Fluorodish 35 mm

In order to assess the differentiation of hUCMSC cells, the qualitative expression of Surfactant Proteins C (SP−C) has been evaluated by immunofluorescence against SPC antibody. hUCMSCs were cultured for 21 days and seeded on fluorodish—35 mm (World Precision Instruments, Inc. Hitchin, UK), were fixed in 10% Formalin for 1 h, permeabilized with 0.1% Triton X-100, blocked with 1% BSA and incubated with SPC rabbit antibody (Abcam, Cambrige, UK) diluted in 1% BSA at 4 °C overnight. After washing with PBS three times, FITC-conjugated anti-rabbit antibody (Millipore, Billerica, MA, USA) was added to the cells for 3 h at room temperature. In parallel, qualitative expression of Cluster of Differentiation 73 (CD-73) has been evaluated by immunofluorescence against CD-73 antibody. hUCMSCs were cultured for 21 days and seeded on fluorodish—35 mm (World Precision Instruments, Inc.), were fixed in 10% Formalin for 1 h, permeabilized with 0.2% Tween 20 for 1 h, blocked with 1% BSA and incubated with CD-73 mouse antibody diluted in 1% PBS/BSA at 1:100 dilution for 2 h at 4 °C. Finally, for all samples, cell nuclei were stained with blue DAPI for 10 min at 37 °C. Samples were observed by confocal microscope system (Leica TCS SP5 MP) with a 63× oil immersion objective. Images acquired with a resolution of 1024 × 1024 pixels.

In order to assess the differentiation of hUCMSC cells, the qualitative expression of surfactant proteins C (SPC) was evaluated by immunofluorescence using SPC antibody. Then, 1 × 10⁴ hUCMSCs were seeded on a fluoro-dish-35 mm (World Precision Instruments, Inc.), HA solubilized in SAGM was used to feed cells while DMEM and SAGM media were used as control (CTR), cell media were changed every 3 days for 21 days of cell culture with fresh medium. After this time, hUCMSC cells were fixed in 10% Formalin for 1 h, permeabilized with 0.1% Triton X-100, blocked with 1% BSA and incubated with SPC rabbit antibody (Abcam) diluted in 1% BSA at 4 °C overnight. After washing with PBS three times, Fitch-conjugated anti-rabbit antibody (Millipore, Billerica, MA, USA) was added to the cells for 3 h at room temperature. Finally, Cell nuclei were stained with blue DAPI for 10 min at 37 °C. Samples were observed by a confocal microscope system (Leica TCS SP5 MP) with 63X oil immersion objectives. Images were acquired with a resolution of 1024 × 1024 pixel. To determine the quantitative expression of human pulmonary surfactant protein A-B-C and D the supernatants for analysis were collected after 21 days of exposure to the biomaterials, and then analyzed using SP A-B-C and D ELISA kits according to the manufacturer’s protocol.

For cell morphology assay, cells were seeded at a density of 1 × 10⁴ cells/mL on fluorodish −35 mm (World Precision Instruments, Inc.), and 30 μg of the NPs formulations SiO₂-Hep_1, SiO₂-Hep_3, and SiO₂-Hep_5 were incubated for 24 h. Then, samples were washed two times with PBS and fixed with 10% formaldehyde for 1 h at 4 °C. The fixed cells were permeabilized with Triton X-100 0.1% in Phosphate-buffered saline (PBS) for 3–5 min. The actin filaments were stained with FITC phalloidin (Cayman Chemical Company, Ann Arbor, MI, USA) in PBS for 30 min at room temperature. Finally, after two washes with PBS to remove unbound phalloidin conjugate, cell nuclei were stained with 4′,6-diamidino-2-phenylindole, DAPI, (Sigma-Aldrich, Milan, Italy). The samples were observed by a confocal microscope system (Leica TCS SP8) with a 63× oil immersion objective. Images were acquired with a resolution of 1024 × 1024 pixel.