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BackgroundAgeing is one of the leading risk factors predisposing cartilage to musculoskeletal diseases, including osteoarthritis. Cumulative evidence suggests that small non-coding RNAs play a role in cartilage-related pathological changes. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs in cartilage. By using small RNA sequencing, we investigated changes in the expression of small non-coding RNAs between young and old equine chondrocytes.MethodsChondrocytes were extracted from five young (4±1 years) and five old (17.4±1.9 years) macroscopically normal equine metacarpophalangeal joints. Following RNA extraction cDNA libraries were prepared and subjected to small RNA sequencing using the Illumina MiSeq platform. Differential expression analysis was performed in R using package DESeq2. For tRNA fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA and small nucleolar RNA findings were validated using qRT-PCR in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low and high grade OA human cartilage tissue.ResultsIn total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including microRNAs, snoRNAs, snRNAs and tRNAs. Of these, 34 were expressed higher and 49 were expressed lower in old chondrocytes compared to young. qRT-PCR analysis confirmed findings in an extended cohort of equine chondrocytes. Ingenuity Pathway Analysis of differentially expressed microRNAs and their predicted target genes linked them to cartilage and OA-related pathways and diseases. tRNA fragment analysis revealed that tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes.ConclusionFor the first time, we have measured the effect of ageing on the expression of small non-coding RNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species, including microRNAs, small nucleolar RNAs and tRNA fragments. This study supports a role for small non-coding RNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.

Osteoarthritis (OA) has long been viewed as a degenerative disease of cartilage, but accumulating evidence indicates that inflammation has a critical role in its pathogenesis. In particular, chondrocyte-mediated inflammatory responses triggered by the activation of innate immune receptors by alarmins (also known as danger signals) are thought to be involved. Thus, toll-like receptors (TLRs) and their signaling pathways are of particular interest. Recent reports suggest that among the TLR-induced innate immune responses, apoptosis is one of the critical events. Apoptosis is of particular importance, given that chondrocyte death is a dominant feature in OA. This review focuses on the role of TLR signaling in chondrocytes and the role of TLR activation in chondrocyte apoptosis. The functional relevance of TLR and TLR-triggered apoptosis in OA are discussed as well as their relevance as candidates for novel disease-modifying OA drugs (DMOADs).

OBJECTIVE: Aging is accompanied by a series of changes in mature tissues that influence their properties and functions. Collagen, as one of the main extracellular components of cartilage, becomes highly crosslinked during aging. In this study, the aim was to examine whether a correlation exists between collagen crosslinking induced by artificial aging and mechanical properties of the temporomandibular joint (TMJ) condyle. To evaluate this hypothesis, collagen crosslinks were induced using ribose incubation. METHODS: Porcine TMJ condyles were incubated for 7 days with different concentrations of ribose. The compressive modulus and stiffness ratio (incubated versus control) was determined after loading. Glycosaminoglycan and collagen content, and the number of crosslinks were analyzed. Tissue structure was visualized by microscopy using different staining methods. RESULTS: Concomitant with an increasing concentration of ribose, an increase of collagen crosslinks was found. The number of crosslinks increased almost 50 fold after incubation with the highest concentration of ribose. Simultaneously, the stiffness ratio of the samples showed a significant increase after incubation with the ribose. Pearson correlation analyses showed a significant positive correlation between the overall stiffness ratio and the crosslink level; the higher the number of crosslinks the higher the stiffness. CONCLUSION: The present model, in which ribose was used to mimic certain aspects of age-related changes, can be employed as an in vitro model to study age-related mechanical changes in the TMJ condyle. ispartof: Archives of Oral Biology vol:87 pages:102-109 ispartof: location:England status: published

OBJECTIVE: Aging can cause an increase in the stiffness of hyaline cartilage as a consequence of increased protein crosslinks. By induction of crosslinking, a reduction in the diffusion of solutions into the hyaline cartilage has been observed. However, there is a lack of knowledge about the effects of aging on the biophysical and biochemical properties of the temporomandibular joint (TMJ) cartilage. Hence, the aim of this study was to examine the biophysical properties (thickness, stiffness, and diffusion) of the TMJ condylar cartilage of horses of different ages and their correlation with biochemical parameters. MATERIALS AND METHODS: We measured the compressive stiffness of the condyles, after which the diffusion of two contrast agents into cartilage was measured using Contrast Enhanced Computed Tomography technique. Furthermore, the content of water, collagen, GAG, and pentosidine was analyzed. RESULTS: Contrary to our expectations, the stiffness of the cartilage did not change with age (modulus remained around 0.7 MPa). The diffusion of the negatively charged contrast agent (Hexabrix) also did not alter. However, the diffusion of the uncharged contrast agent (Visipaque) decreased with aging. The flux was negatively correlated with the amount of collagen and crosslink level which increased with aging. Pentosidine, collagen, and GAG were positively correlated with age whereas thickness and water content showed negative correlations. CONCLUSION: Our data demonstrated that aging was not necessarily reflected in the biophysical properties of TMJ condylar cartilage. The combination of the changes happening due to aging resulted in different diffusive properties, depending on the nature of the solution. ispartof: OSTEOARTHRITIS AND CARTILAGE vol:26 issue:12 pages:1744-1752 ispartof: location:England status: published

The extracellular matrix can trigger cellular responses through its composition and structure. Major extracellular matrix components are the proteoglycans, which are composed of a core protein associated with glycosaminoglycans, among which the small leucine-rich proteoglycans (SLRPs) are the largest family. This review highlights how the codon usage pattern can be used to modulate cellular response and discusses the biological impact of post-translational events on SLRPs, including the substitution of glycosaminoglycan moieties, glycosylation, and degradation. These modifications are listed, and their impacts on the biological activities and structural properties of SLRPs are described. We narrowed the topic to skeletal tissues undergoing dynamic remodeling. ispartof: BIOMOLECULES vol:10 issue:1 ispartof: location:Switzerland status: published

Background: Idiopathic epiretinal membrane (iERM) is a fibrocellular membrane that proliferates on the inner surface of the retina at the macular area. Membrane contraction is an important sight-threatening event and is due to fibrotic remodeling. Methods: Analysis of the current literature regarding the epidemiology, clinical features, and pathogenesis of iERM and fibrotic tissue contraction. Results: Epidemiologic studies report a relationship between iERM prevalence, increasing age, and posterior vitreous detachment. Clinically, iERM progresses through different stages characterized by an increased thickness and wrinkling of the membrane. Pathophysiologically, iERM formation is a fibrotic process in which myofibroblast formation and the deposition of newly formed collagens play key roles. Anomalous posterior vitreous detachment may be a key event initiating the formation of iERM. The age-related accumulation of advanced glycation end products may contribute to anomalous posterior vitreous detachment formation and may also influence the mechanical properties of the iERM. Conclusion: Remodeling of the extracellular matrix at the vitreoretinal interface by aging and fibrotic changes, plays a significant role in the pathogenesis of iERM. A better understanding of molecular mechanisms underlying this process may eventually lead to the development of effective and nonsurgical approaches to treat and prevent vitreoretinal fibrotic diseases.

Background: Idiopathic epiretinal membrane (iERM) is a fibrocellular membrane that proliferates on the inner surface of the retina at the macular area. Membrane contraction is an important sight-threatening event and is due to fibrotic remodeling.Methods: Analysis of the current literature regarding the epidemiology, clinical features, and pathogenesis of iERM and fibrotic tissue contraction.Results: Epidemiologic studies report a relationship between iERM prevalence, increasing age, and posterior vitreous detachment. Clinically, iERM progresses through different stages characterized by an increased thickness and wrinkling of the membrane. Pathophysiologically, iERM formation is a fibrotic process in which myofibroblast formation and the deposition of newly formed collagens play key roles. Anomalous posterior vitreous detachment may be a key event initiating the formation of iERM. The age-related accumulation of advanced glycation end products may contribute to anomalous posterior vitreous detachment formation and may also influence the mechanical properties of the iERM.Conclusion: Remodeling of the extracellular matrix at the vitreoretinal interface by aging and fibrotic changes, plays a significant role in the pathogenesis of iERM. A better understanding of molecular mechanisms underlying this process may eventually lead to the development of effective and nonsurgical approaches to treat and prevent vitreoretinal fibrotic diseases.

The poor regenerative capacity of articular cartilage presents a major clinical challenge and may relate to a limited turnover of the cartilage collagen matrix. However, the collagen turnover rate during life is not clear, and it is debated whether osteoarthritis (OA) can influence it. Using the carbon-14 (C-14) bomb-pulse method, life-long replacement rates of collagen were measured in tibial plateau cartilage from 23 persons born between 1935 and 1997 (15 and 8 persons with OA and healthy cartilage, respectively). The C-14 levels observed in cartilage collagen showed that, virtually, no replacement of the collagen matrix happened after skeletal maturity and that neither OA nor tissue damage, per se, influenced collagen turnover. Regional differences in C-14 content across the joint surface showed that cartilage collagen located centrally on the joint surface is formed several years earlier than collagen located peripherally. The collagen matrix of human articular cartilage is an essentially permanent structure that has no significant turnover in adults, even with the occurrence of disease.

New imaging techniques are valuable for the care of patients with haemophilia. On angiography it is shown that some bleedings in severely damaged joints or after implantation of prostheses are arterial. Effect of clotting factor is often poor. Selective catherization with embolization of the bleeding artery stops the bleed and is clinically effective. From 31 patients with severe haemophilia A or B, 62 knee radiographs were scored according to the Pettersson-scoring system as well as with Knee Digital Image Analysis (KIDA). Using KIDA, good correlation was found for osteoporosis, irregular subchondral surface, narrowing of the joint space, deformity and incongruence. For each of the parameters within one point in the Pettersson score a large variation existed in KIDA grading. MRI is accurate for diagnosis of soft and osteochondral tissue. Nevertheless, it is costly and not very accessible. The use of parallel imaging is more feasible for assessment of multiple joints within a relatively short period of time. Although ultrasonography also holds the potential for being an adjunct to MRI it has the disadvantage that it is operator-dependent. In a cohort of 124 chronically HCV infected haemophilia patients transient elastography was performed to measure liver stiffness. 57 (46%) had no or mild fibrosis, 18 (14.5%) moderate fibrosis and 49 severe or cirrhotic fibrosis. Transient elastography is safe and helpful to refer patients to antiviral therapy.

New imaging techniques are valuable for the care of patients with haemophilia. On angiography it is shown that some bleedings in severely damaged joints or after implantation of prostheses are arterial. Effect of clotting factor is often poor. Selective catherization with embolization of the bleeding artery stops the bleed and is clinically effective. From 31 patients with severe haemophilia A or B, 62 knee radiographs were scored according to the Pettersson-scoring system as well as with Knee Digital Image Analysis (KIDA). Using KIDA, good correlation was found for osteoporosis, irregular subchondral surface, narrowing of the joint space, deformity and incongruence. For each of the parameters within one point in the Pettersson score a large variation existed in KIDA grading. MRI is accurate for diagnosis of soft and osteochondral tissue. Nevertheless, it is costly and not very accessible. The use of parallel imaging is more feasible for assessment of multiple joints within a relatively short period of time. Although ultrasonography also holds the potential for being an adjunct to MRI it has the disadvantage that it is operator-dependent. In a cohort of 124 chronically HCV infected haemophilia patients transient elastography was performed to measure liver stiffness. 57 (46%) had no or mild fibrosis, 18 (14.5%) moderate fibrosis and 49 severe or cirrhotic fibrosis. Transient elastography is safe and helpful to refer patients to antiviral therapy.

Articular cartilage lacks the ability to repair itself and consequently defects in this tissue do not heal. Tissue engineering approaches, employing a scaffold material and cartilage producing cells (chondrocytes), hold promise for the treatment of such defects. In these strategies the limitation of nutrients, such as oxygen, during in vitro culture are of major concern and will have implications for proper bioreactor design. We recently demonstrated that oxygen gradients are indeed present within tissue engineered cartilaginous constructs. Interestingly, oxygen, besides being an essential nutrient, is also a controlling agent of developmental processes including cartilage formation. However, the specific role of oxygen in these processes is still obscure despite the recent advances in the field. In particular, the outcome of published investigations is inconsistent regarding the effect of oxygen tension on chondrocytes. Therefore, this article describes the possible roles of oxygen gradients during embryonic cartilage development and reviews the data reported on the effect of oxygen tension on in vitro chondrocyte proliferation and differentiation from a tissue engineering perspective. Furthermore, possible causes for the variance in the data are discussed. Finally, recommendations are included that may reduce the variation, resulting in more reliable and comparable data.

Articular cartilage lacks the ability to repair itself and consequently defects in this tissue do not heal. Tissue engineering approaches, employing a scaffold material and cartilage producing cells (chondrocytes), hold promise for the treatment of such defects. In these strategies the limitation of nutrients, such as oxygen, during in vitro culture are of major concern and will have implications for proper bioreactor design. We recently demonstrated that oxygen gradients are indeed present within tissue engineered cartilaginous constructs. Interestingly, oxygen, besides being an essential nutrient, is also a controlling agent of developmental processes including cartilage formation. However, the specific role of oxygen in these processes is still obscure despite the recent advances in the field. In particular, the outcome of published investigations is inconsistent regarding the effect of oxygen tension on chondrocytes. Therefore, this article describes the possible roles of oxygen gradients during embryonic cartilage development and reviews the data reported on the effect of oxygen tension on in vitro chondrocyte proliferation and differentiation from a tissue engineering perspective. Furthermore, possible causes for the variance in the data are discussed. Finally, recommendations are included that may reduce the variation, resulting in more reliable and comparable data.

Background and purpose Degenerating cartilage releases potential danger signals that react with Toll-like receptor (TLR) type danger receptors. We investigated the presence and regulation of TLR1, TLR2, and TLR9 in human chondrocytes. Methods We studied TLR1, TLR2, TLR4, and TLR9 mRNA (qRT-PCR) and receptor proteins (by immunostaining) in primary mature healthy chondrocytes, developing chondrocytes, and degenerated chondrocytes in osteoarthritis (OA) tissue sections of different OARSI grades. Effects of a danger signal and of a pro-inflammatory cytokine on TLRs were also studied. Results In primary 2D-chondrocytes, TLR1 and TLR2 were strongly expressed. Stimulation of 2D and 3D chondrocytes with a TLR1/2-specific danger signal increased expression of TLR1 mRNA 1.3- to 1.8-fold, TLR2 mRNA 2.6- to 2.8-fold, and TNF-α mRNA 4.5- to 9-fold. On the other hand, TNF-α increased TLR1 mRNA] expression 16-fold, TLR2 mRNA expression 143- to 201-fold, and TNF-α mRNA expression 131- to 265-fold. TLR4 and TLR9 mRNA expression was not upregulated. There was a correlation between worsening of OA and increased TLR immunostaining in the superficial and middle cartilage zones, while chondrocytes assumed a CD166× progenitor phenotype. Correspondingly, TLR expression was high soon after differentiation of mesenchymal stem cells to chondrocytes. With maturation, it declined (TLR2, TLR9). Interpretation Mature chondrocytes express TLR1 and TLR2 and may react to cartilage matrix/chondrocyte-derived danger signals or degradation products. This leads to synthesis of pro-inflammatory cytokines, which stimulate further TLR and cytokine expression, establishing a vicious circle. This suggests that OA can act as an autoinflammatory disease and links the old mechanical wear-and-tear concept with modern biochemical views of OA. These findings suggest that the chondrocyte itself is the earliest and most important inflammatory cell in OA.

Multipotent mesenchymal stromal or stem cells (MSCs) are likely to be agents of connective tissue homeostasis and repair. Because the hallmark of osteoarthritis (OA) is degeneration and failure to repair connective tissues it is compelling to think that these cells have a role to play in OA. Indeed, MSCs have been implicated in the pathogenesis of OA and, in turn, progression of the disease has been shown to be therapeutically modulated by MSCs. This review discusses current knowledge on the potential of both marrow- and local joint-derived MSCs in OA, the mode of action of the cells, and possible effects of the osteoarthritic niche on the function of MSCs. The use of stem cells for repair of isolated cartilage lesions and strategies for modulation of OA using local cell delivery are discussed as well as therapeutic options for the future to recruit and appropriately activate endogenous progenitors and/or locally systemically administered MSCs in the early stages of the disease. The use of gene therapy protocols, particularly as they pertain to modulation of inflammation associated with the osteoarthritic niche, offer an additional option in the treatment of this chronic disease. In summary, elucidation of the etiology of OA and development of technologies to detect early disease, allied to an increased understanding of the role MSCs in aging and OA, should lead to more targeted and efficacious treatments for this debilitating chronic disease in the future. This work was funded by Science Foundation Ireland, Centre for Science Engineering and Technology award (CSET) in Regenerative Medicine Deposited by bulk import

Many animal models are used to study osteoarthritis (OA). In these models the role of joint loading in the development of CA is not fully understood. We studied the effect of loading on the development of CIA in the canine Groove-model. In ten female beagle dogs OA was induced in one knee according to the Groove-model. The animals were divided in groups with and without forced-loading. Forced-loading was achieved by fixing the contra-lateral limb to the trunk 3 times a week for 4 hours. After 20 weeks joint tissues of all dogs were evaluated. Subjective evaluation revealed less movement with more loading in the forced-loading-group compared to the group without forced-loading. In both groups induction of OA resulted in macroscopical and microscopical CA changes as well as alterations in cartilage metabolism characteristics for OA. Although differences were small, for some parameters they were statistically significant for the forced-loading-group. There were no differences between the contra-lateral healthy joints of both groups. The present study demonstrates that in the Groove-model intensified loading is not a prerequisite for the development of OA, although it adds to some extent to the severity of the OA. (C) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1332-1338, 2009

To evaluate whether subchondral osteoblasts (OB) are involved in the production of cytokines and chemokines in rheumatic diseases.OB were isolated from subchondral bone of rheumatoid arthritis (RA), osteoarthritis (OA) and post-traumatic (PT) patients, cultured in vitro in the presence or absence of interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), and assessed for the production, immunolocalization, and mRNA expression of proinflammatory cytokines (IL-1alpha, IL-1beta, TNF-alpha) and alpha and beta chemokines [IL-8, growth related gene product (GRO-alpha), monocyte chemoattractant protein 1 (MCP-1), RANTES, and macrophage inflammatory proteins MIP-1alpha, MIP-1beta].Cultured OB from different patients did not release IL-1alpha, IL-1beta, or TNF-alpha, and constitutively secreted IL-8, GRO-alpha, and MCP-1, while RANTES, MIP-1alpha, MIP-1beta were undetectable or near the lower level of sensitivity of the immunoenzymatic assay. GRO-alpha was significantly higher in RA than in OA and PT patients. IL-1beta and TNF-alpha alone or in combination strongly stimulated chemokine release by OB. Only RANTES production was not increased by the combination of the 2 cytokines. IL-1alpha, IL-1beta, and TNF-alpha were expressed as cytoplasmic proteins and were not secreted by OB even after stimulation.OB from subchondral bone release chemokines that could be involved in the mechanisms that directly or indirectly cause bone remodelling and cartilage destruction.

Micro Proton Induced X-ray Emission (mu-PIXE) analysis has been employed herein in investigating and quantifying the distribution of a number of essential elements in thin human diseased articular cartilage sections affected by osteoarthritis (OA). Various cations Ca, P and Zn have been reported to play an important role both in the normal growth and remodelling of articular cartilage and subchondral bone as well as in the degenerative and inflammatory processes associated with the disease; they act as co-factors of a class of enzymes known as metalloproteinases which are believed to be active during the initiation, progress and remodelling processes associated with osteoarthritis. Other important enzymes such as alkaline phosphatase are associated with cartilage mineralization. Synchrotron radiation X-ray fluorescence (SR-XRF) for mapping of elemental distributions in bone and cartilage has also been employed by the present group and others. In the current investigations using the cSAXS beamline at the Swiss light source, Small-Angle X-ray Scattering (SAXS) was carried out on decalcified human articular cartilage to explore the structural and organizational changes of collagen networks in diseased articular cartilage. (c) 2009 Elsevier Ltd. All rights reserved.