Morphological and Immunocytochemical Characterization of Osteoblast Cultures from Long Bones of Neonatal Rats.

Abstract

Summary. 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.

High Serum Levels of IGF-I Contribute to Promotion of Endochondral Ossification in Mandibular Condyle and Cause its Specific Elongation in Acromegaly-like Rats

“…The DakoCytomation EnVision + system-HRP (Dako Japan Corp., Kyoto, Japan) was used to identify osteoblasts by osteocalcin immunoreactivity according to Yang et al [11] with modifi cations. Deparaffi nized tissue sections were incubated with diluted antibody to osteocalcin (Bovine Osteocalcin OC4-30, TAKARA BIO INC., Shiga, Japan) (1:2000) and then made visible with 3,3 Јdiaminobenzidine chromogen.…”

Detection of living cells in non-processed but deep-frozen bone allografts

“…Alkaline phosphatase staining was performed using a substrate solution containing Naphtol ASTR phosphate, dimethyl formamide and fast blue BB salt (Sigma) as described by Yang et al (1992). Microscopic evaluation of cell growth, number, and aspect was performed during a A sample was taken from the center of the frozen femoral head, and minced into small fragments, which were either or not incubated with 2 mg/ml (PBS) of collagenase (Type II) for 2 h at 37°C in a shaking water bath.…”

Mechanism of the Morphological Changes Induced by Staurosporine in Rat Osteoblasts

“…Rat osteoblast cultures were prepared as previously described [20][21][22] The long bones of Wistar neonatal rat, including femur, tibia, humerus, radius, and ulna, were dissected with an aseptic technique under pentobarbital anesthesia (7.5 mg/ml, intraperitoneal injection 0.015 ml/g BW). After defleshing, the bones were diced into bare bone fragments and washed in phosphate-buffered saline (PBS) to remove marrow cells.…”