L ascorbic acid 2 phosphate

Human SCAPs (stem cells from the apical papilla) isolated from adult teeth were grown in clonogenic medium: α-Minimum Essential Medium complemented with 15% fetal bovine serum (FBS), 100 μM L-ascorbic acid-2-phosphate, 100 μg/ml streptomycin, 100 U/ml penicillin, and 2 mM L-glutamine (all from Gibco/Invitrogen, Grand Island, NY, US). Aliquots of SCAPs (1 × 10⁶ cells) were used for analyzing their immunophenotype profile, as previously described [29 (link)].

Adipogenic, chondrogenic, and osteogenic differentiation of SHED and ASC were carried out as previously described at the third passage [19 (link)]. The cultures were initiated at a density of 1000 cells/cm² in six-well plates and grown until confluence and then subjected to differentiation into adipogenic, chondrogenic, and osteogenic lineages. Briefly, the adipogenic lineage was initiated by inducing the cells with 10% FBS, 200 μM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine (IMBX), 10 μg/mL insulin, and 1 μM dexamethasone (all from Sigma-Aldrich). Lipid droplets were visualized using oil red staining (Sigma-Aldrich). For chondrogenic differentiation, cells were cultured in medium supplemented with ITS+1 (Sigma-Aldrich), 50 μM L-ascorbic acid 2-phosphate, 55 μM sodium pyruvate (Invitrogen), 25 μM L-proline (Sigma-Aldrich), and 10 ng/mL transformation growth factor-β (TGF-β; Sigma-Aldrich). Assessment of proteoglycan accumulation was visualized by Alcian Blue staining (Sigma-Aldrich). Osteogenic differentiation was stimulated in a 3-week culture period in medium supplemented with 10% FBS, 10–7 M dexamethasone, 10 mM glycerol phosphate (Fluka, Buchs, Switzerland), and 100 μM L-ascorbic acid 2-phosphate. The assessment of calcium accumulation was visualized using von Kossa staining (Sigma-Aldrich).

Primary cells were obtained from healthy tissues (bone marrow and articular cartilage) of a deceased, skeletally mature equine donor (aged 6 years old; n = 1), donated for research by their owner, according to the guidelines of the Institutional Animal Ethical Committee of the veterinary clinic of Utrecht University. Mesenchymal stromal cells (MSCs) were harvested from bone marrow aspirated from the sternum, while articular cartilage-derived chondroprogenitor cells (ACPCs) were obtained from enzymatic digests of cartilage from the metacarpophalangeal joint, following previously reported protocols and following the ethical regulations of the host institution [47 (link)]. MSCs were expanded in minimum essential medium alpha (αMEM, 22561 Gibco, The Netherlands) supplemented with 0.2 mM L-ascorbic acid 2-phosphate (ASAP, Sigma), 10% fetal calf serum (FCS, Lonza, The Netherlands), 100 U/ml penicillin with 100 mg ml⁻¹ streptomycin (Life Technologies, The Netherlands) and 1 ng ml⁻¹ basic fibroblast growth factor (bFGF, Peprotech, UK). ACPCs were expanded in Dulbecco’s modified Eagle medium (DMEM, 31966, Gibco, The Netherlands), supplemented with 10% v/v FCS, 0.2 mM L-ascorbic acid-2-phosphate, 100 U/ml penicillin, 100 mg ml⁻¹ streptomycin and 5 ng ml⁻¹ (bFGF, Peprotech, UK)). Cells were used between passage 3 and 5.

TCs+, TCs- and clones at P4 were seeded at 10⁴ cells/cm² and cultured for 21 days in chondrogenic medium consisting of HG-DMEM supplemented with 1% of FBS, 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, 1 mM sodium pyruvate (all from Sigma-Aldrich), 1% ITS + 1 (1.0 mg/mL insulin from bovine pancreas, 0.55 mg/mL human transferrin, 0.5 μg/mL sodium selenite, 50 mg/mL bovine serum albumin and 470 μg/mL linoleic acid, Sigma-Aldrich), 0.1 mM dexamethasone, 0.1 mM L-ascorbic acid-2-phosphate, 10 ng/ml TGF-β1 (Peprotech) (modified from Barbero et al. 2004 (link)). The cells were fixed with 10% neutral buffered formalin solution, rinsed with distilled water, and stained with Alcian Blue solution (pH 2.5) for 30 min (Sigma-Aldrich) to evaluate glycosaminoglycan deposition. The dye was extracted with guanidine hydrochloride (6 M) and the absorbance read at 650 nm (Perkin Elmer Victor X3 microplate reader) (Ruzzini et al. 2014 (link)).

For chondrogenic differentiation, 5.0 × 10⁵ cells were centrifuged at 250g for 5 min to obtain pellets. The pellets were cultured in four different media: control medium, DMEM supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml L-glutamine, 1 mM sodium pyruvate, 1.25 mg/ml human serum albumin (HAS; Sigma-Aldrich), and 10% FBS; chondrogenic medium, consisting of DMEM supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml L-glutamine, 1 mM sodium pyruvate, 1.25 mg/ml human serum albumin (HAS; Sigma-Aldrich), 1% ITS+1 containing 1.0 mg/ml insulin from bovine pancreas, 0.55 mg/ml human transferrin, 0.5 μg/ml sodium selenite, 50 mg/ml bovine serum albumin and 470 μg/ml linoleic acid (Sigma-Aldrich), 0.1 μM dexamethasone, 0.1 mM L-ascorbic acid-2-phosphate, and 10 ng/ml TGF-β1 (PeproTech, Rocky Hill, NJ, USA) (Lopa S); control medium plus 10% v/v autologous micrografts; chondrogenic medium plus 10% v/v autologous micrografts. The medium was replaced every 3 days and cells cultured at 37 °C under a 5% CO₂ atmosphere for 4 weeks before the following evaluations.