Abstract
Periodontal ligament stem cells (PDLSC) play an important role in periodontal tissue homeostasis/turnover and could be applied in cell-based periodontal regenerative therapy. Bacterial supernatants secreted from diverse periodontal bacteria induce the production of cytokines that contribute to local periodontal tissue destruction. However, little is known about the impact of whole bacterial toxins on the biological behavior of PDLSC. Therefore this study investigated whether proliferation, migration, inflammatory cytokines expression and transcriptional profile would be affected by exposure to endotoxins from bacterial species found in the subgingival plaque. PDLSC were cultured with the following bacterial supernatants: S. mutans, S. anginosus, P. intermedia, F. nucleatum, P. gingivalis and T. denticola. These supernatants were prepared in dilutions of 1:1000, 1:500, 1:300 and 1:50. Using quantitative RT-PCR, gene expression of selected inflammatory cytokines (IL-6, IL-8 and IL-1β) and cell-surface receptors (TLR2, TLR4) showed upregulation of ≈2.0- to 3.0-fold, when exposed to P. intermedia, F. nucleatum, P. gingivalis and T. denticola. However, supernatants did not affect proliferation (MTT) and migration (wound scratch assays) of PDLSC. Next generation RNA sequencing confirmed modified lineage commitment of PDLSC by stimulating chondrogenesis, adipogenesis and inhibition of osteogenesis under P. gingivalis supernatant treatment compared to control. Taken together, this study shows stem cell immunomodulatory response to different periodontal bacteria supernatant and suggests that stem cell transcriptional capacity, migration/proliferation and osteogenesis may differ in the presence of those pathogens. These results bring into question stem cell contribution to periodontal tissue regeneration and onset of inflammation.
Highlights
Periodontitis is a chronic inflammatory disease that leads to the destruction of periodontal tissues and subsequent tooth loss, mainly due to subgingival bacterial infection [1, 2]
RT-PCR analysis determined that 24 h treatment with 1:1000 and 1:500 of all bacterial species had no impact on constitutive gene expression in periodontal ligament stem cells (PDLSCs) (Fig 1)
Alizarin red S staining showed that P. gingivalis supernatant 1:500 dilution impaired the osteogenic differentiation ability of PDLSC, which was demonstrated by the decreased formation number of calcified nodules stained with Alizarin Red compared with the control group after 21 days induction (Fig 2, day 21, p < 0.05)
Summary
Periodontitis is a chronic inflammatory disease that leads to the destruction of periodontal tissues and subsequent tooth loss, mainly due to subgingival bacterial infection [1, 2]. Endotoxins secreted from pathogenic bacteria are crucial virulence factors involved in the initiation and establishment of periodontitis, stimulating proinflammatory cytokine production, monocyte/ lymphocyte infiltration and bone resorption [3, 4]. In both healthy and pathological conditions, periodontal ligament stem cells (PDLSCs) play an important role in maintaining homeostasis and inducing tissue regeneration owing to their ability to differentiate into major cellular types, such as osteoblasts, fibroblasts and cementoblasts [5,6,7]. The success of regenerative periodontal treatment could be improved by understanding the migration and cytokine secretion of PDLSCs when exposed to periodontal bacterial endotoxins
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