Rotina 420r centrifuge

For sample analysis, flat bottom 96-well microtitration plates and Eppendorf tubes were purchased from Greiner (Greiner, Frickenhausen, Germany). Component weighing was performed on a laboratory scale (Ohaus, Parsippany, NJ, USA). Sample centrifugation was performed on a Rotina 420R centrifuge (Hettich, Tuttlingen, Germany). Dynamic cell seeding of scaffolds was performed in a Roto-Therm Plus agitator (Benchmark Scientific, Sayreville, NJ, USA). SDS-Page analyses were performed using a Mini Gel Tank and PowerEase 90W (Thermo Fisher Scientific, Waltham, MA, USA). Gel imaging in white light or in chemiluminescence was performed on a Uvitec Mini HD9 gel imager (Cleaver Scientific, Rugby, UK). Colorimetric and luminescence measurements were performed on a Varioskan LUX multimode plate reader (Thermo Fisher Scientific, Waltham, MA, USA). Immunohistochemistry and Live-Dead imaging were performed on an inverted IX81 fluorescence microscope (Olympus, Tokyo, Japan). Telomerase activity PCR analyses were run on a StepOne Real-time PCR Systems instrument (Thermo Fisher Scientific, Waltham, MA, USA). Sample lyophilization was performed in a LyoBeta Mini freeze-dryer (Telstar, Terrassa, Spain). Sample terminal sterilization by γ-irradiation was performed by Ionisos, Dagneux, France.

Feedstock solution was prepared by mixing 0.4 M aqueous tin chloride pentahydrate (SnCl₄·5H₂O; J. T. Baker, Mexico City, Mexico and urea (CH₄N₂O; Sigma Aldrich, Mexico City, Mexico in 1:2 ratio. Then the mixed solution was vigorously stirred and heated until the solution temperature reaches around 93 ± 5 °C. Unlike the former case (urea), for ammonia (NH₄OH; Sigma Aldrich) as precipitation agent it was added dropwise to 0.4 M aqueous SnCl₄·5H₂O until the pH of the solution reached 12. The resultant precipitates in both cases were centrifuged at 400 rpm for 1 h using a ROTINA-420R centrifuge (Hettich, Toluca, Mexico) and the obtained pastes were dried at 100 °C for 24 h to eliminate any remaining solvent. Finally, the dried powders were calcined in a furnace at 800 °C for 2 h to obtain pure SnO₂ powders. All the synthesis conditions were studied, optimized and reported in our previous works [33 ,34 ].

Component weighing was performed using a laboratory scale (Ohaus, Parsippany, NJ, USA). Sample centrifugation was performed using a Rotina 420R centrifuge (Hettich, Tuttlingen, Germany) or on a Sorvall Legend Micro 21R microcentrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Colorimetric and luminescence measurements were taken using a Varioskan LUX multimode plate reader (Thermo Fisher Scientific, Waltham, MA, USA). Telomerase activity and chondrogenic gene expression quantification assays were run on a StepOne Real-time PCR System instrument (Thermo Fisher Scientific, Waltham, MA, USA). Spectrophotometric analyses were performed using a NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA, USA). Immunohistochemistry imaging was performed using an inverted IX81 fluorescence microscope (Olympus, Tokyo, Japan).

Cells were stained with antibodies for 20 minutes on ice and expression markers were analyzed with a BD LSR II flow cytometer (BD Biosciences). For immunofluorescence staining, 1 × 10⁵ cells in 200 μL/chamber were used, and for flow cytometry, 1 × 10⁵ in 50 μL antibody were used. For intracellular staining, cells were fixed and permeabilized with IntraPrep (Beckman Coulter, Krefeld, Germany) according to the manufacturer’s protocol. For immunofluorescence staining, cells were centrifuged for 5 min at 200× g (4 °C) on culture slides using a Rotina 420R centrifuge (Hettich, Tuttlingen, Germany) and dried overnight. Cytospins were fixed with acetone:methanol (1:1) (AppliChem), blocked with 5% BSA (Sigma-Aldrich, Schnelldorf, Germany) in PBS and stained with primary and secondary antibodies (Table 1) diluted in 0.1% BSA in PBS. Cells were then fixed with 4% PFA (Microcos, Germany) and mounted with ProLong^® Gold antifade reagent with DAPI (Invitrogen). The antibodies used in this study are listed in Table 1. For analysis of dead cells, Propidium iodide (Sigma-Aldrich) was added for 10 min at 4 °C prior to the analysis by flow cytometry.

Frozen samples were shredded using a kitchen grater, and approximately 1.0 g (exact weight listed) was homogenized with 7,5 ml of trichloroacetic acid (TCA, 7% w/v) for 1 min with an Ultra Turrax T25 Basic (Janke & Kunkel IKA^®‐Labortechnik, Staufen, Germany). Potassium hydroxide was then added to the sample solution (KOH, 1 M, 3.25 mL) to achieve a pH of 6.25. The mixing tubes were kept on ice during preparation thereafter centrifuged (18000 rpm, 4°C, 15 min) in a Rotina 420R centrifuge (Hettich Zentrifugen, Germany) before the supernatant was filtered through a nylon filter (0.45 mm) and transferred to HPLC vials (Agilent, 862‐09‐16, 2 ml) for analysis.
The samples were analyzed on a Poroshell 120 porous column (ECC18 3.0 × 100 mm, porous size 2.7 mm, with a Poroshell 120 Fast Guard (3.0 × 5 mm, Sub‐2 mm), Agilent InfinityLab) after a modified method by Sellevold et al. (1986 (link)), as described by Lerfall et al. (2018a (link)). The K value and H value were calculated based on the concentrations of ATP degradations products (Hong et al., 2017 (link); Howgate, 2006 (link)):
$$K=\frac{\left[\mathrm{Ino}\right]+\left[\mathrm{Hx}\right]}{\left[\mathrm{ATP}\right]+\left[\mathrm{ADP}\right]+\left[\mathrm{AMP}\right]+\left[\mathrm{IMP}\right]+\left[\mathrm{Ino}\right]+\left[\mathrm{Hx}\right]}\times 100\%$$
$$H=\frac{\left[\mathrm{Hx}\right]}{\left[\mathrm{IMP}\right]+\left[\mathrm{Ino}\right]+\left[\mathrm{Hx}\right]}\times 100\%$$ where Ino is inosine, Hx is hypoxanthine, ATP is adenosine triphosphate, ADP is adenosine diphosphate, AMP is adenosine monophosphate, and IMP is inosine monophosphate.