In vitro phenotypic effects of Lipoxin A4 on M1 and M2 polarized macrophages derived from THP-1.

BACKGROUND: Lipoxin A4 (LXA4) is a specialized pro-resolving mediator involved in the resolution phase of inflammation that is crucial for the return of tissues to homeostasis, healing, and regenerative processes. LXA4 can modify the microenvironment via its receptor, formyl peptide receptor 2 (FPR2) and thus modulate the inflammatory response. However, the effect of exogeneous LXA4 application on polarized macrophages remains unstudied. The objective of this study was to assess the effect of LXA4 on macrophage activity and on the phenotype modulation of polarized M1 and M2 macrophages derived from THP-1 monocytes. METHODS AND RESULTS: Once differentiated, human macrophages were incubated with interleukin 4 (IL-4) and IL-13 to obtain M2-polarized macrophages or with interferon gamma and lipopolysaccharide for classical macrophage activation. The mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages. LXA4 (0-100 nM) did not affect the viability of M1 and M2 macrophages or the phagocytic activity of these cells. Gene expression of FPR2, referred as a receptor for the LXA4, was higher in M1 compared with M2, and was not modified by the LXA4 at the doses used. Moreover, LXA4 exhibited anti-inflammatory properties illustrated by the decreasing in the gene expression of pro-inflammatory cytokines (IL-6, tumor necrosis factor alpha, IL-1ß) in M1 and by the increase in the expression of anti-inflammatory cytokines (IL-10) in M2 macrophages. CONCLUSIONS: These results provide new insights regarding the potential of LXA4 to regulate the polarization state of macrophages.

Interleukin-33 Deficiency Exacerbates Bone Loss Associated with Porphyromonas Gingivalis-Induced Experimental Periodontitis in Female Mice.

BACKGROUND/AIMS: Interleukin 33 (IL-33) plays a significant role in immunity but its role in bone physiology and periodontitis needs to be further investigated. The aim of this study was to decipher the contribution of IL-33 to bone homeostasis under physiological conditions, and to alveolar bone loss associated with experimental periodontitis (EP) in IL-33 knockout (KO) mice and their wildtype (WT) littermates. METHODS: The bone phenotype of IL-33 KO mice was studied in the maxilla, femur, and fifth lumbar vertebra by micro-computed tomography (micro-CT). EP was induced by a ligature soaked with the periopathogen Porphyromonas gingivalis (Pg) around a maxillary molar. Alveolar bone loss was quantified by micro-CT. The resorption parameters were assessed via toluidine blue staining on maxillary sections. In vitro osteoclastic differentiation assays using bone marrow cells were performed with or without lipopolysaccharide from Pg (LPS-Pg). RESULTS: First, we showed that under physiological conditions, IL-33 deficiency increased the trabecular bone volume/total volume ratio (BV/TV) of the maxillary bone in male and female mice, but not in the femur and fifth lumbar vertebra, suggesting an osteoprotective role for IL-33 in a site-dependent manner. The severity of EP induced by Pg-soaked ligature was increased in IL-33 KO mice but in female mice only, through an increase in the number of osteoclasts. Moreover, osteoclastic differentiation from bone marrow osteoclast progenitors in IL-33-deficient female mice is enhanced in the presence of LPS-Pg. CONCLUSION: Taken together, our data demonstrate that IL-33 plays a sex-dependent osteoprotective role both under physiological conditions and in EP with Pg.

Specialized pro-resolving lipid mediators in endodontics: a narrative review.

Endodontics is the branch of dentistry concerned with the morphology, physiology, and pathology of the human dental pulp and periradicular tissues. Human dental pulp is a highly dynamic tissue equipped with a network of resident immunocompetent cells that play major roles in the defense against pathogens and during tissue injury. However, the efficiency of these mechanisms during dental pulp inflammation (pulpitis) varies due to anatomical and physiological restrictions. Uncontrolled, excessive, or unresolved inflammation can lead to pulp tissue necrosis and subsequent bone infections called apical periodontitis. In most cases, pulpitis treatment consists of total pulp removal. Although this strategy has a good success rate, this treatment has some drawbacks (lack of defense mechanisms, loss of healing capacities, incomplete formation of the root in young patients). In a sizeable number of clinical situations, the decision to perform pulp extirpation and endodontic treatment is justifiable by the lack of therapeutic tools that could otherwise limit the immune/inflammatory process. In the past few decades, many studies have demonstrated that the resolution of acute inflammation is necessary to avoid the development of chronic inflammation and to promote repair or regeneration. This active process is orchestrated by Specialized Pro-resolving lipid Mediators (SPMs), including lipoxins, resolvins, protectins and maresins. Interestingly, SPMs do not have direct anti-inflammatory effects by inhibiting or directly blocking this process but can actively reduce neutrophil infiltration into inflamed tissues, enhance efferocytosis and bacterial phagocytosis by monocytes and macrophages and simultaneously inhibit inflammatory cytokine production. Experimental clinical application of SPMs has shown promising result in a wide range of inflammatory diseases, such as renal fibrosis, cerebral ischemia, marginal periodontitis, and cancer; the potential of SPMs in endodontic therapy has recently been explored. In this review, our objective was to analyze the involvement and potential use of SPMs in endodontic therapies with an emphasis on SPM delivery systems to effectively administer SPMs into the dental pulp space.

Genetic deletion of keratin 8 corrects the altered bone formation and osteopenia in a mouse model of cystic fibrosis.

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-ß-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-ß-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-ß-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis.

Runx2 Controls Bone Resorption through the Down-Regulation of the Wnt Pathway in Osteoblasts.

The transcription factor Runx2 and the Wnt/ß-catenin pathway are major regulators of bone formation. Our aim was to assess the interactions between the Wnt/ß-catenin pathway and Runx2 that contribute to bone resorption. Our results indicate that the activity of the canonical Wnt/ß-catenin pathway depends on Runx2. Runx2 overexpression inhibited ß-catenin levels and activity in vitro and in vivo. Inhibition of Gsk3b using lithium chloride in Runx2-overexpressing osteoporotic female mice rescued the Wnt/ß-catenin signaling in vivo and completely restored trabecular bone volume by increasing bone formation and decreasing bone resorption. The activation of Wnt/ß-catenin signaling by lithium chloride treatment reduced the number and activity of bone marrow-derived osteoclast-like cells in vitro, suggesting that the restoration of trabecular bone in vivo was due to decreased bone resorption, consistent with the reduced receptor activator of NF-κB ligand/osteoprotegerin ratio in Runx2-overexpressing osteoblasts. Lithium chloride also increased osteoblast differentiation and activity in vitro in agreement with the increase in mineral apposition rate and osteocalcin expression detected in vivo. Our results indicate that the activity of the canonical Wnt/ß-catenin pathway in osteoblast is modulated by Runx2. To conclude, our in vivo and in vitro results highlight the role of Runx2 as a negative regulator of Wnt/ß-catenin pathway activity in osteoblasts and indicate that the abnormal Wnt/ß-catenin activity seen in Runx2 transgenic mice affects both osteoblast and osteoclast differentiation and activity.

Increased NF-κB Activity and Decreased Wnt/ß-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice.

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased ß-catenin phosphorylation, reduced osteoblast ß-catenin expression, and altered expression of Wnt/ß-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/ß-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/ß-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis.

Metallome of Pseudomonas aeruginosa: a role for siderophores.

In this paper, we describe the total metal composition (metallome) of Pseudomonas aeruginosa. Inductively coupled plasma atomic emission spectroscopy analyses showed that P. aeruginosa cells concentrate each metal of the metallome from the extracellular media with different efficiencies. Growth in nutrient-restricted media did not substantially affect the overall profile of the metallome; however, the uptake of some metals was strongly stimulated, showing the high potential of some metal acquisition pathways to adapt to changing growth conditions. We also investigated the role of the two major siderophores produced by P. aeruginosa, pyoverdine and pyochelin, in iron uptake and more generally in metallome homeostasis. In addition to their role in iron acquisition, siderophore production also significantly prevented the accumulation of toxic metals in P. aeruginosa cells, thus preserving the equilibrium of the metallome in a polluted environment.

Decreased osteoclastogenesis in serotonin-deficient mice.

Peripheral serotonin, synthesized by tryptophan hydroxylase-1 (TPH(1)), has been shown to play a key role in several physiological functions. Recently, controversy has emerged about whether peripheral serotonin has any effect on bone density and remodeling.We therefore decided to investigate in detail bone remodeling in growing and mature TPH(1) knockout mice (TPH(1)(-/-)). Bone resorption in TPH(1)(-/-) mice, as assessed by biochemical markers and bone histomorphometry, was markedly decreased at both ages. Using bone marrow transplantation, we present evidence that the decrease in bone resorption in TPH(1)(-/-) mice is cell-autonomous. Cultures from TPH(1)(-/-) in the presence of macrophage colony-stimulating factor and receptor activator for NF-KB ligand (RANKL) displayed fewer osteoclasts, and the decreased differentiation could be rescued by adding serotonin. Our data also provide evidence that in the presence of RANKL, osteoclast precursors express TPH(1) and synthesize serotonin. Furthermore, pharmacological inhibition of serotonin receptor 1B with SB224289, and of receptor 2A with ketanserin, also reduced the number of osteoclasts. Our findings reveal that serotonin has an important local action in bone, as it can amplify the effect of RANKL on osteoclastogenesis.

An activating Fgfr3 mutation affects trabecular bone formation via a paracrine mechanism during growth.

The fibroblast growth factor receptor 3 (FGFR3) plays a critical role in the regulation of endochondral ossification. Fgfr3 gain-of-function mutations cause achondroplasia, the most common form of dwarfism, and a spectrum of chondrodysplasias. Despite a significant number of studies on the role of FGFR3 in cartilage, to date, none has investigated the influence of Fgfr3-mediated effects of the growth plate on bone formation. We studied three mouse models, each expressing Fgfr3 mutation either ubiquitously (CMV-Fgfr3(Y367C/+)), in chondrocytes (Col II-Fgfr3(Y367C/+)) or in mature osteoblasts (Col I-Fgfr3(Y367C/+)). Interestingly, we demonstrated that dwarfism with a significant defect in bone formation during growth was only observed in mouse models expressing mutant Fgfr3 in the cartilage. We observed a dramatic reduction in cartilage matrix mineralization and a strong defect of primary spongiosa. Anomalies of primary spongiosa were associated with an increase in osteoclast recruitment and a defect of osteoblasts at the mineralization front. A significant decrease in bone volume, trabecular thickness and number was also observed in the trabecular bone. Interestingly, no anomalies in proliferation and differentiation of primary osteoblasts from CMV-Fgfr3(Y367C/+) mice were observed. Based on these data, we excluded a potential function of Fgfr3 directly on osteoblasts at 3 weeks of age and we obtained evidence that the disorganization of the growth plate is responsible for the anomalies of the trabecular bone during bone formation. Herein, we propose that impaired FGFR3 signaling pathways may affect trabecular bone formation via a paracrine mechanism during growth. These results redefine our understanding of endochondral ossification in FGFR3-related chondrodysplasias.

Targeting Runx2 expression in hypertrophic chondrocytes impairs endochondral ossification during early skeletal development.

Runx2 is a known master transcription factor for osteoblast differentiation, as well as an essential regulator for chondrocyte maturation. Recently, more and more data has shown that Runx2 regulates hypertrophic chondrocyte-specific type X collagen gene (Col10a1) expression in different species. However, how Runx2 regulation of Col10a1 expression impacts chondrocyte maturation, an essential step of endochondral bone formation, remains unknown. We have recently generated transgenic mice in which Flag-tagged Runx2 was driven by a cell-specific Col10a1 control element. Significantly increased level of Runx2 and Col10a1 mRNA transcripts were detected in transgenic mouse limbs at both E17.5 (embryonic day 17.5) and P1 (post-natal day1) stages, suggesting an in vivo correlation of Runx2 and Col10a1 expression. Surprisingly, skeletal staining suggested delayed ossification in both the axial and the appendicular skeleton of transgenic mice from E14.5 until P6. Histological analysis showed elongated hypertrophic zones in transgenic mice, with less von Kossa and TUNEL staining in long bone sections at both E17.5 and P1 stages, suggesting defective mineralization due to delayed chondrocyte maturation or apoptosis. Indeed, we detected increased level of anti-apoptotic genes B-cell leukemia/lymphoma 2, Osteopontin, and Sox9 in transgenic mice by real-time RT-PCR. Moreover, immunohistochemistry and Western blotting analysis also suggested increased Sox9 expression in hypertrophic chondrocytes of transgenic mice. Together, our data suggest that targeting Runx2 in hypertrophic chondrocytes upregulates expression of Col10a1 and other marker genes (such as Sox9). This will change the local matrix environment, delay chondrocyte maturation, reduce apoptosis and matrix mineralization, and eventually, lead to impaired endochondral ossification.

Engineering siderophore production in Pseudomonas to improve asbestos weathering.

Iron plays a key role in microbial metabolism and bacteria have developed multiple siderophore-driven mechanisms due to its poor bioavailability for organisms in the environment. Iron-bearing minerals generally serve as a nutrient source to sustain bacterial growth after bioweathering. Siderophores are high-affinity ferric iron chelators, of which the biosynthesis is tightly regulated by the presence of iron. Pyoverdine-producing Pseudomonas have shown their ability to extract iron and magnesium from asbestos waste as nutrients. However, such bioweathering is rapidly limited due to repression of the pyoverdine pathway and the low bacterial requirement for iron. We developed a metabolically engineered strain of Pseudomonas aeruginosa for which pyoverdine production was no longer repressed by iron as a proof of concept. We compared siderophore-promoted dissolution of flocking asbestos waste by this optimized strain to that by the wild-type strain. Interestingly, pyoverdine production by the optimized strain was seven times higher in the presence of asbestos waste and the dissolution of magnesium and iron from the chrysotile fibres contained in flocking asbestos waste was significantly enhanced. This innovative mineral weathering process contributes to remove toxic iron from the asbestos fibres and may contribute to the development of an eco-friendly method to manage asbestos waste.

New insights into the role of matrix metalloproteinase 3 (MMP3) in bone.

The Matrix Metalloproteinases are important regulators of bone metabolism and can influence bone mass and bone remodeling. We investigate the role of Matrix Metalloproteinase 3 (MMP3) on bone in mice, by using Mmp3 knockout (Mmp3 KO) in the context of estrogen deficiency, and in human, by analyzing the association of promoter polymorphism with bone mineral density in postmenopausal women and with MMP3 expression. We presented evidence in this paper that Mmp3 KO significantly increases trabecular bone mass and trabecular number and does not affect cortical bone thickness. We also found that Mmp3 KO protects from the deleterious effects of ovariectomy on bone mineral density in mice by preventing deterioration of bone microarchitecture. The effect of Mmp3 KO does not involve bone formation parameters but instead acts by inhibition of bone resorption, leading to a reduced bone loss associated to ovariectomy. By studying a human cohort, we found that a polymorphism located in the promoter of the human MMP3 gene is associated with bone mineral density in postmenopausal women and found that MMP3 rs632478 promoter variants are associated with change in promoter activity in transfection experiments. In conclusion MMP3, although weakly expressed in bone cells, could be one of the important regulators of sex hormone action in bone and whose activity could be targeted for therapeutic applications such as in Osteoporosis.

Effects of risedronate in Runx2 overexpressing mice, an animal model for evaluation of treatment effects on bone quality and fractures.

Young mice overexpressing Runx2 specifically in cells of the osteoblastic lineage failed to gain bone mass and exhibited a dramatic increase in bone resorption, leading to severe osteopenia and spontaneous vertebral fractures. The objective of the current study was to determine whether treatment with a bisphosphonate (risedronate, Ris), which reduces fractures in postmenopausal as well as in juvenile osteoporosis, was able to improve bone quality and reduce vertebral fractures in mice overexpressing Runx2. Four-week-old female Runx2 mice received Ris at 2 and 10 µg/kg subcutaneously twice a week for 12 weeks. Runx2 and wild-type mice received vehicle (Veh) as control. We measured the number of new fractures by X-ray and bone mineral density (BMD) by DEXA. We evaluated bone quality by histomorphometry, micro-CT, and Fourier transform infrared imaging (FTIRI). Ris at 20 µg/kg weekly significantly reduced the average number of new vertebral fractures compared to controls. This was accompanied by significantly increased BMD, increased trabecular bone volume, and reduced bone remodeling (seen in indices of bone resorption and formation) in the vertebrae and femoral metaphysis compared to Runx2 Veh. At the femur, Ris also increased cortical thickness. Changes in collagen cross-linking seen on FTIRI confirmed that Runx2 mice have accelerated bone turnover and showed that Ris affects the collagen cross-link ratio at both forming and resorbing sites. In conclusion, young mice overexpressing Runx2 have high bone turnover-induced osteopenia and spontaneous fractures. Ris at 20 µg/kg weekly induced an increase in bone mass, changes in bone microarchitecture, and decreased vertebral fractures.

Osteomorphs as a tool for personalized medicine.

McDonald and colleagues reported osteoclast-related dynamic mechanisms that lead, by fission, to osteomorphs; motile, fusion-competent cells capable of forming bone-resorbing osteoclasts. scRNA-seq analyses revealed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and might be implicated in rare and common bone diseases in humans.

Biodeterioration of asbestos cement by siderophore-producing Pseudomonas.

Since the ban on the use of asbestos due to its carcinogenic properties, the removal of asbestos cement, representing the major asbestos-containing waste, has proven to be a challenge in most industrial countries. Asbestos-containing products are mainly disposed of in landfills and have remained untreated. Bioremediation involving bacteria previously reported the ability of Pseudomonas aeruginosa to release iron from flocking asbestos waste through a siderophore-driven mechanism. We examined the involvement of siderophore-producing Pseudomonas in the biodeterioration of asbestos cement. Iron and magnesium solubilization were evaluated by specific siderophore-producing mutants. The absence of one of the two siderophores affected iron extraction, whereas equivalent dissolution as that of the control was observed in the absence of siderophore. Both pyoverdine and pyochelin biosynthesis was repressed in the presence of asbestos cement, suggesting iron bioavailability from the waste. We compared the efficiency of various pyoverdines to scavenge iron from asbestos cement waste that revealed the efficiency of all pyoverdines. Pyoverdines were efficient in iron removal extracted continuously, with no evident extraction limit, in long-term weathering experiments with these pyoverdines. The optimization of pyoverdine-asbestos weathering may allow the development of a bioremediation process to avoid the disposal of such waste in landfills.

An Extrudable Partially Demineralized Allogeneic Bone Paste Exhibits a Similar Bone Healing Capacity as the "Gold Standard" Bone Graft.

Autologous bone grafts (BGs) remain the reference grafting technique in various clinical contexts of bone grafting procedures despite their numerous peri- and post-operative limitations. The use of allogeneic bone is a viable option for overcoming these limitations, as it is reliable and it has been widely utilized in various forms for decades. However, the lack of versatility of conventional allogeneic BGs (e.g., blocks, powders) limits their potential for use with irregular or hard-to-reach bone defects. In this context, a ready- and easy-to-use partially demineralized allogeneic BG in a paste form has been developed, with the aim of facilitating such bone grafting procedures. The regenerative properties of this bone paste (BP) was assessed and compared to that of a syngeneic BG in a pre-clinical model of intramembranous bone healing in critical size defects in rat calvaria. The microcomputed tridimensional quantifications and the histological observations at 7 weeks after the implantation revealed that the in vivo bone regeneration of critical-size defects (CSDs) filled with the BP was similar to syngeneic bone grafts (BGs). Thus, this ready-to-use, injectable, and moldable partially demineralized allogeneic BP, displaying equivalent bone healing capacity than the "gold standard," may be of particular clinical relevance in the context of oral and maxillofacial bone reconstructions.

A partially demineralized allogeneic bone graft: in vitro osteogenic potential and preclinical evaluation in two different intramembranous bone healing models.

In skeletal surgical procedures, bone regeneration in irregular and hard-to-reach areas may present clinical challenges. In order to overcome the limitations of traditional autologous bone grafts and bone substitutes, an extrudable and easy-to-handle innovative partially demineralized allogenic bone graft in the form of a paste has been developed. In this study, the regenerative potential of this paste was assessed and compared to its clinically used precursor form allogenic bone particles. Compared to the particular bone graft, the bone paste allowed better attachment of human mesenchymal stromal cells and their commitment towards the osteoblastic lineage, and it induced a pro-regenerative phenotype of human monocytes/macrophages. The bone paste also supported bone healing in vivo in a guide bone regeneration model and, more interestingly, exhibited a substantial bone-forming ability when implanted in a critical-size defect model in rat calvaria. Thus, these findings indicate that this novel partially demineralized allogeneic bone paste that combines substantial bone healing properties and rapid and ease-of-use may be a promising alternative to allogeneic bone grafts for bone regeneration in several clinical contexts of oral and maxillofacial bone grafting.

Curdlan-Chitosan Electrospun Fibers as Potential Scaffolds for Bone Regeneration.

Polysaccharides have received a lot of attention in biomedical research for their high potential as scaffolds owing to their unique biological properties. Fibrillar scaffolds made of chitosan demonstrated high promise in tissue engineering, especially for skin. As far as bone regeneration is concerned, curdlan (1,3-ß-glucan) is particularly interesting as it enhances bone growth by helping mesenchymal stem cell adhesion, by favoring their differentiation into osteoblasts and by limiting the osteoclastic activity. Therefore, we aim to combine both chitosan and curdlan polysaccharides in a new scaffold for bone regeneration. For that purpose, curdlan was electrospun as a blend with chitosan into a fibrillar scaffold. We show that this novel scaffold is biodegradable (8% at two weeks), exhibits a good swelling behavior (350%) and is non-cytotoxic in vitro. In addition, the benefit of incorporating curdlan in the scaffold was demonstrated in a scratch assay that evidences the ability of curdlan to express its immunomodulatory properties by enhancing cell migration. Thus, these innovative electrospun curdlan-chitosan scaffolds show great potential for bone tissue engineering.

Bone loss induced by Runx2 over-expression in mice is blunted by osteoblastic over-expression of TIMP-1.

The Runx2 gene is essential for osteoblast differentiation and function. In vivo over-expression of Runx2 in osteoblasts increases bone resorption, and blocks terminal osteoblast differentiation. Several lines of evidence suggest that osteoblastic matrix metalloproteinases (MMPs) could contribute to the increased bone resorption observed in mice over-expressing Runx2 (Runx2 mice). The goal of our study was to use a transgenic approach to find out whether the inhibition of osteoblastic MMPs can reduce the bone loss induced by the over-expression of Runx2. We analyzed the effect of the in vivo over-expression of the TIMP-1 in osteoblasts on the severe osteopenic phenotype in Runx2 mice. Females with the different genotypes (WT, Runx2, TIMP-1 and TIMP-1/Runx2) were analyzed for bone density, architecture, osteoblastic and osteoclastic activity and gene expression using qPCR. TIMP-1 over-expression reduces the bone loss in adult Runx2 mice. The prevention of the bone loss in TIMP-1/Runx2 mice was due to a combination of reduced bone resorption and sustained bone formation. We present evidence that the ability of osteoblastic cells to induce osteoclastic differentiation is lower in TIMP-1/Runx2 mice than in Runx2 mice, probably due to a reduction in the expression of RANK-L and of the Runx2 transgene. Osteoblast primary cells from TIMP-1/Runx2 mice, but not from Runx2 mice, were able to differentiate into fully mature osteoblasts producing high osteocalcin levels. In conclusion, our findings suggest that osteoblastic MMPs can affect osteoblast differentiation. Our work also indicates that osteoblastic MMPs are partly responsible for the bone loss observed in Runx2 transgenic mice.

FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter.

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine-Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric-pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor-ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric-pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric-pyoverdines, i.e. the chromophore, iron, and its chelating groups.