) for 1 hour. Actin (Abcam, Cambridge, MA, USA) was applied as the primary antibody and then reacted with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Sigma, St. Louis, MO, USA). The samples were then reacted with 4,6-diamidino-2-phenylindole (DAPI) (Sigma) for visualizing the nuclei. All the labeled cells were examined using the Confocal Laser Scanning Microscope (FV300, Olympus, Center Valley, PA, USA) equipped with FITC - and DAPI - channel filter systems (Olympus).

Four adult male beagle dogs (average age: 2 years, average weight: 13 kg) received dental prophylaxis for sustaining healthy periodontal conditions. Animal experiments were carried out in accordance with the Guidelines of the National Institute of Health (NIH) regarding the care and use of animals for experimental procedures and with the protocols approved by the Institutional Animal Care and Use Committee of Seoul National University (14-016-BA1402-147-007-01). Under general anesthesia by 2% xylazine hydrochloride (Rumpun, Bayer Korea, Seoul, Korea) with tiletamine hydro-chloride (Zoletil, Virbac, Ltd., Carros, France) and local anesthesia using 2% lidocaine hydrochloride with 1:100,000 epinephrine (Lidocaine, Huons, Seoul, Korea), mandibular 2nd, 3rd and 4th premolars on both sides were extracted. After 12 weeks of healing under the same anesthetic conditions as those of the tooth extraction procedure, SLA-treated titanium implants (diameter × length; 3.1 mm × 9mm), with microgrooves (width × depth, 200 µm × 100 µm; distance between microgrooves, 50 µm) in the coronal portion, were installed according to the guideline of manufacturer (Dentium, co, Seoul, Korea). The experiment was performed with a balanced block design in which different groups of implants were randomly placed in each extraction socket according to the Latin block experimental design: group A: implants installed with 1 mm exposure of coronal microgroove portion; group B: implants installed with 2 mm exposure of coronal microgroove portion (Fig. 1). After connecting healing abutments and suturing, antibiotics (Cefazolin, 30 mg/kg, Chong Kun Dang Pharmaceutical Co., Seoul, Korea) were intramuscularly injected with soft diet feeding. After 2 and 6 weeks, the animals were euthanized for mandibular block sections.

In preparation for histologic specimens, the mandibular blocks were dehydrated in 70-100% ethanol, embedded in methacrylate (Technovit 7200 VCL, Kulzer, Wehrheim, Germany), and sectioned in the mesio-distal plane by using a diamond saw (Exakt, Apparatebau, Norderstedt, Germany). From all the block sections, the central four sections were reduced to a final thickness of 50 µm and stained by hematoxylin and eosin. The specimens were histologically analyzed under an optical microscope (Leica DM 2500, Leica Microsystems, Wetzlar, Germany).

Digital images were captured and a computer-based image analysis system (Image-Pro Plus; Media Cybernetic, Silver Spring, MD, USA) was used to quantify the findings. The soft tissue contact ratio was measured along the continuous line of soft tissue attachment within 3 cycles of micro-grooved pitch from the coronal part of fixture.

All statistical analyses were conducted using the statistical program (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to compare the mean values of the contact angle and surface energy. Mann-Whitney U test was used to compare the mean values from the results of histometry. The threshold for statistical significance was set at P<0.05.