Disposiciones mínimas de seguridad y salud en el trabajo

The description of articular cartilage as a tissue with lubricating properties found on the ends of bones emerged as early as the 4th _{century and the first account of osteoarthritic}

cartilage was recorded in 1741. Up until the late 19th _{century, however, knowledge of the}

structure of articular cartilage remained very limited – fibrillar collagen and chondroitin sulphate were recognised as major components and zonal differences in chondrocyte distribution had been established (rounded cells in the deep tissue and flatter ones near the surface). Though it was also known to be an avascular tissue, its mode of mass transport was in dispute – one faction deemed the synovial fluid to be responsible, while the other favoured the subchondral blood vessels. With the development of the electron microscope and radio isotope technology in the mid-19th _{century, and improved chemical methods}

shortly thereafter, more detailed information about the structure of articular cartilage rapidly began to emerge (Benedek 2006). Around 1960 it was determined that chondroitin sulphate, along with smaller quantities of other polysaccharides such as keratan sulphate, were bound to a core protein and that these chondroitin sulphate-protein complexes probably formed aggregates (Partridge, Davis, and Adair 1961).

From this point a great deal of work, spanning the next five decades (figure 1-4) and pioneered by the likes of Helen Muir, Vincent Hascall, Stanley Sajdera, Dick Heinegård and Tim Hardingham, resulted in the model of articular cartilage that we have today (figure 1-

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5). In 1969 Sajdera and Hascall published their “dissociative method” for extracting protein- polysaccharide complexes from bovine nasal cartilage (Sajdera and Hascall 1969). Unlike the earlier “disruptive” method which involved high speed homogenisation of the tissue and resulted in denaturisation and depolymerisation of its macromolecules, this new technique involved gentle agitation of samples in high ionic strength solutions to yield intact, disaggregated protein-polysaccharide complexes. This allowed for much closer interrogation of the tissue and from then on, a detailed picture of its structure began to emerge. In 1971 collagen type II (COL2) (described by Miller and Matukas in 1969) was identified as the tissue’s predominant collagen (Strawich and Nimni 1971). Bovine articular cartilage was incubated with papain at 4°C and washed with 0.15 M NaCl. Extraction with 0.45 M NaCl resulted in a 20% collagen in solution. After further purification to remove associated GAGs, a triple stranded molecule of COL2 was the only component observed.

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Figure 1-4. Timeline of developing model of articular cartilage. COL2 = collagen type II. GAG = glycosaminoglycan. PG = proteoglycan. HA = hyaluronic acid. PRELP = protein/arginine-rich end leucine-rich repeat protein. CHAD = chondroadherin. COMP = cartilage oligomeric matrix protein. CILP = cartilage intermediate protein. 1_{(Partridge, Davis, and Adair 1961)} 2_{(Sajdera and Hascall 1969)} 3_{(Strawich and Nimni 1971)} 4_{(Rodén et al. 1972)} 5_{(T. E. Hardingham and Muir 1972)} 6_{(Timothy E. Hardingham and Muir 1974)} 7_{(Paulsson and Heinegård 1979)} 8_(Heinegård

et al. 1986) 9_{(Larsson et al. 1991)} 10_{(Hedbom et al. 1992)} 11_{(P. Lorenzo, Bayliss, and Heinegård 1998)} 12_{(P. Lorenzo et al. 2001)} 13_{(Wiberg et al.}

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Figure 1-5. Dick Heinegård’s final model of cartilage structure. CHAD = chondroadherin, COMP = cartilage oligomeric matrix protein, HA = hyaluronic acid, KS = keratan sulphate, CS = chondroitin sulphate, PRELP = protein/arginine-rich end leucine-rich repeat protein. Adaptedfrom Hascall, 2014.

Isolation and characterisation of proteoglycans from cartilage tissue (usually bovine/porcine nasal or tracheal when much of the early work was conducted) via the dissociative method involves incubation with a dissociative solution of 3-4 M guanidine hydrochloride (GuHCl) to extract PGs, which are then re-aggregated with 0.5 M GuHCl. Aggregates are separated from unrelated proteins via caesium chloride (CsCl) density gradient centrifugation to produce an “A1 fraction” of PG aggregates. Further CsCl density centrifugation and incubation with dissociative 4M GuHCl results in a high density PG subunit and a lower density glycoprotein link (necessary for subsequent re-aggregation), which can be further separated into Link 1 and Link 2 with dissociative density gradients (V.

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C. Hascall and Heinegård 1974). This method was utilised for much of the work that followed.

In 1971 Hardingham and Muir showed that small amounts of HA interacted with disaggregated PGs to give a stable increase in hydrodynamic size. Furthermore, this increase peaked at a HA concentration of 1%, suggesting that above this, HA molecules were competing for available PG. This interaction was specific for HA and it was estimated that 10-30 PG molecules were associated with each HA chain (T. E. Hardingham and Muir 1972). The following year the pair determined that each PG must have only one HA binding site, as they were unable to crosslink multiple HA chains. They also observed that each PG binds to a region around the size of a decasaccharide unit (4-5 nm) and, given that each PG occupies around 45 nm on a HA chain, deduced that they must be relatively spread out (Timothy E. Hardingham and Muir 1973). In 1974 they showed that the fraction isolated from cartilage, between the PG subunit and the glycoprotein link was in fact HA and that most PGs associated with it to form aggregates. Smaller, non-aggregating PGs with lower protein and keratan sulphate content were also identified (Timothy E. Hardingham and Muir 1974). Around the same time Hascall and Heinegård also observed large amounts of HA associated with PG aggregates (around 0.8%). These HA molecules were susceptible to degradation by chondroitinase, hyaluronidase and papain, but this was thwarted in the presence of chondroitin sulphate, which has a higher enzyme affinity; all of which suggests that HA is surrounded by PGs (V. C. Hascall and Heinegård 1974). Further work that year confirmed that most of the PGs in cartilage are aggregating, resistant to trypsin and papain digestion, and that link proteins are also present and necessary for their stabilisation (Vincent C. Hascall and Heinegård 1974; Heinegård and Hascall 1974). Further characterisation of

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aggregating PGs followed in subsequent years, but the basic model is much the same as that shown in figure 1-8.

In 1979 Paulsson et al. reverted to the disruptive/associative method of proteoglycan extraction in order to preserve the secondary structure of polypeptides, which can be damaged by chaotropic salts such as GuHCl (Paulsson and Heinegård 1979). A series of chemical analyses revealed a novel molecule, termed “cartilage matrix protein” (later renamed matrilin 1), which formed stable complexes with link proteins and PG monomers and aggregates. This protein, thought to have a role in PG/collagen interactions, had a high molecular mass and was formed of two subunits joined by disulphide bridges. It was later found to interact with biglycan/decorin and COL2 (Wiberg et al. 2003). The discovery of two more novel matrix proteins (protein/arginine-rich end leucine-rich repeat protein (PRELP) and fibromodulin), each formed of a single polypeptide chain, followed a few years later (Heinegård et al. 1986; Vincent C. Hascall 2014). In 1991, Larsson et al. identified yet another matrix protein (later termed chondroadherin (CHAD)), which was subsequently found to interact with integrin α2ß1 (Haglund et al. 2011). Cartilage oligomeric matrix protein (COMP) and its interactions with collagens were described in 1992 (Hedbom et al. 1992) and the discovery of cartilage intermediate protein (CILP) followed in 1998 (P. Lorenzo, Bayliss, and Heinegård 1998). Finally in 2001, the identification of asporin, a protein unable to bind GAGs and thought to have a role in the stabilisation of collagen networks, completed Dick Heinegård’s final model (figure 1-5) (P. Lorenzo et al. 2001).

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