Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs.

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast-cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their 'native' tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

Moderate mechanical stress suppresses the IL-1ß-induced chondrocyte apoptosis by regulating mitochondrial dynamics.

Mitochondrial dysfunction contributes to osteoarthritis (OA) onset and progress. Mitochondrial dynamics, coupled with mitophagy, is critical for the maintenance of mitochondrial fitness, involving many cellular processes, such as proliferation and apoptosis. Excessive mechanical stress induces chondrocyte apoptosis; however, the effects of mechanical stress on mitochondrial dynamics remain elusive. In this study, we performed fluorescence staining, flow cytometry, transmission electron microscope, Western blot analysis, and RNA-sequencing to assess the effects of different strength of mechanical stimulation on mitochondrial functions of chondrocyte treated with interleukin-1ß (IL-1ß). We found that moderate mechanical stress reduced the IL-1ß-induced apoptosis by maintaining mitochondrial function and scavenging the reactive oxygen species, while excessive mechanical stress induced strong mitochondrial dysfunction and apoptosis. Moreover, RNAsequencing revealed that mitophagy and mitochondrial dynamics were involved in the regulation of mechanical stress on chondrocyte biology. In addition to the elevated mitophagy, moderate mechanical stress also promoted mitochondrial dynamics by enhancing the expression of MFN1/2 and OPA1 and the translocation of dynamin-related protein 1 from the cytoplasm to the mitochondria. However, an uncoupling of mitochondrial dynamics, characterized by strongly elevated fission, resulted in the unfavorable apoptosis of excessive mechanical stress-stimulated chondrocytes. This study revealed the effects of mechanical stress upon mitochondrial dynamics in chondrocyte.

High-resolution imaging of the human incudostapedial joint using synchrotron-radiation phase-contrast imaging.

The incudostapedial joint (ISJ) of the middle ear is important for proper transmission of sound energy to the cochlea. Recently, the biomechanics of the ISJ have been investigated using finite-element (FE) modelling, using simplified geometry. The objective of the present study was to investigate the feasibility of synchrotron-radiation phase-contrast imaging (SR-PCI) in visualising the ISJ ultrastructure. Three human cadaveric ISJs were dissected and scanned using SR-PCI at 0.9 µm isotropic voxel size. One of the samples was previously scanned at 9 µm voxel size. The images were visually compared and contrast-to-noise ratios (CNRs) were calculated (of both bone and soft tissues) for quantitative comparisons. The ISJ ultrastructure as well as adjacent bone and soft tissues were clearly visible in images with a 0.9 µm voxel size. The CNRs of the 0.9 µm images were relatively lower than those of the 9 µm scans, while the ratio of bone to soft tissue CNRs were higher, indicating better discernibility of bone from soft tissue in the 0.9 µm scans. This study was the first known attempt to image the ISJ ultrastructure using an SR-PCI scanner at submicron voxel size and results suggest that this method was successful. Future studies are needed to optimise the contrast and test the feasibility of imaging the ISJ in situ. LAY DESCRIPTION: The human middle ear consists of the eardrum, three small bones (the malleus, incus and stapes) and two joints connecting the bones (the incudostapedial joint and the incudomallear joint). The role of the middle ear is to amplify and transfer sound energy to the cochlea, the end organ of hearing. The incudostapedial joint (ISJ) of the middle ear is a synovial joint which is important for proper transmission of sound energy to the cochlea. Similar to other synovial joints it consists of meniscus, fluid and articulating surfaces. Recently, the biomechanics of the ISJ have been investigated using computational models, using grossly simplified geometry. Synchrotron radiation phase contrast imaging (SR-PCI) is a high-resolution imaging technique used to visualise small structures in three dimensions. The objective of the present study was to investigate the feasibility of using SR-PCI in visualising the ISJ ultrastructure. Three human cadaveric ISJs were dissected and scanned using SR-PCI at 0.9 µm isotropic voxel size. One of the samples was previously scanned at 9 µm voxel size. The images were both qualitatively and quantitatively compared. This study was the first known attempt to image the ISJ ultrastructure using an SR-PCI scanner at submicron voxel size and results suggest that this method was successful. Future studies are needed to optimise the contrast and feasibility of imaging the ISJ in situ.

In situ characterization of nanoscale strains in loaded whole joints via synchrotron X-ray tomography.

Imaging techniques for quantifying changes in the hierarchical structure of deforming joints are constrained by destructive sample treatments, sample-size restrictions and lengthy scan times. Here, we report the use of fast low-dose pink-beam synchrotron X-ray tomography in combination with mechanical loading at nanometric precision for in situ imaging, at resolutions below 100 nm, of the mechanical strain in intact untreated joints under physiologically realistic conditions. We show that in young, older and osteoarthritic mice, hierarchical changes in tissue structure and mechanical behaviour can be simultaneously visualized, and that the tissue structure at the cellular level correlates with the mechanical performance of the whole joint. We also use the tomographic approach to study the colocalization of tissue strains to specific chondrocyte lacunar organizations within intact loaded joints and to explore the role of calcified-cartilage stiffness on the biomechanics of healthy and pathological joints.

Bioinspired Hyaluronic Acid/Phosphorylcholine Polymer with Enhanced Lubrication and Anti-Inflammation.

Under pathological conditions, the joint is not well lubricated, which inevitably leads to osteoarthritis. Currently, in clinics injection of hyaluronic acid (HA) as an intra-articular viscosupplement is one of the main methods for alleviation of osteoarthritis. However, the viscosity of HA reduces dramatically under high shear rate due to the shear-thinning effect. Therefore, it is crucial to enhance the lubrication property of HA in order to treat osteoarthritis effectively. In this study, we successfully grafted 2-methacryloyloxyethyl phosphorylcholine (MPC), which is a zwitterionic biomaterial with excellent hydration lubrication, onto the HA with two different molecular weights (HAMPC) to enhance lubrication. The lubrication test performed using an atomic force microscope showed that, compared with HA, the friction coefficient of HAMPC was greatly reduced under various conditions. The in vitro test demonstrated that HAMPC was biocompatible and could upregulate cartilage anabolic genes while simultaneously downregulating cartilage catabolic proteases and pain-related genes. Importantly, high molecular weight HAMPC exhibited improved the capability to regulate these genes compared with low molecular weight HAMPC. In conclusion, the high molecular weight HAMPC developed herein, with enhanced lubrication and anti-inflammation, may be a promising polymer for the treatment of osteoarthritis.

Inhibition of small HA fragment activity and stimulation of A2A adenosine receptor pathway limit apoptosis and reduce cartilage damage in experimental arthritis.

Recent studies have found that the inactivation of small hyaluronan (HA) fragments originating from native HA during inflammation reduced the inflammatory response in models of experimental arthritis. The stimulation of adenosine receptors A2A reduced inflammation by inhibiting NF-kB activation. The combination of both treatments was significantly more effective than either of the individual treatments. The aim of this study was to further investigate the effects of a combined treatment using the HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) on the structure and ultrastructure of the articular cartilage and on apoptosis in a model of collagen-induced arthritis (CIA) in mice. Arthritic mice were treated with Pep-1 and/or CV-1808 intraperitoneally daily for 20 days. At day 35, the hind limbs were processed for light microscopy (hematoxylin/eosin and Safranin-O-Fast Green) and for transmission and scanning electron microscopy. CIA increased IL-6, caspase-3 and caspase-7 mRNA expression and the related protein levels in arthritic articular cartilage, and significantly increased concentrations of Bcl-2-associated X protein (Bax), while B cell-lymphoma-2 protein (Bcl-2) was markedly reduced. The combined Pep-1/CV-1808 treatment significantly reduced CIA injury, particularly at the highest doses, demonstrated by the presence of Safranin-O-positive cartilage, with a smooth surface and normal chondrocytes in the superficial, intermediate and deep zones. Morphological data and histological scoring were strongly supported by the reduction in inflammation and apoptotic markers. The results further support the role of HA degradation and A2A receptors in arthritis.

Interactions between mammalian cells and nano- or micro-sized wear particles: physico-chemical views against biological approaches.

Total joint arthroplasty (TJA) is a more and more frequent approach for the treatment of end-stage osteoarthritis in young and active adults; it successfully relieves joint pain and improves function significantly enhancing the health-related quality of life. Aseptic loosening and other wear-related complications are some of the most recurrent reasons for revision of TJA. This review focuses on current understanding of the biological reactions to prosthetic wear debris comparing in vivo and in vitro results. Mechanisms of interactions of various types of cells with metal, polymeric and ceramic wear particles are summarised. Alternative views based on multidisciplinary approaches are proposed to consider physico-chemical, surface parameters of wear particles (such as: particle size, geometry and charge) and material (particle chemical composition and its nature) with biological effects (cellular responses).

Intravital multiphoton microscopy for dissecting cellular dynamics in arthritic inflammation and bone destruction.

Osteoclasts are giant bone-resorbing polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. They play critical roles not only in normal bone homeostasis (remodeling) but also in the pathogenesis of bone-destructive disorders such as osteoporosis and rheumatoid arthritis. However, how the activity of mature osteoclasts is regulated in vivo remains unclear. To answer this question, we recently developed an advanced imaging system to visualize living bone tissues with intravital multiphoton microscopy. Using this system, we succeeded in visualization of mature osteoclasts in living bones. We herein describe the detailed methodology for visualizing bone resorption of mature osteoclasts in living bone marrow and joints using intravital multiphoton microscopy. This approach would be beneficial for studying the cellular dynamics in arthritic inflammation and bone destruction in vivo and would thus be useful for evaluating novel anti-bone-resorptive drugs.

Involvement of mitochondrial dysfunction and ER-stress in the physiopathology of equine osteochondritis dissecans (OCD).

Osteochondrosis (OC) is a developmental bone disorder affecting several mammalian species including the horse. Equine OC is described as a focal disruption of endochondral ossification, leading to osteochondral lesions (osteochondritis dissecans, OCD) that may release free bodies within the joint. OCD lesions trigger joint swelling, stiffness and lameness and affects about 30% of the equine population. OCD is considered as multifactorial but its physiopathology is still poorly understood and genes involved in genetic predisposition are still unknown. Our study compared two healthy and two OC-affected 18-month-old French Trotters diagnosed with OCD lesions at the intermediate ridge of the distal tibia. A comparative shot-gun proteomic analysis of non-wounded cartilage and sub-chondral bone from healthy (healthy samples) and OC-affected foals (predisposed samples) identified 83 and 53 modulated proteins, respectively. These proteins are involved in various biological pathways including matrix structure and maintenance, protein biosynthesis, folding and transport, mitochondrial activity, energy and calcium metabolism. Transmission electron microscopy revealed typical features of mitochondrial swelling and ER-stress, such as large, empty mitochondria, and hyper-dilated rough endoplasmic reticulum, in the deep zone of both OC lesions and predisposed cartilage. Abnormal fibril organization surrounding chondrocytes and abnormal features at the ossification front were also observed. Combining these findings with quantitative trait loci and whole genome sequencing results identified about 140 functional candidate genes carrying putative damaging mutations in 30 QTL regions. In summary, our study suggests that OCD lesions may result from defective hypertrophic terminal differentiation associated with mitochondrial dysfunction and ER-stress, leading to impaired cartilage and bone biomechanical properties, making them prone to fractures. In addition, 11 modulated proteins and several candidate mutations located in QTL regions were identified, bringing new insight into the molecular physiopathology and genetic basis of OCD.

In vivo X-ray cine-tomography for tracking morphological dynamics.

Scientific cinematography using ultrafast optical imaging is a common tool to study motion. In opaque organisms or structures, X-ray radiography captures sequences of 2D projections to visualize morphological dynamics, but for many applications full four-dimensional (4D) spatiotemporal information is highly desirable. We introduce in vivo X-ray cine-tomography as a 4D imaging technique developed to study real-time dynamics in small living organisms with micrometer spatial resolution and subsecond time resolution. The method enables insights into the physiology of small animals by tracking the 4D morphological dynamics of minute anatomical features as demonstrated in this work by the analysis of fast-moving screw-and-nut-type weevil hip joints. The presented method can be applied to a broad range of biological specimens and biotechnological processes.

The plastic nature of the human bone-periodontal ligament-tooth fibrous joint.

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano-transmission X-ray microscopy (nano-TXM), and microtomography (MicroXCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (µ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by µ-XRF. Interestingly, the hygroscopic 10-30µm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.

Platelets can enhance vascular permeability.

Platelets survey blood vessels, searching for endothelial damage and preventing loss of vascular integrity. However, there are circumstances where vascular permeability increases, suggesting that platelets sometimes fail to fulfill their expected function. Human inflammatory arthritis is associated with tissue edema attributed to enhanced permeability of the synovial microvasculature. Murine studies have suggested that such vascular leak facilitates entry of autoantibodies and may thereby promote joint inflammation. Whereas platelets typically help to promote microvascular integrity, we examined the role of platelets in synovial vascular permeability in murine experimental arthritis. Using an in vivo model of autoimmune arthritis, we confirmed the presence of endothelial gaps in inflamed synovium. Surprisingly, permeability in the inflamed joints was abrogated if the platelets were absent. This effect was mediated by platelet serotonin accumulated via the serotonin transporter and could be antagonized using serotonin-specific reuptake inhibitor antidepressants. As opposed to the conventional role of platelets to microvascular leakage, this demonstration that platelets are capable of amplifying and maintaining permeability adds to the rapidly growing list of unexpected functions for platelets.

An ultrastructural and biochemical analysis of collagen in rat peripheral nerves: the relationship between fibril diameter and mechanical properties.

Mechanical features are distributed heterogeneously within nerve tissue, with compliance increased at articulations. This study explored whether differences in stiffness between joint regions (JRs) and non-joint regions (NJRs) of rat median and sciatic nerves were related to localised variation in collagen content or fibril diameter. There was no significant difference in the amount of collagen detected by biochemical assay in JRs and NJRs of either nerve. Ultrastructural analysis showed collagen fibril diameter ranges of 20-80 nm in the endoneurium and perineurium and 30-130 nm in the epineurium. In the median nerve, but not the sciatic nerve, there were significantly smaller fibrils in JRs compared to NJRs. This corresponded to a greater number density of fibrils in JRs compared to NJRs in the epineurium and endoneurium of the median nerve. We report the presence therefore of a population of thinner collagen fibrils in the JR of the median nerve that corresponds to the location of increased compliance in this tissue, suggesting that localised variation in collagen fibril diameter contributes to the longitudinal heterogeneity of tensile properties in this nerve.

Imaging collagen II using atomic force microscopy (AFM).

Collagen II is a fibrous protein that assembles from basic tropocollagen subunits to form extracellular supramolecular fiber networks within cartilage tissue. Tropocollagen subunits of ~300 nm in length self-assemble first into pentameric uniform microfibrils, which fuse into bigger collagen fibrils that can range from 10 nm to 500 nm in diameter. The collagen fibrils display a characteristic 67-nm repeat because of the staggering of individual collagen molecules with respect to each other. This protocol demonstrates how to prepare collagen protein samples for analysis by atomic force microscopy (AFM). It also describes the steps for generating AFM images of collagen samples during and after manipulation to analyze collagen self-assembly.

Energy storage and synchronisation of hind leg movements during jumping in planthopper insects (Hemiptera, Issidae).

The hind legs of Issus (Hemiptera, Issidae) move in the same plane underneath the body, an arrangement that means they must also move synchronously to power jumping. Moreover, they move so quickly that energy must be stored before a jump and then released suddenly. High speed imaging and analysis of the mechanics of the proximal joints of the hind legs show that mechanical mechanisms ensure both synchrony of movements and energy storage. The hind trochantera move first in jumping and are synchronised to within 30 micros. Synchrony is achieved by mechanical interactions between small protrusions from each trochantera which fluoresce bright blue under specific wavelengths of ultra-violet light and which touch at the midline when the legs are cocked before a jump. In dead Issus, a depression force applied to a cocked hind leg, or to the tendon of its trochanteral depressor muscle causes a simultaneous depression of both hind legs. The protrusion of the hind leg that moves first nudges the other hind leg so that both move synchronously. Contractions of the trochanteral depressor muscles that precede a jump bend the metathoracic pleural arches of the internal skeleton. Large areas of these bow-shaped structures fluoresce bright blue in ultraviolet light, and the intensity of this fluorescence depends on the pH of the bathing saline. These are key signatures of the rubber-like protein resilin. The remainder of a pleural arch consists of stiff cuticle. Bending these composite structures stores energy and their recoil powers jumping.

[Imaging methods in rheumatoid arthritis].

Biological agents revolutionized a therapeutic aspect of rheumatoid arthritis (RA) . It is important to establish early diagnosis and the evaluation method for RA. In addition to conventional X-ray, joint magnetic resonance imaging (MRI) and ultrasonography became to be focused on. Conventional X-ray is not useful for early diagnosis for RA, but important for assessing bone destruction. Joint MRI is able to detect specific joint change such as synovitis and bone oedema, these are early change and strong predictor of bone destruction, hence useful for early diagnosis and prognostic decision for RA. Joint ultrasonography is reported as useful method for both evaluation of disease activity and imaging remission of synovitis. It is important to understand feature of these imaging tools to use in actual practice.

Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid.

The annular ligament across the stapediovestibular joint connects the stapes footplate and the vestibular window and plays an important role in the sound conductive system of the ear. In this study, we investigated the distribution of extracellular matrix components in the ligament by histochemical methods at light and electron microscopic levels. As results, light microscopic immunohistochemistry of fibrillin and 36-kDa microfibril-associated glycoprotein (MAGP-36) showed intense immunoreactivities in the annular ligament between the stapes footplate and vestibular window. In addition, the histochemical localization of hyaluronic acid by using biotinylated hyaluronic acid-binding protein (HABP) clarifi ed the presence of hyaluronic acid in the annular ligament. At the electron microscopic level, the immunogold labeling of fibrillin showed intense labeling on the periphery of the electron-dense mantle. Furthermore, the labeling of fibrillin was preferentially seen on the fibrous components among the electronlucent amorphous substance. The immunogold labeling of MAGP-36 was seen on the electron-dense mantle and scattered on the electron-lucent amorphous substance. The gold labeling with biotinylated HABP clearly showed a distribution of hyaluronic acid throughout the amorphous space in the ligament. The present results provide a histochemical profile of the annular ligament of the rat stapediovestibular joint that may provide clues to elucidation of pathological changes in the ligaments and conductive hearing loss in humans.

Anatomy of the hind legs and actions of their muscles during jumping in leafhopper insects.

The rapid and simultaneous depression of the trochantera about the coxae of both hind legs of leafhoppers are the key joint movements powering a jump. The present study analyses the structure of these joints and the actions of the muscles that move them. The hind coxae are huge and are linked to each other at the midline by a protrusion from one coxa that inserts in a socket of the other and acts like a press-stud (popper) fastener. This asymmetry is not reflected in any left- or right-handed preference either within one species or between species. The movements of the joints in a jump are monitored by a number of possible proprioceptors that should be activated when a hind leg is fully levated in preparation for a jump: a hair row and two hair plates on the coxa, a hair plate on a trochanteral pivot with a coxa, and femoral spines at the femoro-tibial joint. The depressor and levator muscles that move the trochanter are of similar size and together occupy the greater part of the metathorax. Their lever arms are similar when the leg is fully levated, but the lever arm of the depressor increases with initial depression of the coxo-trochanteral joint while that of the levator declines. A jump is preceded by activity in the trochanteral depressor and levator muscles, which results in a forward movement of the coxa and metathorax with the trochanter fully levated. This period of co-contraction could result in storage of energy in skeletal structures in the thorax. Just before the rapid depression of the trochanter in the jump movement the frequency of depressor spikes increases while that in the levator declines, releasing any force stored by the preceding muscle contractions. These bursts of depressor spikes occur at the same time in the left and right muscles but none of the individual motor spikes appeared to be synchronous on the two sides.

Nondegradable hydrogels for the treatment of focal cartilage defects.

Nondegradable materials have long been suggested for the treatment of articular cartilage defects; however, the mechanics of the implant/tissue system necessary to ensure long-term function are unknown. The objective of this study was to explore the performance of nondegradable hydrogel implants in cartilage defects. Our hypothesis was that the structural integrity of the implant and surrounding tissue would be influenced by the compressive modulus of the material used, and that superior results would be obtained with the implantation of a more compliant material. Poly(vinyl alcohol)-poly(vinyl pyrrolidone) hydrogel implants of two different moduli were implanted into osteochondral defects in a rabbit model. Six-month postoperative histological and mechanical data were used to assess the wear and fixation of the implants. The compliant implants remained well fixed and a thin layer of soft tissue grew over the surface of the implants. However, gross deformation of the compliant implants occurred and debris was evident in surrounding bone. The stiffer implants were dislocated from their implantation site, but with no accompanying evidence of debris or implant deformation. Our hypothesis that superior results would be obtained with implantation of a more compliant material was rejected; a compromise between the wear and fixation properties dependent on modulus was found.

Role of RhoC in digit morphogenesis during limb development.

Here we report a new role for the small GTPase RhoC in the control of limb chondrogenesis. Expression of rhoC is a precocious marker of the zeugopodial and digit blastemas and is induced by treatments with TGFbetas preceding the formation of ectopic digits. As development progresses, expression of rhoC outlines the growing distal tip of the digits, and marks the regions of interphalangeal joint formation. Functional experiments show that RhoC is a negative regulator of chondrogenesis, which controls digit outgrowth and joint segmentation. These functions appear to be mediated by reorganization of the actin cytoskeleton and modification of the adhesive properties of the mesenchymal cells.