Demeclocycline hydrochloride

Fifteen mice were divided into five groups: 1) subcutaneously PBS-injected group immediately after intravenous PBS injection from the tail vein (negative control) (n = 3), 2) alizarin complexone (alizarin; Dojindo, Kumamoto, Japan) -injected group (n = 3), 3) calcein (Sigma-Aldrich) -injected group (n = 3), 4) tetracycline hydrochloride (tetracycline; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan)-injected group (n = 3), and 5) demeclocycline hydrochloride (demeclocycline; Sigma-Aldrich, St. Luis, MO, USA) -injected group (n = 3) (Figure 1A). Later four groups received subcutaneous injections of fluorescent reagents using a 27 G needle and a 1 ml syringe (Terumo Corporation, Tokyo, Japan) at a dose of 20 mg/kg immediately after intravenous S. mutans inoculation to the tail vein. All mice were sedated and then sacrificed by cervical dislocation 24 hrs after intravenous PBS injection or S. mutans inoculation. The mice were immersed in 70% ethyl alcohol for several seconds, and then right femurs and liver were dissected for bacteriological analyses. The comparison between alizarin and PBS subcutaneous injections on the colony formation was shown in the Supplementary Section.

For in vivo dynamic fluorescent labeling of the extraction sockets, two animals from each group were administered calcein (20 mg/kg; Sigma-Aldrich, St. Louis, MO, USA), demeclocycline hydrochloride (20 mg/kg; Sigma-Aldrich, St. Louis, MO, USA), and Alizarin complexone (20 mg/kg; ALC, Dojindo, Kumamoto, Japan) subcutaneously on days 6, 15, and 24 after tooth extraction, respectively. The samples were then thoroughly washed with PBS before fixation using 10% PBS-based formaldehyde fixative (pH 7.4) (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) for 48 h at 4 °C under constant shaking motion. The undecalcified frozen blocks were prepared using the same method into 5-μm thick tissue sections retrieved via adhesive Kawamoto film (Section-Lab Co., Ltd., Hiroshima, Japan). Bone formation on the extraction sockets according to the bone-labeling schedule was observed using a BZ-X700 fluorescence microscope (Keyence Corp., Osaka, Japan).