In Vitro Culture Of Osteoblasts And Osteoclasts, And The Effects Of Fluorine On The Cells

A method of cell culture was improved to find out morphological and structural effect of fluorine on osteoblasts and osteoclasts, and on the ability of proliferation and mineralization of osteoblasts in vitro.Calvaria was obtained and scissored into pieces on the super clean blench through sterile method after fetal Kunming mouse within 24 hours were killed by cervical dislocation in the experiment. The bone pieces were digested by 0.25% trypsin for 30 min at 37℃ at first, then digested by 0.2% collagenase at 37℃ in constant temperature oscillator for 60 min, and rinsed repeatedly to disaggregate more single-cells. According to the needed concentration, the cells were seeded with DMEM solution containing 15% fetal cattal serum. The culture solution was changed at second day, and then ones every other day until target experiment. Osteoblasts with typical characteristic were observed completely through this method.Osteoblasts showed different growth phases (lag period, the period of exponential growth, and the period of reduced or zero growth). The part enzymatic disaggregated cells with round shape attached to the dish and outspreaded at 4 hours, but completely and wholly at 24 hours under microscopy. It was observed of osteoblasts semi-confluence at day 3, and complete confluence at day 5, merging growth of road metal at day 7, dense growth at day 9, mineralized matrix deposit at day 11, mineralized nodes at day 13.In vitro effect of fluorine on osteoblasts morphology and structure had dose-time dependence. More than 10-4 mol/l fluorine had significant cytotoxic function on osteoblasts, and acute cell death was observed in 10-2 mol/l and 10-1 mol/l groups. No apparent toxic action was found in groups with fluorine concentration less than 10-4 mol/l. In vitro proliferation of osteoblasts was promoted in groups with fluorine concentration of 10-5mol/l～10-7mol/l, and inhibited in 10-3mol/l and 10-4mol/l fluorine groups.The effect of fluorine on osteoblast ability of mineralization had dose dependence. During the experiment, the ability of mineralization of osteoblasts was promoted in groups with fluorine concentration of 10-5mol/l～10-7mol/l.While in this study, 2 week old Kunming mouse were killed by cervical dislocation. The long bone was collected on the super clean blench through sterile method, then periosteum and epiphyses were discarded. The bone was scissored into two parts in cold DMEM culture solution, bone marrow was obtained by scrape, bone pieces were rinsed with DMEM solution to separate more single-cells. After depositing of bone pieces, bone marrow cells were seeded. The cells unattached to the plate were selected to re-seed on osteoblasts after 30 min. The culture solution was changed every 2 days. Osteoclasts with polynuclear and TRAP positive type were cultured by the method mentioned above.The effects of fluorine on osteoclasts presented dose-time dependence. Cytotoxic action on osteoclasts was significant at 10-1mol/l ～10-3mol/l fluorine, and acute cell death was found in 10-1mol/l and 10-2mol/l fluorine groups under phase-contrast photomicroscope. Inapparent changes were observed in 10-4mol/l～10-7mol/l fluorine groups.The excretion of tartrate-resistant acid phosphatase (TRAP) was affected by fluorine, presenting dose dependence.