L ascorbic acid phosphate

An illustration is provided in Figure 6A. For primary osteoblast isolation, calvaria of neonatal mice were removed on postnatal day 3 (P3) and incubated in 4mM EDTA/PBS solution for 10 minutes at 37°C with agitation. Digestion with EDTA (Sigma, Cat. No. E5134) was repeated. The supernatant was discarded and tissues were placed in 0.1% collagenase I/0.2% dispase solution (Sigma, Cat. No. C2674) for 10 minutes at 37°C with agitation. The supernatant was discarded, and enzymatic digestion was repeated four additional times (fractions II-V). Supernatant from fractions II-V were collected, washed (300xg), and cultured with αMEM supplemented with 10% FBS in 25cm² flasks (1.5x10⁶cells/flask). For osteogenic differentiation to osteocyte-like cells (OLCs), passage 2 osteoblasts were grown to 90-95% confluence, followed by the addition of αMEM supplemented with 10% FBS, 5mM β-glycerophosphate (Sigma, Cat. No. G5422), 100μg/ml L-ascorbic acid phosphate (Sigma, Cat. No. A8960), and 10nM dexamethasone (Sigma, Cat. No. D4902). Osteogenic medium was changed every 3-4 days for 21 days.

AB-MSC spheroids were harvested after 24 h for osteogenic differentiation. Spheroids or monolayer cultured cells were seeded in 6-well plates (Falcon). When cells reached 70–80% confluency, the culture medium was changed to the osteogenic differentiation solution (culture medium supplemented with 100 nM dexamethasone, 50 µM L-ascorbic acid phosphate, and 10 mM β-glycerophosphate [all from Sigma-Aldrich]) for induction. Bone marrow MSCs were also subjected to osteogenic differentiation, and the differences between AB-MSC spheroids and bone marrow MSCs were compared. Osteogenic induction lasted for 14 d, and the medium was changed every 2 d.

Cells were isolated from human resected abdominal flap tissue during abdominoplasty procedure (Papageorgiou Hospital, Hospital Review Board-approved protocols 263-7/12/2016) and with the patient’s consent. Adipose tissue was minced into pieces and enzymatically digested in a mixed solution of 4 mg/mL collagenase/dispase (Roche, Basel, Switzerland) for 5 h at 37 °C. Cells were isolated and expanded in an MEM medium (Invitrogen, Waltham, MA, USA) mixed with 15% v/v FBS (EU-tested, Invitrogen) 2 mM Glutamine, 0.1 mM L-ascorbic acid phosphate (Sigma-Aldrich, Taufkirchen, Germany), 100 U/mL penicillin, and 100 mg/mL streptomycin (all from Sigma-Aldrich). In this study, we refer to this home-made medium as MSC. Isolated cells were expanded until passage 3, and characterized as Mesenchymal Stem Cells by a Guava^® easyCyte 8 HT flow cytometer (Merck-Millipore, Darmstadt, Germany), similarly to our previous work [50 (link)]. Cells expressed CD90 and CD73, whereas CD45 (BioLegend, San Diego, CA, USA) was used as a negative marker.

All experiments were conducted in accordance with the Declaration of Helsinki. Ethical approval was obtained from the Ethics Committee of Human Research from the Institution (CAAE 45748615.4.0000.0075). Third molars (n=3) with incomplete root formation were obtained with the patients’ informed consent. The apical papilla was manually separated from the roots. Tissues were minced and incubated for cell growth in Minimum Essential Medium Eagle – Alpha Modification (α-MEM) (Invitrogen, Thermo Fisher, Waltham, Massachusetts, USA) with 15% fetal bovine serum (FBS) (Gibco, Thermo Fisher, Waltham, Massachusetts, USA), glutamine (2 mM – Invitrogen, Thermofisher, Waltham, Massachusetts, USA), L-ascorbic acid phosphate (0.1 mM – Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (100 µg/mL penicillin, 100 µg/mL streptomycin, 0.5 mg/mL amphotericin B – Invitrogen, Thermofisher, Waltham, Massachusetts, USA) (Proliferation Medium – PM) under standard culture conditions of 37ºC, 100% humidity, 5% CO₂, and 95% air. The cells were used between the second and fourth passages ^19,20 .

A typical process for the fabrication of the tECM-DTSs is presented in Fig. 1. First, the DTSs substrate was fabricated using our previously published protocol [29 (link)]. In short, the Achilles tendons of adult beagle dogs were decellularized through the following procedures: repetitive freeze/thaw treatment, frozen section with a thickness of 300 μm and nuclease treatment (including DNase 150 IU/ml and RNase 100 μg/ml) for 12 h at 37°C. Following washing in 50 ml of 0.1 M PBS (3 × 30 min), the DTSs were lyophilized and sterilized with ethylene oxide (EO). Then, TDSCs were seeded on the top surface of DTSs substrate at 1 × 10⁵ cells per cm² and cultured in complete medium supplemented with 20% fetal bovine serum (FBS). After reaching 90% confluence, 50 μM of L-ascorbic acid phosphate (Sigma) was added for additional culture period of 8 days. At the end of 15-day culture period, the composites of TDSCs-DTSs were re-decellularized as described previously with minor alteration [15 (link)], using 0.5% Triton X-100 supplemented with 20 mM ammonium hydroxide (NH₄OH) at 37°C for 15 min, followed by 100 U/ml DNase I at 37°C for 2 h. Finally, the tECM modified DTSs (hereafter referred to as tECM-DTSs) were washed in 50 ml of 0.1 M PBS (6 × 30 min), frozen at –80°C or lyophilized and sterilized by EO for subsequent use.