Articular Cartilage Phenotype Research Articles

1,25-Dihydroxyvitamin D Deficiency Accelerates Aging-related Osteoarthritis via Downregulation of Sirt1 in Mice.

Emerging observational data suggest that vitamin D deficiency is associated with the onset and progression of knee osteoarthritis (OA). However, the relationship between vitamin D level and OA and the role of vitamin D supplementation in the prevention of knee OA are controversial. To address these issues, we analyzed the articular cartilage phenotype of 6- and 12-month-old wild-type and 1α(OH)ase-/- mice and found that 1,25(OH)2D deficiency accelerated the development of age-related spontaneous knee OA, including cartilage surface destruction, cartilage erosion, proteoglycan loss and cytopenia, increased OARSI score, collagen X and Mmp13 positive chondrocytes, and increased chondrocyte senescence with senescence-associated secretory phenotype (SASP). 1,25(OH)2D3 supplementation rescued all knee OA phenotypes of 1α(OH)ase-/- mice in vivo, and 1,25(OH)2D3 rescued IL-1β-induced chondrocyte OA phenotypes in vitro, including decreased chondrocyte proliferation and cartilage matrix protein synthesis, and increased oxidative stress and cell senescence. We also demonstrated that VDR was expressed in mouse articular chondrocytes, and that VDR knockout mice exhibited knee OA phenotypes. Furthermore, we demonstrated that the down-regulation of Sirt1 in articular chondrocytes of 1α(OH)ase-/- mice was corrected by supplementing 1,25(OH)2D3 or overexpression of Sirt1 in mesenchymal stem cells (MSCs) and 1,25(OH)2D3 up-regulated Sirt1 through VDR mediated transcription. Finally, we demonstrated that overexpression of Sirt1 in MSCs rescued knee OA phenotypes in 1α(OH)ase-/- mice. Thus, we conclude that 1,25(OH)2D3, via VDR-mediated gene transcription, plays a key role in preventing the onset of aging-related knee OA in mouse models by up-regulating Sirt1, an aging-related gene that promotes articular chondrocyte proliferation and extracellular matrix protein synthesis, and inhibits senescence and SASP.

Axial alignment is a critical regulator of knee osteoarthritis.

Although osteoarthritis (OA), a leading cause of disability, has been associated with joint malalignment, scientific translational evidence for this link is lacking. In a clinical case study, we provide evidence of osteochondral recovery upon unloading symptomatic isolated medial tibiofemoral knee OA associated with varus malalignment. By mapping response correlations at high resolution, we identify spatially complex degenerative changes in cartilage after overloading in a clinically relevant ovine model. We further report that unloading diminishes OA cartilage degeneration and alterations of critical parameters of the subchondral bone plate in a similar topographic fashion. Last, therapeutic unloading shifted the articular cartilage and subchondral bone phenotype to normal and restored several physiological correlations disturbed in neutral and varus OA, suggesting a protective effect on the integrity of the entire osteochondral unit. Collectively, these findings identify modifiable trajectories with considerable translational potential to reduce the burden of human OA.

Dickkopf-1 reduces hypertrophic changes in human chondrocytes derived from bone marrow stem cells

The in vitro process of chondrogenic differentiation of mesenchymal stem cells (MSCs) induces a pre-apoptotic hypertrophic phenotype, guided by the active status of the WNT/β‑catenin pathway. To achieve a stable chondrocyte phenotype for cartilage tissue engineering, it is necessary to gain a better understanding of specific genes that regulate the cartilage tissue phenotype. RNA sequencing (RNA-seq) analysis of tissue samples from bone, cartilage, growth plate and muscle show that Dickkopf-1 (DKK1), a natural WNT canonical signaling inhibitor, is expressed in cartilage tissue. This observation reinforces the concept that inhibition of the WNT/β‑catenin pathway is critical for preventing avoid chondrocyte hypertrophy in vitro. We used two doses of DKK1 in a pellet cell culture system to inhibit the terminal differentiation of chondrocytes derived from bone marrow mesenchymal stem cells (MSCs). Bone marrow MSCs were cultured in chondrogenic induction medium with 50 and 200 ng/ml of DKK1 for 21 days. The highest doses of DKK1 reduce β‑catenin expression and nuclear localization at day 21, concomitant with reduced expression and activity of hypertrophy markers collagen type X (COL10A1) and alkaline phosphatase (ALPL), thus decreasing the pre-hypertrophic chondrocyte population. Furthermore, DKK1 stimulated expression of collagen type II (COL2A1) and glycosaminoglycans (GAGs), which represent healthy articular cartilage markers. We conclude that exogenous DKK1 impedes chondrocyte progression into a prehypertrophic stage and stimulates expression of healthy articular cartilage markers by blocking the WNT/β‑catenin pathway. Hence, DKK1 may promote a mature healthy articular cartilage phenotype and facilitate cartilage tissue engineering for joint repair.

Role of IFT88 in icariin‑regulated maintenance of the chondrocyte phenotype.

Maintenance of the chondrocyte phenotype is crucial for cartilage repair during tissue engineering. Intraflagellar transport protein 88 (IFT88) is an essential component of primary cilia, shuttling signals along the axoneme. The hypothesis of the present study was that IFT88 could exert an important role in icariin-regulated maintenance of the chondrocyte phenotype. To this end, the effects of icariin on proliferation and differentiation of the chondrogenic cell line, ATDC5, were explored. Icariin-treated ATDC5 cells and primary chondrocytes expressed IFT88. Icariin has been demonstrated to aid in the maintenance of the articular cartilage phenotype in a rat model of post-traumatic osteoarthritis (PTOA). Icariin promoted chondrocyte proliferation and expression of the chondrogenesis marker genes, COL II and SOX9, increased ciliary assembly, and upregulated IFT88 expression in a concentration- and time-dependent manner. Icariin-treated PTOA rats secreted more cartilage matrix compared with the controls. Knockdown of IFT88 expression with siRNA reduced extracellular signal-regulated kinase (ERK) phosphorylation, and icariin upregulated IFT88 expression by promoting ERK phosphorylation. Thus, IFT88 serves a major role in icariin-mediated maintenance of the chondrocyte phenotype, promoting ciliogenesis and IFT88 expression by increasing ERK phosphorylation. Icariin may therefore be useful for maintenance of the cartilage phenotype during tissue engineering.

Advances in Adult Stem Cell Differentiation and Cellular Reprogramming to Enhance Chondrogenesis

Cell-based therapies to treat articular cartilage and osteochondral defects as a result of osteoarthritis or traumatic injury are a promising approach. Traditional sources of cells have been autologous chondrocytes which are culture expanded and implanted; however, dedifferentiation of these cells results in a type of fibrocartilage which has reduced therapeutic benefit. Advances in cellular reprogramming technology are either through generation of induced pluripotent stem cells (iPSCs) and subsequent chondrogenic or through direct reprogramming of adult cells to chondrocytes. These approaches have the potential to provide an unlimited source of cartilage for therapeutic applications; however, challenges remain in terms of efficient cellular differentiation and ability to integrate and repair tissues. Growth factor-based strategies previously used in chondrogenic differentiation of adult stem cells and embryonic stem cells have been successfully applied to induced pluripotent stem cells, enhancing the ability of iPSCs to produce both patient-specific chondrocytes and to produce large quantities of these cells. A combination of novel biomaterials and additive bioprinting have also opened new approaches to recapitulate zonal cartilage structure and repair of osteochondral defects. The development of innovative protocols to generate chondrocytes from a variety of primary cells continues to proceed rapidly, allowing fine tuning of differentiation processes to produce an articular cartilage phenotype with improved mechanical and tissue integration capabilities.

Transcriptome-Wide Analyses of Human Neonatal Articular Cartilage and Human Mesenchymal Stem Cell-Derived Cartilage Provide a New Molecular Target for Evaluating Engineered Cartilage.

Cellular differentiation comprises a progressive, multistep program that drives cells to fabricate a tissue with specific and site distinctive structural and functional properties. Cartilage constitutes one of the potential differentiation lineages that mesenchymal stem cells (MSCs) can follow under the guidance of specific bioactive agents. Single agents such as transforming growth factor beta (TGF-β) and bone morphogenetic protein 2 in unchanging culture conditions have been historically used to induce in vitro chondrogenic differentiation of MSCs. Despite the expression of traditional chondrogenic biomarkers such as type II collagen and aggrecan, the resulting tissue represents a transient cartilage rather than an in vivo articular cartilage (AC), differing significantly in structure, chemical composition, cellular phenotypes, and mechanical properties. Moreover, there have been no comprehensive, multicomponent parameters to define high-quality and functional engineered hyaline AC. To address these issues, we have taken an innovative approach based on the molecular interrogation of human neonatal articular cartilage (hNAC), dissected from the knees of 1-month-old cadaveric specimens. Subsequently, we compared hNAC-specific transcriptional regulatory elements and differentially expressed genes with adult human bone marrow (hBM) MSC-derived three-dimensional cartilage structures formed in vitro. Using microarray analysis, the transcriptome of hNAC was found to be globally distinct from the transient, cartilage-like tissue formed by hBM-MSCs in vitro. Specifically, over 500 genes that are highly expressed in hNAC were not expressed at any time point during in vitro human MSC chondrogenesis. The analysis also showed that the differences were less variant during the initial stages (first 7 days) of the in vitro chondrogenic differentiation program. These observations suggest that the endochondral fate of hBM-MSC-derived cartilage may be rerouted at earlier stages of the TGF-β-stimulated chondrogenic differentiation program. Based on these analyses, several key molecular differences (transcription factors and coded cartilage-related proteins) were identified in hNAC that will be useful as molecular inductors and identifiers of the in vivo AC phenotype. Our findings provide a new gold standard of a molecularly defined AC phenotype that will serve as a platform to generate novel approaches for AC tissue engineering.

Redifferentiated Chondrocytes for the Repair of Articular Cartilage Lesions

Objectives:Biological therapies such as autologous chondrocyte implantation which use serially passaged chondrocytes to repair cartilage defects, are often unsuccessful as they lead to the formation of fibrocartilage. Fibrocartilage is rich in collagen type 1 (Col1) and lacks the structural complexity, low friction, and load distribution properties of articular cartilage. Fibrocartilage forms as a result of the phenotype of passaged chondrocytes. When serially passaged in monolayer to increase cell numbers, chondrocytes lose their phenotype in a process termed “dedifferentiation”. This is characterized by a loss in the production of collagen type II (Col2) and aggrecan (Acan), the major extracellular matrix molecules of articular cartilage, and an increase in the production of Col1. Therefore, to increase the success of therapies which use passaged chondrocytes, these cells must be redifferentiated to re-establish an articular cartilage phenotype prior to implantation in a cartilage lesion. Transforming growth factor beta (TGFβ) signalling is known to play important roles in both cell differentiation and articular cartilage repair. However, intra-articular injections with TGFβ have been shown to result in osteophyte formation and fibrosis of synovial tissues. Therefore, redifferentiating passaged chondrocytes in vitro using TGFβ prior to their use in vivo may be a better alternative. Fibrin glue is commonly used in orthopaedics to maintain cells in a cartilage lesion, however the effects of fibrin glue on chondrocytes have not been fully investigated. Thus we hypothesize that passaged chondrocytes redifferentiated in vitro with TGFβ will maintain their phenotype and form articular cartilage-like tissue when embedded in fibrin glue in the absence of further exogenous supplementation with TGFβ in vitro.Methods:Chondrocytes were enzymatically isolated from rabbit articular cartilage and serially passaged in monolayer twice (P2) to increase cell number and facilitate dedifferentiation. P2 cells were cultured in scaffold-free 3D using a chondrogenic serum-free media supplemented with 10ng/ml TGFβ3 to facilitate redifferentiation. Redifferentiated chondrocytes were then embedded in fibrin glue (Tisseel, Baxter) at 1.5 x 106 cells/20uL and cultured for 2 weeks in vitro in the absence of TGFβ3. Controls were P2 cells that were not redifferentiated with TGFB3 prior to embedding in fibrin glue. Histology and immunohistochemistry were used to assess tissue formation and cell phenotype.Results:Redifferentiated chondrocytes accumulated more matrix rich in Col2 and Acan than controls (Figure 1). Furthermore, redifferentiated chondrocytes had a round cell shape characteristic of articular chondrocytes. The use of fibrin glue did not impair the ability of redifferentiated chondrocytes to accumulate matrix nor did it affect cell phenotype.Conclusion:Redifferentiated chondrocytes in fibrin glue may be a better alternative to passaged chondrocytes for cell based articular cartilage repair therapies as they accumulate more Col2 and Acan containing matrix than undifferentiated P2. In vivo studies are required to elucidate the potential for redifferentiated chondrocytes in cartilage repair.

Conditional Deletion of the Phd2 Gene in Articular Chondrocytes Accelerates Differentiation and Reduces Articular Cartilage Thickness

Based on our findings that PHD2 is a negative regulator of chondrocyte differentiation and that hypoxia signaling is implicated in the pathogenesis of osteoarthritis, we investigated the consequence of disruption of the Phd2 gene in chondrocytes on the articular cartilage phenotype in mice. Immunohistochemistry detected high expression of PHD2 in the superficial zone (SZ), while PHD3 and HIF-1α (target of PHD2) are mainly expressed in the middle-deep zone (MDZ). Conditional deletion of the Phd2 gene (cKO) in chondrocytes accelerated the transition of progenitors to hypertrophic (differentiating) chondrocytes as revealed by reduced SZ thickness, and increased MDZ thickness, as well as increased chondrocyte hypertrophy. Immunohistochemistry further revealed decreased levels of progenitor markers but increased levels of hypertrophy markers in the articular cartilage of the cKO mice. Treatment of primary articular chondrocytes, in vitro, with IOX2, a specific inhibitor of PHD2, promoted articular chondrocyte differentiation. Knockdown of Hif-1α expression in primary articular chondrocytes using lentiviral vectors containing Hif-1α shRNA resulted in reduced expression levels of Vegf, Glut1, Pgk1, and Col10 compared to control shRNA. We conclude that Phd2 is a key regulator of articular cartilage development that acts by inhibiting the differentiation of articular cartilage progenitors via modulating HIF-1α signaling.

Transplantation of autologous chondrocytes ex-vivo expanded using Thermoreversible Gelation Polymer in a rabbit model of articular cartilage defect

Graft failure due to de-differentiation of the chondrocytes during in vitro culture and after transplantation is a major hurdle in Autologous Chondrocyte Implantation (ACI). We, herein, report the transplantation of autologous chondrocytes ex vivo expanded using a Thermo-reversible Gelation Polymer (TGP) in a rabbit model. A full thickness chondral defect was created in one of the knee joints in each of the six rabbits of the study and autologous chondrocytes in vitro expanded using TGP scaffold were transplanted after 10 weeks. H & E staining of the biopsy after 6 months revealed maintenance of articular cartilage phenotype.

Enhanced articular cartilage by human mesenchymal stem cells in enzymatically mediated transiently RGDS-functionalized collagen-mimetic hydrogels.

Recapitulating aspects of the native tissue biochemical microenvironment faces significant challenges in regenerative medicine and tissue engineering due to the complex and dynamic nature of the tissue. The ability to take advantage of, mimic, and modulate cell-mediated processes within novel naturally-derived hydrogels is of great interest in the field of biomaterials to generate constructs that more closely resemble the biochemical microenvironment and functions of native biological tissues such as articular cartilage. Towards this goal, the temporal presentation of bioactive sequences such as RGDS on the chondrogenic differentiation of human mesenchymal stem cells is considered important as it has been shown to influence the chondrogenic phenotype. Here, a novel and versatile platform to recreate a high degree of biological complexity is proposed, which could also be applicable to other tissue engineering and regenerative medicine applications.

The Key Role of Canonical Wnt/β-catenin Signaling in Cartilage Chondrocytes.

Articular cartilage is a physiologically non-self-renewing avascular tissue with a unique cell type, the chondrocyte, which functions as producing and maintaining the extracellular matrix of cartilage. Cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules. The network sheds light on these mechanisms that appear to be highly relevant to both the identification of pathogenic key factors, and the development of biological approaches for cartilage regeneration. Wnt/β-catenin signaling has been recognized as a key regulator of development and homeostasis in bone, cartilage, and joint. It plays important roles in many biological processes, including the condensation and differentiation of mesenchymal cells, the maintenance of mature articular cartilage phenotype, the hypertrophic maturation in the process of endochondral ossification, and tissue degeneration and regeneration. With regard to the importance of Wnt signaling pathways in regulating chondrocytes physiological and pathological activities, this article reviews the role of Wnt/β-catenin signaling in chondrogenesis, chondrocytes development, degeneration, and the inhibitors of Wnt/β-catenin signaling.

Melanocortin 1 receptor-signaling deficiency results in an articular cartilage phenotype and accelerates pathogenesis of surgically induced murine osteoarthritis.

Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR). MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1) in joint physiology and pathogenesis of osteoarthritis (OA) using MC1-signaling deficient mice (Mc1re/e). OA was surgically induced in Mc1re/e and wild-type (WT) mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months) and 4/8 weeks past surgery. µCT–analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more severe OA-pathology. Our data suggest that MC1-signaling protects against cartilage degradation and subchondral bone sclerosis in OA indicating a beneficial role of the POMC system in joint pathophysiology.

Oleuropein, quercetin and curcumin prevent cartilage breakdown in bovine chondrocytes

Oleuropein, quercetin and curcumin prevent cartilage breakdown in bovine chondrocytes

Mathematical modeling of signaling pathways in osteoarthritis

Purpose: Cartilage homeostasis relies on an intricate balance between anabolic and catabolic processes. In osteoarthritis this balance is shifted towards catabolism, leading to hypertrophy and a gradual degradation of cartilage tissue. So far, drug-based intervention in this process has shown limited progress. We propose to construct a mathematical model of the molecular network that governs key processes in articular cartilage homeostasis. This model can be used as a platform for model expansion by introduction of new experimental findings and hypotheses. Methods: We recently developed ANIMO (Analysis of Networks with Interactive Modeling), an intuitive software tool for modeling molecular networks. Here, we demonstrate a mathematical model of growth plate cartilage using a combination of literature and experimental data. We show how ANIMO allows for intuitive exploration of the model, despite the size and complexity of the model. Results: We constructed a network model of regulatory processes in growth plate chondrocytes. In this model the effects downstream of extracellular growth factors FGF, WNT, IGF-1, PTHrP, Ihh, BMP, and TGF-β are integrated into a cellular response. In silico experiments predict the phenotypic outcome for different inputs and starting states of the model. Osteoarthritic chondrocytes and hypertrophic growth plate chondrocytes show strong parallels in their gene expression profile. We have examined the gene expression profiles of growth plate and articular cartilage. In articular cartilage the expression of the WNT and BMP antagonists DKK1, FRZB and GREM1 is over 10-500 fold higher than in growth plate cartilage. This leads us to think that DKK1, FRZB, GREM1 could act as gatekeepers for preventing hypertrophy. We are currently investigating the range of conditions under which these proteins exert their stabilizing effect on the articular cartilage phenotype in the model. Furthermore, we are interrogating the model to obtain in silico leads to targets for novel combination therapies. Such therapies could be used to intervene in the osteoarthritic state of the network and restore the balanced situation of healthy cartilage. Conclusions: Traditionally, modeling efforts in the realm of molecular cell biology have been the exclusive domain of researchers with a thorough training in mathematics or computer science. We show here that a complex model that is intuitively amenable to exploration and adaptation by biologists is an invaluable asset in cartilage research. Expansion of an existing model with DKK1, FRZB and GREM1 provided evidence for their role in preserving the articular cartilage phenotype.

Local ras in the hypertrophic differentiation of chondrocytes

Local ras in the hypertrophic differentiation of chondrocytes

Inducing articular cartilage phenotype in costochondral cells

IntroductionCostochondral cells may be isolated with minimal donor site morbidity and are unaffected by pathologies of the diarthrodial joints. Identification of optimal exogenous stimuli will allow abundant and robust hyaline articular cartilage to be formed from this cell source.MethodsIn a three factor, two level full factorial design, the effects of hydrostatic pressure (HP), transforming growth factor β1 (TGF-β1), and chondroitinase ABC (C-ABC), and all resulting combinations, were assessed in third passage expanded, redifferentiated costochondral cells. After 4 wks, the new cartilage was assessed for matrix content, superficial zone protein (SZP), and mechanical properties.ResultsHyaline articular cartilage was generated, demonstrating the presence of type II collagen and SZP, and the absence of type I collagen. TGF-β1 upregulated collagen synthesis by 175% and glycosaminoglycan synthesis by 75%, resulting in a nearly 200% increase in tensile and compressive moduli. C-ABC significantly increased collagen content, and fibril density and diameter, leading to a 125% increase in tensile modulus. Hydrostatic pressure increased fibril diameter by 30% and tensile modulus by 45%. Combining TGF-β1 with C-ABC synergistically increased collagen content by 300% and tensile strength by 320%, over control. No significant differences were observed between C-ABC/TGF-β1 dual treatment and HP/C-ABC/TGF-β1.ConclusionsEmploying biochemical, biophysical, and mechanical stimuli generated robust hyaline articular cartilage with a tensile modulus of 2 MPa and a compressive instantaneous modulus of 650 kPa. Using expanded, redifferentiated costochondral cells in the self-assembling process allows for recapitulation of robust mechanical properties, and induced SZP expression, key characteristics of functional articular cartilage.

Induction of chondrogenesis from human embryonic stem cells without embryoid body formation by bone morphogenetic protein 7 and transforming growth factor β1

Human embryonic stem cells (ESCs) provide an unlimited supply of pluripotent cells for articular cartilage tissue engineering and regenerative medicine applications. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: surface, middle, and deep. To date, attempts at differentiating human ESCs into articular chondrocytes have been unsuccessful. The majority of studies have focused on chondrogenic (but not specifically articular cartilage) differentiation. Furthermore, previous investigations of induction of chondrogenesis by human ESCs required embryoid body formation; however, embryoid body formation often results in heterogeneous differentiation. The present study was undertaken to determine the in vitro chondrogenic potential of bone morphogenetic protein 7 (BMP-7) and transforming growth factor beta1 (TGFbeta1)-induced human ESC differentiation toward the articular cartilage phenotype. Dissociated single human ESCs were cultured and passaged on a gelatin-coated flask. The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGFbeta1, CM with BMP-7, or CM with both TGFbeta1 and BMP-7. The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGFbeta1 treatment, CM plus BMP-7 treatment, and CM plus TGFbeta1 and BMP-7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGFbeta1 and BMP-7 were added to the medium. However, expression of the genes for cartilage-specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP-7-treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions. These results demonstrate an efficient and reproducible model system of human ESC-induced chondrogenesis, using a novel direct plating method in which intervening embryoid body formation does not occur. Further work is needed for optimization of conditions to obtain the articular cartilage phenotype that includes the superficial zone marker as demonstrated by SZP/lubricin synthesis.

Metabolism of proteoglycans in tendon

Tendons are dense, fibrous connective tissues that are responsible for transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy (defined as a syndrome of tendon pain, tenderness and swelling that affects function) is very common, both within the sporting arena and in the workplace. Importantly, proteoglycans are essential components of the tendon extracellular matrix (ECM) and changes in their expression and metabolism/turnover have been associated with tendinopathy. Within tendons, the small leucine-rich proteoglycans (SLPRs), decorin, fibromodulin, lumican and keratocan predominate within tensional regions, while in tendon fibrocartilage, increased concentrations of proteoglycans common to the articular cartilage phenotype are present, including aggrecan, biglycan and proteoglycan 4. However, the rate of proteoglycan turnover within tendon is markedly higher than that of cartilage, mediated via the "aggrecanases," which are constitutively active in the tendon matrix. Data suggest that this increased proteoglycan turnover is likely to be required to maintain normal tendon homeostasis, with perturbations in proteoglycan metabolism contributing to tissue dysfunction. Thus, future studies aimed at furthering our fundamental knowledge of tendon proteoglycan metabolism in health and disease are important in the development of improved treatments for tendon disorders.

1095. Gene_Induced Chondrogenesis of Mesenchymal Stem Cells: A Comparative Analysis of Candidate cDNAs

Articular cartilage is a highly specialized tissue that protects the ends of bones in diarthrodal joints. The cartilage extracellular matrix is maintained by chondrocytes that occupy the tissue at low density. Because articular cartilage is avascular, it has a poor capacity to regenerate; thus, the damage resulting from large injuries is considered permanent. Due to their ready availability and capacity to differentiate along numerous lineages, adult mesenchymal stem cells (MSCs) may be useful in the development of cell-based therapies for cartilage repair. In this regard, gene transfer to MSCs can be used to achieve sustained expression of specific protein factors capable of inducing chondrogenic differentiation. In this study, using adenoviral mediated gene transfer and bone marrow derived MSCs in an aggregate culture system, we compared the relative condroinductive capacity of several cDNAs whose protein products have been associated with the induction or maintenance of the articular cartilage phenotype. First generation, E1, E3 deleted, serotype 5 adenoviral vectors carrying the complete cDNAs for TGF-b1, bone morphogenetic protein (BMP) -2, BMP -4 , BMP- 7, Sox 9, indian hedgehog (IHH), and insulin-like growth factor-1 (IGF-1) were constructed using cre-lox recombination. As a source of MSCs, adherent, colony forming cells from the bone marrow of 3-week-old calves were isolated, cultured and used at low passage. Individual flasks of MSCs were infected with Ad.TGF-b1, Ad.BMP-2, Ad.BMP-4, Ad.BMP-7 and Ad.IGF-1 at doses sufficient to provide low ( 100 ng/ml) amounts of transgene expression per 24 hrs. Delivery of Sox-9 and Ad.IHH was performed over a wide range of viral doses. At 24 hours post infection, 2.0 x 10^5 cells of each culture were pelleted by centrifugation. The aggregates were cultured for 3 weeks in conical tubes in a defined, serum-free medium containing ascorbate 2-phosphate and dexamethasone. Chondrogenesis was determined using histologic staining for glycosaminoglycans and by immunohistochemistry for collagen types I and II. By this method, gene transfer and expression of BMP-2 and BMP-4 showed the greatest chondrogenic potential. This was followed to a lesser extent by TGF-b1 and then Sox-9 and IGF-1. Delivery of IHH or BMP-7 was met with only minimal chondrogenic activity which was only slightly greater than uninfected control cells. The potent activity of the secreted factors BMPs-2, -4 and TGF-b1 were expected and are consistent with the literature. The capacity to induce chondrogenesis through delivery of the Sox-9 transcription factor offers a promising intracellular alternative to the overexpression of secretable pleiotropic growth factors. The relative lack of chondroinduction following overexpression of IGF-1 and IHH is perhaps not surprising because these products thought to be more important for maintenance than induction of the chondrocytic phenotype. Future work will involve evaluation these genes in combinatorial studies.

A predominantly articular cartilage-associated gene, SCRG1, is induced by glucocorticoid and stimulates chondrogenesis in vitro

Cartilage tissue engineering using multipotential human mesenchymal stem cells (hMSCs) is a promising approach to develop treatment for degenerative joint diseases. A key requirement is that the engineered tissues maintain their hyaline articular cartilage phenotype and not proceed towards hypertrophy. It is noteworthy that osteoarthritic articular cartilage frequently contains limited regions of reparative cartilage, suggesting the presence of bioactive factors with regenerative activity. Based on this idea, we recently performed cDNA microarray analysis to identify genes that are strongly expressed only in articular cartilage and encode secreted gene products. One of the genes that met our criteria was SCRG1. This study aims to analyze SCRG1 function in cartilage development using an in vitro mesenchymal chondrogenesis system. Full-length SCRG1 cDNA was subcloned into pcDNA5 vector, and transfected into hMSCs and murine C3H10T1/2 mesenchymal cells, placed in pellet cultures and micromass cultures, respectively. The cultures were analyzed by reverse transcription-polymerase chain reaction for the expression of SCRG1 and cartilage marker genes, and by histological staining for cartilage phenotype. Induction of SCRG1 expression was seen during in vitro chondrogenesis, and was dependent on dexamethasone (DEX) known to promote chondrogenesis. Immunohistochemistry revealed that SCRG1 protein was localized to the extracellular matrix. Forced expression of SCRG1 in hMSCs suppressed their proliferation, and stimulated chondrogenesis in C3H10T1/2 cells, confirmed by reduced collagen type I and elevated collagen type IIB expression. These results suggest that SCRG1 is involved in cell growth suppression and differentiation during DEX-dependent chondrogenesis. SCRG1 may be of utility in gene-mediated cartilage tissue engineering.