Adult Human Articular Cartilage Research Articles

Perlecan, A Multi-Functional, Cell-Instructive, Matrix-Stabilizing Proteoglycan With Roles in Tissue Development Has Relevance to Connective Tissue Repair and Regeneration.

This review highlights the multifunctional properties of perlecan (HSPG2) and its potential roles in repair biology. Perlecan is ubiquitous, occurring in vascular, cartilaginous, adipose, lymphoreticular, bone and bone marrow stroma and in neural tissues. Perlecan has roles in angiogenesis, tissue development and extracellular matrix stabilization in mature weight bearing and tensional tissues. Perlecan contributes to mechanosensory properties in cartilage through pericellular interactions with fibrillin-1, type IV, V, VI and XI collagen and elastin. Perlecan domain I - FGF, PDGF, VEGF and BMP interactions promote embryonic cellular proliferation, differentiation, and tissue development. Perlecan domain II, an LDLR-like domain interacts with lipids, Wnt and Hedgehog morphogens. Perlecan domain III binds FGF-7 and 18 and has roles in the secretion of perlecan. Perlecan domain IV, an immunoglobulin repeat domain, has cell attachment and matrix stabilizing properties. Perlecan domain V promotes tissue repair through interactions with VEGF, VEGF-R2 and α2β1 integrin. Perlecan domain-V LG1-LG2 and LG3 fragments antagonize these interactions. Perlecan domain V promotes reconstitution of the blood brain barrier damaged by ischemic stroke and is neurogenic and neuroprotective. Perlecan-VEGF-VEGFR2, perlecan-FGF-2 and perlecan-PDGF interactions promote angiogenesis and wound healing. Perlecan domain I, III and V interactions with platelet factor-4 and megakaryocyte and platelet inhibitory receptor promote adhesion of cells to implants and scaffolds in vascular repair. Perlecan localizes acetylcholinesterase in the neuromuscular junction and is of functional significance in neuromuscular control. Perlecan mutation leads to Schwartz-Jampel Syndrome, functional impairment of the biomechanical properties of the intervertebral disc, variable levels of chondroplasia and myotonia. A greater understanding of the functional working of the neuromuscular junction may be insightful in therapeutic approaches in the treatment of neuromuscular disorders. Tissue engineering of salivary glands has been undertaken using bioactive peptides (TWSKV) derived from perlecan domain IV. Perlecan TWSKV peptide induces differentiation of salivary gland cells into self-assembling acini-like structures that express salivary gland biomarkers and secrete α-amylase. Perlecan also promotes chondroprogenitor stem cell maturation and development of pluripotent migratory stem cell lineages, which participate in diarthrodial joint formation, and early cartilage development. Recent studies have also shown that perlecan is prominently expressed during repair of adult human articular cartilage. Perlecan also has roles in endochondral ossification and bone development. Perlecan domain I hydrogels been used in tissue engineering to establish heparin binding growth factor gradients that promote cell migration and cartilage repair. Perlecan domain I collagen I fibril scaffolds have also been used as an FGF-2 delivery system for tissue repair. With the availability of recombinant perlecan domains, the development of other tissue repair strategies should emerge in the near future. Perlecan co-localization with vascular elastin in the intima, acts as a blood shear-flow endothelial sensor that regulates blood volume and pressure and has a similar role to perlecan in canalicular fluid, regulating bone development and remodeling. This complements perlecan’s roles in growth plate cartilage and in endochondral ossification to form the appendicular and axial skeleton. Perlecan is thus a ubiquitous, multifunctional, and pleomorphic molecule of considerable biological importance. A greater understanding of its diverse biological roles and functional repertoires during tissue development, growth and disease will yield valuable insights into how this impressive proteoglycan could be utilized successfully in repair biology.

Restoring Osteochondral Defects through the Differentiation Potential of Cartilage Stem/Progenitor Cells Cultivated on Porous Scaffolds.

Cartilage stem/progenitor cells (CSPCs) are cartilage-specific, multipotent progenitor cells residing in articular cartilage. In this study, we investigated the characteristics and potential of human CSPCs combined with poly(lactic-co-glycolic acid) (PLGA) scaffolds to induce osteochondral regeneration in rabbit knees. We isolated CSPCs from human adult articular cartilage undergoing total knee replacement (TKR) surgery. We characterized CSPCs and compared them with infrapatellar fat pad-derived stem cells (IFPs) in a colony formation assay and by multilineage differentiation analysis in vitro. We further evaluated the osteochondral regeneration of the CSPC-loaded PLGA scaffold during osteochondral defect repair in rabbits. The characteristics of CSPCs were similar to those of mesenchymal stem cells (MSCs) and exhibited chondrogenic and osteogenic phenotypes without chemical induction. For in vivo analysis, CSPC-loaded PLGA scaffolds produced a hyaline-like cartilaginous tissue, which showed good integration with the host tissue and subchondral bone. Furthermore, CSPCs migrated in response to injury to promote subchondral bone regeneration. Overall, we demonstrated that CSPCs can promote osteochondral regeneration. A monophasic approach of using diseased CSPCs combined with a PLGA scaffold may be beneficial for repairing complex tissues, such as osteochondral tissue.

The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo.

ObjectiveCartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo.DesignWe evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors.ResultsRNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed.ConclusionUsing multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.

Perlecan in the Natural and Cell Therapy Repair of Human Adult Articular Cartilage: Can Modifications in This Proteoglycan Be a Novel Therapeutic Approach?

Articular cartilage is considered to have limited regenerative capacity, which has led to the search for therapies to limit or halt the progression of its destruction. Perlecan, a multifunctional heparan sulphate (HS) proteoglycan, promotes embryonic cartilage development and stabilises the mature tissue. We investigated the immunolocalisation of perlecan and collagen between donor-matched biopsies of human articular cartilage defects (n = 10 × 2) that were repaired either naturally or using autologous cell therapy, and with age-matched normal cartilage. We explored how the removal of HS from perlecan affects human chondrocytes in vitro. Immunohistochemistry showed both a pericellular and diffuse matrix staining pattern for perlecan in both natural and cell therapy repaired cartilage, which related to whether the morphology of the newly formed tissue was hyaline cartilage or fibrocartilage. Immunostaining for perlecan was significantly greater in both these repair tissues compared to normal age-matched controls. The immunolocalisation of collagens type III and VI was also dependent on tissue morphology. Heparanase treatment of chondrocytes in vitro resulted in significantly increased proliferation, while the expression of key chondrogenic surface and genetic markers was unaffected. Perlecan was more prominent in chondrocyte clusters than in individual cells after heparanase treatment. Heparanase treatment could be a means of increasing chondrocyte responsiveness to cartilage injury and perhaps to improve repair of defects.

Molecular characterization of mesenchymal stem cells in human osteoarthritis cartilage reveals contribution to the OA phenotype

Adult human articular cartilage harbors a population of CD166+ mesenchymal stem cell-like progenitors that become more numerous during osteoarthritis (OA). While their role is not well understood, here we report that they are indeed part of cellular clusters formed in OA cartilage, which is a pathological hallmark of this disease. We hypothesize that these cells, termed OA mesenchymal stem cells (OA-MSCs), contribute to OA pathogenesis. To test this hypothesis, we generated and characterized multiple clonally derived stable/immortalized human OA-MSC cell lines, which exhibited the following properties. Firstly, two mesenchymal stem cell populations exist in human OA cartilage. While both populations are multi-potent, one preferentially undergoes chondrogenesis while the other exhibits higher osteogenesis potential. Secondly, both OA-MSCs exhibit significantly higher expression of hypertrophic OA cartilage markers COL10A1 and RUNX2, compared to OA chondrocytes. Induction of chondrogenesis in OA-MSCs further stimulated COL10A1 expression and MMP-13 release, suggesting that they contribute to OA phenotypes. Finally, knocking down RUNX2 is insufficient to inhibit COL10A1 in OA-MSCs and also requires simultaneous knockdown of NOTCH1 thereby suggesting altered gene regulation in OA stem cells in comparison to chondrocytes. Overall, our findings suggest that OA-MSCs may drive pathogenesis of cartilage degeneration and should therefore be a novel cell target for OA therapy.

Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering

The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growth rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering.

Human Cartilage-Derived Progenitor Cells From Committed Chondrocytes for Efficient Cartilage Repair and Regeneration.

Articular cartilage is not a physiologically self-renewing tissue. Injury of cartilage often progresses from the articular surface to the subchondral bone, leading to pathogenesis of tissue degenerative diseases, such as osteoarthritis. Therapies to treat cartilage defects using autologous chondrocyte-based tissue engineering have been developed and used for more than 20 years; however, the challenge of chondrocyte expansion in vitro remains. A promising cell source, cartilage stem/progenitor cells (CSPCs), has attracted recent attention. Because their origin and identity are still unclear, the application potential of CSPCs is under active investigation. Here we have captured the emergence of a group of stem/progenitor cells derived from adult human chondrocytes, highlighted by dynamic changes in expression of the mature chondrocyte marker, COL2, and mesenchymal stromal/stem cell (MSC) marker, CD146. These cells are termed chondrocyte-derived progenitor cells (CDPCs). The stem cell-like potency and differentiation status of CDPCs were determined by physical and biochemical cues during culture. A low-density, low-glucose 2-dimensional culture condition (2DLL) was critical for the emergence and proliferation enhancement of CDPCs. CDPCs showed similar phenotype as bone marrow mesenchymal stromal/stem cells but exhibited greater chondrogenic potential. Moreover, the 2DLL-cultured CDPCs proved efficient in cartilage formation both in vitro and in vivo and in repairing large knee cartilage defects (6-13 cm(2)) in 15 patients. These findings suggest a phenotype conversion between chondrocytes and CDPCs and provide conditions that promote the conversion. These insights expand our understanding of cartilage biology and may enhance the success of chondrocyte-based therapies. Injury of cartilage, a non-self-repairing tissue, often progresses to pathogenesis of degenerative joint diseases, such as osteoarthritis. Although tissue-derived stem cells have been shown to contribute to tissue renewal and homeostasis, the derivation, biological function, and application potential of stem/progenitor cells found in adult human articular cartilage are incompletely understood. This study reports the derivation of a population of cartilage stem/progenitor cells from fully differentiated chondrocytes under specific culture conditions, which have the potential to reassume their chondrocytic phenotype for efficient cartilage regeneration. These findings support the possibility of using in vitro amplified chondrocyte-derived progenitor cells for joint cartilage repair.

Limited evidence of chondrocyte outgrowth from adult human articular cartilage

Cellular outgrowth from articular cartilage tissue has been described in a number of recent experimental studies. The aim of this study was to investigate the occurrence of cellular outgrowth from articular cartilage explants isolated from adult human donors. Macroscopically intact articular cartilage specimens were isolated from adult human donors and cultured either in their native status, or in a cleansed status achieved by forced washing to minimize attaching cells. Additionally, the effect of chemotactic stimuli including cell lysate, High-Mobility-Group-Protein B1 (HMGB-1), Trefoil-factor 3 (TFF3), bone morphogenetic protein-2 (BMP-2), transforming growth factor-ß1 (TGF-ß1), or three-dimensional fibrin or collagen matrices were investigated. Co-cultures with synovial membrane served as a positive control for a source of migratory cells. The occurrence of cellular outgrowth was analyzed by histological examination after a culture period of 4 weeks. Spontaneous cellular outgrowth from cleansed cartilage specimens was not observed at a relevant level and could not significantly be induced by chemotactic stimuli or three-dimensional matrices either. A forming cartilage-adjoining cell layer was only apparent in the case of native cartilage explants with cellular remnants from surgical isolation or in co-culture experiments with synovial membrane. The relevance of cellular outgrowth from cartilage tissue is largely absent in the case of adult human articular cartilage samples. A cartilage-adjoining cell layer forming around the explants may instead originate from still attaching cells that remained from surgical isolation.

Hypoxia-inducible factor 3-alpha expression is associated with the stable chondrocyte phenotype.

The hypoxia-inducible factors HIF-1α and HIF-2α are important regulators of the chondrocyte phenotype but little is known about HIF-3α in cartilage. The objective of this study was to characterize HIF-3α (HIF3A) expression during chondrocyte differentiation in vitro and in native cartilage tissues. HIF3A, COL10A1, and MMP13 were quantified in mesenchymal stem cells (MSCs) and articular chondrocytes from healthy and osteoarthritic (OA) tissue in three-dimensional cultures and in human embryonic epiphyses and adult articular cartilage. HIF3A was found to have an inverse association with hypertrophic markers COL10A1 and MMP13 in chondrogenic cells and tissues. In healthy chondrocytes, HIF3A was induced by dexamethasone and increased during redifferentiation. By comparison, HIF3A expression was extremely low in chondrogenically differentiated MSCs expressing high levels of COL10A1 and MMP13. HIF3A was also lower in redifferentiated OA chondrocytes than in healthy chondrocytes. In human embryonic epiphyseal tissue, HIF3A expression was lowest in the hypertrophic zone. Distinct splice patterns were also found in embryonic cartilage when compared with adult articular cartilage and redifferentiated chondrocytes. These in vitro and in vivo findings suggest that HIF3A levels are indicative of the hypertrophic state of chondrogenic cells and one or more splice variants may be important regulators of the chondrocyte phenotype.

Agrin: A new player in joint biology and a potent growth factor for cartilage regeneration

Purpose: Osteoarthritis is a chronic disabling disease characterized by cartilage breakdown for which there is no cure. Disruption of the gene encoding for the heparan sulphate proteoglycan Agrin results in embryonic skeletal dysplasia suggesting a role for Agrin in cartilage biology and prompting us to study its role in articular cartilage biology and osteoarthritis. The aims of this study are to establish the expression pattern of Agrin in healthy/normal cartilage and compare it to the expression pattern in osteoarthritic or injured cartilage; determine the effects of knockdown and over-expression of Agrin within cartilage in vitro and in vivo and to determine the epistasis of Agrin in articular chondrocytes. Methods: Agrin expression was determined by immunohistochemistry and qPCR. Osteoathritis was induced in 8 week old 129sv mice by destabilisation of the medial meniscus (DMM) and Agrin expression was evaluated by immunofluorescence 8 weeks post-surgery. Paired human samples of preserved cartilage vs severely osteoarthritic cartilage were compared by immunofluorescence. Gain and loss of function experiments were performed using an expression plasmid encoding mammalian Agrin and siRNAs in C28/I2 and bovine chondrocytes in micromass culture. In vivo cartilage formation was assessed using an ectopic implantation model in nude mice; growth-arrested COS7 cells overexpressing Agrin or GFP were combined with bovine chondrocytes (ratio 1:10) and implanted ectopically into nude mice for two weeks. Retrieved implants were characterised by histology and qPCR. Results: Agrin and its known receptors were expressed in healthy adult human articular cartilage and downregulated in human and experimental murine osteoarthritis. Silencing of Agrin by siRNA resulted in reduced GAG production in C28/I2 and in chondrocyte de-differentiation characterised by decreased expression of SOX9, COL2A1 and ACAN mRNA. Overexpression of Agrin in the human chondrocyte cell line C28/I2 and bovine primary articular chondrocytes resulted in enhanced GAG production and SOX9 upregulation. Importantly, in contrast to BMP-2, Agrin over-expression did not induce markers of cartilage hypertrophy including COL10A1 and MMP-13. Delivering Agrin to primary bovine chondrocytes transplanted in the muscle of nude mice resulted in enhanced formation of ectopic cartilage, which did not display signs of hypertrophy, vascular invasion, or endochondral bone formation. Conclusions: Our data show that Agrin is essential for the maintenance of the chondrocytic phenotype and extracellular matrix production whilst exogenous Agrin enhances chondrocyte differentiation and cartilage formation in vitro and in vivo; and therefore may be a valuable chondrogenic molecule in tissue engineering technologies.

Activin receptor-like kinase ALK5 and ALK1 are both required for TGFβ-initiated chondrogenic differentiation of mesenchymal stem cells

s / Osteoarthritis and Cartilage 23 (2015) A26eA81 A79 Conclusions: The administration of stable (i.e., non-radioactive) strontium at sub-therapeutic doses can serve effectively as a dynamic tracer of bone turnover to assess micro-structural changes associated with the pathogenesis of bone disease including osteoarthritis, at high spatial resolution (micron level) not possible with radionuclide-based scintigraphic imaging. In addition, when co-administered with drug interventions, strontium as a dynamic label could be used to gauge the efficacy of disease modifying drugs upon adaptive bone physiology in osteoarthritis or other adaptive bone pathology, such as osteolytic/ sclerotic bone cancer metastases. The KES-SRmCT imaging procedure reported here is the first application of synchrotronmicro-CT to segment and visualize regions of active bone turnover in osteoarthritis in 3-D. Figure 2. Subchondral sclerosis in end-stage osteoarthritis. Note significant incorporation of strontium in subchondral bone detected by EPMA (light blue color) from week 8 (a) to 12 (b) post-surgery. The corresponding histology (Safranin-O stain) indicates sclerotic subchondral bone (note lack of marrow space) and full thickness loss of articular cartilage. 93 ACTIVIN RECEPTOR-LIKE KINASE ALK5 AND ALK1 ARE BOTH REQUIRED FOR TGFb-INITIATED CHONDROGENIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS L.M. de Kroon yz, E. N. Blaney Davidson y, R. Narcisi z, H.M. van Beuningen y, G.J. van Osch z, P.M. van der Kraan y. yRadboud Univ. Med. Ctr., Nijmegen, Netherlands; z Erasmus Univ. Med. Ctr., Rotterdam, Netherlands Purpose: Cartilage matrix defects, due to osteoarthritis or trauma, are not repaired because chondrocytes have poor regenerative capacity. Bone marrow-derived mesenchymal stem cells (BMSCs) are a promising cell source to treat matrix defects due to their capacity to differentiate into chondrocytes. Transforming Growth Factor-b (TGFb) potently initiates chondrogenic differentiation of BMSCs and is known to signal either via Activin receptor-Like Kinase (ALK) receptor ALK5 or ALK1, which activate the intracellular SMAD2/3 and SMAD1/5/8 pathway respectively. Since the role of TGFb receptors in BMSC chondrogenesis is unknown, we investigated whether either ALK5 or ALK1 is crucial to initiate chondrogenic differentiation of BMSCs. Methods: Human fetal BMSCs (purchased from ScienCell) were transduced with either adenoviral constitutive active (ca)ALK5, caALK1 or LacZ (control) for receptor overexpression. To downregulate receptor expression, BMSCs were transfected with lentiviral ALK5-shRNA, ALK1shRNA or a vector without shRNA (control). After viral infection, BMSCs were pellet-cultured in serum-free chondrogenic medium for 7 days. All aforementioned conditions were stimulated with 10 ng/mL TGFb1, which initiates chondrogenic differentiation. Cells overexpressing either caALK5 or caALK1 were cultured without TGFb1 stimulation in order to determine whether constitutive active receptor signaling initiated BMSC chondrogenesis. To verify chondrogenic differentiation, aggrecan gene expression (ACAN) was measured and proteoglycan deposition was evaluated by Safranin O staining. Results: Chondrogenesis was observed in LacZ-transfected BMSC pellets (LacZ-BMSCs) stimulated with TGFb1 as measured by high ACAN expression and positive proteoglycan staining. Overexpressing either caALK5 or caALK1 without stimulating BMSCs with TGFb1 resulted in activation of the SMAD2/3 or SMAD1/5/8 pathway respectively. However, unstimulated caALK1-BMSCs had ~8-fold and caALK5-BMSCs had ~78-fold lower ACAN expression and proteoglycan staining was absent compared to LacZ-BMSCs that were stimulated with TGFb1. Downregulating either ALK1 or ALK5 by shRNA in TGFb1-stimulated BMSCs caused respectively ~18-fold and ~222-fold lower ACAN expression levels than in the control condition and an absence of proteoglycan staining. Conclusions: Our data suggest that TGFb needs to activate both its ALK5 and ALK1 receptor to initiate chondrogenic differentiation of BMSCs. Both receptors seem crucial as BMSC chondrogenesis was not initiated when overexpressing either constitutive active ALK5 or ALK1, and chondrogenesis was inhibited when downregulating either ALK1 or ALK5. This study helps to better understand the molecular events induced by TGFb during chondrogenic differentiation of BMSCs, which is important for improving cartilage matrix formation by mesenchymal stem cells. 94 AGRIN: A NEW PLAYER IN JOINT BIOLOGY AND A POTENT GROWTH FACTOR FOR CARTILAGE REGENERATION S.E. Eldridge. WHRI, London, United Kingdom Purpose: Osteoarthritis is a chronic disabling disease characterized by cartilage breakdown for which there is no cure. Disruption of the gene encoding for the heparan sulphate proteoglycan Agrin results in embryonic skeletal dysplasia suggesting a role for Agrin in cartilage biology and prompting us to study its role in articular cartilage biology and osteoarthritis. The aims of this study are to establish the expression pattern of Agrin in healthy/normal cartilage and compare it to the expression pattern in osteoarthritic or injured cartilage; determine the effects of knockdown and over-expression of Agrin within cartilage in vitro and in vivo and to determine the epistasis of Agrin in articular chondrocytes. Methods: Agrin expression was determined by immunohistochemistry and qPCR. Osteoathritis was induced in 8 week old 129sv mice by destabilisation of the medial meniscus (DMM) and Agrin expression was evaluated by immunofluorescence 8 weeks post-surgery. Paired human samples of preserved cartilage vs severely osteoarthritic cartilage were compared by immunofluorescence. Gain and loss of function experiments were performed using an expression plasmid encoding mammalian Agrin and siRNAs in C28/I2 and bovine chondrocytes in micromass culture. In vivo cartilage formation was assessed using an ectopic implantation model in nude mice; growtharrested COS7 cells overexpressing Agrin or GFP were combined with bovine chondrocytes (ratio 1:10) and implanted ectopically into nude mice for two weeks. Retrieved implants were characterised by histology and qPCR. Results: Agrin and its known receptors were expressed in healthy adult human articular cartilage and downregulated in human and experimental murine osteoarthritis. Silencing of Agrin by siRNA resulted in reduced GAG production in C28/I2 and in chondrocyte de-differentiation characterised by decreased expression of SOX9, COL2A1 and ACAN mRNA. Overexpression of Agrin in the human chondrocyte cell line C28/I2 and bovine primary articular chondrocytes resulted in enhanced GAG production and SOX9 upregulation. Importantly, in contrast to BMP-2, Agrin over-expression did not induce markers of cartilage hypertrophy including COL10A1 and MMP-13. Delivering Agrin to primary bovine chondrocytes transplanted in the muscle of nude mice resulted in enhanced formation of ectopic cartilage, which did not display signs of hypertrophy, vascular invasion, or endochondral bone formation. Conclusions: Our data show that Agrin is essential for the maintenance of the chondrocytic phenotype and extracellular matrix production whilst exogenous Agrin enhances chondrocyte differentiation and cartilage formation in vitro and in vivo; and therefore may be a valuable chondrogenic molecule in tissue engineering technologies.

The Effect of Biomolecular Gradients on Mesenchymal Stem Cell Chondrogenesis under Shear Stress

Tissue engineering is viewed as a promising option for long-term repair of cartilage lesions, but current engineered cartilage constructs fail to match the mechanical properties of native tissue. The extracellular matrix of adult human articular cartilage contains highly organized collagen fibrils that enhance the mechanical properties of the tissue. Unlike articular cartilage, mesenchymal stem cell (MSC) based tissue engineered cartilage constructs lack this oriented microstructure and therefore display much lower mechanical strength. The goal of this study was to investigate the effect of biomolecular gradients and shear stress on MSCs undergoing chondrogenesis within a microfluidic device. Via poly(dimethyl siloxane) soft-lithography, microfluidic devices containing a gradient generator were created. Human MSCs were seeded within these chambers and exposed to flow-based transforming growth factor β1 (TGF-β1) gradients. When the MSCs were both confluent and exposed to shear stress, the cells aligned along the flow direction. Exposure to TGF-β1 gradients led to chondrogenesis of MSCs, indicated by positive type II collagen staining. These results, together with a previous study that showed that aligned MSCs produce aligned collagen, suggest that oriented cartilage tissue structures with superior mechanical properties can be obtained by aligning MSCs along the flow direction and exposing MSCs to chondrogenic gradients.

Effects of insulin-like growth factor-1 and dexamethasone on cytokine-challenged cartilage: relevance to post-traumatic osteoarthritis

Interleukin-1 is one of the inflammatory cytokines elevated after traumatic joint injury that plays a critical role in mediating cartilage tissue degradation, suppressing matrix biosynthesis, and inducing chondrocyte apoptosis, events associated with progression to post-traumatic osteoarthritis (PTOA). We studied the combined use of insulin-like growth factor-1 (IGF-1) and dexamethasone (Dex) to block these multiple degradative effects of cytokine challenge to articular cartilage. Young bovine and adult human articular cartilage explants were treated with IL-1α in the presence or absence of IGF-1, Dex, or their combination. Loss of sulfated glycosaminoglycans (sGAG) and collagen were evaluated by the DMMB and hydroxyproline assays, respectively. Matrix biosynthesis was measured via radiolabel incorporation, chondrocyte gene expression by qRT-PCR, and cell viability by fluorescence staining. In young bovine cartilage, the combination of IGF-1 and Dex significantly inhibited the loss of sGAG and collagen, rescued the suppression of matrix biosynthesis, and inhibited the loss of chondrocyte viability caused by IL-1α treatment. In adult human cartilage, only IGF-1 rescued matrix biosynthesis and only Dex inhibited sGAG loss and improved cell viability. Thus, the combination of IGF-1 + Dex together showed combined beneficial effects in human cartilage. Our findings suggest that the combination of IGF-1 and Dex has greater beneficial effects than either molecule alone in preventing cytokine-mediated cartilage degradation in adult human and young bovine cartilage. Our results support the use of such a combined approach as a potential treatment relevant to early cartilage degradative changes associated with joint injury.

Characterization of colony-forming cells in adult human articular cartilage

Recent studies have shown that adult human articular cartilage contains stem-like cells within the native structure. In this study, we aimed to determine the localization of putative stem cell markers such as CD90, STRO-1, OCT-3/4, CD105 and CD166 in adult human articular cartilage tissue sections and demonstrate the expression of these markers within the expanded surface zone colony-forming (CF) cells and evaluate their differentiation potential. Biopsy samples were either fixed immediately for immunohistochemical analyses or processed for in vitro cell culture. Immunohistochemical and flow cytometry analyses were performed by using CD90, STRO-1, OCT-3/4, CD105 and CD166 antibodies. Isolated colony-forming (CF) cells were further stimulated, by using the appropriate growth factors in their pellet culture, to obtain cartilage, bone and adipose lineages. We observed that the expression of the stem cell markers were in various zones of the human adult cartilage. Flow cytometry results showed that in CF cells the expression of CD90 and CD166 was high, while OCT-3/4 was low. We also determined that CF cells could be stimulated towards cartilage, bone and adipose lineages. The results of this research support the idea that the resident stem-like cells in adult human articular cartilage express these putative stem cell markers, but further experimental investigations are needed to determine the precise localization of these cells.

Heritability assessment of cartilage metabolism. A twin study on procollagen II N-terminal propeptide (PIIANP)

s / Osteoarthritis and Cartilage 21 (2013) S9–S62 S21 knee compared to those without such signs. While replication is pursued, these findings establish CLEC4A as a potential biomarker for early knee OA. 26 HERITABILITY ASSESSMENT OF CARTILAGE METABOLISM. A TWIN STUDY ON PROCOLLAGEN II N-TERMINAL PROPEPTIDE (PIIANP) H.L. Munk y, A.J. Svendsen z, K.O. Kyvik z, G.L. Sorensen z, P. Junker y. yOdense Univ. Hosp., Odense C, Denmark; zUniv. of Southern Denmark, Odense C, Denmark Purpose: The aim of the study was to estimate heritability on circulating collagen IIA N-propeptide (PIIANP) by studying monoand dizygotic healthy twin pairs. PIIANP: Collagen is produced and secreted as a precursormolecule with C-and N-terminal extensions termed procollagen peptides. In the extracellular space these are cleaved off by specific Cand N-peptidases after which collagen can participate in fibril formation. Since procollagen propeptides are released from the parent molecule in a stoichiometric manner, the concentration of these peptides provides an opportunity to assess the current biosynthetic activity. Methods: A total of 602 healthy monozygotic (MZ) and dizygotic (DZ) twin individuals aged 18-55 years were recruited from the Danish Twin Registry. PIIANP was measured by competitive ELISA. The similarity of circulating PIIANP among MZ and DZ twins was assessed by means of intraclass correlations for the traits. Classic twin study methodology is based on the fact that MZ twins have identical segregating genotypes, whereas DZ twins share, on average, one-half of their DNA sequence variation just like ordinary siblings. A greater phenotypic similarity in MZ than in DZ twins is anticipated if there is a significant genetic influence on the trait studied. I agreement with standard practice, we assume no epistasis (genetic inter-locus interaction) and no gene-environment interaction or correlation. The extent to which variation in a trait is attributable to genetics (heritability) can be estimated quantitatively through variance components analysis. The phenotypic (P) variance in a trait can be separated into four variance components: variance due to additive genetic effects (A), genetic dominance (D), shared (family) environment (C) and non-shared(individual) environment (E), e.i., P1⁄4A + D + C + E. It is not possible to test all 4 components of this model at the same time; we therefore tested different models in a stepwise deletion process. Results: The correlation of the logarithmic values of serum PIIANP for the MZ and the DZ twins is presented in figure 1. Figure 1. Correlation diagrams for log(PIIANP) in DZ and in MZ twins. Each dot represents the logarithm of the serum PIIANP level in a pair of twins, with one twin assigned to the abscissa and the other to the ordinate. The intraclass correlation for PIIANP in MZ twins and DZ twins was 0.70(S.E. 0,042) vs. 0.45(S.E. 0,065). This indicates that genetic factors are of aetiological importance for the serum level of PIIANP. In the variance component analysis the most suitable model was an ACE model, where fortyfive percent of the phenotypic variance of PIIANP in serum is determined by genes, while the shared environment accounts for 24% and 31% can be explained by non-shared environment factors. Our study also shows, that among all the tested variables, sex was the only variable whichwas significantly associated with the PIIANP level in serum. Conclusions: Our findings suggest that genetic factors have a considerable impact on the serum level of PIIANP. However, shared and common environmental factors are important modifiers as well. 27 PROTEOMIC ANALYSIS OF CONNEXIN 43 REVEALS NOVEL INTERACTORS, NUCLEAR TRANSLOCATION AND ASSOCIATION WITH PROTEINS DYSFUNCTIONAL RELATED WITH OSTEOARTHROSIS R. Gago-Fuentes, P. Carpintero-Fernandez, P. Fernandez-Puente, J. Mateos, M.D. Mayan, F.J. Blanco. Osteoarticular and Aging Res. Group. Rheumatology Div., BioMed. Res. Ctr. (INIBIC-CHUAC), A Coruna, Spain Purpose: Human adult articular cartilage is composed of a dense extracellular matrix and specialized cells called chondrocytes. The chondrocytes are found to their own lacuna and remain in resting stage refraining formproliferation but displaying amoderatemetabolic activity tomaintain their surroundingmatrix during thewhole adult life.Wehave previously found thathumanadult chondrocytes express thegap junction (GJ) protein connexin 43 and chondrocytes in tissue have long cytoplasmic arms that physically connect two distant cells. GJ channels achieve direct cellular communication by allowing the intercellular exchange of ions, several molecules and second messengers. In addition, several GJ channel-independent functions have been related with Cx43. The interaction of proteins with the C-terminal tail of Cx43 directly modulates different cellular activities such as cell growth and proliferation. Methods: In situ cartilage was immediately frozen after surgery in a Cryomold Standard using Tissue-Tek O.C.TTM Compound and stored at -80 C to keep the pattern of expression and protein levels. Chondrocytes were isolated by sequential digestion with trypsin-EDTA and Collagenase. Isolated cells from healthy and cartilage from osteoarthritis (OA) patients were maintained for stable short-term cell culture in DMEM supplemented with primocin and 15% FCS. For immunohistochemistry (IHC) experiments cells were seeded on chamber slides and fixed with acetone. Co-immunoprecipitation (IP) experiments were performed to identify the proteins that interact with the C-Terminal tail of Cx43. In-gel digestion of immunoprecipitated proteins separated by SDS-PAGE were analysed using the nano-liquid chromatography (Nano-LC, Eksigent) coupled to mass spectrometry (MALDI-TOF/TOF, Applied Biosystem). The identification of proteins was performed using ProteinPilot 3.0 Software. Samples were evaluated by SDS-PAGE followed by Western blotting with specific antibodies. Results: A total of 200 were identified, of which 131 proteins were represented by at least two unique peptides. 103 proteins were specific to the Cx43 IP, not identified in the control IP performed without antibody. Identified interactors show significant enrichment for Gene Ontology (GO) processes directly linked with cytoskeleton dynamics, metabolic pathways, nuclear activity and translation such as s-tubulin, vimentin, GAPDH, histone H3 and H4, and several ribosomal-related proteins. IHC experiments showed that chondrocytes from OA patients in cartilage contain higher levels of Cx43 in the nucleus, the cytoplasm and membrane. However Cx43 was only found in the membrane of healthy chondrocytes in tissue. Conclusions: Lysates of primary articular chondrocytes were subjected to pull-down assays and the identification of proteins by MALDI-TOF/ TOF identify >100 Cx43 associated proteins. Mass-spectrometry results revealed novel functional Cx43 interactors involved in human disease and OA development emphasizing the importance of Cx43-interactions for normal development and physiology. Besides IHC experiments suggest that Cx43 interacts with nuclear and translational components especially in OA cartilage. 28 A NOVEL OA EFFICACY MARKER: CARTILAGE ACTIVITY D.R. Jorgensen y,z, M. Lillholm y, E.B. Dam y. yBiomediq, Copenhagen, Denmark; zUniv. of Copenhagen, Copenhagen, Denmark Purpose: The pathogenesis of OA is complex with multiple events occurring simultaneously in the joint. The opposing cartilage effects of Abstracts / Osteoarthritis and Cartilage 21 (2013) S9–S62 S22s / Osteoarthritis and Cartilage 21 (2013) S9–S62 S22 swelling at focal lesions and thinning at other locations confound traditional cartilage loss markers (volume/thickness). We propose a novel efficacy marker, Cartilage Activity, that quantifies deviation from homeostasis, and validate the Activity marker on three subcohorts with respect to sensitivity to change. Methods: Cartilage Activity was defined as the mean absolute longitudinal change in thickness quantified over one or more compartments. The Activity marker was quantified using thickness maps from a fully automatic segmentation and analysis framework (KneeIQ). The marker was validated on three populations: A) 139 subjects with 0.18T MRI acquired at baseline and after 21 months at the Center for Basic and Clinical Research in Copenhagen (CCBR), B) 500 subjects selected by patient ID order from the Osteoarthritis Initiative (OAI) study with the sagittal DESS sequence acquired for the right knee from 3T MRI at baseline and after 1 year (OAI1), and C) the 455 subjects from OAI with publicly available sub-regional thickness measurements for the right knee provided by Chondrometrics available at baseline and 1 year from same OAI project. For all cohorts, knees with missing radiographic Kellgren & Lawrence score (KL) or KL 4 were removed. On these subcohorts, the sensitivity to change was evaluated by comparing longitudinal marker changes between knees with and without radiographic OA (ROA) and by evaluating standardized response mean (SRM). We considered KL>1 to be ROA, and KL 0-1 to be pre-ROA. The Activity marker was evaluated against the mean thickness marker across the total area of bone (denoted Thickness). For the CCBR cohort, only the medial compartments for tibia (MT) and femur (MF) were computed and combined (MFT). For the OAI sub-cohorts, also lateral compartments (LT and LF) were computed and combined (LFT) and in the whole knee (FTJ). Results: The three sub-cohorts are summarized in the table below. The percentages of females in each sub-cohort were 48%, 64%, and 56%. Sub-cohort characteristics (given as mean std)

Agrin expression is downregulated in OA and is required for chondrocyte differentiation

Purpose: Background: Agrin is a large basement membrane heparan sulphate proteoglycan best known for its function at the neuromuscular junction. It is responsible for synapse formation via binding to the MuSK and LRP4 receptor complex, leading to Acetylcholine receptor aggregation. Mice deficient of Agrin die perinatally as a result of respiratory failure. Agrin is also expressed in other cell types including chondrocytes of the developing growth plate, and agrin-deficient embryos in which agrin expression had been rescued at the neuromuscular junction develop skeletal abnormalities 1. Therefore it is possible that Agrin may play a role in cartilage homeostasis. Aims: The aim of this project is to determine if agrin plays a functional role in the homeostasis of the articular cartilage and in osteoarthritis. Methods: Human adult articular cartilage explants were obtained from preserved or damaged cartilage from individuals undergoing joint replacement for osteoarthritis. Experimental osteoarthritis was induced in 10 week old mice by destabilization of the medial meniscus2. Sham-operated controlateral knees were used as controls. Knees were collected 8weeks after surgery, decalcified and embedded in paraffin. Bovine primary articular chondrocytes (BPAC) were isolated by pronase/collagenase digestion from the articular cartilage of the metatarso-phalangeal joints of 18 month old calves from a local slaughterhouse. The BPAC and the human chondrogenic cell line C28/I2 were cultured in micromass to promote differentiation and extracellular matrix production. For gain- and loss-of-function experiments, chondrocytes were transfected with a mammalian expression vector encoding for full-length Agrin (FL-Agrin) or with Agrin siRNA's respectively. Accumulation of cartilage-specific extracellular matrix production, rich in highly sulphated glycosaminoglycans (GAGs), was quantified by alcian blue staining at pH 0.2 and alizarin red staining. Chondrogenic potential was measured using pellet analysis as previously described3, BPAC pellets were cultured for 14 days, weighed, sectioned and stained with alizarin red and safranin o. Gene expression analysis of Agrin, Col2A1, Aggrecan, Sox9 was performed by real time PCR. Immunofluorescence was used to determine the expression levels of Agrin at protein level in chondrocytes cultured in monoloayer and in paraffin-embedded tissue sections. Results: Agrin was expressed in healthy adult human and bovine articular cartilage. Agrin was signifcantly downregulated in human osteoarthritic cartilage. This downregulation was also replicated in experimental murine osteoarthritis, indicating that it is a consequence rather than a cause of osteoarthritis. Agrin expression was partially retained in the articular cartilage of sham operated knees, indicating that inflammation alone (present both in DMM and in sham) is not sufficient to induce Agrin downregulation, but cartilage damage is necessary. Endogenous expression of Agrin was confirmed in C28/I2 and in BPAC. Overexpression of FL-Agrin resulted in enhanced differentiation as demonstrated by upregulated the cartilage key transcription factor Sox9. . Knock-down of Agrin by siRNA resulted in reduced GAG production in C28/I2 and chondrocyte de-differentiation as documented by decreased expression Sox9, Col2A1 and Aggrecan mRNA. Conclusions: Agrin expressed in healthy adult articular cartilage and is downregulated in osteoarthritis; - Agrin is anabolic in cultured articular chondrocytes; - Agrin is required for chondrocyte differentiation in vitro.

Fibroblast growth factor control of cartilage homeostasis

Osteoarthritis (OA) and degenerative disc disease (DDD) are similar diseases involving the breakdown of cartilage tissue, and a better understanding of the underlying biochemical processes involved in cartilage degeneration may allow for the development of novel biologic therapies aimed at slowing the disease process. Three members of the fibroblast growth factor (FGF) family, FGF-2, FGF-18, and FGF-8, have been implicated as contributing factors in cartilage homeostasis. The role of FGF-2 is controversial in both articular and intervertebral disc (IVD) cartilage as it has been associated with species- and age-dependent anabolic or catabolic events. Recent evidence suggests that FGF-2 selectively activates FGF receptor 1 (FGFR1) to exert catabolic effects in human articular chondrocytes and IVD tissue via upregulation of matrix-degrading enzyme production, inhibition of extracellular matrix (ECM) accumulation and proteoglycan synthesis, and clustering of cells characteristic of arthritic states. FGF-18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating the FGFR3 pathway, inducing ECM formation and chondrogenic cell differentiation, and inhibiting cell proliferation. These changes result in dispersed chondrocytes or disc cells surrounded by abundant matrix. The role of FGF-8 has recently been identified as a catabolic mediator in rat and rabbit articular cartilage, but its precise biological impact on human adult articular cartilage or IVD tissue remains unknown. The available evidence reveals the promise of FGF-2/FGFR1 antagonists, FGF-18/FGFR3 agonists, and FGF-8 antagonists (i.e., anti-FGF-8 antibody) as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future.

Fibroblast growth factor‐2 promotes catabolism via FGFR1‐Ras‐Raf‐MEK1/2‐ERK1/2 axis that coordinates with the PKCδ pathway in human articular chondrocytes

Fibroblast growth factor 2 (FGF-2) has been found to play an anti-anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF-2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF-2 signaling in human articular chondrocytes. Pathway-specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF-2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf-MEK1/2-ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF-2-mediated upregulation of MMP-13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF-2-mediated proteoglycan loss was effectively reversed by individual pathway-specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3-dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF-2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF-2. Such an integration of dual inputs constitutes a novel mechanism of FGF-2 signaling cascade in human articular chondrocytes.

Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis.

Chondrogenesis occurs as a result of mesenchymal cell condensation and chondroprogenitor cell differentiation. Following chondrogenesis, the chondrocytes remain as resting cells to form the articular cartilage or undergo proliferation, terminal differentiation to chondrocyte hypertrophy, and apoptosis in a process termed endochondral ossification, whereby the hypertrophic cartilage is replaced by bone. Human adult articular cartilage is a complex tissue of matrix proteins that varies from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue-engineering strategies is the inability of the resident chondrocytes to lay down a new matrix with the same properties as it had when it was formed during development. Thus, understanding and comparing the mechanisms of cartilage remodeling during development, osteoarthritis (OA), and aging may lead to more effective strategies for preventing cartilage damage and promoting repair. The pivotal proteinase that marks OA progression is matrix metalloproteinase 13 (MMP-13), the major type II collagen-degrading collagenase, which is regulated by both stress and inflammatory signals. We and other investigators have found that there are common mediators of these processes in human OA cartilage. We also observe temporal and spatial expression of these mediators in early through late stages of OA in mouse models and are analyzing the consequences of knockout or transgenic overexpression of critical genes. Since the chondrocytes in adult human cartilage are normally quiescent and maintain the matrix in a low turnover state, understanding how they undergo phenotypic modulation and promote matrix destruction and abnormal repair in OA may to lead to identification of critical targets for therapy to block cartilage damage and promote effective cartilage repair.

Chondrogenesis, joint formation, and cartilage metabolism

Chondrogenesis occurs as a result of mesenchymal cell condensation and chondroprogenitor cell differentiation. The chondrocytes then form the cartilage at the end of the opposing bones with the intervening interzones formed during cavitation or they undergo proliferation, terminal differentiation to chondrocyte hypertrophy, and apoptosis in a process termed endochondral ossification, whereby the hypertrophic cartilage is replaced by bone. A similar sequence of events occurs in the postnatal growth plate and leads to rapid growth of the skeleton. Human adult articular cartilage is a complex tissue of matrix proteins that varies from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue engineering strategies is the inability of the resident chondrocytes to lay down new matrix with the same properties as it had when it was formed during development. This is particularly true of the collagen network, which is susceptible to cleavage once the proteoglycans are depleted. Thus, understanding and comparing the mechanisms of cartilage remodeling during development, osteoarthritis (OA), and aging may lead to more effective strategies for preventing cartilage damage and promoting repair. To identify and characterize mediators of cartilage remodeling common to these processes, we are using culture models of primary human and mouse chondrocytes and cell lines and mouse models to manipulate and compare gene expression with complementary approaches. MMP-13, the major type II collagen-degrading collagenase, is regulated by both stress and inflammatory signals that not only contribute to irreversible joint damage (progression) in OA, but importantly, also to the initiation/onset phase, wherein chondrocytes in articular cartilage leave their natural growth - and differentiation-arrested state. We and other investigators have found that there are common mediators of these processes in human OA cartilage and from early through late stages of OA in mouse models, including the surgical models (good matrix with abnormal loading) and the genetic models during aging (bad matrix with normal loading). We are validating our in vitro analyses of the signaling and transcriptional mechanisms that determine the expression and activities of these mediators by in vivo analyses of the consequences of knockout or transgenic overexpression of these genes in mouse models. In current studies, we are examining the epigenetic mechanisms and using proteomics and genomics approaches to map the signaling networks and microRNA targets that impact on gene expression programs during the onset and progression of OA in both human and murine cartilage. Since the chondrocytes in adult human cartilage are normally quiescent and maintain the matrix in a low turnover stated, understanding how they undergo phenotypic modulation and promote matrix destruction and abnormal repair in OA may to lead to identification of critical targets for therapy to block cartilage damage and promote effective cartilage repair.