Dnase solution

The ECM membrane was constructed using porcine chondrocytes as follows the our previous study [21 ]. In brief, chondrocytes were isolated from the articular cartilage of 2–3 week old porcine knees using 0.2% (wet/vol) collagenase (Worthington Biochemical Corp., Lakewood, NJ) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen Corporation, Grand Island, NY). The primary chondrocytes were cultured at a density of 1.9×10⁵ cells/cm² in 6-well plates for three weeks in order to form a cell-ECM complex membrane. To remove cell components, they underwent decellularization using sequential incubation in a 0.1% SDS solution for 2 h and then in 200 U/ml DNase solution (Sigma, USA) for 24 h at 37°C. The decelluarized ECM membrane was washed with distilled water for 1 h and pressed into a thin membrane, five of which were stacked layer-by-layer. Then, 50, 100 and 200 ng of rhTGF-β₃ was loaded into the middle layers. Next, the rhTGF-β₃-loaded ECM multilayer was dried and cross-linked via UV for 1 h at room temperature (Fig 1(A) and 1(B)). Only 100 ng of rhTGF-ß3 was loaded on the EMLDS because this resulted in a release profile of 10 ng/day consistent with TGF-ß3 concentrations normally used for in vitro chondrogenesis studies.

The decellularization of the PDL strips and tooth particles was carried out by sequential incubation with a 1% SDS solution (Sigma-Aldrich, St. Louis, MO, USA), a 1% Triton X-100 solution (Sigma-Aldrich, USA), and a DNase solution (20 μg/mL; Sigma-Aldrich, USA) in 4.2 mM MgCl₂ (Sigma-Aldrich, USA) [35 (link)]. Further, the samples were incubated in DMEM culture medium (Gibco, Thermo Fisher Scientific, USA) with antibiotics (300 U/mL penicillin, 300 μg/mL streptomycin, and 75 μg/mL amphotericin B) (Gibco, USA) and stored at −70 °C. All treatment steps were applied to samples at room temperature with constant gentle agitation of the samples in an orbital shaker (Corning™ LSE™, USA). Histological sections were stained with VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) to validate the efficiency of decellularization.

Trachea from WT mice were collected in DMEM-F12 GlutaMAX medium (Thermofisher Scientific) containing Penicillin-Streptomycin (100 U/mL, Fisher) cocktail and incubated over night at 4°C with Pronase (1.5 mg/mL, Sigma-Aldrich). Cells were collected and washed 3 times with DMEM-F12 GlutaMAX-10% FBS, 100 μm filtered, and centrifuged at 1,500 rpm for 10 min. Cell pellet was resuspended in DNAse solution (0.5 mg/mL, Sigma-Aldrich) for 5 min. Suspension was centrifuged for 10 min at 1,500 rpm and the pellet was resuspended in DMEM-F12 GlutaMAX media and incubated in Primaria plates (353801, VWR) at 37°C for 4 h to remove potential fibroblasts. Unadherent cells were then collected, centrifuged at 1,500 rpm for 10 min, counted and plated in 24 well-plate coated with rat tail collagen (ThermoFisher Scientific) at 50 000 cells per well. At this step, DMEM-F12 GlutaMAX-5% FBS medium containing Insulin-Transferrin (10 μM ThermoFisher Scientific), Cholera Toxin (0.10 μg/mL, Sigma-Aldrich) Epidermal Growth Factor (0.025 μg/mL, Sigma-Aldrich), Bovine Pituitary Extract (30 μg/mL, Fisher), and Retinoic acid (50 nM, Sigma-Aldrich) was used to maintain epithelial cells prior to stimulation. Supernatant and intracellular fraction were stored at −80°C for further analysis.