**2. Molecular heterogeneity of articular cartilage**

The extracellular phase of cartilage, and all connective tissues, consists of collagen fibres and a polysaccharide-rich ground substance. The polysaccharide constituents have been characterized as proteoglycans containing chains of chondroitin 4 sulphate, chondroitin 6 sulphate and keratin sulphate covalently linked to a central core protein [1].

## **2.1 Types of collagen present in cartilage**

Articular cartilage consists of type II collagen as the major fibril-forming collagen, accompanied by lesser quantities of minor collagen which provide the tensile strength and help in maintaining the fine balance of the extracellular matrix. However, little is known about the processing of these minor collagens and their

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

role in the progression of cartilage degeneration and regeneration. Minor collagens found in articular cartilage along with type II collagen are type VI, IX, X, XI, XII and XIV.

Type VI collagen constitutes only 1–2% of the total collagen in adult articular cartilage and it is mainly rich in the pere-cellular matrix and involved in the integration and attachment of chondrocytes [2]. In articular cartilage, chondrocytes in the middle and deep layers are embedded in pere-cellular matrix enriched with a high content of proteoglycans and hyaluronic acid. Increased levels of type VI collagen are found in the experimental model of osteoarthritis (OA) and human OA [3]. Higher levels of type VI collagen found in OA emphasizes its role as a bridge between the extracellular matrix and the chondrocyte surface, thus influencing the signalling pathways from the extracellular matrix into the cells [4].

 Type IX collagen makes up 1–5% of the total collagen in adult articular cartilage and 10% in foetal cartilage [5]. It is usually present in close association with type II collagen found in growth plate cartilage and adult articular cartilage [6]. Type IX collagen is extensively crosslinked to type II collagen through oxidation of lysyl residue bonds forming a unique hetero-fibrillar structure [7]. Type IX collagen is crucial for the maintenance of cartilage matrix and formation of a collagen fibril meshwork. Decreased expression of type IX collagen in the cartilage was thought to render the matrix more prone to mechanical forces and degradation, resulting in the pathogenesis of OA [8].

 Type X constitutes about 1% of the total collagen found in articular cartilage [9]. It was revealed that 45% of the total collagen produced by the hypertrophic chondrocytes is type X collagen [10]. Type X collagen, as produced exclusively by hypertrophic chondrocytes, indicated its unique role in mineralization. The hypertrophic chondrocytes synthesized a variety of proteins and enzymes which help in the transition of extracellular matrix from cartilage to bone. Apart from type X collagen, hypertrophic chondrocytes also synthesize a variety of matrix metalloproteinases as well as alkaline phosphatase enzymes, which are not usually secreted by the normal proliferating chondrocytes. As type X collagen has a direct role in mineralization, it has been found to be expressed in human OA especially in the vicinity of lesions, but not in the healthy human articular cartilage [11].

Type XI collagen constitutes 3–10 % the total adult articular and foetal cartilage, respectively [2]. Type XI collagen is normally crosslinked to each other in cartilage, this crosslinking results in the formation of mature type XI collagen with the help of type II and type IX collagen. It has been shown that a mutation in type XI collagen caused an increase in degradation of type II collagen in articular cartilage [12]. Lu et al. observed that immunization of rats with homologous type XI collagen led to chronic and relapsing arthritis with different genetics and joint pathology than arthritis induced with homologous type II collagen [12]. The role of type XI collagen in cartilage collagen fibril formation and assembly is not clear; type XI collagen may regulate cartilage formation and it was the first collagen deposited by mesenchymal stem cells undergoing chondrogenic differentiation [13]. Type XII shares structural homologies with type IX and type XIV collagen [14]. Type XII collagen is implicated in fibril formation, cell adhesion, fibrosis and osteogenesis, and in areas of high mechanical stress, it may serve as a protector of tissue integrity [15]. Type XII collagen is associated with articular cartilage and growth plate cartilage during rat forelimb development and may be important for microenvironment that supports the hyaline cartilage formation [16].

Type XIV collagen is a large nonfibrillar extracellular matrix protein structurally similar to type XII collagen. In cartilage, a population of type XIV exists as chondroitin sulphate proteoglycans (PGs) as it is sensitive to chondroitinase ABC and AC treatments [17]. Its association with other cartilage collagens such as type I, II,

V and VI are reported, but it also interacts with heparin CD44 and cartilage oligomeric matrix protein [18]. It is found in areas of high mechanical stress similar to type XII collagen, suggesting its role in fibrillogenesis and maintaining the integrity and mechanical property of the tissue.

#### **2.2 Types of PGs in different layers**

 Proteoglycans have the highest concentrations in the intermediate zone and lowest in the superficial and deep zones. Small PGs comprise of less than 10% of the total PG content in the cartilage matrix. Most are aggrecans (large PGs) with approximately 150 GAG chains (chondroitin sulphate and keratin sulphate and both O-linked and N-linked oligosaccharides attached).The GAGs are heterogeneously distributed along the protein core, with CS-rich and KS-rich regions, respectively. The protein core itself is heterogeneous with three globular regions. Aggrecan varies significantly in length, molecular weight and composition with the amount of KS-rich molecules and ratios of chondroitin 6-sulphate and chondroitin 4-sulphate increasing throughout development and ageing. Most aggrecans in cartilage are attached to a hyaluronic (HA) molecule via a globular (HABR) region; this binding was stabilized by a link protein. Several hundred aggrecans are attached to a single HA core molecule, the latter being a non-sulphated disaccharide chain up to 4 μm in length. PGs are closely associated with collagen fibrils and are thought to be involved in their structural organization and maintaining their compressive stiffness.

 There is now conclusive evidence of the fact that OA is not simply due to wear and tear and a result of ageing; but in numerous studies, it has been reported that early onset of OA is due to activation of inflammatory response. These inflammatory responses could be due to increased oxidative stress to the tissues, resulting in initiation of catabolic enzymes and factor that actively breakdown the major extracellular matrix components of cartilage, namely type II collagen, and the proteoglycans and aggrecan.

### **3. Control of chondrogenesis**

The commitment of mesenchymal cells to the chondrogenic lineage is the key event in bone formation. Work over the past few decades, using both in vivo and in vitro systems, has identified a number of signalling and transcription factors as well as cell shape that regulates the progressive change in chondrocyte phenotype, from their initial induction to their terminal fate. The disruption of these finely tuned pathways for chondrocyte maturation can result in skeletal pathology. A thorough knowledge of these signalling pathways would help us to identify the factors that maintain chondrocyte proliferation and differentiation. Some of the major signalling pathways are described below.

#### **3.1 Bone morphogenic protein signalling**

Bone morphogenic proteins (BMPs) are identified as positive regulators of chondrogenesis and endochondral ossification. BMPs are a member of the transforming growth factor beta (TGβ) superfamily that has wide-ranging biological activity, ranging from cellular regulation of proliferation, apoptosis, differentiation and migration [19, 20]. BMP signalling is mediated by their receptors BMPR1a, BMPR1b and BMPR2, leading to the SMAD signalling pathway [19]. In cartilage, it initiates cartilage synthesis and decreases the activity of catabolic cytokines such as IL-1, IL-6, IL-8, MM1 and MM13 [21, 22]. Though there are several members of

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

 Bone morphogenic protein (BMP) growth factors, most promising among them in the treatment of OA is BMP-7, which promotes the cartilage-specific extracellular matrix proteins such as collagen II and VI, decorin, fibronectin and hyaluronate (HA) by upregulation of hyaluronan synthase [23, 24]. In experiments when it was applied to other types of cells in knee, BMP-7 has shown to increase Extracellular matrix (ECM) in synovial and bone marrow-derived Mesenchymal Stem cell (MSC), both alone and in combination with TGFβ [25]. This profound anabolic effect of BMP-7 is due to its regulatory properties of modulating other growth factors such as insulin-like growth factor 1(ILGF1 and fibroblast growth factor (FGF)) [26]. Despite having anabolic activity, BMP-7 has not shown to induce chondrocyte hypertrophy or other changes in the chondrocyte phenotype, nor did BMP-7 treated animal knee display any proliferation of fibroblast or osteocyte [25]. These properties make it a promising therapy for OA.
