Bone marrow cells were flushed out of the femurs and tibias of 4-week-old green fluorescent protein-transgenic Wistar rats. Cells were treated with a 0.85% NH₄Cl solution for 10 min to lyse the red blood cells and were passed through a 70-μm cell strainer to obtain a single cell suspension. Cells were seeded into 100-mm plastic culture dishes (1 × 10⁶ cells/dish), washed with phosphate buffered saline (PBS), and cultured in growth medium consisting of alpha minimum essential medium (α-MEM; Invitrogen, Grand Island, NY, USA), 20% fetal bovine serum (Equitech-Bio, Kerrville, TX), 2 mM L-glutamine (Invitrogen), 55 μm 2-mercaptoethanol (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Passage 3 (P3) cells were used experimentally in this study.

MSCs (5 × 10⁴ cells/ml) were seeded into 35-mm dishes and incubated for 12 h at 37 °C in 5% CO₂. The slides were then fixed in 4% paraformaldehyde (PFA) for 5 min and blocked with secondary antibody-matched normal serum for 1 h, followed by incubation with mouse anti-rat CD44, CD90, and CD105 antibodies (1:100, Sigma-Aldrich, St. Louis, MO,) overnight at 4 °C. The slides were then treated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:200, Jackson Immuno Research, West Grove, PA) for 1 h at room temperature and mounted using VECTASHIELD® Mounting Medium containing 4′6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA).

Single piece, screw-type pure Ti (Japan Industrial Standards Class 1; equivalent to ASTM Grade 1) experimental implants with a machine-polished surface (Sky blue, Fukuoka, Japan) were used in accordance to our previous study [15] (Fig. 1a). Implant surface roughness was measured using a laser scanning microscope (VK-9710, Keyence, Osaka, Japan), and the arithmetic mean roughness (Ra) was found to be 0.16 μm. The implants were treated with 100% acetone and autoclave sterilized before use.

All experimental procedures were approved by the Ethics Committee on Animal Experimentation at Kyushu University (Approval Number: A25-133-0), Japan, in accordance with the ARRIVE guidelines and the Guidelines of the Japanese Physiological Society. Surgical implantation was performed in accordance with previously published protocols [4, 16]. Briefly, 6-week-old Wistar rats (27 males, 120–150 g) underwent immediate implant placement as follows: the maxillary right first molars were removed and an experimental implant was screwed into the socket under systemic chloral hydrate under systemic anesthesia.