• Articular cartilage is a biphasic material that is an A-list basic science topic with predictable questions, with themes applying structure to function.
• It is a regular component for Part 1 SBA and Part 2 basic science viva, with some overlap in the adult pathology viva section, especially osteoarthritis.

• There are three main types of cartilage that are distinguished by their prototeoglycan content and collagen structure: (1) hyaline articular cartilage characterised by type II collagen and aggregating proteoglycans; (2) elastic cartilage characterised by a high proportion of elastic fibres; and (3) fibrocartilage characterised by type I collagen and less proteoglycan content (meniscus).
• The main function of hyaline articular cartilage is to provide a smooth lubricated weight-bearing joint with minimal friction.
• It is devoid of nerves, vessels and lymphatics, with the synovial fluid providing nutrition and oxygen via diffusion.
• pH of cartilage: 7.4.
• Consists of extracellular matrix (ECM) and chondrocytes.
• ECM primarily contains water, collagen and proteoglycans. It is responsible for the mechanical properties of cartilage.
• The ECM is described as four zones:

1. Superficial
2. Middle
3. Deep
4. Calcified

• The matrix is also divided into three distinct regions with respect to the distance from the chondrocyte cell membrane:

1. Pericellular
2. Territorial
3. Interterritorial

• The superficial (tangential) zone is the thinnest layer and provides most of the cartilage’s tensile strength and protects the deeper zones; it is primarily formed from type II and IX collagens, with fibres aligned parallel to the articular surface forming a dense mat.
• At the very surface of the gliding zone is a layer called the lamina splendens. This contains no cells, a clear film of fine collagen fibrils with little proteoglycan.
• The superficial zone has a high density of chondrocytes that synthesise high concentrations of collagen and a much lower concentration of proteoglycans.
• Good resistance to shear forces during joint movement due to tangential arrangement, greatest tensile strength.
• Low metabolic activity hence low healing potential.
• The highest concentration of collagen and the lowest concentration of proteoglycan

Figure 1. Collagen and cellular arrangement of articular cartilage

Figure 2. H&E stain and schematic representation of hyaline cartilage morphology and structure. SZ, superficial zone; MZ, middle zone; DZ, deep zone; CZ, calcified zone; SB, subchondral bone. Picture used with permission obtained from J Cytochem Biochem.

• The middle (transitional) zone is the largest layer and acts to limit compressive force, with collagen fibrils aligned obliquely to the articular surface.
• High concentration of proteoglycans.
• Lowest concentration of water.

• The deep zone provides the greatest resistance to compression, helped by its collagen fibrils being perpendicular to the articular surface and a higher concentration of proteoglycans.
• Compared to the other layers, the deep zone contains thicker collagen fibrils.
• Cells spherical arranged in vertical columns.

• The calcified zone contains fewer cells and acts to anchor the deep zone collagen to the subchondral bone. The tidemark is the term to describe the boundary between the deep and calcified zones.

• A basophil line marking the precipitation of calcium salts).
• Provides resistant to shear.
• It is the boundary between the calcified and uncalcified cartilage. It is cell free and represents a calcification front.
• The collagen fibres in the deep zone penetrate through the tidemark into the calcified cartilage to provide structural stability for articular cartilage on the subchondral bone.

• Matrix regions differ in their proximity to chondrocytes, collagen content, collagen fibril diameter, collagen fibril orientation and proteoglycan and noncollagenous protein content and organisation.
• The pericellular matrix is a thin layer adjacent to the cell membrane, and it completely surrounds the chondrocyte. It contains mainly proteoglycans, as well as glycoproteins and other non-collagenous proteins. This matrix region may play a functional role to initiate signal transduction within cartilage with load bearing.
• The territorial matrix surrounds the pericellular matrix; it is composed mostly of fine collagen fibrils, forming a basket-like network around the cells. This region is thicker than the pericellular matrix, and it has been proposed that the territorial matrix may protect the cartilage cells against mechanical stresses and may contribute to the resilience of the articular cartilage structure and its ability to withstand substantial loads.
• The interterritorial region is the largest of the three matrix regions; it contributes most to the biomechanical properties of articular cartilage. This region is characterised by the randomly oriented bundles of large collagen fibrils, arranged parallel to the surface of the superficial zone, obliquely in the middle zone, and perpendicular to the joint surface in the deep zone. Proteoglycans are abundant in the interterritorial zone.

Figure 3. Three matrix regions with increasing distance from the chondrocyte

Figure 4. Proteoglycan aggrecan and aggregate

Table 1. Constituents of articular cartilage

• Derived from mesenchymal stem cells, chondrocytes produce and maintain ECM, and are the main cell type of articular cartilage.
• Chondrocytes have no contact with neighbouring cells and are spheroidal.
• There are distinct subpopulations of chondrocytes in the different zones of cartilage whose properties differ in terms of their morphology, metabolism, phenotypic stability and their response to cytokines.
• The morphology of the cell varies according to the zone; for instance, the superficial zone contains a high density of flat cells, compared to the larger cells of the deeper zone.

Figure 5. Cell morphology of chondrocytes.Superficial zone where flattened chondrocytes are located,middle zone containing elongated chondrocytes ,the deep zone where chondrocytes are arranged in columns and at the bottom calcified zone.The tide mark and calcified zone forms a junction between bone and non-calcified cartilage tissue..  

• ECM is produced and maintained by chondrocytes.
• Water occupies up to 80% of the total weight, with 65% in the deep zone and 80% in the superficial zone. Several inorganic ions such as calcium, sodium and potassium are dissolved. Water permits load-dependent deformation, nutrition and lubrication.
• In osteoarthritis, the water content increases due to increased permeability.
• Collagen represents up to 60% of the dry weight, and the ECM is predominately composed of type II collagen (~90%), although other types of collagens are also present.
• Proteoglycans are glycosolated monomers that increase the resilience to compressive forces. ECM contains several proteoglycans that include aggrecan, decorin and fibromodulin.

• Water makes up 65–80% of the ECM.
• The distribution is 80% at the superficial layers and 65% at the deeper layers
• Water permits load-dependent deformation of articular cartilage by its movement both in and out and within cartilage.
• Increased water content leads to increased permeability, decreased strength and decreased elasticity.
• Water provides a medium for lubrication.

• Water is attracted and retained in articular cartilage by the ionic pressure created by the high level of negative charges on glycosaminoglycan chains on proteoglycan molecules.
• The ionic pressure creates a swelling pressure which will keep imbibing water until it is resisted by the tension in the fibrous component of cartilage, the collagen fibres.
• As proteogylcans are bound closely, the closeness of the negative charges creates a repulsion force that must be neutralised by positive ions in the surrounding fluid. The amount of water present in cartilage depends on the concentration of proteoglycans and the stiffness and strength of the collagen network. The proteoglycan aggregates are basically responsible for the turgid nature of the cartilage and in articular cartilage they provide the osmotic properties needed to resist compressive loads. If the collagen network is degraded, as in the case of osteoarthritis, the amount of water in the cartilage increases, because more negative ions are exposed to draw in fluid. The increase in fluid can significantly alter the mechanical behaviour of the cartilage.

• Collagen provides indirect support of articular cartilage by balancing the osmotic swelling effect of the proteoglycans.
• The collagen network in articular cartilage is highly anisotropic which confers complex mechanical properties.
• The Benninghoff model illustrates the variation of collagen alignment with depth into cartilage. There is a tangential orientation at the articular surface, passing through a gothic arch arrangement into a radial alignment.
• The collagen content also varies with depth, being most abundant at the surface and least abundant in the deep zone.

Collagen Structure

• Collagen consists of three polypeptide chains that form a triple helix along part of their length.
• They can be divided into fibrillar and non-fibrillar collagens.
• The fibrillar collagen consists of triple helix molecules, arranged head to tail in linear arrays and side to side in a quarter staggered manner.
• Cartilage consists of several types of collagen.
• The main collagen in articular cartilage is type II accounting for 90–95% of the collagen.
• Types II, IX and XI form a mesh that serves to trap proteogylcans providing for stiffness and strength.
• Type VI helps chondrocytes adhere to the matrix. Increases in early osteoarthritis.
• Type XI constrains the proteoglycan matrix.
• Type X is only found near the calcified zone.
• The triple helix of type II collagen is made up of three amino acid chains with a repeating tripeptide pattern: glycine-X-Y, where X is mainly proline and Y is mainly hydroxyproline. The molecules coil around each other, because glycine is always at the centre of the helix.
• In some inherited osteoarthritic conditions there is abnormal spacing of the glycines stopping the normal tight twist of the three helices. Therefore, collagens are weaker and when high stresses are applied to areas of a joint this will damage the collagen fibres.

Figure 6. Diagram illustrating the ansiotopic alignment of collagen fibers in articular cartilage.

• Proteoglycans are complex macromolecules that trap and hold water providing the tissue with its turgid nature that resists compression.
• They give articular cartilage its compressive strength and elasticity.
• They are secreted by chondrocytes and composed of subunits called glycosaminoglycans.
• The most common glycosaminoglycan in articular cartilage is chondroitin sulphate (of which there are two subtypes, chondroitin-4 sulphate and chondroitin-6 sulphate), then the keratin sulphate and dermatan sulphate.
• Chondroitin-4 sulphate is the most abundant and decreases over the years; chondroitin-6 sulphate remains constant; and keratin sulphate increases with age.
• Glycosaminoglycan link to a protein core by sugar bonds to form a proteoglycan aggrecan.

• Aggrecan molecules do not exist in isolation within the ECM, but as proteoglycan aggregates.
• Each aggregate is composed of a central filament of hyaluronic acid with up to 100 aggrecan molecules radiating from it, with each interaction stabilised by the presence of a link protein.

Figure 8. Diagram illustrating the ansiotopic alignment of collagen fibers in articular cartilage.

• Proteoglycans have an average lifespan of 3 months and have a great capacity for retaining water, which gives elasticity to the tissue.

• Articular cartilage undergoes significant structural, matrix composition and mechanical changes with age.
• There is disruption of the collagen–proteoglycan matrix. This reduces its compressive stiffness. Leeching out of the proteoglycan leads to increased permeability and reduced stiffness. Increased permeability leads to loss of lubricant.
• There is an alteration in the distribution of chondrocytes within each of the zones. Moreover, the water content of the ECM decreases, with loss of its ability to withstand deformation.

Growth plate

Zones of the growth plate

• Reserve zone
• Proliferative zone
• Hypertrophic zone: Maturation, Degeneration, Provisional calcification
• Primary spongiosa
• Secondary spongiosa

Physeal-metaphyseal junction

Weakest link of the growth plate-need to decrease shearing stress
Mamillary processes - Microscopic irregularities
Undulations -Macroscopic contouring

Periphery of the physis

Groove of ranvier

• Wedge of cells laterally, supplies chondrocytes to the periphery of the growth plate for lateral growth [Increases the width of the growth plate]

Perichondral ring of lacroix

• Dense fibrous band at the periphery growth plate, anchors and supports the physis

Reserve zone

Resting zone, involved in matrix production, storage lipids, glycogen and proteoclygan aggregates.
Contains germinal cells [stem cell population] existing singly or in pairs separated by an abundant extracellular matrix and not clearly ordered in columns      
Cells are in a relatively quiescent state
Low PO₂ tension as epiphyseal arteries pass through this region but do not form terminal capillaries.
Injury to this layer results in cessation of growth

Proliferative zone

Chondrocytes are highly ordered in columns directed along the axis of growth of the long bone.
Longitudinal growth occurs with stacking of chondrocytes
The top cell is the dividing mother cell or progenitor cell on which the entire growth in length of bone depends 
This zone is involved with cell division and matrix production
High PO₂ tension and high proteogylcan concentration [inhibits minerisation]
Failure of progenitor cells to thrive results in termination of growth at the long bone end

Hypertrophic zone

Zone involved in the maturation of cells
Cells abruptly increase in size [x5], accumulate calciumin their mitochondria and then die releasing calcium from matrix vesicles[ which allows calcification of the matrix]
Columns of cartilage cells extend toward the metaphysis being constantly lengthened by cell division occurring at the base[Proliferative zone]
Cells nearer the metaphysis begin to undergo changes that ultimately lead to their destruction
Low PO₂ tension
The rate of chondrocyte maturation is regulated by systemic hormones and local growth factors
Marked increase in alkaline phosphatase enzyme activity
This enzyme increases the concentration of phosphate ions, which are required in the calcification process
In hypophosphatasia there is an absence of alkaline phosphatase and deficient mineralization of the matrix resulting in widening of the growth plate                
Physeal fractures are classically believed to occur through the zone of provisional calcification [within the hypertrophic zone]
Although it complicates matters slightly it is probably best to learn the three zones of subdivision of this layer

Maturation zone

Preparation of matrix for calcification

Degeneration zone

Cell deterioration and death

Provisional calcification zone

The last two or three cells in  the column of cartilage cells are in the zone of provisional calcification.

Primary spongiosa

Vascular invasion
Ingress of vascular channels and bone-marrow stromal cells
Calcified cartilage bars resorbed by osteoblasts, formation woven bone

Secondary spongiosa

Remodelling to lamellar bone

Growth plate signalling pathways

• The height of the growth plate is tightly controlled by a local feedback loop between parathyroid hormone-related protein (PTHrP) and Indian hedgehog (IHH).
• PTHrP produced at the resting zone suppresses the differentiation of chondrocytes while IHH produced at the maturation zone stimulates the differentiation of chondrocytes and positively regulates the production of PTHrP.

Fibroblast growth factor (FGF) signalling mediated by FGFR3 is a negative regulator for chondrocytic proliferation.
BMP-2 expressed within the perichondrium promotes proliferation of chondrocytes through BMP type I receptors

• IHH and BMP signalling pathways act in parallel to induce chondrocyte proliferation and differentiation. In contrast, FGF signalling inhibits BMP signalling and negatively regulates IHH expression, suggesting that BMP signalling coordinately works with PTHrP/IHH and FGF signalling to control the balance between proliferation and differentiation of chondrocytes.
• PTHrP also acts on osteoblasts and osteoclasts in the calcifying zone, thus also enhancing bone formation and remodelling the newly constructed metaphysis.