Calcein

Lead nitrate, calcium phosphate, Tricaine and paraformaldehyde used in the experiments were purchased from Biopped, Paterson, NJ, USA. Alizarin red, calcein and acridine orange were purchased from Solarbio Co., Ltd. (Beijing, China). Trizol was purchased from TaKaRa, Kyoto, Japan.

All the lipids (PCs and PEs) used in preparing liposomes (Table 1) were purchased from Corden Pharma Switzerland LLC, Liestal, CH, except that DHPC (1,2-diheptanoyl-sn-glycero-3-phosphocholine), DPHPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) were from Avanti Lipids Polar, Inc., Alabaster, AL. Calcein (Solarbio, Beijing, China) was used as a fluorescent dye to mark the contents of GVs. Organic solvents, including mineral oil (liquid paraffin, d = 0.840 g/mL), were from Adamas-Beta Reagent (Shanghai, China), heavy mineral oil from Sigma–Aldrich (St. Louis, MO, USA), and chloroform and methanol to dissolve the lipids from Sinopharm Chemical Reagent (Shanghai, China). N-(2 Hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES), sucrose, glucose, and KOH were purchased from Sigma–Aldrich (St. Louis, MO, USA).

As shown in Figure 5A, premolars and first molars of beagle dogs were removed under general anesthesia. At 3 months after tooth extraction, titanium rods (3 mm in diameter) were implanted in the alveolar bone of the TNT and NTNF groups. Calcein (20 mg/kg, Solarbio, China) and alizarin red (30 mg/kg, Solarbio, China) were injected into the beagles for double-fluorescence labeling at 9 and 11 weeks after implantation. When osseointegration was completed 3 months later, all beagle dogs were euthanized. The jaws with titanium rod implants were excised and fixed with 4% paraformaldehyde for 48 h and then soaked in 75° alcohol after rinsing overnight with running water.