L tryptophan

Sodium acetate trihydrate (pro analysis), acetic acid (glacial), acetonitrile (HPLC grade), and methanol (HPLC grade) were purchased from Merck KGaA (Darmstadt, Germany). Disodium hydrogen orthophosphate heptahydrate (98.0–102.0%), ethylenediamine tetraacetic acid (EDTA) (>99%), and orthophosphoric acid (>85%) were purchased from VWR Chemicals BDH (Radnor, PA, USA). Triethylamine (>99%), DL-norleucine (98%), L-asparagine (>98%), L-glutamine (>99%), L-tryptophan (>98%), DL-dithiothreitol (>985), and phenylisothiocyanate (>99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The following reference materials were purchased from Sigma-Aldrich: Amino acid standard solution AAS18 (2.5 μmol mL⁻¹ of 18 proteinogenic amino acids in 0.1 N HCl), Amino acid standard solution A6407 (2.5 μmol mL⁻¹ of 26 physiological amino acids in 0.1 N HCl), Amino acid standard solution A6282 (2.5 μmol mL⁻¹ of 14 physiological, basic amino acids in 0.1 N HCl).
A proteinogenic amino acid standard consisting of Amino acid standard solution AAS18 and 0.05 μmol mL⁻¹ of L-asparagine, L-glutamine and L-tryptophan was prepared. A composite physiological amino acid standard was prepared from equal volumes of amino acid standard solutions A6407 and A6282.

Five-week-old C57BL/6 mice were used in the experiments, according to the Guidelines for Animal Research of Okayama University Dental School, under the approval of the Okayama University Ethical Committee (OKU-2013125). For analysis of the effect of L-tryptophan or L-kynurenine in vivo, mice were injected intraperitoneally with 50 mg/kg/day of L-tryptophan (Sigma-Aldrich) or L-kynurenine sulfate (Sigma-Aldrich) for consecutive three weeks, and then femurs were dissected from mice for mBMSC isolation or further micro-CT and histological analysis.
For the surgical experiment, mice were anesthetized by inhalation of Isoflurane (Isoflu: Dainippon Sumitomo Pharma Co., Osaka, Japan). The left lower limb was shaved and aseptically cleaned with 70% ethanol. An incision of approximately 15 mm in the frontal skin of the mid-femur was performed to expose the muscle. The muscle was then elevated and the periosteum was separated to expose the femur surface. A drill was used to make a surgical defect of 1 mm in diameter in the anterior portion of the diaphysis, 5 mm above the knee joint [27 (link)]. The diaphysis was irrigated with saline during the surgery. Thereafter, the muscles were replaced in the original position and the incision line was sutured.

Primary fibroblasts were isolated from the skin of nonstressed Swiss mice (1–2 months) by the standard explant technique as previously described [6 (link), 37 ]. The cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Cultilab Ltda, Campinas, Brazil) and antibiotics (100 UI/ml penicillin, 50 μg/ml kanamycin, 100 μg/ml streptomycin, and 6 μg/ml amphotericin B) (Sigma-Aldrich) at 37°C and 5% CO₂. Cells were seeded for experiments when they reached about 95% confluence. Experiments were performed at passages 3 to 10, at least three times, in triplicate.
In all experiments, cells were treated with epinephrine (100 μM) (Hipolabor, Minas Gerais, Brazil), L-tryptophan (10 μM) (Sigma-Aldrich), or epinephrine plus L-tryptophan [38 (link)]. The dose of 10 μM of L-tryptophan was based on a dose-dependent and time-dependent study (data not shown) and previous study [39 (link)]. Drugs were prepared in DMEM with 2% FBS and antibiotics.