In-situ visualization and quantification of mineralization of cultured osteogenetic cells.
An osteoblastic cell line (HOS cells) produces a prominent osteoid matrix with mineralization. Fibroblasts, on the other hand, do not exhibit this mineralization. To evaluate the degree of mineralization, we added calcein to the culture medium and then observed the culture wells by using an image analyzer. The calcein uptake into the cell/matrix layer was detected in the HOS cells but not in the fibroblasts. The calcein uptake was also quantified in situ by using an image analyzer, which revealed high levels in the HOS cells, which correlated well with the calcium content of the mineralized matrix. Rat marrow cells were also cultured in media containing calcein, fetal bovine serum, beta-glycerophosphate, L-ascorbic acid 2-phosphate, and with or without dexamethasone. With the dexamethasone, the cells exhibited osteogenic differentiation that resulted in mineralized matrix formation after about 10 days. The matrix formation coincided with the appearance of calcein uptake into the cell/matrix layer, with the amount of calcein uptake increasing with time. By contrast, the culture without the dexamethasone did not exhibit matrix formation and the calcein uptake was negligible. In the case of both HOS cell and rat marrow cell cultures in vitro, calcein did not affect expressions of their alkaline phosphatase activity or osteocalcin production. Furthermore, histologic observation revealed that rat marrow cells subcultured with calcein could show osteogenic ability after in vivo implantation. These results suggest that the current method of detecting calcein uptake in a culture allows the monitoring of the osteogenic capacity of cultured cells, as well as the measurement of the amount of mineralization produced by the osteogenic cells. Given that osteogenic cultured cells/mineralized matrices are used in bone reconstruction surgery, the in situ monitoring method is invaluable in that it allows us to evaluate the osteogenic capacity of in vitro constructs.
Uchimura
E
E
Tissue Engineering Research Center, National Institute of Advanced Industrial Science and Technology, Nakoji 3-11-46, Amagasaki, Hyogo 661-0974, Japan.
Machida
H
H
Kotobuki
N
N
Kihara
T
T
Kitamura
S
S
Ikeuchi
M
M
Hirose
M
M
Miyake
J
J
Ohgushi
H
H
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000201:enzymology; D016026:Bone Marrow Transplantation; D002113:Calcification, Physiologic / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D017690:Cell Transplantation; D003907:Dexamethasone / Q000494:pharmacology; D004338:Drug Combinations; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008297:Male; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344
D004338:Drug Combinations; D005452:Fluoresceins; D003907:Dexamethasone; D000469:Alkaline Phosphatase; C007740:fluorexon