Effects Of IGF-I And BMP-2 Combined Application On Promoting Proliferation, Differentiation And Calcification Of MC 3T3-E1 And NIH 3T3 Cells

Periodontal disease is a disease initiated from periodontal tissues(gingival, periodontal ligament, alveolar bone and cementum). In Chinese population, the incidence rate of this disease arrives 73%. Periodontitis is the most common in different kinds of periodontal diseases. Alveolar bone resorption and attachment loss of periodontal epithelium are considered to be the main pathological changes in periodontitis, and they are difficult to be repaired naturally. So how to make the periodontal tissues regenerate and repair is a key subject in periodontology study.The periodontal tissues are mostly comprised by periodontium and alveolar bone. The fibroblast is the most of the total cell population which exerts the most important biology function. Now people considered the key of the regeneration of the hard periodontal tissues is that the proliferation and differentiation of the cells which have osteoplasticpotential, and the regeneration of the soft tissues depend on proliferation, moving and differentiation of the remain periodontium cells.Recombinant human insulin-like growth factor-I (rhIGF-I) and recombinant human bone morphogenetic protein-2 (rhBMP-2), secreted from multiple types of cells, are growth factors belonging to polypeptides. IGF-I can promote the proliferation and differentiation of the cells, and BMP-2 is major on promoting osteogenic. But we hardly have seen literatures about the combined effect of IGF-I and BMP-2 on promoting cell proliferate, differentiate and calcificate.In this study, we make the mouse osteoblast-like line MC 3T3-E1 and the mouse fibroblast line NIH 3T3 as the effect cells, and observe if IGF-I and BMP-2 have the combined effects on proliferation, differentiation and calcification of the two cells for providing experiment evidences to apply the two cytokines repairing the hard and soft periodontal tissues in clinic.Objective: To demonstrate the effects of recombinant human insulin-like growth factor-I (rhIGF-I) or recombinant human bone morphogenetic protein-2 (rhBMP-2) and combined application of the two cytokines on proliferation, differentiation and calcification of MC 3T3-E1 cells and NIH 3T3 cells.Methods: Mouse osteobalast-like cell MC3T3-E1 and mouse fibroblast cell NIH 3T3 were treated with different dosages of rhIGF-I orrhBMP-2 and rhIGF-I plus rhBMP-2. Proliferation of the two cells were measured by methylthiazol tetrazolium ( MTT ) method and flow cytometry. Differentiation of the two cells were examined by using alkaline phosphatase (ALP) measurement kit. Radioimmunoassay was applied to detect levels of osteocalcin (OC) secreted by the two cells. Von kossa staining method was used to study the calcification effects of the two cells. Results:1. Results of MTT methodMC 3T3-E1 cells treated with 1-50 ng/ml rhIGF-I and NIH 3T3 cells treated with 5~75 ng/ml rhIGF-I showed obvious effects of promoting proliferation (P<0.01), 10-100 ng/ml rhBMP-2 were also able to promote proliferation for both the two cells (P<0.01); rhIGF-I plus rhBMP-2 could show stronger effects(P<0.01) on promoting proliferation for both the two cells than using any one of the two cytokines (P<0.05). The results of MTT examinations were similar by using the same different concentrations of rhIGF-I and rhBMP-2 treated for 8, 24 and 48 h, respectively.2. Results of mensuration of cell cycle50 ng/ml rhIGF-I and 100 ng/ml rhBMP-2 and combination of the two cytokines at above cocentration showed obvious effects to MC 3T3-E1 cells on increasing the percentages of S-phase cells, decreasingthe percentages of G1-phase cells. 75ng/ml rhIGF-I and 10ng/ml rhBMP-2 and combination of the two cytokines at above cocentration showed obvious effects to NIH 3T3 cells on increasing the percentages of S-phase cells, decreasing the percentages of G1-phase cells. RhIGF-I plus rhBMP-2 could show stronger effects on cell cycle for both the two cells than using any one of the two cytokines. The results were similar by using the same different concentrations of rhIGF-I and rhBMP-2 treated for 24 and 48 h, respectively.3. Re...