Hypoxia Regulates the Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Cells Under Cyclic Tensile Stress via Mitogen-Activated Protein Kinase Pathways

• Autores: Li Lu
• Localización: Journal of periodontology, ISSN 0022-3492, Nº. 3, 2014, págs. 498-508
• Idioma: inglés
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• Resumen

  • Background: Previous studies have shown that periodontal ligament exists in a hypoxic microenvironment, especially under the condition of periodontitis or physical stress. The present study is designed to investigate the effects and mechanisms of hypoxia on regulating the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs) under cyclic tensile stress (CTS).

    Methods: hPDLCs were cultured in 2% O2 (hypoxia) or 20% O2 (normoxia) and then subjected to a cyclic in-plane tensile deformation of 10% at 0.5 Hz. The following parameters were measured: 1) cell proliferation by flow cytometry; 2) cell ultrastructure by transmission electron microscopy; 3) expression of hypoxia-inducible factor-1a (HIF-1a) and osteogenic relative factors (i.e., secreted phosphoprotein 1 [SPP1; also known as bone sialoprotein I/osteopontin], runt-related transcription factor 2 [RUNX2], and transcription factor Sp7 [SP7]) by real-time polymerase chain reaction and Western blot; and 4) involvement of mitogen-activated protein kinase (MAPK) signaling pathways by Western blot with specific inhibitor.

    Results: Proliferation index in the hypoxia with CTS group was significantly higher than in other groups. Significant increases in HIF-1a, SPP1, RUNX2, and SP7 occurred in the presence of hypoxia for 24 hours. In addition, MAPK inhibitor (PD 98,059) significantly attenuated hypoxia and CTS-induced phosphor-ERK1/2 (extracellular regulated kinase 1/2), phosphor-JNK (c-jun N-terminal kinase), and phosphor-P38 expression.

    Conclusions: Hypoxia regulates CTS-responsive changes in proliferation and osteogenic differentiation of hPDLCs via MAPK pathways. Hypoxia-treated hPDLCs may serve as an in vitro model to explore the molecular mechanisms of periodontitis.

    Hypoxia, a common pathologic process due to inadequate supply of oxygen or an oxygen barrier, has been known as a prominent microenvironment in many diseases, such as solid tumors, ischemic cardiovascular disease, and bone or soft tissue injury.1-3 Hypoxia has also been associated with the pathogenesis of periodontitis, which is one of the most common oral infectious diseases in humans.4-6 Periodontal ligament (PDL), a highly vascularized tissue that connects cementum and alveolar bone, acts as a critical element in maintaining periodontal homeostasis and remodeling under mechanical loading from occlusion and orthodontic tooth movement.7,8 It is well known that physiologic blood flow in PDL is slightly lower than that in either dental pulp or cerebral tissue and is easily reduced by mechanical forces.9 Also, smoking and the growth of anaerobic bacterial biofilm may further reduce oxygen tension in the vicinity of periodontal tissues.10-12 Furthermore, hypoxia-inducible factor 1a (HIF-1a), a key transcription factor responding to hypoxia, has been proven to be expressed in diseased periodontium.13,14 Therefore, it has been hypothesized that PDL exists in a hypoxic microenvironment.

    Periodontal ligament cells (PDLCs), the highly specialized and predominant cells in PDL, constitutively receive mechanical stress, such as compressive stress or tensile stress, during occlusion and tooth movement.15 Numerous findings have indicated that proper tensile stress applied to PDLCs in a normoxic environment could upregulate bone-related genes, such as runt-related transcription factor 2 (RUNX2), transcription factor Sp7 (SP7; also known as osterix), and secreted phosphoprotein 1 (SPP1; also known as bone sialoprotein I/osteopontin), resulting in bone formation in alveolar bone.16,17 On the other hand, bone-resorbing factors such as receptor activator of nuclear factor-kappa B ligand (RANKL), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a) have been detected in PDLCs under compressive stress.18,19 However, research on the effects of hypoxic microenvironment on the behavior of PDLCs is very limited.2,4-6,9,13 In the present study, the authors establish a model of human PDLCs (hPDLCs) under hypoxia and cyclic tensile stress (CTS) in vitro to analyze the effect of hypoxia on proliferation and osteogenic differentiation and clarify the molecular mechanisms in this process.