For apoptosis detection, the 10-μm bucco-palatal sections from around the experimental implant were incubated overnight with FITC-conjugated anti-rat GFP (1:100, Sigma-Aldrich) and 7-amino actinomycin D (7-AAD, Apoptosis Detection Kit; BD Biosciences, Franklin Lakes, NJ) at 4 °C. Apoptotic cells were then counted and calculated as a percentage of the total cells.

MSCs were cultured in osteogenic culture medium containing 1.8 mM KH₂PO₄ and 10 nM dexamethasone (Sigma-Aldrich). After 28 days of osteogenic induction, an expression of the osteogenic marker runt-related transcription factor 2 (Runx2, 1:100, Santa Cruz Biotechnology) was determined by immunofluorescent staining.

MSCs were cultured in adipogenic culture medium containing 0.5 mM isobutylmethylxanthine, 60 μM indomethacin, 0.5 μM hydrocortisone, and 10 μg/ml insulin (all Sigma-Aldrich). After 14 days of adipogenic induction, expression of the adipogenic marker, peroxisome proliferator-activated receptor gamma (PPARγ, 1:100, Santa Cruz Biotechnology), was determined by immunofluorescent staining.

Oral mucosa epithelial cell (OEC) cultures were performed based on a previous report [19]. Briefly, oral mucosa derived from 4-day-old Wistar rats was incubated with dispase (1 × 10³ IU/ml) in Mg²⁺ and Ca²⁺-free PBS for 12 h at 4 °C. The oral epithelium was then peeled from the connective tissue using tweezers. The epithelium was dispersed by pipetting ten times and seeded onto dishes or Ti plates placed on the bottom of dishes. OECs were cultured in defined keratinocyte serum-free medium (DK-SFM; Invitrogen, Grand Island, NY) and gentamicin (50 μg/ml) on plastic in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.

OECs were cocultured indirectly with MSCs using Transwell® insert as a separator between the two cell types. Briefly, OECs were cultured at a density of 5 × 10⁵ cells/mL on mirror-surfaced pure Ti plates [15 mm diameter by 1 mm thickness, 0.19 μm roughness (Ra), Japan Industrial Standards Class 1 (equivalent for ASTM Grade I)] (KS40, Kobelco, Kobe, Japan). Transwell inserts without MSCs in the upper chamber served as controls (Fig. 7a). As shown in Fig. 7c, the upper chamber contained either 5 × 10², 5 × 10³, 5 × 10⁴, or 5 × 10⁵ MSCs (in 0.5 ml of culture medium), while the bottom chamber contained 5 × 10⁴ OECs (in 1.5 ml of medium) for the various assays described below.