1457.3.2.2 Osteogenesis
There appears to be a complex relationship between adipogenesis and osteogen-
esis. Both adipocytes and osteoblasts are derived from a common multipotent
MSC, thus leading one to believe that the two pathways are interconnected
(Fig. 7.1). Obesity increases adipocyte differentiation and fat accumulation and
is believed to decrease osteoblast differentiation and bone formation. Several
recent investigators have reviewed the complex relationship between adipogen-
esis and osteogenesis (Cao 2011 ; Liao 2014; Nuttall et al. 2014 ; Romagnoli and
Brandi 2014 ; Atashi et al. 2015 ).
The osteogenic maturation pathway involves cell proliferation, differentia-
tion and matrix deposition followed by mineralization. Differentiation of ASCs
into osteoblasts in vitro involves incubating a monolayer of ASCs with ascorbic
acid, β-glycerophosphate and dexamethasone for 3 weeks. The composition of
the osteogenic induction medium is provided in the supplementary material.
Ascorbic acid acts as a cofactor for collagen synthesis through the hydroxyl-
ation of proline and lysine residues in collagen and induces extracellular matrix
(ECM) production through the synthesis of non-collagenous bone matrix pro-
teins. β-Glycerophosphate is needed for calcification and mineralization of the
ECM. Dexamethasone regulates osteoblastic gene expression (Fiorentini et al.
2011 ). Dexamethasone treatment has been shown to increase alkaline phospha-
tase activity in vitro which is required for matrix mineralization and morpho-
logical transformation to cuboidal- shaped cells (Cheng et al. 1994 ). Alizarin
Red S staining is commonly used for identifying calcific deposition during
matrix mineralization in osteogenic differentiation cultures which is an early
marker for differentiation (Fig. 7.5). Please refer to the supplementary material
for a brief description of the qualitative method used to evaluate osteogenic
induction in vitro.
Fig. 7.3 Visualization of adipocyte formation following adipocyte differentiation using fluores-
cence microscopy. (a) ASCs were stained with Nile Red (2 μg/ml) and DAPI. (b) ASCs were
stained with Bodipy 493/503 (20 μg/ml) and DAPI. Both images were captured at Day 21 after
adipocyte differentiation was induced
7 Isolation and Characterization of Adipose-Derived Stromal Cells