Molecular mechanisms modulating chondrogenesis

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.