Regulatory Aspects Of Fluoride On Proliferation, Differentiation And Apoptosis Of Caprine Osteoblasts Cultured In Vitro

The present study is designed to analyze the regulations of fluoride on ruminant bone metabolism, and to discover the processes and mechanism of how fluoride affects caprine periosteum derived osteoblasts, which were isolated by means of tissue culture, complete and incomplete enzyme digestion, respectively. They then routinely cultured in DMEM supplemented with 15% heat-inactivated fetal bovine serum under 37℃, humidified and 5% CO₂ conditions. On such a basis, osteoblasts were then subcultured until they had come semi-confluent, and identified by morphology (Giemsa and H.E. stain), scanning electron microscope (SEM), ALP and mineralized nodule(alizarin red and von kossa) stain.The effects of different doses of fluoride on cellular morphology were investigated by inverted phase-contrast microscope and SEM, while the regulations on proliferation, differentiation and calcification of osteoblasts were measured by tetrazolium salt test (MTT method), ALP activity detection and mineralized nodule count. To det ermine whether fluoride would induce apoptotic cell death, we monitored DNA fragmentation and the cellular morphology with DNA ladder, Hoechst33342/PI double staining and transmission electron microscope (TEM). The level of apoptosis was analyzed with a fluorescence-activated cell sorter (FACS) by the Annexin-V-FITC and the propidium iodine (PI) double staining method. Cell-cycle analysis was also examined with FACS using PI staining. The experimental results are as follows:1. The results indicated that adhesion time and complete-confluence time of periosteum derived osteoblasts with incomplete enzyme digestion was at 4hrs and 10 days, and the merging osteoblasts and apparent mineralized nodules on day 4 and 14 from passage cultures, respectively. Triangular, polyangular cells and obvious cell processes were observed. The cells have large nuclei and 1 ³ nucleoli. ALP and mineralized nodule stain showed positive reaction.Rough cell processes were observed, which join and overlap cells under SEM.2. After freezing-thawing, the viability of osteoblasts determined using trypan blue stain is more than 90%. Compared with primary culture, no bio-characteristic changes were observed from the 15th filial cells.3. On 48 and 96 hrs of exposure to fluoride, 1.0×10^-71.0×10^-5 mol/L concentration promotes osteoblast proliferation, and the role of promoting osteoblast proliferation was obvious with 1.0×10^-7 and 1.0 ×10^-6 mol/L fluoride (P<0.01) compared with controls. Higher concentration of fluoride with 1.0×10^-3 mol/L significantly suppressed the cell proliferation (P<0.01). 1.0×10^-4 and 5.0×10^-4 mol/L fluoride had no effects on osteoblast proliferation after 48hrs of exposure, but significant suppression was observed after 96hrs (P<0.01) .4. ALP activity and mineralization ability were obviously increased with 1.0×10^-71.0×10^-6mol/L fluoride (P<0.01), but decreased with 5.0 X 10^{- 4} mol/L doses.5. The SEM of osteoblasts showed abundant microvilli and cell protrusions which join and overlap each other (control). Incomplete membrane and shrinkage, detachment or break-up of cell protrusion was observed under higher than 1.0 X 10^{- 4} mol/L fluoride.6. Ultrastructural features of osteoblasts indicated clear nuclear envelope,large nucleolus, even karyoplasms, abundant endoplasmic reticulum and mitochondria (control). When fluoride is more than 1.0 X 10^{- 4} mol/L, cellular membrane disintegration, cytoplasm condensation, chromatin compaction or fragmentation, endoplasmic reticulum (ER) expansion, nucleus shrinkage or periphery was observed.7. Fluoride at 1.0X 10^{- 52}.5X 10"5 mol/L induced apoptosis with DNA fragmentation. FACScell-cycle analysis demonstrated that fluoride resulted in potent Go/G₁ arrest. Few cells could be found in either the S phases or the G2/M, thus inhibiting the transformation from S phase into G2/M phase under high levels of fluoride. The percentage of earlier and later stage of apoptosis was highest under 1.0X 10^{- 4} mol/L: 3.33% and 2.92%, respectively...