Julien Braux

A new insight into the dissociating effect of strontium on bone resorption and formation.

Calcium phosphates are widely used as biomaterials and strontium (Sr) is known to have the ability to modify the bone balance towards osteosynthesis. In the present study we investigated the capacity of Sr-substituted sol-gel calcium phosphate to modify the expression of genes and proteins involved in extracellular matrix synthesis by primary bone cells. We first determined the most effective concentration of strontium using human primary bone cells. Sol-gel biphasic calcium phosphate (BCP) powders were then synthesised to obtain release of the optimal concentration of strontium. Finally, human osteoblasts obtained from explant cultures were cultured in the presence of sol-gel BCP, Sr-substituted BCP (5% Sr-substituted BCP, corresponding to a release of 5×10(-5)M [Sr(2+)] under the culture conditions (BCP(5%))) and medium containing strontium chloride (SrCl(2)). Viability, proliferation, cell morphology, protein production and protein activity were studied. We demonstrated that 5×10(-5)M SrCl(2) and BCP(5%) increased the expression of type I collagen and SERPINH1 mRNA and reduced the production of matrix metalloproteinases (MMP-1 and MMP-2) without modifying the levels of the tissue inhibitors of MMPs (TIMPs). Thus strontium has a positive effect on bone formation.

Inflammatory cell response to calcium phosphate biomaterial particles: an overview.

Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite (HA) and amorphous calcium phosphate (CaP) crystals deposited in an organic matrix. One objective of bone tissue engineering is to mimic the chemical and structural properties of this complex tissue. CaP ceramics, such as sintered HA and beta-tricalcium phosphate, are widely used as bone substitutes or prosthesis coatings because of their osteoconductive properties. These ceramic interactions with tissues induce a cell response that can be different according to the composition of the material. In this review, we discuss inflammatory cell responses to CaP materials to provide a comprehensive overview of mechanisms governing the integration or loosening of implants, which remains a major concern in tissue engineering. A focus on the effects of the functionalization of CaP biomaterials highlights potential ways to increase tissue integration and limit rejection processes.

The effect of zinc on hydroxyapatite-mediated activation of human polymorphonuclear neutrophils and bone implant-associated acute inflammation.

Hydroxyapatite (HA) is widely used as coating biomaterial for prosthesis metal parts and as bone substitute. The release of HA particles induces an inflammatory response and, if uncontrolled, could result in implant loss. At the inflamed site, the polymorphonuclear cells (PMNs) represent the earliest phagocytic cells that predominate the cellular infiltrate. We have recently proposed that HA wear debris activate polymorphonuclear cells (PMNs) initiating and/or amplifying thereby the acute inflammatory response. Previous studies have shown that activation of monocytes by HA could be modulated by supplementing this latter with the divalent cation, Zinc. The purpose of this work was to investigate the modulation of PMNs activation following exposure to zinc-substituted HA. Our study demonstrate that addition of zinc to HA particles resulted in decreased levels of the pro-inflammatory mediator interleukin-8 (IL-8) and the matrix metallo-proteinase-9. We also show that these changes involve IL-8 receptors (CXCR-1 and CXCR-2).

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates the production of osteoprotegerin (OPG) and prostaglandin (PG) E2 in human bone

Bone loss is an important clinical issue in patients with cystic fibrosis (CF). Whether the cystic fibrosis transmembrane conductance regulator (CFTR) plays a direct role in bone cell function is yet unknown. In this study, we provide evidence that inhibition of CFTR-Cl(-) channel function results in a significant decrease of osteoprotegerin (OPG) secretion accompanied with a concomitant increase of prostaglandin (PG) E(2) secretion of primary human osteoblast cultures (n=5). Our data therefore suggest that in bone cells of CF patients, the loss of CFTR activity may result in an increased inflammation-driven bone resorption (through both the reduced OPG and increased PGE(2) production), and thus might contribute to the early bone loss reported in young children with CF.

Bone disease in cystic fibrosis: new pathogenic insights opening novel therapies

Mutations within the gene encoding for the chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) results in cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease that causes a number of long-term health problems, as the bone disease. Osteoporosis and increased vertebral fracture risk associated with CF disease are becoming more important as the life expectancy of patients continues to improve. The etiology of low bone density is multifactorial, most probably a combination of inadequate peak bone mass during puberty and increased bone losses in adults. Body mass index, male sex, advanced pulmonary disease, malnutrition and chronic therapies are established additional risk factors for CF-related bone disease (CFBD). Consistently, recent evidence has confirmed that CFTR plays a major role in the osteoprotegerin (OPG) and COX-2 metabolite prostaglandin E2 (PGE2) production, two key regulators in the bone formation and regeneration. Several others mechanisms were also recognized from animal and cell models contributing to malfunctions of osteoblast (cell that form bone) and indirectly of bone-resorpting osteoclasts. Understanding such mechanisms is crucial for the development of therapies in CFBD. Innovative therapeutic approaches using CFTR modulators such as C18 have recently shown in vitro capacity to enhance PGE2 production and normalized the RANKL-to-OPG ratio in human osteoblasts bearing the mutation F508del-CFTR and therefore potential clinical utility in CFBD. This review focuses on the recently identified pathogenic mechanisms leading to CFBD and potential future therapies for treating CFBD.

Overexpression of RANKL in osteoblasts: a possible mechanism of susceptibility to bone disease in cystic fibrosis.

Bone fragility and loss are a significant cause of morbidity in patients with cystic fibrosis (CF), and the lack of effective therapeutic options means that treatment is more often palliative rather than curative. A deeper understanding of the pathogenesis of CF‐related bone disease (CFBD) is necessary to develop new therapies. Defective CF transmembrane conductance regulator (CFTR) protein and chronic inflammation in bone are important components of the CFBD development. The receptor activator of nuclear factor kappa‐B ligand (RANKL) and osteoprotegerin (OPG) drive the regulation of bone turnover. To investigate their roles in CFBD, we evaluated the involvement of defective CFTR in their production level in CF primary human osteoblasts with and without inflammatory stimulation, in the presence or not of pharmacological correctors of the CFTR. No major difference in cell ultrastructure was noted between cultured CF and non‐CF osteoblasts, but a delayed bone matrix mineralization was observed in CF osteoblasts. Strikingly, resting CF osteoblasts exhibited strong production of RANKL protein, which was highly localized at the cell membrane and was enhanced in TNF (TNF‐α) or IL‐17‐stimulated conditions. Under TNF stimulation, a defective response in OPG production was observed in CF osteoblasts in contrast to the elevated OPG production of non‐CF osteoblasts, leading to an elevated RANKL‐to‐OPG protein ratio in CF osteoblasts. Pharmacological inhibition of CFTR chloride channel conductance in non‐CF osteoblasts replicated both the decreased OPG production and the enhanced RANKL‐to‐OPG ratio. Interestingly, using CFTR correctors such as C18, we significantly reduced the production of RANKL by CF osteoblasts, in both resting and TNF‐stimulated conditions. In conclusion, the overexpression of RANKL and high membranous RANKL localization in osteoblasts are related to defective CFTR, and may worsen bone resorption, leading to bone loss in patients with CF. Targeting osteoblasts with CFTR correctors may represent an effective strategy to treat CFBD. Copyright

Strontium-Substituted Bioceramics Particles: A New Way to Modulate MCP-1 and Gro-α Production by Human Primary Osteoblastic Cells

Background: To avoid morbidity and limited availability associated with autografts, synthetic calcium phosphate (CaP) ceramics were extensively developed and used as bone filling materials. Controlling their induced-inflammatory response nevertheless remained a major concern. Strontium-containing CaP ceramics were recently demonstrated for impacting cytokines’ secretion pattern of human primary monocytes. The present study focuses on the ability of strontium-containing CaP to control the human primary bone cell production of two major inflammatory and pro-osteoclastogenic mediators, namely MCP-1 and Gro-α, in response to ceramics particles. Methods: This in vitro study was performed using human primary osteoblasts in which their response to ceramics was evaluated by PCR arrays, antibody arrays were used for screening and real-time PCR and ELISA for more focused analyses. Results: Study of mRNA and protein expression highlights that human primary bone cells are able to produce these inflammatory mediators and reveal that the adjunction of CaP in the culture medium leads to their enhanced production. Importantly, the current work determines the down-regulating effect of strontium-substituted CaP on MCP-1 and Gro-α production. Conclusion: Our findings point out a new capability of strontium to modulate human primary bone cells’ communication with the immune system.

Antimicrobial peptide gene expression in periodontitis patients: A pilot study

AIM Antimicrobial peptides (AMPs) are one of the most active components of innate immunity and have characteristics that could place them at the heart of the pathogenesis of periodontal disease. This study investigated differences in the expression of AMP coding genes obtained using a simple harvesting technique, gingival smear, between two groups of patients: chronic periodontitis subjects versus healthy ones. MATERIALS AND METHODS Twenty-three patients were enrolled in two groups: 12 were diagnosed with moderate or severe generalized chronic periodontitis, and 11 were diagnosed as clinically healthy. Gingival smears were retrieved and studied using reverse transcription-quantitative PCR (RT-qPCR) after mRNA purification. RESULTS Fifteen gene expressions were obtained using real-time RT-qPCR. Three AMP genes, histatin 3 (HTN3), α-defensin 4 (DEFA4) and lysozyme C (LYZ), presented different expression levels in periodontitis patients compared with healthy subjects. The relative expression level of DEFA4 appeared to be a protective factor against periodontitis. CONCLUSION Gingival smears studied by RT-qPCR may be used to assess the expression of AMPs coding genes. A lack of expression of DEFA4 could be a potential indicator of periodontitis status.

An Optimized Method to Generate Human Active Osteoclasts From Peripheral Blood Monocytes

Osteoclasts (OCs), the bone-resorbing cells, play a key role in skeletal development and adult bone remodeling. They also participate in the pathogenesis of various bone disorders. One of the major technical difficulties in the generation of OCs, when working on human material, is the ability to achieve large differentiation of mature OCs from human peripheral blood mononuclear cells (PBMCs). Access to a standardized source of active OCs is needed to better analyze the roles of human OCs. The aim of this study was to develop a procedure yielding active and mature OCs from fresh human PBMCs. We therefore examined the differentiation of PBMCs to OCs in different cell culture media, using non-stripped and charcoal-stripped sera in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). We also studied the effects of vitamin D3 in the differentiation level of PBMCs to OCs. Phalloidin-AlexaFluor®488/DAPI fluorescent stainings and dentin resorption analyses by scanning electron microscopy were used to identify the number and size of differentiated OCs, number of nuclei per cell and resorption activities of OCs for a 7–14–21-day culture period. This study reports an optimized method for an efficient production of human active OCs from a low seeding density of PBMCs, after a 14-day culture period by using a medium containing fetal bovine charcoal-stripped serum in the presence of M-CSF and RANKL, and in the absence of vitamin D3.