**4. Cell signalling in chondrogenesis**

Gene expression changes during different stages of endochondral ossification. The immature chondrocytes in the resting zone express the transcription factors Sox 5, Sox6, Sox9 and the structural protein type II collagen and aggrecan. The pre-hypertrophic zone is characterized by the presence of parathyroid receptor 1(PTH-1R) and Indian hedgehog expression (Ihh). The next stage goes into the early hypertrophic zone, which is characterized by type X collagen and alkaline phosphatase enzyme expression, and, subsequently, the reduced amount of type II collagen and reduced expression of Sox5, Sox6 and Sox9 transcription factors. Finally, the chondrocytes proceed to their final phase of a late hypertrophic stage, which is characterized by the expression of vascular endothelial growth factor A (VEGFA), matrix metalloproteinase 13(MM13) and osteopontin. These changes in gene expression herald the cartilage ECM being replaced by bone.

Wnt signalling is important for many developmental processes. It has been shown that activation of Wnt signalling promotes osteoblast differentiation but inhibits chondrocyte differentiation of MSC [42, 43]. Wnt signalling acts through β-catenin to promote chondrocyte hypertrophy and reports suggested that genetic inactivation of β-catenin increased Sox9 expression both in the intramembranous bone formation and endochondral ossification [44, 45]. It was also reported that osteoblast precursor lacking β-catenin expression can develop into chondrocytes [42]. Wnt signalling is also important for proper orientation of chondrocyte column in growth plate cartilage.

Ihh signalling is a key regulator of pre-hypertrophic and early hypertrophic chondrocytes. Ihh signalling directly affects chondrocyte proliferation, premature chondrocyte hypertrophy and failure of osteoblast development and endochondral bone.

Runx2 and Runx3 are members of the Runx transcriptional factors family important for chondrocyte hypertrophy. Several studies demonstrated that ectopic

#### *Epigenetics and Cartilage Regeneration DOI: http://dx.doi.org/10.5772/intechopen.82362*

expression of Runx2 in immature chondrocytes leads to the expression of hypertrophic markers such as COLX α1, MM13 and VEGF [46–48].

As cartilage is an avascular tissue and its nutritional needs are met by surrounding synovial fluids, chondrocytes are adapted to survive in low oxygen levels and they secrete hypoxia-inducible factor 1 (HIF-1) which insures its survival and maintenance in low oxygen tension. Synthesis of type II collagen and aggrecan is upregulated in low oxygen levels, and also, it is associated with the rounded chondrocytic morphology. In the presence of high oxygen tension, chondrocytes become more spindle shaped. HF-1 also showed inhibition of type I collagen synthesis by inhibiting its promoter activity [49].
