Absorption-based assays for the analysis of osteogenic and chondrogenic yield.
The typical characteristics of cartilage and bone tissue are their unique extracellular matrices on which our body relies for structural support. In the respective tissue, the cells that create these matrices are the chondrocyte and the osteoblast. During in vitro differentiation from an embryonic or any other stem cell, specific cell types must be unequivocally identifiable to be able to draw the conclusion that a specific cell type has indeed been generated. Here, gene expression profiling can be helpful, but examining functional properties of cells is a lot more conclusive. As proteoglycans are found in and are part of the function of cartilage tissue, their detection and quantification becomes an important diagnostic tool in tissue engineering. Likewise, in bone regeneration therapy and in research, alkaline phosphatase is a known marker to detect the degree of development and function of differentiating osteoblasts. Calcification of the maturing osteoblast is the last stage in its development, and thus, the quantification of deposited calcium can aid in determining how many cells in a given culture have successfully matured into fully functioning osteoblasts. This chapter describes methods ideal for testing of proteoglycan content, alkaline phosphatase activity, and calcium deposit during in vitro chondro- and osteogenesis.
Department of Surgery, Cambridge Institute for Medical Research, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK.
Lesley A
LA
Davis
Anke
A
Dienelt
Nicole I
NI
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D000042:Absorption; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002138:Calibration; D002458:Cell Fractionation; D049109:Cell Proliferation; D020219:Chondrogenesis; D053595:Embryonic Stem Cells / Q000166:cytology / Q000201:enzymology; D006801:Humans; D008751:Methylene Blue / Q000031:analogs & derivatives / Q000378:metabolism; D051379:Mice; D010012:Osteogenesis; D012015:Reference Standards
C435946:dimethylmethylene blue; D000469:Alkaline Phosphatase; D002118:Calcium; D008751:Methylene Blue