L thyroxine

A two-part ABS mold was recapitulated from a 3D digitized image of a temporomandibular joint (TMJ) condyle, as described above. The two-part ABS mold was infiltrated with a 4% agarose/50 mM CaCl₂ solution and allowed to set. The resulting two-part 4% agarose/50 mM CaCl₂ mold was assembled and injected with MSC-laden 2% alginate at a cell density of 20×10⁶ MSCs/mL. TMJ condyle constructs were cultured in a CM at 5% pO₂ for a period of 5 weeks, followed by culture in a hypertrophic medium consisting of hgDMEM GlutaMAX supplemented with 100 U/mL penicillin/streptomycin, 100 μg/mL sodium pyruvate, 40 μg/mL l-proline, 50 μg/mL l-ascorbic acid-2-phosphate, 4.7 μg/mL linoleic acid, 1.5 mg/mL bovine serum albumin, 1× insulin–transferrin–selenium, 1 nM dexamethasone, 2.5 μg/mL amphotericin B, 1 nM l-thyroxine, and 20 μg/mL β-glycerophosphate (both Sigma-Aldrich) at 20% pO₂ for an additional 3 weeks.

Oli-neu cells were grown in poly-L-lysine coated dishes and expanded in proliferation media consisting of DMEM ((life tec 41965062), N2 supplement, Pen/Strep Glu (life tec 10378016), T3 (sigma T6397) 340ng/ml, L-thyroxine (sigma 89430) 400 ng/ml, bFGF 10ng/ml and Pdgf-BB 1ng/ml. For Oli-neu differentiation, media without growth factors was added for two days in vitro and in the presence of 1 μM of erb inhibitor (PD174265, St Cruz, ref sc-204170).

The chondrogenic conditions applied in this study are defined as culture at 5% pO₂ in a chondrogenic medium consisting of high glucose DMEM GlutaMAX supplemented with 100 U/mL penicillin/streptomycin (both Gibco), 100 µg/mL sodium pyruvate, 40 µg/mL L-proline, 50 µg/mL L-ascorbic acid-2-phosphate, 4.7 µg/mL linoleic acid, 1.5 mg/mL bovine serum albumine, 1×insulin–transferrin–selenium, 100 nM dexamethasone (all from Sigma-Aldrich) and 10 ng/mL of human transforming growth factor-β3 (TGF-β3) (Prospec-Tany TechnoGene Ltd., Israel) [33] (link). The hypertrophic conditions applied are defined as culture at 20% pO₂ in a hypertrophic medium consisting of high glucose DMEM GlutaMAX supplemented with 100 U/mL penicillin/streptomycin, 100 µg/mL sodium pyruvate, 40 µg/mL L-proline, 50 µg/mL L-ascorbic acid-2-phosphate, 4.7 µg/mL linoleic acid, 1.5 mg/mL bovine serum albumine, 1×insulin–transferrin–selenium, 1 nM dexamethasone, 1 nM L-thyroxine and 20 µg/mL β-glycerophosphate (both Sigma-Aldrich) [13] (link), [34] (link).

The cell line Oli-neu was cultured in modified Sato media [28 (link)]. Cell culture dishes were coated with poly-L-lysine (Sigma). HEK293T cells were cultured in DMEM (Sigma) with 10% Horse serum (Biochrom) and 1% Sodium-pyruvate (Sigma). Transfection of HEK293T cells with the NG2del constructs was effected by a standard protocol using the GenePulserXcell (Bio-Rad). Transcription was increased by including 4 mM sodium butyrate for the protease assay.
Cerebella of postnatal day 8–9 homozygous NG2-EYFP(NG2-KO) mice [29 (link)] or C57BL/6N mice (as control) were dissociated in 1% trypsin, 0.05% DNase in HBSS using a fire-polished Pasteur pipette to obtain a single-cell suspension, followed by seeding on poly-L-lysine-coated dishes. The cells were cultured in B27 medium containing DMEM, pyruvate, triiodo-L-thyronine, L-thyroxine (Sigma), B27 supplement (Gibco), 10ng/ml PDGF, 5ng/ml FGF (PrepoTech) and 1% HS. The medium was changed on the following day and renewed every 3–4 d. After 10-14d (after morphological assessment) cultures were stressed with H₂O₂ in B27 medium without growth factors.
Glioblastoma cells (R10) were cultured on ECM (Sigma) gel-coated dishes. ECM Gel was diluted 1/10 with Neurobasal media. Growth medium was Neurobasal medium (Invitrogen) with N2 supplement, B27 supplement, L-glutamine, EGF, FGF and 1% [v/v] penicillin/streptomycin (Serva).

For preparation of the GH, porcine GH (Alpharma, Victoria, Australia) was dissolved in 0.1M NaHCO3 for stability, then 0.9% saline was added to make a single injection concentration of 21μg/50μL for the average animal body weight of 7g (3μg/g of body weight); pH was adjusted from 8.3 to 7.8. GH injections were performed twice a day on weekdays with each subcutaneous injection administering half the daily dose (~3μg/g); the first injection was ~9am and the second was ~4pm (~6μg/g/day total GH treatment). On weekends, only one injection was performed with the full daily dosage (~6μg/g/day).
For the preparation of the T4, L-thyroxine (Sigma, St Louis, MO) was dissolved in a 0.9% saline solution with a pH of 7.8. It was administered three times per week (Monday, Wednesday, and Friday) via subcutaneous injections (0.1μg/g body weight; 0.7 μg/50μL dose) and administered in conjunction with the respective morning GH doses, albeit delivered in separate injections.
The doses were selected on the basis of previous studies in Ames dwarf mice [42 , 43 ].

Fourteen two-month-old female Wistar rats were housed at a density of four rats per cage on a 12 h light-dark cycle, temperature of 18–22 °C and received food and water ad libitum. The females of all groups were submitted to vaginal cytology and the rats in proestrus were housed in plastic cages with adult male rats for 12 h during the night. The next morning, vaginal smears were performed and gestation was confirmed by the presence of sperm in the vaginal cytology. That day was designated as gestation day 0, and rats were separated, randomly, into individual boxes, comprising the treated (n = 7) and control groups (n = 7).
Treated rats received daily L-thyroxine (Sigma-Aldrich, St. Louis, MO, USA), administered using an orogastric tube, at a dose of 50 μg/animal/day, diluted in 5 mL of distilled water according to previously established protocols [13 (link),23 (link),49 (link)]; this treatment was performed throughout the gestation period and for three days of lactation. Females in the control group received the same volume of distilled water using an orogastric tube.
Three days after birth, neonates from each mother were euthanized. After intraperitoneal anesthesia with ketamine (100 mg/kg) (Sintec, São Paulo, Brasil) and xylazine (10 mg/kg) (Sintec, São Paulo, Brasil), rats were euthanized by cardiac puncture.

Male Wistar rats (n = 20; initial weight: 323.1 ± 13.49 g, Harlan Ibérica SL, Barcelona, Spain) were housed in the Animal Facility of the Universidad Autónoma de Madrid (Registration number EX-021U) and held in groups of 2 in appropriate cages, in controlled environmental conditions (20–24 °C, 55% relative humidity, 12 h light–dark cycle). The animals had access to fresh water and specific rat chow ad libitum.
Animals were randomly divided into two groups: (1) control rats (CT, n = 10); and (2) rats with induced hyperthyroidism (HT; n = 10). HT rats were infused with L-Thyroxine (1.5 µg/100 g per day, diluted in 0.02 N NaOH dissolved in sterile saline buffer for 14 days; Sigma-Aldrich Co., Madrid, Spain) with subcutaneously implanted Alzet osmotic minipumps (Durect Corp., Cupertino, CA, USA) [61 (link)]. Pumps containing only vehicle were implanted in CT rats.