Abstract:

BACKGROUND: In vitro research of bone cells were difficult for result analysis due to various factors. The separated bone cells could obtain single cell line. Therefore, the single cell line was used to observe direct effect of various materials or methods and avoid some interfering factors.
OBJECTIVE: To investigate a feasible method to mass-produce, culture, and reserve normal osteoblast of people. 
METHODS: Diaphysis of extremities from normal fetus aged four months were harvested to digest in collagenase and pancreatin to generate single cell suspensions, the cells and tissue segments were cultured for establishing normal osteoblast system of people. Morphology and histology were observed; type Ⅰ collagen produce was measured using immunohistochemistry; cells were reserved using liquid nitrogen frozen method; cells in good logarithmic growth phase were collected for preparation of genetics-chromosome.
RESULTS AND CONCLUSION: Osteoblast was fusiformed-shaped and had plentiful processes. Nucleus was orbicular-ovate and leaning to lateral side. Soma was large, and plasma was abundant. Alkaline phosphatase staining suggested that a great number of gray-black particles were observed in plasma, and some region was darkly stained. Quantitative analysis demonstrated that levels of alkaline phosphatase and osteocalcin in the plasma were significantly higher than fibroblast; immunohistochemical staining suggested that the type Ⅰ collagen was mainly produced. Chromosome analysis indicated that there were 23 pairs of chromosomes, and abnormal chromosome was not detected, suggesting that the obtained samples were normal human cells which were still normal following various cultures. Fluorochrome-tetracycline staining demonstrated that the cultured cells in this study had the osteogenesis ability. In this study, a normal osteoblast system of people was established and could be reserved with liquid nitrogen for a long time. The cells had stable biological characteristics following both various passages and frozen reservation.