Neutrophils modulate natural killer-mediated osteoclastogenesis during Aggregatibacteractinomycetemcomitans (JP2 clone) infection.

The study investigates the interplay of neutrophils and natural-killer cells (NK) in mediating osseoresorption during infection of molar-incisor-pattern-periodontitis (MIPP). Human neutrophils from periodontally healthy and MIPP patients were inoculated with the periopathogen Aggregatibacter-actinomycetemcomitans (JP2) and their supernatants were exposed to NK to study their function and osteoclastogenesis promotion. A mouse MIPP model was used to compare disease progression following NK versus neutrophils depletion. The exposure of primary NK to supernatants of neutrophils inoculated with JP2 led to NK cell arrest and activation with enhanced osteoprotegerin expression. Incubation of monocytes with NK led to osteoclastogenesis, whereas NK that were pre-exposed to healthy neutrophil supernatant showed reduced osteoclastogenesis. In mice, NK depletion led to the similar bone phenotype as the neutrophil's depletion highlighting their role on osseoprotection. The present study portrays a key crosstalk between neutrophils and NK cells during JP2 infection as a central mechanism that regulates bone loss.

Resolvin D1 improves allograft osteointegration and directly enhances osteoblasts differentiation.

Introduction: Allografts are the most common bone grafts for repairing osseous defects. However, their use is associated with an increased risk for infections, donor disease transmission and osteointegration deficiency. Resolvin D1 (RvD1) is an endogenous lipid with a scientifically proven pivotal role in inflammation resolution and osteoclastogenesis inhibition. Yet, its biological relevance as a potential bone regenerative drug has been scarcely studied. Here, we aim to investigate the RvD1 effect on allograft osteointegration in the alveolar bone regeneration (ABR) murine model. Methods: ABR model consisted of osseous defects that were generated by the extraction of the maxillary first molar in C57BL/6 mice. The sockets were filled with allograft and analyzed via RNA sequencing. Then they were locally injected with either RvD1 or saline via single or repeated administrations. The mice were sacrificed 2W after the procedure, and regenerated sites were analyzed using µCT and histology. First, MC3T3-E1 preosteoblasts were plated with IL-17 pro-inflammatory medium, and RANKL/OPG ratio was measured. Secondly, the MC3T3-E1 were cultured w/o RvD1, for 3W. Osteoblasts' markers were evaluated in different days, using qRT-PCR and Alizarin Red staining for calcified matrix. Results: In vivo, neither allograft alone nor single RvD1 administration promote bone regeneration in comparison to the control of spontaneous healing and even triggered an elevation in NR1D1 and IL1RL1 expression, markers associated with inflammation and inhibition of bone cell differentiation. However, repeated RvD1 treatment increased bone content by 135.92% ± 45.98% compared to its specific control, repeated sham, and by 39.12% ± 26.3% when compared to the spontaneous healing control group (n=7/group). Histologically, repeated RvD1 reduced the number of TRAP-positive cells, and enhanced allograft osteointegration with new bone formation. In vitro, RvD1 rescued OPG expression and decreased RANKL/OPG ratio in IL-17 pro-inflammatory conditions. Furthermore, RvD1 increased the expression of RUNX2, OSX, BSP and OC/BGLAP2 and the mineralized extracellular matrix during MC3T3-E1 osteoblasts differentiation. Conclusions: Repeated administrations of RvD1 promote bone regeneration via a dual mechanism: directly, via enhancement of osteoblasts' differentiation and indirectly, through reduction of osteoclastogenesis and RANKL/OPG ratio. This suggests that RvD1 may be a potential therapeutic bioagent for osseous regeneration following allograft implantation.

Resolvin D1 shows osseous-protection via RANK reduction on monocytes during orthodontic tooth movement.

The study aimed to investigate the role of RvD1 in acute and prolonged sterile inflammation and bone remodeling. A mouse model of sterile inflammation that involves bone resorption was used to examine endogenous RvD1 kinetics during inflammation. Application of exogenous RvD1 significantly inhibited bone remodeling via osteoclast reduction, alongside an anti-inflammatory secretome shift, increased macrophages recruitment and reduction of T-cytotoxic cells. In vitro and in vivo, RvD1 led to significant reduction in RANK expression which reduce osteoclastogenesis in a dose-dependent manner. Taken together, the data shows a dual role for RvD1, as a potent immunoresolvent agent alongside an osteoresolvent role, showing a potential therapeutic agent in bone resorption associated inflammatory conditions.

Specific inflammatory osteoclast precursors induced during chronic inflammation give rise to highly active osteoclasts associated with inflammatory bone loss.

Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6ChiCD11bhi inflammatory OCPs (iOCPs) induced during chronic inflammation, and homeostatic Ly6ChiCD11blo OCPs (hOCPs) which remained unchanged. Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions, they expanded during chronic inflammation and generated OCs with enhanced activity. In contrast, hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment. Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs, highlighting that different osteoclast populations are formed by distinct precursors. We further identified the TNF-α and S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation. Furthermore, we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α-/- mice, in correlation with attenuated IBL. Our findings suggest a central role for iOCPs in IBL induction. iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.

Analytical methodology to measure periodontal bone morphometry following orthodontic tooth movement in mice.

INTRODUCTION: Basic research in orthodontics is commonly conducted in rodents. However, experimental studies on orthodontic tooth movement (OTM) lack a standard method to examine OTM and periodontal changes. This study describes a unifying protocol for the analysis of OTM and associated bone microarchitectural changes in mice using microcomputed tomography (µCT). METHODS: Mice (10 animals/group) were divided into control and OTM groups. OTM was generated by anchoring a nickel-titanium closed-coil spring to the upper incisors to pull the upper left first molar. A third group of TNFα -/- mice was added since these are known to have slower OTM. Using µCT, we implemented and tested a number of methods to measure OTM distance and examine 3D bone morphometric parameters associated with OTM in mice. RESULTS: In total, we tested five methods to measure the OTM distance in mice. The results indicated that measuring the intermolar diastema, and assessing tooth movement relative to the anterior root of the zygomatic arch, displayed the lowest standard deviation and enabled optimal detection of intergroup differences. We also developed two protocols for µCT analysis of the periradicular bone that yielded no false-positive results. Our results revealed that including the width of the periodontal ligament rather than excluding it from the region of interest in mice detected more statistically significant differences in the morphometric parameters between the OTM and control sides and between WT and TNFα -/- mice despite more subtle differences. CONCLUSIONS: We, therefore, propose new guidelines for a standardized µCT-based method to analyse OTM and the extent of the periradicular bone structural changes in mice.

The Balance between Orthodontic Force and Radiation in the Jawbone: Microstructural, Histological, and Molecular Study in a Rat Model.

Irradiation of facial bones is associated with a lifelong risk of osteonecrosis. In a rat model, maxillae were exposed to a single 5 Gy dose of external beam radiation and orthodontic force was applied for 2 weeks on the first maxillary molar; control rats were treated identically without radiation. Tooth movement in irradiated jaws was 30% less than in controls, representing radiation-related damage. Micro-CT, histological, and molecular outcomes of orthodontic tooth movement were studied. Microstructurally, bone parameters (trabecular thickness, bone volume fraction, bone mineral density) were significantly affected by orthodontic force but not by radiation. Histological parameters were influenced only by orthodontic force, especially by an increase in osteoclasts. A molecular study revealed a differential distribution of cells expressing pre-osteoclast markers (RANK+-majority, CD11b+, CD14+-minority), with changes being influenced by orthodontic force (increased CD11b+ and CD14+ cells) and also by radiation (decreased RANK+ cells). The activation status of osteoclasts (TRAP staining) showed an orthodontic-force-related increase, which probably could not fully compensate for the radiation-associated impairment. The overall balance showed that orthodontic force had elicited a substantial microstructural, histological, and functional normalization process in irradiated maxillae but a radiation-induced impact was still conspicuous. Additional studies are needed to validate these findings.

A Mouse Model for Studying the Development of Apical Periodontitis with Age.

Older age is associated with reduced immune function. Our aim was to study how age affects the development of apical periodontitis (AP). AP was induced in two age groups of mice (young vs. adult). Histological samples were stained by Hematoxylin Eosin, Brown and Brenn, and Tartrate-Resistant Acid Phosphatase. In addition, the samples were scanned by Micro-Computerized-Tomography (micro-CT) to evaluate apical constriction and periapical lesion size. Cell density in the periapical region was computationally assessed. Moreover, lesion immune cell populations were characterized by flow cytometry and immunofluorescence. The young group presented more canals with necrotic radicular pulp compared to the adults. There was no difference in bacteria location in the canals between the groups. Apical constriction size was larger in the young mice compared to the adults. The periapical cell density was higher in the young group, while the dominant immune cells in the lesions were neutrophils, which also exhibited the highest young/adult ratio. Immunofluorescence demonstrated neutrophils in the lesion. More osteoclasts were present in the lesions of the young mice, in correlation to the higher volume of bone resorption in this group. Overall, we conclude that the immune reaction to AP stimuli was attenuated in the adult mice compared to the young.

Salivary Cytokines in Children with Nephrotic Syndrome versus Healthy Children: A Comparative Study.

BACKGROUND: The aims of this study were to compare salivary cytokines and total protein between children with nephrotic syndrome (NS) and healthy children, and to examine whether saliva parameters can differentiate between steroid sensitivity and resistance and between disease remission and relapse. METHODS: Twenty-seven children with nephrotic syndrome were classified according to steroid sensitivity and resistance, and disease remission and relapse. Twenty healthy children served as controls. Whole saliva samples were collected from all the participants. Urine and blood tests done on the same day as the saliva collection were recorded. Salivary total protein was quantified using bicinchoninic acid and IFNγ, IL-4, IL-8, IL-6, and IL1ß levels using ELISA. RESULTS: The mean ages of the nephrotic syndrome and control groups were 11.3 ± 2.4 and 9 ± 4.2, respectively. Compared to the control group, for the nephrotic syndrome group, total salivary protein was significantly lower, as were the levels of all the cytokines examined except IFNγ. Statistically significant differences were not found in any of the salivary markers examined between the children with nephrotic syndrome who were treatment sensitive (n = 19) and resistant (n = 8). Protein and IL-8 salivary levels were lower in the active (n = 7) than in the remission (n = 20) group. CONCLUSIONS: Salivary parameters distinguished children with nephrotic syndrome in relapse from healthy children. This may be due to decreased salivary protein excretion, which reflects decreased plasma levels, consequent to proteinuria. Accordingly, salivary markers may be developed as a diagnostic or screening tool for NS activity.

Immunorthodontics: in vivo gene expression of orthodontic tooth movement.

Orthodontic tooth movement (OTM) is a "sterile" inflammatory process. The present study aimed to reveal the underlying biological mechanisms, by studying the force associated-gene expression changes, in a time-dependent manner. Ni-Ti springs were set to move the upper 1st-molar in C57BL/6 mice. OTM was measured by µCT. Total-RNA was extracted from tissue blocks at 1,3,7 and 14-days post force application, and from two control groups: naïve and inactivated spring. Gene-expression profiles were generated by next-generation-RNA-sequencing. Gene Set Enrichment Analysis, K-means algorithm and Ingenuity pathway analysis were used for data interpretation. Genes of interest were validated with qRT-PCR. A total of 3075 differentially expressed genes (DEGs) were identified, with the greatest number at day 3. Two distinct clusters patterns were recognized: those in which DEGs peaked in the first days and declined thereafter (tissue degradation, phagocytosis, leukocyte extravasation, innate and adaptive immune system responses), and those in which DEGs were initially down-regulated and increased at day 14 (cell proliferation and migration, cytoskeletal rearrangement, tissue homeostasis, angiogenesis). The uncovering of novel innate and adaptive immune processes in OTM led us to propose a new term "Immunorthodontics". This genomic data can serve as a platform for OTM modulation future approaches.

The impact of alloplast and allograft on bone homeostasis: Orthodontic tooth movement into regenerated bone.

BACKGROUND: The aim of the study is to examine bone healing following augmentation with allograft or ß-tricalcium phosphate (ß-TCP) and evaluate orthodontic tooth movement (OTM) into the augmented sites. METHODS: The study included two parts. Part I included the alveolar bone regeneration model. Osseous defects were created by extraction of the maxillary first molars in C57BL/6 mice, and the sockets were filled with allograft, ß-TCP, or left unfilled (n = 6/group). Mouse allograft was prepared by a novel method using long bones. Maxillae were collected at 2, 4, and 6 weeks for microcomputed tomography (µCT) and histological analysis. In Part II, OTM was performed after full bone healing, through grafted and unfilled sockets (n = 10/group), and the second molar shift was assessed using µCT. RESULTS: Bone volume and trabeculation were reduced in ß-TCP compared with allograft and non-grafted groups at 2 and 4 weeks post-grafting, but similar at 6 weeks. Graft particles could be detected at 2 weeks post-grafting for ß-TCP, and at 2 and 4 weeks for allograft. Increased osteoclasts' presence was observed in the ß-TCP group at 2 and 4 weeks compared with allograft and control. OTM was similar in the two graft groups, but impaired versus the non-grafted controls. CONCLUSION: ß-TCP and allograft induce full normal healing but alter OTM into the regenerated sites.

Bone regeneration with bovine bone impairs orthodontic tooth movement despite proper osseous wound healing in a novel mouse model.

BACKGROUND: The aim of this study was to investigate the biological mechanisms underlying alveolar bone regeneration (ABR) and orthodontic tooth movement into bovine bone (BB) regenerated sites. METHODS: Two mouse models were established in C57BL/6 mice. The ABR model was based on osseous defects filled with BB. The orthodontic tooth movement-ABR model was used to move a molar into the regenerated site. Osseous morphometric analysis and tooth movement distance were evaluated with micro-CT. Histologic characteristics and osteoclast (OCS) accumulation were evaluated by hematoxylin and eosin and tartrate-resistant acid phosphatase staining (TRAP). Expression and location of the receptor activator of nuclear factor-kappa B (RANKL) and of osteoprotegerin (OPG) were evaluated by immunofluorescent staining. RESULTS: Bone healing peaked at 4 weeks. The distance of the orthodontic tooth movement into the bovine bone was significantly reduced versus that of the nonbovine bone controls. BB particles accumulated along the root's pressure side during orthodontic treatment. Despite the osteoclasts' presence adjacent to the BB particles, no BB resorption was observed. Increased RANKL expression was seen at the orthodontic tooth movement pressure zone, without any change in OPG expression. CONCLUSION: The two novel mouse models show that the lack of resorption of BB xenografts renders them inadequate for proper orthodontic tooth movement at a later stage.