Fibroblast Matrix Metalloproteinase Production After Exposure to Actinobacillus actinomycetemcomitans

Objectives:  Oral bacteria may regulate fibroblast activities in both periodontal pathogenesis and wound healing.  The purpose of this study was to determine the effects of Actinobacillus actinomycetemcomitans (Aa) on the production of matrix metalloproteinases (MMPs) by human gingival fibroblasts (HGF).

Methods:  Primary cultures of HGF were grown under standard culture conditions in DMEM.  Aa (strains 1169, Y4) were cultured under both aerobic and anaerobic conditions in BHI medium.  Bacteria were then sonicated, and cell free supernatants were collected.  HGF were exposed to bacterial extracts (diluted 1:5 in DMEM), DMEM alone (control) and BHI/DMEM (control).  Culture medium was collected at 3, 6, 12, 24, and 48 hrs.  Production of MMPs 1, 3, and 9 was quantified using ELISA.  Experiments were conducted in duplicate for each experimental condition at each time point. 

Results:  HGF production of MMP-1 and 3 was increased by both strains of Aa (1169, Y4) for both aerobic and anaerobic culture.  MMP-9 production was below detection limits for both control and experimental conditions.  Under control conditions at 48hrs, HGF produced 4.77 ng/ml of MMP-1, whereas with exposure to Aa strain 1169 (anaerobic) 344ng/ml of MMP-1 was produced.  For Aa 1169 (aerobic) MMP-1 was above detection limit.  Aa strain Y4 (aerobic) increased MMP-1 production to 432ng/ml, and for Aa Y4 (anaerobic) it was above detection limit.  MMP-3 production by HGF under control conditions at 48hrs was 4.45ng/ml, and after exposure to all four Aa extracts was above detection limit.  BHI medium was also shown to stimulate HGF production of both MMP-1 and 3.

Conclusion:  HGF production of MMP-1 and 3, but not MMP-9, was stimulated by sonicated extracts of Aa.  The interaction of Aa with resident fibroblasts may have important effects on periodontal matrix turnover and degradation.  Supported by NIH/NCRR P20RR018741 and the J. Dean Robertson Society.