Gene expression analysis of subchondral bone, cartilage, and synovium in naturally occurring equine palmar/plantar osteochondral disease.

Osteoarthritis (OA) is a disease of the entire joint but the relationship between pathological events in various joint tissues is poorly understood. We examined concurrent changes in bone, cartilage, and synovium in a naturally occurring equine model of joint degeneration. Joints (n = 64) were grossly assessed for palmar/plantar osteochondral disease (POD) in racehorses that required euthanasia for unrelated reasons and assigned a grade of 0 (n = 34), 1 (n = 17), 2 or 3 (n = 13) using a recognized grading scheme. Synovium, cartilage, and subchondral bone were collected for histological and gene expression analysis. Relations between POD grade, cartilage histological score, and gene expression levels were examined using one-way analysis of variance or Kruskal-Wallis test and Spearman's correlation coefficient with corrections for multiple comparisons. Cartilage histological score increased in joints with POD grade 1 (p = 0.002) and 2 or 3 (p < 0.001) compared to 0. At grade 1, expression of COL1A1, COL2A1, and MMP1 increased and BGN decreased in subchondral bone while expression of BGN and ACAN decreased in cartilage. These changes further progressed at grades 2 and 3. POD grades 2 and 3 were associated with decreased expression of osteoclast inhibitor OPG and increased markers of cartilage degeneration (MMP13, COL1A1). Expression of the vascular endothelial growth factor decreased with POD grade and negatively correlated with cartilage histological score. Synovium showed no histological or transcriptomic changes related to pathology grade. Cartilage degeneration in POD is likely to be secondary to remodeling of the subchondral bone. Limited activation of proinflammatory and catabolic genes and moderate synovial pathology suggests distinct molecular phenotype of POD compared with OA.

Mouse microRNA signatures in joint ageing and post-traumatic osteoarthritis.

OBJECTIVE: This study investigated mice serum and joint microRNA expression profiles in ageing and osteoarthritis to elucidate the role of microRNAs in the development and progression of disease, and provide biomarkers for ageing and osteoarthritis. DESIGN: Whole joints and serum samples were collected from C57BL6/J male mice and subjected to small RNA sequencing. Groups used included; surgically-induced post-traumatic osteoarthritis, (DMM; 24 months-old); sham surgery (24 months-old); old mice (18 months-old); and young mice (8 months-old). Differentially expressed microRNAs between the four groups were identified and validated using real-time quantitative PCR. MicroRNA differential expression data was used for target prediction and pathway analysis. RESULTS: In joint tissues, miR-140-5p, miR-205-5p, miR-682, miR-208b-3p, miR-499-5p, miR-455-3p and miR-6238 were differentially expressed between young and old groups; miR-146a-5p, miR-3474, miR-615-3p and miR-151-5p were differentially expressed between DMM and Sham groups; and miR-652-3p, miR-23b-3p, miR-708-5p, miR-5099, miR-23a-3p, miR-214-3p, miR-6238 and miR-148-3p between the old and DMM groups. The number of differentially expressed microRNAs in serum was higher, some in common with joint tissues including miR-140-5p and miR-455-3p between young and old groups; and miR-23b-3p, miR-5099 and miR-6238 between old and DMM groups.We confirmed miR-140-5p, miR-499-5p and miR-455-3p expression to be decreased in old mouse joints compared to young, suggesting their potential use as biomarkers of joint ageing in mice. CONCLUSIONS: MiR-140-5p, miR-499-5p and miR-455-3p could be used as joint ageing biomarkers in mice. Further research into these specific molecules in human tissues is now warranted to check their potential suitability as human biomarkers of ageing.

Ex Vivo Equine Cartilage Explant Osteoarthritis Model: A Metabolomics and Proteomics Study.

Osteoarthritis is an age-related degenerative musculoskeletal disease characterized by loss of articular cartilage, synovitis, and subchondral bone sclerosis. Osteoarthritis pathogenesis is yet to be fully elucidated with no osteoarthritis-specific biomarkers in clinical use. Ex vivo equine cartilage explants (n = 5) were incubated in tumor necrosis factor-α (TNF-α)/interleukin-1ß (IL-1ß)-supplemented culture media for 8 days, with the media removed and replaced at 2, 5, and 8 days. Acetonitrile metabolite extractions of 8 day cartilage explants and media samples at all time points underwent one-dimensional (1D) 1H nuclear magnetic resonance metabolomic analysis, with media samples also undergoing mass spectrometry proteomic analysis. Within the cartilage, glucose and lysine were elevated following TNF-α/IL-1ß treatment, while adenosine, alanine, betaine, creatine, myo-inositol, and uridine decreased. Within the culture media, 4, 4, and 6 differentially abundant metabolites and 154, 138, and 72 differentially abundant proteins were identified at 1-2, 3-5, and 6-8 days, respectively, including reduced alanine and increased isoleucine, enolase 1, vimentin, and lamin A/C following treatment. Nine potential novel osteoarthritis neopeptides were elevated in the treated media. Implicated pathways were dominated by those involved in cellular movement. Our innovative study has provided insightful information on early osteoarthritis pathogenesis, enabling potential translation for clinical markers and possible new therapeutic targets.

Cross platform analysis of transcriptomic data identifies ageing has distinct and opposite effects on tendon in males and females.

The development of tendinopathy is influenced by a variety of factors including age, gender, sex hormones and diabetes status. Cross platform comparative analysis of transcriptomic data elucidated the connections between these entities in the context of ageing. Tissue-engineered tendons differentiated from bone marrow derived mesenchymal stem cells from young (20-24 years) and old (54-70 years) donors were assayed using ribonucleic acid sequencing (RNA-seq). Extension of the experiment to microarray and RNA-seq data from tendon identified gender specific gene expression changes highlighting disparity with existing literature and published pathways. Separation of RNA-seq data by sex revealed underlying negative binomial distributions which increased statistical power. Sex specific de novo transcriptome assemblies generated fewer larger transcripts that contained miRNAs, lincRNAs and snoRNAs. The results identify that in old males decreased expression of CRABP2 leads to cell proliferation, whereas in old females it leads to cellular senescence. In conjunction with existing literature the results explain gender disparity in the development and types of degenerative diseases as well as highlighting a wide range of considerations for the analysis of transcriptomic data. Wider implications are that degenerative diseases may need to be treated differently in males and females because alternative mechanisms may be involved.

A proteomic analysis of chondrogenic, osteogenic and tenogenic constructs from ageing mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have prospective applications in regenerative medicine and tissue engineering but to what extent phenotype and differentiation capacity alter with ageing is uncertain. Consequently, any loss in functionality with age would have profound consequences for the maintenance of tissue viability and the quality of tissues. Proteomics enables the set of proteins responsible for a particular cell phenotype to be identified, as well as enabling insights into mechanisms responsible for age-related alterations in musculoskeletal tissues. Few proteomic studies have been undertaken regarding age-related effects on tissue engineered into cartilage and bone, and none for tendon. This study provides a proteome inventory for chondrogenic, osteogenic and tenogenic constructs synthesised from human MSCs, and elucidates proteomic alterations as a consequence of donor age. METHODS: Human bone-marrow derived MSCs from young (n = 4, 21.8 years ± 2.4SD) and old (n = 4, 65.5 years ± 8.3SD) donors were used to make chondrogenic, osteogenic and tenogenic tissue-engineered constructs. We utilised an analytical method relying on extracted peptide intensities as a label-free approach for peptide quantitation by liquid chromatography-mass spectrometry. Results were validated using western blotting. RESULTS: We identified proteins that were differentially expressed with ageing; 128 proteins in chondrogenic constructs, 207 in tenogenic constructs and four in osteogenic constructs. Differentially regulated proteins were subjected to bioinformatic analysis to ascertain their molecular functions and the signalling pathways. For all construct types, age-affected proteins were involved in altered cell survival and death, and antioxidant and cytoskeletal changes. Energy and protein metabolism were the principle pathways affected in tenogenic constructs, whereas lipid metabolism was strongly affected in chondrogenic constructs and mitochondrial dysfunction in osteogenic constructs. CONCLUSIONS: Our results imply that further work on MSC-based therapeutics for the older population needs to focus on oxidative stress protection. The differentially regulated proteome characterised by this study can potentially guide translational research specifically aimed at effective clinical interventions.

Resveratrol and N-acetylcysteine influence redox balance in equine articular chondrocytes under acidic and very low oxygen conditions.

Mature articular cartilage is an avascular tissue characterized by a low oxygen environment. In joint disease, acidosis and further reductions in oxygen levels occur, compromising cartilage integrity.This study investigated how acidosis and very low oxygen levels affect components of the cellular redox system in equine articular chondrocytesand whether the antioxidants resveratrol and N-acetylcysteine could modulate this system. We used articular chondrocytes isolated from nondiseased equine joints and cultured them in a 3-D alginate bead system for 48h in <1, 2, 5, and 21% O2 at pH 7.2 or 6.2 in the absence or presence of the proinflammatory cytokine, interleukin-1ß (10ng/ml).In addition, chondrocytes were cultured with resveratrol (10µM) or N-acetylcysteine (NAC) (2mM).Cell viability, glycosaminoglycan (GAG) release, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), GSH:GSSG ratio, and SOD1 and SOD2 protein expression were measured. Very low levels of oxygen (<1%), acidosis (pH 6.2), and exposure to IL-1ß led to reductions in cell viability, increased GAG release, alterations in ΔΨm and ROS levels, and reduced GSH:GSSG ratio. In addition, SOD1 and SOD2 protein expressions were reduced. Both resveratrol and NAC partially restored ΔΨm and ROS levels and prevented GAG release and cell loss and normalized SOD1 and SOD2 protein expression. In particular NAC was highly effective at restoring the GSH:GSSG ratio.These results show that the antioxidants resveratrol and N-acetylcysteine can counteract the redox imbalance in articular chondrocytes induced by low oxygen and acidic conditions.

Tendon extracellular matrix damage, degradation and inflammation in response to in vitro overload exercise.

The role of inflammation in tendon injury is uncertain and a topic of current interest. In vitro studies of tendon accelerated overload damage can serve as a valuable source of information on the early stages of tendinopathy. Viable fascicle bundles from bovine flexor tendons were subjected to cyclic uniaxial loading from 1-10% strain. Immuno-staining for inflammatory markers and matrix degradation markers was performed on the samples after mechanical testing. Loaded samples exhibited visible extracellular matrix damage, with disrupted collagen fibers and fiber kinks, and notable damage to the interfascicular matrix. Inflammatory markers COX-2 and IL-6 were only expressed in the cyclically loaded samples. Collagen degradation markers MMP-1 and C1,2C were colocalized in many areas, with staining occurring in the interfascicular matrix or the fascicular tenocytes. These markers were present in control samples, but staining became increasingly intense with loading. Little MMP-3 or MMP-13 was evident in control sections. In loaded samples, some sections showed intense staining of these markers, again localized to interfascicular regions. This study suggests that inflammatory markers may be expressed rapidly after tendon overload exercise. Interestingly, both inflammation and damage-induced matrix remodeling seem to be concentrated in, or in the vicinity of, the highly cellular interfascicular matrix.

The role of polyphosphates in the sequestration of matrix metalloproteinases.

This study outlines the potential of a novel therapeutic dressing for the management of chronic wounds. The dressing incorporates polyphosphate, a non toxic compound with a number of beneficial characteristics in terms of wound healing, in a foam matrix. The aim of this study was to identify the potential of polyphosphate incorporated in the foam dressing to sequester the activity of matrix metalloproteinases (MMPs) and proteases derived from Pseudomonas aeruginosa. Methods used included gelatin zymography and milk-casein agar plate analysis. Results have shown that this dressing is effectively capable of reducing the levels of MMP-2 and MMP-9 in both their active and latent forms using an in vitro model. The dressing also demonstrated the compound's potential in the regulation of P. aeruginosa derived proteases.

Matrix assisted laser desorption ionization mass spectrometry imaging identifies markers of ageing and osteoarthritic cartilage.

INTRODUCTION: Cartilage protein distribution and the changes that occur in cartilage ageing and disease are essential in understanding the process of cartilage ageing and age related diseases such as osteoarthritis. The aim of this study was to investigate the peptide profiles in ageing and osteoarthritic (OA) cartilage sections using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). METHODS: The distribution of proteins in young, old and OA equine cartilage was compared following tryptic digestion of cartilage slices and MALDI-MSI undertaken with a MALDI SYNAPT™ HDMS system. Protein identification was undertaken using database searches following multivariate analysis. Peptide intensity differences between young, ageing and OA cartilage were imaged with Biomap software. Analysis of aggrecanase specific cleavage patterns of a crude cartilage proteoglycan extract were used to validate some of the differences in peptide intensity identified. Immunohistochemistry studies validated the differences in protein abundance. RESULTS: Young, old and OA equine cartilage was discriminated based on their peptide signature using discriminant analysis. Proteins including aggrecan core protein, fibromodulin, and cartilage oligomeric matrix protein were identified and localised. Fibronectin peptides displayed a stronger intensity in OA cartilage. Age-specific protein markers for collectin-43 and cartilage oligomeric matrix protein were identified. In addition potential fibromodulin and biglycan peptides targeted for degradation in OA were detected. CONCLUSIONS: MALDI-MSI provided a novel platform to study cartilage ageing and disease enabling age and disease specific peptides in cartilage to be elucidated and spatially resolved.

Myocardial cytokine expression in dogs with systemic and naturally occurring cardiac diseases.

OBJECTIVE: To compare myocardial cytokine expression in dogs with naturally occurring cardiac or systemic diseases and dogs without cardiac or systemic diseases (control dogs) SAMPLE: Myocardial tissue samples from 7 systemic disease-affected dogs (SDDs), 7 cardiac disease-affected dogs (CDDs), and 8 control dogs. PROCEDURES: mRNA expression of interleukin (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, transforming growth factor (TGF)-ß1, TGF-ß2, TGF-ß3, and growth differentiation factor-15 in myocardial tissue samples obtained from CDDs, SDDs, and control dogs were analyzed via quantitative PCR assays. RESULTS: In control dogs, only mRNA for TNF-α, TGF-ß1, and TGF-ß3 was detected; concentrations were significantly higher in male than in female dogs. In SDDs and CDDs, all cytokines, growth factors, and growth differentiation factor-15 were expressed. Compared with findings in SDDs, IL-1, IL-6, IL-8, IL-10, TNF-α, and IFN-γ expression was significantly increased in CDDs; specifically, IL-1, IL-8, TNF-α, TGF-ß1, and TGF-ß3 expression was increased in the atria and IL-8, IL-10, TNF-α, and IFN-γ expression was increased in the ventricles of CDDs. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggested that the alterations in cytokine expression in SDDs and CDDs, compared with control dog findings, were a result of inflammatory system activation. The differences in cytokine expression in atria and ventricles between SDDs and CDDs were suggestive of different remodeling processes. A better knowledge of myocardial involvement in SDDs and of immune regulation in CDDs might beneficially affect morbidity and mortality rates and provide new treatment approaches.

Collection and propagation methods for mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are derived from adult mesenchymal tissues and have the ability to undergo differentiation into bone, cartilage, and fat, and have therefore attracted great interest in regenerative medicine. Many isolation and culture methods have been described, making comparison between laboratories and quality-control protocols difficult. A uniform protocol to characterize equine MSC has recently been proposed, aiming to introduce consistency across the equine stem cell research field. This article reviews the published techniques for collection and propagation of equine MSC, focusing on bone marrow-derived and adipose-derived cells.

Stem cell-based therapies for bone repair.

This article provides an overview of the cellular and molecular events involved in bone repair and the current approaches to using stem cells as an adjunct to this process. The article emphasizes the key role of osteoprogenitor cells in the formation of bone and where the clinical applications of current research may lend themselves to large animal orthopaedics. The processes involved in osteogenic differentiation are presented and strategies for bone formation, including induction by osteogenic factors, bioscaffolds, and gene therapy, are reviewed.

Evidence-based medicine and stem cell therapy: how do we know such technologies are safe and efficacious?

Evidence-based medicine (EBM) refers to the conscientious, explicit, and judicious use of current best evidence from research for the care of an individual patient. Central to the adoption of EBM is both producing and identifying the best possible evidence for a particular intervention or therapy. This article identifies and reviews the approaches to producing and identifying the best possible evidence that is necessary for the full acceptance of stem cell therapies in the horse and reviews the approaches that will allow future clinical studies in stem cell therapies to provide the best evidence for determining efficacy.

Analysis of post transcriptional regulation of SOX9 mRNA during in vitro chondrogenesis.

Marker genes are used to monitor chondrogenic differentiation, but little is known about the turnover of their mRNA during this process. We set out to measure the half life of mRNA encoding the transcription factor SOX9, an important marker of chondrocytic phenotype. We dedifferentiated human articular chondrocytes in monolayer culture before placing them in chondrogenic three-dimensional pellet cultures. At the same time, we induced chondrocytic differentiation of human bone marrow-derived mesenchymal stem cells under the same three-dimensional conditions. Pellets were cultured in standard chondrogenic media with and without BMP7. We found that SOX9 mRNA half life exhibited an inverse correlation with total SOX9 mRNA levels in both dedifferentiating human articular chondrocytes and chondrogenic pellet cultures. There was no evidence for a specific effect of BMP7 on SOX9 mRNA decay. Our findings provide an insight into a level of gene control rarely explored in regenerative medicine, which could be important in the optimization of in vitro cartilage production.

Post-transcriptional gene regulation in chondrocytes.

The control of gene expression in articular chondrocytes is an essential factor in maintaining the homoeostasis of extracellular matrix synthesis and turnover necessary in healthy articular cartilage. Although much is known of how steady-state levels of gene expression and rates of transcription are altered, there has been a poorer understanding of gene control at the post-transcriptional level and its relevance to cartilage health and disease. Now, an emerging picture is developing of the importance of this tier of gene regulation, driven by in vitro studies and mouse genetic models. This level of cellular regulation represents an as yet unexplored area of potential intervention for the treatment of degenerative cartilage disorders such as osteoarthritis.

Computed tomography to identify preoperative guidelines for internal fixation of the distal sesamoid bone in horses: an in vitro study.

OBJECTIVE: To assess the reliability of computed tomography (CT) to identify the direction of implant insertion for cortical screws along the longitudinal axis of intact (nonfractured) distal sesamoid bones. STUDY DESIGN: In vitro study. SAMPLE POPULATION: Cadaveric paired equine forelimbs (n=16). METHODS: Insertion of a cortical screw in lag fashion along the longitudinal axis of intact (nonfractured) distal sesamoid bones was evaluated in 2 groups (3.5 and 4.5 mm) of 8 paired limbs. In each group, the direction of the distal sesamoid bone was determined by CT (Equine XTC 3000 pQCT scanner). Screw placement was verified by specimen dissection. Implant direction was considered satisfactory if the entire screw length was within the distal sesamoid bone and not damaging the articular or flexural surfaces. RESULTS: In our sample and according to our criteria, the proportion of satisfactory direction of screws was 0.63 (5/8) for 4.5 mm implants, and 0.87 (7/8) for 3.5 mm implants. CONCLUSIONS: CT is a useful imaging modality to identify anatomic landmarks for insertion of a 3.5 mm cortical screw in the distal sesamoid bone.

MMP-mediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroids.

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.

Use of computed tomography in standing position to identify guidelines for screw insertion in the distal phalanx of horses: an ex vivo study.

OBJECTIVES: To compare the precision of radiography and computed tomography (CT) preoperatively in the standing position for identification of guidelines for screw insertion in the distal phalanx, and to identify whether standing CT might improve operative time compared with preoperative radiographic planning. STUDY DESIGN: Experimental ex vivo study. ANIMALS: Cadaveric equine thoracic limb pairs (n=10). METHODS: Insertion of a 4.5 mm cortex screw in lag fashion into an intact distal phalanx was evaluated in 2 groups (n=10) of cadaveric equine thoracic limbs. In 1 group, the site, direction, and length of the implant were determined by radiography, and in the other group, by CT. Accuracy of screw placement was verified by specimen dissection. Outcomes were (1) absence of penetration of the articular surface, the solar surface, or the semilunar canal (2) appropriate length and direction of the screw. Surgical time was also measured. RESULTS: No screw penetrated the articular surface, the solar surface, or the semilunar canal in either group. CT was more accurate to identify guidelines for screw insertion (U=23.50, P=.049). With CT, surgical time (mean, 7.7 minutes) was significantly shorter (U=0.000, P=.000) than with radiography (mean, 12.7 minutes). CONCLUSION: Standing CT can be used to accurately determine anatomic landmarks for screw insertion in lag fashion in sagittal fractures of the distal phalanx. CLINICAL RELEVANCE: This study has a clear clinical relevance for improved internal fixation of sagittal fractures of the distal phalanx.

Use of computed tomography to diagnose new bone formation associated with desmitis of the proximal aspect of the suspensory ligament in third metacarpal or third metatarsal bones of three horses.

CASE DESCRIPTION: 3 horses with lameness localized to the proximal aspect of the metacarpus or metatarsus. CLINICAL FINDINGS: All horses had evidence of problems that originated from the proximal aspect of the suspensory ligament (PASL), including signs of pain on palpation, positive results of diagnostic nerve blocks, ultrasonographic detection of enlargement and diffuse areas of reduced echogenicity in the proximal region of insertion of the ligament, and radiographic detection of increased mineral opacity in the proximal aspect of the metacarpus or metatarsus. Desmitis of the PASL was diagnosed in each horse; however, conservative treatment failed to improve the lameness. The horses were taken to surgery for splitting of the PASL and osteostixis of the proximal aspect of the third metacarpal or metatarsal bone. At that time, the proximal aspect of the metacarpus or metatarsus was evaluated via computed tomography (CT), which identified new bone formation at the proximal aspect of the third metacarpal or metatarsal bone that had not already been identified. TREATMENT AND OUTCOME: In all horses, the newly formed bone was removed surgically under radiographic and CT guidance, and the splitting and osteostixis that had been planned were performed. After rehabilitation, all horses returned to full training at 6 months after surgery. All horses responded well to the surgical treatment and were sound 8 months afterward. CLINICAL RELEVANCE: Use of CT imaging should be considered in lame horses with pain associated with the proximal aspect of the third metacarpal or metatarsal bones that does not improve with conservative treatment.

SOX9 transduction of a human chondrocytic cell line identifies novel genes regulated in primary human chondrocytes and in osteoarthritis.

The transcription factor SOX9 is important in maintaining the chondrocyte phenotype. To identify novel genes regulated by SOX9 we investigated changes in gene expression by microarray analysis following retroviral transduction with SOX9 of a human chondrocytic cell line (SW1353). From the results the expression of a group of genes (SRPX, S100A1, APOD, RGC32, CRTL1, MYBPH, CRLF1 and SPINT1) was evaluated further in human articular chondrocytes (HACs). First, the same genes were investigated in primary cultures of HACs following SOX9 transduction, and four were found to be similarly regulated (SRPX, APOD, CRTL1 and S100A1). Second, during dedifferentiation of HACs by passage in monolayer cell culture, during which the expression of SOX9 progressively decreased, four of the genes (S100A1, RGC32, CRTL1 and SPINT1) also decreased in their expression. Third, in samples of osteoarthritic (OA) cartilage, which had decreased SOX9 expression compared with age-matched controls, there was decreased expression of SRPX, APOD, RGC32, CRTL1 and SPINT1. The results showed that a group of genes identified as being upregulated by SOX9 in the initial SW1353 screen were also regulated in expression in healthy and OA cartilage. Other genes initially identified were differently expressed in isolated OA chondrocytes and their expression was unrelated to changes in SOX9. The results thus identified some genes whose expression appeared to be linked to SOX9 expression in isolated chondrocytes and were also altered during cartilage degeneration in osteoarthritis.