**5. Alterations of the ECM in the skeletal tissue: Injuries and pathologies**

The extracellular matrix has structural and functional relevance, it's a highly organized and assembled macromolecular structure, also provide cellular adhesion environments, activation and inactivation of growth factors and regulatory cytokines. The proteolytic processing of ECM components, results in the production of fragments with biological effects on migration, proliferation and cellular organization.

When any component of the ECM has a disorder, could generate chondrodysplasia, it means alterations in the development and growth of cartilage. Chondrodysplasias are caused by various mutations in genes involved in cartilage development and finally in the formation and growth of the long bones. These mutations also often alter the formation of other tissues.

Achondrogenesis type II, is a chondrodysplasia classified as collagenopathy type II. In this family are located several chondrodysplasia caused by mutations in the gene for collagen II, which is the most abundant protein in cartilage [42]. These dysplasias are, achondrogenesis type II, hypochondrogenesis, congenital espondiloepiphysial dysplasia and Kniest dysplasia, among others. Collagen II is a homotrimer (three identical chains encoded by the COL2A1 gene located on chromosome 12. This collagen is mainly found in the hyaline cartilage and vitreous humor, so its deficiency is associated with abnormalities of the spine, of the epiphysis and eye problems. Despite their differences these dysplasias share clinical and radiological manifestations, so the axial skeleton is affected more than the limbs, cleft palate, myopia and retinal degeneration [43].

RGD motifs allows a precise spatial distribution pattern of integrins for specific cellular

Integrins can activate several signaling pathways independently and frequently they act synergistically with other growth factor receptors as insulin receptor, type 1 insulin-like growth factor receptor, VEGF receptor, TGF-b receptor, platelet-derived growth factor-b

The heparan sulfate proteoglycans (HSPGs) contribute to the organization of the matrix by binding to the many core matrix molecules via HS chains as laminin, fibronectin and collagen. The chondroitin sulphate proteoglycans (CSPGs) as aggrecan, versican, brevican and the small, leucine-rich proteoglycans such as decorin and biglycan also bind to and regulate a number of growth factors, such as members of the TGF family. The hyaluronic acid is a glycosamino‐ glycan synthesized on the cell surface and is responsible for the gel-like consistency of cartilage

During normal or pathologic physiology of the cartilage, the ECM must be remodeling and degraded to allow the chondrocytes for processing and deposition of new matrix by specific proteases. There are two well-known families of proteases that are involved in the biology of the ECM, the matrix metalloproteinase (MMP) and the desintegrins and metalloproteinases with thrombospondin motif (ADAMTS) families. The MMP-13 is involved in the cleavage of fibromodulin and type IX collagen and is present and active in the pathological process of cartilage as OA and rheumatoid arthritis. The aggrecanases family's ADAMTS-4 and ADAMTS-5 play an important role in cartilage damage during early OA which cleavage the glycosaminoglycans chains that are the key contributors to the maintenance of the charge density, the osmotic environment and water retain important characteristics of the mechanical

**5. Alterations of the ECM in the skeletal tissue: Injuries and pathologies**

The extracellular matrix has structural and functional relevance, it's a highly organized and assembled macromolecular structure, also provide cellular adhesion environments, activation and inactivation of growth factors and regulatory cytokines. The proteolytic processing of ECM components, results in the production of fragments with biological effects on migration,

When any component of the ECM has a disorder, could generate chondrodysplasia, it means alterations in the development and growth of cartilage. Chondrodysplasias are caused by various mutations in genes involved in cartilage development and finally in the formation and growth of the long bones. These mutations also often alter the formation of other tissues.

(PDGF-b) receptor and epidermal growth factor (EGF) receptor [37,38].

response among ligand molecules [36, 37].

370 Regenerative Medicine and Tissue Engineering

**4.1. Role of proteoglycans in signal regulation**

by its hydroscopic properties [36, 39].

properties of the cartilage [40, 41].

proliferation and cellular organization.

**4.2. Remodelation and degradation of ECM**

Furthermore, other disorders of matrix components such as collagen IX and XI, which interact with the collagen II to form supramolecular structures, are closely related phenomena.

It is found that the Osteogenesis Imperfecta (OI) is caused by molecular defects of collagen type I[44] and metaphyseal chondrodysplasia Schmid type is caused by errors in collagen type X biosynthesis [45], the latter is characterized by alterations in vertebrae and in the metaphysis of long bones, also show reduction of the area of reserve cartilage in growth plate and in the articular cartilage, alters the contents of bone and there is an atypical distribution of the matrix components of the growth plate.

The cartilage oligomeric matrix protein (COMP) is a member trombospondins family, and its alteration causes pseudoachondroplasia, this disorder shows short limbs and lax ligaments [46], the growth plate is shorter and the area of hypertrophic cartilage is reduced.

Cartilage needs molecular signals for development and maintenance, such as growth factors, which in many cases are regulating the synthesis of the ECM, and may be found active or latent in the extracellular matrix. Bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-β), growth and differentiation factor 5 (GDF-5), are signals related to the develop‐ ment and growth of cartilage, alterations in these molecules cause some malformations, such as the brachypodism (short limbs) [47].

Cartilage matrix is rich in sulfated proteoglycans and the gene encoding for sulfate transporter called DTDST (Dystrophic Dysplasia Sulfate Transporter) in patients with dystrophic dyspla‐ sia was found mutations in this gene, and shown to be deficient cartilage sulfating [48].

Campomelic dysplasia is a rare disease associated with XY individuals who possess varying degrees of sex reversal. SOX-9 is a transcription factor structurally related to the gene SRY (sexdetermining region Y) required for testicular development. However, SOX-9 also directly regulates the gene for type II collagen, the main molecule of the cartilage matrix and therefore of chondrocyte differentiation [49, 50, 51].

The inactivation of the gene coding for the mouse gelatinase B, defined the mechanism that controls the final step of the chondrocyte maturation [52]. Gelatinase B is an enzyme present in the extracellular matrix of cartilage and its activity is related to the control of apoptosis of hypertrophic chondrocytes and the vascular tissue. This study hypothesized the existence of chondroclast, these cells of myeloid origin express gelatinase-B and are located in the cartilage/ bone region and resorb cartilage matrix.

Based on the above is to emphasize the importance of the extracellular matrix as a modulator of cellular differentiation of chondrocytes, the extracellular components correlate with the differentiation state. That is, collagen I is present at early stages of differentiation and matu‐ ration, in mesenchyme and perichondrium; collagen II is on mature cartilage and collagen X is exclusive of hypertrophic cartilage also collagen type I are expressed in terminal stages of chondrocytes [53].

The ECM not only serves as a binder that gives form to tissues in addition to their structural role has physiological functions. The chondrocytes are in the array a series of signals that allows them to gain some cell shape and organization of the cytoskeletal network. Cell morphology that can modulate many physiological functions such as proliferation, differentiation, cell death and gene expression. This transmembrane receptor-mediated would be able to receive the extracellular signal from the ECM and transduce the signal into the cell, triggering a response by the chondrocyte differentiation [54].

Integrins are transmembrane receptor consisting of one α subunit and a β, are only functional to form the α-β heterodimer on the cell membrane. β1 family of integrins are major receptors of ECM molecules and have the ability to allow cell adhesion and simultaneously issuing an intracellular signal to which the cell responds in different ways, as also interact with integrins the cytoskeleton and molecules involved in signal transduction.

It has been shown that integrins interaction with extracellular matrix molecules affects cytoskeleton organization, proliferation, differentiation and gene expression in fibroblasts and epithelial cells.

In addition we have studied the survival and differentiation of chondrocytes, including the deposit in the interstitial matrix of collagen type X could be mediated by integrins [55]. Inhibition of integrin b1 subunit with a neutralizing antibody blocks the deposition of collagen X in the interstitial matrix and growth of the breastbone is decreased. Moreover, the chondro‐ cytes are significantly smaller, show a disorganization of the actin cytoskeleton and show increased apoptosis.

There is also evidence that blocking the β1 subunit of integrins in an in vitro model of differentiation of cartilage inhibits cartilage nodule formation and the synthesis of collagen type II [56].

However, the study of the role of these receptors in the process of chondrocyte differentiation is not yet well established, but it would be of significant importance in determining the relationship of the extracellular matrix to the chondrocyte.

#### **5.1. The extracellular matrix and chondrocyte differentiation in osteoarthritis**

Articular cartilage mineralization frequently accompanies and complicates osteoarthritis and aging. Several works has demonstrated that certain features of growth cartilage development are shared in degenerative cartilage. These include chondrocyte proliferation, hypertrophy, matrix mineralization and apoptosis. Development of growth plate is regulated by growth factors signaling and cellular interactions with the extracellular matrix (ECM). Parathyroid hormone related protein (PthrP) and Indian Hedgehog (Ihh) are central mediators of endo‐ chondral development; PthrP is abundant in synovial fluid of osteoarthritic patient but Ihh expression is diminish in OA cartilage, Fgf-18 is a regulator of chondrocyte proliferation and its intra-synovial application in OA rat results in cartilage generation. Also, Wnt signaling plays an important role in chondrocyte differentiation in growth plate, Wnt-5a promotes chondrocyte prehypertrophy and inhibits chondrocyte hypertrophy unlike Wnt-4 that induces chondrocyte hypertrophy and increases its expression in early stage of osteoarthritis. On the other hand, is pronounced imbalance of cartilage matrix turnover in osteoarthritic cartilage, and results in mayor deposition of collagen type I and X, reduced expression of collagen type II. Thus, the rate of chondrocyte hypertrophy is higher on growth plate and OA articular cartilage than healthy articular cartilage, it recap the signaling in cartilage growth plate. But, although articular and growth plate cartilages share several features, there are one important difference, the rate of cartilage hypertrophy. What is the signal that makes the difference? In the ECM we could find some elements to answer this question.

#### *5.1.1. Alterations in the extracellular matrix of articular cartilage during OA*

Based on the above is to emphasize the importance of the extracellular matrix as a modulator of cellular differentiation of chondrocytes, the extracellular components correlate with the differentiation state. That is, collagen I is present at early stages of differentiation and matu‐ ration, in mesenchyme and perichondrium; collagen II is on mature cartilage and collagen X is exclusive of hypertrophic cartilage also collagen type I are expressed in terminal stages of

The ECM not only serves as a binder that gives form to tissues in addition to their structural role has physiological functions. The chondrocytes are in the array a series of signals that allows them to gain some cell shape and organization of the cytoskeletal network. Cell morphology that can modulate many physiological functions such as proliferation, differentiation, cell death and gene expression. This transmembrane receptor-mediated would be able to receive the extracellular signal from the ECM and transduce the signal into the cell, triggering a

Integrins are transmembrane receptor consisting of one α subunit and a β, are only functional to form the α-β heterodimer on the cell membrane. β1 family of integrins are major receptors of ECM molecules and have the ability to allow cell adhesion and simultaneously issuing an intracellular signal to which the cell responds in different ways, as also interact with integrins

It has been shown that integrins interaction with extracellular matrix molecules affects cytoskeleton organization, proliferation, differentiation and gene expression in fibroblasts and

In addition we have studied the survival and differentiation of chondrocytes, including the deposit in the interstitial matrix of collagen type X could be mediated by integrins [55]. Inhibition of integrin b1 subunit with a neutralizing antibody blocks the deposition of collagen X in the interstitial matrix and growth of the breastbone is decreased. Moreover, the chondro‐ cytes are significantly smaller, show a disorganization of the actin cytoskeleton and show

There is also evidence that blocking the β1 subunit of integrins in an in vitro model of differentiation of cartilage inhibits cartilage nodule formation and the synthesis of collagen

However, the study of the role of these receptors in the process of chondrocyte differentiation is not yet well established, but it would be of significant importance in determining the

Articular cartilage mineralization frequently accompanies and complicates osteoarthritis and aging. Several works has demonstrated that certain features of growth cartilage development are shared in degenerative cartilage. These include chondrocyte proliferation, hypertrophy, matrix mineralization and apoptosis. Development of growth plate is regulated by growth factors signaling and cellular interactions with the extracellular matrix (ECM). Parathyroid

**5.1. The extracellular matrix and chondrocyte differentiation in osteoarthritis**

chondrocytes [53].

372 Regenerative Medicine and Tissue Engineering

epithelial cells.

increased apoptosis.

type II [56].

response by the chondrocyte differentiation [54].

the cytoskeleton and molecules involved in signal transduction.

relationship of the extracellular matrix to the chondrocyte.

Traditionally it has been thought that osteoarthritis is a disease of wear or tears consequence of articular cartilage due to aging or following injury. The limited regenerative capacity of cartilage cannot reverse its destruction, it is sometimes triggered by an inflammatory response from the synovial, inflammation occurs when the condition is called osteoarthritis [57]. Until recent years genetic mutations were excluded as a risk factor or predisposition to osteoarthritis. The first genes identified to OA encode components of the extracellular matrix, such as Collagen *COL2A1*, *COL9A2* and *COL11A2*, which were studied in transgenic mouse models [58]. It has been found that the substitution of glycine destabilizes the triple helix structure of collagen type II making it more susceptible to degradation by MMP-13 [59]. Other ECM molecules related to OA are ADAMTS-4 and ADAMTS-5 enzymes which degrade aggrecan, the most abundant proteoglycan in articular cartilage [60]. When aggrecan is degraded, the collagen II is exposed to the DDR-2 enzyme which is able to degrade it [61]. The alteration of the ECM of articular cartilage in the first instance causes cell proliferation and the formation of fibrous tissue that forms a scar in response to injury, there are produced growth factors such as TGF-β could promote chondrocyte hypertrophy, so that recapitulates OA cartilage differ‐ entiation mechanisms of the growth plate to form ultimately bone nodules at the edges of articular cartilage called osteophytes [62]. Clearly the importance of ECM in the differentiation of articular cartilage, but there are various growth factors and transcription factors that regulate the maturation and proliferation of chondrocytes in articular cartilage and cartilage growth plate, which also control the expression of many of the components of the ECM, and also direct the skeletal morphogenesis. Genes has recently been determined as Smad-3, Dkk, Wnt4, Mig-6 etc [63- 66], OA generated in murine models, these molecules regulate different cellular processes such as cell proliferation, cell differentiation, cell death, degradation and synthesis of ECM. We can group the molecules according to the governing process: Chondro‐ genesis, Proliferation, Differentiation and Cell Death. Many of these molecules can be good genetic markers of predisposition to OA, and are fundamental to how to design a strategy for articular cartilage repair.

#### *5.1.2. Differentiation of articular cartilage chondrocyte*

Although exists different types of cartilage, they are very similar but have different functions. Articular cartilage and cartilage growth plate are good examples. In general, the molecular mechanisms of chondrocyte differentiation in both cartilages are equivalent. However, for the function of synovial joints is essential that chondrocytes maintenance in prehypertrophic state differentiation, while the longitudinal growth of bone depends on the proliferation and differentiation of chondrocytes in the growth plate to the hypertro‐ phy and bone formation [67, 68]. We can even talk about a model that relates the structure and function of cartilage based on histological and functional differences of both cartilag‐ es. Both in the cartilage growth plate and in articular cartilage chondrocytes can be found at various stages of differentiation, but the organization and activity of chondrocytes differ in each stage of both cartilage.

In the growth plate chondrocytes reserves represent an immature state and are organ‐ ized in tiny rows of small round cells, embedded in an abundant extracellular matrix rich in collagen type II and aggrecan, proliferating chondrocytes are stacked as "coins" several rows forming compact occupying a large area of the growth plate, the first rows are more proliferation activity than the rows deep; prehypertrofic chondrocytes (mature) are larger cells that have exited the cell cycle and express Ihh, a key molecule in cartilage differentia‐ tion, these cells secrete and accumulate a large amount of carbohydrates and finally the hypertrophic chondrocytes are cells of highest volume and high alkaline phosphatase activity, the ECM is mainly composed of collagen type X and begins to calcify, some cells degenerate and die by apoptosis leaving the spaces occupied to consolidate osteoblasts and bone tissue. This process is known as endochondral ossification which regulates the growth of bone in terms of cartilage differentiation. It is noteworthy that an important signaling center in this process is the perichondrium, which are very small and flattened cells surrounding the cartilage and expressed PTHrP [69] and Fgf-18 [70], which respectively induce and inhibit the proliferation of chondrocytes, the receiver PPR and PTHrP [71] is expressed in the upper rows, whereas the Fgf-18 receptor and FGF-R3 is found in the deeper cell layers of proliferating chondrocytes. Patch is Ihh receptor and is expressed in the perichondrium, so that Ihh induces the expression of PTHrP and this in turn induces proliferation and expression of Ihh in the growth plate. This regulatory loop promotes the longitudinal growth of the mold of cartilage, but it is necessary that the mold is rigid. For this, the FGF18 inhibits the proliferation of cartilage to regulate expression of Ihh and this result in the differentiation of chondrocyte hypertrophy up. This signaling cascade also occurs during the formation of joint cartilage, where bone formation is more limited as in the secondary ossification centers.

Articular cartilage has apparently different stages of differentiation of chondrocytes, only that which corresponds to the resting chondrocytes have important differences in the composition of the ECM, as the presence of lubricin, the Collagen type IIa the aggrecan, CD44, ASC, [72, 73] these cells are most abundant in the articular cartilage cells for proliferation area are not organized in rows and have very low proliferation rate, making them more similar to the prehypertrophic cartilage, as the rate of is very slow maturation, hypertrophic chondrocytes make up a small area of just one or two cell lines the border between cartilage and bone, known as "water mark" (tide mark).

#### **5.2. Endochondral ossification during skeletal development and OA**

*5.1.2. Differentiation of articular cartilage chondrocyte*

in each stage of both cartilage.

374 Regenerative Medicine and Tissue Engineering

the secondary ossification centers.

Although exists different types of cartilage, they are very similar but have different functions. Articular cartilage and cartilage growth plate are good examples. In general, the molecular mechanisms of chondrocyte differentiation in both cartilages are equivalent. However, for the function of synovial joints is essential that chondrocytes maintenance in prehypertrophic state differentiation, while the longitudinal growth of bone depends on the proliferation and differentiation of chondrocytes in the growth plate to the hypertro‐ phy and bone formation [67, 68]. We can even talk about a model that relates the structure and function of cartilage based on histological and functional differences of both cartilag‐ es. Both in the cartilage growth plate and in articular cartilage chondrocytes can be found at various stages of differentiation, but the organization and activity of chondrocytes differ

In the growth plate chondrocytes reserves represent an immature state and are organ‐ ized in tiny rows of small round cells, embedded in an abundant extracellular matrix rich in collagen type II and aggrecan, proliferating chondrocytes are stacked as "coins" several rows forming compact occupying a large area of the growth plate, the first rows are more proliferation activity than the rows deep; prehypertrofic chondrocytes (mature) are larger cells that have exited the cell cycle and express Ihh, a key molecule in cartilage differentia‐ tion, these cells secrete and accumulate a large amount of carbohydrates and finally the hypertrophic chondrocytes are cells of highest volume and high alkaline phosphatase activity, the ECM is mainly composed of collagen type X and begins to calcify, some cells degenerate and die by apoptosis leaving the spaces occupied to consolidate osteoblasts and bone tissue. This process is known as endochondral ossification which regulates the growth of bone in terms of cartilage differentiation. It is noteworthy that an important signaling center in this process is the perichondrium, which are very small and flattened cells surrounding the cartilage and expressed PTHrP [69] and Fgf-18 [70], which respectively induce and inhibit the proliferation of chondrocytes, the receiver PPR and PTHrP [71] is expressed in the upper rows, whereas the Fgf-18 receptor and FGF-R3 is found in the deeper cell layers of proliferating chondrocytes. Patch is Ihh receptor and is expressed in the perichondrium, so that Ihh induces the expression of PTHrP and this in turn induces proliferation and expression of Ihh in the growth plate. This regulatory loop promotes the longitudinal growth of the mold of cartilage, but it is necessary that the mold is rigid. For this, the FGF18 inhibits the proliferation of cartilage to regulate expression of Ihh and this result in the differentiation of chondrocyte hypertrophy up. This signaling cascade also occurs during the formation of joint cartilage, where bone formation is more limited as in

Articular cartilage has apparently different stages of differentiation of chondrocytes, only that which corresponds to the resting chondrocytes have important differences in the composition of the ECM, as the presence of lubricin, the Collagen type IIa the aggrecan, CD44, ASC, [72, 73] these cells are most abundant in the articular cartilage cells for proliferation area are not organized in rows and have very low proliferation rate, making them more similar to the prehypertrophic cartilage, as the rate of is very slow maturation, hypertrophic chondrocytes The joints that separate from each other skeletal elements serve as important signaling centers during skeletal development, and regulate the proliferation and maturation of chondrocytes. It is well known that chondrocyte maturation is crucial for endochondral ossification and to define the final size of each skeletal element. In the end, the processes of the formation of joints and cartilage differentiation of skeletal elements are strongly related. The limb skeletal elements are formed by endochondral ossification, the process begins with the aggregation of mesenchymal cells that form the pre-cartilaginous conden‐ sation, this condensation increases the proliferation of chondrocytes and forms a "bar" initial cartilage [74]. It has been proposed that the first step for the formation of the joint is that it inhibits differentiation of prehypertrofic chondrocytes in cells located in the region of the joint prospecting, outside the influence of signals that promote maturation of the cartilage, while neighboring cells continue their differentiation process to form bone hypertrophy and subsequently by endochondral ossification, so contributing to the formation of adjacent skeletal elements [75]. Cells suspected joint region form the inter‐ zone, characterized by a highly packed region of flattened cells, these cells produce other types of collagen and collagen type I and III, unlike chondrocytes that produce collagen type II. The interzone also expressed molecules such as Wnt-9a [76] and Bmp antago‐ nists like noggin [77], which remain the property of these cells not chondrogenic. Some cell adhesion molecules such as integrin α5β1 also regulate the formation of joints by controlling the differentiation of chondrocytes [78], whereas other signaling molecules that are expressed in the interzone as Wnt-4, Fgf-18, Gdf (5, 6 and 7) and several members of the Bmp, promote growth and differentiation of adjacent cartilaginous elements [79]. It is likely that different cell types present in a mature synovial joint, including synovial cells, articular chondrocytes and permanent joint capsule cells originate in the interzone. Permanent articular chondrocytes originating from the interzone, are very similar to chondrocytes in the growth plate, and although both cell types are hyaline cartilage and functions have important differences. The most important difference is that articular chondrocytes decrease its maturation toward hypertrophy of chondrocytes unlike the growth plate which we observed a wide region of hypertrophic chondrocytes, as this process allows for the ossification and growth of long bones. Hypertrophic chondrocytes are the highest volume and produce a very specific extracellular matrix rich in collagen type X. The hypertrophy of chondrocytes is followed by apoptosis, the invasion of blood vessels, osteoclasts and other mesenchymal cells from the perichondrium and production of bone matrix. Therefore, the size and fine structure of the long bones depends on the coordinated regulation of proliferation, maturation and hypertrophy of chondrocytes in response to many extracellular signals. The protein Indian hedgehog (Ihh) and peptide related to Thyroid Hormone (PTHrP) play a critical role in these processes, Ihh is pro‐ duced by prehypertrophic chondrocytes and induces the expression of PTHrP in the perichondrium which in turn regulates the rate of chondrocytes which exit the cell cycle and continue to hypertrophy [80]. Ihh also stimulates proliferation of chondrocytes and controls the differentiation of mesenchymal cells into osteoblasts in the collar bone. Thus, when the chondrocytes stop expressing Ihh activates the expression of Runx-2 and Runx-3 [81], some transcription factors required for hypertrophy of chondrocytes and differentia‐ tion of osteoblasts. On the contrary, in particular FGF-18 [82] expressed in the perichondri‐ um and through its receptor Fgf-R3 expressed in cartilage prehypertrofic cartilage negatively regulates cell proliferation and promotes the hypertrophy of chondrocytes, the constitutive activation of FGFR3 results in dwarfism [83] and may inhibit the formation of joints, this confirms the idea that proliferating chondrocytes may have two possible destinations, become pre-articular chondrocytes or prehypertrophic chondrocytes.

#### **5.3. Control of chondrocyte differentiation and two destinations, Ihh vs Wnt signaling and its role in OA**

During the formation of the skeleton some chondrocytes are involved in the growth of long bones and ossification. At this early stage, the GDF-5 signaling is essential for the formation of joints and articular cartilage [84, 85], its expression is delimited in the interzone and begins just before forming the joints, on the other hand, the Bmp-7 is important for the chondrocyte maturation and bone formation and is expressed in the perichondrium of the skeletal elements in formation and growth [86], but not expressed in the perichondrium of the developing cartilage. Although the induction of the joint is initiated by the expression of Wnt-9a in the interzone and the interzone chondrocytes lose their phenotype [76], GDF-5 signaling is essential for the joint and articular cartilage formation. Ihh is another important molecule for skeletal development, Ihh inhibits Wnt-9a expression and is maintained in skeletal growth and endochondral ossification, as when it reaches a certain size decreases the expression of Ihh and thereby activates the expres‐ sion of Wnt patway induces hypertrophy of chondrocytes and bone formation [87]. It is noteworthy that during the OA Wnt signaling is overactivated [65] and GDF-5 is downregulated, which suggests a recapitulation of endochondral ossification during OA. Furthermore, when the receptor Bmp-RIA is inactivated in mouse generated phenotypes similar to human osteoarthritis and when activated the Wnt pathway by blocking antago‐ nist Dkk [64], reverse the process of articular cartilage destruction and endochondral ossification, this suggests that these pathways permit the maintenance of adult articular cartilage.

#### **5.4. Proliferation, hypertrophy and cell death are activated during OA**

Not only in the embryonic stages imbalance of proliferative signals and bring important consequences hypertrophy in articular cartilage, osteoarthritis is a striking example of this imbalance of signals. There are animal models that recapitulate this degenerative joint disease, as in the case of the mutant mice of Smad-3 [63], a molecule that transduces the TGF-β signal. Molecular analysis of these mice shows ectopic expression of type X collagen in the articular cartilage and increased hypertrophy of chondrocytes; this shows the TGF-β as an inhibitor of differentiation of articular chondrocytes. Similarly, the cancellation of Mig-6 in mice results in early degeneration of joints [66], as evidenced by degradation of articular cartilage, fibrous tissue formation and growth of osteophytes. It is well known that articular cartilage injuries may result in osteoarthritis, fibrous tissue formation is an immediate healing response to a traumatic injury, and the healing is often promoted by TGF-β, which in turn could induce osteophyte formation that recapitulates chondrogenesis and endochondral ossification in adult articular cartilage.

#### **5.5. Why not articular cartilage regenerate**

duced by prehypertrophic chondrocytes and induces the expression of PTHrP in the perichondrium which in turn regulates the rate of chondrocytes which exit the cell cycle and continue to hypertrophy [80]. Ihh also stimulates proliferation of chondrocytes and controls the differentiation of mesenchymal cells into osteoblasts in the collar bone. Thus, when the chondrocytes stop expressing Ihh activates the expression of Runx-2 and Runx-3 [81], some transcription factors required for hypertrophy of chondrocytes and differentia‐ tion of osteoblasts. On the contrary, in particular FGF-18 [82] expressed in the perichondri‐ um and through its receptor Fgf-R3 expressed in cartilage prehypertrofic cartilage negatively regulates cell proliferation and promotes the hypertrophy of chondrocytes, the constitutive activation of FGFR3 results in dwarfism [83] and may inhibit the formation of joints, this confirms the idea that proliferating chondrocytes may have two possible

destinations, become pre-articular chondrocytes or prehypertrophic chondrocytes.

**5.4. Proliferation, hypertrophy and cell death are activated during OA**

Not only in the embryonic stages imbalance of proliferative signals and bring important consequences hypertrophy in articular cartilage, osteoarthritis is a striking example of this imbalance of signals. There are animal models that recapitulate this degenerative joint disease, as in the case of the mutant mice of Smad-3 [63], a molecule that transduces the TGF-β signal. Molecular analysis of these mice shows ectopic expression of type X collagen in the articular

**its role in OA**

376 Regenerative Medicine and Tissue Engineering

cartilage.

**5.3. Control of chondrocyte differentiation and two destinations, Ihh vs Wnt signaling and**

During the formation of the skeleton some chondrocytes are involved in the growth of long bones and ossification. At this early stage, the GDF-5 signaling is essential for the formation of joints and articular cartilage [84, 85], its expression is delimited in the interzone and begins just before forming the joints, on the other hand, the Bmp-7 is important for the chondrocyte maturation and bone formation and is expressed in the perichondrium of the skeletal elements in formation and growth [86], but not expressed in the perichondrium of the developing cartilage. Although the induction of the joint is initiated by the expression of Wnt-9a in the interzone and the interzone chondrocytes lose their phenotype [76], GDF-5 signaling is essential for the joint and articular cartilage formation. Ihh is another important molecule for skeletal development, Ihh inhibits Wnt-9a expression and is maintained in skeletal growth and endochondral ossification, as when it reaches a certain size decreases the expression of Ihh and thereby activates the expres‐ sion of Wnt patway induces hypertrophy of chondrocytes and bone formation [87]. It is noteworthy that during the OA Wnt signaling is overactivated [65] and GDF-5 is downregulated, which suggests a recapitulation of endochondral ossification during OA. Furthermore, when the receptor Bmp-RIA is inactivated in mouse generated phenotypes similar to human osteoarthritis and when activated the Wnt pathway by blocking antago‐ nist Dkk [64], reverse the process of articular cartilage destruction and endochondral ossification, this suggests that these pathways permit the maintenance of adult articular During development are constantly chondrocytes proliferation and differentiation, thus skeletal elements grow in length and ossify, as mentioned earlier, articular cartilage chondro‐ cytes have a low rate of proliferation and differentiation, this makes them different and allows articular cartilage is kept almost throughout life. What keeps the ever-growing cartilage during development is the molecular signals that modulate the rate of growth and differentiation, these signals are regulated by the perichondrium. The perichondrium has progenitor cells that are very useful for cartilage repair, its similar to bone, the periosteum is important for bone repair, such as fractures. While the perichondrium is maintained until adult stages, the perichondrium is disappearing from the stage young individuals, which is why the low capacity of regeneration of cartilage [88].
