Osteoarthritis – The Challenge
Symposium of the Deutsches Rheuma-Forschungszentrum Berlin and the Pitzer Stiftung, Bad Nauheim
February 3rd, 2014
Berlin-Brandenburgische Akademie der Wissenschaften, Jägerstrasse 22/23, 10117 Berlin
The challenge – understanding the molecular pathogenesis of osteoarthritis and developing innovative therapeutic concepts
The German Rheumatism-Research Center, Berlin (DRFZ), an Institute of the Leibniz Association, organized the Symposium OSTEOARTHRITIS – THE CHALLENGE, with gratefully acknowledged support of the Willy-Robert Pitzer Foundation. Venue of this event was the Berlin-Brandenburgische Akademie der Wissenschaften, located in the middle of Berlin, at the Gendarmenmarkt.
With this symposium, an overview on cutting-edge and in-depth research, as well as key challenges in the field of osteoarthritis was provided by more than 20 international experts. Experienced leaders in the field and promising young investigators met. The organizers set the stage for presentations of new and bright concepts of the current understanding and treatment strategies of osteoarthritis. 102 participants from Germany, Europe and the US used the occasion for the exchange of ideas.
The chairpersons of each symposia session have provided a brief synopsis of their session, as follows.
- What Is Osteoarthritis: Mediators Involved in the Pathogenesis of Osteoarthritis
summarized by: Steven Goldring, Gerd-Rüdiger Burmester
Mary B. Goldring, Weill Cornell Medical College, New York, USA opened the symposium with her introduction to osteoarthritis. Osteoarthritis (OA) is a whole joint disease, involving cartilage articular degeneration, synovitis, disruption of meniscus, tendons, and ligaments, osteophyte formation, calcified cartilage expansion, and subchondral bone sclerosis. Thinning and loss of cartilage are critical determinants in OA progression, and once it has broken down, the complexity of its structure and cellular composition presents a therapeutic challenge for regenerative medicine. The disruption of cartilage homeostasis, which is due to multiple potential causes related to aging, genetic predisposition, trauma, or metabolic disorder, induces profound phenotypic modifications of chondrocytes. Early changes involve disruption of the pericellular matrix through signaling events mediated by chondrocyte receptors such as discoidin domain receptor-2 (DDR-2) and syndecan-4 and stress- and inflammation-induced kinase cascades that are also involved in mechanotransduction, including IKKs and MAPKs. These pathways converge on transcriptional regulation of the cartilage-degrading enzymes, aggrecanases and collagenases, especially MMP-13, by NF-kB, C/EBPb, ETS, Runx2, and hypoxia-inducible factor (HIF) 2a. Epigenetic regulation by CpG methylation, HDACs, and microRNAs and elucidation of mechanisms over the time course of the disease initiation and progression in preclinical mouse models of OA will inform us about new approaches for designing therapies.
- Chondrocyte differentiation and Cartilage Development
summarized by: Andrea Vortkamp and Frank Luyten
In the second session basic mechanisms of cartilage differentiation were discussed. Manuela Wuelling from the University Duisburg-Essen started out with an excellent presentation giving new insights into the role of the transcription factor Trps1, which activates Histone deacetylases. Surprisingly, loss of Trps1 leads to defects in chromatin condensation and consequently a delay in G2/Mphase of mitosis linking the regulation of transcription to cell cycle progression. She further discussed new insight into the interaction of Trps1 and HDACs in regulating chondrocyte hypertrophy by epigenetically modifying the Runx2 promoter, a main regulator of hypertrophic differentiation. Extending these studies will be important to decipher the epigenetic status of the articular cartilage, and the potential impact on the development of osteoarthritis.
Francesco Dell’Accio from Barts and the London School of Medicine, UK, then followed with new unpublished insights into the unexpected function of the chemokine CXCL6 in maintaining articular cartilage homeostasis. CXCL6 is stored in the pericellular space, at least in part in a heparan sulfate dependent manner, and is released in OA cartilage. Mice deficient in CXCR2, the main ELR+ CXC chemokine receptor in mice, developed more severe osteoarthritis than wild-type controls following destabilization of the medial meniscus with an increased rate of chondrocyte apoptosis. In vitro, disruption of CXCR1/2 signaling in human and mouse chondrocytes led to a decrease in extracellular matrix production, reduced expression of chondrocyte differentiation markers, and increased chondrocyte apoptosis. CXCR2-dependent chondrocyte homeostasis was mediated by AKT signaling since forced expression of constitutively active AKT rescued both, the expression of phenotypic markers and the apoptosis induced by CXCR2 blockade. Therefore, these studies revealed an unexpected homeostatic role for CXCR1/2 signaling in cartilage dependent on AKT/SOX9 signaling.
Jürgen Rohwedel from the University of Lübeck discussed experiments identifying the level of Notch signaling as a critical balancer for the differentiation of chondrocytes from ES cells. He continued with stimulating insights into the differentiation of iPS cells. Whereas FGF combined with BMP signals will initiate the differentiation of growth plate like chondrocytes, FGF treatment together with GDF5 will drive them into a more stable, articular chondrocyte phenotype, making them potentially more suitable for replacement therapies.
The importance of Integrin signaling in organizing chondrocyte morphology and growth plate organization was eluted on by Attila Aszodi from the University of Munich. His data demonstrate that Integrin-Rac1/Cdc42 dependent organization of the cytoskeleton is required for the columnar shape of chondrocytes in the growth plate and consequently for its organized elongation. In addition, using Atomic Force Microscopy he demonstrated a reduced stiffness of the articular matrix in Integrin mutants. Surprisingly, although ColX is upregulated, the articular cartilage is not eroded, identifying integrin signaling as an important aspect to maintain cartilage homeostasis as part of the degradation program.
In the last presentation of this session, Aimee Zuniga from the University of Basel discussed the earliest stages of chondrocyte differentiation in the developing embryo, introducing VLK as an activator of chondrocyte differentiation functionally interacting with the Ihh dependent transcription factor Gli3. Searching for the initiation of the chondrocytes differentiation program she presented data that inactivation of the Tgf-ß depended transcription factor Smad4 in prechondrogenic mesenchyme inhibits the differentiation of chondrocytes. In contrast to earlier studies in which inactivation of Smad4 in differentiating chondrocytes only resulted in a mild chondrocyte differentiation defect, her data identified TGF-ß dependent Smad4 signaling as a critical inductive factor of the chondrogenic fate.
- Endochondral Development
summarized by: Ulf Müller-Ladner and Thorsten Schinke
The session started with a presentation by Bent Brachvogel from the Institute of Experimental Neonatology at Cologne University. Brachvogel focused on the potential relevance of the growth plate extracellular matrix (ECM) as a modulator of the immune response. He described findings obtained in two different mouse models lacking specific matrix proteins of the growth plate, either Col-X or Col-IX. The major hypothesis was that the ECM template of the growth plate is remodeled during development to create the bone marrow cavity and to provide the appropriate microenvironment for hematopoiesis.
Consistent with this hypothesis, Col-X-deficient mice, despite displaying essentially no abnormalities of skeletal growth, showed a slight imbalance in the adult T cell response. Moreover, the more severe changes in the organization of the cartilage ECM in Col-IX-deficient mice had a profound effect on myeloid progenitor cell differentiation and function. A proteomic analysis further identified several key components of the ECM that may promote the differentiation of bone marrow progenitor cells to dendritic cells and macrophages.
Although future studies are now required to explain how these ECM components can affect the differentiation of hematopoietic stem cells, it is evident, that these novel interactions expand our knowledge regarding the relevance of the cartilage ECM for hematopoietic cell function. Since similar interactions are likely involved in the pathogenesis of arthritis, they could further lead to novel therapeutic strategies to prevent articular cartilage loss in the respective patients.
Jean-Pierre David of the Institute for Osteology and Biomechanics of the University Medical Center Hamburg-Eppendorf addressed the role of a unique ribosomal kinase (Rsk2) in bone metabolism and inflammatory arthritis, especially rheumatoid arthritis. Rsk2 is an ERK- and Pi3 kinase- dependent activated kinase that his group originally characterized as a positive regulator of bone formation by osteoblasts. In addition, it could be shown that Rsk2 is stabilizing c-Fos via phosphorylation thereby promoting the growth of c-Fos-dependent osteosarcomas. Aside the function of Rsk2 in regulating osteoblast activity and transformation they could also show that Rsk2 was highly activated in the inflamed pannus of rheumatoid arthritis (RA) patients, which stimulated to further analyze the function of Rsk2 in mediating joint destruction and systemic bone loss in RA. They inactivated Rsk2 in mice overexpressing the human TNF-a, which has already proven to be a reliable animal model for inflammatory arthritis that displays all clinical and histological signs of human RA. The clinical analysis revealed a substantial acceleration and exacerbation of arthritis in the absence of Rsk2. This exacerbation was confirmed by histological analyses that demonstrated a strong increased of inflammation of the affected joints, and an earlier destruction of the cartilage and of the subchondral bone. Rsk2 deficiency was also associated with an accelerated systemic bone loss due to nearly complete absence of bone formation and an increased bone resorption. Bone marrow transfer of Rsk2 deficient cells into hTNFtg recipient mice excluded a participation of the hematopoietic cells to the accelerated development of the disease. Mechanistically, the increased general osteoporosis was caused by a cell autonomous sensitization of osteoblasts and osteocytes to TNF-induced apoptosis thereby increasing the recruitment of bone resorbing osteoclasts. Similarly, the increased local destruction of joint was associated with a more aggressive phenotype of the synovial fibroblasts, including the decreased expression of genes known to protect articular cartilage as well as increased expression of pro-inflammatory and pro-osteoclastogenic cytokines as well as metalloproteinases. In addition, increased proliferation of Rsk2 deficient fibroblasts augmented the pool of activated fibroblasts contributing to the accelerated joint destruction. Taken together, it could be shown that Rsk2 is a kinase, which, when activated in disease-driving mesenchymal cells such as osteoblasts and synovial fibroblasts, is able to protect to some extent against TNF-mediated joint and bone destruction in inflammatory arthritis.
- Stress to the joint
summarized by: Elisabeth Märker-Hermann and Francis Berenbaum
Mechanical stress to the joint may also be directly involved in the initiation and progression of osteoarthritis along with the remodeling of subchondral bone and articular cartilage. Bettina Willie, Julius Wolff Instiut, Charité- Universitätsmedizin Berlin, introduced her model of age-related bone loss in mice. Based on the well-known loss of bone formation response to exercise with aging, she used anin vivo loading model and investigated the loss in trabecular and cortical bone in young and old mice. Her data show that trabecular bone loss is greater than cortical bone loss, and that only the formation – but not the resorption – response of remodeling is reduced by loading in older mice.
Cartilage is sensible to mechanical forces such as compression, tension and shear. Frederic Mallein-Gerin, Institute for Biology and Chemistry of Proteins, Lyon, studied the chondrocyte mechanotransduction pathways at the cell, protein and gene expression levels. A lack of b1-integrins at the surface of chondrocytes results in an altered cellmorphology. Moreover, diffusion properties of the cartilage are altered. At the protein level, TGF-b signaling is activated by dynamic but not by static compression. Finally, he has shown that on the gene level, mechanical stress leads to downregulation of many genes (for example Fzd10) rather than to upregulation (Fos, Jun and others) of expression.
Pain is a characteristic clinical hallmark of osteoarthritis and is already present in early and activated osteoarthritis. In chronic osteoarthritic pain, peripheral sensitization, spinal sensitization with glia activation, supraspinal reactions with atrophy of the grey matter and reduction of descending inhibition can be seen. Hans-Georg Schaible, Universitätsklinikum Jena, discussed data on nerve fibres and neuropeptides in OA synovitis. The synovitis of patients with activated OA shows a reduced number of nerve fibres with less release of the neuropeptide CGRP. This might predispose to neuropathic pain. Candidate mediators of chronic osteoarthritis pain involve Nerve Growth Factor, Interleukin-6 and prostaglandin receptors.
- Degeneration and Regeneration – Vicious and Beneficial Circles
summarized by Bettina Willie and Wolfgang Rüther
Wim van den Berg, University Medical Center St. Radboud, Nijmegen, reviewed how animal models of OA have provided insight into pathogenic pathways. He summarized key players involved in OA caritlage damage and focused on the role of Wnt signaling pathway proteins in OA.
Thomas Pap, Universitätsklinikum Münster, reviewed several pathways involved in the pathogenesis of OA including the interplay between these pathways. He further focused on the role of syndecan-4 as a key player in cartilage degradation during OA. This is facilitated by regulating the expression of matrix degrading enzymes, by mediating IL-1 signaling and additionally, by directly binding ADAMTS-5 and thereby participating directly in the cartilage degradation cascade.
Raluca Niesner, Deutsches Rheuma-Forschungszentrum Berlin, discussed the use of longitudinal intravital imaging to identify cellular processes involving the immune system during bone healing. She described the novel imaging technology called longitudinal intra-vital micro-endoscopy of the bone marrow (LIMB). Hereby, the endoscope is implanted into the cortex of long bones using an external fixation system, which allows her group to analyze the dynamics of the immune reactions due to various bone injury models and characterize the role of immune cells and bone marrow stromal cells in bone healing.
Nicolai Miosge, Universitätsklinikum Göttingen, summarized current knowledge concerning stem cell related cells in cartilage tissue that are thought to be involved in the regeneration of both, cartilage tissue and the periodontal tissue. He discussed in detail the idea of mesenchymal stem cells as therapeutic tool to enhance the microenvironment of the stem cells already present in the diseased tissue in order to direct regeneration.
- Tissue Engineering
summarized by: Michael Sittinger and Henning Madry
Today, cell-based cartilage tissue engineering treatments are routinely applied for joint cartilage repair. So far, all established procedures are based on autologous chondrocytes isolated from biopsies from non-load bearing cartilage. In the original approach of autologous chondrocyte implantation described by Mats Brittberg in 1994, a suspension of chondrocytes is injected into cartilage lesions covered by a periosteal flap. Later, matrix assisted engineered tissues were developed consisting of autologous chondrocytes and scaffolds for improved surgical handling and quality of preformed tissue. In both technologies, chondrocytes secrete new extracellular matrix to form the repair cartilage. However, highly porous scaffolds consisting of resorbable synthetic polymers or natural materials support and guide de-novo tissue formation by improving cell distribution and initial mechanical stability.
Meanwhile, thousands of patients with articular cartilage defects have been treated by tissue engineering therapies and autologous chondrocyte transplantation which is now considered as an effective and durable treatment for large full-thickness cartilage lesions of the knee joint. However, currently available clinical options in cartilage tissue engineering are limited to treatment of focal cartilage lesions and exclude application in progressed osteoarthritis. Therefore, further research and new tissue engineering technologies are required to develop suitable therapies for chronic degenerative cartilage diseases.
Many different porous biocompatible and resorbable biomaterials proved to be interesting for cartilage repair in preclinical research. Such materials either provide a scaffold for cells to be implanted or alternatively allow cells to migrate into the space filling or covering material in vivo. Marcel Karperien, a biologist and expert in biomaterials research in Twente, presented his invention of a novel injectable gel-matrix which may be applied for arthroscopic repair of a wide range of cartilage lesions. His in situ forming dextran-tyramine hydrogel was shown to self-attach to cartilage matrix and to attract cells for tissue regeneration in preclinical testing. His technology is presently developed towards an injectable plaster for joint cartilage repair.
The field of regenerative medicine has rather quickly moved from bench to bedside in the area of tissue engineering. Here, tissue-engineered cartilage can be created using articular chondrocytes that seeded onto three-dimensional matrices which are cultivated in bioreactors. Ivan Martin, a pioneer in tissue engineering for articular cartilage repair, presented progress of his work on the repair of focal, non-osteoarthritic articular cartilage defect by engineered grafts based on nasal chondrocytes. Investigations of fundamental aspects of cell differentiation and osteochondral development showed that developmental paradigms can be successfully applied to the challenge of tissue engineering of cartilage. Data on osteochondral repair using engineered cartilage in a goat model were also reported. These efforts led to a clinical phase I trial to proof the safety and feasibility of using tissue-engineered cartilage based on nasal chondrocytes for reconstructive knee cartilage surgery. Research is now in progress to test the applicability of such engineered grafts based on nasal chondrocytes for the treatment of osteoarthritis.
SOX9 is a critical transcription factor for chondrocyte differentiation that activates the gene for type-II collagen and other cartilage-specific genes. Especially because of its role in chondrogenesis, SOX9 may be of great value for osteoarthritis therapy. Others have already shown, that chondrogenic differentiation was successfully enhanced when overexpressed via replication-defective recombinant adeno-associated virus (rAAV) vectors in human MSCs, in vitro and in vivo. Moreover, in human MSCs, markers of hypertrophy were reduced. In the studies on adipogenic and osteogenic differentiation presented by Susanne Grässel, a stable SOX9 knockdown in undifferentiated rat MSC resulted in a marked decrease in proliferation rate and an increase in apoptotic activity. In addition, the major transcription factor for adipogenic differentiation, C/EBPβ, was repressed after silencing SOX9. These data confirm the important role of SOX9 as a link between differentiation, proliferation and apoptosis in undifferentiated adult rat mesenchymal stem cells. When in a similar approach SOX9 was silenced in adipose-derived stem cells (ASC), proliferation and expression of osteogenic markers was modulated.