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Regensburg, Germany

Leyh M.,University of Regensburg | Leyh M.,Center for Medical Biotechnology | Seitz A.,University of Ulm | Durselen L.,University of Ulm | And 6 more authors.
Stem Cell Research and Therapy

Introduction. In the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a "tri-culture" model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants. Methods. Gene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures). Results: In general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young's and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1β), IL-6, and IL-8. Stimulation of monocultures with IL-1β and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only. Conclusions: Our results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from co- and tri-cultured cells and leads to impaired mechanical and biochemical properties of the matrix because of an altered fibrillar network. We suggest that soluble factors, including IL-1β and IL-6, released from OA cartilage partly mediate these effects. Thus, neighbored OA cartilage provides inhibitory signals with respect to BMSCs' chondrogenic differentiation and matrix composition, which need to be accounted for in future cell-based OA treatment strategies. © 2014 Leyh et al.; licensee BioMed Central Ltd. Source

Muschter D.,University of Regensburg | Muschter D.,Center for Medical Biotechnology | Schafer N.,University of Regensburg | Stangl H.,University of Regensburg | And 3 more authors.

Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors. Copyright: © 2015 Muschter et al. Source

Ratzinger S.,University of Regensburg | Ratzinger S.,Center for Medical Biotechnology | Eble J.A.,Goethe University Frankfurt | Pasoldt A.,University of Regensburg | And 5 more authors.
Matrix Biology

In Crohn's disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF) provided by intact tissue is disturbed due to inflammation and thus, cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original ECM leads ISEMF to exit this cycle. In contrast, during fibrosis, ISEMF persist. It is known that ISEMF produce and deposit collagen types I, III, IV and V; however synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed the distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell-matrix interactions in different phases of the inflammatory process and their effect on cell spreading, proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall where it co-localizes with fibrillin-1 and integrin α1. ISEMF reveal increasing gene and protein expression of collagen XVI concurrent to increasing inflammation. ISEMF reveal more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This involves recruitment of α1β1 integrin, which shows increased cell surface expression on ISEMF in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and, even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell-matrix interaction preferentially through recruitment of α1ß1 integrin into the tips of the focal adhesion contacts. © 2009 Elsevier B.V. Source

Grassel S.,University of Regensburg | Grassel S.,Center for Medical Biotechnology | Bauer R.J.,Center for Medical Biotechnology | Bauer R.J.,University of Regensburg
Matrix Biology

Collagen XVI, by structural analogy a member of the FACIT- (fibril-associated collagens with interrupted triple helices) family of collagens, is described as a minor collagen component of connective tissues. Collagen XVI is expressed in various cells and tissues without known occurrence of splice variants or isoforms. For skin and cartilage tissues its suprastructure is known. Presumably, there it acts as an adaptor protein connecting and organizing large fibrillar networks and thus modulates integrity and stability of the extracellular matrix (ECM).Collagen XVI is produced by myofibroblasts in the normal intestine and its synthesis is increased in the inflamed bowel wall where myofibroblasts develop increased numbers of focal adhesion contacts on collagen XVI. Consequently, recruitment of α1 integrin into the focal adhesions at the tip of the cells is induced followed by increased cell spreading on collagen XVI. This presumably adds to the maintenance of myofibroblasts in the inflamed intestinal regions and thus promotes fibrotic responses of the tissue. Notably, α1/α2 integrins interact with collagen XVI through an α1/α2β1 integrin binding site located in the COL 1-3 domains.Collagen XVI may act as a substrate for adhesion and invasion of connective tissue tumor cells. In glioblastoma it induces tumor invasiveness by modification of the β1-integrin activation pattern. Thus, altering the cell-matrix interaction through collagen XVI might be a molecular mechanism to further augment the invasive phenotype of glioma cells. In this line, in oral squamous cell carcinoma collagen XVI expression is induced which results in an upregulation of Kindlin-1 followed by an increased interaction with beta1-integrin. Consequently, collagen XVI induces a proliferative tumor phenotype by promoting an early S-phase entry.In summary, collagen XVI plays a decisive role in the interaction of connective tissue cells with their ECM, which is impaired in pathological situations. Alteration of tissue location and expression level of collagen XVI appears to promote tumorigenesis and to perpetuate inflammatory reactions. © 2012 International Society of Matrix Biology. Source

Ratzinger S.,Center for Medical Biotechnology | Ratzinger S.,University of Regensburg | Grassel S.,University of Regensburg | Grassel S.,Center for Medical Biotechnology | And 4 more authors.
Matrix Biology

Type XVI collagen belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). Recently, high affinity to integrin alpha1beta1 has been shown allowing cells expressing those integrins to attach and spread on recombinant type XVI collagen. Here, we show that type XVI collagen is overexpressed in dysplastic areas of mucosal epithelium from oral squamous cell carcinoma (OSCC) patients. Induction of its expression in OSCC cell lines (COLXVI cells) leads to an increased expression of Kindlin-1. Moreover, we demonstrate a significantly increased Kindlin-1/beta1-integrin interaction. Additionally, we detected a higher number of activated beta1-integrins in COLXVI cells and found a neo-expression of alpha1 integrin subunit on these cells. FACS analysis revealed a significantly higher amount of COLXVI cells in S-phase and G2/M-phase 6. h after synchronisation leading to a markedly higher proliferation activity. Blocking beta1-integrins with a specific antibody resulted in reduced proliferation of COLXVI cells. In summary, we demonstrate that overexpression of type XVI collagen in aberrant oral keratinocytes leads to Kindlin-1 induction, increased Kindlin-1/beta1-integrin interaction, integrin activation and subsequently to a proliferative cellular phenotype. © 2011 International Society of Matrix Biology. Source

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