Anne Vaughan-Thomas

University of Liverpool, Liverpool, ENG, United Kingdom

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Publications (30)84.77 Total impact

  • J F Innes, C Gordon, A Vaughan-Thomas, N P Rhodes, P D Clegg
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    ABSTRACT: Osteochondral lesions are a major cause of pain and disability in several species including dogs, horses and human beings. The objective of this study was to assess three potential sources of canine cells for their osteochondral regenerative potential. Cartilage, synovium and adipose tissue cells were grown in pellet culture in chondrogenic or osteogenic media. Cartilage-derived pellets displayed the best chondrogenic differentiation as indicated by significantly higher COL2A1 and SOX9 mRNA expression, greater glycosaminoglycan content, and higher retention of Safranin-O stain compared to the synovium and adipose-derived cells. Following application of the osteogenic media, all three cell sources exhibited small areas of positive alizarin red staining. Poor intracellular alkaline phosphatase activity was found in all three cell types when stimulated although osteocalcin and RUNX2 expression were significantly increased. Cells isolated and cultured from canine articular cartilage retained their specific chondrocytic phenotype. Furthermore, canine adipocytes and synovial cells did not undergo chondrogenic differentiation and did not exhibit evidence of multipotency. Although osteogenic differentiation was initiated at a genomic level, phenotypic osteoblastic differentiation was not observed. The findings of this study suggest that cells isolated from canine adipose tissue and synovium are sub-optimal substitutes for chondrocytes when engineering articular cartilage in vitro.
    The Veterinary Journal 07/2013; · 2.42 Impact Factor
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    ABSTRACT: Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4',6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy. Both cranial and caudal CLs contained cells of heterogenous morphologies. Cells were arranged between collagen bundles and frequently had cytoplasmic processes. Some of these processes were long (type A cells), others were shorter, thicker and more branched (type B cells), and some had no processes (type C cells). Processes were frequently shown to contact other cells, extending longitudinally and transversely through the CLs. Cells with longer processes had fusiform nuclei, and those with no processes had rounded nuclei and were more frequent in the mid-substance of both CLs. Cells with long processes were more commonly noted in the CLs of the Greyhound. As contact between cells may facilitate direct communication, variances in cell morphology between breeds at a differing risk of CCL rupture may reflect differences in CL physiology.
    The Veterinary Journal 03/2012; 193(2):561-6. · 2.42 Impact Factor
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    ABSTRACT: Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the α1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library. 73% of the interacting clones encoded fibronectin. The interaction was confirmed using in vitro immunoprecipitation and was further characterized by surface plasmon resonance. Using whole and pepsin-derived preparations of type IX collagen, the interaction was shown to be specific for the NC4 domain with no interaction with the triple helical collagenous domains. The interaction was shown to be of high affinity with nanomolar K(d) values. Analysis of the fibronectin-interacting clones indicates that the constant domain is the likely site of interaction. Type IX collagen and fibronectin were shown to co-localize in cartilage. This novel interaction between the NC4 domain of type IX collagen and fibronectin may represent an in vivo interaction in cartilage that could contribute to the matrix integrity of the tissue.
    Journal of Biological Chemistry 07/2011; 286(40):34986-97. · 4.65 Impact Factor
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    ABSTRACT: Although elastin fibres and oxytalan fibres (bundles of microfibrils) have important mechanical, biochemical and cell regulatory functions, neither their distribution nor their function in cruciate ligaments has been investigated. Twelve pairs of cruciate ligaments (CLs) were obtained from 10 adult dogs with no evidence of knee osteoarthritis. Elastic fibres were identified using Verhoeff's and Miller's staining. Fibrillins 1 and 2 were immunolocalised and imaged using confocal laser scanning microscopy. Hydrated, unfixed tissue was analysed using Nomarski differential interference microscopy (NDIC), allowing structural and mechanical analysis. Microfibrils and elastin fibres were widespread in both CLs, predominantly within ligament fascicles, parallel to collagen bundles. Although elastin fibres were sparse, microfibrils were abundant. We described abundant fibres composed of both fibrillin 1 and fibrillin 2, which had a similar pattern of distribution to oxytalan fibres. NDIC demonstrated complex interfascicular and interbundle anatomy in the CL complex. The distribution of elastin fibres is suggestive of a mechanical role in bundle reorganisation following ligament deformation. The presence and location of fibrillin 2 in oxytalan fibres in ligament differs from the solely fibrillin 1-containing oxytalan fibres previously described in tendon and may demonstrate a fundamental difference between ligament and tendon.
    Journal of Anatomy 04/2011; 218(6):600-7. · 2.36 Impact Factor
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    ABSTRACT: Osteoarthritis is the most common arthropathy of mammalian species including cats. Cartilage degradation is central to the disorder and here we present, for the first time, an in vitro model of feline cartilage degradation which will be useful for further studies in this target species. Feline articular cartilage explant cultures were maintained for 28 days and in the presence of oncostatin M with and without interleukin (IL)-17, tumour necrosis factor (TNF), IL-1alpha, or IL-1beta. Media samples and digested cartilage explants were analysed for glycosaminoglycan (GAG) and collagen content. The combination of IL-1beta and OSM, both at 20 ng/ml, was able to promote GAG release to the greatest extent at 14 days. At 28 days, all groups showed relatively high release of GAG. At 14 days, only IL-1beta and OSM in combination were associated with a statistically significant increase in collagen release over and above control tissue. IL-1beta dose-response studies showed that an IL-1beta dose of 10 ng/ml promotes a statistically significant increase in GAG breakdown when used with OSM, and higher doses of IL-1beta did not result in significantly greater response. The model demonstrated both GAG and collagen degradation and will be of use for further understanding of feline cartilage metabolism and for screening of potential structure-modifying agents to be used in cats.
    Journal of feline medicine and surgery. 08/2010; 12(8):614-20.
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    ABSTRACT: Type II collagen is a major component of articular cartilage and its breakdown is a key feature of osteoarthritis. Products of cartilage collagen metabolism can be detected in the blood, synovial fluid and urine. Several biomarker assays have been developed which can be used to measure the synthesis and degradation of collagen, and therefore provide information regarding cartilage turnover. This is the first part of a two-part review and describes the need for accurate, reliable information regarding collagen turnover, the processes by which the biomarker epitopes are generated, their application to the study of both healthy and diseased cartilage and the results of currently published studies, with particular reference to the veterinary species. The second part of the review considers the non-collagenous biomarkers of cartilage matrix turnover.
    The Veterinary Journal 07/2010; 185(1):36-42. · 2.42 Impact Factor
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    ABSTRACT: Osteoarthritis (OA) results in the destruction and breakdown of articular cartilage matrix. Breakdown of the cartilage proteoglycan component results in the generation of constituent fragments that can be detected in the blood, synovial fluid or urine. Non-collagenous, non-proteoglycan components of cartilage can also be detected following their release as a result of turnover and disease. OA also alters the circulating profile of metabolites in the body. Metabolomic strategies have been used to distinguish populations with OA from normal populations by the creation of a metabolomic 'fingerprint' attributable to the disease. This paper is the second part of a two-part review and describes some of the techniques used to measure the concentrations of some of these 'non-collagenous' biomarkers, and how the application of these measurements assists the study of joint disease. Collagen-based biomarkers were discussed in part one.
    The Veterinary Journal 07/2010; 185(1):43-9. · 2.42 Impact Factor
  • International Journal of Experimental Pathology. 01/2010; 91(2):A43-A43.
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    ABSTRACT: The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1beta (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or tumor necrosis factor-alpha (TNFalpha), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro-matrix metalloproteinases (MMP)-2 but not pro-MMP-9, as assessed by gelatin zymography. APC did not directly activate pro-MMP-13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10(- 5)M and 10(-10)M. High concentrations significantly increased GAG release from IL-1+APC-treated explants. With the exception of MPA at 10(-10)M, all concentrations of corticosteroids caused significant decreases in IL-1+APC-driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP-1, -3, and -13 mRNA. The collagenolysis associated with IL-1+APC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively.
    Journal of Orthopaedic Research 09/2009; 28(3):370-8. · 2.88 Impact Factor
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    ABSTRACT: There is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis. Expression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative PCR (qPCR). Expression levels of genes associated with tendon phenotype were then evaluated in healthy, including developmental, and diseased equine tendon tissue and in tendon fibroblasts maintained in both monolayer culture and in three dimensional (3D) collagen gels. Significantly increased expression of scleraxis was found in tendon compared with bone (P = 0.002) but not compared to cartilage. High levels of COL1A2 and scleraxis and low levels of tenascin-C were found to be most representative of adult tensional tendon phenotype. While, relative expression of scleraxis in developing mid-gestational tendon or in acute or chronically diseased tendon did not differ significantly from normal adult tendon, tenascin-C message was significantly upregulated in acutely injured equine tendon (P = 0.001). Relative scleraxis gene expression levels in tendon cell monolayer and 3D cultures were significantly lower than in normal adult tendon (P = 0.002, P = 0.02 respectively). The findings of this study indicate that high expression of both COL1A2 and scleraxis, and low expression of tenascin-C is representative of a tensional tendon phenotype. The in vitro culture methods used in these experiments however, may not recapitulate the phenotype of normal tensional tendon fibroblasts in tissues as evidenced by gene expression.
    BMC Musculoskeletal Disorders 03/2009; 10:27. · 1.88 Impact Factor
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    Lisa Macrory, Anne Vaughan-Thomas, Peter D Clegg, John F Innes
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    ABSTRACT: To study the ability of tepoxalin, a dual inhibitor of cyclooxygenase (COX) and lipoxygenase (LOX) and its active metabolite to reduce the catabolic response of cartilage to cytokine stimulation in an in vitro model of canine osteoarthritis (OA).Grossly normal cartilage was collected post-mortem from seven dogs that had no evidence of joint disease. Cartilage explants were cultured in media containing the recombinant canine interleukin-1beta (IL-1beta) at 100 ng/ml and recombinant human oncostatin-M (OSM) at 50 ng/ml. The effects of tepoxalin and its metabolite were studied at three concentrations (1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) M). Total glycosaminoglycan (GAG) and collagen (hydroxyproline) release from cartilage explants were used as outcome measures of proteoglycan and collagen depletion respectively. PGE2 and LTB4 assays were performed to study the effects of the drug on COX and LOX activity. Treatment with IL-1beta and OSM significantly upregulated both collagen (p = 0.004) and proteoglycan (p = 0.001) release from the explants. Tepoxalin at 10(-5) M and 10(-6) M caused a decrease in collagen release from the explants (p = 0.047 and p = 0.075). Drug treatment showed no effect on GAG release. PGE2 concentration in culture media at day 7 was significantly increased by IL-1beta and OSM and treatment with both tepoxalin and its metabolite showed a trend towards dose-dependent reduction of PGE2 production. LTB4 concentrations were too low to be quantified. Cytotoxicity assays suggested that neither tepoxalin nor its metabolite had a toxic effect on the cartilage chondrocytes at the concentrations and used in this study. This study provides evidence that tepoxalin exerts inhibition of COX and can reduce in vitro collagen loss from canine cartilage explants at a concentration of 10(-5) M. We can conclude that, in this model, tepoxalin can partially inhibit the development of cartilage degeneration when it is available locally to the tissue.
    BMC Veterinary Research 02/2009; 5:25. · 1.86 Impact Factor
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    ABSTRACT: Articular chondrocytes exist in an environment lacking in oxygen and nutrients due to the avascular nature of cartilage. The main source of metabolic energy is glucose, which is taken up by glucose transporters (GLUTs). In diseased joints, oxygen tensions and glucose availability alter as a result of inflammation and changes in vascularisation. Accordingly, in this study we examined the effects of hypoxia and the hypoxia mimetic cobalt chloride (CoCl(2)) on glucose transport in equine chondrocytes and compared expression of the hypoxia responsive GLUT1 gene in normal and diseased cartilage. Monolayers of equine chondrocytes were exposed to 20% O(2), 1% O(2), CoCl(2) (75 microM), or a combination of 1% O(2) and CoCl(2). Glucose uptake was measured using 2-deoxy-D-[2,6-(3)H] glucose. GLUT1 protein and mRNA expression were determined by FACS analysis and qPCR, respectively. GLUT1 mRNA expression in normal and diseased cartilage was analyzed using explants derived from normal, OA, and OCD cartilage. Chondrocytes under hypoxic conditions exhibited a significantly increased glucose uptake as well as upregulated GLUT1 protein expression. GLUT1 mRNA expression significantly increased in combined hypoxia-CoCl(2) treatment. Analysis of clinical samples indicated a significant reduction in GLUT1 mRNA in OA samples. In OCD samples GLUT1 expression also decreased but did not reach statistical significance. The increase in glucose uptake and GLUT1 expression under hypoxic conditions confirms that hypoxia alters the metabolic requirements of chondrocytes. The altered GLUT1 mRNA expression in diseased cartilage with significance in OA suggests that reduced GLUT1 may contribute to the failure of OA cartilage repair.
    Journal of Orthopaedic Research 11/2008; 27(4):529-35. · 2.88 Impact Factor
  • E R Garvican, A Vaughan-Thomas, C Redmond, P D Clegg
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    ABSTRACT: Our objective was to characterize the variation in gene expression for key genes associated with chondrogenic phenotype of osteochondrosis (OC)-affected and normal chondrocytes, and to identify whether OC chondrocytes can redifferentiate and regain a phenotype similar to normal chondrocytes if appropriate chondrogenic signals are given. Equine articular cartilage removed at surgery to treat clinically significant OC lesions was collected (n = 10), and the gene expression evaluated and compared to aged-matched normal samples (n = 10). Cartilage was harvested from normal (n = 4) and OC (n = 3) joints from horses at necropsy. Chondrogenic pellet cultures were established following monolayer proliferation. After 14 days in culture, the pellets were assessed by histochemical and pellet weight analysis, assay of glycosaminoglycan (GAG) content, and gene expression. Chondrocytes from OC cartilage expressed significantly more Coll-I, -II, -III, and -X than chondrocytes from normal cartilage (all p < 0.0001). Furthermore, OC chondrocytes expressed significantly more MMP-13, ADAMTS-4 (both p < 0.0001), and TIMP-1 (p < 0.001) and significantly less TIMP-2 and TIMP-3. Pellets created from OC chondrocytes contained significantly less GAG (p = 0.0069) and expressed significantly less Sox9 and significantly more superficial zone protein (SZP) (p = 0.0105) than pellets created from normal cartilage. The results suggest that chondrocytes from OC cartilage at the time of surgical treatment have perturbations in phenotype compared to cells from normal cartilage. Despite these differences, following monolayer expansion and pellet culture under chondrogenic conditions, chondrocytes derived from OC cartilage retain some ability to undergo chondrogenic differentiation and synthesize an appropriate cartilage-like matrix. However, this chondrogenic differentiation potential is inferior to that seen in aged-matched normal chondrocytes.
    Journal of Orthopaedic Research 09/2008; 26(8):1133-40. · 2.88 Impact Factor
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    ABSTRACT: Fatigue fracture of cuboidal bones occurs in the human foot as well as the equine carpus. The racehorse provides a naturally-occurring model to study the effects of high-intensity exercise on the morphology and metabolism of cuboidal bones. We studied both the mineral and the collagenous matrix of the third (C(3)) and radial (C(r)) carpal bones of raced and non-raced Thoroughbred (TB) horses. We hypothesised that racehorses would show increases in the mineral component of these bones and post-translational modifications of the collagenous matrix alongside changes in markers of collagen remodelling and bone formation. C(3) and C(r) carpal bones were retrieved from raced TB horses (n=14) and non-raced TB horses (n=11). Standardised proximal-distal sections were taken from each bone and these were sliced transversely to study the proximal-distal differences in bone metabolism from the subchondral plate through to trabecular bone. Histomorphometry and bone mineral density measurements were performed in parallel with biochemical analyses including total collagen, collagen synthesis and cross-links, matrix metalloproteinases-2 and 9 and their inhibitors, calcium and phosphate, and bone alkaline phosphatase. The results of this study show that, while there is a net increase in bone formation in the racehorses, there is additionally an increase in bone collagen synthesis and remodelling, particularly within the trabecular regions of the bone. The increase in bone density would lead to greater stiffness, particularly in the cortical bone, and failure of this 'stiffer' cortical bone may result from its lack of support from the rapidly remodelling and structurally weakened underlying trabecular bone.
    Bone 07/2008; 43(4):724-33. · 4.46 Impact Factor
  • E R Garvican, A Vaughan-Thomas, C Redmond, P D Clegg
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    ABSTRACT: Matrix degradation by metalloproteinases is considered a key feature in the loss of articular cartilage seen in many joint diseases. Membrane-type matrix metalloproteinase-3 (MT3-MMP) expression is elevated in human cartilage in end-stage osteoarthritis. We investigated whether MT3-MMP is similarly regulated in cartilage in two naturally occurring arthropathies in vivo and whether proinflammatory cytokines regulate its expression in vitro. MT3-MMP expression was evaluated in cartilage from horses with osteoarthritis and osteochondrosis and compared with age- and site-matched normal cartilage. MT3-MMP also was measured in normal cartilage stimulated with proinflammatory cytokines. MT3-MMP expression was not significantly altered in either osteoarthritis or osteochondrosis cartilage. However, gene expression was significantly downregulated by the addition of recombinant human interleukin-1beta, oncostatin M, or tumor necrosis factor-alpha to normal cartilage explants. The results suggest that MT3-MMP may not have a role in matrix destruction in equine cartilage diseases. Further work is required to characterize its regulation and function.
    Connective tissue research 02/2008; 49(2):62-7. · 1.55 Impact Factor
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    ABSTRACT: Recent studies have identified a range of interactions between type IX collagen and other cartilage matrix components. To determine the extent to which these interactions are important in maintaining the integrity of ageing articular cartilage, we analyzed an age range of normal healthy articular cartilage samples by Western blotting, immunohistochemical, and PCR analyses. Reduced levels of type IX collagen were detected in post adolescence cartilage. Type IX collagen epitopes were evident throughout the matrix in all cartilage samples up to 19 years of age. Post adolescence, however, the pattern of immunoreactivity revealed territorial staining only. Type IX collagen expression at the transcriptional level is maintained at all ages. Type IX collagen fragments were extracted from young tissue, supporting the hypothesis that young cartilage is continually remodelled, while mature cartilage maintains relatively low levels of collagen turnover. Clearly the age changes we observed may have significant effects on the integrity of the tissue as the chondrocytes in ageing articular cartilage have limited capacity to turnover the interterritorial matrix. However, this study provides evidence that even in old age, the chondrocyte attempts to maintain its pericellular environment and hence its mechanical role. Therefore, the potential of type IX collagen to interact with other matrix components continues to be of importance in the territorial environment, and these interactions may have significant roles in mechanotransduction.
    Connective tissue research 02/2008; 49(5):374-82. · 1.55 Impact Factor
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    ABSTRACT: Felid herpesvirus 1 (FeHV-1) is an important feline pathogen of the upper respiratory tract which can be identified in clinical cases by virus isolation and PCR. Studies on the effect and mode of spread of FeHV-1 in the respiratory epithelium, however, have previously only been performed in infected cats. Feline tracheal organ cultures which were inoculated with FeHV-1 at varying multiplicity of infection (MOI) were established. A dose-dependent response was observed. Low MOIs induced multifocal infection in the otherwise viable respiratory epithelium, which allowed monitoring of viral growth over several days. Therefore, tracheal organ cultures represent a suitable model for further study of the morphological and functional effects of FeHV-1 on respiratory epithelium, mimicking the in vivo situation.
    Journal of Virological Methods 01/2007; 138(1-2):191-5. · 1.90 Impact Factor
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    ABSTRACT: To evaluate the hypothesis that the concentration of the 1/20/5D4 epitope of keratan sulphate, cartilage oligomeric matrix protein and total sulphated glycosaminoglycans in synovial fluids from dogs with cranial cruciate ligament disease would be affected by tibial plateau levelling osteotomy. In addition, to evaluate the hypothesis that medial meniscal release or meniscal injury would alter the expression of these candidate biomarkers. Forty-one dogs with naturally occurring cranial cruciate ligament disease were recruited prospectively. Synovial fluids were collected from the index joint before surgery and six weeks and six months postsurgery. Following tibial plateau levelling osteotomy, synovial fluids were assayed for 1/20/5D4 epitope of keratan sulphate and cartilage oligomeric matrix protein concentration using an inhibition ELISA and for sulphated glycosaminoglycans using a direct dye-binding assay. The sulphated glycosaminoglycans ratio did not change significantly during the study. Medial meniscal injury at entry was associated with lower concentrations of synovial fluid cartilage oligomeric matrix protein (P<0.05, unpaired t test). There was no association between medial meniscal release and the changes in marker concentrations, either from 0 to six weeks or 0 to six months. Tibial plateau levelling osteotomy did not significantly alter the expression of the named candidate biomarkers. These findings reflect the limited nature of the arthrotomy or indicate that tibial plateau levelling osteotomy does not influence the progression of osteoarthritis (OA). From these studies, there is no evidence that tibial plateau levelling osteotomy affects cartilage metabolism.
    Journal of Small Animal Practice 01/2007; 47(12):708-14. · 1.18 Impact Factor
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    ABSTRACT: While intervertebral disc (IVD) degeneration is associated with the majority of cases of low back pain, current treatments are symptomatic rather than curative. Tissue engineering offers a treatment that both cures the problem of disc degeneration and restores normal disc function. One of the major problems for any tissue engineering strategy, however, is ensuring that both the cells and matrices used are suitable for the target tissue. In this study, we have developed and studied a potential system for tissue engineering of the nucleus pulposus (NP) of the severely degenerate IVD. While cells from degenerate discs are not suitable for tissue engineering, bone-marrow-derived mesenchymal stem cells, which are capable of differentiating into chondrocyte-like cells such as those found within the NP of the disc, offer a potential source of cells. We have used transfection with adenoviral SOX-9, a transcription factor involved in differentiation of MSCs along the chondrogenic lineage, combined with culture in a specialised medium, to differentiate monolayer MSCs to NP-like (chondrocyte-like) cells, as shown by real-time quantitative polymerase chain reaction for NP-marker genes. We have also replicated these findings on porous, biodegradable three-dimensional (3D) poly-l-lactic acid scaffolds and shown expression and deposition of NP matrix markers such as type II collagen and aggrecan. We are therefore proposing pre-differentiation of human MSCs and seeding on porous, biodegradable 3D synthetic polymer scaffolds as a realistic tissue engineering strategy for regeneration of the degenerate human IVD.
    Biomaterials 09/2006; 27(22):4069-78. · 8.31 Impact Factor
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    ABSTRACT: To assess 2 methods of RNA purification by use of different quality metrics and identify the most useful metric for quality assessment of RNA extracted from articular cartilage from dogs with osteoarthritis. 40 articular cartilage specimens from the femoral heads of 3 clinically normal dogs and 37 dogs with osteoarthritis. RNA was extracted from articular cartilage by 2 purification methods. Quality metrics of each sample were determined and recorded by use of a UV spectrophotometer (Spec I; to determine the 260 to 280 nm absorbance ratio [A(260):A(280) ratio]), a second UV spectrophotometer (Spec II; to determine A(260):A(280) and A(260):A(230) absorbance ratios), and a microfluidic capillary electrophoresis analyzer (to determine the ribosomal peak ratio [RR], degradation factor [DF], and RNA integrity number [RIN]). The RNA was extracted from affected (osteoarthritic) articular cartilage and assessed with the same quality metrics. Metric results were compared with visual analysis of the electropherogram to determine the most useful RNA quality metric. No differences in methods of RNA purification were determined by use of quality metrics. The RNA extracted from unaffected (normal) cartilage was of higher quality than that extracted from affected (osteoarthritic) cartilage, as determined by the RIN and Spec II A(260):A(230) ratio. The RIN and RR were the most sensitive metrics for determining RNA quality, whereas the DF was most specific. A significant proportion (32%) of RNA extracted from osteoarthritic articular cartilage specimens was determined as being of low quality. No single metric provided a completely sensitive and specific assessment of the quality of RNA recovered from articular cartilage.
    American Journal of Veterinary Research 09/2006; 67(8):1438-44. · 1.35 Impact Factor

Publication Stats

387 Citations
84.77 Total Impact Points

Institutions

  • 2004–2013
    • University of Liverpool
      • • Department of Musculoskeletal Biology
      • • School of Veterinary Science
      Liverpool, ENG, United Kingdom
  • 2001–2002
    • Cardiff University
      • School of Biosciences
      Cardiff, WLS, United Kingdom
  • 1998
    • University of Wales
      • School of Molecular and Medical Biosciences
      Cardiff, Wales, United Kingdom