Identification of components in Fusobacterium nucleatum chemostat-culture supernatants that are potent inhibitors of human gingival fibroblast proliferation

Abstract

The present investigation concerned the effect of chemostat-culture cell-free supernatants of Fusobacterium nucleatum on the growth and synthetic activity of human gingival fibroblasts in vitro. Human gingival fibroblasts were cultured in fetal calf serum supplemented Dulbecco-Vogt medium containing various dilutions of conditioned or unconditioned bacterial culture medium. Cell proliferation was monitored by assessing cell growth over 5 days or incorporation of [3H]-thymidine into DNA. Protein and proteoglycan synthesis were monitored by the incorporation of [3H]-proline and [35S]-sulfate, respectively, into macromolecules. While the conditioned culture medium caused a complete inhibition of cell growth and incorporation of [3H]-thymidine DNA, there was no discernible effect on protein or proteoglycan synthesis. This indicated that the cells remained viable yet unable to divide. Such a view was supported by the observation that the inhibitory effect was reversible upon removal of the conditioned medium. This activity had a molecular size less than 30,000, was heat-stable and nonvolatile. Chemical analysis of the conditioned bacterial culture supernatants indicated that high proportions of butyrate, ammonium, and acetate were present. When these components were added to unconditioned medium and tested, most of the inhibitory activity could be attributed to ammonium and butyrate. Since many bacteria which constitute the subgingival microflora release ammonium and butyrate, a very high concentration of these metabolites may well accumulate. Clearly, the potential for inhibition of fibroblast proliferation has ramifications related to diminished tissue repair following bacterially-induced periodontal destruction.