Osteogenic phenotype of normal osteoblast vs human osteosarcoma cell lines

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.Thesis (M.S.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2002 (Orthodontics).Includes bibliography (leaves 103-117).Osteosarcoma cells have been widely used in bone research as experimental models for investigating particular osteoblast phenotypes. However, the phenotypic similarities to normal human osteoblast are not established. The objective of this study was to perform a systematic comparison of normal human osteoblast cells (HOB) and three widely used osteosarcoma cell lines (OS), Saos-2, Mg-63, and HOS-TE 85, to ascertain their relevance as experimental models for investigating particular osteoblastic phenotypes. We compared their cell attachment efficiency, cell proliferation rate, and differentiated functions such as expression of alkaline phosphatase, osteocalcin and mineralization in the presence and absence of 1.25-dihydroxyvitamin D subscript 3(Vitamin D subscript 3). Normal human osteoblast cell cultures were established by harvesting alveolar bone obtained during tooth removal of impacted 3 superscript rd molar. OS cells were obtained from American Type CuIture Collection (ATCC). HOB and OS cells were cultured in growth medium containing DMEM/F12 (1:1), 10% (V/V) heat inactivated fetal bovine serum (FBS), 10%antibiotics (100 U/ml), and 2mM L-Glutamine for different time intervals: 16hrs, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days and 24 days. HOB and OS cells were cultured in the growth medium for 3 days until confluence at which time the culture medium was supplemented with differentiation media containing DMEM/F12 (1:1), 1% FBS, 10% antibiotic (100∪/mI), 2mM L-Glutamine, 10mM β gIycerophospate 50 mega/ml ascorbic acid, and 10 superscipt -8 M Vitamin K for 24 hours. Fresh Differentiation Media with 10 superscipt -8 M Vitamin D subscript 3 was then added and incubated for 48 hrs at 37 degrees C, 100% ethanoI was added as the vehicle control in the control group, for 4 different time intervals at 3 days, 9 days, 16 days and 20 days. At each time interval, all the cell lines were done with Vitamin D subscript 3 and without Vitamin D subscript.The cells were maintained at 37 degrees C in 5% CO subscript 2 and 95% air under humidified atmosphere. Cell attachment efficiency and proliferation rate were examined by measuring the optical density of crystal violet dye. Alkaline phosphatase (ALP) activity of the culture was measured using the P-nitro phenol reaction to the fixed cultures. superscript 125 l radioisotope was used for labeling the expression of osteocalcin (OC). Alizarin red S staining was used to measure the amount of mineralization. [TRUNCATED