Aluminum inhibits the growth of hydroxyapatite crystals developed on a biomimetic methacrylic polymer

ProjectAluminum (Al) is an increasing problem in biomedicine since it can interact with phosphates. Bone is one of the preferential target tissues of Al deposition: Al interacts with mineralization and/or bone cell activities. We searched the influence of Al deposition in hydroxyapatite developed on a biomimetic polymer (carboxymethylated poly(2-hydroxyethyl-methacrylate)) which mimics bone mineralization in the absence of cells.ProceduresPellets of polymer were incubated for 5 days in a synthetic body fluid (SBF) to induce mineralization, then 21 days in SBF containing 20, 40 and 60 μg/L Al³⁺. Other pellets were incubated in SBF containing commercial Al foil (33 mg/vial) either in 1, 2 or 6 pieces. The mineral deposits were dissolved in HCl and Ca²⁺, PO₄^3? and Al³⁺ content was measured. Hydroxyapatite was characterized by SEM and X energy-dispersive X-ray analysis (EDX).ResultsThe amount of Al³⁺ was dose-dependently increased in Ca/P deposits on the polymer pellets. At high concentration (or with the 6 Al foils) growth of hydroxyapatite calcospherite was inhibited; only calcified plates emerging from the polymer were observed. Pellets incubated with 1 and 2 Al foils exhibited a reduction in calcospherite diameter and an increase in the Al³⁺/Ca²⁺ ratio. EDX identified Al in the mineral deposits.ConclusionsIn this acellular model, Al³⁺ altered the growth of calcospherites at low concentration and inhibited their development at high concentration. In SBF, a release of Al³⁺ from aluminum foils also inhibited mineralization. This study emphasizes the importance of Al in bone pathology and stresses the question of its release from biomaterials.     

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.

Kinetic analysis of mineral formation during in vitro modeling of matrix vesicle mineralization: effect of annexin A5, phosphatidylserine, and type II collagen

Matrix vesicles (MVs) are involved in de novo mineral formation by nearly all vertebrate tissues. The driving force for MV mineralization is a nucleational core composed of three principal constituents: (i) amorphous calcium phosphate (ACP), complexed in part with phosphatidylserine (PS) to form (ii) calcium-phosphate-lipid complexes (CPLX), and (iii) annexin A5 (AnxA5), the principal lipid-dependent Ca(2+)-binding protein in MVs. We describe methods for reconstituting the nucleational core using a biomimetic approach and for analyzing the kinetics of its induction of mineral formation. The method is based on light scattering by the nascent crystallites at 340 nm and monitors mineral formation at regular intervals without disturbing the system using an automated plate reader. It yields precise replicate values that typically agree within less than 5%. As with MVs, mineral formation by the synthetic complex follows a sigmoidal pattern; following a quiescent induction period, rapid formation ensues for a limited time, followed by a distinct decline in rate that continues to slow, ultimately reaching a maximal asymptotic value. Key to quantization of mineral formation is the use of first-derivative analysis, which defines the induction time, the rate and the amount of initial mineral formation. Furthermore, using a five-parameter logistic curve-fitting algorithm, the maximal amount of mineral formation can be predicted accurately. Using these methods, we document the dramatic finding that AnxA5 synergistically activates PS-CPLX, transforming it from a very weak nucleator of mineral formation to a potent one. The methods presented should enable systematic study of the effects of numerous other factors thought to contribute to mineral formation.     

Effects of fibrin on the integration hydroxyapatite coating implants: experimental study in a rabbit model

The purpose of this study was to investigate the effects of the addition of fibrin (SAF) to titanium alloy implants coated with hydroxyapatite (HAP) on osteogenesis in rabbits. A titanium (Ti) alloy implant was inserted into the femoral neck of twenty-four adult rabbits. Six rabbits were included on each of the following groups: Ti control, HAP-coated Ti module, HAP-coated Ti module with added fibrin glue and Ti module also with added fibrin glue. After seven weeks, bone growth was examined radiographically and by histo-morphometry. The SAF/HAP mixture did caused to a significant increase in bone growth compared to the other groups. The addition of fibrin did not result in an increase in new-bone growth and increase the formation of fibrous tissue in contact with the implant. We concluded that SAF did not demonstrate osteoinductive properties.     

Biomimetic self-assembly of apatite hybrid materials: From a single molecular template to bi-/multi-molecular templates

The self-assembly of apatite and proteins is a critical process to induce the formation of the bones and teeth in vertebrates. Although hierarchical structures and biomineralization mechanisms of the mineralized tissues have been intensively studied, most researches focus on the self-assembly biomimetic route using one single-molecular template, while the natural bone is an outcome of a multi-molecular template co-assembly process. Inspired by such a mechanism in nature, a novel strategy based on multi-molecular template co-assembly for fabricating bone-like hybrid materials was firstly proposed by the authors. In this review article we have summarized the new trends from single-molecular template to bi-/multi-molecular template systems in biomimetic fabrication of apatite hybrid materials. So far, many novel apatite hybrid materials with controlled morphologies and hierarchical structures have been successfully achieved using bi-/multi-molecular template strategy, and are found to have multiple common features in comparison with natural mineralized tissues. The carboxyl, carbonyl and amino groups of the template molecules are identified to initiate the nucleation of calcium phosphate during the assembling process. For bi-/multi-molecular templates, the incorporation of multiple promotion sites for calcium and phosphate ions precisely enables to regulate the apatite nucleation from the early stage. The roles of acidic molecules and the synergetic effects of protein templates have been significantly recognized in recent studies. In addition, a specific attention is paid to self-assembling of apatite nanoparticles into ordered structures on tissue regenerative scaffolds due to their promising clinical applications ranging from implant grafts, coatings to drug and gene delivery.     

A supra-cellular model for coupling of bone resorption to formation during remodeling: lessons from two bone resorption inhibitors affecting bone formation differently

The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is the physical site where coupling of bone resorption to bone formation occurs.     

Quantitative studies of the non-productive binding of lysozyme to partially N-acetylated chitosans: Binding of large ligands to a one-dimensional binary lattice studied by a modified McGhee and von Hippel model

This paper considers the non-productive (inhibitory) binding of chitosans to lysozyme from chicken egg white. Chitosans are linear, binary heteropolysaccharides consisting of 2-acetamido-2-deoxy-β-d-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-d-glucose (GlcN, D-unit). The active site cleft of lysozyme can bind six consecutive sugar residues in subsites named A–F, and specific binding of chitosan sequences to lysozyme occurs with A-units in subsite C. Chitosans with different fractions of A-units (F_A) induced nearly identical changes in the ¹H NMR spectrum of lysozyme upon binding, and the concentration of bound lysozyme could be determined. The data were analysed using a modified version of the McGhee and von Hippel model for binding of large ligands to one-dimensional homogeneous lattices. The average value of the dissociation constant for different sequences that may bind to lysozyme (K^ave_D) was estimated, as well as the number of chitosan units covered by lysozyme upon binding. K^ave_D decreased with increasing F_A-values at pH* 3 and 4.5, while the opposite was true at pH* 5.5. Contributions from different hexamer sequences to K^ave_D of the chitosans were considered, and the data revealed interesting features with respect to binding of lysozyme to partially N-acetylated chitosans. The relevance of the present data with respect to understanding lysozyme degradation kinetics of chitosans is discussed.     

Dimethyl sulfide emissions from a sedimental microbial ecosystem subject to diel variations of oxic and anoxic conditions: a simple mathematical model

This modelling study relates dimethylsulfide emission from a microbial mat to the flux of dimethylsulfoniopropionate that is exuded into the interstitial space of the mat by phototrophs. Dimethylsulfoniopropionate may be either cleaved or demethylated. Only cleavage results in the production of dimethylsulfide, which itself is further oxidized or escapes from the mat. The fate of dimethylsulfoniopropionate depends on the functional group composition of the mat, the physiological characteristics of these groups, and the eco-physiological conditions oxic/anoxic and light/dark, which both vary in a diel cycle. These three factors are accounted for in a mathematical model of a microbial mat typical of the Wadden Islands of The Netherlands and Germany. Model simulations quantify increased dimethylsulfide production under alkaline stress as well as additional dimethylsulfoniopropionate loads.     

The reduction of trimethylarsine oxide to trimethylarsine by thiols: a mechanistic model for the biological reduction of arsenicals

Trimethylarsine oxide is reduced to trimethylarsine in aqueous solution by a variety of thiols and dithiols including cysteine, glutathione, and lipoic acid. Kinetic results and other observations suggest that the rate-determining step is the production of [Me₃AsSR]⁺ from an initially formed Me₃As(SR)OH species, and that the reduction occurs via a two-electron transfer from Me₃As(SR)₂ affording Me₃As and RS-SR. A simple model for the biological methylation of arsenic is proposed based on oxidative methylation of arsenic(III) by S-adenosylmethionine and reduction by a thiol such as lipoic acid.     

Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: How to reliably produce a biomimetic 3D model

To obtain a multicellular MCF-7 spheroid model to mimic the three-dimensional (3D) of tumors, the microwell liquid overlay (A) and hanging-drop/agar (B) methods were first compared for their technical parameters. Then a method for embedding spheroids within collagen was optimized. For method A, centrifugation assisted cells form irregular aggregates but not spheroids. For method B, an extended sedimentation period of over 24 h for cell suspensions and increased viscosity of the culture medium using methylcellulose were necessary to harvest a dense and regular cell spheroid. When the number was less than 5000 cells/drop, embedded spheroids showed no tight cores and higher viability than the unembedded. However, above 5000 cells/drop, cellular viability of embedded spheroids was not significantly different from unembedded spheroids and cells invading through the collagen were in a sun-burst pattern with tight cores. Propidium Iodide staining indicated that spheroids had necrotic cores. The doxorubicin cytotoxicity demonstrated that spheroids were less susceptible to DOX than their monolayer cells. A reliable and reproducible method for embedding spheroids using the hanging-drop/agarose method within collagen is described herein. The cell culture model can be used to guide experimental manipulation of 3D cell cultures and to evaluate anticancer drug efficacy.     

Molecular design,synthesis and in vitro biological evaluation of thienopyrimidine–hydroxamic acids as chimeric kinase HDAC inhibitors: a challenging approach to combat cancer

A series of thieno[2,3-d]pyrimidine-based hydroxamic acid hybrids was designed and synthesised as multitarget anti-cancer agents, through incorporating the pharmacophore of EGFR, VEGFR2 into the inhibitory functionality of HDAC6. Three compounds (12c, 15b and 20b) were promising hits, whereas (12c) exhibited potent VEGFR2 inhibition (IC₅₀=185 nM), potent EGFR inhibition (IC₅₀=1.14 µM), and mild HDAC6 inhibition (23% inhibition). Moreover, compound (15c) was the most potent dual inhibitor among all the synthesised compounds, as it exhibited potent EGFR and VEGFR2 inhibition (IC₅₀=19 nM) and (IC₅₀=5.58 µM), respectively. While compounds (20d) and (7c) displayed nanomolar selective kinase inhibition with EGFR IC₅₀= 68 nM and VEGFR2 IC₅₀= 191 nM, respectively. All of the synthesised compounds were screened in vitro for their cytotoxic effect on 60 human NCI tumour cell lines. Additionally, molecular docking studies and ADMET studies were carried out to gain further insight into their binding mode and predict the pharmacokinetic properties of all the synthesised inhibitors.     

The common bile duct ligation in rat: a relevant in vivo model to study the role of mechanical stress on cell and matrix behaviour

Common bile duct ligation leads to bile accumulation and liver fibrosis. In this model, little attention has been dedicated to the modification of the common bile duct. We have studied by histochemistry and immunohistochemistry, 3 and 5 days after ligation, the connective tissue modifications of the common bile duct wall. After bile duct ligation, compared with normal bile duct, a strong increase of the bile duct diameter, due to bile stasis, and a thickness of the bile duct wall were observed; numerous myofibroblasts expressing α-smooth muscle actin appeared in parallel with the detection of many proliferating connective tissue cells. These myofibroblasts secreted very early high amount of elastic fibre components, elastin and fibrillin-1. Elastic fibre increase was also observed close to the epithelial cell layer. Procollagen type III deposition was also induced 3 days after ligation but decreased thereafter, underlining that myofibroblasts modify their synthesis of extracellular matrix components to comply with the request. We show here that common bile duct ligation represents an invaluable model to study myofibroblastic differentiation and extracellular matrix adaptation produced by an acute mechanical stress.     

Studies of xylan interactions and cross-linking to synthetic lignins formed by bulk and end-wise polymerization: a model study of lignin carbohydrate complex formation

The mechanism of lignin carbohydrate complex formation by addition of polysaccharides on quinone methide (QM) generated during lignin polymerisation was investigated using a model approach. Dehydrogenation polymers (DHPs, lignin model compounds) were synthesized from coniferyl alcohol in the presence of a glucuronoarabinoxylan (GAX) extracted from oat spelts, by Zutropfverfahren (ZT) and Zulaufverfahren (ZL) methods. The methods ZT and ZL differed in their distribution of QM over the reaction period but generated roughly the same QM amount. Steric exclusion chromatography of the ZT and ZL reaction products showed that only the ZT reaction produced high molar mass compounds. Covalent linkages in the ZT reaction involving ether bonds between GAX moiety and α carbon of the lignin monomer were confirmed by ¹³C NMR and xylanase-based fractionation. The underlying phenomena were further investigated by examining the interactions between GAX and DHP in sorption experiments. GAX and DHPs were shown to interact to form hydrophobic aggregates. In the ZT process, slow addition permitted polymer reorganisation which led to dehydration around the lignin-like growing chains thereby limiting the addition of water on the quinone methide formed during polymerisation and thus favoured lignin–carbohydrate complex (LCC) formation.     

Retracted: Effects of microRNA-370 on mesangial cell proliferation and extracellular matrix accumulation by binding to canopy 1 in a rat model of diabetic nephropathy

As one major diabetic complication, diabetic nephropathy (DN) has been reported to be associated with various kinds of microRNA (miRNA). Thus, we conducted this study to explore the potential of miR-370 in a rat model of DN through investigation of mesangial cell proliferation and extracellular matrix (ECM). A total of 40 healthy adult male Sprague–Dawley rats were enrolled and assigned into normal (n = 10) and DN ( n = 30, DN rat model) groups. Dual-luciferase reporter assay was performed for the targeting relationship between miR-370 and canopy 1 (CNPY1). Mesangial cells were collected and transfected with prepared mimic, inhibitor or small interfering RNA (siRNA) for analyzing the effect of miR-370 on DN mice with the help of expression and cell biological processes detection. CNPY1 was confirmed as a target gene of miR-370. DN mice had increased expression of miR-370, fibronectin, type I collagen (Col I), type IV collagen (Col IV), and plasminogen activator inhibitor-1 (PAI-1) but reduced CNPY1 expression. Cells transfected with miR-370 mimic and siRNA–CNPY1 had increased expression of fibronectin, Col I, Col IV, and PAI-1 but decreased CNPY1 expression. The miR-370 mimic and siRNA–CNPY1 groups showed increased cell proliferation, as well as elevated ECM accumulation and declined cell apoptosis rate as compared with the blank and negative control groups, with reverse trends observed in the miR-370 inhibitor group. Our study concludes that overexpression of miR-370 promotes mesangial cell proliferation and ECM accumulation by suppressing CNPY1 in a rat model of DN.     

Regulation of cell growth and the expression of extracellular matrix proteins in colorectal adenocarcinoma: a fibroblast-tumor cell coculture model to study tumor-host interactions in vitro

The production of abundant connective tissue within malignant tumors, the so-called desmoplastic stromal reaction, is a hallmark of colorectal adenocarcinomas. This stroma is produced to a large extent by myofibroblasts and contains various amounts of collagens (type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. In this study we have established a monolayer coculture model between two different colorectal adenocarcinoma cell lines (HRT-18, and CX-2) and colonic fibroblasts (CCD-18) to investigate the mechanisms regulating (i) the production of extracellular matrix (ECM) components, (ii) the induction of myofibroblastic differentiation, and (iii) cellular proliferation. We found that TGFbeta1 and FGF-2 stimulated ECM synthesis of fibroblasts. Myofibroblastic differentiation was stimulated by TGFbeta1 but suppressed by FGF-2. There was a mutual stimulation of proliferation between fibroblasts and carcinoma cells. The analogies with ECM components expressed in cocultures and colorectal adenocarcinoma samples suggest that the coculture model used in this study is useful to study tumor cell-fibroblast interactions.