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Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death.
CYR61 and WISP3 belong to the family of CCN-proteins. These proteins are characterised by 10% cysteine residues whose positions are strictly conserved. The proteins are extracellular signalling molecules that can be associated with the extracellular matrix. CCN-proteins function in a cell- and tissue specific overlapping yet distinct manner. CCN-proteins are expressed and function in several cells and tissues of the musculoskeletal system. In this study the impact of the angiogenic inducer cysteine-rich protein 61 (CYR61/CCN1) on endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) as well as the wnt1 inducible signalling pathway protein 3 (WISP3/CCN6) on MSCs were elucidated. EPCs are promising cells to induce neovascularisation in ischemic regions as tissue engineered constructs. A major drawback is the small amount of cells that can be obtained from patients; therefore a stimulating factor to induce in vitro propagation of EPCs is urgently needed. In this study, mononuclear cells obtained from peripheral blood were treated with 0.5 µg/ml CYR61, resulting in an up to 7-fold increased cell number within one week compared to untreated control cells. To characterise if EPCs treated with CYR61 display altered or maintained EPC phenotype, the expression of the established markers CD34, CD133 and KDR as well as the uptake of acLDL and concurrent staining for ulex lectin was analysed. Both CYR61 treated and untreated control cells displayed EPCs characteristics, indicating that CYR61 treatment induces EPC number without altering their phenotype. Further studies revealed that the stimulating effect of CYR61 on EPCs is due to enhanced adhesion, rather than improved proliferation. Usage of mutated CYR61-proteins showed that the adhesive effect is mediated, at least partly, by the integrin α6β1, while the integrin αυβ3 has no influence. Endogenous expression of CYR61 was not detectable in EPCs, which indicated that control cells are not influenced by endogenous secretion of CYR61 and also could explain the dose-dependent effect of CYR61 that is measured at a low concentration of 0.05 µg/ml. MSCs were treated with 0.5 µg/ml CYR61, a combination of growth factors including VEGF, both together and compared to untreated control cells. Matrigel angiogenesis assay revealed an induction of angiogenesis, detected by induced sprouting of the cells, after CYR61 treatment of the MSC. Induced sprouting and vessel like structure formation after CYR61 treatment was similar to the results obtained after treatment with growth factors including the established angiogenesis inducer VEGF. This result clearly demonstrates the angiogenic potential of CYR61 on MSCs. Further studies revealed a migrative and proliferative effect of CYR61 on MSCs. Both properties are crucial for the induction of angiogenesis thus further strengthening the view of CYR61 as an angiogenic inducer. MSCs and EPCs are promising cells for tissue engineering applications in bone remodelling and reconstruction. MSCs due to their potential to differentiate into other lineages; EPCs induce neovascularisation within the construct. Both cell types respond to CYR61 treatment. Furthermore EPCs home to sides were CYR61 expression is detectable and both are induced by similar stimulators. Therefore CYR61 is a promising factor for tissue engineered bone reconstruction applications. WISP3 is expressed in cartilage in vivo and in chondrocytes in vitro. Loss of function mutations in the WISP3 gene are associated to the inherited human disease progressive pseudorheumatoid dysplasia (PPD), that is characterised by cartilage loss and bone and joint destruction. Since MSCs also express the protein, the aim of this study was to elucidate if recombinant protein targets MSCs. A migratory effect of WISP3 treatment on MSCs and osteogenic differentiated MSCs has been proven in this study. To elucidate if global gene expression patterns are influenced by WISP3, cells were treated with 0.5 µg/ml WISP3 and compared to untreated control MSCs. Gene expression study by using affymetrix technology revealed an induction of interferon inducible genes including CXCL chemokines and members of the TNFSF family. Reevaluation by RT-PCR on identical RNA and an additional time series confirmed the results. Although no established cartilage associated genes were detected as regulated genes within this 24h treatment, anti-angiogenic and immunosuppressive genes indicate a protective role of WISP3 for the cartilage, which is sensitive to inflammatory processes. Both CCN-proteins CYR61 and WISP3 are valuable for the musculoskeletal system. This and previous studies revealed the role of CYR61 for osteogenesis and angiogenesis of tissue engineered applications. WISP3 is responsible for development, protection and maintenance of cartilage. Therefore further studies with the proteins in the musculoskeletal system are of high relevance.
WISP3 is a member of the CCN family which comprises six members found in the 1990’s: Cysteine-rich,angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) and the Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6).They are involved in the adhesion, migration, mitogenesis, chemotaxis, proliferation, cell survival, angiogenesis, tumorigenesis, and wound healing by the interaction with different integrins and heparan sulfate proteoglycans. Until now the only member correlated to the musculoskeletal autosomal disease Progressive Pseudorheumatoid Dysplasia (PPD) is WISP3. PPD is characterised by normal embryonic development followed by cartilage degradation over time starting around the age of three to eight years. Animal studies in mice exhibited no differences between knock out or overexpression compared to wild type litter mates, thus were not able to reproduce the symptoms observed in PPD patients. Studies in vitro and in vivo revealed a role for WISP3 in antagonising BMP, IGF and Wnt signalling pathways. Since most of the knowledge of WISP3 was gained in epithelial cells, cancer cells or chondrocyte cell lines, we investigated the roll of WISP3 in primary human mesenchymal stem cells (hMSCs) as well as primary chondrocytes.
WISP3 knock down was efficiently established with three short hairpin RNAs in both cell types, displaying a change of morphology followed by a reduction in cell number. Simultaneous treatment with recombinant WISP3 was not enough to rescue the observed phenotype nor increase the endogenous expression of WISP3. We concluded that WISP3 acts as an essential survival factor, where the loss resulted in the passing of cell cycle control points followed by apoptosis. Nevertheless, Annexin V-Cy3 staining and detection of active caspases by Western blot and immunofluorescence staining detected no clear evidence for apoptosis. Furthermore, the gene expression of the death receptors TRAILR1 and TRAILR2,important for the extrinsic activation of apoptosis, remained unchanged during WISP3 mRNA reduction. Autophagy as cause of cell death was also excluded, given that the autophagy marker LC3 A/B demonstrated to be uncleaved in WISP3-deficient hMSCs. To reveal correlated signalling pathways to WISP3 a whole genome expression analyses of WISP3-deficient hMSCs compared to a control (scramble) was performed. Microarray analyses exhibited differentially regulated genes involved in cell cycle control, adhesion, cytoskeleton and cell death. Cell death observed by WISP3 knock down in hMSCs and chondrocytes might be explained by the induction of necroptosis through the BMP/TAK1/RIPK1 signalling axis. Loss of WISP3 allows BMP to bind its receptor activating the Smad 2/3/4 complex which in turn can activate TAK1 as previously demonstrated in epithelial cells. TAK1 is able to block
caspase-dependent apoptosis thereby triggering the assembly of the necrosome resulting in cell death by necroptosis.
Together with its role in cell cycle control and extracellular matrix adhesion, as demonstrated in human mammary epithelial cells, the data supports the role of WISP3 as tumor suppressor and survival factor in cells of the musculoskeletal system as well as epithelial cells.
Chondrogenic differentiation of human mesenchymal stem cells and articular cartilage reconstruction
(2008)
Articular cartilage defects are still one of the major challenges in orthopedic and trauma surgery. Today, autologous chondrocyte transplantation (ACT), as a cell-based therapy, is an established procedure. However, one major limitation of this technique is the loss of the chondrogenic phenotype during expansion. Human mesenchymal stem cells (hMSCs) have an extensive proliferation potential and the capacity to differentiate into chondrocytes when maintained under specific conditions. They are therefore considered as candidate cells for tissue engineering approaches of functional cartilage tissue substitutes. First in this study, hMSCs were embedded in a collagen type I hydrogel to evaluate the cartilaginous construct in vitro. HMSC collagen hydrogels cultivated in different culture media showed always a marked contraction, most pronounced in chondrogenic differentiation medium supplemented with TGF-ß1. After stimulation with chondrogenic factors (dexamethasone and TGF-ß1) hMSCs were able to undergo chondrogenesis when embedded in the collagen type I hydrogel, as evaluated by the temporal induction of cartilage-specific gene expression. Furthermore, the cells showed a chondrocyte-like appearance and were homogeneously distributed within a proteoglycan- and collagen type II-rich extracellular matrix, except a small area in the center of the constructs. In this study, chondrogenic differentiation could not be realized with every hMSC preparation. With the improvement of the culture conditions, e.g. the use of a different FBS lot in the gel fabrication process, a higher amount of cartilage-specific matrix deposition could be achieved. Nevertheless, the large variations in the differentiation capacity display the high donor-to-donor variability influencing the development of a cartilaginous construct. Taken together, the results demonstrate that the collagen type I hydrogel is a suitable carrier matrix for hMSC-based cartilage regeneration therapies which present a promising future alternative to ACT. Second, to further improve the quality of tissue-engineered cartilaginous constructs, mechanical stimulation in specific bioreactor systems are often employed. In this study, the effects of mechanical loading on hMSC differentiation have been examined. HMSC collagen hydrogels were cultured in a defined chondrogenic differentiation medium without TGF-ß1 and subjected to a combined mechanical stimulation protocol, consisting of perfusion and cyclic uniaxial compression. Bioreactor cultivation neither affected overall cell viability nor the cell number in collagen hydrogels. Compared with non-loaded controls, mechanical loading promoted the gene expression of COMP and biglycan and induced an up-regulation of matrix metalloproteinase 3. These results circumstantiate that hMSCs are sensitive to mechanical forces, but their differentiation to chondrocytes could not be induced. Further studies are needed to identify the specific metabolic pathways which are altered by mechanical stimulation. Third, for the development of new cell-based therapies for articular cartilage repair, a reliable cell monitoring technique is required to track the cells in vivo non-invasively and repeatedly. This study aimed at analyzing systematically the performance and biological impact of a simple and efficient labeling protocol for hMSCs. Very small superparamagnetic iron oxide particles (VSOPs) were used as magnetic resonance (MR) contrast agent. Iron uptake was confirmed histologically with prussian blue staining and quantified by mass spectrometry. Compared with unlabeled cells, VSOP-labeling did neither influence significantly the viability nor the proliferation potential of hMSCs. Furthermore, iron incorporation did not affect the differentiation capacity of hMSCs. The efficiency of the labeling protocol was assessed with high resolution MR imaging at 11.7 Tesla. VSOP-labeled hMSCs were visualized in a collagen type I hydrogel indicated by distinct hypointense spots in the MR images, resulting from an iron specific loss of signal intensity. This was confirmed by prussian blue staining. In summary, this labeling technique has great potential to visualize hMSCs and track their migration after transplantation for articular cartilage repair with MR imaging.