Xylenol

D-fructose was purchased from PhytoTechnology laboratories, Lenexa KS, The United States. A one-step RT-PCR kit (TransGen EasyScript®) was purchased from TransGen Biotech Co. Ltd (Beijing, China). All other chemicals, unless stated otherwise, including nicotinamide adenine dinucleotide phosphate (NADPH), sulphanilamide, 1-chloro-2,4-dinitrobenzene (CDNB), xylenol, 5.5-dithio bis 2-nitrobenzoic acid (DTNB), bovine serum albumin (BSA), pyrogallol, HEPES, N-(1-naphthyl) ethylenediamine, and reduced glutathione (GSH) were purchased from Sigma-Aldrich, Germany.

During the 12 weeks healing period, fluorescent bone markers were injected 55 (link) to observe the dynamics of new bone formation. Three weeks after the operation, 20 mg alizarin red S/kg of body weight was administered intraperitoneally (IP) (Sigma-Aldrich), 20 mg oxytetracycline HCl/kg of body weight (Sigma-Aldrich) was injected IP at 6 weeks post-surgery, and 20 mg xylenol/kg of body weight (Sigma-Aldrich) was injected IP at 9 weeks post-surgery. All dyes were prepared immediately before use in saline.

After 8 weeks of healing, the dynamics of the newly formed bones were observed by injecting them with fluorescent bone marker labels. At 2 weeks, alizarin red S (20 mg/kg body weight), oxytetracycline HCl (20 mg/kg body weight), and xylenol (20 mg/kg body weight) (Sigma-Aldrich) were administered via intraperitoneal injection. All dyes were prepared with saline immediately before use. To visualize remodeled bone, an intravital multiphoton microscope (SP8-MP; Leica, Wetzlar, Germany) with a water immersion lens (25×, 0.9 NA) was used. The fluorophores were excited using the InSight DS Plus laser system (Spectra-Physics, Santa Clara, USA) at the following wavelengths: xylenol (excitation = 800 nm, emission = 375-570 nm), oxytetracycline (excitation = 1040 nm, emission = 365-490 nm), and alizarin (excitation = 1180 nm, emission = 538-580 nm). Alizarin -, oxytetracycline-, and xylenol-positive areas were quantified using the ImageJ software. All values are expressed as mean ± SD (n = 4).