Elsevier

Archives of Oral Biology

Volume 59, Issue 2, February 2014, Pages 167-175
Archives of Oral Biology

Porphyromonas gingivalis LPS inhibits osteoblastic differentiation and promotes pro-inflammatory cytokine production in human periodontal ligament stem cells

https://doi.org/10.1016/j.archoralbio.2013.11.008Get rights and content

Abstract

Objective

Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) induces pro-inflammatory cytokines, such as interleukin-1 β (IL-1β), IL-6, and IL-8, which induce periodontal tissue destruction. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration and are expected to have future applications in cellular therapies for periodontitis. However, no studies have examined the effects of P. gingivalis LPS on PDLSCs. The aim of this study was to investigate how P. gingivalis LPS affects the osteoblastic differentiation and pro-inflammatory cytokine production of PDLSCs.

Design

PDLSCs were obtained from healthy adult human mandibular third molars. The identification of PDLSCs was confirmed by immunohistochemical evaluations of the mesenchymal stem cell markers STRO-1 and SSEA-4. Cell proliferation and osteoblastic differentiation were investigated by culturing the PDLSCs in a normal or osteogenic medium with P. gingivalis LPS (0, 1, or 10 μg/mL) and then measuring the alkaline phosphatase (ALP) activity and the production of collagen type 1 Alpha 1 (COL1A1), osteocalcin production, and mineralisation. Additionally, we examined the production of IL-1β, IL-6, and IL-8 in the PDLSCs.

Results

P. gingivalis LPS inhibited the ALP activity, COL1A1 and osteocalcin production, and mineralisation in the PDLSCs, which are positive for STRO-1 and SSEA-4. P. gingivalis LPS also promoted cell proliferation and produced IL-1β, IL-6, and IL-8.

Conclusions

This study provides the first findings that P. gingivalis LPS inhibits osteoblastic differentiation and induces pro-inflammatory cytokines in PDLSCs. These findings will help clarify the relationship between periodontitis and periodontal tissue regeneration.

Introduction

One of the most common causes of tooth loss is periodontal disease, which is defined as the destruction of the alveolar bone and periodontal ligament by bacterial infection.1 Many of the bacteria that cause periodontal disease are Gram-negative bacilli, such as Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans.2, 3 P. gingivalis has been implicated as the causal pathogen in most cases of periodontal disease.4, 5, 6 Lipopolysaccharide (LPS) is an outer membrane component of Gram-negative bacterial cell walls and an endotoxin with a wide range of biological activities. LPS induces the production of inflammatory cytokines, such as interleukin-1 β (IL-1β), IL-6, and IL-8.7, 8 These cytokines and LPS induce alveolar bone resorption and destroy periodontal tissue.9

Periodontal tissue is composed of the periodontal ligament (PDL), cementum, alveolar bone, and gingiva. The PDL is composed of fibrous connective tissue that connects the tooth root to the alveolar bone.10 PDL cells have roles in maintaining tissue regeneration11. PDL cells produce bone-related proteins; they also express alkaline phosphatase (ALP) activity, collagen type 1 alpha 1, osteocalcin, and possess mineralisation capacity.12, 13, 14 Although PDL cells are a component of connective tissues, they also exhibit osteoblastic differentiation capacity.

Periodontal ligament stem cells (PDLSCs) exist in periodontal tissues and participate in periodontal tissue regeneration.15 Similar to other mesenchymal stem cells (MSCs), PDLSCs have the potential to differentiate into osteoblasts.16, 17 PDLSCs have tissue regeneration capacity.18, 19, 20 PDLSCs exhibit greater potential to regenerate periodontal tissues than other MSCs, such as bone marrow stromal cells.21 Therefore, PDLSCs play a particularly important role in periodontal tissue regeneration. Moreover, PDLSCs are predicted to be an important source of cells in periodontal tissue regeneration therapy. However, no studies have reported the effects of LPS on PDLSCs; therefore, this area of investigation is critical.

The aim of this study was to characterize the effects of P. gingivalis LPS on osteoblastic differentiation and mineralisation in PDLSCs. Additionally, we examined the production of inflammatory cytokines, such as IL-1β, IL-6, and IL-8 in the PDLSCs.

Section snippets

Culture and isolation of human PDLSCs

The human tissue experiments were performed in accordance with the guidelines of the Osaka Dental University for Medical Ethics, and all experiments were approved by the Osaka Dental University Medical Ethics Committee (approval no. 110712). All participants provided written informed consent to participate in this study, and the study design was approved by the appropriate ethics review boards. PDLSCs were isolated and cultured as described in our previous study.22 After extraction, the teeth

Isolation and characterisation of PDLSCs

The PDL-derived cells formed clonogenic clusters of fibroblast-like cells (Fig. 1A). No staining was detected in control (Fig. 1B). The PDLSCs derived from the PDL cells were positive for vimentin (Fig. 1C). The PDLSCs were also positive for STRO-1 (Fig. 1D) and SSEA-4 (Fig. 1E), which are MSC markers. The isolated PDLSCs were capable of differentiating into osteogenic cells (Fig. 1F).

Cell proliferation

The proliferation of the PDLSCs in the normal culture medium containing P. gingivalis LPS was significantly

Discussion

The results of this study indicated that P. gingivalis LPS enhanced the proliferation of PDLSC cells. The osteoblastic differentiation assay revealed that the ALP activity, COL1A1 production, osteocalcin production, and mineralisation were significantly suppressed by P. gingivalis LPS. P. gingivalis LPS also enhanced the production of IL-1β, IL-6, and IL-8.

PDLSCs express STRO-1, a marker of bone marrow mesenchymal stem cells (BMSCs).16 SSEA-4 is also a marker of MSCs23 and PDLSCs.24 In this

Funding

No additional external funding was received for this study. The funders played no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.

Competing interests

The authors report no conflicts of interest related to this study.

Ethical approval

Not required.

Acknowledgments

This study was partially supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (C) (no. 24593138 to A.T. and no. 24792345 to Y.T.).

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