Twenty-four hours after implantation, rats were lightly anesthetized with chloral hydrate and lidocaine hydrochloride, and ex vivo expanded P3 green fluorescent protein (GFP)-MSCs (1 × 10⁶ cells) were administrated via one of the following modes: (1) systemic injection via the tail vein (systemic group), (2) local injection into the gingivobuccal fold around the dental implant (local group), and (3) no MSC injection (control group).

Peripheral blood was collected from the retro-orbital plexus of the model rats. Samples were spread onto slides, dried for 30 min, and fixed in 4% PFA for 10 min. For fluorescence staining, samples were incubated with FITC-conjugated rat anti-GFP antibodies for 2 h at 37 °C. Imaging was performed using an Axiotech Microscope (Carl Zeiss, Göttingen, Germany).

Peripheral blood was centrifuged to obtain the blood serum, corrected 24 h after the MSC administration. The supernatants from the blood were extracted using M-PER® (Thermo Fisher Scientific, Waltham, MA) mammalian protein extraction reagent. The samples were centrifuged and used in an enzyme-linked immunosorbent assay (ELISA) for detection of interleukin (IL)-2, IL-4, and IL-10 (R&D Systems).

The procedure for topical application of horseradish peroxidase (HRP) was similar to that reported previously [16, 17]. Four weeks after implantation, rats were placed under deep anesthesia, and 50 mg/ml of HRP (type 11, molecular weight 40,000 Da, Sigma-Aldrich) was instilled into the oral mucosa surrounding the implant for 60 min. The optimal length of HRP penetration was estimated using peroxidase 3,3′-diaminobenzidine (DAB, Nacalai tesque, Kyoto, Japan) and hematoxylin staining.

Tissues were prepared in accordance to the methods described in our previous studies [8, 15]. After each experimental period, the rats were deeply anesthetized and perfused intracardially with heparinized PBS followed by 4% PFA (pH 7.4). Tissue samples (lung, liver, heart, and maxilla) were collected at each experimental period, dissected, and immersed in 4% PFA for 48 h at 4 °C. The oral mucosa surrounding the implant and tooth site was carefully removed from the bone, implant, or tooth, and cut into 10-μm bucco-palatal sections using a cryostat at − 20 °C. The sections were then stained immunohistochemically using mouse anti-rat GFP (1:100, Sigma-Aldrich), CD90 (1:100, Sigma-Aldrich), and Ln-332 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies (1:100, Sigma-Aldrich) overnight at 4 °C. Samples were then treated with FITC-conjugated secondary antibody (1:200, Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature and mounted with DAPI (Vector Laboratories), as described previously [17, 18].