Background: Periodontitis is an infectious, inflammatory disease resulting in destruction of the supporting tissues of the teeth. While bacterial biofilm is central to disease pathogenesis, the host response plays an important role in the severity and progression of periodontitis. Studies have shown a strong genetic influence, accounting for as much as 50% of disease presentation. Previous work in our laboratory utilized a Genome-wide Association Study (GWAS) with a Lipopolysaccharide (LPS)-induced periodontitis murine model to identify candidate genes associated with periodontitis. Using GWAS, mRNA, and protein expression data, chemokines Cxcl9 and Cxcl10 were found to be associated and significantly upregulated in a high bone loss strain C57BL/6J compared to bone loss resistant strain A/J. Cxcl9 and Cxcl10 both exert their function through the receptor CXCR3.

Objective: The purpose of this study was to investigate the influence of Cxcl9 and Cxcl10 on LPS-induced periodontal bone loss by blocking their action utilizing a CXCR3 knockout mouse.

Materials and Methods: 12 wild-type (WT) and 12 CXCR3 knockout (KO) C57BL/6J mice were included in this study. Periodontitis was induced using P.gingivalis-LPS injections between the maxillary first and second molars 2x/week for 6 weeks. Following sacrifice, maxillae were scanned (microCT) and bone loss quantified. Histologic analysis of osteoclasts and pro-inflammatory mediators was performed.

Results: Deleting CXCR3 demonstrated ~50% reduction in bone loss after LPS- injections compared to WT mice. 3D volumetric analysis showed no significant differences in initial bone volume/tissue volume (BV/TV) between CXCR3 KO and WT animals, indicating the changes observed were due to LPS treatment and not inherent differences in bone quality. Histologically, an increase in cellular infiltrates was seen in WT LPS treated mice compared to CXCR3 KO LPS treated mice, visualized through H&E and COX-2 immunostaining. Quantification of osteoclasts through TRAP staining revealed significantly more TRAP+ cells in WT LPS treated compared to CXCR3 KO LPS treated mice, correlating with the increased bone loss seen in the WT LPS treated animals.

Conclusion: CXCR3 and binding chemokines Cxcl9 and Cxcl10 are likely key players in the maintenance and amplification of inflammatory pathways. CXCR3 may be a possible target for modulating the host response in periodontitis by dampening the inflammatory cascade following LPS-stimulation. Further work is needed to characterize the CXCR3 pathway and validate other candidate genes associated with LPS-induced bone loss. The ultimate goal is to identify patients at high risk for periodontal disease and manage them with individualized treatment.

Background: Peri-implantitis is an inflammatory disease process that presents with bone loss around implant fixtures. Without the proper treatment, it can lead to the eventual loss of the implant. However, controversy lies over the effectiveness of treatment modalities as the criteria for disease diagnosis and success of treatment varies in the literature. With an increasing global burden of disease comes incentive to find predictable treatment options for peri-implantitis. Wnt4 has been shown in prior studies to attenuate osteoporotic bone loss and prior studies in our lab using a murine model for peri-implantitis, found transgenic Wnt4 mice to exhibit the same pattern of attenuated bone loss in the maxilla.

Objective: To identify whether experimental recombinant Wnt4 (rWnt4) injection shows attenuated bone loss compared to a control PBS injection, in a murine model of ligature induced peri-implantitis.

Methods: Four-week-old male C57BL/6J mice had their left maxillary molars extracted. Following eight weeks of healing, titanium implants were placed in the extraction sites and allowed to integrate for four weeks. After osseointegration, evaluated by presence and lack of mobility in the implants, a 6-0 ligature was tied under the head of the implant in half of the mice to induce peri-implantitis. Half of the mice did not receive ligature placement around the implants. Each group was then divided again to either receive daily control PBS injections or experimental rWnt4 injections intraperitoneally for 30 days. Four weeks after ligature placement and daily injections, the mice were sacrificed. The maxillae were imaged using micro-computerized tomography (μCT) scanning and linear and volumetric bone loss were analyzed. Tissues from the representative samples (based on proximity to mean measurements) were stained with hematoxylin and eosin (H&E), Picrosirius Red for collagen, and Tartrate Resistant Acid Phosphatase (TRAP) for osteoclasts. Immunohistochemistry (IHC) was also performed and analyzed for NF-κB, MMP-9, and Cox-2.

Results: Clinically, four weeks after ligature placement there was increased soft tissue edema present around implants from the ligature induced peri-implantitis group compared to the no-ligature group. Radiographically, linear bone loss analysis showed statistically greater bone loss in the ligature compared to no ligature group for both the PBS and rWnt4 injection groups. The same finding was observed for the volumetric bone loss analysis around implants. There was no statistical difference found in bone loss between the rWnt4 injected groups compared to PBS injections for both the ligature and no ligature groups. TRAP staining for osteoclasts showed statistically greater numbers of osteoclasts in the PBS injection ligature group compared to PBS injection no ligature group. Picrosirius red staining showed more organized collagen fibril distribution in rWnt4 ligature vs. PBS ligature groups. There was also a decreased amount of immunoreactivity for MMP-9, NF-κB and Cox 2 on IHC staining for the rWnt4 ligature group compared to the PBS ligature group.

Conclusion: The findings from this research study validate the ligature-induced peri-implantitis murine model and show that ligature samples have greater tissue and bone loss, making this an ideal animal model to study this disease process. Although the bone loss exhibited by rWnt4 injections was not statistically significant, it provides a preliminary framework for future studies of Wnt4 effects on bone metabolism in the maxilla. The decreased amount of immunoreactivity shown in IHC staining of MMP-9, NF-κB and Cox-2 show a positive effect of decreased inflammatory response in the soft tissues surrounding implants in the injected rWnt4 injection groups, which could be translated to the treatment of peri-implant mucositis. Given that there was a decrease in inflammatory markers, a dose response study may allow us to identify a Wnt4 dose that has a positive therapeutic effect on peri-implantitis, which could be further translated to the treatment of peri-implantitis in humans.

Periodontitis (PD) is characterized by bacterial infection and inflammation of supporting tissues of the teeth. If left untreated, PD can lead to tooth loss. PD affects ~47% of the U.S. population over 30 and, interestingly, twin studies have shown PD to be 50% heritable. While the host immunoinflammatory response and genetic background play a role in PD, few studies have mechanistically interrogated genetic targets to validate candidate genes associated with PD.

Objective: Identify genes that mediate Lipopolysaccharide (LPS)-induced periodontitis.

Methods: P. gingivalis (P.g.)-LPS was injected between maxillary molars in 104 strains of the Hybrid Mouse Diversity Panel (HMDP) 2x/week for 6 weeks. Following sacrifice, maxillae were scanned (microCT) and bone loss was quantitated. FaST-LMM was used to identify genetic loci associated to bone loss. Gene expression (microarray) and protein (histology) were further assessed in A/J and C57BL/6J. CX-C motif chemokine receptor 3 (CXCR3) knockout (KO) and wild-type (WT) mice were analyzed radiographically and histologically after LPS-injections. AMG-487, an in vivo CXCR3 inhibitor, was injected systemically and locally and maxillae were analyzed radiographically and histologically after LPS-injections to investigate the therapeutic potential of CXCR3 inhibition.

Results: 50% heritability and a strain-dependent 6-fold difference in LPS-induced bone loss were observed across the HMDP. Our FaST-LMM and RNA expression data identified Cxcl family members (inflammatory immune cell chemoattractants essential in immune responses) as associated with PD. Additionally, Cxcl10 protein, as well as, an increase in immune cells and pro-inflammatory cytokines were observed in C57BL/6J (high bone loss) and not in A/J (low bone loss) after LPS-injections. Most interestingly, deleting CXCR3 (Cxcl10 receptor), demonstrated ~50% reduction in bone loss and a decrease in osteoclasts after LPS-injections compared to WT mice. Furthermore, mice treated with AMG-487 systemically and locally resulted in ~50% reduction in bone loss and decreased osteoclasts after LPS-injections.

Conclusions: Using a genome-wide association approach, we have identified CXCR3 as a possible target for modulating the host response in PD susceptibility. Our future work will characterize the CXCR3 pathway and validate other candidate genes associated with LPS-induced bone loss with the ultimate goal to identify patients at high risk to PD.

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