Tl led

Primary osteogenic cells were seeded in six-well plates (1 × 10⁵ cells per well) and incubated for 24 h. Cells cultured in the medium without the addition of CCN were used as the control. CCN were added to the cells at increasing concentrations (5, 10, 20, 50, and 100 mL/L). After 24 h, the cells were fixed with 4% paraformaldehyde and stained with a 2% Alizarin red solution (Merck, Warsaw, Poland) [28 (link)]. Cell morphology was recorded using an optical microscope (TL-LED, Leica Microsystems, Wetzlar, Germany).

Primary mesenchymal cells originating from a hind limb (PMC), primary heart cells (PHC), primary neuronal cells originating from the brain (PNC), primary eye cells (PEC), and primary blood vessel cells isolated from the chorioallantoic membrane (PVC) were assessed using an optical inverted microscope (TL-LED, Leica Microsystems GmbH, Wetzlar, Germany) connected to a digital camera (Leica MC190 HD) using LAS V4.10 software. Cells were seeded (1 × 10⁵ cells per well) in 35 mm diameter Petri dishes with and without GO scaffolds. After incubation for 24 h, samples were imaged.
PMC were additionally visualized using a Quanta 200 SEM (FEI, Hillsboro, OR, USA). Cells were seeded (1 × 10⁵ cells per well) in 35 mm diameter Petri dishes with and without GO scaffolds. After a 96-h incubation period, samples were prepared as described by Heckman et al. [46 (link)]. Cells were fixed using 2.5% glutaraldehyde in phosphate-buffered saline (PBS) at pH 7.2, contrasted with 1% osmium tetroxide (Sigma-Aldrich) and 1% carbohydrazide (Sigma-Aldrich). Subsequently, cells were dehydrated in increasing concentrations of hexylene glycol (Sigma-Aldrich). Drying was performed using a Polaron CPD 7501 critical point dryer (Quorum Technologies, Laughton, UK).

Cell morphology was visualised with a light optical inverted microscope (TL-LED, Leica Microsystems, Germany) and a scanning electron microscope (SEM; Quanta 200, FEI, Hillsboro, USA), on day 5th of primary cell culture on the 6-well plates. For light microscope observation, cells were fixed in ice-cold methanol for 10 min, and then eosin/hematoxylin stained, according to the standard protocol. In turn, cells imaged in SEM were first fixed in 2.5% l-glutaraldehyde in phosphate-buffer saline (PBS; Life Technologies, Houston, USA), subsequently contrasted with osmium tetroxide (Sigma-Aldrich, St Louis, USA) and carbohydrazide (Sigma-Aldrich, St Louis, USA). Next, the cells were dehydrated in hexylene glycol (Sigma-Aldrich, St Louis, USA). Finally, drying was performed using a Polaron CPD 750 l critical point dryer (Quorum Technologies, Laughton, UK).