Porphyromonas gingivalis Reduces Mitogenic and Chemotactic Responses of Human Periodontal Ligament Cells to Platelet‐Derived Growth Factor In Vitro

Abstract

The effects of a sonicated Porphyromonas gingivalis ATCC 33277 protein extract on the mitogenic and chemotactic responses of human periodontal ligament (PDL) cells to the recombinant human platelet‐derived growth factor‐BB homodimer (PDGFBB) were examined in vitro. Proliferation of PDL cells was inhibited by P. gingivalis extract at concentrations higher than 10 μg/mL protein. At 100 μg/mL of P. gingivalis extract, cells did not proliferate. DNA synthesis in PDL cells, as revealed by [3H]thymidine incorporation, was also inhibited by approximately 50% in the presence of 50 μg/mL P. gingivalis extract for 24 hours. In contrast, PDGF‐BB at 1 ng/mL enhanced DNA synthesis in PDL cells, followed by maximum enhancement at concentrations higher than 10 ng/mL PDGF‐BB. However, this mitogenic response to PDGF‐BB was markedly reduced in the presence of 20 μg/mL of P. gingivalis extract and did not reach the maximum level even if PDGF‐BB concentrations were increased to 250 ng/mL. PDL cells exhibited a chemotactic response to PDGF‐BB at 1 ng/mL, which was also inhibited by pretreatment of the cells with P. gingivalis extract at 10 to 50 pg/mL. Scatchard analysis of a [125I]‐PDGF binding assay demonstrated that PDL cells have both high and low PDGF binding affinity sites. Treatment of the cells with P. gingivalis extract decreased the number of PDGF‐binding sites to approximately 35% of the control level, while it caused only a slight change in the affinities of both types of binding site. These results indicated that the P. gingivalis extract reduced mitogenic and chemotactic responses of human PDL cells, possibly through mechanisms involving a decrease in PDGF‐binding capacity of these cells. Due to this inhibitory effect of P. gingivalis, the normal levels of PDGF in periodontal lesions may not be sufficient to promote periodontal regeneration through activation of PDL cell proliferation and migration. Therefore, the therapeutic use of PDGF‐BB, as a supplement to pre‐existing PDGF and as an adjunct, while also eliminating P. gingivalis from periodontal lesions, would help periodontal tissue regeneration. J Periodontol 1996;67:1335–1341.

Inhibitory Effects of Tumor Necrosis Factor‐Alpha on Migration of Human Periodontal Ligament Cells

“…PDL cells were washed with phosphate buffered saline (PBS) and treated with a lysis buffer *** (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 1% surfactant ††† ) and protease inhibitor cocktail, ‡‡‡ followed by centrifugation at 14,000 × g for 10 minutes at 4°C. Equal amounts of protein (60 μg/lane) from each cell lysate were subjected to electrophoresis on a 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gel, after which the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, as described previously 17 . The membrane was incubated in a blocking buffer (Tris buffered saline, pH 7.4, containing 0.1% Tween‐20 and 5% non‐fat dry milk) for 1 hour, and then reacted with rabbit polyclonal anti‐human α5 integrin antibodies §§§ (1:1,000), rabbit monoclonal anti‐human β1 integrin antibody ‖‖‖ (1:1,000), rabbit polyclonal anti‐human phospho‐FAK (pTyr 397 ) antibodies ¶¶¶ (1:1,000), rabbit polyclonal anti‐human phospho‐paxillin (pTyr 118 ) antibodies ### (1:1,000), mouse monoclonal FAK antibody **** (1:1,000), or paxillin antibody †††† (1:1,000).…”

“…Several factors, such as bacterial products and growth factors, were reportedly shown to influence PDL cell migration 2,16,17 . However, little is known about the influence of TNF‐α on PDL cells.…”