Benzo[a]pyrene/aryl hydrocarbon receptor signaling inhibits osteoblastic differentiation and collagen synthesis of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Cigarette smoking has detrimental effects on periodontal tissue, and is known to be a risk factor for periodontal disease, including the loss of alveolar bone and ligament tissue. However, the direct effects of cigarette smoking on periodontal tissue remain unclear. Recently, we demonstrated that benzo[a]pyrene (BaP), which is a prototypic member of polycyclic aryl hydrocarbons and forms part of the content of cigarettes, attenuated the expression of extracellular matrix remodeling-related genes in human periodontal ligament (PDL) cells (HPDLCs). Thus, we aimed to examine the effects of BaP on the osteoblastic differentiation and collagen synthesis of HPDLCs. MATERIAL AND METHODS: HPDLCs were obtained from healthy molars of three patients, and quantitative reverse transcription-polymerase chain reaction were performed for gene expression analyses of cytochrome P450 1A1 and 1B1, alkaline phosphatase, bone sialoprotein and aryl hydrocarbon receptor (AhR), a receptor for polycyclic aryl hydrocarbons. We have also analyzed the role of the AhR, using 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), which is an AhR antagonist. RESULTS: The treatment of HPDLCs with BaP reduced mRNA expression of osteogenic genes, alkaline phosphatase activity, mineralization and collagen synthesis. The treatment with CH-223191 subsequently restored the observed suppressive effects of BaP on HPDLCs. CONCLUSIONS: The present results suggest that BaP exerts inhibitory effects on the maintenance of homeostasis in HPDL tissue, such as osteoblastic differentiation and collagen synthesis of HPDLCs, and that this signaling pathway could be suppressed by preventing the transactivity of AhR. Future studies may unveil a role for the inhibition of AhR as a promising therapeutic agent for periodontal disease caused by cigarette smoking.

Mechanical induction of interleukin-11 regulates osteoblastic/cementoblastic differentiation of human periodontal ligament stem/progenitor cells.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is continually exposed to mechanical loading caused by mastication or occlusion. Physiological loading is thus considered a key regulator of PDL tissue homeostasis; however, it remains unclear how this occurs. We recently reported that an appropriate magnitude of mechanical stretch can maintain PDL tissue homeostasis via the renin-angiotensin system. In the present study, we investigated the expression of interleukin-11 (IL-11) in human primary PDL cells (HPDLCs) exposed to stretch loading, the contribution of angiotensin II (Ang II) to this event and the effects of IL-11 on osteoblastic/cementoblastic differentiation of human PDL progenitor cells (cell line 1-17). MATERIAL AND METHODS: Human primary PDL cells, derived from human tissues, with or without antagonists against the Ang II receptors AT1 or AT2, were subjected to cyclical stretch loading with 8% elongation for 1 h. Expression of IL-11 was measured by ELISA in these cultures and by immunohistochemistry in the sectioned maxillae of rats. The osteoblastic/cementoblastic potential of cell line 1-17 was determined using cell proliferation, gene expression and Alizarin Red staining. RESULTS: Positive staining for IL-11 was observed in the PDL of rat maxillae and in cultures of HPDLCs. In HPDLCs exposed to stretch, expression of the IL11 gene and the IL-11 protein were up-regulated, concomitant with an increase in Ang II and via AT2. Recombinant human IL-11 (rhIL-11) stimulated an increase in expression of mRNA for the cementoblast-specific marker, CP-23, and for the osteoblastic markers, osteopontin and bone sialoprotein, and promoted proliferation in cell line 1-17. In addition, rhIL-11 also increased the degree of mineralized nodule formation in cell line 1-17 cultures treated with CaCl2 . CONCLUSION: Mechanical loading appears to control proliferation and osteoblastic/cementoblastic differentiation of human PDL stem/progenitor cells through the regulation of Ang II and AT2 by IL-11.

An in vitro evaluation of two resin-based sealers on proliferation and differentiation of human periodontal ligament cells.

AIM: To evaluate the effects of a polymethyl methacrylate resin-based sealer [Superbond sealer (SB)] on the proliferation and osteogenic differentiation of human periodontal ligament cells (HPDLCs) in vitro, compared with a methacrylate resin-based sealer [Epiphany SE sealer (EP)]. METHODOLOGY: Human periodontal ligament cells were obtained from of healthy third molar teeth of two participants with informed consent. To determine the effects of the eluent from set resin sealers on HPDLCs, the 7-day-washed (washed) or non-washed freshly prepared (fresh) set SB or EP discs were prepared. Cells cultured on these discs were evaluated by the WST-1 proliferation assay and scanning electron microscopy (SEM). The osteogenic differentiation of HPDLCs on washed SB discs was then evaluated by gene expression analysis of osteopontin (OPN) and osteocalcin (OCN) by using quantitative RT-PCR. RESULTS: Human periodontal ligament cells exhibited growth on washed SB discs, whereas fresh SB and EP discs and washed EP discs inhibited proliferation of HPDLCs. SEM observation revealed that HPDLCs tightly attached and spread on the surface of washed SB discs, whilst no HPDLCs were observed on the surface of fresh and washed EP discs. Furthermore, HPDLCs significantly upregulated gene expressions of OPN and OCN when cultured on washed SB discs in osteogenic differentiation medium for 2 weeks. CONCLUSIONS: Although Superbond sealer initially exerted cytotoxic effects on HPDLCs, these effects were reduced during washing for 7 days compared to EP, which continued to be cytotoxic even though the specimens were washed for the same period of time. Washed Superbond allowed HPDLCs to differentiate into osteogenic cells.

The roles of angiotensin II in stretched periodontal ligament cells.

The loading caused by occlusion and mastication plays an important role in maintaining periodontal ligament (PDL) tissues. We hypothesized that a loading magnitude would be involved in the production of biological factors that function in the maintenance of PDL tissues. Here, we identified up-regulated gene expressions of transforming growth factor-ß1 (TGF-ß1), alkaline phosphatase (ALP), and angiotensinogen in human PDL fibroblastic cells (HPLFs) that were exposed to 8% stretch loading. Immunolocalization of angiotensin I/II (Ang I/II), which was converted from angiotensinogen, was detected in rat PDL tissues. HPLFs that were stimulated by Ang II also increased their gene expressions of TGF-ß1 and ALP. Furthermore, the antagonist for Ang II type 2 receptor, rather than for type 1, significantly inhibited gene expressions induced by the stretch loading. Analysis of these data suggests that Ang II mediates the loading signal in stretched HPLFs to induce expressions of TGF-ß1 and ALP.