Morphological and immunocytochemical characterization of osteoblast cultures from long bones of neonatal rats.
The present study reports a successful culture system of rat bone cells obtained from the bare long bone fragments of neonatal Wistar rats. The morphology, histochemical and immunocytochemical properties of the cultured cells were characterized by phase contrast microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), histochemical staining with alkaline phosphatase (ALP), and immunocytochemical staining with the anti-osteocalcin antibodies. Under phase contrast microscopy, the cultured rat bone cells changed their morphology from being slender with relatively-short cytoplasmic processes to becoming polygonal with longer processes, and subsequently flattened. These cultured bone cells formed bone nodules after 3 weeks. Under SEM, the cultured bone cell appeared polygonal with some microvilli. The TEM showed that their cytoplasm contained abundant endoplasmic reticulum and well-developed Golgi apparatus. Positive histochemical staining of ALP was also detected as a blue coloration and granular appearance in the cultured bone cells. Immunocytochemistry with the polyclonal and monoclonal anti-osteocalcin antibodies showed the localization of osteocalcin in both the cytoplasm and nucleus. All these data lead us to consider that the cultured bone cells in our system were probably osteoblasts from growing bone tissue in vitro. This provides a convenient test system for further studies on the regulation of the growth and metabolism of rat bone cells.
Yang
R S
RS
Department of Orthopaedics, College of Medicine, National Taiwan University, Taipei.
Liu
T K
TK
Tsai
K S
KS
Lin-Shiau
S Y
SY
Lu
K S
KS
D000469:Alkaline Phosphatase; D000818:Animals; D000831:Animals, Newborn / Q000033:anatomy & histology* / Q000378:metabolism; D001842:Bone and Bones / Q000737:chemistry / Q000166:cytology*; D002478:Cells, Cultured; D006651:Histocytochemistry; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D010006:Osteoblasts / Q000737:chemistry / Q000648:ultrastructure*; D015675:Osteocalcin / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar
D015675:Osteocalcin; D000469:Alkaline Phosphatase