*2.6.2. Alcian blue staining*

Alcian blue is used to stain sulfate glycosaminoglycans and proteoglycans in the extracellular matrix. The same procedure, as indicated above, was followed using Alcian blue solution as the dying reagent and destained samples were collected. These samples were later analyzed using a Thermo Scientific NanoDrop 2000c spectrophotometer.

## *2.6.3. Alkaline phosphatase (AP) assay*

During *in vitro* bone formation, AP activity is greatly enhanced and hence used as a marker to evaluate the differentiation of MSCs towards osteoblastic lineage or maintenance of the osteoblast phenotype. It is easily detected using BCIP/NBT as a substrate, which stains cells blue-violet when AP is present. Alcohol fixed plates were washed with a buffer and carefully aspirated without disrupting the monolayer of cells. Enough BCIP/ NBT substrate solution was added to just cover the monolayer and left in the dark for 5–10 min. It was then washed and destained using 7% glacial acetic acid for 5 min to quantify the AP activity. The solution was aspirated and analyzed using a NanoDrop 2000c spectrophotometer.
