Passaging of gingival fibroblasts from periodontally healthy and diseased sites upregulates osteogenesis-related genes.

To investigate biological processes of the periodontium, in vitro primary cell models have been established. To study the biology of the gingiva, primary gingival fibroblast cell models are widely used. For such experiments, cells need to be expanded and passaged. A key assumption is that primary cells maintain most of their original characteristics they have in situ. The aim of this research is to explore the impact of early passaging on selected gene expression of human gingival fibroblast cells. For this purpose, gene expression from the outgrowth of the resected tissues until the fourth passage was followed for nine tissue samples, from both healthy and diseased sites. Micrographs were taken from the cultures, RNA was extracted from the samples of each passage and quantitative PCR was performed for selected genes representing various biological processes. Epithelial cells were present during the first outgrowth, but were no longer present in the second passage. Our results indicate that the morphology of the gingival fibroblast cells does not change with passaging and that passages 2-4 contain only gingival fibroblasts. Gene expression of M-CSF, TNF-α, TLR4, POSTN and FAPα was unchanged by passaging, the expression of IL-6, IL-1ß and TLR2 decreased due to passaging and the expression of in particular the selected osteogenesis genes (ALP, RUNX2, Osteonectin, COL1A), OPG and MKI67 increased with passaging. Worldwide, use of the same passage in laboratory experiments using primary cell cultures is the standard. Our results support this, since for certain genes, in particular osteogenesis genes, expression may alter solely due to passaging.

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

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.

T Cell Proliferation Is Induced by Chronically TLR2-Stimulated Gingival Fibroblasts or Monocytes.

During inflammation of the gums, resident cells of the periodontium, gingival fibroblasts (GFs), interact with heterogeneous cell populations of the innate and adaptive immune system that play a crucial role in protecting the host from pathogenic infectious agents. We investigated the effects of chronic inflammation, by exposing peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocyte (PBL) cultures, and GF-PBMC cocultures to Toll-like receptor 2 (TLR2) and TLR4 activators for 21 days and assessed whether this influenced leukocyte retention, survival, and proliferation. Chronic stimulation of PBMC-GF cocultures with TLR2 and TLR4 agonists induced a reduction of NK (CD56+CD3-), T (CD3+), and B (CD19+) cells, whereas the number of TLR-expressing monocytes were unaffected. TLR2 agonists doubled the T cell proliferation, likely of a selective population, given the net decrease of T cells. Subsequent chronic exposure experiments without GF, using PBMC and PBL cultures, showed a significantly (p < 0.0001) increased proinflammatory cytokine production of TNF-α and IL-1ß up to 21 days only in TLR2-activated PBMC with concomitant T cell proliferation, suggesting a role for monocytes. In conclusion, chronic TLR activation mediates the shift in cell populations during infection. Particularly, TLR2 activators play an important role in T cell proliferation and proinflammatory cytokine production by monocytes, suggesting that TLR2 activation represents a bridge between innate and adaptive immunity.