Harvard School of Dental Medicine

Runx2 is a transcription factor that has a crucial role in the development of bone; haploinsufficiency of Runx2 leads to the autosomal-dominant disorder, cleidocranial dysplasia (CCD) characterized by various skeletal abnormalities. Zinc finger protein 521 (Zfp521) is a transcription factor that is expressed in several cell types including bone. Recent studies demonstrated that Zfp521 interacts with Runx2 and regulates osteoblast maturation at least in part by repressing the transcriptional activity of Runx2; furthermore, it was demonstrated that the repression of Runx2 by Zfp521 involves the recruitment of HDAC3. The interaction of Runx2 with HDAC3 is strongly enhanced by Zfp521. Zfp521 may regulate osteoblast commitment and differentiation by modulating the Runx2 transcriptional activity by decreasing the levels of Runx2 acetylation. Objective: Runx2 is a key regulator of osteoblast differentiation; Zfp521 may regulate osteoblast commitment and differentiation, at least in part by decreasing the levels of Runx2 acetylation. We investigated 1) Effect of Zfp521 on Runx2 acetylation in HEK293 cells 2) The endogenous Runx2 acetylation levels in MC3T3-E1 cells during osteoblast differentiation. Results: These studies demonstrated that Runx2 acetylation is decreased when Zfp521 is stable expressed in HEK293 cells. Runx2 acetylation levels were detected using immunoprecitation analyses. Sodium butyrate (NaB) prevents protein deacetylation by inhibiting HDACs. The treatment of cells with NaB increased global protein acetylation levels. Importantly, stable expression of Zfp521 did not change global protein acetylation. Therefore, this study suggested that Zfp521 specifically influences Runx2 acetylation. In addition, our findings suggest that Zfp521 impairs Runx2 acetylation by HDACs. Preliminary results show that transient transfection of P300 in HEK293 increased Runx2 acetylation levels. However, stable expression of Zfp521 can still partially decrease Runx2 acetylation levels. This study suggests that Zfp521 function can be linked to P300. Runx2 acetylation levels were then assessed in MC3T3-E1 cells during osteoblast differentiation. While Runx2 protein levels increase by 7 days in culture, and gradually decreases by days 14, Runx2 acetylation was undetectable.
Conclusion: Runx2 is the transcription factor that has essential role in osteoblast commitment and differentiation. Zfp521 represses the transcriptional activity of Runx2 by recruiting HDAC3. These studies suggest that Zfp521 modulates Runx2 activity by decreasing Runx2 acetylation level. These studies have extended our knowledge of the mechanisms by which Zfp521 regulates osteoblast differentiation and bone formation, which could have important implications for on the development of future osteo-anabolic treatments.

Background: Halofuginone (HF) is a natural product that has been shown to have therapeutic benefits in a variety of pathologic conditions, from cancer to autoimmune diseases. These beneficial effects are mainly through inhibition of pro-inflammatory cytokines. Inflammatory cytokines have been recently shown to exert a crucial role in development of osteoarthritis and one of their most important targets is Matrix metallopeptidase-13 (MMP-13). In this preclinical study, we investigated the effect of HF on the progression of Osteoarthritis. Methods: The effect of adding HF ± IL-1β/TNF-α on mRNA expression of MMP-13 on the C28/I2 Chondrocytes was evaluated with qPCR in vitro. To study the effect of HF in vivo, a mice destabilization of the medial meniscus (DMM) osteoarthritis model was employed and untreated control group were compared with early treatment with HF (starting 48 hours post-surgery for 12 weeks) and late treatment (4 weeks post-surgery for 8 weeks). After sacrificing the animal, joint destruction in the knee tissue was assessed with Safranine O/Fast Green staining and MMP13 expression was evaluated by immunohistochemical staining. Results: In chondrocytes, MMP-13 expression was significantly increased with IL-1β or TNF-α (13.77-fold, p-value<0.05 and 5.8-fold, p-value<0.05, respectively). Addition of HF Significantly reduced MMP13 expression close to baseline levels (1.73-fold, p-value<0.05 when co-incubated with IL-1β and 1.6-fold p-value<0.05 when co-incubated with TNF-α). Injection of HF in the mice osteoarthritis model in vivo, significantly reduced Osteoarthritis progression according to OARSI scoring (3.8 vs. 1.16 vs. 1.07, p-value<0.05) and there was no difference between early vs. late administration of HF. While MMP-13 was overexpressed in the control (DMM surgery without HF treatment) groups in IHC staining, expression of MMP13 was suppressed by injection of HF in both groups. Conclusion: Halofuginone inhibits MMP-13 expression and diminishes joint destruction. These preclinical findings provide supporting data for clinical investigation of HF as a therapeutic target for osteoarthritis.

Objectives: Graft versus host disease (GVHD), infections and disease relapse continue to be major complications of allogeneic hematopoietic cell transplantation (HCT). B cell abnormalities have been associated with these complications, and the objective of these studies was to identify and quantify abnormalities of B cell recovery after allogeneic HCT. Methods: Flow cytometry was used to analyze peripheral blood from healthy donors (HD) and samples obtained at 1, 2, 3, 6, 9, 12, 18, 24, 30, and 36 months post HCT. A panel of 8 fluorophore-conjugated monoclonal antibodies specific for B cell surface markers was used to identify levels of total B cells, antigen naïve, antigen experienced, transitional B cells, naïve B cells, IgD memory, pre germinal, post germinal, and plasmablasts. Results: 224 male patients and 175 female patients (total N=399) with a median age of 58 years (range 19-74) were included in this study. 308 patients received reduced intensity and 91 received myeloablative conditioning for allogeneic HCT in the management of various hematological malignancies. The 2-year overall survival was 65% and progression free survival was 55%. The absolute number of total B cells in patients compared to HD remained low for 9 months (118.8 x 106/L vs 204 x 106/L, P < 0.02) and normal levels were reached by month 12. Recovering cells were primarily antigen naïve B cells and the frequency of antigen experienced B cells remained below normal when compared to HD for all time points post HCT (P < 0.0001). Within the antigen naïve compartment, the frequency of transitional B cells remained elevated and naïve B cells remained below normal until normal levels were reached by month 9. Within the antigen experienced compartment, the frequency of pre germinal cells was elevated compared to HD from 6 months (4.8% vs 0.70%; P < 0.0002) post HCT. Plasmablast frequencies were elevated compared to HD from 6 months (7.3% vs 1.1%; P < 0.0009) post HCT. IgD memory cells did not reach normal frequency for most time points. Post germinal cells did not reach normal frequencies from 6 to 12 months post HCT. Conclusion: Disturbances in B cell maturation after HCT persist for prolonged periods despite recovery of normal numbers of circulating B cells. Naïve and transitional B cells that predominate have low affinity BCRs and have been implicated in cGVHD. Reduced numbers of high affinity antigen experienced B cells likely predispose patients to infectious complications post HCT.

Objective: This project aimed to determine whether single tooth implant restorations fabricated with CAD/CAM zirconia abutments/porcelain fused to zirconia crowns reveal different biological and esthetic outcomes compared with prefabricated anatomic titanium abutments/porcelain fused to metal crowns in the esthetic zone. Materials and Methods: Thirty patients who needed a single implant restoration in the esthetic zone were included in the study. Twenty-nine patients completed screening, baseline, crown insertion, one-month, six-month and one-year follow-up visits. At the screening visit, the patients were randomly allocated into two groups: the prefabricated anatomic titanium abutments/ porcelain fused to metal crown (Ti) group and the CAD/CAM zirconia abutments/ porcelain fused to zirconia crowns (Zr) group. Plaque and bleeding scores, microbial profiles, probing depth, width of keratinized tissue, vertical bone changes, pink and white esthetic scores, papilla height, and clinical crown height were evaluated through several study time points. Furthermore, patients’ self-esteem, satisfaction, and esthetic evaluations were assessed using visual analog scores (VAS). A simple scale called subjective and objective esthetic classification (SOE) was created to assess the esthetic success of treatment. Statistical analyses were performed using the Mann Whitney U test, Chi-square test and a generalized linear mixed model. Results: All implants were successfully osseointegrated with a 100% survival rate over one year. Biologically, both groups had comparable outcomes except that the mean prevalence of the bacteria in the Zr group was significantly greater than in the Ti group at the final visits for Streptococcus intermedius (p< 0.0001). Also, the Treponema denticola DNA probe signal was lower in the Zr group than the in Ti group at the final visits (p= 0.0007). In addition, the mean of probing depth of the mesial tooth at the mesio-lingual site (p= 0.02) was less in the Zr group. All the esthetic parameters showed no statistically significant differences between both groups. Patients’ self-esteem, satisfaction, and esthetic evaluations did not differ between groups. Conclusion: After one year of clinical performance, the Zr group showed comparable results to the Ti group. This indicated that good clinical, biological and esthetic outcomes could be achieved by either treatment option. Further observations and follow-up are required to evaluate long-term results.

Objective: The goal of this project was to determine the impact of local inflammation on changes in the subgingival biofilm composition in ligature-induced periodontitis in rats using the specialized pro-resolving mediator (SPM), resolvin E1 (RvE1). Materials and Methods: The impact of RvE1 on the microbiota of ligature-induced periodontitis was assessed in two separate experiments; treatment of established periodontitis and prevention of ligature-induced periodontitis. In the treatment study, eighteen rats were separated into four groups comprising no ligature, ligature alone (no treatment), ligature with topical RvE1 treatment (ligature+RvE1) and, ligature with topical vehicle treatment (ligature + Vehicle). 3-0 silk ligatures were tied around maxillary second molars bilaterally for three weeks to induce disease. After three weeks, the treatment phase began with the application of RvE1 or vehicle (ethanol) every other day for an additional three weeks. Subgingival plaque samples were collected every four days throughout the experiment. The composition of the subgingival microbiota was initially screened by checkerboard DNA-DNA hybridization using probes on 40 subgingival species. Definitive, unbiased characterization of the subgingival microbiota was accomplished with next-generation sequencing using the Illumina MiSeq® platform. Six rats were sacrificed on Days 1, 21 and 42 and maxillae were dissected to collect samples for gingival RNA extraction, bone morphometric measurements, and histomorphometric analysis. Local tissue gene expression (Cxcl-1, Ptgs2, Nos2) was detected using qRT-PCR. Tissue specimens were prepared for histology and stained with H&E and tartrate resistant acid phosphatase (TRAP). In the prevention study, sixteen rats were separated into four groups (no ligature, ligature + RvE1 (0.1µg/µl), ligature + RvE1 (0.5 µg/µl), ligature + Vehicle). 5-0 silk ligatures were placed around maxillary second molars bilaterally to induce disease. At the time of ligature placement, animals received assigned treatment thrice weekly (M, W, F) for four weeks. Subgingival plaque samples were collected every four days (M and F). Four rats were sacrificed at baseline (Day 1) and the vehicle and two treatment groups (four each) were sacrificed at day 28 and samples processed as described above. The two-group comparisons were assessed by Student’s t-test. The multiple-group comparison was assessed by one-way ANOVA and post hoc tests. Results: In the first study (treatment), topical application of RvE1 significantly reversed the bone loss associated with periodontitis compared to the vehicle. RvE1 application significantly reduced the expression of Cxcl1 and osteoclast density compared to the vehicle application. In the prevention study, RvE1 treatment significantly prevented the bone loss during the disease progression. RvE1 application significantly reduced the expression of Ptgs2, Nos2 compared to the vehicle application. Osteoclast density and inflammatory cell infiltration in the RvE1 groups were significantly lower than these in the Vehicle group. The cell counts of bacterial species gradually increased and the subgingival microbiota shifted during the disease progression. In the treatment study, RvE1 treatment significantly reduced cell counts compared to the vehicle application at the end of treatment phase. The shift of subgingival microbiota was limited by the RvE1 treatment. In the prevention study, the taxonomic composition and diversity of subgingival microbiota was controlled by the RvE1 application. The change of subgingival microbiota appeared to be associated with the state of inflammation in the periodontal environment. Conclusion: Resolvin E1 treatment of existing ligature-induced periodontitis significantly regenerates lost alveolar bone and prevents alveolar bone loss. Resolvin E1 treatment limits microbial shifts and reduces total bacterial load by inhibiting inflammation of local environment in experimental periodontitis.

This thesis investigated the efficacy of two techniques, digital spectrophotometry and narrow-band endoscopy, in observing and describing gingival tissue in a patient population. The efficacy of existing gingival restorative materials in mimicking the optical qualities of the participant pool was also investigated using spectrophotometric analysis. The spectrophotometric analysis examined the ability of a digital, non-contact, full-tooth spectrophotometer (Crystaleye; Olympus, Japan) to evaluate objectively gingival color, thickness, and translucency in 100 participants drawn from the patient population of a dental school. Color metrics of gingival soft tissue were obtained from all teeth in the maxillary sextant in the participant population and stratified with respect to tooth location, type, and patient sex and ethnicity. It was concluded that: • In the anterior maxillary gingiva, the luminance (brightness) of the periodontal gingival tissues decreased, whereas the chromacity (color saturation) increased, as the site progressed more posteriorly in the mouth. • Gingival color varied significantly with patient sex, with males exhibiting lower value (darker/grayer) gingiva of a more purple hue. • Gingival color varied significantly with patient ethnicity, with White participants exhibiting brighter, yet less chromatic gingiva than Black, Asian, and Hispanic participants. • Gingival thickness measured via cast analysis was found to be 13% thicker in the male population. • Gingival translucency was observed to be correlated linearly to gingival thickness calculated using two existing metrics and one novel metric. Female participants were found to have significantly more translucent gingiva than male participants. A spectrophotometric coverage error analysis investigating the efficacy with which 5 different brands of gingival acrylics replicated the optical properties of the participant population was performed. It was determined that significant differences existed in the coverage errors of different materials, with some of the shade guide sets exhibiting average best-matches outside of the range of clinical acceptability. It was observed that the coverage errors of the shade guide sets were not determined by the number of tabs in the set, but rather by the fitness of a few isolated tabs within the participant population. In a proof-of-concept analysis, a two-tab shade guide set was conceived that was optimized for the sample participant population that outperformed all other sets in coverage errors. This may have implications in the development of future spectrophotometrically guided shade sets optimized for patient populations. Lastly, using a clinical endoscope (CV-190; Olympus, Tokyo, Japan) equipped with narrow-band imaging capabilities, the microvasculature of the marginal gingiva in the esthetic zone was observed and categorized. The analysis focused on the complexity of the intrapapillary capillary loop (IPCL), a diagnostic indicator of mucosal inflammation. No significant correlations were determined between the classifications of the observed IPCLs and the spectral data obtained, suggesting that within the limitations of this study, endoscopic imaging may be limited in quantitative or diagnostic applications, although it is useful for subjective visualization of the IPCLs.

Background The goal of this study is to understand role of transforming growth factor beta 1 (TGF-β1) in development of osteoarthritis (OA). Results from studies indicate that the genetic inactivation of Smad-3, or the disruption of the interaction of Tgf-β1 with its receptor Tgf-β type II receptor (Tgfbr2), in germline cells results in OA-like knee joints in mice at one month of age. However, other studies suggest that the increased expression of Tgf-β1 in mature knee joints causes OA in animal models. A human genetic study reports that a two-nucleotide deletion, 741-742del AT, and/or a nucleotide change, 859C>T or 782C>T in SMAD-3 are associated with early-onset OA. This observation is consistent with the finding that the lack of Tgf-β1 signaling in the germline cell results in OA in developing joints and that increased Tgf-β1 signaling causes OA in mature joints. The plausible explanation for this “conflicting” role of TGF-β1 in the pathogenesis of OA is that the effective TGF-β1 signaling acts in either a dose-dependent or a developmental stage-dependent manner. The present study addresses the question as to whether inhibition of Tgf-β1 signaling prevents mature knee joints from being degenerated in mouse models of OA.

Methods

1. Using conditional knock out techniques with aggrecan-CreERt2 mice and floxed Tgfbr2 mice, Tgfbr2 was removed from articular cartilage of knee joints in 2-month-old mice. Mice without Tgfbr2 were kept for another 6 months or longer. Knee joints from the mice (n=8) and their corresponding control (n=4) were collected for morphological analysis.
2. Mice without Tgfbr2 at two months old were subjected to DMM to induce articular cartilage degeneration. Knee joints from the mice at 4 and 8 weeks post surgery (n=8 in each group) were collected for morphological analysis.

Results

1. We did not find the initiation and acceleration of articular cartilage degeneration by the genetic inactivation of Tgfbr2 in knee joints of mice at the age of 9 months or older. We also did not find hypertrophic chondrocytes in the articular cartilage of the mice.
2. We found that removal of Tgfbr2 in articular cartilage of knee joints delayed articular cartilage degeneration, at least 6 weeks, compared to that in wild-type littermates.

Conclusion Inhibition of Tgf-β1 signaling attenuated articular cartilage degeneration in mature knee joints of mouse models of OA. Therefore, inhibition activity of TGF-β1, not application of TGF-β1, may be considered in treatment of OA in mature joints.

Esthetic outcomes with implants begin with proper implant placement, but the predictability of the peri-implant esthetic outcome is also affected by patient’s pre-existing or reconstructed local tissue. An unpleasant optical phenomenon where the peri-implant mucosa appears gray has been documented in the literature. However, it’s etiology and solutions have not yet been fully investigated. The overall goal of this project is to perform comprehensive optical examination and to establish the clinical guideline to achieve optimal peri-implant mucosa.

A. Specific Aim 1: Assess the optical properties of the peri-implant mucosa.
A total of 40 patients who has a healthy, single bone level implant in the maxillary anterior zone is recruited at HSDM. For each patient, the test site (peri-implant mucosa) and the control site (adjacent natural gingiva) are identified. Using a dental spectrophotometer, CIELAB color coordinates, translucency parameter (TP), and thickness of test and control site are measured. We found that the color of peri-implant mucosa of bone level implants is significantly different from adjacent gingiva (p=0.0003). We further found that while color of the peri-implant mucosa are significantly different from those of the adjacent gingiva, the thickness and TP do not contribute to this color difference.

B. Specific Aim 2: Evaluate the vascular morphology change of the peri-implant mucosa. Studies have shown that a significant vascular reconstruction takes place around a dental implant. Therefore, using a narrow band imaging endoscope, interpapillary capillary loops (IPCL) around a dental implant are compared to those around a natural tooth. We found that there are more interpapillary capillary loops in peri-implant mucosa compared to gingiva (p=0.02).

C. Specific Aim 3: Determine the threshold for soft tissue color discernment
While many studies have demonstrated the color threshold for shades of teeth and restorations, there is very little information with respect to soft tissue colors. Therefore, in controlled in-vivo and ex-vivo settings, color threshold of soft tissue was investigated, and a correlation between the objective color threshold (ΔE) and subjective color threshold for soft tissue color were also determined. For soft tissue, objective threshold is found to be ΔE=6.50-6.99, and the correlation between subjective and objective evaluations is significant (r=0.67) in ex-vivo setting.

D. Specific Aim 4: Evaluate the efficacy of the newly developed colored abutment on improving the optical property. In order to improve this gray optical phenomenon, a pink colored abutment system has been developed. In a randomized manner, we investigated the color of the peri-implant mucosa with pink and gray abutment. We found that this pink colored abutment can significantly improve the aforementioned optical phenomenon, especially in those with thin tissue (<2mm) (p=0.04) and those with pink neck implant (p=0.04). The clinical significance, however, still needs to be determined.

Abstract: Osteoarthritis (OA) affects 250 million people worldwide. Currently, no targets for disease-modifying osteoarthritis drugs exist. Matrix metalloproteinase-13 (MMP-13) would make it an ideal target; however, its broad biological effects restrict its application as a target enzyme of inhibitory drugs in the treatment of OA. The expression and activation of discoidin domain receptor 2 (DDR2) is increased in human OA tissues and mouse models of OA and was co-localized with elevated expression of MMP-13 in degenerative articular cartilages. In healthy articular cartilage, DDR2 is kept inactivated by the pericellular matrix, which separates the receptor from its ligand, type II collagen. Once enzymes capable of degrading the pericellular molecules expose chondrocytes to type II collagen, DDR2 is activated and induces expression of MMP-13 leading to degradation of type II collagen and proteoglycans resulting in joint destruction and OA. We tested the hypothesis that complete removal of Ddr2 from the knee joint of mouse adult articular cartilage can delay progression of osteoarthritis prior to or after initiation of articular cartilage degeneration. To accomplish this goal, conditional knock out techniques were used with Aggrecan-CreERT2 mice and floxed Ddr2 mice, Ddr2 was removed from articular cartilage of knee joints in mice at 8 weeks of age via intraperitoneal Tamoxifen injection (2mg/10g body weight) for 5 consecutive days (Group A). Mice were subjected to destabilization of the medial meniscus (DMM) or sham surgery at 10 weeks of age. An additional experimental group was subjected to DMM or sham surgery at 10 weeks of age and then DDR2 was removed by intraperitoneal Tamoxifen injection 8 weeks later (Group B). Knee joints from mice in Group A and their corresponding controls were harvested at 8 weeks or 16 weeks post-surgery and mice from Group B and their controls were harvested at 16 weeks post surgery. Histology was performed and the OARSI Modified Mankin Score was used to evaluate articular cartilage degeneration. Statistically significant differences were determined via T-test. We found the average modified score for Group A 8 week control was 1.64 (n=7) whereas with Ddr2 removed was 0.64 (n=7) [P<0.05]. 2) The average modified score for Group A 16 week control was 4.67 (n=7) and with Ddr2 removed was 1.27 (n=9) [P<0.05]. 3) The average modified score for Group B was 1.1 (n=5). In conclusion, conditional removal of Ddr2 in articular cartilage attenuated articular cartilage degeneration in mature knee joints of mouse models of OA.

Objectives The aim of this thesis was to study the biosynthesis of Maresin 1 (MaR1) in human macrophage and to investigate the potential defect of this pathway in patients with localized aggressive periodontitis as well as the role of MaR1 in regulating phagocyte functions and leukocyte-platelet aggregation using LAP as a disease model. Methods Human recombinant enzyme (12-lipoxygenase and soluble epoxide hydrolyse) and synthetic intermediates were used to investigate the maresin pathway by LC/MS/MS-based lipid mediator (LM) metabololipidomics. Human macrophage 12-LOX sequence was crosschecked with platelet-type 12-LOX. Enzyme expression levels in human myeloid mononuclear lineage were determined with flow cytometry. Peripheral blood was collected from LAP patients and age/gender-matched healthy controls (HC). Neutrophils and monocytes were isolated, and macrophages were obtained from monocytes with GM-CSF incubation. LAP and HC macrophage 12-LOX expression levels were monitored by flow cytometry and endogenous maresin metabolite levels were investigated using LM metabololipidomics. Periodontal pathogens including Porphyromonas gingivalis (P. g) and Aggregatibacter actinomycetemcotimans (A. a) were incubated with neutrophils and macrophages to investigate phagocyte functions. Phagocytosis and intracellular anti-microbial reactive oxygen species (ROS) production were determined using fluorescence plate reader. Bacterial killing by macrophages and in whole blood was evaluated by assessing residual colony forming units following incubation with bacteria. Results We demonstrated that human macrophage 12-LOX is identical with platelet-type 12-LOX and is upregulated from monocyte to different mononuclear lineage. 12-LOX converts DHA to 14S-HpDHA, which is a key intermediate in MaR1 biosynthesis. In addition, soluble epoxide hydrolyze (sEH) is responsible for the further production of novel anti-inflammatory and pro-resolving Maresin 2 (MaR2; 13, 14S, di-HDHA). Since 12-LOX is the key-initiating enzyme, investigation begin with macrophages from LAP patients, which have lower 12-LOX expression (~30% lower) and reduced MaR1 (87.8±50 vs. 239.1±32 pg/ 106 cells) levels compared to macrophages from healthy controls (HC). Functionally, LAP macrophages gave impaired phagocytosis (~40% lower) and killing of periodontal pathogens including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. In addition, neutrophils from LAP patients also displayed diminished kinetics (~30% slower) and reduced maximal phagocytosis (~20% lower) of these pathogens. Exogenous MaR1 rescued the impairment with LAP phagocytes enhancing phagocytosis (31-65% increase, 1nM), intracellular anti-microbial reactive oxygen species production (26-71% increase, 1nM) and bacterial killing of these periodontal pathogens in vitro. Additionally, MaR1 retained biological action and enhanced bacterial killing in the whole blood ex vivo from HC and LAP patients (22-37% reduction of bacterial titers, 10nM). MaR1 counter-regulated leukocyte-platelet aggregation against two stimulus (platelet activating factor (PAF) and P. gingivalis) and regulated the expression levels of surface adhesion molecules (CD18 and P-selectin). Patients with LAP had elevated leukocyte-platelet aggregation (40% higher compared to HC) in the whole blood that could be reduced by MaR1 in a dose-dependent manner (40-60% reduction of the excess aggregates, 1-100nM). Conclusions These results indicate that 12-LOX is the key enzyme in maresin biosynthetic pathway. LAP macrophages, had lower 12-LOX expression levels and dysregulated maresin biosynthesis. Reduced phagocyte functions were established with both LAP macrophages and neutrophils; exogenous MaR1 rescued this impairment with LAP patients and also enhanced the functions of HC phagocytes. Additionally, MaR1 enhanced bacterial killing and reduced elevated leukocyte-platelet aggregates in LAP whole blood. Together these results suggest that therapeutics targeting maresin pathway may have clinical application treating LAP and oral diseases associated with infection and uncontrolled inflammation.

Current modeling postulates that the development of cancer is driven by the accumulation of genetic mutations and epigenetic modifications, resulting in a clonal population of cells with deregulated growth characteristics. Three epigenetic mechanisms have been well documented, including DNA methylation, histone modifications and non-coding RNAs. Of these, DNA methylation is the best studied epigenetic alteration in cancer. Characterization of the expression of the epigenetic marker 5-hydroxymethylcytosine (5-hmC) in human and experimental melanoma and its precursors has established it as an important functionally relevant epigenetic biomarker in cancer. We therefore posited that 5-hmC in oral premalignant lesions and oral squamous cell carcinomas represents an opportunity to identify a novel epigenetic molecular pathway that may serve as a biomarker for early diagnosis of oral neoplasms and subsequent improvements in the prognosis of oral cancer. A total of sixty-six histologic samples (N=66) were obtained from the Harvard School of Dental Medicine affiliated bio-archives. Immunohistochemistry for 5-hmC was performed on nine cases diagnosed as fibromas (F, nF=9) with uninvolved margins used to evaluate non-inflamed/non-cancerous mucosa; nine cases diagnosed as frictional keratosis (FK, nFK=9); ten cases diagnosed as lichen planus (LP, nLP=10); fifteen cases diagnosed as moderate-to-severe oral epithelial dysplasia (OED, nOED=15) and twenty-three cases diagnosed as oral squamous cell carcinoma (oSCC, noSCC=23). Human cell lines of oral keratinocytes from benign oral mucosa, oral epithelial dysplasia and four types of oral squamous cell carcinoma were also evaluated to detect the expression of 5-hmC genomic DNA. Finally, immunohistochemistry for 5-hmC was also performed in histologic samples of a murine model where oral squamous cell carcinoma was induced with the carcinogen 4-nitroquinoline-1-oxide (N=10). Five samples consisted of non-inflamed/non-neoplastic normal dorsal tongue mucosa (T, nT=5) and five consisted of oral squamous cell carcinoma (mSCC, nmSCC=5). Progressive loss from benign and reactive/inflammatory oral mucosal lesions to oral epithelial dysplasia and oral squamous cell carcinoma was observed in patient samples. Likewise, distinct patterns with regard to the distribution of staining was noted in benign non-inflamed lesions versus reactive inflammatory lesions such as frictional keratosis and lichen planus, providing further explanation germane for the understanding of the responses of the oral mucosa to tissue injury. The finding in human tissue of loss of 5-hmC immunoreactivity within dysplasia and carcinoma were substantiated in human cell lines at a DNA level, and further validated in an in vivo experimental model of murine oral SCC. In aggregate, our results confirm the potential importance of loss of 5-hmC as a novel epigenetic mark in oral SCC at both diagnostic and biological levels, and establish a murine model of oral SCC induction as a potentially informative approach to understanding the role of epigenetic regulation in oral carcinogenesis.

Objectives: The objectives of this research are to investigate the biological response of the body to different magnitude of force, and to investigate if the response varies among individuals. Therefore this research has 3 Specific Aims: Aim 1 is to investigate the biological response to different magnitudes of orthodontic forces at molecular and cellular levels in animals; Aim 2 is to investigate whether an equal magnitude of force can stimulate different levels of biological response among individuals; Aim 3 is to investigate whether the limit of biological response to different magnitudes of orthodontic force varies among individuals.

Methods and Materials: For Aim 1, different magnitudes (0 to 100 cN) of constant, continuous force were applied on the maxillary first molar of Sprague Dawley rats. The maxillae were collected for RNA and protein analysis, immunohistochemistry, and micro CT at different time points. For Aim 2, human subjects in different age groups (age 11-14 and 21-45) were recruited. Canine retraction was rendered with a constant force of 50 cN, and gingival crevicular fluid (GCF) was collected at different time points up to 28 days after retraction. The activity of inflammatory markers in GCF including IL-1β, CCL2, TNF-α, RANKL, and MMP-9 were measured using protein arrays. The rate of canine retraction in 28 and 56 days was measured on study models. For Aim 3, human subjects in different age groups (same as Aim 2) were recruited. Each subject in both age groups was randomly assigned to receive certain magnitude of constant force (50 to 200 cN) for canine retraction. The activity of different inflammatory markers in GCF one day after retraction was measured using protein arrays. The rate of canine retraction in 28 days was measured on study models.

Results: In the animal study, there was a linear relation between the force and the level of cytokine expression at lower magnitudes of force. Higher magnitudes of force did not increase the expression of cytokines. Activity of CCL2, CCL5, IL-1, TNF, RANKL, and number of osteoclasts reached a saturation point in response to higher magnitudes of force, with unchanged rate of tooth movement. In the clinical studies, activities of IL-1β, CCL2, TNF-α, RANKL, and MMP-9 increased significantly one day after retraction in both age groups. Inflammatory marker activities were significantly higher in adults compared with adolescents at 50-cN force. However, the rate of tooth movement was greater in adolescents than adults during the 56-day study period. At higher force magnitudes, the inflammatory marker activities were higher in adolescents than adults. Both age groups demonstrated saturation in biological response, with higher saturation point in adolescents than adults.

Conclusion:

1. After a certain magnitude of force, there is a saturation in the biological response, where higher magnitude of force does not increase inflammatory markers, osteoclasts, nor amount of tooth movement. Therefore, higher forces to accelerate the rate of tooth movement are not justified and other methods should be considered.
2. The level of biological response varies among individuals to an identical magnitude of force. Therefore, one should compare the level of biological response within the same individual.
3. Saturation of biological response to higher magnitude of orthodontic force exists in both rats and humans, and the saturation point varies among individuals. Adolescents exhibit higher saturation point than adults. Therefore it is not justified to apply higher magnitudes of force in adults.

Objectives: It has been shown that inadequate sleep has negative effect on health including obesity, diabetes, and cardiovascular diseases. Current evidence showed that little sleep impairs immunity, promotes systemic inflammation, and increases the risk of pathological change in many body organs. Aims of this study were to assess the relations between insufficient sleep in developing obesity and gingivitis among a cohort of Kuwaiti children. We also identified the risk behaviors contributed to decreased sleep among those children. This is the first study that has examined the relation between sleep duration and the oral inflammatory condition; gingivitis. In addition, the majority of research related to sleep has been conducted in western countries. Therefore, it seems logical to examine sleep duration as a risk factor for obesity in a population having one of the highest adult prevalence levels of obesity in the world; the Kuwaiti population. Methods: Longitudinal data were collected from 6,316 children 8-14 years old at two time points. Children were approximately equally distributed from 138 elementary schools and representing the 6 governorates of Kuwait. Calibrated examiners in the selected schools conducted oral examinations, sleep evaluation interviews, body weight measurements, nutritional analysis, physical activity assessments, and collected saliva samples. A cross sectional model, and four longitudinal multilevel models were conducted to determine four different outcomes; waist circumference, obesity, gingivitis, and sleep duration at three levels; time, individual, and school. The main independent variables examined were daily sleep hours and salivary glucose levels. Other explanatory variables and confounders assessed were: dental caries, tooth filling, trouble breathing at night, TV and videogame use, and governorate; adjusted for snacking and gender. Results: There was a statistically significant increase in abdominal obesity and gingivitis with shorter sleep duration with significant change over time (P<0.05). Children who watched TV or played videogames just before bedtime, and those who used screen activities more than two hours a day, slept significantly less than other children. The magnitude of gingivitis was significantly different between the six governorates of Kuwait (P<0.05). There was a statistically significant variation in the observations between schools over time. Conclusion: Longitudinal analysis of Kuwaiti children revealed that obesity and gingivitis increased with shorter sleep duration overtime. Screen based activities including TV and videogame use were major factors contributed to decrease night sleep hours. There was a strong clustering effect within the schools in shaping the three health conditions; obesity, gingivitis, and short sleep duration in Kuwaiti children. Public health intervention programs should target children and their families in schools at higher risk to focus on improving night sleep behavior as well as limit their screen time, in addition to maintaining healthy eating habits, practicing proper oral hygiene measures, and being physically active. Supporting agency and grant number: The Dasman Institute in Kuwait funded this study. The Grant number is: (RA/065/2011 and RA/005/2011).

Burning mouth syndrome (BMS) is a chronic pain condition reported to affect up to 7.9% of the general population, with associated detrimental impact on patients’ quality of life. Currently employed treatment regimens follow therapy for other neuropathic pain conditions. Very few placebo-controlled randomized trials (RCTs) have been conducted to evaluate the efficacy of these regimens, with a wide range of placebo responses documented. Low-dose clonazepam is considered first-line therapy for BMS, either in a topical or systemic mode of administration. An innovative formulation of topical clonazepam in the form of a compounded oral solution has been used at the Division of Oral Medicine and Dentistry at Brigham and Women’s Hospital (DOM-BWH) since 2008 for the management of BMS and other oral dysesthesias. An initial concentration of 0.5 mg/mL was used until 2012, when this was changed to a 0.1 mg/mL solution. The objectives of this project were to 1) quantify the magnitude of placebo response in BMS, 2) evaluate the tolerability, safety, and efficacy of the two concentrations of topical clonazepam solution for the management of BMS, and 3) compare their effectiveness in improving burning symptoms. We first conducted a systematic review of published randomized, blinded, placebo-controlled trials of therapies for BMS and evaluated the magnitude of the placebo response compared with the response to the treatment. Twelve RCTs were included. Ten studies (83%) reported at least some improvement in the symptomatology of patients receiving active treatment compared with baseline. In six of these studies (60%), there was also a positive response to placebo. On average, treatment with placebos produced a response that was 72% as large as the response to active drugs. Next, we conducted a retrospective chart review of all patients with oral dysesthesia, including BMS, managed with topical clonazepam solution (0.1 mg/mL or 0.5 mg/mL) in DOM-BWH from 2008 to 2015. The relative safety of the two concentrations of the solution was evaluated in terms of occurrence of adverse drug reactions (ADRs) and occurrence of change to treatment plan secondary to ADRs. A total of 541 charts were reviewed. 162 subjects met the inclusion criteria, 84 patients in the 0.1 mg/mL cohort and 78 in the 0.5 mg/mL cohort, evaluated at a median follow-up of 6 weeks. Thirty-eight (23%) patients developed ADRs. The most frequently reported ADR was sedation (62% of ADRs), followed by altered mental status and dizziness (7% each). In total, dose adjustments were required in nine patients (6%), and treatment was discontinued in 13 patients (8%). ADRs were more frequently reported in the 0.5 mg/mL cohort, but no significant difference was found between the two concentrations, either in terms of occurrence of ADRs or change to treatment secondary to ADRs, or in terms of types of ADRs (p>0.05). Finally, we conducted a retrospective chart review of all patients diagnosed specifically with BMS and managed with topical clonazepam solution (0.1 mg/mL or 0.5 mg/mL) from 2008 to 2015. The efficacy of the two concentrations in improving burning symptoms was compared using patient-reported outcome measures, including the percentage improvement in burning symptoms as reported at first follow-up, and the change from baseline to first follow-up in the worst burning severity over the week prior to evaluation, ranked on an 11-point numeric rating scale (NRS). The study included 57 subjects, with 32 patients in the 0.1 mg/mL cohort and 25 patients in the 0.5 mg/mL cohort, who were evaluated at a median follow-up of 7 weeks. The median overall percentage improvement associated with the 0.1 mg/mL solution was 32.5% (range 0-100%), not significantly higher than the commonly used 30% cut-off. Treatment with the 0.5 mg/mL solution was associated with median overall percentage improvement of 75% (range 0-100%), significantly higher than the more conservative 50% cut-off (p<0.01). The median reduction in NRS score was 6 points in the 0.5 mg/mL concentration, and 0.5 points in the 0.1 mg/mL concentration. Using either outcome measure, response to treatment with the 0.5 mg/mL solution was superior to that associated with the 0.1 mg/mL solution (p<0.01). This thesis is the first to suggest a potentially considerable role for placebo effect in treatments for BMS. Our results suggest that treatment with topical clonazepam solution is generally safe and well-tolerated, with a similar safety profile for both concentrations. A 0.5 mg/mL concentration is highly effective in the management of burning dysesthesia in patients with BMS, significantly more than a 0.1 mg/mL concentration.

We aimed to evaluate longitudinal clinical outcomes and cost-effectiveness of a comprehensive school-based caries prevention program, ForsythKids. In collaboration with the Massachusetts Department of Health, we solicited all principals and nurses from Massachusetts’s elementary schools in which greater than 50% of students received free or reduced meals, to participate. Dentists were calibrated at baseline and hygienists trained to deliver standardized dental care. Dentists clinically examined children following guidelines provided by the National Institute of Dental and Craniofacial Research. We assessed trends in the proportion of sound surfaces (PrSS) and teeth (PrST) remaining sound over subjects' number of visits in the program. We fit multivariable linear regression models with visit number as a predictor, adjusting for age, baseline untreated decay, gender, and previous dental care and accounting for the repeated measures by subject by using a generalized estimating equations (GEE) approach. We stratified models on the presence of untreated decay at baseline. For cost-effectiveness analysis, an individual micro-simulation decision-analytic model was implemented to assess the cost-effectiveness of the ForsythKids program over 6 years in terms of cost per quality-adjusted life-year (QALY) and cost per sound tooth year. Analyses were conducted from a societal perspective. On average, the proportion of sound surfaces remaining sound ranged from 95% to 99%, depending on type of dentition, baseline untreated decay, and type of surface. Further, the per- visit trend was almost flat (0.07% to 1%) in PrST and PrSS. Regarding cost analysis, the annual cost per child in the ForsythKids program was $520. In terms of cost per QALY, over a six-year time horizon, implementing the ForsythKids program led to an ICER of $40,454 per QALY. Moreover, in terms of cost per sound tooth year, an ICER of $1,095 per sound tooth year was estimated. In summary, while the results may be subject to attrition or selection bias, they are consistent with a protective effect of school-based comprehensive caries prevention programs. Further, the ForsythKids program appears to be a good value for money in terms of cost per QALY and cost per sound tooth year.

Objectives – To investigate the anatomy of the mouse palate, the midpalatal suture, and the cellular characteristics in the sutures before and immediately after midpalatal suture expansion, and as well as, to study the effects of compression force on the midpalatal suture.

Materials and Methods – Wild-type C57BL ⁄ 6 male mice, aged between 6 weeks and 12 months, were chosen for all the experiments. The complete palate of the non-operated group and the midpalatal suture-expanded or -compressed group at different ages was used for histological, micro-CT, immunohistochemistry, and sutural cell analyses. Animals in the experimental group for compression were subjected to palatal suture compression force by closing loops for the periods of 1, 3, 5, 7, 14, and 28 days.

Results – This study documents precise morphological and histological characteristics of the mouse palatal sutures. In addition to the opening of the midpalatal suture caused by expansion, both transverse and interpalatine sutures were also seen to be affected. Cellular density was decreased in different types of sutures following the application of expansion force. In respect to compression study, suture width, maxillary width, and bone volume to total volume were significantly decreased compared to control at 14 and 28 days following the application of compressive force. Compression force caused an increased cell apoptosis in midpalatal suture area. Osteoclast activity and expression of MMP-9 were increased, specifically on the nasal side.

Conclusions – The detailed morphology and histology of the mouse palate and the cellular changes that occur following midpalatal suture expansion or compression, as described here, will be helpful as a basis for further investigations of palatal suture tissue responses to mechanical force.

Abstract: Background: alphav (av) integrin is the most common integrin alpha subunit, it is expressed in different cells and involved in many cellular process including adhesion, migration, survival and growth factor signaling. alphav on immune cells has a role in promoting T regulatory (Treg) cells production and differentiation of T helper 17 (Th17) through activating TGF-beta as shown in conditional knockout of alphav in immune cells. Osteopontin (OPN) is a ligand for alphav and involved in modulation of inflammation through its interaction with alphav in immune cells. OPN has been shown to have a protective role in endodontic infection, so we predict alphav to have similar role. The aim of the study is to establish the role of αv integrin in the immune response to endodontic polymicrobial infection, using an established mouse model of endodontic infection Methods: alphavfl/fl mice were mated with mice expressing Cre under the control of the Tie2 promoter, which is expressed in all cells of the hematopoietic lineage as well as in endothelial cells. These mice were subjected to pulp exposure and infection with a mixture of four endodontic pathogens. Bone loss was assessed 0, 7, 14, and 21 days after infection using micro CT. Inflammation and neutrophils enumeration in periapical area evaluated using immunohistochemistry, also osteoclast presence in periapical area examined with TRAP staining. Microarray and qPCR used to investigate cellular and cytokines activity changes. Blood plasma collected from mice during sacrificing was evaluated using ELISA for level of IgG’s and antibody activity against the specific bacteria used for the infection. Results: Lack of alphav expression was confirmed on neutrophils by FACS. Twenty-one days after pulp exposure and infection with oral pathogens, 6 of 16 av-tie2cre KO mice developed swelling and abscesses in the mandible, while no abscesses were observed in control mice. However, the periapical bone resorption was similar in the two groups. Strikingly, ectopic bone formation on the buccal and lingual aspects of the mandibular surface was observed only in the av-tie2cre animals, suggesting a severe disruption of bone turnover caused by the inflammatory process in these animals. Neutrophils showed decrease in number in periapical area and microarray and the observe disseminating infection suggested a B cells defect either in function or migration. Conclusions: Together, these results demonstrate a critical role for the alphav integrin in the suppression of inflammation during the process of endodontic infection.

Introduction: Periradicular periodontitis is an inflammatory disease caused by perpetual microbial infection in the root canal system which, if left untreated, results in the destruction of tooth supporting tissues. Tenacious infection of virulent bacteria can lead to continuous activation of host immune response which results in excess production of proinflammatory mediators, including the bone resorption factor RANKL. To date, attempts to minimize the destruction of tooth supporting tissues have included laser therapy and phototherapy, but these approaches have been confined to treating and monitoring the exogenous etiology, i.e., bacterial infection. Therefore, to improve the diagnosis and treatment of periradicular periodontitis, new technologies are needed to control host cell biological responses and evaluate the level of inflammation in the root canal system and periradicular area. Objectives: Intracellular generated adenosine triphosphate (ATP) and its metabolite, adenosine (ADO), act as major energy currencies in the cell system. Only recently has it been reported that extracellular ATP (eATP) released from activated neutrophils elicits proinflammatory responses, while ADO, which is generated via enzymatic conversion of ATP by cell surface-expressed ecto-5′-nucleotidase (CD73) and Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), functions as anti-inflammatory mediator. Accordingly, this study investigated the effects of CD73 and ADO on osteoimmunological bone resorption in periradicular periodontitis. Methods: The possible anti-bone loss role of ADO was examined in vitro by reacting RANKL-stimulated RAW264.7 osteoclast precursors with ADO receptor agonist 5′-(N-Ethylcarboxamido) adenosine (NECA). More specifically, the effects of NECA on RANKL-induced osteoclastogenesis were evaluated by monitoring TRAP+ multinucleated cell number and pit formation. Since activated T cells are the major cellular source of RANKL in periradicular periodontitis, the effects of NECA on proliferation and sRANKL production by TCR/CD28-stimulated T cells were examined in vitro. The therapeutic efficacy of NECA on the pathogenic bone loss induced in periradicular periodontitis was evaluated in a pulp exposure-induced periradicular periodontitis model using C57BL/6 wild-type (WT) mice. On the other hand, the impact of ADO loss-of-function was compared between CD73-/-Foxp3-GFP+/+ mice and WT mice, both with experimentally induced periradicular lesion. Periradicular tissues were isolated from sacrificed mice, and the mRNAs of inflammation-associated and bone remodeling-related genes, amount of ATP and size of periradicular lesions were monitored up to 3 weeks using qPCR and µCT. Expressions of CD73 and other proteins in the periradicular tissues were examined by immunohistochemistry and immunofluorescence staining. Results: As determined from the in vitro experiments, NECA significantly suppressed not only RANKL-mediated osteoclastogenesis from RAW264.7 cells (P<0.05), but also significantly downregulated proliferation and RANKL production from TCR/CD28-activated T cells (P<0.05), suggesting that the elicited signal could downregulate RANKL-induced osteoclastogenesis. As determined from the in vivo experiments, the amount of ATP and expression of CD73 mRNA were significantly increased in the mouse periradicular lesion in a time-dependent manner. NECA-treated wild-type (WT) mice demonstrated significantly elevated mRNAs for CD39, CD73, alkaline phosphatase, and RUNX2, while showing decreased expressions of RANKL and IL1-β mRNAs, accompanied by diminished size of periradicular lesion, compared to nondrug-treated control WT mice experimentally induced with periradicular lesion. Finally, the amount of ATP and mRNAs for RANKL, IL1-β, IL-6, and TNF-α, as well as the size of periradicular lesion, were significantly larger (P<0.05) in CD73-/-Foxp3-GFP+/+ compared to control WT-mice. Conclusion: By its catalytic action to convert ATP to ADO, increased CD73, by its catalytic action to convert ATP to ADO, in the periradicular lesion may play a role in downregulating inflammatory bone loss, suggesting the CD73/ADO axis as a novel therapeutic target for periradicular periodontitis, leading to the development of a therapeutic regime which, beyond controlling bacterial infection, can control host cell biological responses and, hence, the level of inflammation in the affected root apex.

ABSTRACT

Objectives: Owing to the limited number and scope of studies in dentistry regarding quality and patient safety, the overarching goal of this thesis was to understand the nature and occurrence of quality and safety events in dentistry using a two-pronged approach – biomedical literature and patient-reported experiences.

Methods: I conducted two exploratory studies: the first was a detailed retrospective review of published case reports on dental patient safety events; the second was a cross-sectional study of 450 patients at a large dental teaching practice in South Africa about their quality and safety experiences using a self-adminstered questionnaire.

Results: 180 case reports (270 cases) were identified through the literature search. Most reports came from Europe and North America. The most commonly-reported safety event was wrong treatment or unnecessary treatment following misdiagnosis (23%). 11% of case reports ended in the death of the patient. On the other hand, the patient survey revealed that 45.5% had experienced one or more safety events during dental visits in South Africa (1.6 events per respondent). Intra-oral hard tissue injury, such as adjacent tooth damage during treatment, was the most commonly reported event by patients (30.4%). ‘Never events’ such as wrong tooth extractions or wrong-site procedures, occurred in 7% of patients. The combined quality rating was fair; about 41.4% of participants rated the quality of dental care they received as sub-optimal. Access to care was ranked the lowest among patient-defined quality dimensions.

Conclusions: Quality and safety events occur in dentistry, and are quite common.

Published case reports offer a window into the types and severity of quality and safety events in dentistry. Although the literature is very skewed to reports of significant events and is thus not representative of all AEs that happen, it is a valuable source of information especially in the absence of a centralized reporting system. Patient reports much better let us understand the effects and sequelae of AEs. More work is needed to move the profession forward in our understanding of these events so that we may prevent them.

Background: A periapical lesion is an infection-induced inflammation around the apex of the tooth caused by bacterial invasion and ultimately results in the destruction of bone in the area. To date, the relationship between pathogens and the host immune system in the development of periapical lesions have been extensively investigated. However, a detailed understanding of the host factors that determine successful periapical wound healing is currently lacking. The long-term goal of the present study is to identify the molecular network that regulates wound healing and tissue regeneration in dentoalveolar infections in order to develop rational therapeutic approaches. Although a rodent model of periapical lesion was well established in Forsyth, adequate models for periapical wound healing are currently unavailable. Since the nature of apical periodontitis is infection-induced inflammation, the aim of this study was to systematically examine the effect of azithromycin treatment compared to ampicillin as a wound-healing treatment approach in mouse periapical lesions. Azithromycin is a macrolide antibiotic that specifically accumulates in macrophages exhibiting an anti-inflammatory effect via alteration of macrophage phenotype from pro-inflammatory M1 to anti-inflammatory M2 macrophages.

Material and Methods: Dental pulps of mandibular first molars in C57BL/6J mice (6 weeks of age) were surgically exposed and inoculated with a cocktail of common human endodontic pathogens, which are sensitive to employed antibiotics. Mice received daily doses of azithromycin (8 mg/kg), ampicillin (20 mg/kg) or phosphate-buffered saline (PBS) from day 10 to 20 post infection. No root canal treatments were provided. Mandibles were isolated on days 10 (disease baseline) and 21. Right hemi mandibles were subjected to micro-computed tomography (μCT) to determine the extent of periapical bone loss and subsequent histological and immunohistochemistry analysis. Left hemi mandibles were used for RNA extraction for gene expression analysis through quantitative RT-PCR. Moreover, primary peritoneal macrophages stimulated with LPS and treated with either azithromycin or ampicillin were evaluated for pro-inflammatory cytokines expression (using ELISA) and nitric oxide production (iNOS assay). The effect of azithromycin in the level of NF-kB gene expression was assessed through luciferase reporter assay using Luciferase Stable RAW 264.7 cells.

Results: The μCT results indicated that azithromycin treatment suppressed the extent of periapical bone loss compared to PBS treatment with a significant difference (p<0.05) in the average size of periapical lesions. In addition, the extent of bone loss on Day 21 in azithromycin group was significantly “recovered” compared with the baseline disease group (p<0.05). In histological observations, azithromycin-treated animals showed less neutrophil infiltration and a polarization of M2 macrophages in periapical lesions, while PBS treatment resulted in moderate neutrophil infiltration and an M1/M2-mixed macrophage profile. mRNA expression of pro-inflammatory cytokine IL-6 and chemokine CXCL2, as well as colony stimulating factor 2 (CSF2) had a significant down regulation in the  azithromycin group when compared to baseline disease (p<0.05). Up regulation of IL-4-mRNA was also observed. In vitro experiments confirmed the down regulation of IL-1α and IL-1β (p>0.05) and nitric oxide production (p<0.05) in LPS stimulated peritoneal macrophages treated with azithromycin. Finally, the luciferase activity was decreased when cells were stimulated with azithromycin 50uM. 

Conclusions: These results taken together suggest that the suppression of periapical bone loss by azithromycin appears to be dependent on its immunomodulatory properties. This report is the first finding on pro-resolving M2 macrophage polarization during periapical wound healing. This research can shed light to the development of a mouse model of periapical wound healing. In addition, this dual role of azithromycin, microbicidal and anti-inflammatory effects, could pose as a great adjuvant on the root canal therapy being highly translational.