Expression And Effects Of IGF-1 And BFGF In Fibroblast And Osteoblast Induced By Fluoride

Objective: The skeletal phase damage of chronic fluorosis includes bone sclerosis, osteomalasia, bone rarefaction and extraperiosteal ossification. The soft tissue calcification and ossification, which plays an important role in the development of severe disabled skeletal fluorosis, belongs to the category of ectopic ossification. The bone formation intimately correlates with the precursor cell, the stimulating -ossification factor and the osteoplastic surroundings. Fibroblast (FB), as the main type of cell exists in the extra-periosteal soft tissue, is in the category of inducible osteogenic precursor cell (IOPC) because of its osteogenesis function under the non-physiological conditions. Recently, reseachers paid close attention to the condition and the mechanism of ossification. As our previous reseach indicate, fluoride can induce expression of osteogenic phenotype and enhance the potent osteogenesis function of fibroblast. The interaction between fluoride and fibroblast would be strongly analogous to the mechanism of extraperiosteal ossification. IGF-1 and bFGF as bone derived growth factors (BDGFs), are the important growth factors for bone growth and metabolism. Both can influence the development, differentiation and synthesis of fibroblastand osteoblast (OB), and play important roles in promoting the osteogenetic action of fibroblast and osteoblast. However, reseach on the causal relations of fluoride and IGF-1, bFGF and osteogenetic function of fibroblast is scarce. To study the osteogenic action of osteoblast induced by fluoride, the osteogenesis stimulated by fluoride on fibroblast, the effects and importance of fluoride on fibroblast in the development of extraperiosteal calcification and ossification of skeletal fluorosis, the present study investigated the expression of IGF-1 and bFGF in Fluoride-treated fibroblast and osteoblast.Methods: Fibroblast cell line (L929) and primary cultured osteoblast-like cells from mouse were exposed to different concentrations of fluoride (control group 0 mg/L, experimental group 0.0001, 0.01, 0.1, 1, 10, 20mg/L) for certain time (2, 4, 24, 48, 72h). The levels of IGF-1 and bFGF mRNA and protein were measured by using the methods of Elisa, IHC, and PCR. In addition, three-dimension culture system was established by rat tail collagen as three-dimensional scaffold, in which fibroblasts were planted and cultured, for better observing the effects of fluoride really near to the physiological conditions. 0.01mg/L fluoride-concentration group was added to the experimental group and the fluoride-infuluence time was changed (4, 24, 48, 72, 96h).Results: Compared with the control group, the expression of IGF-1 and bFGF mRNA and protein increased significantly in fluoride-treated fibroblast. The same tendency was found in osteoblast and stronger enhancement was observed. Fluoride increased the expression of IGF-1 and bFGF in fibroblasts cultured in the FPCL, showed the sametendency as traditional cultivate system.Conclusions: The higher expression of IGF-1 and bFGF in both fibroblast and osteoblast demonstrated that fluoride is an effectual stimulating factor, may play an important role in enhancing the osteogenesitic function of fibroblast and osteoblast. This study suggested that fluoride can induce expression of osteogenic phenotype, differentiation to osteoblast and enhance the potent osteogenesis function of fibroblast. In all probability it is one of the reasons for development of extraperiosteal ossification.The difference of effective fluoride-concentration and fluoride-induced time to enhance the expression of IGF-1 and bFGF in fluoride-treated fibroblasts indicated that the two maybe have some complementary action in enhancing the osteogenesitic function of fibroblast. The same higher expression tendency of IGF-1 and bFGF in three-dimension culture system and traditional cultivate system showed more evidence to prove stronger osteogenesitic function in fluoride-treated fibroblast.To conclude, the change of IGF-1 and bFGF played important role in promoting the osteogenesitic function in fibroblast induced by fluoride. The interaction between fluoride and IGF-1, bFGF; IGF-1 and bFGF;or the two factors and other factors is complicated.Many details remain to be studied for better understanding of mechanism of extraperiosteal ossification.The project brought some new ideas:1. The present study observed the higher expression of IGF-1 and bFGF mRNA and protein in Fluoride-treated fibroblast and suggested that it play an important role in expression of osteogenic phenotype and theosteogenesis stimulated by fluoride on fibroblast2. The present study investigated the change of IGF-1 and bFGF in fluoride-treated osteoblast under the same conditions.The results showed fluoride could stimulate the expression of the two factors in osteoblast, not only indicated that fluoride play important roles in promoting the osteogenetic action of osteoblast but also gave important evidences to prove the possibility of fibroblast-differentiation to osteoblast induced by fluoride.3. This study compared the expression of IGF-1 with bFGF in fibroblast and osteoblast treated by fluoride of different concentration at different period, indicated that IGF-1 and bFGF maybe have some complementary action in enhancing the osteogenesitic function of fibroblast and osteoblast.,which will give further clues to the studies of mechanism of extraperiosteal ossification of skeletal fluorosis.4. Three-dimension culture system was established by rat tail collagen as three-dimensional scaffold for better observing the effects of fluoride really near to the physiological conditions, provided one original method to study the osteogenesis stimulated by fluoride on fibroblast.