In vitro effect of calcium on osteogenesis of human osteoblasts
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Bioactive glasses, mainly composed of silicon, calcium, and phosphorous have been frequently used as synthetic bone graft material. However, the osteogenic effect of released ions from bioactive glass still remains largely unknown. This in vitro study was designed to test the effects of calcium ions (Ca) alone or combined with silicon ions (Si) or/and phosphorous ions (P), the major components of bioactive glasses, on human osteoblast cell attachment cell proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) expression, and mineralization. The second passages of normal human osteoblast like cells, derived from human alveolar bone obtained from impacted third molar extractions, were cultured in media supplemented with Ca alone (50, 100, 200, 300, 400 ppm), or combined with Si (50, 100 ppm) and/or P (8.3, 16.7 ppm) for three different time intervals of 16 hours, 12 and 20 days. Culture medium already containing a physioIogic range of Ca and P was used as a baseline control. Cells were screened for osteoblast phenotype prior to all experiments. The culture medium was observed for pH change. Cell attachment efficiency and proliferation rate were examined by measuring the optical density of crystal violet dye stained in the cultures. ALP activity was determined by measuring the optical density of a p-nitrophenol mixture in a fixed cellular layer. 125 superscipt I radioisotope was applied for labelling the expression of OC. Alizarin red S stain was used to measure the amount of calcium deposit. The results showed that the cell attachment rate was not affected by various ion compositions of culture media. Supplemented Ca at 100 ppm significantly increased cell proliferation rate, ALP activity, OC expression, and mineralization of the cultures by 2.5-fold (1.5-fold on OC expression) at both 12- and 20-day intervals, compared to the control. It was statistically different from other groups supplemented with 50, 200, 300, 400 ppm Ca on cell proliferation rate, ALP activity, OC expression, and mineralization at both time intervals except the 12-day culture for which there are no differences in cell proliferation rate, ALP activity, OC expression between 100 and 200 ppm groups and in mineralization between the 100-400 ppm groups, considered to be the optimal dose of Ca on the osteogenesis of human osteoblast cells. While concentrations of Ca higher than 300 ppm failed to increase alkaline phosphatase activity and osteocalcin expression, it did significantly increase calcium deposits. The addition of 100 ppm Si and 16.7 ppm P to the supplemented Ca at 100 ppm significantly increased cell proliferation rate, ALP activity, OC expression and mineralization of the cultures by 3.5-fold at both time intervals, compared with the baseline cultures. It was statistically different from other groups on cell proliferation rate, ALP activity, OC expression, and mineralization at both time intervals. This in vitro study demonstrated both an important role of Ca ion in stimulating osteogenesis of normal human osteoblast cells, and that the combination of Ca, Si, and P ions significantly multiplies the osteogenesis of human osteoblast cells.
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 email@example.com.Thesis (M.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2000 (Prosthodontics).Includes bibliographical references (leaves 96-105).
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