Yulia Merkher

Liposomes act as effective biolubricants for friction reduction in human synovial joints.

Phospholipids (PL) form the matrix of biological membranes and of the lipoprotein envelope monolayer, and are responsible for many of the unique physicochemical, biochemical, and biological properties of these supermolecular bioassemblies. It was suggested that phospholipids present in the synovial fluid (SF) and on the surface of articular cartilage have major involvement in the low friction of cartilage, which is essential for proper mobility of synovial joints. In pathologies, such as impaired biolubrication (leading to common joint disorders such as osteoarthritis), the level of phospholipids in the SF is reduced. Using a human-sourced cartilage-on-cartilage setup, we studied to what extent and how phospholipids act as highly effective cartilage biolubricants. We found that large multilamellar vesicles (MLV), >800 nm in diameter, composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or of a mixture of DMPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) are superior lubricants in comparison to MLV composed of other phosphatidylcholines. Introducing cholesterol into liposomes resulted in less effective lubricants. DMPC-MLV was also superior to small unilamellar vesicles (SUV), <100 nm in diameter, composed of DMPC. MLV are superior to SUV due to MLV retention at and near (<200 microm below) the cartilage surface, while SUV penetrate deeper into the cartilage (450-730 microm). Superiority of specific PL compositions is explained by the thermotropic behavior (including compressibility) of the lipid bilayer. Correlating physicochemical properties of the MLV with the friction results suggests that MLV having lipid bilayers in the liquid-disordered phase and having a solid-ordered to liquid-disordered phase transition temperature slightly below physiological temperature are optimal for lubrication. High phospholipid headgroup hydration, high compressibility, and softness are the common denominators of all efficient PL compositions. The high efficiency of DMPC-MLV and DMPC/DPPC-MLV as cartilage lubricants combined with their resistance to degradation at 37 degrees C supports further evaluation of these MLV for treatment of joint impairments related to poor lubrication. This work also demonstrates the relevance of basic physicochemical properties of phospholipids to their activities in biological systems.

Articular Cartilage Proteoglycans As Boundary Lubricants: Structure and Frictional Interaction of Surface-Attached Hyaluronan and Hyaluronan–Aggrecan Complexes

Mammalian synovial joints are extremely efficient lubrication systems reaching friction coefficient μ as low as 0.001 at high pressures (up to 100 atm) and shear rates (up to 10(6) to 10(7) Hz); however, despite much previous work, the exact mechanism responsible for this behavior is still unknown. In this work, we study the molecular mechanism of synovial joint lubrication by emulating the articular cartilage superficial zone structure. Macromolecules extracted and purified from bovine hip joints using well-known biochemical techniques and characterized with atomic force microscope (AFM) have been used to reconstruct a hyaluronan (HA)--aggrecan layer on the surface of molecularly smooth mica. Aggrecan forms, with the help of link protein, supramolecular complexes with the surface-attached HA similar to those at the cartilage/synovial fluid interface. Using a surface force balance (SFB), normal and shear interactions between a HA--aggrecan-coated mica surface and bare mica have been examined, focusing, in particular, on the frictional forces. In each stage, control studies have been performed to ensure careful monitoring of the macromolecular surface layers. We found the aggrecan--HA complex to be a much better boundary lubricant than the HA alone, an effect attributed largely to the fluid hydration sheath bound to the highly charged glycosaminoglycan (GAG) segments on the aggrecan core protein. A semiquantitative model of the osmotic pressure is used to describe the normal force profiles between the surfaces and interpret the boundary lubrication mechanism of such layers.

Biochemical composition and turnover of the extracellular matrix of the normal and degenerate intervertebral disc

BackgroundThe intervertebral disc (IVD) is a complex cartilaginous structure which functions to resist biomechanical loads during spinal movement. It consists of the highly viscous cartilaginous nucleus pulposus, which is surrounded laterally by a thick outer ring of fibrous cartilage—the annulus fibrosus—and sandwiched inferiorly and superiorly by the cartilage end-plates. The main extracellular matrix molecules of the disc are collagens, proteoglycans, glycoproteins and elastin. The disc also contains appreciable amounts of water, matrix-degrading protease enzymes and their inhibitors, soluble signalling molecules and various metabolic breakdown products.MethodsThis review provides a comprehensive description of the biochemical composition of the extracellular matrix of the IVD and, specifically, the proteases involved in its molecular turnover. Quantitation of the turnover rates using racemization of aspartic acid as a molecular clock is also discussed.ConclusionsMolecular turnover rates of the major constituent matrix macromolecules of the IVD are found to be particularly slow, especially in the case of collagen. Over a normal human life span, this slow turnover may compromise the structural integrity of the IVD extracellular matrix essential for normal physiological functioning.

Normal and Shear Interactions between Hyaluronan−Aggrecan Complexes Mimicking Possible Boundary Lubricants in Articular Cartilage in Synovial Joints

Using a surface force balance, normal and shear interactions have been measured between two atomically smooth surfaces coated with hyaluronan (HA), and with HA/aggrecan (Agg) complexes stabilized by cartilage link protein (LP). Such HA/Agg/LP complexes are the most abundant mobile macromolecular species permeating articular cartilage in synovial joints and have been conjectured to be present as boundary lubricants at its surface. The aim of the present study is to gain insight into the extremely efficient lubrication when two cartilage surfaces slide past each other in healthy joints, and in particular to elucidate the possible role in this of the HA/Agg/LP complexes. Within the range of our parameters, our results reveal that the HA/Agg/LP macromolecular surface complexes are much better boundary lubricants than HA alone, likely because of the higher level of hydration, due to the higher charge density, of the HA/Agg/LP layers with respect to the HA alone. However, the friction coefficients (μ) associated with the mutual interactions and sliding of opposing HA/Agg/LP layers (μ ≈ 0.01 up to pressure P of ca. 12 atm, increasing sharply at higher P) suggest that such complexes by themselves cannot account for the remarkable boundary lubrication observed in mammalian joints (up to P > 50 atm).

Advances in the diagnosis of degenerated lumbar discs and their possible clinical application

PurposeOne possible source of chronic low back pain is a degenerated intervertebral disc. In this review, various diagnostic methods for the assessment of the presence of degenerative changes are described. These include clinical MRI, a number of novel MRI techniques and nuclear magnetic resonance spectroscopy.MethodsNon-systematic literature review.ResultsClinical MRI is the most commonly employed technique to determine the general “health status” of the intervertebral disc. Novel MRI techniques, such as quantitative MRI, T1ρ MRI, sodium MRI and nuclear magnetic resonance spectroscopy, are more sensitive in quantifying the biochemical changes of disc degeneration, as measured by alteration in collagen structure, as well as water and proteoglycan loss. As potential future diagnostic alternatives, miniature sensors are currently being developed to measure parameters associated with the disc degeneration cascade, such as intradiscal pressure and PG concentration. However, none of the methods listed above show sufficient specificity to identify a degenerated disc as the actual source of the pain. Provocative discography is the only test aimed at a direct diagnosis of discogenic pain, but it has a high false positive rate and there is some evidence of long-term adverse effects. Imaging techniques have also been tested for this purpose, but their validity has not been confirmed and they do appear to be problematic.ConclusionsA reliable diagnostic tool that could help a clinician to determine if a disc is the source of the pain in patients with chronic LBP is still not available. New MRI techniques are under investigation that could result in a significant improvement over current methods, particularly as they can allow monitoring, not only of morphological but also of biochemical changes.

Longevity of elastin in human intervertebral disc as probed by the racemization of aspartic acid

BACKGROUND Aging and degeneration of human intervertebral disc (IVD) are associated with biochemical changes, including racemization and glycation. These changes can only be counteracted by protein turnover. Little is known about the longevity of IVD elastin in health or disease. Yet, such knowledge is important for a quantitative understanding of tissue synthesis and degradation. METHODS We have measured the accumulation of d-Asp and pentosidine in IVD elastin. Samples representing a broad range of ages (28-82years) and degeneration grades (1-5) were analyzed. RESULTS d/l-Asp for elastin increased linearly with age from 3.2% (early 30s) to 14.8% (early 80s) for normal tissue (grades 1-2) and from 1.7% (late 20s) to 6.0% (until the mid 50s) for degenerate tissue (grades 3-5) with accumulation rates of 16.2±3.1×10(-4) and 11.7±3.8×10(-4)year(-1), respectively; no significant difference was found between these values (p<0.05). Above the mid 50s, a decrease in d-Asp accumulation was recorded in the degenerate tissue. d-Asp accumulation correlated well with pentosidine content for elastin from healthy and degenerate tissues combined. We conclude that IVD elastin is metabolically-stable and long-lived in both healthy and degenerate human IVDs, with signs of new synthesis in the latter. The correlation of d-Asp with pentosidine content suggests that both these agents may be used as markers in the overall aging process of IVD. GENERAL SIGNIFICANCE Accumulation of modified IVD elastin argues for its longevity and may have a negative impact on its role in disc function. Weak signs of newly synthesized molecules may act to counteract this effect in degenerate tissue.

Prolyl hydroxylation in elastin is not random

BACKGROUND This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. METHODS Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. RESULTS It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. CONCLUSIONS The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. GENERAL SIGNIFICANCE The study for the first time shows that prolyl hydroxylation is highly regulated in elastin.

Proximity of Metastatic Cells Strengthens the Mechanical Interaction with Their Environment

The main cause of cancer-related deaths is metastasis-spreading of cancer cells to different sites in the body. A critical step in metastasis formation is invasion of cells through the surrounding tissue. During invasion, cancer cells change their shape and apply forces. We have previously identified that about 30% of single, metastatic, breast cancer cells will indent impenetrable synthetic, non-degradable, polyacrylamide gels, when gel stiffness is in the range 1–10 kPa. By measuring the depth of indentation of an initially flat gel and monitoring time-dependent microscopic changes in cell morphology, we were able to distinguish between benign and metastatic cells, also identifying their metastatic potential (MP); benign cells do not indent the gels. Recent works have indicated that metastases from solid tumors occur predominantly by collective cell invasion. Hence, in the current study we evaluate the mechanical interactions of cell clusters with the impenetrable gel. We observe that indenting subpopulations of metastatic cells are doubled in clusters, and cells are also indent more deeply; this increases likelihood to successfully form metastasis in the body. Concurrently, double the fraction of high MP cells indent gels as compared to low MP cells, while benign cells do not indent even in clusters. We also show that the gel platform can be used to determine the time-dependent impact of chemotherapeutics on the cells’ ability to apply forces and indent gels. Our approach can provide a rapid, mechanical prediction of the likelihood for invasiveness of cancer cells and can further be applied in a patient-specific approach, thus providing a personalized prognosis that may improve treatment of cancer patients and increase their life expectancy.

Wear Between Human Cartilage Surfaces

The present paper describes wear tests performed using human cartilage on cartilage under various working conditions. Wear was assessed by determining the concentration of proteoglycans (PG) and hydroxyproline in the lubricating solution.Copyright

Techniques for Assessment of Wear Between Human Cartilage Surfaces

Osteoarthritis (OA) is a disease of joints, affecting a large number of people worldwide. One of the symptoms of OA is wear of articular cartilage; it is thought that among other factors this may be due to failure of lubrication. Injection of bio-lubricants into a joint may remedy this problem. Wear of cartilage and its prevention is a focus of much interest. The present paper describes wear tests performed using cartilage on cartilage under various working conditions. Several techniques assessing wear are described, such as changes in surface morphology using optical profilometry and variation in the concentration of proteoglycans (PG) and hydroxyproline in the lubricating solution.Copyright