Sml0223

Two-month-old wild-type C57BL/6 J male mice were purchased from Jax laboratory. Itch−/− mice on a C57BL/6 J background were generated and genotyped by PCR51 (link). Although both male and female patients take anti-depressants, we used male mice in the study because we wanted to avoid the potential influence of estrogen on bone in female mice. Three sets of experiments were performed. 1) To study the role of Clomipramine (CLP, SIGMA, C-7291) on bone mass, mice (n = 5–7/group) were treated with water as control (Ctrl) or CLP (10 mg/kg body weight) by intraperitoneal (i.p.) injection daily for two weeks. 2) To study the effect of Zoledronic acid on CLP-induced bone loss, mice were pre-treated with saline or Zoledronic acid25 (link) (ZA, SIGMA, SML0223; 0.25 mg/kg body weight) by i.p. injection twice/week for two weeks, then followed by CLP for another 2 weeks (n = 4–6/group). 3) To study if itch is required for CLP-induced bone loss, age- (3-month-old) and gender- (male) matched Itch−/− mice (n = 4–5/group) were treated with CLP (10 mg/kg body weight) by i.p. injection daily for two weeks. Mice were sacrificed 4 hours after the last treatment and samples were harvested for analyses.

With regard to the antioxidative drugs, N-acetyl-L-cysteine (NAC) and amifostine (2-(3-Aminopropyl)aminoethyl phosphorothioate, WR2721, Ethyol) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France; #9165 and #A5922, respectively). Cells were incubated with these two last drugs separately for 24 h at 37 °C at 10–300 mM NAC or at 24–96 mM for amifostine. The choice of these drug concentrations was motivated by preliminary experiments based on the assessment of γH2AX foci when drug was applied alone to radioresistant fibroblasts. All the conditions leading to the production of more than 2 γH2AX foci per cell were excluded. With regard to the pro-episkevic drugs, cells were incubated with either 1 µM pravastatin (#P4498, Sigma-Aldrich) for 24 h at 37 °C alone or 1 µM zoledronate (#SML0223, Sigma-Aldrich) for 12 h at 37 °C alone (to permit intercomparisons) or together (ZOPRA treatment) [46 (link)]. The choice of these drug concentrations was notably motivated by our previous data published in [46 (link)].

Rabbits were maintained in a conventional room for 1 week to adapt to the environment. The BP, HA, and ADSC groups then received zoledronic acid (800 μg/kg, sml-0223, Sigma, USA) and dexamethasone (Dex, 10 mg/kg, Sinopharm Group, China) intravenously (I.V.) once per week for 8 weeks. The bilateral premolars were extracted under deep anesthesia by I.V. injection of pentobarbital (20 mg/kg, P3761, Sigma, USA) and xylazine (50 μl/kg, Jilin Huamu Animal Health Product Co., Ltd., China) after the sixth dose. For the ADSC group, ADSCs (passage 3–5) incubated on coral hydroxyapatite (HA) were immediately filled into the extraction sockets of animals with a medication history (ADSC group). Non-medication-treated animals and nonfilled animals (healthy group), medication-treated and nonfilled animals (BP group), and medication-treated and non-ADSC hydroxyapatite-filled animals (HA group) were used as controls. Then, at 2 and 8 weeks post extraction, the tooth extraction socket and the adjacent first molar were harvested at each indicated time. The unhealed area, necrotic bone, and new bone formation were measured based on clinical appearance and histology. All parameters mentioned above were quantified by ImageJ software (Version 1.51 s, US National Institutes of Health, Bethesda, Maryland).