| 1. |
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| 2. |
- Barreto Henriksson, Helena, et al.
(författare)
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Support of Concept that Migrating Progenitor Cells from Stem Cell Niches Contribute to Normal Regeneration of the Adult Mammal Intervertebral Disc: A Descriptive study in the New Zeeland white Rabbit.
- 2012
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Ingår i: Spine. - 0362-2436 .- 1528-1159. ; 37:9, s. 722-732
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: Study Design. Descriptive experimental study performed in rabbits of two age groups.Objective. To study and investigate presence of prechondrocytic cells and cell migration routes in the IVD region, to gain knowledge about the normal IVD regeneration pattern.Summary of Background Data. Disc degeneration is believed to play a major role in patients with chronic lumbar pain. Regeneration processes and cell migration within the intervertebral disc (IVD) have been sparsely described. Therefore it is of interest to increase knowledge of these processes in order to understand pathological conditions of the IVD.Methods. 5-bromo-2-deoxyuridine (BrdU) in vivo labelling was performed in two groups of rabbits, 3 and 9 months old at the beginning of the experiment, in total 27 rabbits. BrdU is incorporated into DNA during mitosis and then it is gradually diluted with each cell division until it finally disappears. Incorporation of BrdU was then visualized by immunohistochemistry (IHC) at different time points providing cell division pattern and presence of slow-cycling cells in the IVD region. IVD tissue was investigated by IHC for: Growth- and differentiation-factor-5 (GDF5), SOX9 (chondrogenic lineage markers), SNAIL homolog1 (SNAI1), SNAIL homolog2 (SLUG)(migration markers) and β1-INTEGRIN (cellular adhesion marker). In addition, GDF5, SOX9 and BMPRIB expression were investigated on genetic level.Results. BrdU+ cells were observed in early time points in the IVD niche, adjacent to the epiphyseal plate, at later time points mainly in outer region of the annulus fibrosus (AF) for both age groups of rabbits, indicating a gradual migration of cells. The presence of SLUG, SNAI1, GDF5, SOX9 and β1-INTEGRIN were found in same regions.Conclusion. The results suggest a cellular migration route from the IVD stem cell niche toward the AF and the inner parts of the IVD. These findings may be of importance for understanding IVD regenerative mechanisms and for future development of biological treatment strategies.
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| 3. |
- Ekman, Stina, et al.
(författare)
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Effect of circadian rhythm, age, training and acute lameness on serum concentrations of cartilage oligomeric matrix protein (COMP) neo-epitope in horses
- 2019
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Ingår i: Equine Veterinary Journal. - : Wiley. - 0425-1644 .- 2042-3306. ; 51:5, s. 674-680
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Tidskriftsartikel (refereegranskat)abstract
- Background Molecular serum markers that can identify early reversible osteoarthritis (OA) in horses are lacking. Objectives We studied serum concentrations of a novel cartilage oligomeric matrix protein (COMP) neo-epitope in horses subjected to short-term exercise and with acute lameness. The effects of circadian rhythm and age were also evaluated. Study design Longitudinal studies in healthy horses and cross-sectional comparison of lame and non-lame horses. Methods Sera were collected from five horses before and after short-term interval exercise and during full-day box rest. Sera from 32 acutely lame horses were used to evaluate age-related effects. Independent samples from control horses (n = 41) and horses with acute lameness (n = 71) were included. COMP neo-epitope concentrations were analysed using custom-developed inhibition ELISAs validated for equine serum. The presence of COMP neo-epitope was delineated in healthy and osteoarthritic articular cartilage with immunohistochemistry. Results COMP neo-epitope concentrations decreased after speed training but returned to baseline levels post-exercise. No correlations between age and serum COMP neo-epitope concentrations were found (r = 0.0013). The mean (+/- s.d.) serum concentration of COMP neo-epitope in independent samples from non-lame horses was 0.84 +/- 0.38 mu g/mL, and for lame horses was 5.24 +/- 1.83 mu g/mL (P<0.001). Antibodies against COMP neo-epitope did not stain normal articular cartilage, but intracytoplasmic staining was found in superficial chondrocytes of mild OA cartilage and in the extracellular matrix of moderately osteoarthritic cartilage. Main limitations ELISA was based on polyclonal antisera rather than a monoclonal antibody. There is a sex and breed bias within the groups of horses, also it could have been of value to include horses with septic arthritis and tendonitis and investigated joint differences. Conclusions This COMP neo-epitope can be measured in sera, and results indicate that it could be a biomarker for pathologic fragmentation of cartilage in connection with acute joint lameness.
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| 4. |
- Ley, Cecilia, et al.
(författare)
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Effects of high mobility group box protein-1, interleukin-1β, and interleukin-6 on cartilage matrix metabolism in three-dimensional equine chondrocyte cultures.
- 2011
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Ingår i: Connective tissue research. - : Informa UK Limited. - 1607-8438 .- 0300-8207. ; 52:4, s. 290-300
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Tidskriftsartikel (refereegranskat)abstract
- The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.
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| 5. |
- Löfgren, Maria, et al.
(författare)
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Cell and matrix modulation in prenatal and postnatal equine growth cartilage, zones of Ranvier and articular cartilage
- 2014
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Ingår i: Journal of Anatomy. - : Wiley. - 0021-8782 .- 1469-7580. ; 225:5, s. 548-568
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Tidskriftsartikel (refereegranskat)abstract
- Formation of synovial joints includes phenotypic changes of the chondrocytes and the organisation of their extracellular matrix is regulated by different factors and signalling pathways. Increased knowledge of the normal processes involved in joint development may be used to identify similar regulatory mechanisms during pathological conditions in the joint. Samples of the distal radius were collected from prenatal and postnatal equine growth plates, zones of Ranvier and articular cartilage with the aim of identifying Notch signalling components and cells with stem cell-like characteristics and to follow changes in matrix protein localisation during joint development. The localisation of the Notch signalling components Notch1, Delta4, Hes1, Notch dysregulating protein epidermal growth factor-like domain 7 (EGFL7), the stem cell-indicating factor Stro-1 and the matrix molecules cartilage oligomeric matrix protein (COMP), fibromodulin, matrilin-1 and chondroadherin were studied using immunohistochemistry. Spatial changes in protein localisations during cartilage maturation were observed for Notch signalling components and matrix molecules, with increased pericellular localisation indicating new synthesis and involvement of these proteins in the formation of the joint. However, it was not possible to characterise the phenotype of the chondrocytes based on their surrounding matrix during normal chondrogenesis. The zone of Ranvier was identified in all horses and characterised as an area expressing Stro-1, EGFL7 and chondroadherin with an absence of COMP and Notch signalling. Stro-1 was also present in cells close to the perichondrium, in the articular cartilage and in the fetal resting zone, indicating stem cell-like characteristics of these cells. The presence of stem cells in the articular cartilage will be of importance for the repair of damaged cartilage. Perivascular chondrocytes and hypertrophic cells of the cartilage bone interface displayed positive staining for EGFL7, which is a novel finding and suggests a role of EGFL7 in the vascular infiltration of growth cartilage.
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| 6. |
- Löfgren, Maria, et al.
(författare)
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Conditioned serum in vitro treatment of chondrocyte pellets and osteoarthritic explants
- 2023
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Ingår i: Equine Veterinary Journal. - : Wiley. - 0425-1644 .- 2042-3306. ; 55:2, s. 325-335
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Tidskriftsartikel (refereegranskat)abstract
- Background: Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. Objectives: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1 beta and cartilage explants with mild osteoarthritis. Study design: In vitro experimental study. Methods: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h] and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1 beta and IL-1 receptor antagonist (IL-1Ra) concentrations were measured in all sera. In model 1, chondrocyte pellet cultures were stimulated with IL-1 beta prior to treatment with the serum preparations for 2 and 48 h. Microarray, polymerase chain reaction, and matrix metallopeptidase-13 analyses were performed. In model 2, cartilage explants from horses with structural osteoarthritis were treated with PS or PCS on days 0, 6 and 12, or left untreated, and evaluated at day 24 using the OARSI grading scale for histological evaluation of articular cartilage. Results: The IL-1Ra concentration in PS24h and PCS was significantly higher than in PS. In model 1, inflammation- and cartilage matrix degradation-related genes were upregulated after 48 h in all treatment groups versus untreated controls. Cartilage matrix molecules, aggrecan and collagens, were downregulated in PS24h- and PCS-treated pellets versus untreated controls. Growth factor signalling genes were upregulated-FGF7 in all treatment groups, BMP2 in PS24h-, and INHBA in PCS-treated-compared with untreated controls. In model 2, the OARSI score at day 24 was not significantly different between treatment groups. Main limitations: Results from in vitro models cannot be directly translated to in vivo situations. Conclusions: In vitro treatment with conditioned serum did not alleviate IL-1 beta-induced responses in chondrocyte pellets or lead to morphological improvement in osteoarthritic cartilage explants.
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| 7. |
- Löfgren, Maria, et al.
(författare)
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Time-dependent changes in gene expression induced in vitro by interleukin-1 beta in equine articular cartilage
- 2018
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Ingår i: Research in Veterinary Science. - : Elsevier BV. - 0034-5288 .- 1532-2661. ; 118, s. 466-476
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Tidskriftsartikel (refereegranskat)abstract
- Osteoarthritis is an inflammatory and degenerative joint disease commonly affecting horses. To identify genes of relevance for cartilage pathology in osteoarthritis we studied the time-course effects of interleukin (IL)-1 beta on equine articular cartilage. Articular cartilage explants from the distal third metacarpal bone were collected postmortem from three horses without evidence of joint disease. The explants were stimulated with IL-1 beta for 27 days and global gene expression was measured by microarray. Gene expression was compared to that of unstimulated explants at days 3, 9, 15, 21 and 27. Release of inflammatory proteins was measured using Proximity Extension Assay. Stimulation with IL-1 beta led to time-dependent changes in gene expression related to inflammation, the extracellular matrix (ECM), and phenotypic alterations. Gene expression and protein release of cytokines, chemokines, and matrix-degrading enzymes increased in the stimulated explants. Collagen type II was down regulated from day 15, whereas other ECM molecules were downregulated earlier. In contrast molecules involved in ECM signaling (perlecan, chondroitin sulfate proteoglycan 4, and syndecan 4) were upregulated. At the late time points, genes related to a chondrogenic phenotype were downregulated, and genes related to a hypertrophic phenotype were upregulated, suggesting a transition towards hypertrophy later in the culturing period. The data suggest that this in vitro model mimics time course events of in vivo inflammation in OA and it may be valuable as an in vitro tool to test treatments and to study disease mechanisms.
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| 8. |
- Nystrom, S., et al.
(författare)
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The expression of nerve growth factor in healthy and inflamed equine chondrocytes analysed by capillary western immunoassay
- 2022
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Ingår i: Research in Veterinary Science. - : Elsevier BV. - 0034-5288 .- 1532-2661. ; 151, s. 156-163
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Tidskriftsartikel (refereegranskat)abstract
- Nerve Growth Factor (NGF) is a signalling molecule for pain and inflammation. NGF is increased in synovial fluid from osteoarthritic humans and animals, compared to healthy controls. Monoclonal antibody therapy directed against NGF has been approved to treat pain in osteoarthritic dogs but despite many years of trialling, therapy has not been approved for human use. One reason for this is that adverse reactions with rapidly progressing osteoarthritis has occurred in some individuals. More detailed knowledge of NGF expression in joints is needed. In this study, capillary-based Simple Western was used to analyse NGF in cultured equine chondrocytes. Chondrocytes were collected post mortem from three macroscopically healthy intercarpal joints and three intercarpal joints with mild osteoarthritic changes. The chondrocytes were expanded to passage one and seeded in chondrogenic medium to maintain the phenotype. On day four, cells were either stimulated with LPS or kept untreated in medium. All cells were harvested on day five. Wes analysis of lysates did not show mature NGF but two proforms, 40 and 45 kDa, were identified. Results were confirmed with western blot. The same proforms were expressed in chondrocytes from healthy and osteoarthritic joints. Acute inflammation induced by LPS stimulation did not change the forms of expressed NGF. Capillary Simple Western offers a sensitive and sample -sparing alternative to traditional western blot. However, confirmation of peaks is imperative in order to avoid misinterpretation of findings. In addition, in this case the method did not offer the possibility of quantification advertised by the manufacturers.
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| 9. |
- Rotter Sopasakis, Victoria, 1972, et al.
(författare)
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Elevated Glucose Levels Preserve Glucose Uptake, Hyaluronan Production, and Low Glutamate Release Following Interleukin-1 beta Stimulation of Differentiated Chondrocytes
- 2019
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Ingår i: Cartilage. - : SAGE Publications. - 1947-6035 .- 1947-6043. ; 10:4, s. 491-503
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Tidskriftsartikel (refereegranskat)abstract
- Objective Chondrocytes are responsible for remodeling and maintaining the structural and functional integrity of the cartilage extracellular matrix. Because of the absence of a vascular supply, chondrocytes survive in a relatively hypoxic environment and thus have limited regenerative capacity during conditions of cellular stress associated with inflammation and matrix degradation, such as osteoarthritis (OA). Glucose is essential to sustain chondrocyte metabolism and is a precursor for key matrix components. In this study, we investigated the importance of glucose as a fuel source for matrix repair during inflammation as well as the effect of glucose on inflammatory mediators associated with osteoarthritis. Design To create an OA model, we used equine chondrocytes from 4 individual horses that were differentiated into cartilage pellets in vitro followed by interleukin-1 beta (IL-1 beta) stimulation for 72 hours. The cells were kept at either normoglycemic conditions (5 mM glucose) or supraphysiological glucose concentrations (25 mM glucose) during the stimulation with IL-1 beta. Results We found that elevated glucose levels preserve glucose uptake, hyaluronan synthesis, and matrix integrity, as well as induce anti-inflammatory actions by maintaining low expression of Toll-like receptor-4 and low secretion of glutamate. Conclusions Adequate supply of glucose to chondrocytes during conditions of inflammation and matrix degradation interrupts the detrimental inflammatory cycle and induces synthesis of hyaluronan, thereby promoting cartilage repair.
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| 10. |
- Skiöldebrand, Eva, et al.
(författare)
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A randomized, triple-blinded controlled clinical study with a novel disease-modifying drug combination in equine lameness-associated osteoarthritis
- 2023
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Ingår i: Osteoarthritis and Cartilage Open. - 2665-9131. ; 5:3
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Tidskriftsartikel (refereegranskat)abstract
- Objective: This study aimed to test a novel treatment combination (TC) (equivalent to sildenafil, mepivacaine, and glucose) with disease-modifying properties compared to Celestone® bifas® (CB) in a randomized triple-blinded phase III clinical study in horses with mild osteoarthritis (OA). Joint biomarkers (reflecting the articular cartilage and subchondral bone remodelling) and clinical lameness were used as readouts to evaluate the treatment efficacy. Methods: Twenty horses with OA-associated lameness in the carpal joint were included in the study and received either TC (n = 10) or CB (n = 10) drug intra-articularly-twice in the middle carpal joint with an interval of 2 weeks (visit 1 & 2). Clinical lameness was assessed both objectively (Lameness locator) and subjectively (visually). Synovial fluid and serum were sampled for quantification of the extracellular matrix (ECM) neo-epitope joint biomarkers represented by biglycan (BGN262) and cartilage oligomeric matrix protein (COMP156). Another two weeks later clinical lameness was recorded, and serum was collected for biomarkers analysis. The overall health status was compared pre and post-intervention by interviewing the trainer. Results: Post-intervention, SF BGN262 levels significantly declined in TC (P = 0.002) and COMP156 levels significantly increased in CB (P = 0.002). The flexion test scores improved in the TC compared to CB (P =0.033) and also had an improved trotting gait quality (P =0.044). No adverse events were reported. Conclusion: This is the first clinical study presenting companion diagnostics assisting in identifying OA phenotype and evaluating the efficacy and safety of a novel disease-modifying osteoarthritic drug.
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