Cellsens entry 1

After 21 days of differentiation, the representative cells were stained using EZStain^TM Osteocyte staining kit (cat. #CCK030; Himedia, Mumbai, India) to understand the deposition of mineral matrix in the differentiated cells. In brief, spent media from the wells were discarded and the cells were washed with PBS and fixed by incubating in ice-cold 70% ethanol for 1 h. The ethanol from the cell surface was washed away by rinsing with sufficient water. The staining solution (pH 4.1–4.3) from the kit was added and incubated for 45 min at room temperature in dark. Finally, the excess staining solution was removed by rinsing with distilled water. The stained cell images were captured by DP-73 camera using 200x magnifications in CellSens Entry 1.8 software (Olympus Soft Imaging Solutions, GmbH, Germany).

Passage 4 cells of different media were seeded in 1 ml of respective media at a density of 5×10³ cells/cm² per well in a sterile 24-well plate separately for all pigs (n=3). The media was changed with aDMEM containing all the supplements for 48 h uniformly and allowed to be grown until about 90–95% confluence as per the subjective evaluation. The spent media was removed and replaced with equal volume of osteogenic differentiation medium in each of the wells. Fresh induction media were replaced at every 3 days until 21 days of differentiation. Changes in morphology and mineral matrix formation were recorded every day at 100x magnification using Olympus IX51 inverted microscope (Olympus, Japan). Cell images were captured at every 3 days interval using DP-73 camera and CellSens Entry 1.8 software (Olympus Soft Imaging Solutions, GmbH, Germany) and images were evaluated for the comparative differences in morphology, progression of osteogenic differentiation based on the criteria for formation of mineral matrix or nodular structures, appearance of adipocytes and the undifferentiated cells remained in culture. The images were analyzed by a single person from the sequential cell images captured at different time points.

In this test, the safety of the PSO nasal system containing 25% PSO at a PSO:PL ratio of 3:1 on the nasal cavity and mucosa was evaluated in rats using a method previously described by Duchi et al. [20 (link)]. In brief, nine male HSD rats (220–250 g) were divided equally into two treatment groups and one untreated control group. Rats in the treatment groups received nasally 15 μL PSO phospholipid oily system or normal saline into both nostrils twice a day, for one week. At the end of the experiment, the animals were sacrificed and the nasal cavities were removed and fixed in 3.7% formaldehyde PBS. Sections of the nasal cavity were cut serially at 5 μm thickness and stained with hematoxylin and eosin. The sections were examined by a histopathologist (Authority for Animal Facilities, Hebrew University of Jerusalem, Jerusalem, Israel) using an Olympus light microscope BX43 and an Olympus digital camera DP21 with Olympus cellSens Entry 1.13 software (Olympus, Tokyo, Japan) using a magnification of ×10. Local toxicity was assessed by evaluating the histopathological alterations in different regions of the nasal cavity including cartilage and turbinate bone, lamina propria and submucosa, mucosal epithelium, and lumen.