Periodontal Fibroblasts Modulate Proliferation and Osteogenic Differentiation of Embryonic Stem Cells Through Production of Fibroblast Growth Factors

• Localización: Journal of periodontology, ISSN 0022-3492, Nº. 4, 2014, págs. 645-654
• Idioma: inglés
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• Resumen

  • Background: Periodontal ligament fibroblasts (PLFs) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. It is hypothesized that PLFs induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GFs) and skin fibroblasts (SFs).

    Methods: PLFs and GFs were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SFs were purchased commercially. mES cells were incubated with culture supernatants of these fibroblasts or cocultured directly with the cells. Proliferation and mineralization in mES cells were determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured.

    Results: In cocultures, PLFs stimulated proliferation of mES cells more effectively than GFs or SFs. Similarly, the addition of culture supernatant of PLFs induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor (FGF)4 or the c-Jun N-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of mES cells in the order PLFs > GFs > SFs. These activities of PLFs were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pretreatment of mES cells with the extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin.

    Conclusion: PLFs regulate the proliferation and osteogenic differentiation of mES cells more strongly than GFs and SFs via the secretion of FGF through a mechanism that involves mitogen-activated protein kinase-mediated signaling.

    Human periodontal tissue is mainly composed of gingiva, periodontal ligaments (PDL), and alveolar bone. PDL fibroblasts (PLFs) are the most abundant cells in the PDL1,2 and play a critical role in repair and regeneration of the PDL.3 PLFs have osteoblast-like properties and maintain PDL homeostasis during alveolar bone remodeling.3,4 It is commonly accepted that gingival fibroblasts (GFs) and PLFs share common functions in alveolar bone remodeling and tooth movement, although they are heterogeneous cell populations. However, the present authors demonstrated previously that PLFs are more efficient at osteoclast induction than GFs, suggesting that PLFs regulate bone resorption and formation more sensitively than GFs during orthodontic tooth movement.5 It is also worth considering that the PDL contains a multipotent stem cell population that can differentiate into osteoblasts and cementoblasts.6-8 It is postulated that PLFs stimulate stem cells to differentiate into various types of cells rather than the differentiation of PLFs themselves into osteoblasts during bone remodeling in responses to mechanical stresses.9 In these processes, PLFs may stimulate proliferation and/or differentiation of stem-like cells through the production of various factors, such as fibroblast growth factors (FGFs), transforming growth factors (TGFs), bone morphogenetic proteins (BMPs), and several cytokines.10-13 However, little information is available on the mechanisms by which PLFs have greater osteogenic potential than GFs. The mechanisms by which PLFs stimulate proliferation and osteogenic differentiation of stem cells also remain unclear.

    Therefore, the present authors investigate the effects of PLFs and GFs on proliferation and osteogenic differentiation in mouse embryonic stem (mES) cells and then compare their activity with that of skin fibroblasts (SFs). The possible mechanisms by which PLFs regulate proliferation and differentiation of mES cells are also explored, especially the roles of FGFs in proliferation and osteogenic differentiation.