One of the major constituents of the synovial fluid that is thought to be responsible for chondroprotection and boundary lubrication is the glycoprotein lubricin (PRG4); however, the molecular mechanisms by which lubricin carries out its critical functions still remain largely unknown. We hypothesized that the interaction of lubricin with type II collagen, the main component of the cartilage extracellular matrix, results in enhanced tribological and wear properties. In this study, we examined: i) the molecular details by which lubricin interacts with type II collagen and how binding is related to boundary lubrication and adhesive interactions; and, ii) whether collagen structure can affect lubricin adsorption and its chondroprotective properties. We found that lubricin adsorbs strongly onto denatured, amorphous, and fibrillar collagen surfaces. Furthermore, we found large repulsive interactions between the collagen surfaces in presence of lubricin, which increased with increasing lubricin concentration. Lubricin attenuated the large friction and also the long-range adhesion between fibrillar collagen surfaces. Interestingly, lubricin adsorbed onto and mediated the frictional response between the denatured and native amorphous collagen surfaces equally and showed no preference on the supramolecular architecture of collagen. However, the coefficient of friction was lowest on fibrillar collagen in the presence of lubricin. We speculate that an important role of lubricin in mediating interactions at the cartilage surface is to attach to the cartilage surface and provide a protective coating that maintains the contacting surfaces in a sterically repulsive state.
Colloidal Probe Microscopy; Lateral Force Microscopy; Atomic Force Microscope; Friction; Tribology; PRG4; Wear; Glycoproteins; Collagen; Type II Collagen Lubricin; SAMs; Boundary Lubrication
Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive loss of articular cartilage. While macroscale degradation of the cartilage extracellular matrix (ECM) has been extensively studied, microscale changes in the chondrocyte pericellular matrix (PCM) and immediate microenvironment with OA are not fully understood. The objective of this study was to quantify osteoarthritic changes in the micromechanical properties of the ECM and PCM of human articular cartilage in situ using atomic force microscopy (AFM).
AFM elastic mapping was performed on cryosections of human cartilage harvested from both condyles of macroscopically normal and osteoarthritic knee joints. This method was used to test the hypotheses that both ECM and PCM regions exhibit a loss of mechanical properties with OA and that the size of the PCM is enlarged in OA cartilage as compared to normal tissue.
Significant decreases were observed in both ECM and PCM moduli of 45% and 30%, respectively, on the medial condyle of OA knee joints as compared to cartilage from macroscopically normal joints. Enlargement of the PCM, as measured biomechanically, was also observed in medial condyle OA cartilage, reflecting the underlying distribution of type VI collagen in the region. No significant differences were observed in elastic moduli or their spatial distribution on the lateral condyle between normal and OA joints.
Our findings provide new evidence of significant site-specific degenerative changes in the chondrocyte micromechanical environment with OA.
Atomic force microscopy; chondron; type VI collagen; scanning probe microscopy; proteoglycan
Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle-vesicle and vesicle-substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates.
In this study, we present a technique to create a complex, high cholesterol-containing supported lipid bilayers (SLBs) using α-helical (AH) peptide-induced vesicle fusion. Vesicles consisting of POPC : POPE : POPS : SM : Chol (9.35 : 19.25 : 8.25 : 18.15 : 45.00) were used to form a SLB that models the native composition of the human immunodeficiency virus-1 (HIV-1) lipid envelope. In the absence of AH peptides, these biomimetic vesicles fail to form a complete SLB. We verified and characterized AH peptide-induced vesicle fusion by quartz crystal microbalance with dissipation monitoring, neutron reflectivity, and atomic force microscopy. Successful SLB formation entailed a characteristic frequency shift of −35.4 ± 2.0 Hz and a change in dissipation energy of 1.91 ± 0.52 × 10−6. Neutron reflectivity measurements determined the SLB thickness to be 49.9 +1.9−1.5 Å, and showed the SLB to be 100 +0.0−0.1% complete and void of residual AH peptide after washing. Atomic force microscopy imaging confirmed complete SLB formation and revealed three distinct domains with no visible defects. This vesicle fusion technique gives researchers access to a complex SLB composition with high cholesterol content and thus the ability to better recapitulate the native HIV-1 lipid membrane.
Our work is motivated by the observation that rare, broadly neutralizing antibodies (NAbs), 4E10 and 2F5, associate with HIV-1 lipids as part of a required first step in neutralization before binding to membrane-proximal antigens. Subsequently, induction of these types of NAbs may be limited by immunologic tolerance due to autoreactivity with host cell membranes. Despite the significance of this lipid reactivity there is little experimental evidence detailing NAb-membrane interactions. Simple and efficient screening assays are needed to select antibodies that have similar lipid reactivity as known NAbs. To this end we have developed a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol self-assembled monolayers (SAMs) that replicate salient lipid surface chemistries and NAb binding to lipid surfaces. Specifically, we probed the relative importance of charge and hydrophobicity on antibody-surface interactions. We found that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies (mAbs). Furthermore, we confirmed the importance of charge-mediated antibody surface interactions, originally suggested by results from mAb interactions with conventional lipid vesicle/bilayer surfaces. Our approach, using self-assembled thiol monolayers that replicate the binding behavior of NAbs on lipid surfaces, thus provides an efficient and useful tool to screen interactions of mAbs and lipid-reactive NAbs.
HIV-1; Neutralizing antibody; Thiol model surface; Surface plasmon resonance
The mechanical properties of organic and biomolecular thin films on surfaces play an important role in a broad range of applications. Although force-modulation microscopy (FMM) is used to map the apparent elastic properties of such films with high lateral resolution in air, it has rarely been applied in aqueous media. In this letter we describe the use of FMM to map the apparent elastic properties of self-assembled monolayers and end-tethered protein thin films in aqueous media. Furthermore, we describe a simple analysis of the contact mechanics that enables the selection of FMM imaging parameters and thus yields a reliable interpretation of the FMM image contrast.
acoustic atomic force microscopy; biomolecules; elastic modulus mapping; nanomechanical characterization; self-assembled monolayers
We exploit a series of robust, but simple and convenient colloidal lithography (CL) approaches, using a microsphere array as a mask or as a guiding template, and combine this with surface-initiated atom-transfer radical polymerization (SI-ATRP) to fabricate patterned polymer-brush microstructures. The advantages of the CL technique over other lithographic approaches for the fabrication of patterned polymer brushes are (i) that it can be carried out with commercially available colloidal particles at a relatively low cost, (ii) that no complex equipment is required to create the patterned templates with micro- and nanoscale features, and (iii) that polymer brush features are controlled simply by changing the size or chemical functionality of the microspheres or the substrate.
atom-transfer radical polymerization; colloidal lithography; patterning; self-assembled microsphere monolayer
Mutation or loss of collagen VI has been linked to a variety of musculoskeletal abnormalities, particularly muscular dystrophies, tissue ossification and/or fibrosis, and hip osteoarthritis. However, the role of collagen VI in bone and cartilage structure and function in the knee is unknown. In this study, we examined the role of collagen VI in the morphology and physical properties of bone and cartilage in the knee joint of Col6a1−/− mice by micro-computed tomography (microCT), histology, atomic force microscopy (AFM), and scanning microphotolysis (SCAMP). Col6a1−/− mice showed significant differences in trabecular bone structure, with lower bone volume, connectivity density, trabecular number, and trabecular thickness but higher structure model index and trabecular separation compared to Col6a1+/+ mice. Subchondral bone thickness and mineral content increased significantly with age in Col6a1+/+ mice, but not in Col6a1−/− mice. Col6a1−/− mice had lower cartilage degradation scores, but developed early, severe osteophytes compared to Col6a1+/+mice. In both groups, cartilage roughness increased with age, but neither the frictional coefficient nor compressive modulus of the cartilage changed with age or genotype, as measured by AFM. Cartilage diffusivity, measured via SCAMP, varied minimally with age or genotype. The absence of type VI collagen has profound effects on knee joint structure and morphometry, yet minimal influences on the physical properties of the cartilage. Together with previous studies showing accelerated hip osteoarthritis in Col6a1−/− mice, these findings suggest different roles for collagen VI at different sites in the body, consistent with clinical data.
To assess the role of Prg4 in joint lubrication and chondroprotection by measuring friction, stiffness, surface topography, and subsurface histology of the hip joints of Prg4-/- and wild-type mice.
Friction and elastic modulus were measured on cartilage of femoral heads of Prg4-/- and wild-type mice aged 2, 4, 10, and 16 weeks using atomic force microscopy and the surface microstructure was imaged. Histologic sections of each femoral head were stained and graded.
Histologic analysis of Prg4-/- joints showed an enlarged, fragmented surface layer of variable thickness with Safranin-O positive formations sometimes present, a roughened underlying articular cartilage surface, and a progressive loss of pericellular proteoglycans. Friction was significantly higher on Prg4-/- cartilage at 16 weeks but statistically significant differences in friction were not detected at younger ages. The elastic modulus of the cartilage was similar between Prg4-/- and wild-type cartilage surfaces at young ages but wild-type cartilage showed increasing stiffness with age with significantly higher moduli than Prg4-/- cartilage at later ages.
Deletion of Prg4 results in significant structural and biomechanical changes in the articular cartilage with age, some of which are consistent with osteoarthritic degeneration. These findings suggest that Prg4 plays a significant role in preserving normal joint structure and function.
Lubricin and hyaluronic acid (HA), molecular constituents of synovial fluid, have long been theorized to play a role in joint lubrication and wear protection. While lubricin has been shown to function as a boundary lubricant, conflicting evidence exists as to the boundary lubricating ability of hyaluronic acid. Here, we use colloidal force microscopy to explore the friction behavior of these two molecules on the microscale between chemically uniform hydrophilic (hydroxyl-terminated) and hydrophobic (methyl-terminated) surfaces in physiological buffer solution. Behaviors on both surfaces are physiologically relevant since the heterogeneous articular cartilage surface contains both hydrophilic and hydrophobic elements. Friction between hydrophobic surfaces was initially high (μ=1.1, at 100nN of applied normal load) and was significantly reduced by lubricin addition while friction between hydrophilic surfaces was initially low (μ=0.1) and was slightly increased by lubricin addition. At lubricin concentrations above 200 µg/ml, friction behavior on the two surfaces was similar (μ=0.2) indicating that nearly all interaction between the two surfaces was between adsorbed lubricin molecules rather than between the surfaces themselves. In contrast, addition of HA did not appreciably alter the frictional behavior between the model surfaces. No synergistic effect on friction behavior was seen in a physiological mixture of lubricin and HA. Lubricin can equally mediate the frictional response between both hydrophilic and hydrophobic surfaces, likely fully preventing direct surface-to-surface contact at sufficient concentrations, whereas HA provides considerably less boundary lubrication.
Colloidal force microscopy; Lateral force microscopy; Atomic force microscope; Friction; Tribology; PRG4; Wear; Glycoproteins; Hyaluronic acid; Lubricin; Joint lubrication; Cartilage lubrication; Self-assembled monolayer; Synovial lubricants
Chondrocytes in articular cartilage normally exhibit high expression of collagen II and aggrecan but rapidly dedifferentiate to a fibroblastic phenotype if passaged in culture. Previous studies have suggested that the loss of chondrocyte phenotype is associated with changes in the structure of the F-actin cytoskeleton, which also controls cell mechanical properties. In this study, we examined how dedifferentiation in monolayer influences the mechanical properties of chondrocytes isolated from different zones of articular cartilage. Atomic force microscopy was used to measure the mechanical properties of superficial and middle/deep zone chondrocytes as they underwent serial passaging and subsequent growth on fibronectin-coated, micropatterned self-assembled monolayers (MSAMs) that restored a rounded cell shape in 2D culture. Chondrocytes exhibited significant increases in elastic and viscoelastic moduli with dedifferentiation in culture. These changes were only partially ameliorated by the restoration of a rounded shape on micropatterned surfaces. Furthermore, intrinsic zonal differences in cell mechanical properties were rapidly lost with passage. These findings indicate that cell mechanical properties may provide additional measures of phenotypic expression of chondrocytes as they undergo dedifferentiation and possibly redifferentiation in culture.
atomic force microscope; cell mechanics; micropattern; cartilage; monolayer expansion; morphology; chondrocyte; indentation; differentiation
The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or “biomarkers,” of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations.
cell mechanics; atomic force microscopy; osteoblast; chondrocyte; adipocyte; ADAS cell; MSC; mechanotransduction
We present an experimental analysis of the plasmonic scattering properties of gold nanoparticles controllably placed nanometers away from a gold metal film. We show that the spectral response of this system results from the interplay between the localized plasmon resonance of the nanoparticle and the surface plasmon polaritons of the gold film, as previously predicted by theoretical studies. In addition, we report that the metal film induces a polarization to the single nanoparticle light scattering, resulting in a doughnut-shaped point spread function when imaged in the far-field. Both the spectral response and the polarization effects are highly sensitive to the nanoparticle–film separation distance. Such a system shows promise in potential biometrology and diagnostic devices.
A polymer brush consisting of 70% poly(N-isopropylacrylamide) (PNIPAAM) and 30% polymethacrylic acid (PMAA) was synthesized from gold substrates with a grafting-from AIBN type free-radical initiator. Fractionation of two peptides, Bradykinin and Buccalin, was accomplished in less than 120 seconds by placing a 30 pM (pH∼6.2) droplet onto the polymer brush substrate. The eluant containing the anionic Buccalin is pipetted away for MALDI analysis while the cationic Bradykinin adsorbed to the swollen anionic brush and was subsequently released by adding a droplet of formic acid to the substrate. This caused the brush to collapse and release the Bradykinin, much like squeezing a sponge; these nanosponge substrates exhibited very high loading capacity (>2.0 mg/ml) compared to plasma-polymer-modified MALDI substrates. Ellipsometric measurements showed that complementary peptides adsorb rapidly while those of the same charge do not and MALDI-MS analysis of the two fractions showed separation of both peptides. The adsorption of Bradykinin was monitored over time and 85% of the peptide had been adsorbed to the nanosponge in 1 minute from a 0.5 mg/ml aqueous solution.
We investigated the effect of temperature, ionic strength, solvent polarity, and type of guest residue on the force—extension behavior of single, end-tethered elastin-like polypeptides (ELPs), using single molecule force spectroscopy (SMFS). ELPs are stimulus-responsive polypeptides that contain repeats of the five amino acids Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. We fitted the force—extension data with a freely jointed chain (FJC) model which allowed us to resolve small differences in the effective Kuhn segment length distributions that largely arise from differences in the hydrophobic hydration behavior of ELP. Our results agree qualitatively with predictions from recent molecular dynamics simulations and demonstrate that hydrophobic hydration modulates the molecular elasticity for ELPs. Furthermore, our results show that SMFS, when combined with our approach for data analysis, can be used to study the subtleties of polypeptide—water interactions and thus provides a basis for the study of hydrophobic hydration in intrinsically unstructured biomacromolecules.
Articular cartilage provides a low-friction, wear resistant surface for the motion of diarthrodial joints. The objective of this study was to develop a method for in situ friction measurement of murine cartilage using a colloidal probe attached to the cantilever of an atomic force microscope. Sliding friction was measured between a chemically functionalized microsphere and the cartilage of the murine femoral head. Friction was measured at normal loads ranging incrementally from 20 nN to 100 nN with a sliding speed of 40 μm/s and sliding distance of 64 μm. Under these test conditions, hydrostatic pressurization and biphasic load support in the cartilage were minimized, providing frictional measurements that predominantly reflect boundary lubrication properties. Friction coefficients measured on murine tissue (0.25±0.11) were similar to those measured on porcine tissue (0.23±0.09) and were in general agreement with measurements of boundary friction on cartilage by other researchers. Using the colloidal probe as an indenter, the elastic mechanical properties and surface roughness were measured in the same configuration. Interfacial shear was found to be the principal mechanism of friction generation, with little to no friction resulting from plowing forces, collision forces, or energy losses due to normal deformation. This measurement technique can be applied to future studies of cartilage friction and mechanical properties on genetically altered mice or other small animals.
Scanning probe microscopy; Boundary lubrication; Lubricin; Tribology
To assess the role of the glycoprotein PRG4 in joint lubrication and chondroprotection by measuring friction, stiffness, surface topography, and subsurface histology of the hip joints of Prg4−/− and wild-type (WT) mice.
Friction and elastic modulus were measured in cartilage from the femoral heads of Prg4−/− and WT mice ages 2, 4, 10, and 16 weeks using atomic force microscopy, and the surface microstructure was imaged. Histologic sections of each femoral head were stained and graded.
Histologic analysis of the joints of Prg4−/− mice showed an enlarged, fragmented surface layer of variable thickness with Safranin O–positive formations sometimes present, a roughened underlying articular cartilage surface, and a progressive loss of pericellular proteoglycans. Friction was significantly higher on cartilage of Prg4−/− mice at age 16 weeks, but statistically significant differences in friction were not detected at younger ages. The elastic modulus of the cartilage was similar between cartilage surfaces of Prg4−/− and WT mice at young ages, but cartilage of WT mice showed increasing stiffness with age, with significantly higher moduli than cartilage of Prg4−/− mice at older ages.
Deletion of the gene Prg4 results in significant structural and biomechanical changes in the articular cartilage with age, some of which are consistent with osteoarthritic degeneration. These findings suggest that PRG4 plays a significant role in preserving normal joint structure and function.