Cartilage Biochemistry and Morphology

The hyaline cartilage could be regarded as a composite gel with relatively low percentage chondrocytes (5%) embedded in a rich extracellular matrix consisting in negatively charged hydrophilic proteoglycans constrained by a three-dimensional collagen network.

The negatively charged proteoglycans have the ability to form large aggregates, which can bind water molecules within the positively charged collagen fibrils, thus generating a high osmotic pressure within the gel.

The collagen fibers are responsible for the structure of cartilage and consist mainly of collagen type II. They are highly cross-linked via collagen type IX fibers.²

Chondrocytes are the producers of the surrounding ground substance: matrix.

The cells have different appearances depending on where in the cartilage they are situated. The cells in the top layer appear flattened, whereas the cells in the deeper layer are more rounded and aligned along vertically orientated type II collagen.³

Collagen is the most important scaffolding material in the body, existing in several types. The major type in hyaline cartilage is named type II. It is built by three identical polypeptide alpha-chains. These chains are coiled to form a triple-helix and are produced by the chondrocyte in the form of procollagen. Outside the cell, this procollagen is transformed to tropocollagen, and these molecules aggregate to form the much larger molecule: collagen.

In the hyaline cartilage there also exist minor collagens like types IX, XI, V, and VI. Type IX contributes with covalent cross-linking of the type II fibrils, whereas type XI is thought to control the diameter of type II fibrils. The collagen gives the cartilage its strength and tensile stiffness.

Proteoglycans are large protein-polysaccharide molecules making up 5% to 10% of the wet weight of the cartilage.⁴ They are composed by chains of the glycosaminoglycans keratan sulfate and chondroitin sulfate covalently bound to a central protein core molecule. Large aggregates are formed with several proteoglycan monomers via a link protein connecting the central protein cores to a chain of hyaluronic acid.

All the components of the proteoglycan aggregates are synthesized by the chondrocytes.

The proteoglycans are unevenly distributed throughout the cartilage layers with the highest concentration in the middle part and the lowest concentration in the superficial layers.⁵ The proteoglycans give the cartilage its elasticity and resilience.

There is a difference in cartilage composition between the cartilage surface and the subchondral bone plate. These structural differences give rise to four separate layers or zones (see Fig. 1-1).

FIGURE 1-1 Schematic drawing of the different layers in a full-thickness osteocartilaginous biopsy.

In the top zone, the superficial zone, there is first a cell-free fibril-layer, called the lamina splendens.⁶ Beneath this thin layer, chondrocytes are dispersed in an elongated manner parallel to the surface, reflecting as well the tangential orientation of the collagen fibers. This is the tangential layer.

In the second zone, often called the transitional layer, the cells are larger, rounded, and randomly distributed between the oblique-oriented collagen fiber. In the third zone, the chondrocytes are even larger and arranged in typical columns because of the radial collagen fiber courses, the radial zone.

The fourth layer, finally, which is mineralized, is called the calcified zone. There exists a visible border between the third and fourth zone, the tidemark with a special affinity for basic dyes (e.g., toluidine blue).

The calcified zone provides an important transition to the less resilient subchondral bone. For a long time this was regarded more or less as an inactive zone, until Hunziker (1992)⁷ noted that also the chondrocytes here could take up and incorporate (35S) sulfate into the pericellular and territorial matrix. Hunziker speculated that, following trauma, the metabolic activity here becomes temporarily impaired.⁷

Regarding experimental animals, it is important to know that it is only in adult animals that the division into zone I to zone III is possible.⁸ In the immature animal, the cells are more randomly distributed with a gradient in cell size from the top to the calcified zone, with the cells in the deeper parts being largest. Thus, the articular cartilage organization during prepubertal growth imitates the structure of the growth plate, and during that time the biomechanical properties of the cartilage change with an increase in stiffness and in shearing and compressive resistance.^7,9

Metabolic Events in the Cartilage

Under normal conditions, the components of the matrix have a slow turnover. The collagen has the slowest turnover rate compared to the much faster turnover of the proteoglycans.

The majority of the proteoglycans have a life span of about 600 days, but a small proportion of the proteoglycans in adult cartilage act as a fast fraction with a half-life of about 8 days. The proteoglycans are thus also more vulnerable to enzymatic degradation.^10,11

The chondrocytes secrete different enzymes called metalloproteinases (collagenases, gelatinases, and stromelysin), which all control the degree of degradation. The degradation of proteoglycans is followed by an increased synthesis of proteoglycans, which then become integrated into the matrix.

This is a sophisticated and well-balanced process regulated by the chondrocytes, and a disturbance of any of these events could lead to destruction of the cartilaginous matrix. This happens in osteoarthritis where an early sign is an imbalance in synthesis and degradation of the matrix.

Pathology Evaluation

With a biopsy from a cartilage area and with the help of a microscope, a lot of information about the composition and organization of the cartilage matrix can be made.

It is important to know that a biopsy with histology provides information about a very small part of the cartilage tissue and a small part of a repaired area. It is subsequently very important to know the following:

It is necessary to know the exact location from where the biopsy is taken and how it was taken. The International Cartilage Society (ICRS) has developed a mapping system, which is useful to utilize when describing the location.¹² A biopsy should be taken in the following manner:

A 2-mm biopsy going through all layers and down through bone is recommended. There are several types of instruments on the market, but it is easiest to employ a biopsy instrument used for bone marrow biopsies such as a Jamshidi needle.

A pathologist with special interest in cartilage tissue evaluation is involved in the assessment of the biopsies.

Biopsies can be either wax embedded for sections or frozen for cryo sections.

As recommended by the ICRS Histology Endpoint Committee,¹³ cartilage morphology can be evaluated by hematoxylin and eosin staining. Normal and polarized light are used.

Additional stainings with toluidine blue, alcian blue, or safranin O are used for evaluation of glycosaminoglycan content.

Immunohistochemical staining is done for evaluation of degree of collagen types I and II (best done on frozen sections).

Mineralization is studied with von Kossa technology.

Molecular Biology

In situ hybridization studies the genes that the chondrocyte expresses and indicates what the cells are capable to synthesize.¹⁴

Histological Repair Scores

Various histological scoring systems exists for analysis of osteoarthritic or normal, in vivo repaired, or tissue-engineered cartilage, but only a few have been validated:¹⁵

Imaging Evaluation of Cartilage Repair

To get a better total idea of the repair area, imaging techniques need to be used.

The clinical doctors need to know more about the type of cartilage lesion before arthroscopy in order to select the best repair choice about the quality of the induced repair, when to evaluate the symptoms post surgery, and when to intervene and how.

Arthrography combined with computed tomography (CT) provides information about the contour and surface characteristics of the cartilage and cartilage repair.²³ Combined with three-dimensional reconstruction of the bone structure, useful information is yielded.

Recent developments in MRI technology used in the field of musculoskeletal research have created new possibilities by providing precise and reliable quantitative information on the joint structure as well as changes over time.

The main advantages of MRI as a method for cartilage imaging are as follows:

Quantitative measurement of morphology can be used to monitor loss of cartilage tissue, but there is extensive interest in using MRI to detect changes that precede gross tissue degradation that may occur in early disease.

Such mapping techniques to image compositional changes that may be sensitive to early cartilage damage include T2 mapping, delayed gadolinium enhanced MRI of cartilage (dGEMRIC), and T1rho.

Fat-suppressed three-dimensional gradient echo (3D-GRE)²⁴ allows the exact description of the thickness and surface of cartilage.

T2-weighted (dual) fast spin echo (FSE) techniques with or without fat-suppression²⁴ give the information about the normal and abnormal internal structure of hyaline cartilage.

dGEMRIC²⁵ relies on intravenous injection of a negatively charged MR contrast agent and the acquisition of a T1 map after equilibration of the contrast agent in the cartilage to estimate the glycosaminoglycan distribution within cartilage.

Quantitative T2 MR²⁶ mapping of articular cartilage is a noninvasive imaging technique that has the potential to characterize hyaline articular cartilage and repair tissue.²⁴ Normal articular cartilage demonstrates an increase in T2 values from the subchondral bone to the articular surface that has been correlated with type II collagen fiber matrix organization (anisotropy) in these zones.

Qualitative and quantitative T2 mapping helped differentiate hyaline cartilage from reparative fibrocartilage after cartilage repair at 1.5-T MR imaging.²⁴

Cartilage T2 mapping at 1.5-T MR imaging shows promise as a noninvasive tool to study and differentiate cartilage composition after surgical cartilage repair procedures.²⁶

One MRI technique, magnetization transfer (MT) imaging,²⁷ is known to generate a useful image contrast in cartilage in vitro, which is sensitive to the macromolecular content of the cartilage. Palmieri and coworkers²⁷ have studied cartilage repair with microfracture, comparing the repair with autologous chondrocyte implantation (ACI) repair. The differences between damaged and repaired cartilage magnetization transfer ratio (MTR) were too small to enable MT imaging to be a useful tool for postoperative follow-up of cartilage repair procedures. However, there was an evolution toward normal MTR values in the cartilage repair tissue (especially after ACI repair), while the MTR of microfracture repaired cartilage still showed a significant difference from normal cartilage at a 24-month follow-up.²⁷

Furthermore, another technique called T1rho can potentially be used to noninvasively to quantitatively assess the biochemical and biomechanical characteristics of articular cartilage in humans during the progression of osteoarthritis.²⁸