Functional performance of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold: a pre-clinical in vitro tribological study.

Osteochondral grafts are used for repair of focal osteochondral lesions. Autologous grafts are the gold standard treatment; however, limited graft availability and donor site morbidity restrict use. Therefore, there is a clinical need for different graft sources/materials which replicate natural cartilage function. Chitosan has been proposed for this application. The aim of this study was to assess the biomechanics and biotribology of a bioresorbable chitosan/chitosan-nano-hydroxyapatite osteochondral construct (OCC), implanted in an in vitro porcine knee experimental simulation model. The OCC implanted in different surgical positions (flush, proud and inverted) was compared to predicate grafts in current clinical use and a positive control consisting of a stainless steel graft implanted proud of the cartilage surface. After 3 h (10 800 cycles) wear simulation under a walking gait, subsidence occurred in all OCC samples irrespective of surgical positioning, but with no apparent loss of material and low meniscus wear. Half the predicate grafts exhibited delamination and scratching of the cartilage surfaces. No graft subsidence occurred in the positive controls but wear and deformation of the meniscus were apparent. Implanting a new chitosan-based OCC either optimally (flush), inverted or proud of the cartilage surface resulted in minimal wear, damage and deformation of the meniscus.

Reducing the dehydrogenation temperature of lithium hydride through alloying with germanium.

LiH is a highly stable light metal hydride with a hydrogen capacity of 12.5 wt%. However, having a dehydrogenation enthalpy, ΔH(dehy), of 181.2 kJ mol(-1)(H2) and a resultant T(1 bar) of 944 °C, it is not a practical hydride for most hydrogen storage applications. In the work presented here, germanium has been found to dramatically reduce the dehydrogenation temperature for LiH down to just 270 °C. The enthalpy of dehydrogenation was reduced through the formation of lithium germanides. The reaction pathway was identified in this study using in situ powder neutron diffraction, showing the successive formation of more Li-rich germanides, following the series: LiGe, Li4Ge2H, Li9Ge4, and Li7Ge2. The enthalpy of formation for these germanides provides the thermodynamic tuning to reduce the ΔH(dehy) for the system. The 3LiH-Ge system investigated is found to be reversible with a maximum capacity of 3.0 ± 0.1 wt%.

Solid-state 13C NMR investigations of cyclophanes: [2.2]paracyclophane and 1,8-dioxa[8](2,7)pyrenophane.

Solid-state NMR (ssNMR) and ab initio quantum mechanical calculations are used in order to understand and to better characterize the molecular conformation and properties of [2.2]paracyclophane and 1,8-dioxa[8](2,7)pyrenophane. Both molecules are cyclophanes, consisting of an aromatic ring assembly and a cyclic aliphatic chain connected to both ends of the aromatic portion. The aliphatic chain causes curvature in the six-membered aromatic ring structures. This led us to examine how the ring strain due to curvature affects the chemical shifts. Using X-ray structures of both [2.2]paracyclophane and 1,8-dioxa[8](2,7)pyrenophane as our starting model, we calculate the chemical shielding tensors and compare these data with those collected from the (13)C ssNMR FIREMAT experiment. We define curvature of [2.2]paracyclophane and 1,8-dioxa[8](2,7)pyrenophane using the π-orbital axis vector (POAV) pyramidalization angle (θ(p)).

Development and in vitro assessment of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold.

An innovative approach was developed to engineer a multi-layered chitosan scaffold for osteochondral defect repair. A combination of freeze drying and porogen-leaching out methods produced a porous, bioresorbable scaffold with a distinct gradient of pore size (mean = 160-275 µm). Incorporation of 70 wt% nano-hydroxyapatite (nHA) provided additional strength to the bone-like layer. The scaffold showed instantaneous mechanical recovery under compressive loading and did not delaminate under tensile loading. The scaffold supported the attachment and proliferation of human mesenchymal stem cells (MSCs), with typical adherent cell morphology found on the bone layer compared to a rounded cell morphology on the chondrogenic layer. Osteogenic and chondrogenic differentiation of MSCs preferentially occurred in selected layers of the scaffold in vitro, driven by the distinct pore gradient and material composition. This scaffold is a suitable candidate for minimally invasive arthroscopic delivery in the clinic with potential to regenerate damaged cartilage and bone.

The effect of H2 partial pressure on the reaction progression and reversibility of lithium-containing multicomponent destabilized hydrogen storage systems.

It is known that the reaction path for the decomposition of LiBH(4):MgH(2) systems is dependent on whether decomposition is performed under vacuum or under a hydrogen pressure (typically 1-5 bar). However, the sensitivity of this multicomponent hydride system to partial pressures of H(2) has not been investigated previously. A combination of in situ powder neutron and X-ray diffraction (deuterides were used for the neutron experiments) have shed light on the effect of low partial pressures of hydrogen on the decomposition of these materials. Different partial pressures have been achieved through the use of different vacuum systems. It was found that all the samples decomposed to form Li-Mg alloys regardless of the vacuum system used or sample stoichiometry of the multicomponent system. However, upon cooling the reaction products, the alloys showed phase instability in all but the highest efficiency pumps (i.e., lowest base pressures), with the alloys reacting to form LiH and Mg. This work has significant impact on the investigation of Li-containing multicomponent systems and the reproducibility of results if different dynamic vacuum conditions are used, as this affects the apparent amount of hydrogen evolved (as determined by ex situ experiments). These results have also helped to explain differences in the reported reversibility of the systems, with Li-rich samples forming a passivating hydride layer, hindering further hydrogenation.

Long-Term Culture of Stem Cells on Phosphate-Based Glass Microspheres: Synergistic Role of Chemical Formulation and 3D Architecture.

Phosphate-based glasses (PBGs) are biomaterials that degrade under physiological conditions and can be modified to release various ions depending on end applications. This study utilized slow-degrading (P45:45P2O5-16CaO-24MgO-11Na2O-4Fe2O3, mol %) and comparatively faster degrading (P40:40P2O5-16CaO-24MgO-20Na2O, mol %) PBG microspheres with or without porosity, to evaluate the combined effect of chemical formulation and geometry on human mesenchymal stem cells (MSCs), a clinically relevant cell source for orthopedic applications. Scanning electron microscopy showed 2, 46, and 29% of P45 bulk (P45-B), P40 bulk (P40-B), and P40 porous (P40-P) microspheres, respectively, that had cracks or peeling off surfaces after 42 days of incubation in culture medium. Cytotoxicity assessment showed that glass debris released into the culture medium may interact with cells and affect their survival. Direct-contact cell experiments up to 42 days showed that P45-B microspheres did not sustain viable long-term cell cultures and did not facilitate extracellular matrix formation. On the other hand, P40-B microspheres enhanced alkaline phosphatase activity, calcium deposition, and collagen and osteocalcin production in MSCs. Introduction of porosity in P40 glass further enhanced these parameters and proliferation at later time points. The small pore windows (<5 µm wide) and interconnection (<10 µm wide) may have allowed limited cell penetration into the porous structures. P40-B and P40-P have potential for bone repair and reinforcement therapy based on their chemical formulation and porous geometry.

A combined solid-state NMR and synchrotron X-ray diffraction powder study on the structure of the antioxidant (+)-catechin 4.5-hydrate.

Analyses combining X-ray powder diffraction (XRD) and solid-state NMR (SSNMR) data can now provide crystal structures in challenging powders that are inaccessible by traditional methods. The flavonoid catechin is an ideal candidate for these methods, as it has eluded crystallographic characterization despite extensive study. Catechin was first described nearly two centuries ago, and its powders exhibit numerous levels of hydration. Here, synchrotron XRD data provide all heavy-atom positions in (+)-catechin 4.5-hydrate and establish the space group as C2. SSNMR data ((13)C tensor and (1)H/(13)C correlation) complete the conformation by providing catechin's five OH hydrogen orientations. Since 1903, this phase has been erroneously identified as a 4.0 hydrate, but XRD and density data establish that this discrepancy is due to the facile loss of the water molecule located at a Wyckoff special position in the unit cell. A final improvement to heavy-atom positions is provided by a geometry optimization of bond lengths and valence angles with XRD torsion angles held constant. The structural enhancement in this final structure is confirmed by the significantly improved fit of computed (13)C tensors to experimental data.

Solid-state 13C NMR investigations of 4,7-dihydro-1H-tricyclopenta[def,jkl,pqr]triphenylene (sumanene) and indeno[1,2,3-cd]fluoranthene: Buckminsterfullerene moieties.

4,7-Dihydro-1H-tricyclopenta[def,jkl,pqr]triphenylene (sumanene) and indeno[1,2,3-cd]fluoranthene (indenofluoranthene) are structural moieties related to Buckminsterfullerene (C(60)). As such, understanding their structural characteristics is of great interest because of the insight they shed upon C(60). Hence, solid-state NMR (ssNMR) and ab initio quantum mechanical calculations with Gaussian03 are used in order to understand and to better characterize the molecular conformation and properties of sumanene and indenofluoranthene. Sumanene has bowl shaped curvature in its natural conformation and indenofluoranthene is planar in its natural conformation, which led us to examine how altering the curvature affects the chemical shifts in relation to those of C(60). Using X-ray structures of both sumanene and indenofluoranthene as our starting model, we calculate the energy and chemical shielding tensors and compare these data with those collected utilizing the (13)C ssNMR FIREMAT experiment. We define curvature of sumanene and indenofluoranthene using the pi-orbital axis vector (POAV) pyramidalization angle (theta(p)). We calculate the energy of varying conformations of indenofluoranthene versus their theta(p) associated with each deformed conformation.

Solid-State NMR spectra and long, intra-dimer bonding in the pi-[TTF](2)(2+) (TTF = tetrathiafulvalene) dication.

The (13)C chemical-shift tensor principal values for TTF and pi-[TTF](2)(2+) (TTF = tetrathiafulvalene) dimer dications have been measured in order to better understand the electronic structure and long intradimer bonding of these TTF-based dimer structures. The structure of pi-[TTF](2)(2+) is abnormal due to its two C-C and four S-S ca. 3.4 A intradimer separations, which is less than the sum of the sulfur van der Waals radii, and has a singlet (1)A(1g) electronic ground state. This study of TTF and [TTF](2)(2+) was conducted to determine how the NMR chemical-shift tensor principal values change as a function of electronic structure. This study also establishes a better understanding of the interactions that lead to spin-pairing of the monomeric radical units. The density functional theory (DFT) calculated nuclear shielding tensors are correlated with the experimentally determined principal chemical-shift values. The embedded ion method (EIM) was used to investigate the electrostatic lattice potential in [TTF](2)(2+). These theoretical methods provide information on the tensor magnitudes and orientations of their tensor principal values with respect to the molecular frame. The experimental chemical-shift principal values agree with the calculated quantum mechanical chemical-shielding principal values, within typical errors commonly seen for this class of molecular system. Relatively weak Wiberg bond orders between the two [TTF](+) components of the dimer dication correlate with the long bonds linking the two [TTF](+) monomers and substantiate the claim that there is weak multicenter bonding present.

What instruments should we use to assess paediatric decision-making interventions? A narrative review.

There is an increasing number of shared decision-making (SDM) interventions in paediatrics. However, there is little consensus as to the best instruments to assess the feasibility and impact of these interventions. This narrative review aims to answer: (1) what feasibility, knowledge and decision-making instruments have been used to assess paediatric SDM interventions and (2) what are the psychometric properties of used decision-making instruments, guided by the 'consensus-based standards for the selection of health measurement instrument' criteria. We conducted a review of the peer-reviewed literature. We identified 23 studies that evaluated a paediatric intervention to facilitate SDM for a specific health decision. Eighteen studies assessed intervention feasibility, with a wide variability in assessment between studies. Twelve studies assessed objective knowledge, and four studies assessed subjective knowledge with all but one study aggregating correct responses. We identified nine decision-making instruments that had been assessed psychometrically, although few had been thoroughly evaluated. The Decisional Conflict Scale was the most commonly-used instrument and the only instrument evaluated in paediatrics. Our study revealed a lack of consistency in the instruments used to evaluate decision-making interventions in paediatrics, making it difficult to compare interventions. We provide several recommendations for researchers to improve the assessment of SDM interventions in paediatrics.

Extracting an Empirical Intermetallic Hydride Design Principle from Limited Data via Interpretable Machine Learning.

An open question in the metal hydride community is whether there are simple, physics-based design rules that dictate the thermodynamic properties of these materials across the variety of structures and chemistry they can exhibit. While black box machine learning-based algorithms can predict these properties with some success, they do not directly provide the basis on which these predictions are made, therefore complicating the a priori design of novel materials exhibiting a desired property value. In this work we demonstrate how feature importance, as identified by a gradient boosting tree regressor, uncovers the strong dependence of the metal hydride equilibrium H2 pressure on a volume-based descriptor that can be computed from just the elemental composition of the intermetallic alloy. Elucidation of this simple structure-property relationship is valid across a range of compositions, metal substitutions, and structural classes exhibited by intermetallic hydrides. This permits rational targeting of novel intermetallics for high-pressure hydrogen storage (low-stability hydrides) by their descriptor values, and we predict a known intermetallic to form a low-stability hydride (as confirmed by density functional theory calculations) that has not yet been experimentally investigated.

Developing highly nanoporous titanate structures via wet chemical conversion of DC magnetron sputtered titanium thin films.

Titanate structures have been widely investigated as biomedical component surfaces due to their bioactive, osteoinductive and antibacterial properties. However, these surfaces are limited to Ti and its alloys, due to the nature of the chemical conversion employed. The authors present a new method for generating nanoporous titanate structures on alternative biomaterial surfaces, such as other metals/alloys, ceramics and polymers, to produce bioactive and/or antibacterial properties in a simple yet effective way. Wet chemical (NaOH; 5 M; 60 °C; 24 h) conversion of DC magnetron sputtered Ti surfaces on 316L stainless steel were investigated to explore effects of microstructure on sodium titanate conversion. It was found that the more equiaxed thin films (B/300) generated the thickest titanate structures (ca. 1.6 µm), which disagreed with the proposed hypothesis of columnar structures allowing greater NaOH ingress. All film parameters tested ultimately generated titanate structures, as confirmed via EDX, SEM, XPS, XRD, FTIR and Raman analyses. Additionally, the more columnar structures (NB/NH & B/NH) had a greater quantity of Na (ca. 26 at.%) in the top portion of the films, as confirmed via XPS, however, on average the Na content was consistent across the films (ca. 5-9 at.%). Film adhesion for the more columnar structures (ca. 42 MPa), even on polished substrates, were close to that of the FDA requirement for plasma-sprayed HA coatings (ca. 50 MPa). This study demonstrates the potential of these surfaces to be applied onto a wide variety of material types, even polymeric materials, due to the lower processing temperatures utilised, with the vision to generate bioactive and/or antibacterial properties on a plethora of bioinert materials.

Preclinical biological and physicochemical evaluation of two-photon engineered 3D biomimetic copolymer scaffolds for bone healing.

A major challenge in orthopedics is the repair of large non-union bone fractures. A promising therapy for this indication is the use of biodegradable bioinspired biomaterials that stabilize the fracture site, relieve pain and initiate bone formation and healing. This study uses a multidisciplinary evaluation strategy to assess immunogenicity, allergenicity, bone responses and physicochemical properties of a novel biomaterial scaffold. Two-photon stereolithography generated personalized custom-built scaffolds with a repeating 3D structure of Schwarz Primitive minimal surface unit cell with a specific pore size of ∼400 µm from three different methacrylated poly(d,l-lactide-co-ε-caprolactone) copolymers with lactide to caprolactone monomer ratios of 16 : 4, 18 : 2 and 9 : 1. Using in vitro and in vivo assays for bone responses, immunological reactions and degradation dynamics, we found that copolymer composition influenced the scaffold physicochemical and biological properties. The scaffolds with the fastest degradation rate correlated with adverse cellular effects and mechanical stiffness correlated with in vitro osteoblast mineralization. The physicochemical properties also correlated with in vivo bone healing and immune responses. Overall these observations provide compelling support for these scaffolds for bone repair and illustrate the effectiveness of a promising multidisciplinary strategy with great potential for the preclinical evaluation of biomaterials.

Hydrogen storage in high surface area carbons: experimental demonstration of the effects of nitrogen doping.

The influence of nitrogen doping on the hydrogen uptake and storage capacity of high surface area carbon materials is presented in this report. To generate suitable study materials, we have exploited the relationship between synthesis conditions and textural properties of zeolite-templated carbons to generate a range of high surface area carbons with similar pore size distribution but which are either N-doped or N-free. For N-doped carbons, the nitrogen content was kept within a narrow range of between 4.7 and 7.7 wt %. The carbon materials, irrespective of whether they were doped or not, exhibited high surface area (1900-3700 m(2)/g) and pore volume (0.99 and 1.88 cm(3)/g), a micropore surface area of 1500-2800 m(2)/g, and a micropore volume of 0.65-1.24 cm(3)/g. The hydrogen uptake varied between 4.1 and 6.9 wt %. We present experimental data that indicates that the effect of N-doping on hydrogen uptake is only apparent when related to the surface area and pore volume associated with micropores rather than total porosity. Furthermore, by considering the isosteric heat of hydrogen adsorption and excess hydrogen uptake on N-free or N-doped carbons, it is shown that N-doping can be beneficial at lower coverage (low hydrogen uptake) but is detrimental at higher coverage (higher hydrogen uptake). The findings are consistent with previous theoretical predictions on the effect of N-doping of carbon on hydrogen uptake. The findings, therefore, add new insights that are useful for the development of carbon materials with enhanced hydrogen storage capacity.

Redetermination of 1,4-dimethoxy-benzene.

The structure of the centrosymmetric title compound, C(8)H(10)O(2), originally determined by Goodwin et al. [Acta Cryst.(1950), 3, 279-284], has been redetermined to modern standards of precision to aid in its use as a model compound for (13)C chemical-shift tensor measurements in single-crystal NMR studies. In the crystal structure, a C-H⋯O inter-action helps to establish the packing.

Structural, mechanical and swelling characteristics of 3D scaffolds from chitosan-agarose blends.

This study aimed to explore the correlation between mechanical and structural properties of chitosan-agarose blend (Ch-Agrs) scaffolds. Porosity of Ch-Agrs scaffolds was constant at 93%, whilst pore sizes varied between 150 and 550 µm. Pore sizes of the blend scaffolds (150-300 µm) were significantly smaller than for either agarose or chitosan scaffolds alone (ca. 500 µm). Ch50-Agrs50 blend scaffold showed the highest compressive modulus and strength values (4.5 ± 0.4 and 0.35 ± 0.03 MPa) due to reduction in the pore size. The presence of agarose improved the stability of the blends in aqueous media. The increase in compressive properties and residual weight after the TGA test, combined with the reduction in the swelling percentage of the blend scaffolds suggested an interaction between chitosan and agarose via hydrogen bonding which was confirmed using FTIR analysis. All wet blend scaffolds exhibited instant recovery after full compression. This study shows the potential of Ch-Agrs scaffolds for repairing soft tissue.

In vitro cellular testing of strontium/calcium substituted phosphate glass discs and microspheres shows potential for bone regeneration.

Phosphate-based glasses (PBGs) are ideal materials for regenerative medicine strategies because their composition, degradation rates, and ion release profiles can easily be controlled. Strontium has previously been found to simultaneously affect bone resorption and deposition. Therefore, by combining the inherent properties of resorbable PBG and therapeutic activity of strontium, these glasses could be used as a delivery device of therapeutic factors for the treatment of orthopaedic diseases such as osteoporosis. This study shows the cytocompatibility and osteogenic potential of PBGs where CaO is gradually replaced by SrO in the near invert glass system 40P2 O5 ·(16-x)CaO·20Na2 O·24MgO·xSrO (x = 0, 4, 8, 12, and 16 mol%). Direct seeding of MG63 cells onto glass discs showed no significant difference in cell metabolic activity and DNA amount measurement across the different formulations studied. Cell attachment and spreading was confirmed via scanning electron microscopy (SEM) imaging at Days 3 and 14. Alkaline phosphatase (ALP) activity was similarly maintained across the glass compositions. Follow-on studies explored the effect of each glass composition in microsphere conformation (size: 63-125 µm) on human mesenchymal stem cells (hMSCs) in 3D cultures, and analysis of cell metabolic activity and ALP activity showed no significant differences at Day 14 over the compositional range investigated, in line with the observations from MG63 cell culture studies. Environmental SEM and live cell imaging at Day 14 of hMSCs seeded on the microspheres showed cell attachment and colonisation of the microsphere surfaces, confirming these formulations as promising candidates for regenerative medicine strategies addressing compromised musculoskeletal/orthopaedic diseases.

Porous Phosphate-Based Glass Microspheres Show Biocompatibility, Tissue Infiltration, and Osteogenic Onset in an Ovine Bone Defect Model.

Phosphate-based glasses (PBGs) are bioactive and fully degradable materials with tailorable degradation rates. PBGs can be produced as porous microspheres through a single-step process, using changes in their formulation and geometry to produce varying pore sizes and interconnectivity for use in a range of applications, including biomedical use. Calcium phosphate PBGs have recently been proposed as orthobiologics, based on their in vitro cytocompatibility and ion release profile. In this study, porous microspheres made of two PBG formulations either containing TiO2 (P40Ti) or without (P40) were implanted in vivo in a large animal model of bone defect. The biocompatibility and osteogenic potential of these porous materials were assessed 13 weeks postimplantation in sheep and compared to empty defects and autologous bone grafts used as negative and positive controls. Histological analysis showed marked differences between the two formulations, as lower trabeculae-like interconnection and higher fatty bone marrow content were observed in the faster degrading P40-implanted defects, while the slower degrading P40Ti material promoted dense interconnected tissue. Autologous bone marrow concentrate (BMC) was also incorporated within the P40 and P40Ti microspheres in some defects; however, no significant differences were observed in comparison to microspheres implanted alone. Both formulations induced the formation of a collagen-enriched matrix, from 20 to 40% for P40 and P40Ti2.5 groups, suggesting commitment toward the bone lineage. With the faster degrading P40 formulation, mineralization of the tissue matrix was observed both with and without BMC. Some lymphocyte-like cells and foreign body multinucleated giant cells were observed with P40Ti2.5, suggesting that this more durable formulation might be linked to an inflammatory response. In conclusion, these first in vivo results indicate that PBG microspheres could be useful candidates for bone repair and regenerative medicine strategies and highlight the role of material degradation in the process of tissue formation and maturation.

Multiplexed FRET to image multiple signaling events in live cells.

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.

Purification, identification and activity of phomodione, a furandione from an endophytic Phoma species.

Phomodione, [(4aS(*),9bR(*))-2,6-diacetyl-7-hydroxy-4a,9-dimethoxy-8,9b-dimethyl-4a.9b-dihydrodibenzo[b,d]furan-1,3(2H,4H)-dione], an usnic acid derivative, was isolated from culture broth of a Phoma species, discovered as an endophyte on a Guinea plant (Saurauia scaberrinae). It was identified using NMR, X-ray crystallography, high resolution mass spectrometry, as well as infrared and Raman spectroscopy. In addition to phomodione, usnic acid and cercosporamide, known compounds with antibiotic activity, were also found in the culture medium. Phomodione exhibited a minimum inhibitory concentration of 1.6 microg/mL against Staphylococcus aureus using the disk diffusion assay, and was active against a representative oomycete, ascomycete and basidiomycete at between three and eight micro-grams per mL.