Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

SOCS2 regulates alveolar bone loss in Aggregatibacter actinomycetemcomitans-induced periodontal disease.

INTRODUCTION: The role of suppressor of cytokine signaling 2 (SOCS2) in Aggregatibacter actinomycetemcomitans (Aa)-induced alveolar bone loss is unknown; thus, it was investigated in this study. METHODS: Alveolar bone loss was induced by infecting C57BL/6 wild-type (WT) and Socs2-knockout (Socs2-/-) mice with Aa. Bone parameters, bone loss, bone cell counts, the expression of bone remodeling markers, and cytokine profile were evaluated by microtomography, histology, qPCR, and/or ELISA. Bone marrow cells (BMC) from WT and Socs2-/- mice were differentiated in osteoblasts or osteoclasts for analysis of the expression of specific markers. RESULTS: Socs2-/- mice intrinsically exhibited irregular phenotypes in the maxillary bone and an increased number of osteoclasts. Upon Aa infection, SOCS2 deficiency resulted in the increased alveolar bone loss, despite decreased proinflammatory cytokine production, in comparison to the WT mice. In vitro, SOCS2 deficiency resulted in the increased osteoclasts formation, decreased expression of bone remodeling markers, and proinflammatory cytokines after Aa-LPS stimulus. CONCLUSIONS: Collectively, data suggest that SOCS2 is a regulator of Aa-induced alveolar bone loss by controlling the differentiation and activity of bone cells, and proinflammatory cytokines availability in the periodontal microenvironment and an important target for new therapeutic strategies. Thus, it can be helpful in preventing alveolar bone loss in periodontal inflammatory conditions.

A Biosafety Level 2 Mouse Model for Studying Betacoronavirus-Induced Acute Lung Damage and Systemic Manifestations.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Methotrexate promotes recovery of arthritis-induced alveolar bone loss and modifies the composition of the oral-gut microbiota.

OBJECTIVES: The impact of rheumatoid arthritis (RA) on the shaping of the oral and gut microbiome raises the question of whether and how RA treatment modifies microbial communities. We examined changes in the oral and gut microbiota in a mouse model of antigen-induced arthritis (AIA) treated or not with methotrexate (MTX). METHODS: Maxillae and stools were evaluated by the MiSeq platform of the V4 region of the 16S rRNA gene. Alveolar bone parameters were analysed by micro-computed tomography. Moreover, arthritis-induced changes in hyperalgesia and oedema were assessed, along with the impact on periodontal bone health. RESULTS: Microbial communities in MTX-treated AIA mice revealed distinct clusters compared to the control and AIA groups. Overall, MTX impacted the richness and variability of microorganisms in the oral-gut axis microbiome at the phylum level. Regarding the oral microbiome, while in the control group the most dominant phylum was Firmicutes, in the AIA group there was a shift towards the predominance of Campilobacteriota and Bacteroidetes associated with the disease. MTX treatment led to greater dominance of the health-associated phylum Proteobacteria. In the gut microbiome, AIA induction resulted in increased abundance of the Verrucomicrobiota phylum, and MTX treatment restored its levels compared to control. Importantly, the MTX-treated AIA animals had significantly less periodontal bone loss, as well as decreased hyperalgesia and joint oedema compared to the AIA animals. CONCLUSION: Data suggest the benefit of MTX treatment in protecting alveolar bone, in addition to providing new insights on the drug-microbiome interaction in the course of RA.

Osteogenic potential of sol-gel bioactive glasses containing manganese.

Bioactive glasses (BGs) are widely used for bone regeneration, and allow the incorporation of different ions with therapeutic properties into the glass network. Amongst the different ions with therapeutic benefits, manganese (Mn) has been shown to influence bone metabolism and activate human osteoblasts integrins, improving cell adhesion, proliferation and spreading. Mn has also been incorporated into bioceramics as a therapeutic ion for improved osteogenesis. Here, up to 4.4 mol% MnO was substituted for CaO in the 58S composition (60 mol% SiO2, 36 mol% CaO, 4 mol% P2O5) and its effects on the glass properties and capability to influence the osteogenic differentiation were evaluated. Mn-containing BGs with amorphous structure, high specific surface area and nanoporosity were obtained. The presence of Mn2+ species was confirmed by X-ray photoelectron spectroscopy (XPS). Mn-containing BGs presented no cytotoxic effect on human mesenchymal stem cells (hMSCs) and enabled sustained ion release in culture medium. hMSCs osteogenic differentiation stimulation and influence on the mineralisation process was also confirmed through the alkaline phosphatase (ALP) activity, and expression of osteogenic differentiation markers, such as collagen type I, osteopontin and osteocalcin, which presented higher expression in the presence of Mn-containing samples compared to control. Results show that the release of manganese ions from bioactive glass provoked human mesenchymal stem cell (hMSC) differentiation down a bone pathway, whereas hMSCs exposed to the Mn-free glass did not differentiate. Mn incorporation offers great promise for obtaining glasses with superior properties for bone tissue regeneration.

Bone strength is reduced in a neonatal androgenized rat model.

Background: Whether polycystic ovary syndrome (PCOS) affects bone health during a woman's lifespan remains controversial. An androgenized rodent model replicated many metabolic and reproductive features of women with PCOS, and we aimed to use it to investigate the impact of androgens on microarchitecture (by micro-CT), bone mechanical strength, bone formation and resorption markers in rats with intact ovaries (SHAM) who underwent oophorectomy. Methods: Wistar rats (Rattus norvegicus albinus) were employed for the experiments in this study. The protocol of androgenization consisted of the application of 1.25 mg s.c. testosterone propionate beteween days 2-5 of life, while the controls received the same amount of corn oil s.c. as previously established. Androgenized SHAM rats exhibited chronic anovulation identified by vaginal cytology and a reduction in the proportion of corpus luteum in the ovary in comparison to control SHAM rats. The realization of the ovariectomy or SHAM procedure occurred on Day 100 of life. All groups (n = 8) were followed-up for 180 days to address the study endpoints. Results: Micro-CT from androgenized female rats (SHAM) showed a divergence between the trabecular and cortical bone profiles. Compared to SHAM controls, these rats had an increase in trabecular bone mass with a diminution in bone resorption C-terminal telopeptide of type 1 collagen (CTX) (p < 0.05), a concomitant decrease in cortical area and thickness in the femur, and a reduction in the strength of the femur on the mechanical test (p < 0.01). Conclusions: Our results suggest that a reduction in the cortical thickness and cortical area observed in PCOS model rats was associated with a reduced strength of the femur, despite increased trabecular formation. Ovariectomy in the androgenized OVX group limited the progression rate of cortical bone loss, resulting in bone resistance and cortical thickness comparable to those observed in the control OVX group.

Dysbiotic oral microbiota contributes to alveolar bone loss associated with obesity in mice.

INTRODUCTION: Periodontal diseases (PD) are inflammatory conditions that affect the teeth supporting tissues. Increased body fat tissues may contribute to activation of the systemic inflammatory response, leading to comorbidities. Some studies have shown that individuals with obesity present higher incidence of PD than eutrophics. OBJECTIVE: To investigate the impact of obesity on periodontal tissues and oral microbiota in mice. METHODOLOGY: Two obesity mice models were performed, one using 12 weeks of the dietary protocol with a high-fat (HF) diet in C57BL/6 mice and the other using leptin receptor-deficient mice (db/db-/-), which became spontaneously obese. After euthanasia, a DNA-DNA hybridization technique was employed to evaluate the microbiota composition and topical application of chlorhexidine (CHX), an antiseptic, was used to investigate the impact of the oral microbiota on the alveolar bone regarding obesity. RESULTS: Increased adipose tissue may induce alveolar bone loss, neutrophil recruitment, and changes in the oral biofilm, similar to that observed in an experimental model of PD. Topical application of CHX impaired bone changes. CONCLUSION: Obesity may induce changes in the oral microbiota and neutrophil recruitment, which are associated with alveolar bone loss.

Cobalt-containing bioactive glass mimics vascular endothelial growth factor A and hypoxia inducible factor 1 function.

Bioactive glasses (BGs) have shown great potential for tissue regeneration and their composition flexibility allows the incorporation of different ions with physiological activities and therapeutic properties in the glass network. Among the many ions that could be incorporated, cobalt (Co) is a significant one, as it mimics hypoxia, triggering the formation of new blood vessels by the vascular endothelial growth factor A (VEGFA), due to the stabilizing effect on the hypoxia inducible factor 1 subunit alpha (HIF1A), an activator of angiogenesis-related genes, and is therefore of great interest for tissue engineering applications. However, despite its promising properties, the effects of glasses incorporated with Co on angiogenesis, through human umbilical cord vein endothelial cells (HUVECs) studies, need to be further investigated. Therefore, this work aimed to evaluate the biocompatibility and angiogenic potential of a new sol-gel BG, derived from the SiO2 -CaO-P2 O5 -CoO system. The structural evaluation showed the predominance of an amorphous glass structure, and the homogeneous presence of cobalt in the samples was confirmed. in vitro experiments showed that Co-containing glasses did not affect the viability of HUVECs, stimulated the formation of tubes and the gene expression of HIF1A and VEGFA. in vivo experiments showed that Co-containing glasses stimulated VEGFA and HIF1A expression in blood vessels and cell nuclei, respectively, in the deep dermis layer of the dorsal region of rats, featuring considerable local stimulation of the angiogenesis process due to Co-release. Co-containing glasses showed therapeutic effect, and Co incorporation is a promising strategy for obtaining materials with superior angiogenesis properties for tissue engineering applications.

In vitro effects of the co-release of icariin and strontium from bioactive glass submicron spheres on the reduced osteogenic potential of rat osteoporotic bone marrow mesenchymal stem cells.

Osteoporosis is a metabolic disease that affects bone tissue and is highly associated with bone fractures. Typical osteoporosis fracture treatments, such as bisphosphonates and hormone replacement, present important challenges because of their low bioavailability on the site of action. Options to overcome this issue are systems for the local release of therapeutic agents such as bioactive glasses containing therapeutic molecules and ions. These agents are released during the dissolution process, combining the drugs and ion therapeutic effects for osteoporosis treatment. Among the therapeutic agents that can be applied for bone repair are strontium (Sr) ion and phytopharmaceutical icariin, which have shown potential to promote healthy bone marrow stem cells osteogenic differentiation, increase bone formation and prevent bone loss. Submicron Sr-containing bioactive glass mesoporous spheres with sustained ion release capacity were obtained. Icariin was successfully incorporated into the particles, and the glass composition influenced the icariin incorporation efficiency and release rates. In this work, for the first time, Sr and icariin were incorporated into bioactive glass submicron mesoporous spheres and the in vitro effects of the therapeutic agents release were evaluated on the reduced osteogenic potential of rat osteoporotic bone marrow mesenchymal stem cells, and results showed an improvement on the reduced differentiation potential.

Aerobic and resistance training improve alveolar bone quality and interferes with bone-remodeling during orthodontic tooth movement in mice.

The direct effects of physical activity on long bones are already recognized. However, little information is available regarding distant osseous sites, such as maxillary bone. We evaluated the influence of physical training on alveolar bone quality, with and without mechanically-induced load during orthodontic tooth movement in mice. Forty-two C57BL/6 mice were divided into sedentary, resistance and aerobic training groups. Training period lasted for eight weeks and mechanical loads (orthodontic tooth movement - OTM) were applied during the last 14 days of training. Both types of training enhanced the quality of maxillary bone, increasing bone mineral density (BMD), trabecular bone volume (BV) and bone volume/total volume ratio (BV/TV). OTM significantly reduced in trained groups. Consistently, the number of osteoblasts increased whereas the number of osteoclasts decreased on the OTM side in trained groups in comparison to the sedentary group. IGF-1, RUNX2 and OPG genes expression were also increased. The RANKL/OPG ratio and IL-6 expression were reduced in the maxillary bone. Similar results were verified in the femoral bone. In line with these findings, physical training resulted in a decrease of osteoclast differentiation from femoral bone marrow; as well as the force required to fracture the tibia of trained animals increased. Physical training also caused EDL muscle hypertrophy and increased expression of IGF-1 and IGF-1/Myostatin ratio in the gastrocnemius muscle, whereas FNDC5 gene expression was similar among groups in femur, but decreased in alveolar bone submitted to OTM. In conclusion, physical training increased bone quality, not only on long bones, but also in a distant site such as the maxilla. Differences were more evident in the course of maxillary mechanical loading. Mechanisms involve systemic and local effects on bone cells and target molecules as RANKL, OPG, IL-6 and IGF-1.

Maxillary suture expansion: A mouse model to explore the molecular effects of mechanically-induced bone remodeling.

The aim of this study was to analyze the effect of rapid maxillary expansion (RME) on hard tissues. Opening loops bonded to the first and second maxillary molars on both sides were used to apply distracting forces of 0.28 N, 0.42 N and 0.56 N at the midpalatal suture for 7 and 14 days. Microcomputed tomography (MicroCT), histomorphometry and quantitative polymerase chain reaction (qPCR) analysis were performed to evaluate RME effectiveness, midpalatal suture remodeling, cell counting of osteoblasts, osteoclasts and chondrocytes and the expression of bone remodeling markers, respectively. All forces at the two different time points resulted in similar RME and enhanced of bone remodeling. Accordingly, increased number of osteoblasts and reduced chondrocytes counting and no difference in osteoclasts were seen after all RME protocols. RME yielded increased expression of bone remodeling markers as osteocalcin (Ocn), dentin matrix acidic phosphoprotein-1 (Dmp1), runt-related transcription factor 2 (Runx2), collagen type I Alpha 1 (Col1a1), alkaline phosphatase (ALP), receptor activator of nuclear factor kappa B (RANK), receptor activator of nuclear factor kappa B ligand (Rankl), osteoprotegerin (Opg), cathepsin K (Ctsk), matrix metalloproteinases 9 and 13 (Mmp9 and 13), transforming growth fator beta 1, 2 and 3 (Tgfb 1, Tgfb 2 and Tgfb3), bone morphogenetic protein 2 (Bmp-2), sclerostin (Sost), beta-catenin-like protein 1 (Ctnnbl) and Wnt signaling pathways 3, 3a and 5a (Wnt 3, Wnt 3a and Wnt 5a). These findings characterize the cellular changes and potential molecular pathways involved in RME, proving the reliability of this protocol as a model for mechanical-induced bone remodeling.

Estrogen protects dental roots from orthodontic-induced inflammatory resorption.

OBJECTIVE: Root resorption is a side effect of orthodontic tooth movement (OTM). Despite the recognized role of estrogen on bone, there is little information about their effects on orthodontic-induced inflammatory root resorption (OIIRR). We aimed to investigate if estrogen deficiency affects OIIRR in two mice strains. METHODS: Female Balb/C (Balb) and C57BL6/J (C57) mice were ovariectomized (OVX) and replaced with estradiol (E2). Tooth samples subjected or not to OTM were collected and analyzed by microCT, histomorphometry and qPCR. RESULTS: OVX resulted in decreased root volume (RV/TV) and root mineral density (RMD) in Balb mice without OTM. In contrast, OVX did not modify physiological root structure of C57 mice. OTM and OIIRR were increased after OVX in both mice strains after 30 days. E2 replacement reversed this phenotype in Balb, but not in C57 mice. Due to the significant increase of OIIRR in OVX Balb mice, the expression of key molecules was investigated in periodontium. Accordingly, these mice showed increased expression of receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor alpha, matrix metalloproteinases-2 and -13 and decreased osteoprotegerin (OPG) and interleukin-10 expression after OTM. E2 replacement reversed the changes of these markers. CONCLUSION: The lack of estrogen in Balb mice without OTM triggered loss of root structure which was positively correlated to RANKL/OPG ratio. Regardless of mouse strain, the absence of estrogen following OTM induced OIIRR. Mechanisms involve the imbalance of RANKL/OPG system, inflammatory and osteoclastic makers.

Dysbiotic oral microbiota contributes to alveolar bone loss associated with obesity in mice

Abstract Periodontal diseases (PD) are inflammatory conditions that affect the teeth supporting tissues. Increased body fat tissues may contribute to activation of the systemic inflammatory response, leading to comorbidities. Some studies have shown that individuals with obesity present higher incidence of PD than eutrophics. Objective: To investigate the impact of obesity on periodontal tissues and oral microbiota in mice. Methodology: Two obesity mice models were performed, one using 12 weeks of the dietary protocol with a high-fat (HF) diet in C57BL/6 mice and the other using leptin receptor-deficient mice (db/db-/-), which became spontaneously obese. After euthanasia, a DNA-DNA hybridization technique was employed to evaluate the microbiota composition and topical application of chlorhexidine (CHX), an antiseptic, was used to investigate the impact of the oral microbiota on the alveolar bone regarding obesity. Results: Increased adipose tissue may induce alveolar bone loss, neutrophil recruitment, and changes in the oral biofilm, similar to that observed in an experimental model of PD. Topical application of CHX impaired bone changes. Conclusion: Obesity may induce changes in the oral microbiota and neutrophil recruitment, which are associated with alveolar bone loss.

Synthesis and characterization of biodegradable polyurethane films based on HDI with hydrolyzable crosslinked bonds and a homogeneous structure for biomedical applications.

Synthetic biodegradable polymers are considered strategic in the biomaterials field and are used in various applications. Among the polymers used as biomaterials, polyurethanes (PUs) feature prominently due to their versatility and the ability to obtain products with a wide range of physical and mechanical properties. In this work, new biodegradable polyurethane films were developed based on hexamethylene diisocyanate (HDI) and glycerol as the hard segment (HS), and poly(caprolactone) triol (PCL triol) and low-molecular-weight poly(ethylene glycol) PEG as the soft segment (SS) without the use of a catalyst. The films obtained were characterized by structural, mechanical and biological testing. A highly connected network with a homogeneous PU structure was obtained due to crosslinked bonds. The films showed amorphous structures, high water uptake, hydrogel behavior, and susceptibility to hydrolytic degradation. Mechanical tests indicated that the films reached a high deformation at break of up to 425.4%, an elastic modulus of 1.6 MPa and a tensile strength of 3.6 MPa. The materials presented a moderate toxic effect on MTT assay and can be considered potential materials for biomedical applications.