Osteogenic Differentiation Of Periodontal Ligament Cells Induced By BMP-2、bFGF And Dex In Dogs

Periodontal ligament cells(PDLCs) have been applied extensively in thearea of periodontal tissue regeneration. With the modulation of some induciblefactors, PDLCs can be promoted in differentiating into osteoblasts andcementoblasts in vitro. Nevertheless, previous research has largely focused onthe different effect on the biological behavior of PDLCs which were broughtabout by different factors. Moreover, they were often limited to the single factor.So far no study on the changes of biological behavior in PDLCs with thecombined use of various inducible factors has been reported. This researchobserved the cell proliferation, the osteogenic and adipogenic differentiation andthe expression of some part of osteogenic factors with the combined applicationof BMP-2、 bFGF and Dex on inducing PDLCs, which can providing atheoretical basis on the promotion on the osteogenic differentiation in PDLCs ofthese3factors. Objective: To observe the changes of biological properties（osteogenicdifferentiation）of PDLCs induced BMP-2、bFGF and Dex.Methods: The fourth generation of PDLCs cultured in vitro were randomlydivided into5groups:(1) the control group (with no inducible factors);(2) thegroup with10μg/L bFGF and200μg/L BMP-2added;(3) the group with200μg/L BMP-2and10-8mol/L Dex added;(4) the group with10μg/L bFGF and10-8mol/L Dex added;(5) the group with200μg/LBMP-2,10μg/L bFGF and10-8mol/L Dex added. The effect of combined use of BMP-2, bFGF and Dexonon the proliferation of PDLCs in dogs were observed by MTT test, theosteogenic and adipogenic differentiation test and ALP assay. Meanwhile, theexpression of the key osteogenic gene including OPN, Col-I and BSP wereevaluated by RT-PCR analysis and immunofluorescence test.Results:(1)After7,14days, the induced PDLCs differentiated intofibroblast-like cells with polygon in shape including triangle and fusiform.Vimentin expressed positive and keratin was negative;(2)MTT testdemonstrated that all of the four experimental groups promoted the proliferationof PDLCs in dogs and the effect were time-dependent;(3) Adipogenic,osteogenic experimental results show that: bFGF+Dex, BMP-2+bFGF,BMP-2+Dex, bFGF+BMP-2+Dex composed of14days after adipogenicinduction, observed after the induction of the oil red O staining, cells in eachgroupto the positive fat droplets stained.Osteogenesis induced by31d,bFGF+BMP-2+Dex mineralized nodule staining deep, larger cell density andconcentrated. The osteogenic and adipogenic differentiation test and ALP assayshowed that the promotion of two differentiation were observed in all four experimental groups and ALP test in the group with BMP-2, bFGF and Dexadded increased than others remarkably;(4) RT-PCR results were seen that OPNand Col-I gene in the group with BMP-2, bFGF and Dex added expressed mostremarkably than others. The results of immunofluorescence test on BSP weresame and it expressed most dramatically in the group with BMP-2, bFGF andDex added than others.Conclusion: These results indicate that combined application BMP-2,bFGF and Dex can remarkably promote the osteogenic differentiation ofPDLCs.