Anti-apoptotic effects of human gingival mesenchymal stromal cells on polymorphonuclear leucocytes.

OBJECTIVES: Polymorphonuclear leucocytes (PMNs) constitute the first line of host defence and are crucial in maintaining periodontal health. Their survival and function are modulated by mesenchymal stromal cells (MSCs) from different origin. Gingival MSCs (GMSCs) play an important role in maintaining oral health and in the initial inflammatory response. The present study aimed to investigate the effects of GMSCs on PMNs apoptosis and reactive oxygen species (ROS) production. METHODS: PMNs were either directly incubated with untreated, interleukin (IL)-1ß- or tumour necrosis factor (TNF)-α-treated GMSCs or stimulated with their conditioned media. Resulting ROS production was evaluated by dichlorofluorescin diacetate staining, whereas PMNs apoptosis was assessed by Annexin V staining, followed by flow cytometry analysis. RESULTS: While conditioned media of untreated and TNF-α-treated GMSCs did not affect apoptosis of PMNs, it was significantly delayed by conditioned media of GMSCs treated with IL-1ß. In direct co-culture, GMSCs exerted anti-apoptotic effects on PMNs independently of the previous stimulation. However, the strongest impact was observed by IL-1ß-treated GMSCs. ROS production of PMNs was not influenced by GMSCs or their conditioned media. CONCLUSION: This study demonstrates for the first time the immunomodulatory properties of GMSCs towards PMNs, revealing that IL-1ß enhances anti-apoptotic effects of GMSCs.

Effect of vitamin D3 on the osteogenic differentiation of human periodontal ligament stromal cells under inflammatory conditions.

OBJECTIVES: Vitamin D3 is known to activate osteogenic differentiation of human periodontal ligament stromal cells (hPDLSCs). Recently, inflammatory stimuli were shown to inhibit the transcriptional activity of hPDLSCs, but their effect on vitamin D3 -induced osteogenic differentiation is not known. The present study aimed to investigate whether the effects of 1,25-dihydroxvitamin D3 (1,25(OH)2 D3 ) and 25-hydroxvitamin D3 (25(OH)D3 ) on the osteogenic differentiation of hPDLSCs are also altered under inflammatory conditions. Furthermore, the expression of osteogenesis-related factors by hPDLSCs under osteogenic conditions was assessed in the presence of inflammatory stimuli. MATERIALS AND METHODS: Primary hPDLSCs of six donors were cultured in osteogenic induction medium containing either 1,25(OH)2 D3 (0-10 nM) or 25(OH)D3 (0-100 nM) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) or Pam3CSK4 for 7, 14 and 21 days. Osteogenic differentiation of hPDLSCs was evaluated by analysis of mineralization as assessed by Alizarin Red S staining and gene expression levels of osteogenesis-related factors osteocalcin, osteopontin and runt-related transcription factor 2 (RUNX2) were analysed with qPCR. RESULTS: Treatment with 1,25(OH)2 D3 significantly enhanced the osteogenic differentiation of hPDLSCs and their expression of osteocalcin and osteopontin. The 1,25(OH)2 D3 -triggered expression of osteogenesis-related factors was significantly lower in the presence of Pam3CSK4, but not P. gingivalis LPS. None of the inflammatory stimuli had significant effects on the 1,25(OH)2 D3 -induced osteogenic differentiation. 25(OH)D3 neither affected gene expression levels nor osteogenic differentiation of hPDLSCs cultured in osteogenic induction medium. CONCLUSION: The results of this study indicate that inflammatory stimuli also diminish the 1,25(OH)2 D3 -induced expression of osteogenesis-related factors in hPDLSCs under osteogenic conditions, while having no effect on the osteogenic differentiation.

Response of Human Mesenchymal Stromal Cells from Periodontal Tissue to LPS Depends on the Purity but Not on the LPS Source.

Human periodontal ligament stromal cells (hPDLSCs) and gingival mesenchymal stromal cells (hGMSCs) are resident mesenchymal stromal cells (MSCs) of the periodontal tissue. The lipopolysaccharide (LPS) from Porphyromonas gingivalis is structurally distinct from that of other Gram-negative bacteria, and earlier studies linked this structural difference to a distinct virulence activity and the ability to activate toll-like receptor 2 (TLR-2), besides TLR-4 as commonly occurring upon LPS challenge. Later studies, in contrast, argue that TLR-2 activation by P. gingivalis LPS is due to lipoprotein contamination. In the present study, we aimed to define the influence of structure versus purity of P. gingivalis LPS on the immune response of hPDLSCs and hGMSCs. Cells were stimulated with commercially available "standard" P. gingivalis LPS, "ultrapure" P. gingivalis LPS, or "ultrapure" Escherichia coli LPS, and the expression of interleukin- (IL-) 8, IL-6, monocyte chemoattractant protein- (MCP-) 1, TLR-2, and TLR-4 was evaluated. The contribution of TLR-4 to the LPS-induced response was assessed using the specific TLR-4 inhibitor TAK-242. "Standard" P. gingivalis LPS induced significantly higher IL-8, IL-6, and MCP-1 production compared to the "ultrapure" LPS preparations, with no significant difference detectable for "ultrapure" LPS from P. gingivalis and E. coli. By using TAK-242, the response of hPDLSCs and hGMSCs to "ultrapure" LPS preparations was effectively inhibited to the levels comparable to those of nonstimulated controls. In contrast, high levels of response to "standard" LPS were observed, even in the presence of TAK-242. Our data show that the response of MSCs from periodontal tissue to LPS depends more on the purity of the LPS preparation than on the LPS source. Even a small amount of contaminating lipoproteins can drastically enhance the hPDLSCs' and hGMSCs; responsiveness to P. gingivalis LPS, which might also contribute to the progression of periodontal disease.

Soluble CD14 Enhances the Response of Periodontal Ligament Stem Cells to Toll-Like Receptor 2 Agonists.

Human periodontal ligament stem cells (hPDLSCs) do not express membrane-bound CD14, and their responsiveness to bacterial lipopolysaccharide (LPS) is drastically enhanced by soluble CD14 (sCD14), which is due to the facilitation of the interaction between LPS and Toll-like receptor- (TLR-) 4. Several studies also show that sCD14 enhances the responsiveness of different immune cells to TLR-2, but such effect in hPDLSCs has not been studied so far. In the present study, we investigated for the first time the potential effect of sCD14 on the hPDLSC response to two different TLR-2 agonists, in vitro. Primary hPDLSCs were stimulated with synthetic lipopeptide Pam3CSK4 or lipoteichoic acid (LTA) in concentrations 1-1000 ng/ml in the presence/absence of sCD14 (250 ng/ml). Additionally, the effect of different sCD14 concentrations (2.5-250 ng/ml) on the TLR-2 response was determined in Pam3CSK4- or LTA-triggered hPDLSCs. The resulting expression of interleukin- (IL-) 6, chemokine C-X-C motif ligand 8 (CXCL8), and chemokine C-C motif ligand 2 (CCL2) was measured by qPCR and ELISA. The production of IL-6, CXCL8, and CCL2 was gradually increased by both TLR-2 agonists and was significantly enhanced by sCD14. The response of hPDLSCs to low and submaximal concentrations of TLR-2 agonists (1-100 ng/ml) was most effectively enhanced by sCD14. The effect of sCD14 on TLR-2 response in hPDLSCs was concentration-dependent and was already detectable at low sCD14 levels. Our data showed that exogenous sCD14 significantly enhanced the responsiveness of hPDLSCs to TLR-2 agonists and enabled the detection of their small amounts. This effect was already detectable at low sCD14 levels, which are comparable to those in saliva and gingival crevicular fluid. Changes in the local sCD14 level may be considered as a crucial factor influencing the susceptibility of hPDLSCs to different pathogens and thus may contribute to the progression of periodontitis.

Transcriptional activity of vitamin D receptor in human periodontal ligament cells is diminished under inflammatory conditions.

BACKGROUND: Although vitamin D3 deficiency is considered as a risk factor for periodontitis, supplementation during periodontal treatment has not been shown to be beneficial to date. Human periodontal ligament cells (hPDLCs) are regulated by vitamin D3 and play a fundamental role in periodontal tissue homeostasis and inflammatory response in periodontitis. The aim of this study is to investigate possible alterations of the vitamin D3 activity in hPDLCs under inflammatory conditions. METHODS: Cells isolated from six different donors were treated with either 1,25(OH)2 D3 (0 to 10 nM) or 25(OH)D3 (0 to 100 nM) in the presence and absence of ultrapure or standard Porphyromonas gingivalis lipopolysaccharide (PgLPS), Pam3CSK4, or interferon-γ for 48 hours. Additionally, nuclear factor (NF)-κB inhibition was performed with BAY 11-7082. The bioactivity of vitamin D in hPDLCs was assessed based on the gene expression levels of vitamin D receptor (VDR)-regulated genes osteocalcin and osteopontin. Additionally, VDR and CYP27B1 expression levels were measured. RESULTS: The vitamin D3 -induced increase of osteocalcin and osteopontin expression was significantly decreased in the presence of standard PgLPS and Pam3CSK4, which was not observed by ultrapure PgLPS. Interferon-y had diverse effects on the response of hPDLCs to vitamin D3 metabolites. NF-kB inhibition abolished the effects of standard PgLPS and Pam3CSK4. Standard PgLPS and Pam3CSK4 increased VDR expression in the presence of vitamin D3 . CYP27B1 expression was not affected by vitamin D3 and inflammatory conditions. CONCLUSIONS: This study indicates that the transcriptional activity of VDR is diminished under inflammatory conditions, which might mitigate the effectiveness of vitamin D3 supplementation during periodontal treatment.

Effect of implant surface material and roughness to the susceptibility of primary gingival fibroblasts to inflammatory stimuli.

OBJECTIVES: The impact of the implant surface material and roughness on inflammatory processes in peri-implantitis is not entirely clear. Hence, we investigated how titanium and zirconia surfaces with different roughness influence the susceptibility of primary human gingival fibroblasts to different inflammatory stimuli. METHODS: Primary human gingival fibroblasts were isolated from 8 healthy individuals and cultured on following surfaces: smooth titanium machined surface (TiM), smooth zirconia machined surface (ZrM), moderately rough titanium surface (SLA), or moderately rough zirconia surface (ZLA). Subsequently, stimulation with one of the following stimuli was performed: Porphyromonas gingivalis lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß. The resulting production of IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 was measured by qPCR and ELISA. RESULTS: P. gingivalis LPS induced IL-6 and MCP-1 production was slightly higher on titanium surfaces compared to zirconia surfaces. IL-1ß induced IL-6 production was not affected by any surface characteristic. The production of MCP-1 in response to IL-1ß was higher on smooth compared to rough surfaces and was not affected by the material. The production of IL-6 and MCP-1 in response to TNF-α was most strongly affected by surface characteristics. Higher production of these cytokine was observed on smooth compared to rough surfaces and on titanium compared to zirconia surfaces. Surface characteristics had only minor effects on IL-8 production. SIGNIFICANCE: The susceptibility of primary gingival fibroblasts to inflammation depends on various factors, such as surface material, surface roughness and the nature of inflammatory stimuli. All these factors might determine susceptibility to peri-implantitis.