Molecular mechanisms modulating chondrogenesis

Catalina Galeano Garcés is a PhD student in the research group Developmental BioEngineering (DBE). Her supervisor is prof.dr. H.B.J. Karperien from the faculty of Science and Technology.

In the last decade various tissue engineering strategies have emerged for articular cartilage repair. MSCs are currently being used in various clinical trials to exploit the multilineage capacity and differentiation potential of these cells. Altough promising clinical results have been seen, controlling the commitment and differentiation of the cells to the expected pathway remains challenging. Our area of investigation will focus its efforts in achieving the understanding of proper molecular mechanisms that could control the commitment and differentiation of the cells whilst avoiding a hypertrophic or fibrous phenotype. The first and second chapters will provide a background, significance, and an overview on the use of environmental conditions, such as hypoxia, for cartilage repair. Chapter three focuses on validating chondrogenesis of adipose derived stem cells (aMSCs) in low oxygen cultures. Cell type specific effects of low oxygen and 3D environments indicated that genetic programming of aMSCs to a chondrocytic phenotype is effective under hypoxic conditions, as evidenced by increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan positive matrix. Chapter four and five draw major attention to the molecular mechanisms by which miRNAs could direct chondrogenesis during the hypoxic response of MSCs and explores the potential of microRNA-210 (miR-210) to enhance in vitro chondrogenic differentiation of stem cells. Hypoxic regulated miR-210 was found to be essential in the regulation of genes in charge of several functions crucial for cartilage development, chondrogenic differentiation and the oxidative stress response. Exogenous miR-210 expression can potentially be utilized instead of inducing chondrogenic differentiation using TGFβ1 in a three dimensional culture under low oxygen, to promote chondrogenesis of MSCs while inhibiting their hypertrophic differentiation. Chondrogenesis improvement was evidenced by increased expression of cartilaginous markers, proteoglycan deposition and collagen II protein content. Chapter six will reveal the effects of synovial fluid on in vitro models of primary chondrocytes and mesenchymal stem cells. Metabolic activity assays on primary chondrocytes and aMSCs showed both cell types survived and proliferated during culture with synovial fluid (SF). Moreover, synovial fluid seems to be permissive for chondrogenic differentiation of aMSCs in the presence of chondrogenic cocktail, which was confirmed by positive type II collagen immunohistochemistry. Our results serve as an initial screening of the possibilities of SF to replace fetal bovine serum as a culture supplement for in vitro expansion of human primary chondrocytes and aMSCs. Chapter seven explores the role of ZNF648, a cartilage specific transcription factor, expressed in immature cartilage and growth plate, to understand its role during cartilage development and to provide a molecular mechanism to create a competent tissue for cartilage repair. In the growth plate, this ZNF648 protein seems to maintain a chondrocytic phenotype of immature cells, whereas in later stages of cartilage maturation its expression is reduced. An enhanced expression of zinc finger 648 (ZNF648) increased collagen type II (COL2A1) expression in growth plate derived chondrocytes while reducing its expression in articular chondrocytes, which suggested an important regulatory mechanism during early development of the chondrocytes in the growth plate but not in terminally differentiated chondrocytes such as the ones in articular cartilage. Thus, development of novel approaches using ZNF648 to improve and maintain cartilage homeostasis is thought to play an essential role in future clinical therapies. Ultimately, chapter eight will provide a general discussion of the results presented in this thesis and presents future perspectives for the use of stem cells in cartilage regeneration therapies.