The Characterization and Distribution of Magnesium Whitlockite Crystals in Human Articular Cartilage

The occurrence of crystals (previously termed 'cuboid crystals': 50-500nmsize range) not apparent by light microscopy, in human articular cartilage has been confirmed by transmission electron microscopy of tissue prepared by various techniques, including anhydrous and cryo processing. Earlier reports of such crystal deposition had been limited to osteoarthritic and elderly femoral head articular cartilage. In this study crystals have been reported in articular cartilage across an age range from five to ninety two years in normal and osteoarthritic tissue from a variety of joint sites. The distribution of crystal deposition within articular cartilage was described both qualitatively and quantitatively; normal femoral head tissue was investigated in most detail. Over 90 % of crystals were commonly deposited within the first 50μm below the articular surface; crystals appeared either in a band parallel to the surface or in a pericellular distribution. In deeper zones crystal deposition was restricted to pericellular distribution, and areas of chondrocyte necrosis. Quantitative analysis of crystal deposition distribution in articular cartilage at sites around the femoral head revealed a significantly greater deposition in the superior (zenith) region than the inferior (infrafoveal) region. Elemental analysis of crystals confirmed a calcium, phosphorus and magnesium content. It also demonstrated no variation in the mean calcium to phosphorus ratio with crystal size, specimen age, or between normal and osteoarthritic specimens. A crystal isolation technique involving collagenase digestion, centrifugation and sodium hypochlorite treatment was developed, enabling crystal characterization by electron and x-ray diffraction. Crystals were identified as magnesium whitlockite; the first report of this mineral in articular cartilage. The mode of formation and role of these crystals remain unknown, although histological and histochemical investigations revealed a consistent association with intramatrical lipid, containing a phospholipid component. The results of this study are most tenable with a concept of opportunistic crystal deposition

Oxidation state of a polyurethane membrane after plasma etching

Low moduli cell culture substrates can be used to apply dynamic mechanical strain to cells, by surface deformation. Understanding the surface interaction with cells is critical to improving cell adhesion and normal growth. A medical grade polyurethane (PU), Chronoflex AL 80A, was modified by oxygen plasma etching and characterised by X-ray photoelectron spectroscopy. Etching resulted in increased cross-linking at the isocyanate bond and formation of new oxygen moieties. The model, derived from patent data and XPS data of the unetched PU, indicated that the additional oxygen was likely to be hydroxyl and carbonyl groups. Etched membranes enhanced protein adhesion, resulting in full surface coverage compared to unetched PU. The etched PU supported cell adhesion and spreading, while the unetched PU was not conducive to monolayer formation

Topographical and chemical effects of electrochemically assisted deposited hydroxyapatite coatings on osteoblast-like cells

A recently commercialised hydroxyapatite electrochemically assisted chemical deposition technique (BoneMaster) has been shown to induce increased bone apposition; whether this response is caused by the surface topography or chemistry is unknown. An in-vitro examination using human osteoblast-like cells was performed on a series of BoneMaster-coated surfaces. The chemistry was separated from the topography using a thin gold coating; Thermanox coverslips were used as a control. BoneMaster surfaces showed significantly greater alkaline phosphatase activity and osteocalcin production compared with controls; however, no difference was found between the gold-coated and uncoated BoneMaster samples, indicating topography is the main contributing factor

Impact of Serum Source on Human Mesenchymal Stem Cell Osteogenic Differentiation in Culture

Human mesenchymal stem cells (MSCs) show promise for musculoskeletal repair applications. Animal-derived serum is extensively used for MSC culture as a source of nutrients, extracellular matrix proteins and growth factors. However, the routine use of fetal calf serum (FCS) is not innocuous due to its animal antigens and ill-defined composition, driving the development of alternatives protocols. The present study sought to reduce exposure to FCS via the transient use of human serum. Transient exposure to animal serum had previously proved successful for the osteogenic differentiation of MSCs, but had not yet been tested with alternative serum sources. Here, human serum supported proliferation of MSCs, which retained surface marker expression and presented higher alkaline phosphatase activity than those in FCS-based medium. Addition of osteogenic supplements supported strong mineralisation over a 3-week treatment. When limiting serum exposure to the first 5 days of treatment, MSCs achieved higher differentiation with human serum than FCS. Finally, human serum analysis revealed significantly higher levels of osteogenic components such as alkaline phosphatase and 25-Hydroxyvitamin D, consistent with the enhanced osteogenic effect. These results indicate that human serum used at the start of the culture offers an efficient replacement for continuous FCS treatment, and could enable short-term exposure to patient-derived serum in the future

Integrated multi-assay culture model for stem cell chondrogenic differentiation

Recent osteochondral repair strategies highlight the promise of mesenchymal progenitors, an accessible stem cell source with osteogenic and chondrogenic potential, used in conjunction with biomaterials for tissue engineering. For this, regenerative medicine approaches require robust models to ensure selected cell populations can generate the desired cell type in a reproducible and measurable manner. Techniques for in vitro chondrogenic differentiation are well-established but largely qualitative, relying on sample staining and imaging. To facilitate the in vitro screening of pro-chondrogenic treatments, a 3D micropellet culture combined with three quantitative GAG assays has been developed, with a fourth parallel assay measuring sample content to enable normalisation. The effect of Transforming Growth Factor beta (TGF-?) used to validate this culture format produced a measurable increase in proteoglycan production in the parallel assays, in both 2D and 3D culture configurations. When compared to traditional micropellets, the monolayer format appeared less able to detect changes in cell differentiation, however in-well 3D cultures displayed a significant differential response. Effects on collagen 2 expression confirmed these observations. Based on these results, a microplate format was optimised for 3D culture, in a high-throughput in-well configuration. This model showed improved sensitivity and confirmed the 3D micropellet in-well quantitative assays as an effective differentiation format compatible with streamlined, high-throughput chondrogenic screens

In vitro degradation and mechanical properties of PLA-PCL copolymer unit cell scaffolds generated by two-photon polymerization

The manufacture of 3D scaffolds with specific controlled porous architecture, defined microstructure and an adjustable degradation profile was achieved using two-photon polymerization (TPP) with a size of 2  ×  4  ×  2 mm3. Scaffolds made from poly(D,L-lactide-co-ɛ-caprolactone) copolymer with varying lactic acid (LA) and ɛ -caprolactone (CL) ratios (LC16:4, 18:2 and 9:1) were generated via ring-opening-polymerization and photoactivation. The reactivity was quantified using photo-DSC, yielding a double bond conversion ranging from 70% to 90%. The pore sizes for all LC scaffolds were see 300 μm and throat sizes varied from 152 to 177 μm. In vitro degradation was conducted at different temperatures; 37, 50 and 65 °C. Change in compressive properties immersed at 37 °C over time was also measured. Variations in thermal, degradation and mechanical properties of the LC scaffolds were related to the LA/CL ratio. Scaffold LC16:4 showed significantly lower glass transition temperature (T g) (4.8 °C) in comparison with the LC 18:2 and 9:1 (see 32 °C). Rates of mass loss for the LC16:4 scaffolds at all temperatures were significantly lower than that for LC18:2 and 9:1. The degradation activation energies for scaffold materials ranged from 82.7 to 94.9 kJ mol−1. A prediction for degradation time was applied through a correlation between long-term degradation studies at 37 °C and short-term studies at elevated temperatures (50 and 65 °C) using the half-life of mass loss (Time (M1/2)) parameter. However, the initial compressive moduli for LC18:2 and 9:1 scaffolds were 7 to 14 times higher than LC16:4 (see 0.27) which was suggested to be due to its higher CL content (20%). All scaffolds showed a gradual loss in their compressive strength and modulus over time as a result of progressive mass loss over time. The manufacturing process utilized and the scaffolds produced have potential for use in tissue engineering and regenerative medicine applications

Metallic skeleton promoted two-phase durable icephobic layers

HypothesisThe accretion of ice on component surfaces often causes severe impacts or accidents in modern industries. Applying icephobic surface is considered as an effective solution to minimise the hazards. However, the durability of the current icephobic surface and coatings for long-term service remains a great challenge. Therefore, it is indeed to develop new durable material structures with great icephobic performance.ExperimentsA new design concept of combining robust porous metallic skeletons and icephobic filling was proposed. Nickel/polydimethylsiloxane (PDMS) two-phase layer was prepared using porous Ni foam skeletons impregnated with PDMS as filling material by a two-step method.FindingsGood icephobicity and mechanical durability have been verified. Under external force, micro-cracks could easily initiate at the ice/solid interface due to the small surface cavities and the difference of local elastic modulus between the ice and PDMS, which would promote the ice fracture and thus lead to low ice adhesion strength. The surface morphology and icephobicity almost remain unchanged after water-sand erosion, showing greatly improved mechanical durability. By combining the advantages of the mechanical durability of porous Ni skeleton and the icephobicity of PDMS matrix, the Ni foam/PDMS two-phase layer demonstrates great potentials for ice protection with long-term service time

Effect of surface adsorption on icing behaviour of metallic coating

Icephobicity of materials has received intensive attention in recent years due to the increasing requirement of ice protection in aerospace, wind energy and power lines. However, the influencing factors of material icephobicity have not been well identified. In this work, the effect of surface gaseous adsorption on icing behaviour of materials was investigated for the first time. Ni-Cu-P coatings with different surface morphologies were fabricated and used as the objects of the study. Environmental scanning electron microscopy (ESEM) was utilized to observe the water condensation and ice formation on the coatings. X-ray photoelectron spectroscopy (XPS) was employed to analyse the variations of surface adsorption. Droplets icing time and ice adhesion strength of the coatings were also studied. The results showed that the icing time of water droplets on the Ni-Cu-P coatings increased significantly, and the ice adhesion strength decreased sharply with the spontaneous surface adsorption of gaseous species (mainly hydrocarbon groups) in air. The adsorbed hydrocarbon species would promote the formation of air pockets between the ice-coating interface, which could effectively reduce the interfacial contact of the formed ice with the coating. When the adsorbed hydrocarbon species were removed by plasma cleaning, water droplets tended to have more direct contacts with the coatings prior to icing, leading to the formation of interlocked ice and significantly increased the ice adhesion on the surface. The variation of surface icephobicity can also be attributed to the changes of surface energy due to the surface adsorption. The results indicated that the surface gaseous adsorption in air played an important role in determining the surface icing behaviour and the icephobicity of the materials

Investigation on time-dependent wetting behavior of Ni-Cu-P ternary coating

Hydrophobic metallic coatings have attracted increasing interest in the recent years due to their excellent mechanical durability. But the wetting behavior and hydrophobic mechanism of metallic coatings are far from clear. In this work, Ni-Cu-P ternary coatings with hierarchical structure were prepared on 304 stainless steel by electrodeposition method. The surface morphologies, phase compositions, and wetting behavior were studied systemically. Time-dependent wetting behaviour of Ni-Cu-P coatings had been clearly observed, and the surface of the as-deposited coatings changed from hydrophilic state to hydrophobic state after aging in ambient air. The related surface wetting mechanism was investigated with the assistance of plasma cleaning to study the possible surface adsorption contributing to the time-dependent wetting behavior. The variations of the surface species were analyzed using X-ray photoelectron spectroscopy (XPS), showing the composition change of both carbon and the oxygen. The atomic ratio of hydrocarbon on the Ni-Cu-P coating first increased from 78.7% to 86.5% when stored in ambient air and then decreased from 82.3% to 65.9% after the plasma cleaning treatment; while the variation of oxygen content was an opposite trend. The results indicated that the observed time-dependent wettability was a combined result of the adsorption of airborne hydrocarbon and the change of lattice oxygen on the coating surface

Performance of multiphase scaffolds for bone repair based on two-photon polymerized poly(d,l-lactide-co-ɛ-caprolactone), recombinamers hydrogel and nano-HA

Multiphase hybrids were fabricated from poly(d,l-lactide-co-ɛ-caprolactone) (PLCL) copolymer scaffolds impregnated with silk-elastin-like recombinamers (SELRs) hydrogel containing 2 wt% hydroxyapatite nanoparticles (nHA). The PLCL scaffolds, triply-periodic minimal surface geometry, were manufactured using two-photon stereolithography. In vitro degradation studies were conducted on PLCL scaffolds in inflamed tissue mimic media (pH ~ 4.5–6.5) or phosphate buffered saline (PBS) at 37 °C. Compression test revealed instant shape recovery of PLCL scaffolds after compression to 70% strain, ideal for arthroscopic delivery. Degradation of these scaffolds was accelerated in acidic media, where mass loss and compressive properties at day 56 were about 2–6 times lower than the scaffolds degraded in PBS. No significant difference was seen in the compressive properties between PLCL scaffolds and the hybrids due to the order of magnitude difference between the hydrogels and the PLCL scaffolds. Moreover, degradation properties of the hybrids did not significantly change by inclusion of SELR+/−nHA nanocomposite hydrogels. The hybrids lost approximately 40% and 84% of their initial weight and mechanical properties, respectively after 112 days of degradation. Cytotoxicity assessment revealed no cytotoxic effects of PLCL or PLCL-SELR+/−2%nHA scaffolds on bone marrow-derived human Mesenchymal Stem Cells. These findings highlight the potential of these hybrid constructs for bone and cartilage repair