Cryoreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.

The effects of bioactive akermanite on physiochemical, drug-delivery and biological properties of poly (lactide-co-glycolide) beads

Poly(lactide-co-glycolide) (PLGA) beads have been widely studied as a potential drug/protein carrier. The main shortcomings of PLGA beads are that they lack bioactivity and controllable drug-delivery ability, and their acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Akermanite (AK) (Ca(2) MgSi(2) O(7) ) is a novel bioactive ceramic which has shown excellent bioactivity and degradation in vivo. This study aimed to incorporate AK to PLGA beads to improve the physiochemical, drug-delivery, and biological properties of PLGA beads. The microstructure of beads was characterized by SEM. The effect of AK incorporating into PLGA beads on the mechanical strength, apatite-formation ability, the loading and release of BSA, and the proliferation, and differentiation of bone marrow stromal cells (BMSCs) was investigated. The results showed that the incorporation of AK into PLGA beads altered the anisotropic microporous structure into homogenous one and improved their compressive strength and apatite-formation ability in simulated body fluids (SBF). AK neutralized the acidic products from PLGA beads, leading to stable pH value of 7.4 in biological environment. AK led to a sustainable and controllable release of bovine serum albumin (BSA) in PLGA beads. The incorporation of AK into PLGA beads enhanced the proliferation and alkaline phosphatase activity of BMSCs. This study implies that the incorporation of AK into PLGA beads is a promising method to enhance their physiochemical and biological property. AK/PLGA composite beads are a potential bioactive drug-delivery system for bone tissue repair.

Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in Vitro redifferentiation

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. ----- ----- Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).----- ----- Study Design: Controlled laboratory study.----- ----- Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).----- ----- Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.----- ----- Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.

Homodimerization controls the fibroblast growth factor 9 subfamily's receptor binding and heparan sulfate-dependent diffusion in the extracellular matrix

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.

Sulfate anion templation of macrocycles, capsules, interpenetrated and interlocked structures

This review examines the potential of anions, in particular sulfate, to template the formation of complex molecular architectures. Until recently, sulfate has been largely overlooked in this area and the examples described herein demonstrate this anion’s versatility in templating the formation of a diverse range of molecular systems including macrocycles, helixes, molecular capsules, interpenetrated and interlocked assemblies such as catenanes. In addition sulfate has been shown to template the formation of interpenetrated structures on a range of solid surfaces including gold, polystyrene beads and silicate nanoparticles, highlighting the potential of this anion in the fabrication of functional sensory devices exhibiting highly selective binding behaviour.

Electrochemically induced actuation of liquid metal marbles

Controlled actuation of soft objects with functional surfaces in aqueous environments presents opportunities for liquid phase electronics, novel assembled super-structures and unusual mechanical properties. We show the extraordinary electrochemically induced actuation of liquid metal droplets coated with nanoparticles, so-called “liquid metal marbles”. We demonstrate that nanoparticle coatings of these marbles offer an extra dimension for affecting the bipolar electrochemically induced actuation. The nanoparticles can readily migrate along the surface of liquid metals, upon the application of electric fields, altering the capacitive behaviour and surface tension in a highly asymmetric fashion. Surprising actuation behaviours are observed illustrating that nanoparticle coatings can have a strong effect on the movement of these marbles. This significant novel phenomenon, combined with unique properties of liquid metal marbles, represents an exciting platform for enabling diverse applications that cannot be achieved using rigid metal beads.

‘Click’ functionalised polymer resins : a new approach to the synthesis of surface attached bipyridinium and naphthalene diimide [2]rotaxanes

Herein we describe the design and synthesis of a series of solid-tethered [2]rotaxanes utilising crown ether-naphthalene diimide or crown ether- bipyridinium host guest interactions. TentaGel polystyrene resins were initially modified in a two-stage procedure to azide functionalised beads before the target supramolecular architectures were attached using a copper catalysed “click” procedure. The final assembly was examined using IR spectroscopy and gel-phase 1H High Resolution Magic Angle Spinning (HR MAS) NMR spectroscopy. The HR MAS technique enabled a direct comparison between the solid-tethered architectures and the synthesis and characterisation of analogous solution-based [2]rotaxanes to be made.

A micro-level investigation of the solid displacement method for porosity determination of dried food

Porosity is one of the key parameters of the macroscopic structure of porous media, generally defined as the ratio of the free spaces occupied (by the volume of air) within the material to the total volume of the material. Porosity is determined by measuring skeletal volume and the envelope volume. Solid displacement method is one of the inexpensive and easy methods to determine the envelope volume of a sample with an irregular shape. In this method, generally glass beads are used as a solid due to their uniform size, compactness and fluidity properties. The smaller size of the glass beads means that they enter into the open pores which have a larger diameter than the glass beads. Although extensive research has been carried out on porosity determination using displacement method, no study exists which adequately reports micro-level observation of the sample during measurement. This study set out with the aim of assessing the accuracy of solid displacement method of bulk density measurement of dried foods by micro-level observation. Solid displacement method of porosity determination was conducted using a cylindrical vial (cylindrical plastic container) and 57 µm glass beads in order to measure the bulk density of apple slices at different moisture contents. A scanning electron microscope (SEM), a profilometer and ImageJ software were used to investigate the penetration of glass beads into the surface pores during the determination of the porosity of dried food. A helium pycnometer was used to measure the particle density of the sample. Results show that a significant number of pores were large enough to allow the glass beads to enter into the pores, thereby causing some erroneous results. It was also found that coating the dried sample with appropriate coating material prior to measurement can resolve this problem.

Effects of particle size distribution in the response of model granular materials in multi-ring shear

Rail track undergoes complex loading patterns under moving traffic conditions compared to roads due to its continued and discontinued multi-layered structure, including rail, sleepers, ballast layer, sub-ballast layer, and subgrade. Particle size distributions (PSDs) of ballast, subballast, and subgrade layers can be critical in cyclic plastic deformation of rail track under moving traffic on frequent track degradation of rail tracks, especially at bridge transition zones. Conventional test approaches: static shear and cyclic single-point load tests are however unable to replicate actual loading patterns of moving train. Multi-ring shear apparatus; a new type of torsional simple shear apparatus, which can reproduce moving traffic conditions, was used in this study to investigate influence of particle size distribution of rail track layers on cyclic plastic deformation. Three particle size distributions, using glass beads were examined under different loading patterns: cyclic sin-gle-point load, and cyclic moving wheel load to evaluate cyclic plastic deformation of rail track under different loading methods. The results of these tests suggest that particle size distributions of rail track structural layers have significant impacts on cyclic plastic deformation under moving train load. Further, the limitations in con-ventional test methods used in laboratories to estimate the plastic deformation of rail track materials lead to underestimate the plastic deformation of rail tracks.

Amber teething necklaces: Is there any basis to their use in teething infants?

Introduction. Baltic amber-bead necklaces or bracelets are commonly used for managing teething symptoms in infants. The effectiveness of these beads is claimed to be from succinic acid release (a compound said to have analgesic and anti-inflammatory properties), which is then absorbed through the skin. Aims. To investigate whether succinic acid is contained in Baltic amber teething necklaces purchased in Australia, whether it can be released from the beads, and whether it has anti-inflammatory activity. Methods. Infrared spectroscopy was used to confirm that the teething necklaces were made of Baltic amber. The amount of succinic acid contained within the beads was quantified, and succinic acid release from intact beads was measured in phosphate buffered saline (PBS) pH 5.5 or octanol. Anti-inflammatory activity of succinic acid was compared with ibuprofen, paracetamol and hydrocortisone in vitro using THP-1 human macrophages stimulated with LPS. Secretion of the cytokines IL-1a, IL-1b, IL-8 and TNF-α were determined by ELISA. Results. Each necklace (33 beads in length) contained 19.17 ± 4.89 mg of succinic acid (mean ± se). Over a 6-month period, no succinic acid was detected in PBS. While 0.13 ± 0.09 mg of succinic acid per necklace was released in octanol, this was due to only one replicate of amber beads which had fragmented into shards free-floating in the solvent. Succinic acid had no effect on cytokine secretion unless extremely high concentrations were used and changes were likely to be associated with cell apoptosis and death. Discussion. No evidence for anti-inflammatory activity was found in the cytokines studied. It is possible that succinic acid could exert an effect via some other mechanism, but while the teething necklaces do contain small quantities of succinic acid, it is highly unlikely to be released from intact beads.

Amber teething necklaces – medical marvel or maternal myth?

BACKGROUND: Baltic amber-bead necklaces or bracelets are commonly used for managing teething symptoms in infants. The effectiveness of these beads is claimed to be from succinic acid release (a compound with analgesic and anti-inflammatory properties), which is then absorbed through the skin. AIM: To investigate whether Baltic amber teething necklaces purchased in Australia contained succinic acid, and to quantify succinic acid release from the beads. METHODS: Infrared spectroscopy was used to confirm that the teething necklaces were made of Baltic amber. The amount of succinic acid contained within the beads was quantified, and succinic acid release from intact beads was measured in phosphate buffered saline (PBS) pH 5.5 or octanol to simulate aqueous or oily skin environments. RESULTS: Each necklace (33 beads in length) contained 19.17±4.89 mg of succinic acid (mean±se). Over a 6-month period, no succinic acid was detected in PBS, while 0.13±0.09 mg of succinic acid per necklace was released in octanol. Only one replicate of amber beads in octanol released succinic acid, and they had fragmented, with shards free-floating in the solvent. DISCUSSION: It is likely succinic acid was only detected because the beads were breaking down in octanol, which does not occur when worn around the neck of a child. Furthermore, the hydrophilic properties of succinic acid would not favour its absorption across hydrophobic layers of the skin and into the bloodstream. CONCLUSION: While the teething necklaces do contain small quantities of succinic acid, it is highly unlikely to be released from intact beads.

Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays

Aberrant glycosylation of proteins is a hallmark of tumorigenesis, and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is non-invasive, technically straightforward and the sample collection and storage is relatively easy. Although, differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimised a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analysed with LC-MS/MS to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.