Bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin

Abstract

Gingival inflammation, bacterial infection, alveolar bone destruction, and subsequent tooth loss are characteristic features of periodontal disease, but the precise mechanisms of bone loss are poorly understood. Most animal models of the disease require injury to gingival tissues or teeth, and the effects of microorganisms are thus complicated by host responses to tissue destruction. To determine whether three putative periodontal pathogens, Porphyromonas gingivalis, Campylobacter rectus, and Fusobacterium nucleatum, could cause localized bone resorption in vivo in the absence of tissue injury, we injected live or heat-killed preparations of these microorganisms into the subcutaneous tissues overlying the calvaria of normal mice once daily for 6 days and then examined the bones histologically. We found that all three microorganisms (both live and heat killed) stimulated bone resorption and that the strain of F. nucleatum used appeared to be the strongest inducer of osteoclast activity. Treatment of the mice concomitantly with indomethacin reduced but did not completely inhibit bone resorption by these microorganisms, suggesting that their effects were mediated, in part, by arachidonic acid metabolites (e.g., prostaglandins). Our findings indicate that these potential pathogens can stimulate bone resorption locally when placed beside a bone surface in vivo in the absence of prior tissue injury and support a role for them in the pathogenesis of bone loss around teeth in periodontitis.

FIG. 1

Effects of local injections of F. nucleatum on murine calvarial bone and soft tissue. Live F. nucleatum bacteria (2 × 10⁷) were injected once daily for 6 days into the s.c. tissues overlying the calvaria of mice, and the animals were sacrificed 24 h later. Edema and abscesses (A) developed in the soft tissue overlying the calvarium of this mouse injected with F. nucleatum. In addition, increased numbers of osteoclasts (arrows) were seen inside the calvarium, and these caused an increase in the size of the bone marrow spaces (see Fig. 2C for comparison) due to increased endosteal bone resorption. Hematoxylin-eosin stain was used.

FIG. 2

Effects of local injections of F. nucleatum and P. gingivalis on mouse calvaria. Live bacteria were injected once daily for 6 days into the s.c. tissues overlying the calvaria of mice, and the animals were sacrificed 24 h later. (A) Increased numbers of osteoclasts (arrows) were seen in the bone marrow spaces, which were enlarged as a result of the increased osteoclast activity in the calvarium of a mouse injected with 2 × 10⁷ F. nucleatum bacteria. (B) In contrast, only occasional osteoclasts (arrows) were seen in the bone marrow spaces of the calvarium of a mouse injected with 2 × 10⁷ P. gingivalis bacteria, and few of these marrow spaces were enlarged. These appearances (B) are similar to those of the calvarium of a control mouse (C) given daily injections of the vehicle (RTF). A moderately heavy acute inflammatory infiltrate and moderate edema are present in the soft tissues overlying the calvaria of the mice injected with bacteria. Mild edema and a few inflammatory cells are present in the soft tissue overlying the calvarium of the control mouse. Hematoxylin-eosin stain was used.

FIG. 3

Effects of injections of live or heat-killed bacteria on osteoclast numbers in mouse calvaria. Organisms were injected once daily for 6 days at the doses indicated. The mice were sacrificed 24 h later, and osteoclast numbers in decalcified calvarial sections were counted. Values are means ± standard errors of means. ∗, Significantly different from mean value for vehicle-treated (RTF) mice (P < 0.05). #, Dose response significantly different from that to P. gingivalis (P < 0.01). +, Dose response significantly different from that to C. rectus (P < 0.001). $, Significantly different from mean value for mice treated with live F. nucleatum bacteria (P < 0.01).

FIG. 4

Effects of indomethacin and live bacteria on osteoclast numbers in mouse calvaria. Injections of indomethacin (40 μg in PBS) were given s.c. every 8 h for 6 days, beginning 2 h before the first bacterial injection and continuing until 4 h after the last injection in groups of mice (n = 5 for bacteria alone; n = 5, n = 6, and n = 7 for indomethacin with P. gingivalis, F. nucleatum, and C. rectus, respectively; n = 8 and n = 9 for RTF alone and RTF with indomethacin, respectively). Values are means ± standard errors of means. All values for the bacterium-injected mice (with and without indomethacin) were significantly different from values for the vehicle-treated (RTF) mice (P < 0.05). ∗, Significantly different from values for mice given bacteria alone (P < 0.01).