Effect of cholesterol on the mechanical stability of gel-phase phospholipid bilayers studied by AFM force spectroscopy.

The remarkably low sliding friction of articular cartilage in the major joints such as hips and knees, which is crucial for its homeostasis and joint health, has been attributed to lipid bilayers forming lubricious boundary layers at its surface. The robustness of such layers, and thus their lubrication efficiency at joint pressures, depends on the lipids forming them, including cholesterol which is a ubiquitous component, and which may act to strengthen of weaken the bilayer. In this work, a systematic study using an atomic force microscope (AFM) was carried out to understand the effect of cholesterol on the nanomechanical stability of two saturated phospholipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphatidlycholine) and DPPC (1,2-dipalmitoyl-sn-glycero- phosphatidylcholine), that differ in acyl chain lengths. Measurements were carried out both in water and in phosphate buffer solution (PBS). The nanomechanical stability of the lipid bilayers was quantitatively evaluated by measuring the breakthrough force needed to puncture the bilayer by the AFM tip. The molar fractions of cholesterol incorporated in the bilayers were 10% and 40%. We found that for both DSPC and DPPC, cholesterol significantly decreases the mechanical stability of the bilayers in solid-ordered (SO) phase. In accordance with the literature, the strengthening effect of salt on the lipid bilayers was also observed. For DPPC with 10 mol % cholesterol, the effect of tip properties and the experimental procedure parameters on the breakthrough forces were also studied. Tip radius (2-42 nm), material (Si, Si3N4, Au) and loading rate (40-1000 nm/s) were varied systematically. The values of the breakthrough forces measured were not significantly affected by any of these parameters, showing that the weakening effect of cholesterol does not result from such changes in experimental conditions. As we have previously demonstrated that mechanical robustness improves the tribological performance of lipid layers, this study helps to shed light on the mechanism of physiological lubrication. Nanoindentation of SDPC bilayers.

Patient-Reported Prevalence, Characteristics, and Impact of Leg Cramps in an Urban Primary Care Clinic.

This study addresses the prevalence and characteristics of leg cramps in 294 primary care patients (mean age = 46.5 years), with 51.7% reporting leg cramps. Patients who experience resting or exercise-induced leg cramps were more likely to be older and female. Cramp severity averaged 5.6 on a scale of 1-10 and disturbed sleep "sometimes" or "often" in 55% of patients. Most patients did not discuss cramps with their clinician. Our study reveals a possible shift in patients who experience leg cramps to younger age and chronicity. Resting leg cramps should be reviewed by clinicians as a symptom of declining health and advancing aging.

Organohydrogels with High-Speed Lubrication by Confining Polymer Chain Mobility by an Interpenetrated Heteronetwork.

Hydrogels with pure hydrophilic network have received much attention due to their excellent low frictional behavior. However, the lubrication performance of hydrogels is not satisfied under high-speed condition due to the energy dissipation caused by adsorbed polymer chains as well as the failure of lubricating mechanisms accompanied by the transition of lubrication regime. In this work, interpenetrating double-network organohydrogels were constructed by combining hydrophilic and oleophilic polymer networks to modify the physiochemical properties of surface polymer chains, especially the chain mobility. The oleophilic polymer network spatially restricting the mobility of the swollen hydrophilic network in water, resulted in a low coefficient of friction (ca. 0.01) compared with conventional hydrogels at high speed (0.1 m s-1 ). Meanwhile, the organohydrogels had superior wear resistance, with almost no wear observed on the sliding track after 5 k cycles of rubbing at high speed. The design concept of organohydrogels can be extended to a variety of low-wear, highly-lubricating materials.

Geometric tumor embolic budding characterizes inflammatory breast cancer.

PURPOSE: Inflammatory breast cancer (IBC) is characterized by numerous tumor emboli especially within dermal lymphatics. The explanation remains a mystery. METHODS: This study combines experimental studies with two different IBC xenografts with image algorithmic studies utilizing human tissue microarrays (TMAs) of IBC vs non-IBC cases to support a novel hypothesis to explain IBC's sina qua non signature of florid lymphovascular emboli. RESULTS: In the human TMAs, compared to tumor features like nuclear grade (size), mitosis and Ki-67 immunoreactivity which show that IBC is only modestly more proliferative with larger nuclei than non-IBC, what really sets IBC apart is the markedly greater number of tumor emboli and distinctly smaller emboli whose numbers indicate geometric or exponential differences between IBC and non-IBC. In the experimental xenograft studies, Mary-X gives rise to tight spheroids in vitro which exhibit dynamic budding into smaller daughter spheroids whereas Karen-X exhibits only loose non-budding aggregates. Furthermore Mary-X emboli also bud dramatically into smaller daughter emboli in vivo. The mechanism that regulates this involves the generation of E-cad/NTF1, a calpain-mediated cleavage 100 kDa product of 120 kDa full length membrane E-cadherin. Inhibiting this calpain-mediated cleavage of E-cadherin by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides that both penetrate the cell membrane and mimic either the cleavage site or the binding site on E-cadherin, inhibits the generation of E-cad/NTF1 in a dose-dependent manner, reduces spheroid compactness and decreases budding. CONCLUSION: Since E-cad/NFT1 retains the p120ctn binding site but loses the α-and ß-catenin sites, promoting its 360° distribution around the cell's membrane, the vacilating levels of this molecule trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of emboli seen in IBC.

Dehydration does not affect lipid-based hydration lubrication.

Phosphatidylcholine (PC) lipid bilayers at surfaces massively reduce sliding friction, via the hydration lubrication mechanism acting at their highly-hydrated phosphocholine headgroups, a central paradigm of biological lubrication, particularly at articular cartilage surfaces where low friction is crucial for joint well-being. Nanotribological measurements probed the effect on such lubrication of dehydration by dimethyl sulfoxide (DMSO), known to strongly dehydrate the phosphocholine headgroups of such PC bilayers, i.e. reduce the thickness of the inter-bilayer water layer, and thus expected to substantially degrade the hydration lubrication. Remarkably, and unexpectedly, we found that the dehydration has little effect on the friction. We used several approaches, including atomic force microscopy, small- and wide-angle X-ray scattering and all-atom molecular dynamics simulations to elucidate this. Our results show that while DMSO clearly removes hydration water from the lipid head-groups, this is offset by both higher areal head-group density and by rigidity-enhancement of the lipid bilayers, both of which act to reduce frictional dissipation. This sheds strong light on the robustness of lipid-based hydration lubrication in biological systems, despite the ubiquitous presence of bio-osmolytes which compete for hydration water.

Designer Liposomic Nanocarriers Are Effective Biofilm Eradicators.

Drug delivery via nanovehicles is successfully employed in several clinical settings, yet bacterial infections, forming microbial communities in the form of biofilms, present a strong challenge to therapeutic treatment due to resistance to conventional antimicrobial therapies. Liposomes can provide a versatile drug-vector strategy for biofilm treatment, but are limited by the need to balance colloidal stability with biofilm penetration. We have discovered a liposomic functionalization strategy, using membrane-embedded moieties of poly[2-(methacryloyloxy)ethyl phosphorylcholine], pMPC, that overcomes this limitation. Such pMPCylation results in liposomic stability equivalent to current functionalization strategies (mostly PEGylation, the present gold-standard), but with strikingly improved cellular uptake and cargo conveyance. Fluorimetry, cryo-electron, and fluorescence microscopies reveal a far-enhanced antibiotic delivery to model Pseudomonas aeruginosa biofilms by pMPC-liposomes, followed by faster cytosolic cargo release, resulting in significantly greater biofilm eradication than either PEGylation or free drug. Moreover, this combination of techniques uncovers the molecular mechanism underlying the enhanced interaction with bacteria, indicating it arises from bridging by divalent ions of the zwitterionic groups on the pMPC moieties to the negatively charged lipopolysaccharide chains emanating from the bacterial membranes. Our results point to pMPCylation as a transformative strategy for liposomal functionalization, leading to next-generation delivery systems for biofilm treatment.

Hydration Lubrication in Biomedical Applications: From Cartilage to Hydrogels.

In the course of evolution, nature has achieved remarkably lubricated surfaces, with healthy articular cartilage in the major (synovial) joints being the prime example, that can last a lifetime as they slide past each other with ultralow friction (friction coefficient µ = the force to slide surfaces past each other/load compressing the surfaces < 0.01) under physiological pressures (up to 10 MPa or more)). Such properties are unmatched by any man-made materials. The precise mechanism of low friction between such sliding cartilage tissues, which is closely related to osteoarthritis (OA), the most widespread joint disease, affecting hundreds of millions worldwide, has been studied for nearly a century, but is still not fully understood. Traditionally, the roles of load bearing by interstitial fluid within the cartilage bulk and that of thin exuded fluid films at the interface between the sliding cartilage surfaces have been proposed as the main lubrication mechanism. More recent work, however, suggests that molecular boundary layers at the surfaces of articular cartilage and other tissues play a major role in their lubrication. In particular, in recent years hydration lubrication has emerged as a new paradigm for boundary lubrication in aqueous media based on subnanometer hydration shells which massively reduce frictional dissipation. The vectors of hydration lubrication include trapped hydrated ions, hydrated surfactants, biological macromolecules, biomimetic polymers, polyelectrolytes and polyzwitterionic brushes, and close-packed layers of phosphatidylcholine (PC) vesicles, all having in common the exposure of highly hydrated groups at the slip plane. Among them, vesicles (or bilayers) of PC lipids, which are the most widespread lipid class in mammals, are exceptionally efficient lubricating elements as a result of the high hydration of the phosphocholine headgroups they expose. Such lipids are ubiquitous in joints, leading to the proposal that macromolecular surface complexes exposing PC bilayers are responsible for the remarkable lubrication of cartilage. Cartilage, comprising ∼70% water, may be considered to be a complex biological hydrogel, and studying the frictional properties of hydrogels may thus provide new insights into its lubrication mechanisms, leading in turn to novel, highly lubricious hydrogels that may be used in a variety of biomedical and other applications. A better understanding of cartilage lubrication could moreover lead to better treatments for OA, for example, through intra-articular injections of appropriate lubricants or through the creation of low-friction hydrogels that may be used as tissue engineering scaffolds for diseased cartilage. In this Account, we begin by introducing the concept and origin of hydration lubrication, extending from the seminal study of lubrication by hydrated simple ions to more complex systems. We then briefly review different modes of lubrication in synovial joints, focusing primarily on boundary lubrication. We consider modes of hydrogel lubrication and different kinds of such low-friction synthetic gels and then focus on cartilage-inspired, boundary-lubricated hydrogels. We conclude by discussing challenges and opportunities.

Poly-phosphocholination of liposomes leads to highly-extended retention time in mice joints.

Surface-attached layers of phosphatidylcholine (PC) lipid vesicles (liposomes) may reduce the friction coefficient µ (= force-to-slide/load) between the sliding surfaces down to µ ≈ 10-3-10-4 up to tens of atm contact pressures, as high as those in the major joints (hips or knees). Such friction reduction is attributed to hydration lubrication by the highly-hydrated phosphocholine head-groups exposed at the outer vesicle surfaces. It has been suggested therefore that intra-articular (IA) administration of liposomes as potential boundary lubricants may alleviate degenerative, friction-associated joint conditions such as osteoarthritis (OA), which is associated with insufficient lubrication at the articular cartilage surface. To overcome the problem, common to all nanoparticles, of rapid removal by the mononuclear phagocyte system, as well as to ensure long-term colloidal stability during storage, functionalizing liposomes with poly(ethylene glycol) moieties, PEGylation, is often used. Here we describe a different liposome functionalization approach, using poly(2-methacryloyloxyethyl phosphorylcholine), PMPC, moieties (strictly, lipid-PMPC conjugates), and compare the retention time in mice joints of such PMPCylated liposomes with otherwise-identical but PEGylated vesicles following IA administration. We find, using fluorescence labeling and in vivo optical imaging, that when PMPC-stabilized liposomes are injected into mice knee joints, there is a massive increase of the vesicles' retention half-life in the joints of about (4-5)-fold (ca. 300-400% increase in retention time) compared with the PEGylated liposomes (and some 100-fold longer than the retention time of intra-articularly injected hyaluronan or HA). Such PMPCylated liposomes are therefore promising candidates as potential long-lived boundary lubricants at the articular cartilage surface, with implication for friction-associated pathologies. Moreover, as lipid vesicles are well known to be efficient drug carriers, such long retention in the joints may enable analgesic or anti-inflammatory agents for joint pathologies to be more efficiently delivered via IA administration using PMPCylated liposomal vehicles relative to PEGylated ones.

Direct measurement of the viscoelectric effect in water.

The viscoelectric effect concerns the increase in viscosity of a polar liquid in an electric field due to its interaction with the dipolar molecules and was first determined for polar organic liquids more than 80 y ago. For the case of water, however, the most common polar liquid, direct measurement of the viscoelectric effect is challenging and has not to date been carried out, despite its importance in a wide range of electrokinetic and flow effects. In consequence, estimates of its magnitude for water vary by more than three orders of magnitude. Here, we measure the viscoelectric effect in water directly using a surface force balance by measuring the dynamic approach of two molecularly smooth surfaces with a controlled, uniform electric field between them across highly purified water. As the water is squeezed out of the gap between the approaching surfaces, viscous damping dominates the approach dynamics; this is modulated by the viscoelectric effect under the uniform transverse electric field across the water, enabling its magnitude to be directly determined as a function of the field. We measured a value for this magnitude, which differs by one and by two orders of magnitude, respectively, from its highest and lowest previously estimated values.

Recent Progress in Cartilage Lubrication.

Healthy articular cartilage, covering the ends of bones in major joints such as hips and knees, presents the most efficiently-lubricated surface known in nature, with friction coefficients as low as 0.001 up to physiologically high pressures. Such low friction is indeed essential for its well-being. It minimizes wear-and-tear and hence the cartilage degradation associated with osteoarthritis, the most common joint disease, and, by reducing shear stress on the mechanotransductive, cartilage-embedded chondrocytes (the only cell type in the cartilage), it regulates their function to maintain homeostasis. Understanding the origins of such low friction of the articular cartilage, therefore, is of major importance in order to alleviate disease symptoms, and slow or even reverse its breakdown. This progress report considers the relation between frictional behavior and the cellular mechanical environment in the cartilage, then reviews the mechanism of lubrication in the joints, in particular focusing on boundary lubrication. Following recent advances based on hydration lubrication, a proposed synergy between different molecular components of the synovial joints, acting together in enabling the low friction, has been proposed. Additionally, recent development of natural and bio-inspired lubricants is reviewed.

Modulating Interfacial Energy Dissipation via Potential-Controlled Ion Trapping.

As a metal (gold) surface at a given, but variable potential slides past a dielectric (mica) surface at a fixed charge, across aqueous salt solutions, two distinct dissipation regimes may be identified. In regime I, when the gold potential is such that counterions are expelled from between the surfaces, which then come to adhesive contact, the frictional dissipation is high, with coefficient of friction µ ≈ 0.8-0.9. In regime II, when hydrated counterions are trapped between the compressed surfaces, hydration lubrication is active and friction is much lower, µ = 0.05 ± 0.03. Moreover, the dissipation regime as the surfaces contact is largely retained even when the metal potential changes to the other regime, attributed to the slow kinetics of counterion expulsion from or penetration into the subnanometer intersurface gap. Our results indicate how frictional dissipation between such a conducting/nonconducting couple may be modulated by the potential applied to the metal.

Lipid-Bilayer Assemblies on Polymer-Bearing Surfaces: The Nature of the Slip Plane in Asymmetric Boundary Lubrication.

Phospholipid-macromolecule complexes have been proposed to form highly efficient, lubricating boundary layers at artificial soft surfaces or at biological surfaces such as articular cartilage, where the friction reduction is attributed to the hydration lubrication mechanism acting at the exposed, hydrated head groups of the lipids. Here we measure, using a surface force balance, the normal and frictional interactions between model mica substrates across several different configurations of phosphatidylcholine (PC) lipid aggregates and adsorbed polymer (PEO) layers, to provide insight into the nature of such lubricating boundary layers in both symmetric and especially asymmetric configurations. Our results reveal that, irrespective of the configuration, the slip plane between the sliding surfaces reverts wherever possible to a bilayer-bilayer interface where hydration lubrication reduces the friction strongly. Where such an interface is not available, the sliding friction remains high. These findings may account for the low friction observed between both biological and synthetic hydrogel surfaces which may be asymmetrically coated with lipid-based boundary layers and fully support the hydration lubrication mechanism attributed to act at such boundary layers.

Cartilage-inspired, lipid-based boundary-lubricated hydrogels.

The lubrication of hydrogels arises from fluid or solvated surface phases. By contrast, the lubricity of articular cartilage, a complex biohydrogel, has been at least partially attributed to nonfluid, lipid-exposing boundary layers. We emulated this behavior in synthetic hydrogels by incorporating trace lipid concentrations to create a molecularly thin, lipid-based boundary layer that renews continuously. We observed a 80% to 99.3% reduction in friction and wear relative to the lipid-free gel, over a wide range of conditions. This effect persists when the gels are dried and then rehydrated. Our approach may provide a method for sustained, extreme lubrication of hydrogels in applications from tissue engineering to clinical diagnostics.

Effects of Hyaluronan Molecular Weight on the Lubrication of Cartilage-Emulating Boundary Layers.

Osteoarthritic joints contain lower-molecular-weight (MW) hyaluronan (hyaluronic acid, HA) than healthy joints. To understand the relevance of this HA size effect for joint lubrication, the friction and surface structure of cartilage-emulating surfaces with HA of different MWs were studied using a surface force balance (SFB) and atomic force microscopy (AFM). Gelatin (gel)-covered mica surfaces were coated with high-MW HA (HHA), medium-MW HA (MHA), or low-MW HA (LHA), and lipids of hydrogenated soy l-α-phosphatidylcholine (HSPC) in the form of small unilamellar vesicles, using a layer-by-layer assembly method. SFB results indicate that the gel-HHA-HSPC boundary layer provides very efficient lubrication, attributed to hydration lubrication at the phosphocholine headgroups exposed by the HA-attached lipids, with friction coefficients (COF) as low as 10-3-10-4 at contact stresses at least up to P = 120 atm. However, for the gel-MHA-HSPC and gel-LHA-HSPC surfaces, the friction, initially low, increases sharply at much lower pressures (up to 30-60 atm at most). This higher friction with the shorter chains may be due to their weaker total adhesion energy to the gelatin, where the attraction between the negatively charged HA and the weakly positively charged gelatin is attributed largely to counterion-release entropy. Thus, the complexes of LHA and MHA with the lubricating HSPC lipids are more easily removed by shear during sliding, especially at high stresses, than the HHA-HSPC complex, which is strongly adhered to gelatin. This is ultimately the reason for lower-pressure lubrication breakdown with the shorter polysaccharides. Our results provide molecular-level insight into why the decrease in HA molecular weight in osteoarthritic joints may be associated with higher friction at the articular cartilage surface, and may have relevance for treatments of osteoarthritis involving intra-articular HA injections.

The Role of Hyaluronic Acid in Cartilage Boundary Lubrication.

Hydration lubrication has emerged as a new paradigm for lubrication in aqueous and biological media, accounting especially for the extremely low friction (friction coefficients down to 0.001) of articular cartilage lubrication in joints. Among the ensemble of molecules acting in the joint, phosphatidylcholine (PC) lipids have been proposed as the key molecules forming, in a complex with other molecules including hyaluronic acid (HA), a robust layer on the outer surface of the cartilage. HA, ubiquitous in synovial joints, is not in itself a good boundary lubricant, but binds the PC lipids at the cartilage surface; these, in turn, massively reduce the friction via hydration lubrication at their exposed, highly hydrated phosphocholine headgroups. An important unresolved issue in this scenario is why the free HA molecules in the synovial fluid do not suppress the lubricity by adsorbing simultaneously to the opposing lipid layers, i.e., forming an adhesive, dissipative bridge between them, as they slide past each other during joint articulation. To address this question, we directly examined the friction between two hydrogenated soy PC (HSPC) lipid layers (in the form of liposomes) immersed in HA solution or two palmitoyl-oleoyl PC (POPC) lipid layers across HA-POPC solution using a surface force balance (SFB). The results show, clearly and surprisingly, that HA addition does not affect the outstanding lubrication provided by the PC lipid layers. A possible mechanism indicated by our data that may account for this is that multiple lipid layers form on each cartilage surface, so that the slip plane may move from the midplane between the opposing surfaces, which is bridged by the HA, to an HA-free interface within a multilayer, where hydration lubrication is freely active. Another possibility suggested by our model experiments is that lipids in synovial fluid may complex with HA, thereby inhibiting the HA molecules from adhering to the lipids on the cartilage surfaces.

Automated Detection of Helium Bubbles in Irradiated X-750.

Imaging nanoscale features using transmission electron microscopy is key to predicting and assessing the mechanical behavior of structural materials in nuclear reactors. Analyzing these micrographs is often a tedious and labour intensive manual process. It is a prime candidate for automation. Here, a region-based convolutional neural network is adapted to detect helium bubbles in micrographs of neutron-irradiated Inconel X-750 reactor spacer springs. We demonstrate that this neural network produces analyses of similar accuracy and reproducibility to that produced by humans. Further, we show this method as being four orders of magnitude faster than manual analysis allowing for generation of significant quantities of data. The proposed method can be used with micrographs of different Fresnel contrasts and magnification levels.

Designer Nanoparticles as Robust Superlubrication Vectors.

Phosphatidylcholine lipid bilayers or liposomes at interfaces in aqueous environments can provide extremely efficient lubrication. This is attributed to the hydration lubrication mechanism acting at the highly hydrated phosphocholine-headgroup layers exposed at the outer surface of each bilayer. Micelles exposing such phosphocholine groups could be an attractive alternative to liposomes due to their much easier preparation and structure control, but all studies to date of surfactant micelles have revealed that at relatively low normal stresses the surface layers rupture and friction increases abruptly. Here, we examine surface interactions between three kinds of phosphocholine-exposing micelles with different designed structures: single-tail surfactant micelles, homo-oligomeric micelles, and block copolymer micelles. Normal and shear forces between mica surfaces immersed in solutions of these micelles were measured using a surface force balance. The adsorbed layers on the mica were imaged using atomic force microscope, revealing surface structures ranging from wormlike to spherical micelles. The block copolymer micelles showed relatively low coverage arising from their stabilizing corona and consequently poor lubrication (µ ∼ 10-1). In contrast, the surfactant and homo-oligomeric micelles fully covered the mica surface and demonstrated excellent lubrication (µ ∼ O(10-3)). However, while the boundary layer of single-tailed surfactant micelles degraded under moderate pressure, the homo-oligomeric micellar boundary layer was robust at all applied contact pressures in our study (up to about 5 MPa). We attribute the difference to the much greater energy required to remove a homo-oligomeric molecule from its micelle, resulting in far greater stability under pressure and shear.