Lesions of articular cartilage are still leading to irreversible degenerative joint diseases including osteoarthritis (OA). Aside pain, the disease results in loss of function of the affected joint and severe disability and a substantial reduction of quality of life of the affected patient. The human does not have the endogenous capacity to restore the structure and the function of the osteoarthritic knee joint damage. In contrast, animals such as the red-spotted newt (Notophthalmus viridescens viridescens) are able to repair complete organs and tissues including joints after artificial damage by collagenase treatment and surgery. The underlying mechanisms of this regenerative process can be further investigated by cDNA microarray showing distinct genes, which were differentially upregulated in both models for OA.Based on these results, in this doctoral project, the genes, that were found to be upregulated in the cDNA microarray study and additional genes from the vitamin A pathway, and the innate immune system, both known to be involved in repair processes were selected for real-time PCR analysis to primarily quantify their deregulation status. Here, real-time PCR study confirmed the deregulation of distinct molecules including secreted protein acidic and rich in cysteine (SPARC), periostin (POSTN), decorin (DCN), complement factor B (CFB), tenascin-C (TN-C), toll-like receptor 2 (TLR2), and retinoic acid receptor responder 1 (RARRES1). Among these selected factors, TN-C was the most interesting one, because the deregulation of this matricellular protein was the most prominent among the other selected candidates. IHC analysis revealed that the spatial expression of TN-C during the knee joint regeneration process was unique in each OA model. At day 10 and 20 after surgery treatment, TN-C appeared in the defected articular cartilage of the treated knee joints in two of three analyzed animals. In contrast, in the animals of the collagenase model, the TN-C expression was absent in the defected articular cartilage. It supported the idea of a differential initiation of the repair process depending on the type of the damage inflicted. The appearance of this matricellular protein in the defected articular cartilage in the animals of the surgery model can be seen as a part of an inflammatory response during the injury process. At day 20 after treatment, undifferentiated tissue emerged in the regenerating newt knee joint of all analyzed animals. TN-C was expressed abundantly in this tissue, which is a further indication of its involvement in the knee joint regeneration. At day 40 after treatment, all of the three analyzed animals in the surgery model and one animal of the two analyzed animals in the collagenase model demonstrated expression of TN-C in the newly formed articular cartilage. Here, TN-C could promote the formation of this tissue. In the regenerating knee joint of one analyzed animal in the surgically-treated animals, TN-C appeared only in the newly formed articular cartilage, but it was absent in the old-defected cartilage. In order to investigate whether the involvement of TN-C was functionally important for the knee joint regeneration process, gene silencing experiments were conducted in newt primary chondrocytes using TN-C esiRNAs in vitro, introduced into the primary newt chondrocytes by nucleofection. In two independent experiments, TN-C expression could be reduced to 0.55-fold (45% reduction) and 0.29-fold (71% reduction), the effect of one-time TN-C gene silencing on regenerative capacity of newt chondrocytes with respect to migration and adhesive capacity of these cells did not result in a statistically significant effect. In addition, in order to elucidate the effect of TN-C knockdown on other dysregulated candidate genes including SPARC and DCN, relative quantification of the transcription product of both genes was performed. The result of the measurement demonstrated that TN-C knockdown in newt chondrocytes did not alter the expression levels of these molecules. SPARC, another prominent candidate gene, which was selected from the cDNA microarray, was present in several tissues in untreated newt legs including osteocytes, articular cartilage, blood vessels and skin. As SPARC is known to be important for the normal function of these tissues, in the regenerating newt knee joint already at day 20 after surgery treatment, this matricellular protein was localized abundantly in distinct areas in the skeletal muscle and periosteum. Of note, this expression was not directly related to the regeneration process since these tissues were not primarily damaged by the surgical treatment.Similar to the results obtained from the TN-C and SPARC experiments, several other matrix repair associated molecules were found to be upregulated and expressed at different time points during the newt knee joint regeneration process, providing interesting new insights into this fascinating phenomenon. However, the experiments of this doctoral thesis also showed the still existing limitations of an experimental approach in an organism, for which the technical systems still need to be fully established.
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