Methionine down-regulates TLR4/MyD88/NF-kappa B signalling in osteoclast precursors to reduce bone loss during osteoporosis

Background and PurposeStudies have demonstrated that a moderate intake of amino acids is associated with development of bone health. Methionine, a sulphur-containing essential amino acid, has been largely implicated for improving cartilage formation, however its physiological significance on bone integrity and functionality have not been elucidated. We investigated whether methionine can prevent osteoporotic bone loss. Experimental ApproachThe anti-resorptive effect of methionine, (250mgkg(-1) body wt administered in drinking water for 10 weeks), was evaluated in ovariectomized (OVX) rats by monitoring changes in bone turnover, formation of osteoclasts from blood-derived mononuclear cells and changes in the synthesis of pro-osteoclastogenic cytokines. Key resultsMethionine improved bone density and significantly decreased the degree of osteoclast development from blood mononuclear cells in OVX rats, as indicated by decreased production of osteoclast markers tartarate resistant acid phosphatase b (TRAP5b) and MIP-1. siRNA-mediated knockdown of myeloid differentiation primary response 88 MyD88], a signalling molecule in the toll-like receptor (TLR) signalling cascade, abolished the synthesis of both TRAP5b and MIP-1 in developing osteoclasts. Methionine supplementation disrupted osteoclast development by inhibiting TLR-4/MyD88/NF-B pathway. Conclusions and ImplicationsTLR-4/MyD88/NF-B signalling pathway is integral for osteoclast development and this is down-regulated in osteoporotic system on methionine treatment. Methionine treatment could be beneficial for the treatment of postmenopausal osteoporosis.

Multifunctional Properties of Multistage Spark Plasma Sintered HA-BaTiO3-Based Piezobiocomposites for Bone Replacement Applications

This work reports the processing-microstructure-property correlation of novel HA-BaTiO3-based piezobiocomposites, which demonstrated the bone-mimicking functional properties. A series of composites of hydroxyapatite (HA) with varying amounts of piezoelectric BaTiO3 (BT) were optimally processed using uniquely designed multistage spark plasma sintering (SPS) route. Transmission electron microscopy imaging during in situ heating provides complementary information on the real-time observation of sintering behavior. Ultrafine grains (0.50m) of HA and BT phases were predominantly retained in the SPSed samples. The experimental results revealed that dielectric constant, AC conductivity, piezoelectric strain coefficient, compressive strength, and modulus values of HA-40wt% BT closely resembles with that of the natural bone. The addition of 40wt% BT enhances the long-crack fracture toughness, compressive strength, and modulus by 132%, 200%, and 165%, respectively, with respect to HA. The above-mentioned exceptional combination of functional properties potentially establishes HA-40wt% BT piezocomposite as a new-generation composite for orthopedic implant applications.

Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-beta and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation. Published by Elsevier Ltd.

Critical cysteines in Akt1 regulate its activity and proteasomal degradation: implications for neurodegenerative diseases

Impaired Akt1 signaling is observed in neurodegenerative diseases, including Parkinson's disease (PD). In PD models oxidative modification of Akt1 leads to its dephosphorylation and consequent loss of its kinase activity. To explore the underlying mechanism we exposed Neuro2A cells to cadmium, a pan inhibitor of protein thiol disulfide oxidoreductases, including glutaredoxin 1 (Grx1), or downregulated Grx1, which led to dephosphorylation of Akt1, loss of its kinase activity, and also decreased Akt1 protein levels. Mutation of cysteines to serines at 296 and 310 in Akt1 did not affect its basal kinase activity but abolished cadmium- and Grx1 downregulation-induced reduction in Akt1 kinase activity, indicating their critical role in redox modulation of Akt1 function and turnover. Cadmium-induced decrease in phosphorylated Akt1 correlated with increased association of wild-type (WT) Akt1 with PP2A, which was absent in the C296-310S Akt1 mutant and was also abolished by N-acetylcysteine treatment. Further, increased proteasomal degradation of Akt1 by cadmium was not seen in the C296-310S Akt1 mutant, indicating that oxidation of cysteine residues facilitates degradation of WT Akt1. Moreover, preventing oxidative modification of Akt1 cysteines 296 and 310 by mutating them to serines increased the cell survival effects of Akt1. Thus, in neurodegenerative states such as PD, maintaining the thiol status of cysteines 296 and 310 in Akt1 would be critical for Akt1 kinase activity and for preventing its degradation by proteasomes. Preventing downregulation of Akt signaling not only has long-range consequences for cell survival but could also affect the multiple roles that Ala plays, including in the Akt-mTOR signaling cascade. (C) 2014 Elsevier Inc. All rights reserved.

Perovskite ceramic nanoparticles in polymer composites for augmenting bone tissue regeneration

There is increasing interest in the use of nanoparticles as fillers in polymer matrices to develop biomaterials which mimic the mechanical, chemical and electrical properties of bone tissue for orthopaedic applications. The objective of this study was to prepare poly(epsilon-caprolactone) (PCL) nanocomposites incorporating three different perovskite ceramic nanoparticles, namely, calcium titanate (CT), strontium titanate (ST) and barium titanate (BT). The tensile strength and modulus of the composites increased with the addition of nanoparticles. Scanning electron microscopy indicated that dispersion of the nanoparticles scaled with the density of the ceramics, which in turn played an important role in determining the enhancement in mechanical properties of the composite. Dielectric spectroscopy revealed improved permittivity and reduced losses in the composites when compared to neat PCL. Nanofibrous scaffolds were fabricated via electrospinning. Induction coupled plasma-optical emission spectroscopy indicated the release of small quantities of Ca+2, Sr+2, Ba+2 ions from the scaffolds. Piezo-force microscopy revealed that BT nanoparticles imparted piezoelectric properties to the scaffolds. In vitro studies revealed that all composites support osteoblast proliferation. Expression of osteogenic genes was enhanced on the nanocomposites in the following order: PCL/CT>PCL/ST>PCL/BT>PCL. This study demonstrates that the use of perovskite nanoparticles could be a promising technique to engineer better polymeric scaffolds for bone tissue engineering.

Hydroxyapatite-titanium bulk composites for bone tissue engineering applications

The research work on bulk hydroxyapatite (HA)-based composites are driven by the need to develop biomaterials with better mechanical properties without compromising its bioactivity and biocompatibility properties. Despite several years of research, the mechanical properties of the HA-based composites still need to be enhanced to match the properties of natural cortical bone. In this regard, the scope of this review on the HA-based bulk biomaterials is limited to the processing and the mechanical as well as biocompatibility properties for bone tissue engineering applications of a model system that is hydroxyapatite-titanium (HA-Ti) bulk composites. It will be discussed in this review how HA-Ti based bulk composites can be processed to have better fracture toughness and strength without compromising biocompatibility. The advantages of the functionally gradient materials to integrate the mechanical and biocompatibility properties is a promising approach in hard tissue engineering and has been emphasized here in reference to the limited literature reports. On the biomaterials fabrication aspect, the recent results are discussed to demonstrate that advanced manufacturing techniques, like spark plasma sintering can be adopted as a processing route to restrict the sintering reactions, while enhancing the mechanical properties. Various toughening mechanisms related to careful tailoring of microstructure are discussed. The in vitro cytocompatibilty, cell fate processes as well as in vivo biocompatibility results are also reviewed and the use of flow cytometry to quantify in vitro cell fate processes is being emphasized. (C) 2014 Wiley Periodicals, Inc.

A new approach to depict bone surfaces in finger imaging using photoacoustic tomography

Imaging the vasculature close around the finger joints is of interest in the field of rheumatology. Locally increased vasculature in the synovial membrane of these joints can be a marker for rheumatoid arthritis. In previous work we showed that part of the photoacoustically induced ultrasound from the epidermis reflects on the bone surface within the finger. These reflected signals could be wrongly interpreted as new photoacoustic sources. In this work we show that a conventional ultrasound reconstruction algorithm, that considers the skin as a collection of ultrasound transmitters and the PA tomography probe as the detector array, can be used to delineate bone surfaces of a finger. This can in the future assist in the localization of the joint gaps. This can provide us with a landmark to localize the region of the inflamed synovial membrane. We test the approach on finger mimicking phantoms.

Photoacoustic tomography of the human finger: towards the assessment of inflammatory joint diseases

Inflammatory arthritis is often manifested in finger joints. The growth of new or withdrawal of old blood vessels can be a sensitive marker for these diseases. Photoacoustic (PA) imaging has great potential in this respect since it allows the sensitive and highly resolved visualization of blood. We systematically investigated PA imaging of finger vasculature in healthy volunteers using a newly developed PA tomographic system. We present the PA results which show excellent detail of the vasculature. Vessels with diameters ranging between 100 mu m and 1.5 mm are visible along with details of the skin, including the epidermis and the subpapillary plexus. The focus of all the studies is at the proximal and distal interphalangeal joints, and in the context of ultimately visualizing the inflamed synovial membrane in patients. This work is important in laying the foundation for detailed research into PA imaging of the phalangeal vasculature in patients suffering from rheumatoid arthritis.

A Method for Delineation of Bone Surfaces in Photoacoustic Computed Tomography of the Finger

Photoacoustic (PA) imaging of interphalangeal peripheral joints is of interest in the context of using the synovial membrane as a surrogate marker of rheumatoid arthritis. Previous work has shown that ultrasound (US) produced by absorption of light at the epidermis reflects on the bone surfaces within the finger. When the reflected signals are backprojected in the region of interest, artifacts are produced, confounding interpretation of the images. In this work, we present an approach where the PA signals known to originate from the epidermis are treated as virtual US transmitters, and a separate reconstruction is performed as in US reflection imaging. This allows us to identify the bone surfaces. Furthermore, the identification of the joint space is important as this provides a landmark to localize a region-of-interest in seeking the inflamed synovial membrane. The ability to delineate bone surfaces allows us to identify not only the artifacts but also the interphalangeal joint space without recourse to new US hardware or a new measurement. We test the approach on phantoms and on a healthy human finger.

Conceptual design of three-dimensional scaffolds of powder-based materials for bone tissue engineering applications

Purpose - The purpose of this paper is to investigate the possibility to construct tissue-engineered bone repair scaffolds with pore size distributions using rapid prototyping techniques. Design/methodology/approach - The fabrication of porous scaffolds with complex porous architectures represents a major challenge in tissue engineering and the design aspects to mimic complex pore shape as well as spatial distribution of pore sizes of natural hard tissue remain unexplored. In this context, this work aims to evaluate the three-dimensional printing process to study its potential for scaffold fabrication as well as some innovative design of homogeneously porous or gradient porous scaffolds is described and such design has wider implication in the field of bone tissue engineering. Findings - The present work discusses biomedically relevant various design strategies with spatial/radial gradient in pore sizes as well as with different pore sizes and with different pore geometries. Originality/value - One of the important implications of the proposed novel design scheme would be the development of porous bioactive/biodegradable composites with gradient pore size, porosity, composition and with spatially distributed biochemical stimuli so that stem cells loaded into scaffolds would develop into complex tissues such as those at the bone-cartilage interface.

Engineering a multi-biofunctional composite using poly(ethylenimine) decorated graphene oxide for bone tissue regeneration

Toward preparing strong multi-biofunctional materials, poly(ethylenimine) (PEI) conjugated graphene oxide (GO_PEI) was synthesized using poly(acrylic acid) (PAA) as a spacer and incorporated in poly( e-caprolactone) (PCL) at different fractions. GO_PEI significantly promoted the proliferation and formation of focal adhesions in human mesenchymal stem cells (hMSCs) on PCL. GO_PEI was highly potent in inducing stem cell osteogenesis leading to near doubling of alkaline phosphatase expression and mineralization over neat PCL with 5% filler content and was approximate to 50% better than GO. Remarkably, 5% GO_ PEI was as potent as soluble osteoinductive factors. Increased adsorption of osteogenic factors due to the amine and oxygen containing functional groups on GO_ PEI augment stem cell differentiation. GO_ PEI was also highly efficient in imparting bactericidal activity with 85% reduction in counts of E. coli colonies compared to neat PCL at 5% filler content and was more than twice as efficient as GO. This may be attributed to the synergistic effect of the sharp edges of the particles along with the presence of the different chemical moieties. Thus, GO_ PEI based polymer composites can be utilized to prepare bioactive resorbable biomaterials as an alternative to using labile biomolecules for fabricating orthopedic devices for fracture fixation and tissue engineering.