The actions of resolvin E1 on osteoblast function

Thesis (Ph.D.)--Boston UniversityResolvins are endogenous anti-inflammatory I pro-resolving lipid mediators derived from omega-3 fatty acids. Resolvin E1 (RvE1) reverses periodontitis and promotes regeneration of alveolar bone in vivo. The goal of this project was to determine the mechanism of RvE1 impact on bone formation. RvE1 significantly enhanced bone formation relative to a vehicle control in a mouse craniotomy model of bone healing. Since RvE1 is reported to act through receptors expressed by cells of the innate immune system, the initial hypothesis tested was that RvE1 actions are mediated through bone macrophages. The hypothesis was rejected, as no impact of RvE1 on macrophage mediated bone formation was demonstrable. The alternative hypothesis was that RvE1 acts directly on osteoblasts. Using mouse neonatal osteoblasts, calcification of osteoblast cultures was demonstrated. Osteoblasts express the RvE1 receptor, ChemR23, at the mRNA and protein level. Examination of intracellular signaling by RvE1 demonstrated increased phosphorylation of rpS6 through the AKT-mTOR pathway. The specificity of RvE1 signaling through ChemR23 was demonstrated with ChemR23 specific blocking antibody that abrogated the phosphorylation of rpS6. Rapamycin, an inhibitor of mTOR, also blocked rpS6 phosphorylation. To examine the mechanism of RvE1 treated osteoblast enhanced bone formation, secretion of bone specific proteins by osteoblasts after pro-inflammatory stimulation (IL-6) was examined with a focus on the osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) axis, which regulates osteoclast differentiation. Secretion of RANKL and OPG by mouse neonatal osteoblasts stimulated with IL-6 and treated with RvE1 was measured by ELISA. IL-6 stimulation did not impact RANKL levels but decreased OPG production, thereby changing the RANKL/OPG to favor osteoclast activation and bone resorption. RvE1 blocked OPG changes, however, maintaining a RANKL/OPG more favorable to bone formation. In conclusion, RvE1 has anabolic actions in a mouse model of bone healing mediated through RANKL/OPG. RvE1 signals the receptor ChemR23 on the osteoblast surface through the mTOR pathway and phosphorylation of rpS6. Functionally, RvE1 shifts the balance between OPG and RANKL to favor bone formation. Mediators of innate immunity thus also directly regulate bone cells

FOXO1 Modulates Osteoblast Differentiation

Forkhead box O1 (FOXO1) is upregulated during bone formation and in response to stimulation by bone morphogenetic proteins. Studies presented here examined the functional role of FOXO1 in a well defined culture system in which pre-osteoblastic cells undergo terminal differentiation in vitro. Mineralizing cultures of MC3T3-E1 cells were examined with or without FOXO1 knockdown by RNAi. Normal cells show the upregulation of FOXO1 and RUNX2 DNA binding activity, alkaline phosphatase activity, and mRNA levels of FOXO1, RUNX2, type 1 collagen, osteocalcin and MMP13 during formation of mineralizing nodules. In FOXO1 depleted cells each of these measurements was significantly reduced compared to values in control cells transfected with scrambled siRNA (P \u3c 0.05). Depletion of FOXO1 also reduced the number of mineralized nodules formed. Moreover, chromatin immunoprecipitation assays revealed a direct interaction of FOXO1 with the RUNX2 promoter. Overexpression of FOXO1 reduced the MC3T3-E1 cell number and the number of PCNA positive cells with little effect on apoptosis. These findings indicate that FOXO1 plays an important role in promoting osteoblast differentiation and suppressing proliferation in differentiating cells

Mineral maturity and crystallinity index are distinct characteristics of bone mineral

The purpose of this study was to test the hypothesis that mineral maturity and crystallinity index are two different characteristics of bone mineral. To this end, Fourier transform infrared microspectroscopy (FTIRM) was used. To test our hypothesis, synthetic apatites and human bone samples were used for the validation of the two parameters using FTIRM. Iliac crest samples from seven human controls and two with skeletal fluorosis were analyzed at the bone structural unit (BSU) level by FTIRM on sections 2–4 lm thick. Mineral maturity and crystallinity index were highly correlated in synthetic apatites but poorly correlated in normal human bone. In skeletal fluorosis, crystallinity index was increased and maturity decreased, supporting the fact of separate measurement of these two parameters. Moreover, results obtained in fluorosis suggested that mineral characteristics can be modified independently of bone remodeling. In conclusion, mineral maturity and crystallinity index are two different parameters measured separately by FTIRM and offering new perspectives to assess bone mineral traits in osteoporosis

Synthesis and kinetic resolution of N-Boc-2-arylpiperidines

The chiral base n-BuLi/(-)-sparteine or n-BuLi/(+)-sparteine surrogate promotes kinetic resolution of N-Boc-2-arylpiperidines by asymmetric deprotonation. The enantioenriched starting material was recovered with yields 39-48% and ers up to 97:3. On lithiation then electrophilic quench, 2,2-disubstituted piperidines were obtained with excellent yields and enantioselectivities. © 2014 the Partner Organisations

Classification of fracture and non-fracture groups by analysis of coherent X-ray scatter

Osteoporotic fractures present a significant social and economic burden, which is set to rise commensurately with the aging population. Greater understanding of the physicochemical differences between osteoporotic and normal conditions will facilitate the development of diagnostic technologies with increased performance and treatments with increased efficacy. Using coherent X-ray scattering we have evaluated a population of 108 ex vivo human bone samples comprised of non-fracture and fracture groups. Principal component fed linear discriminant analysis was used to develop a classification model to discern each condition resulting in a sensitivity and specificity of 93% and 91%, respectively. Evaluating the coherent X-ray scatter differences from each condition supports the hypothesis that a causal physicochemical change has occurred in the fracture group. This work is a critical step along the path towards developing an in vivo diagnostic tool for fracture risk prediction

Characterization of Mullite Ceramic Membranes and their Application in the Removal Escherichia Coli

This paper aims the morphological and structural characterization of ceramic membranes of mullite and their application in the removal of Escherichia coli. A complex irregular structure presented by the pores of the membrane was verified by scanning electron microscopy (SEM). The average pore size and distribution were determined by mercury intrusion porosimetry. The average pore size of the material presented was 0,39 μm. Microfiltration tests resulted in a protein retention of 46, 76 and 89% for trypsin (TR), egg albumin (EA) and bovine serum albumin (BSA), proving the efficiency of the membrane microfiltration tests for molecular weight of 69 kDa. The application of the membranes on the retention of gram-negative bacterium E. coli resulted in a 66% efficiency at a pressure of 200 kPa and a 98% efficiency when applied a pressure of 50 kPa. Therefore, the use of mullite membranes show limited efficiency towards bacteria retention. Nevertheless, they present fluxes similar to other materials proposed in the literature

Superhydrophilic Functionalization of Microfiltration Ceramic Membranes Enables Separation of Hydrocarbons from Frac and Produced Water

The environmental impact of shale oil and gas production by hydraulic fracturing (fracking) is of increasing concern. The biggest potential source of environmental contamination is flowback and produced water, which is highly contaminated with hydrocarbons, bacteria and particulates, meaning that traditional membranes are readily fouled. We show the chemical functionalisation of alumina ceramic microfiltration membranes (0.22 μm pore size) with cysteic acid creates a superhydrophilic surface, allowing for separation of hydrocarbons from frac and produced waters without fouling. The single pass rejection coefficients was >90% for all samples. The separation of hydrocarbons from water when the former have hydrodynamic diameters smaller than the pore size of the membrane is due to the zwitter ionically charged superhydrophilic pore surface. Membrane fouling is essentially eliminated, while a specific flux is obtained at a lower pressure (<2 bar) than that required achieving the same flux for the untreated membrane (4–8 bar)

Towards new material biomarkers for fracture risk

Osteoporosis is a prevalent bone condition, characterised by low bone mass and increased fracture risk. Currently, the gold standard for identifying osteoporosis and increased fracture risk is through quantification of bone mineral density (BMD) using dual energy X-ray absorption (DEXA). However, the risk of osteoporotic fracture is determined collectively by bone mass, architecture and physicochemistry of the mineral composite building blocks. Thus DEXA scans alone inevitably fail to fully discriminate individuals who will suffer a fragility fracture. This study examines trabecular bone at both ultrastructure and microarchitectural levels to provide a detailed material view of bone, and therefore provides a more comprehensive explanation of osteoporotic fracture risk. Physicochemical characterisation obtained through X-ray diffraction and infrared analysis indicated significant differences in apatite crystal chemistry and nanostructure between fracture and non-fracture groups. Further, this study, through considering the potential correlations between the chemical biomarkers and microarchitectural properties of trabecular bone, has investigated the relationship between bone mechanical properties (e.g. fragility) and physicochemical material features

Mineral Composition is Altered by Osteoblast Expression of an Engineered Gs-Coupled Receptor

Activation of the Gs G protein–coupled receptor Rs1 in osteoblasts increases bone mineral density by 5- to 15-fold in mice and recapitulates histologic aspects of fibrous dysplasia of the bone. However, the effects of constitutive Gs signaling on bone tissue quality are not known. The goal of this study was to determine bone tissue quality in mice resulting from osteoblast-specific constitutive Gs activation, by the complementary techniques of FTIR spectroscopy and synchrotron radiation micro-computed tomography (SRμCT). Col1(2.3)-tTA/TetO-Rs1 double transgenic (DT) mice, which showed osteoblast-specific constitutive Gs signaling activity by the Rs1 receptor, were created. Femora and calvariae of DT and wild-type (WT) mice (6 and 15 weeks old) were analyzed by FTIR spectroscopy. WT and DT femora (3 and 9 weeks old) were imaged by SRμCT. Mineral-to-matrix ratio was 25% lower (P = 0.010), carbonate-to-phosphate ratio was 20% higher (P = 0.025), crystallinity was 4% lower (P = 0.004), and cross-link ratio was 11% lower (P = 0.025) in 6-week DT bone. Differences persisted in 15-week animals. Quantitative SRμCT analysis revealed substantial differences in mean values and heterogeneity of tissue mineral density (TMD). TMD values were 1,156 ± 100 and 711 ± 251 mg/cm3 (mean ± SD) in WT and DT femoral diaphyses, respectively, at 3 weeks. Similar differences were found in 9-week animals. These results demonstrate that continuous Gs activation in murine osteoblasts leads to deposition of immature bone tissue with reduced mineralization. Our findings suggest that bone tissue quality may be an important contributor to increased fracture risk in fibrous dysplasia patients