Current clinical opinion on surgical approaches and rehabilitation of hand flexor tendon injury-a questionnaire study.

The management of flexor tendon injury has seen many iterations over the years, but more substantial innovations in practice have been sadly lacking. The aim of this study was to investigate the current practice of flexor tendon injury management, and variation in practice from the previous reports, most troublesome complications, and whether there was a clinical interest in potential innovative tendon repair technologies. An online survey was distributed via the British Society for Surgery of the Hand (BSSH) and a total of 132 responses were collected anonymously. Results showed that although most surgeons followed the current medical recommendation based on the literature, a significant number of surgeons still employed more conventional treatments in clinic, such as general anesthesia, ineffective tendon retrieval techniques, and passive rehabilitation. Complications including adhesion formation and re-rupture remained persistent. The interest in new approaches such as use of minimally invasive instruments, biodegradable materials and additive manufactured devices was not strong, however the surgeons were potentially open to more effective and economic solutions.

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.

Therapeutic Potential of Dental Pulp Stem Cells and Leukocyte- and Platelet-Rich Fibrin for Osteoarthritis.

Osteoarthritis (OA) is a degenerative and inflammatory joint disorder with cartilage loss. Dental pulp stem cells (DPSCs) can undergo chondrogenic differentiation and secrete growth factors associated with tissue repair and immunomodulation. Leukocyte- and platelet-rich fibrin (L-PRF) emerges in regenerative medicine because of its growth factor content and fibrin matrix. This study evaluates the therapeutic application of DPSCs and L-PRF in OA via immunomodulation and cartilage regeneration. Chondrogenic differentiation of DPSCs, with or without L-PRF exudate (ex) and conditioned medium (CM), and of bone marrow-mesenchymal stem cells was compared. These cells showed differential chondrogenesis. L-PRF was unable to increase cartilage-associated components. Immature murine articular chondrocytes (iMACs) were cultured with L-PRF ex, L-PRF CM, or DPSC CM. L-PRF CM had pro-survival and proliferative effects on unstimulated and cytokine-stimulated iMACs. L-PRF CM stimulated the release of IL-6 and PGE2, and increased MMP-13, TIMP-1 and IL-6 mRNA levels in cytokine-stimulated iMACs. DPSC CM increased the survival and proliferation of unstimulated iMACs. In cytokine-stimulated iMACs, DPSC CM increased TIMP-1 gene expression, whereas it inhibited nitrite release in 3D culture. We showed promising effects of DPSCs in an in vitro OA model, as they undergo chondrogenesis in vitro, stimulate the survival of chondrocytes and have immunomodulatory effects.

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.

Isothiocyanates are detected in human synovial fluid following broccoli consumption and can affect the tissues of the knee joint.

Osteoarthritis is a major cause of disability and there is no current pharmaceutical treatment which can prevent the disease or slow its progression. Dietary advice or supplementation is clearly an attractive option since it has low toxicity and ease of implementation on a population level. We have previously demonstrated that sulforaphane, a dietary isothiocyanate derived from its glucosinolate precursor which is found in broccoli, can prevent cartilage destruction in cells, in in vitro and in vivo models of osteoarthritis. As the next phase of this research, we enrolled 40 patients with knee osteoarthritis undergoing total knee replacement into a proof-of-principle trial. Patients were randomised to either a low or high glucosinolate diet for 14 days prior to surgery. We detected ITCs in the synovial fluid of the high glucosinolate group, but not the low glucosinolate group. This was mirrored by an increase in ITCs and specifically sulforaphane in the plasma. Proteomic analysis of synovial fluid showed significantly distinct profiles between groups with 125 differentially expressed proteins. The functional consequence of this diet will now be tested in a clinical trial.

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.

Decoding the Regulatory Landscape of Ageing in Musculoskeletal Engineered Tissues Using Genome-Wide DNA Methylation and RNASeq.

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based regenerative medicine and tissue engineering. Epigenetic mechanisms, like DNA methylation, contribute to the changes in gene expression in ageing. However there was a lack of sufficient knowledge of the role that differential methylation plays during chondrogenic, osteogenic and tenogenic differentiation from ageing MSCs. This study undertook genome level determination of the effects of DNA methylation on expression in engineered tissues from chronologically aged MSCs. We compiled unique DNA methylation signatures from chondrogenic, osteogenic, and tenogenic engineered tissues derived from young; n = 4 (21.8 years ± 2.4 SD) and old; n = 4 (65.5 years±8.3SD) human MSCs donors using the Illumina HumanMethylation 450 Beadchip arrays and compared these to gene expression by RNA sequencing. Unique and common signatures of global DNA methylation were identified. There were 201, 67 and 32 chondrogenic, osteogenic and tenogenic age-related DE protein-coding genes respectively. Findings inferred the nature of the transcript networks was predominantly for 'cell death and survival', 'cell morphology', and 'cell growth and proliferation'. Further studies are required to validate if this gene expression effect translates to cell events. Alternative splicing (AS) was dysregulated in ageing with 119, 21 and 9 differential splicing events identified in chondrogenic, osteogenic and tenogenic respectively, and enrichment in genes associated principally with metabolic processes. Gene ontology analysis of differentially methylated loci indicated age-related enrichment for all engineered tissue types in 'skeletal system morphogenesis', 'regulation of cell proliferation' and 'regulation of transcription' suggesting that dynamic epigenetic modifications may occur in genes associated with shared and distinct pathways dependent upon engineered tissue type. An altered phenotype in engineered tissues was observed with ageing at numerous levels. These changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients. In addition we have identified a number of tissue-dependant pathways, which warrant further studies.

Characterization of neopeptides in equine articular cartilage degradation.

Osteoarthritis is characterized by a loss of extracellular matrix that leads to cartilage degradation and joint space narrowing. Specific proteases, including the aggrecanases ADAMTS-4 and matrix metalloproteinase 3, are important in initiating and promoting cartilage degradation in osteoarthritis. This study investigated protease-specific and disease-specific cleavage patterns of particular extracellular matrix proteins by comparing new peptide fragments, neopeptides, in specific exogenous protease-driven digestion of a crude cartilage proteoglycan extract and an in-vitro model of early osteoarthritis. Additionally, equine cartilage explants were treated with interleukin-1 and the media collected. Proteolytic cleavage products following trypsin digestion were then identified using tandem mass spectrometry. Complete sequences of proteolytically cleaved neopeptides were determined for the major cartilage proteoglycans aggrecan, biglycan, decorin, fibromodulin plus cartilage oligomeric matrix protein. The generation of neopeptides varied with enzyme specificity; however, some peptides were common to all samples. Previous known and novel cleavage sites were identifies. The identification of novel peptide fragments provides a platform for the development of antibodies that could assist in the identification of biomarkers for osteoarthritis (OA), as well as the identification of basic biochemical processes underlying OA.

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.

Magnetic resonance imaging (MRI) anatomy of the ovine stifle.

OBJECTIVE: To describe magnetic resonance imaging (MRI) anatomy of the ovine stifle and investigate meniscotibial and cruciate ligaments anatomy. STUDY DESIGN: Descriptive ex vivo study. ANIMALS: Pelvic limbs (n = 44) from 22 adult Texel ewes. METHODS: Forty limbs (n = 40) were scanned using 3 Tesla MRI before gross anatomic dissection. Two other limb pairs were frozen and transected to obtain sections that were compared with MRI images for identification of anatomic structures. RESULTS: In all stifles, the craniomedial bundle of the cranial cruciate ligament inserted caudally to the cranial attachment of the medial meniscus. No transverse intermeniscal ligament was identified in 80% of stifles, whereas a few small ligamentous fibers were seen crossing from 1 cranial horn to the other in 20% of stifles. There was good differentiation of menisci, ligaments, and synovial cavities on MRI images. Two bundles were identified in all cranial cruciate ligaments on MRI. Sensitivity and specificity of 3T MRI for detection of transverse intermeniscal ligament were 42% and 84%, respectively. CONCLUSION: 3T MRI provided well defined reference images for menisci, synovial cavities, and most ligaments.

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.