Influence of Aerobic/Anaerobic Actinobacillus actinomycetemcomitans on In Vitro Wound Healing

Objective:  Periodontal bacteria may influence tissue cell behavior during both pathogenesis and wound healing.  The purpose of this study was to examine the effects of aerobic and anaerobic metabolism of Actinobacillus actinomycetemcomitans (Aa) on fibroblast behavior in an in vitro wound model.

Methods:  Primary cultures of human gingival fibroblasts (HGF) were grown to confluence in 35mm culture dishes and then subjected to an in vitro wound procedure in which cells from one half of the culture dish were removed.  Wound repopulation was assessed by recording the number of cells and distance traveled from the wound edge to the leading cell front at 24, 48, 72, and 96hr.  Aa (strain Y4) was cultured under both aerobic and anaerobic conditions in BHI medium.  Bacterial cultures were sonicated and cell free supernatant was collected.  Immediately after wounding, HGF were exposed to DMEM alone (control), or sonicated extracts of aerobic or anaerobic AaY4 (diluted 1:5 in DMEM) for 30min, 45min or 96hr.

Results:  Progressive wound repopulation by HGF occurred for all conditions, but was impaired by Aa extracts.  Under control conditions, by 96hr HGF leading cell front was 6.38 mm from the wound edge, with an average of 374.43 cells per field.  Continuous exposure to anaerobic Aa resulted in the greatest impairment of wound healing, with a mean leading front of 1.45 mm from the wound edge (96hr), and a mean of 43.1 cells (96hr) per field.  In comparison, at 96hr continuous exposure of HGF wounds to aerobic Aa produced a mean leading front of 1.7 mm and a mean of 77.07 cells/field.

Conclusion:  In vitro wound healing of HGF was impaired by both anaerobic and aerobic products of Aa, but to a greater extent with anaerobic metabolism.  Supported by NIH/NCRR P20RR018741 and the J. Dean Robertson Society.