Ae2000

At the completion of the study the animals were sacrificed and the femur bone was extracted and used to study the bone structure. The bone samples were fixed in cyanuric chloride (0.5%) in methanol containing 1% N-methyl morpholine (0.1 M) for two days [27 (link)], then decalcified using formic acid (10%) and stained with hematoxylin and eosin. A digital compound microscope (Motic AE2000, Motic Asia, Kowloon, Hong Kong) was used to examine the slides and histopathological changes; the structure and morphology of the trabecular bone were especially assessed. Histomorphometry variables were analyzed using an image analyzing computer software (Motic images plus 2.0) linked to a microscope [28 (link)].

A metal wire was inserted into the injection capillary, and it was connected to the positive electrode of a direct current (DC) high voltage power supply. The device was grounded by connecting a metal tube wrapping the collection capillary to the negative end of the power supply. The direction of the electric field generated was the same as the flow direction of the inner liquid. The injected liquid phase was charged through the injection nozzle. The applied electric field intensity was controlled by adjusting the potential value of the power supply, U, while the distance between electrodes were kept constant. The applied electric field intensity typically ranged from 0 kV/cm to 6 kV/cm. The pulsating modes of the charged liquid jets were visualized and recorded using a high speed camera (Phantom M110) coupled with an inverted microscope (Motic AE2000).

Oil Red O Stock solution was prepared by mixing Oil Red O powder (Sigma-Aldrich, St Louis, MO, USA) (500 mg : 100 ml, w–v) in isopropanol. A working solution of 60% (v:v) ORO was prepared fresh by diluting in distilled water. Cells were rinsed with ice-cold PBS once then treated with paraformaldehyde (4%) (w:v) in PBS for 10 min at room temperature. Thereafter, the paraformaldehyde was then removed, cells were washed with cold PBS twice, and ORO solution (60%) was added to each of the cells. Cells were incubated in the ORO working solution for 15 min, rinsed in ice-cold PBS twice, and dried before visualization under light microscopy (Motic AE2000, Motic, Richmond, BC, Canada).

The mineralization of microgels was quantified by the opacity of microgels. In brief, after 1, 4, 7, 14, and 21 days of culture, cell-laden microgels were observed with a phase contrast microscope (Motic AE2000, China), and the gray value of microgels was evaluated using ImageJ. The mineralization of cell-laden alginate microgels was also characterized with the Alizarin Red S staining (Solarbio, China). In brief, after 21 days of culture in the osteogenic medium, microgels were collected and washed with PBS and then fixed with 4% PFA for 15 min; microgels were then stained with 0.1% Alizarin Red S for 15 min before being observed with the phase contrast microscope.

The brain tissues were fixed in 4% paraformaldehyde for 24 h, processed, embedded in paraffin, and subsequently cut into 4–8 μm-thick sections. Following deparaffinization, tissue samples were hydrated, hematoxylin stained, differentiated with bluing reagent, eosin stained, and dehydrated. Sections were air-dried, coverslipped, and photographed under a light microscope (Motic AE2000, Japan). Typical sections of the hippocampus and cerebrum were made in each group of animals.

The brain tissues were fixed in 4% paraformaldehyde, washed, dehydrated, transparent, waxed, embedded, and sliced. Then, they were gradient dewaxed, antigen repaired, cooled to room temperature, and washed in a wet box with PBS 3 times for 5 min each. Each section was dripped with endogenous peroxidase and incubated for 20–30 min in a 37°C incubator. It was washed with PBS 3 times, 5 min each. Next, we added primary antibody dilutions to each section and placed them in a humidified box at 4°C overnight. After returning to room temperature for 30 min, it was washed with PBS three times for 5 min each. The second antibody was added and incubated at 37°C for 30 min. Chromogenic time (3–5 min) was determined according to the titer intensity of the antibody. The newly prepared DAB chromogenic solution was added dropwise until we observed the specific staining under a microscope. The reaction was stopped with distilled water. After counterstaining with hematoxylin for 1 min, rinsed with running water, dried naturally at room temperature, the sheets were sealed with neutral gum, and baked overnight in an oven. Images were taken under a light microscope (Motic AE2000, Japan). Five regions of each specimen were randomly collected to calculate the average number and the average optical density of positive cells by Image J.