Enhanced chondrogenesis from human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potential for the repair of damaged articular cartilage. We developed a serum-free 14-day protocol for hESC differentiation into chondrocyte progenitors, which surprisingly lacked strong cartilage matrix production in in vitro tests. In order to direct these progenitors to a more mature phenotype, we investigated substituting different members of the TGFß family in the protocol. Initially, we supplemented, or substituted GDF5 (day 11-14), with combinations of BMP7 and TGFß-1, or -3, but these modifications yielded no improvement in matrix gene expression. However, replacing BMP4 with BMP2 (days 3-10 of the protocol) resulted in a more rapid increase in SOX9 gene expression and increased expression of chondrogenic genes SOX5, ACAN and COL2A1. The replacement of BMP4 with BMP2 also enhanced the formation of chondrogenic cell aggregates, with greater deposition of type II collagen. This change was not accompanied by hypertrophic chondrocyte marker COL10A1 expression. The results demonstrate that BMP2 has greater specificity for the generation of chondrogenic cells from hESCs than BMP4 and this was consistent in two hESC lines (HUES1 and MAN7). hESC-chondrogenic cells derived with either BMP2 or BMP4 were tested in vivo by implanting them in fibrin into osteochondral defects in the femur of RNU rats. Repaired cartilage tissue, positive for Safranin O and type II collagen was detected at 6 and 12 weeks with both cell sources, but the BMP2 cells scored higher for tissue quality (Pineda score). Therefore, BMP2 is more effective at driving chondrogenic differentiation from human pluripotent stem cells than BMP4 and the effect on the resulting chondroprogenitors is sustained in an in vivo setting.

The development of a mature collagen network in cartilage from human bone marrow stem cells in Transwell culture.

Damaged hyaline cartilage shows a limited capacity for innate repair. Potential sources of cells to augment the clinical repair of cartilage defects include autologous chondrocytes and mesenchymal stem cells. We have reported that culture of human bone marrow mesenchymal stem cells with specific growth and differentiation factors as shallow multilayers on Transwell permeable membranes provided ideal conditions for chondrogenesis. Rigid translucent cartilaginous disks formed and expressed cartilage-specific structural proteins aggrecan and type II collagen. We report here the analysis of the collagen network assembled in these cartilage constructs and identify key features of the network as it became mature during 28 days of culture. The type II collagen was co-polymerized with types XI and IX collagens in a fibrillar network stabilized by hydroxylysyl pyridinoline cross-links as in epiphyseal and hyaline cartilages. Tandem ion-trap mass-spectrometry identified 3-hydroxylation of Proline 986 and Proline 944 of the α1(II) chains, a post-translational feature of human epiphyseal cartilage type II collagen. The formation of a type II collagen based hydroxy-lysyl pyridinoline cross-linked network typical of cartilage in 28 days shows that the Transwell system not only produces, secretes and assembles cartilage collagens, but also provides all the extracellular mechanisms to modify and generate covalent cross-links that determine a robust collagen network. This organized assembly explains the stiff, flexible nature of the cartilage constructs developed from hMSCs in this culture system.

PhenomeExpress: a refined network analysis of expression datasets by inclusion of known disease phenotypes.

We describe a new method, PhenomeExpress, for the analysis of transcriptomic datasets to identify pathogenic disease mechanisms. Our analysis method includes input from both protein-protein interaction and phenotype similarity networks. This introduces valuable information from disease relevant phenotypes, which aids the identification of sub-networks that are significantly enriched in differentially expressed genes and are related to the disease relevant phenotypes. This contrasts with many active sub-network detection methods, which rely solely on protein-protein interaction networks derived from compounded data of many unrelated biological conditions and which are therefore not specific to the context of the experiment. PhenomeExpress thus exploits readily available animal model and human disease phenotype information. It combines this prior evidence of disease phenotypes with the experimentally derived disease data sets to provide a more targeted analysis. Two case studies, in subchondral bone in osteoarthritis and in Pax5 in acute lymphoblastic leukaemia, demonstrate that PhenomeExpress identifies core disease pathways in both mouse and human disease expression datasets derived from different technologies. We also validate the approach by comparison to state-of-the-art active sub-network detection methods, which reveals how it may enhance the detection of molecular phenotypes and provide a more detailed context to those previously identified as possible candidates.

Cartilage repair using human embryonic stem cell-derived chondroprogenitors.

In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions.

Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin.

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D'D3; NT5B) and subdomains (D1, D1-D2, D2-D'-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.

Generating cartilage repair from pluripotent stem cells.

The treatment of degeneration and injury of articular cartilage has been very challenging for scientists and surgeons. As an avascular and hypocellular tissue, cartilage has a very limited capacity for self-repair. Chondrocytes are the only cell type in cartilage, in which they are surrounded by the extracellular matrix that they secrete and assemble. Autologous chondrocyte implantation for cartilage defects has achieved good results, but the limited resources and complexity of the procedure have hindered wider application. Stem cells form an alternative to chondrocytes as a source of chondrogenic cells due to their ability to proliferate extensively while retaining the potential for differentiation. Adult stem cells such as mesenchymal stem cells have been differentiated into chondrocytes, but the limitations in their proliferative ability and the heterogeneous cell population hinder their adoption as a prime alternative source for generating chondrocytes. Human embryonic stem cells (hESCs) are attractive as candidates for cell replacement therapy because of their unlimited self-renewal and ability for differentiation into mesodermal derivatives as well as other lineages. In this review, we focus on current protocols for chondrogenic differentiation of ESCs, in particular the chemically defined culture system developed in our lab that could potentially be adapted for clinical application.

The characterisation of mesenchymal stem cells: a stem cell is not a stem cell is not a stem cell.

There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Although there have been developments in almost all surgical disciplines, the greatest advances are being made in orthopaedics. This is due to many factors including the familiarity with bone marrow derived mesenchymal stem cells. Unfortunately significant hurdles remain to be overcome in many areas before tissue engineering becomes more routinely used in clinical practice. Stem cells have been identified in a number of adult tissues, albeit in small numbers. In addition to bone marrow, mesenchymal stem cells have been identified in a number of tissues including adipose tissue and fat pad. The mesenchymal stem cells are generally isolated from the tissue and expanded in culture. These cells are characterised or defined using a set of cell surface markers; mesenchymal stem cells are generally positive for CD44, CD90 and CD105, and are negative for haematopoetic markers CD34 and CD45, and the neurogenic marker CD56. In this paper the characterisation of stem cells is discussed followed by preliminary evidence suggesting that pericytes may be a candidate stem cell.

Mesenchymal stem cells, sources of cells and differentiation potential.

Tissue is frequently damaged or lost in injury and disease. There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Tissue engineering is an exciting strategy being explored to deal with damaged or lost tissue. It is the science of generating tissue using molecular and cellular techniques, combined with material engineering principles, to replace tissue. This could be in the form of cells with or without matrices. Although there have been developments in almost all surgical disciplines, the greatest advances are being made in orthopaedics, especially in cartilage repair. This is due to many factors including the familiarity with bone marrow derived mesenchymal stem cells and cartilage being a relatively simpler tissue to engineer. Unfortunately significant hurdles remain to be overcome in many areas before tissue engineering becomes more routinely used in clinical practice. Cells used in tissue engineering could be autologous, allogeneic or xenogeneic. The cells could be stem cells or cells further down the differentiation pathway. The use of embryonic stem cells is associated with religious, political and social concerns, but the use of adult stem cells is generally well accepted. Stem cells have been identified in a number of adult tissues, albeit in small numbers. In addition to bone marrow, mesenchymal stem cells have been identified in a number of tissues including adipose tissue and fat pad. The mesenchymal stem cells are generally isolated from the tissue and expanded in culture. These cells can be differentiated down a particular differentiation pathway e.g. osteoblast or chondrocyte, using predefined culture conditions before being used for clinical applications. In this paper stem cells are discussed including their various sources and their differentiation potential.

Cartilage tissue engineering approaches applicable in orthopaedic surgery: the past, the present, and the future.

Tissue is frequently damaged or lost in injury and disease. There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Although there have been developments in almost all surgical disciplines, the greatest advances are being made in orthopaedics, especially in cartilage repair. This is due to many factors including the familiarity with bone marrow derived mesenchymal stem cells and cartilage being a relatively simpler tissue to engineer. Unfortunately significant hurdles remain to be overcome in many areas before tissue engineering becomes more routinely used in clinical practice. In this paper we discuss the structure, function and embryology of cartilage and osteoarthritis. This is followed by a review of current treatment strategies for the repair of cartilage and the use of tissue engineering.

Directed differentiation of human embryonic stem cells toward chondrocytes.

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95-97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

Order within disorder: aggrecan chondroitin sulphate-attachment region provides new structural insights into protein sequences classified as disordered.

Structural investigation of proteins containing large stretches of sequences without predicted secondary structure is the focus of much increased attention. Here, we have produced an unglycosylated 30 kDa peptide from the chondroitin sulphate (CS)-attachment region of human aggrecan (CS-peptide), which was predicted to be intrinsically disordered and compared its structure with the adjacent aggrecan G3 domain. Biophysical analyses, including analytical ultracentrifugation, light scattering, and circular dichroism showed that the CS-peptide had an elongated and stiffened conformation in contrast to the globular G3 domain. The results suggested that it contained significant secondary structure, which was sensitive to urea, and we propose that the CS-peptide forms an elongated wormlike molecule based on a dynamic range of energetically equivalent secondary structures stabilized by hydrogen bonds. The dimensions of the structure predicted from small-angle X-ray scattering analysis were compatible with EM images of fully glycosylated aggrecan and a partly glycosylated aggrecan CS2-G3 construct. The semiordered structure identified in CS-peptide was not predicted by common structural algorithms and identified a potentially distinct class of semiordered structure within sequences currently identified as disordered. Sequence comparisons suggested some evidence for comparable structures in proteins encoded by other genes (PRG4, MUC5B, and CBP). The function of these semiordered sequences may serve to spatially position attached folded modules and/or to present polypeptides for modification, such as glycosylation, and to provide templates for the multiple pleiotropic interactions proposed for disordered proteins.

Fell-Muir lecture: cartilage 2010 - the known unknowns.

Over the past 40 years there have been giant steps forward in our understanding of cellular and molecular biology that have given us the framework by which to understand tissue organization and tissue function on a range of scales. However, although the progress has been great, the more we have discovered, the more we are aware of what we don't yet know. In this article, I would like to flag up some issues of cartilage biology, function and pathology where we still have significant ignorance. As scientists we all provide contributions to add to the greater understanding of science and progress is on a broad front, but gaps are left where particular difficulty is encountered and in life sciences it is no different. Progress is fast where new knowledge and techniques pave the way, but where study is complex and relevant techniques poorly developed the gaps are left behind. In cartilage research and matrix biology, the gaps can particularly be seen at interfaces between disciplines and where technology development has lagged behind and in the particular challenges of understanding how molecular properties can explain tissue macro properties.

The potential of stem cells in the treatment of knee cartilage defects.

Cartilage is frequently damaged but only shows a limited capacity for repair. There are a number of treatment strategies currently available for the repair of articular cartilage defects including abrasion chondroplasty, subchondral drilling, microfracture and mosaicplasty but these show variable results. For the younger patients, there is great interest in the potential of cell-based strategies to provide a biological replacement of damaged cartilage using autologous chondrocytes. The results of clinical studies using these cell-based techniques do not conclusively show improvement over conventional techniques. These techniques also do not consistently result in the formation of the desired hyaline cartilage rather than fibrocartilage. Mesenchymal stem cells present a promising cell source for cartilage repair. Mesenchymal stem cells have been isolated from a number of adult tissues including the bone marrow and the synovial fat pad. These cells have the ability to proliferate in culture and differentiate down different pathways including the chondrogenic pathway. In the first instance, differentiated stem cells can be used for the repair of localised cartilage defects by producing hyaline cartilage. In the future, this strategy has the potential to be extended to treat more generalised cartilage defects, especially as the cell source is not a limiting factor. The use of cell-based therapies also allows the versatility of using scaffolds and growth factors, with recombinant proteins or gene therapy. A number of challenges however still need to be overcome including further work on identifying the optimal source of stem cells, along with refining the conditions that enhance expansion and chondrogenesis.

Bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5 in culture and show enhanced chondrogenesis in hypoxic conditions.

Bone marrow-derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in some cells. In this study, bone marrow-derived stem cells were characterized and the effects of hypoxia on chondrogenesis investigated. Adherent bone marrow colony-forming cells were characterized for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. The cells stained strongly for markers of adult mesenchymal stem cells, and a high number of cells were also positive for the pericyte marker 3G5. The cells showed a chondrogenic response in cell aggregate cultures and, in lowered oxygen, there was increased matrix accumulation of proteoglycan, but less cell proliferation. In hypoxia, there was increased expression of key transcription factor SOX6, and of collagens II and XI, and aggrecan. Pericytes are a candidate stem cell in many tissue, and our results show that bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension. This has important implications for tissue engineering applications of bone marrow-derived stem cells.

Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes.

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471-39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24-48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

Stem cell applications and tissue engineering approaches in surgical practice.

There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Although there have been developments in almost all surgical disciplines, the greatest advances are being made in orthopaedics, especially in bone repair. Significant hurdles however remain to be overcome before tissue engineering becomes more routinely used in surgical practice.

The influence of donor and hypoxic conditions on the assembly of cartilage matrix by osteoarthritic human articular chondrocytes on Hyalograft matrices.

Resurfacing of cartilage defects using cell-seeded, biomaterial grafts is a promising approach for articular cartilage repair and in this study we investigated the ability of human chondrocytes from osteoarthritic joints to generate cartilage tissue under standard conditions in cultured over 21 days on Hyalograft matrices under normoxic (20% O(2)) and hypoxic (5% O(2)) conditions. The results showed that constructs were more chondrogenic when cultured under hypoxic conditions, which resulted in greater production of sulphated glycosaminoglycan and collagen type II within the constructs and the cells expressed higher levels of genes encoding cartilage matrix proteins and chondrocyte transcription factors. However, there were very wide differences in the chondrogenic potential amongst donors as the weight ratio of total sulphated glycosaminoglycan to DNA in constructs varied from above 200 to below 10. These results establish that the generation of cartilage from human OA chondrocytes on biodegradable supports is favoured in lowered oxygen, but that under standard conditions, even at low passage, there is a large variation in the chondrogenic potential amongst chondrocytes from different donors. Further analysis of this variation suggested that the gene expression ratio of COL2A1/COL1A1 mRNA in the chondrocytes in monolayer culture may predict their subsequent performance in forming cartilage matrix on the Hyalograft scaffold.

Hyaluronan binding to link module of TSG-6 and to G1 domain of aggrecan is differently regulated by pH.

The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA(8) and HA(10) oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 to HA was maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0-8.0 and moderate to strong binding affinity over a much broader pH range (5.5-8.0). The K(D) determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1.

Human infrapatellar fat pad-derived stem cells express the pericyte marker 3G5 and show enhanced chondrogenesis after expansion in fibroblast growth factor-2.

INTRODUCTION: Infrapatellar fat pad (IPFP) is a possible source of stem cells for the repair of articular cartilage defects. In this study, adherent proliferative cells were isolated from digests of IPFP tissue. The effects of the expansion of these cells in fibroblast growth factor-2 (FGF-2) were tested on their proliferation, characterisation, and chondrogenic potential. METHODS: IPFP tissue was obtained from six patients undergoing total knee replacement, and sections were stained with 3G5, alpha smooth muscle actin, and von Willebrand factor to identify different cell types in the vasculature. Cells were isolated from IPFP, and both mixed populations and clonal lines derived from them were characterised for cell surface epitopes, including 3G5. Cells were expanded with and without FGF-2 and were tested for chondrogenic differentiation in cell aggregate cultures. RESULTS: 3G5-positive cells were present in perivascular regions in tissue sections of the IPFP, and proliferative adherent cells isolated from the IPFP were also 3G5-positive. However, 3G5 expression was on only a small proportion of cells in all populations and at all passages, including the clonally expanded cells. The cells showed cell surface epitope expression similar to adult stem cells. They stained strongly for CD13, CD29, CD44, CD90, and CD105 and were negative for CD34 and CD56 but were also negative for LNGFR (low-affinity nerve growth factor receptor) and STRO1. The IPFP-derived cells showed chondrogenic differentiation in cell aggregate cultures, and prior expansion with FGF-2 enhanced chondrogenesis. Expansion in FGF-2 resulted in greater downregulation of many cartilage-associated genes, but on subsequent chondrogenic differentiation, they showed stronger upregulation of these genes and this resulted in greater matrix production per cell. CONCLUSION: These results show that these cells express mesenchymal stem cell markers, but further work is needed to determine the true origin of these cells. These results suggest that the expansion of these cells with FGF-2 has important consequences for facilitating their chondrogenic differentiation.

SOX9 transduction increases chondroitin sulfate synthesis in cultured human articular chondrocytes without altering glycosyltransferase and sulfotransferase transcription.

The transcription factor SOX9 (Sry-type high-mobility-group box 9) is expressed in all chondrocytes and is essential for the expression of aggrecan, which during biosynthesis is substituted with more than 10 times its weight of CS (chondroitin sulfate) and is secreted by chondrocytes to form the characteristic GAG (glycosaminoglycan)-rich ECM (extracellular matrix) of cartilage. SOX9 expression rapidly falls during monolayer culture of isolated chondrocytes and this turns off aggrecan and associated CS synthesis. We therefore investigated whether SOX9 transduction of cultured human articular chondrocytes had any effect on the gene expression of the glycosyltransferases and sulfotransferases necessary for GAG biosynthesis. Retroviral SOX9 transduction of passaged chondrocytes increased the endogenous rate of GAG synthesis and the total capacity for GAG synthesis assessed in monolayer culture with beta-xyloside. Both the endogenous rate and the total capacity of GAG biosynthesis were increased further in chondrogenic cell aggregate cultures. The GAG synthesized was predominantly CS and the hydrodynamic size of the newly synthesized chains was unchanged by SOX9 transduction. Aggrecan gene expression was increased in the SOX9-transduced chondrocytes and increased further in chondrogenic culture, but no comparable effects were found in SOX9 transduced dermal fibroblasts. However, the expression of CS glycosyltransferase and sulfotransferase genes in chondrocytes was unaffected by SOX9 transduction. Therefore SOX9 transduction in chondrocytes increased their CS synthetic capacity, but this was not accompanied by changes in the transcription of the CS biosynthetic enzymes and must occur by indirect regulation of enzyme activity through control of enzyme protein translation or enzyme organization.