Principales riesgos de la industria

Chondrogenesis and the resulting production of articular cartilage is a complex process which varies depending on physiological location. During early embryonic development, human embryonic stem cells (hESC) in the limb buds give rise to a bi-potent mesendoderm population which develop into a mesenchymal core surrounded by an ectoderm (A. Cheng, Hardingham, and Kimber 2014). Condensed populations of mesenchymal stem cells form chondrification centres and subsequently differentiate into chondroprogenitors. Chondroblasts are then formed and they start to produce extracellular matrix (ECM) with an abundance of type II collagen and proteoglycans such as aggrecan (A. Cheng, Hardingham, and Kimber 2014). Eventually these cells lose contact with each other, become chondrocytes and organise into zones that form the growth plates of bones. Much of this initial cartilage model is replaced by bone via a process of endochondral ossification during foetal and postnatal development, but cartilaginous regions persist in the growth plates until early adulthood and remain at the ends of long bones as articular cartilage throughout life. (Oseni et al. 2011; A. Cheng, Hardingham, and Kimber 2014; Foster et al. 2015).

Sex determining region Y-Box 9 (SOX9) is a master transcription factor for all cartilage elements; its expression is switched on in both chondrogenic and osteogenic bone marrow- derived stromal cells (BMSC) prior to condensation and remains high in pre-chondrocytes and chondroblasts (Véronique Lefebvre and Smits 2005). Experiments with murine ESC have shown that there can be no expression of the key ECM proteins COL2 and ACAN when SOX9 expression is blocked (Bi et al. 1999). Furthermore, in its absence, mesenchymal cells are unable to differentiate into chondroblasts at all (Mori-Akiyama et al. 2003). The SOX9 protein has an Sry-related high-mobility group (HMG) box domain via which it binds to DNA

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in order to initiate transcription of other genes such as L-SOX5 (SOX5), SOX6 and later, in cooperation with these two, COL2A1 (Véronique Lefebvre and Smits 2005). No other transcription factors, upstream of SOX9, have been identified that may determine chondrocyte cell fate in all regions of cartilage. However, various homoebox transcription factors (Hox genes) coordinate the expression of genes involved in limb patterning during embryogenesis and may also be transducers of signalling pathways in chondrogenesis. For example, the COL2A1 gene has multiple transcription factor recognition motifs in addition to those for SOX proteins. BMP2 signalling has been shown to activate the Hox gene DLX-2, which in turns leads to upregulation of COL2A1 (Xu et al. 2001). The formation of a mesenchymal condensation in the developing limb bud requires successful cell contact, aggregation and fusion; all of which is reliant upon the expression of cell adhesion molecules (CAMs) such as N-cadherin (N-CAM), tenascin-C (Tnc), versican and thrombospondin-4 (Meech et al. 2005). BARX2 is another Hox gene which helps to orchestrate chondrogenesis; it does so by regulating the expression of CAMs and its necessity for the formation of mesenchymal condensations and cartilage differentiation in the developing limbs of mice has been demonstrated (Meech et al. 2005). Furthermore, its expression is regulated by growth factors BMP4 and GDF5, and it was shown to act in conjunction with other targets of BMP signalling such as SOX9. BARX2 and COL2 proteins were shown to be co-expressed at the joint interzone and in the articular cartilage and, in vitro, BARX2 caused enhanced aggregation of bone marrow-derived cells (Meech et al. 2005). Additional proteins have been identified as transcriptional co-activators of SOX9 and their ablation shown to result in reduced expression of COL2. These include ZNF219 which brings about increased SOX9 activity on the COL2A1 gene promoter (Takigawa et al. 2010); PGC-1 alpha which directly

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interacts with SOX9 (Kawakami et al. 2005); and p300 which binds to the COL2A1 promoter region (Tsuda et al. 2003).

SOX5 and SOX6 expression are activated in pre-chondrocytes and are very high in fully committed chondroblasts. Although they are not required for the determination of lineage commitment (normal precartilaginous condensations can form in their absence), low SOX5/SOX6 expression results in poor chondrogenic differentiation even when SOX9 expression is high (Smits et al. 2001). The three proteins bind to enhancer regions on the

COL2A1 gene and, together, can stimulate non-chondrogenic cells to express COL2A1, ACAN

and other cartilage markers and are able to suppress hypertrophy; addition of transforming growth factor-ß (TGF-ß) and BMP4 can enhance this effect (Ikeda et al. 2004). Other cartilage matrix and regulatory genes have been shown to possess SOX binding regions, including collagen type XI alpha 2 (COL11A2) and ACAN (Véronique Lefebvre and Smits 2005). Thus, the SOX trio are master transcription factors for chondrogenesis and high expression of SOX5/SOX6 is indicative of a more mature phenotype than expression of SOX9 alone.

Prior to endochondral ossification, chondrocytes must first undergo a process of hypertrophy, which is characterised by an increase in cell volume, down-regulation of chondrogenic markers such as SOX9 and COL2, and upregulation of osteogenic markers such as collagen type X , RUNX2 and collagenases such as matrix metallopeptidase 13 (MMP13) (Mackie et al. 2008). Eventually, all chondrocytes in the growth plates of long bones become hypertrophic but, under normal (non-pathogenic) circumstances, articular chondrocytes do not. Quite how they escape growth plate maturation is unclear, but there are some well documented differences which make them distinct from those destined for

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endochondral ossification. Pre-chondrocytes differentiate into chondroblasts which, unlike mature chondrocytes, are highly proliferative and responsible for laying down large amounts of ECM. Articular chondroblasts express high levels of lubricin (PRG4) and by the time they become mature chondrocytes (at the end of postnatal development) they also express high levels of ACAN, whereas both proliferation and COL2 expression are reduced (Rhee et al. 2005; Véronique Lefebvre and Smits 2005). Differential expression of the chicken Erg (chErg) transcription activator has also been observed between articular chondrocytes and growth plate chondrocytes (Iwamoto et al. 2000). Pre-hypertrophic chondrocytes express chErg, but a short-spliced variant was found to be expressed in cells taken from the articular cartilage of developing chicks. Furthermore, virally driven expression of the variant in the growth plate resulted in a failure of the tissue to undergo endochondral ossification, which indicates that chErg has a role in chondrocyte maturation. These differences suggest that the lineage commitment of chondrocytes in the growth plates and those in the epiphyses diverges early on in the process of joint formation.