**2. Anatomy of articular cartilage**

Understanding the form and function of articular cartilage is the cornerstone to developing successful treatments for articular cartilage damage. Articular cartilage is a tissue that bears load and forms the articulating surfaces of diarthrodial joints. Articular cartilage dissipates loads, has low friction, provides lubrication, and can last up to 8 decades.

Articular cartilage is predominately composed of extracellular matrix (ECM) with a sparse population of chondrocytes that help to maintain the ECM. The major components of the

Articular Cartilage Regeneration with Stem Cells 131

molecules for example bind with hyaluronic acid to create macromolecules (proteoglycan aggregate). These macromolecules are effectively immobilized in the collagen ECM. There tends to be a higher concentration of proteoglycans in the ECM closest to the chondrocytes. Proteoglycans are made up of repeating dissacharaide subunits called glycosaminoglycans (GAG). There are 3 types of GAGs found in cartilage 1. chondroitin sulfate, 2. keratin sulfate, and 3. dermatan sulfate. Chondroitin sulfate is most abundant and with age chondroitin-4 sulfate is found to decrease while keratin sulfate increases. These changes with age or arthritis can directly affect the properties of the cartilage. For example, with age, articular cartilage has decreased water content but with osteoarthritis it has increased water

To image articular cartilage, standard hematoxylin and eosin (H&E) is sufficient to visualize cartilage damage and clinical use (Fig 2). However, additional stains can provide more specific information about the ECM, proteoglycans, and chondrocytes. For proteoglycans cationic dyes such as Safranin-O and Alcian blue are typically used. Safranin-O stains polysaccharides (both carboxylated and sulfated) orange. Alcian blue can stain for both types of polysaccharides (pH 2.5) or be more specific for sulfated polysaccharides (pH 1.0) such as chondroitin sulfate, turning them turquoise (Horvai 2011). A Trichrome (Gomori or Masson's) stain highlights the orientation of collagen fibrils with a bright blue stain, while staining cytoplasm and other proteins red. Additionally, elastin fibers which are abundant in elastic cartilage are typically visualized with a silver stain such as a Verhoff stain where the fibers stain black. Cartilage staining can provide clear visualization of the cartilage profile in a specific location, however it is more qualitative than quantitative. To obtain more quantitative measurements of protein content Polymerase Chain Reaction (PCR) and

Partial and full thickness cartilage defects have a limited ability for healing. This is in part attributed to the avascular properties of cartilage, limited stem cell population, as well as the hypoxic environment of diarthrodial joints. Additionally the mechanical loads in the joint can make articular cartilage healing a challenge. The full natural history of full thickness chondral defects is not well documented in the literature. However it is thought that full thickness defects left untreated lead to joint space narrowing and degenerative arthritis

Cartilage injuries can occur with a twisting, shearing type injury in combination with axial loading. These defects are commonly associated with concomitant knee pathology such as meniscus tears, anterior cruciate ligament tears, medial collateral or lateral collateral ligament tears. There is a 5-10% incidence of full thickness chondral lesions following acute hemarthrosis (Noyes et al, 1980). In athletes, there is approximately a 36% prevalence of full thickness focal chondral defects, with 14% being asymptomatic

Patients who have symptomatic chondral lesions typically present with pain localized to the compartment of injury, increased with weight-bearing of that compartment. They may also have recurrent effusions, catching, locking, or other mechanical symptoms. The physical examination typically will demonstrate crepitance, joint effusion, tenderness along ipsilateral

content (Mankin et al, 2000).

**2.1 Histology of cartilage** 

other molecular techniques are needed.

**3. Cartilage injury** 

(Messner & Maletius, 1996).

(Flanigan et al, 2010).

ECM are water, collagen, and proteoglycans. These combine with the chondrocytes to form the complex structure of articular cartilage which varies throughout its depth. The structure of articular cartilage is typically divided into 4 zones (superficial, middle, deep, and zone of calcified cartilage). The superficial zone or tangential zone is adjacent to the joint cavity and forms a gliding surface. The superficial zone is characterized by thin collagen fibrils that are aligned parallel with the articular surface. This zone also has disk shaped chondrocytes, low proteoglycan content, with high collagen and water contents. The middle (transitional) zone is characterized by large diameter collagen fibers which are oriented obliquely, round chondrocytes, and an increased proteoglycan content. The deep (radial) zone has the highest proteoglycan content with collagen fibers oriented perpendicular to the joint surface. The chondrocytes in the deep zone are round and organized into columns. The deepest layer is the zone of calcified cartilage and separates the hyaline cartilage from the subchondral bone. This zone is characterized by collagen fibrils that are radially aligned with round chondrocytes that are buried in calcified matrix. This zone has a low concentration of proteoglycans and a high concentration of calcium salts (Fig 1).

Fig. 1. This figure illustrates the zones of hyaline cartilage: superficial, middle, deep, and zone of calcified cartilage. Lacunae are present in hyaline cartilage and typically contains 1-2 chondrocytes. There is a periphery of increased proteoglycan content around each chondrocyte and lacuna.

Articular cartilage is avascular and obtains its nutrition from the diffusion of synovial fluid through the ECM and from underlying bone. Chondrocytes produce ECM components in response to chemical (growth factor and cytokines) and physical (mechanical load, hydrostatic pressure) stimuli. The ECM is composed primarily of water 65-80%, collagen (type II) 10-20%, and aggrecan 4-7%. Other components of the ECM make up less than 5% of articular cartilage. These include proteoglycans, biglycan, decorin, fibromodulin, various collagen types (V, VI, IX, X, XI), link protein, hyaluronate, fibronectin, and lipids. The role of each of these molecules is not fully understood. Collagen functions to provide shear and tensile strength to the cartilage. Proteoglycans are produced and secreted by the chondrocytes. Proteoglycans are tangled in between collagen fibers creating an ECM that inhibits the movement of water and provides compression strength of cartilage. Aggrecan molecules for example bind with hyaluronic acid to create macromolecules (proteoglycan aggregate). These macromolecules are effectively immobilized in the collagen ECM. There tends to be a higher concentration of proteoglycans in the ECM closest to the chondrocytes. Proteoglycans are made up of repeating dissacharaide subunits called glycosaminoglycans (GAG). There are 3 types of GAGs found in cartilage 1. chondroitin sulfate, 2. keratin sulfate, and 3. dermatan sulfate. Chondroitin sulfate is most abundant and with age chondroitin-4 sulfate is found to decrease while keratin sulfate increases. These changes with age or arthritis can directly affect the properties of the cartilage. For example, with age, articular cartilage has decreased water content but with osteoarthritis it has increased water content (Mankin et al, 2000).
