Chronic Exposure of Gingival Fibroblasts to TLR2 or TLR4 Agonist Inhibits Osteoclastogenesis but Does Not Affect Osteogenesis

Abstract

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines.

Diabetes Medication Metformin Inhibits Osteoclast Formation and Activity in In Vitro Models for Periodontitis

“…Figure 1A ). As in previous studies from our group ( Karlis et al, 2020 ; Loo-Kirana et al, 2021 ), the observed Alizarin Red staining was due to so-called nodules or hot-spots of mineralization that formed on top of the fibroblasts ( Figure 1D ). Interestingly, mineralization medium only induced osteopontin expression when compared to normal medium, whereas RUNX-2, Collagen-I expression and the activity of Alkaline phosphatase was not influenced, not by mineralization medium nor by metformin.…”

“…These promising results with metformin in animal studies as well as in clinical studies urge mechanistic insight using a relevant cell system. Periodontal ligament fibroblasts anchor teeth in bone and also play a crucial role in both osteogenesis and osteoclastogenesis, processes that can be elegantly mimicked in vitro (De Vries et al, 2017;Ruppeka-Rupeika et al, 2018;De Vries et al, 2019;Karlis et al, 2020;Loo-Kirana R.;Gilijamse M.;Hogervorst J., 2021). Support for the relevance of periodontal ligament fibroblasts comes from an animal study where periodontal ligament fibroblasts were shown to play a key role in alveolar bone regeneration (Ren et al, 2015).…”

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

“…gingivalis LPS and Pam3CSK4, which have been observed in our previous study, could not be confirmed at this level 22 . However, our results are in accordance with the recently published study of Karlis et al, who showed that TLR2 and TLR4 activation has no effect on the osteogenic differentiation of gingival fibroblasts 37 …”

Although Anatomically Micrometers Apart: Human Periodontal Ligament Cells Are Slightly More Active in Bone Remodeling Than Alveolar Bone Derived Cells

“…This staining was performed after 21 days, also visualizing the nodular mineral deposition ( Figure 6A ). We recently demonstrated that this nodular aspects further branch into smaller nodules, possibly connected with threads that may resemble collagen fibrils, when examined by scanning electron microscopy ( de Vries et al, 2019 ; Karlis et al, 2020 ). For all patients, except for patient 2, the strongest staining was observed for PDL compared to bone A and bone B.…”