Ma1 26771

L2 to L3 vertebral bone tissue proteins were extracted using a cell lysate buffer as described previously.⁽²⁸⁾ p53, p21, p16, collagen 1, NFATc1, cathepsin K, and Nox4 protein expression in bone tissue were assessed by standard Western immunoblotting using antibodies recognizing these proteins, followed by incubation with secondary antibodies conjugated with horseradish peroxidase. Anti‐p53, Ms, monoclonal, #ab28 (Abcam, Cambridge, UK); anti‐p21, Rb, monoclonal, #ab109199 (Abcam); anti‐p16, Rb, monoclonal, #ab108349 (Abcam); anticollagen 1 (Col‐1a), Ms, monoclonal, #MA1‐26771 (Thermo Fisher Scientific, Waltham, MA, USA); anti‐NFATc1, Ms, monoclonal, #sc‐7294 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anticathepsin K, Rb, polyclonal, #ab19027 (Abcam); anti‐Nox4, Rb, polyclonal, #ABC459 (Millipore, Billerica, MA, USA); and anti‐βActin, Ms, monoclonal, #A1978 (Sigma‐Aldrich, St. Louis, MO, USA). β‐actin protein in bone tissue was analyzed by immunoblotting, using mouse monoclonal antibody recognizing β‐actin (Sigma‐Aldrich), followed by incubation with a secondary antimouse antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology). Immunoblots were visualized using SuperSignal West Pico chemiluminescent (Pierce, Rockford, IL, USA). Quantitation of the intensity of the bands in the autoradiograms was performed using a VersaDoc imaging system (Bio‐Rad).

The production of type I collagen and osteopontin, two relevant bone ECM-specific proteins, by the hBM-MSCs seeded on the different scaffolds was evaluated after 21 days of osteogenic differentiation through immunofluorescence imaging. PFA-fixed MSC-seeded scaffolds were first washed in PBS and 1% BSA (Sigma Aldrich); after which, they were immersed in a 1% BSA, 10% FBS and 0.3% Triton X-100 permeabilization/blocking solution for 45 min at room temperature. The samples were then incubated overnight with the primary antibodies for osteopontin (ab8448; abcam, Cambridge, UK) and type I collagen (MA1-26771, ThermoFisher) (1:150 in 1% BSA, 10% FBS and 0.3% Triton X-100 solution) at 4 °C. Afterwards, the nanofibrous scaffolds were incubated for 1 h in solutions containing the secondary antibodies (prepared in 1% BSA at a dilution factor of 1:150)—goat anti-rabbit AlexaFluor™ 448 for osteopontin and goat anti-mouse AlexaFluor™ 546 for type I collagen—at room temperature protected from light. After being washed in PBS, the cell-seeded scaffolds were imaged using a fluorescence microscope (LEICA DMI3000B, Leica Microsystems).

The distribution of type I collagen and osteopontin (important macromolecules produced during bone ECM formation) within the structure of the different piezoelectric scaffolds after 21 days of osteogenic differentiation was evaluated through immunofluorescence analysis. Previously fixed cell-seeded electrospun fibers were washed twice in PBS, after which the scaffolds were immersed in a permeabilization/blocking solution comprising 1% BSA (Sigma-Aldrich), 10% FBS and 0.03% Triton X-100 for 45 min at room temperature. Solutions containing primary antibodies for type I collagen (MA1–26771, Thermo-Fischer) and osteopontin (ab8448, Abcam) (1:200 in 1% BSA, 10% FBS, 0.03% Triton X-100 solution) were then incubated with the respective samples overnight at 4°C. The fibrous scaffolds were then incubated with a secondary antibody (1:200 in 1% BSA) for 1 h at room temperature in the dark. Following two washes with PBS, the fibers were counterstained with DAPI (1.5 μg/mL in PBS) for 5 min at room temperature, washed twice with PBS and imaged using a fluorescence microscope (LEICA DMI3000B, Leica Microsystems).