Search

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.

Aim: The purpose of this study was to investigate the effect of normal synovial fluid (SF) on exposed endogenous tendon-derived cells (TDCs) and engrafted mesenchymal stem cells (MSCs) within the tendon extracellular matrix., Methods: Explants from equine superficial digital flexor (extra-synovial) and deep digital flexor tendons (DDFTs) from the compressed, intra-synovial and the tensile, extra-synovial regions were cultured in allogeneic or autologous SF-media. Human hamstring explants were cultured in allogeneic SF. Explant viability was assessed by staining. Proliferation of equine monolayer MSCs and TDCs in SF-media and co-culture with DDFT explants was determined by alamarblue®. Non-viable Native Tendon matrices (NNTs) were re-populated with MSCs or TDCs and cultured in SF-media. Immunohistochemical staining of tendon sections for the apoptotic proteins caspase-3, -8, and -9 was performed., Results: Contact with autologous or allogeneic SF resulted in rapid death of resident tenocytes in equine and human tendon. SF did not affect the viability of equine epitenon cells, or of MSCs and TDCs in the monolayer or indirect explant co-culture. MSCs and TDCs, engrafted into NNTs, died when cultured in SF. Caspase-3, -8, and -9 expression was the greatest in SDFT explants exposed to allogeneic SF., Conclusions: The efficacy of cells administered intra-synovially for tendon lesion repair is likely to be limited, since once incorporated into the matrix, cells become vlnerable to the adverse effects of SF. These observations could account for the poor success rate of intra-synovial tendon healing following damage to the epitenon and contact with SF, common with most soft tissue intra-synovial pathologies.

The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease., (Copyright © 2014. Published by Elsevier B.V.)

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Study Design: Analysis of proteoglycan synthesis, distribution and assembly of notochordal cells and small nucleus pulposus cells embedded in alginate beads and cultured in presence of [S]-Na2SO4., Objective: To determine whether the degeneration of the nucleus pulposus of the intervertebral disc is associated with a change in the cell phenotype., Summary of Background Data: The loss of the notochordal cell from the nucleus pulposus is associated with ageing and disc degeneration. The reduction in their numbers after birth in humans and in the chondrodystrophoid dog has been suggested to result from cell death and replacement or differentiation by chondrocytes. The almost total disappearance of the notochordal cells in the nucleus pulposus correlates with early degenerative changes in the disc and a concomitant reduction in proteoglycan content, increased collagen, and loss of water content. The basic mechanism of this accelerated degeneration with ageing is poorly understood., Methods: Nucleus pulposus and anulus fibrosus cells were isolated from the lumbar intervertebral discs of chondrodystrophoid and nonchondrodystrophoid dogs. The cells from the nucleus pulposus were further separated by size into notochordal cells and small nucleus pulposus cells. Cells were embedded in alginate beads and cultured in the presence of [S]-Na2SO4 to measure proteoglycan size, rate of synthesis, and distribution into the pericellular and intercellular compartments., Results: Large notochordal cells in the nucleus pulposus of chondrodystrophoid dogs formed 13% of the cell population in young dogs and fell to 0.4% in adults, whereas they were the predominant cell type in the nonchondrodystrophoid dogs at all ages. These cells were capable of 1.5-fold greater rate of synthesis of proteoglycans than the small nucleus pulpous cells. Proteoglycans secreted by the large cells were evenly distributed between the pericellular and intercellular compartments,whereas the small cells distributed 3-fold more proteoglycan into the intercellular phase. By size exclusion chromatography, the proteoglycans synthesized by the small cells of the chondrodystrophoid dogs formed large-size aggregates (Kav = 0.1) within the pericellular region, which then moved to the intercellular region over 5 to 10 days. In contrast, proteoglycans secreted by the notochordal cells were capable of rapid migration to the intercellular phase before assembly into large-sized aggregates. The ability to form aggregates was independent of age of the animal., Conclusions: Our model shows that a change in intervertebral disc cell phenotype correlates with the grade of disc degeneration and that the notochordal cells synthesize proteoglycans, which exhibit delayed aggregation than those synthesized by the small nucleus pulposus cells. This implies that the cell type composition of the nucleus pulposus of the chondrodystrophoid and nonchondrodystrophoid dogs produces an extracellular matrix that is assembled in a distinct manner, which may affect tissue integrity.

Hyaluronan and versican-rich pericellular matrices form around arterial smooth muscle cells (ASMC) preferentially during the detachment phase of proliferation and migration. PDGF is a potent mitogen and chemotactic agent for ASMC and also stimulates the production of extracellular matrix molecules which may regulate the proliferative and migratory capacity of the cells. We have examined the effect of PDGF on the formation of hyaluronan-dependent pericellular matrices, and on the synthesis and interaction of several major pericellular coat constituents. As demonstrated using a particle exclusion assay, PDGF stimulated the formation of pericellular matrices and was seen both in an increased proportion of cells with a coat and a greater coat size. This increase was accompanied by a transient increase in hyaluronan synthase 2 (HAS2) expression and an increase in hyaluronan synthesis and polymer length. PDGF also increased the synthesis of versican and link protein as measured at the mRNA and protein levels. The amount of native versican-hyaluronan aggregates and link-stabilized aggregate was also increased following PDGF treatment. Time lapse imaging showed that pericellular matrix formation occurred around trailing cell processes prior to their detachment. These data suggest that PDGF modulates the synthesis and organization of ASMC pericellular coat-forming molecules such as versican, hyaluronan, and link protein, which leads to extracellular matrix expansion and alterations in ASMC phenotype., (Copyright 2001 Academic Press.)

Coordinate differentiation of the chondrocytes plays a crucial role during skeletal development. In the cascade of endochondral bone formation, mature chondrocytes of the fetal growth plate represent metabolically highly active cells. They show high expression levels of the major cartilage matrix genes, collagen types II, IX, and XI, the major cartilage proteoglycan aggrecan, and proteoglycan link protein. The strongest signals are found in areas of maximal growth, the proliferative and upper hypertrophic zones. The major cartilage matrix components are co-expressed by the chondrocytes of the resting and proliferative zones. Type X collagen is restricted to lower hypertrophic chondrocytes. Interestingly, in the lower hypertrophic zone type IX collagen, but not type II and XI collagen, mRNA expression is downregulated, indicating a discoordinate expression of these collagen types in hypertrophic chondrocytes. The results of this study confirm the strict zonal differentiation pattern of chondrocytes in the developing fetal growth plate, which can be monitored by the expression patterns of its major expression products, the collagen subtypes and aggrecan and proteoglycan link protein.