Skeletal tissues

Introduction

The skeletal system is formed from highly specialised types of supporting/connective tissue. The tissues are made up of collagen and acellular matrix, as well as the cells which synthesise them. Bone provides a rigid protective and supporting framework, the rigidity resulting from the deposition of calcium salts within the collagen and matrix. Cartilage occurs in different forms and provides a smooth articular surface at bone ends, as well as structural support in special areas (e.g. trachea, pinna). It is also important in one form of new bone formation.

Joints are composite structures which join the bones of the skeleton and, depending on the function and structure of individual joints, permit varying degrees of movement. Ligaments are robust but flexible bands of collagenous tissue which contribute to the stability of joints. Tendons provide strong, pliable connections between muscles and their points of insertion into bones.

The functional differences between the various tissues of the skeletal system relate principally to the different nature and proportion of the ground substance and fibrous elements of the extracellular matrix. The cells of all the skeletal tissues, like the cells of the less specialised supporting/connective tissues, have close structural and functional relationships and a common origin from primitive mesenchymal cells (see Ch. 4).

Cartilage

The semi-rigid nature of cartilage stems from the predominance of proteoglycan ground substance in the extracellular matrix.

Proteoglycans (see Ch. 4), disposed in proteoglycan aggregates of 100 or more molecules, make up the ground substance and account for the solid, yet flexible, consistency of cartilage. Sulphated glycosaminoglycans (GAGs, chondroitin sulphate and keratan sulphate) predominate in the proteoglycan aggregates, with molecules of the non-sulphated GAG hyaluronic acid forming the central backbone of the complex. The different types of cartilage vary in the amount and nature of fibres in the ground substance: hyaline cartilage contains few fibres, fibrocartilage contains abundant collagen fibres and elastic cartilage contains elastin fibres.

Cartilage formation commences with the differentiation of stellate-shaped primitive mesenchymal cells (see Fig. 4.2) to form rounded cartilage precursor cells called chondroblasts. Subsequent mitotic divisions give rise to aggregations of closely packed chondroblasts which grow and begin synthesis of ground substance and fibrous extracellular material. Secretion of extracellular material traps each chondroblast within the cartilaginous matrix, thereby separating the chondroblasts from one another. Each chondroblast then undergoes one or two further mitotic divisions to form a small cluster of mature cells separated by a small amount of extracellular material.

Mature cartilage cells, known as chondrocytes, maintain the integrity of the cartilage matrix. Most mature cartilage masses acquire a surrounding layer called the perichondrium, composed of collagen fibres and spindle-shaped cells which resemble fibroblasts. These have the capacity to transform into chondroblasts and form new cartilage by appositional growth. There is also very limited capacity in mature cartilages masses for interstitial growth. This occurs by further division of chondrocytes trapped within the previously formed matrix and subsequent deposition of more matrix material. The hyaline cartilage of the articular surfaces of joints does not have perichondrium on the surface and has no capacity to regenerate new cartilage after damage. In general, mature cartilage has a very limited capacity to repair and regenerate, partly because of its poor blood supply.

Most cartilage is devoid of blood vessels, and consequently the exchange of metabolites between chondrocytes and surrounding tissues depends on diffusion through the water of the ground substance. This limits the thickness to which cartilage may develop while maintaining viability of the innermost cells. In sites where cartilage is particularly thick (e.g. costal cartilage), cartilage canals convey small vessels into the centre of the cartilage mass.

The role of cartilage in bone formation is discussed in Figs 10.18 to 10.21.

Cartilage

The various different forms of cartilage are specialised to perform distinct functions. The nature of the extracellular matrix differs in these subtypes.

Hyaline cartilage is found on the articular surfaces of many joints, lying over the surface of the bone ends and forming a smooth, firm, but slightly flexible surface. Hyaline cartilage has very abundant gel-like ground substance.

Fibrocartilage is found in the intervertebral discs as well as in sites such as the pubic symphysis. Fibrocartilage resembles dense connective tissue and has very abundant collagen fibres but with intervening bands of extracellular matrix.

Elastic cartilage is important in the structure of the external ear and the larynx, being firm but very flexible due to the presence of many elastin fibres within an extracellular matrix which is similar to that of hyaline cartilage.

FIG. 10.1 Hyaline cartilage
(a) H&E (MP) (b) Thin epoxy resin section, toluidine blue (HP)
Hyaline cartilage is the most common type of cartilage. It is found in the nasal septum, larynx, tracheal rings, most articular surfaces and the sternal ends of the ribs. It also forms the precursor of bone in the developing skeleton. Mature hyaline cartilage is characterised by small aggregates of chondrocytes embedded in an amorphous matrix of ground substance, reinforced by collagen fibres.
Micrograph (a) shows a hyaline cartilage mass with its outer perichondrium P. The chondrocytes of the formed cartilage Cc are arranged in clusters, usually of 2 to 4 cells, each cluster being separated from its neighbours by amorphous cartilage matrix M. The perichondrium is composed of parallel collagen fibres containing a few spindle-shaped nuclei of inactive fibrocytes but, on its inner surface, these cells are transforming into small chondroblasts Cb which are in the process of enlarging, dividing and synthesising new cartilage matrix.
The matrix of hyaline cartilage appears fairly amorphous, since the ground substance and collagen have similar refractive properties. With the exception of articular cartilage, the collagen of hyaline cartilage, designated as type II collagen (see Ch. 4), is not cross-banded and is arranged in an interlacing network of fine fibrils. This collagen cannot be demonstrated by light microscopy.
The thin epoxy resin section of hyaline cartilage in micrograph (b) shows the cellular details of mature chondrocytes. Note that the chondrocytes fully occupy the spaces in the matrix M, each space containing a single chondrocyte. Mature chondrocytes are characterised by small nuclei N with dispersed chromatin and basophilic granular cytoplasm, reflecting a well-developed rough endoplasmic reticulum. Lipid droplets L, often larger than the nuclei, are a prominent feature of larger chondrocytes. The cytoplasm is also rich in glycogen. These characteristics reflect the active role of chondrocytes in synthesis of both the ground substance and fibrous elements of the cartilage matrix. In fully formed cartilage, the constituents of the extracellular matrix are continuously turned over, the integrity of the matrix being thus absolutely dependent on the viability of the chondrocytes.

FIG. 10.2 Fibrocartilage
H&E/Alcian blue (HP)
Fibrocartilage has features intermediate between cartilage and dense fibrous supporting tissue. It is found in intervertebral discs, some articular cartilages, the pubic symphysis, in association with dense collagenous tissue in joint capsules, in ligaments and in the connections of some tendons to bone. It consists of alternating layers of hyaline cartilage matrix with thick layers of dense collagen fibres, orientated in the direction of the functional stresses.
This micrograph is taken from the same specimen of intervertebral disc illustrated in Fig. 10.30. Pink-stained collagen characteristically permeates the blue-stained cartilage ground substance. Chondrocytes Cc are arranged in rows between the dense collagen layers within lacunae in the glycoprotein matrix.

Cb chondroblast Cc chondrocyte L lipid droplet M cartilage matrix N nucleus P perichondrium

FIG. 10.3 Elastic cartilage
Elastic van Gieson (HP)
Elastic cartilage occurs in the external ear and external auditory canal, the epiglottis, parts of the laryngeal cartilages and in the walls of the Eustachian tubes.
The histological structure of elastic cartilage is similar to that of hyaline cartilage. Its elasticity is derived from the presence of numerous bundles of branching elastin fibres in the cartilage matrix. This network of elastin fibres (stained black in this preparation) is particularly dense in the immediate vicinity of the chondrocytes. Collagen (stained red) is also a major constituent of the cartilage matrix and makes up the bulk of the perichondrium P, intermingled with a few elastic fibres. Development and growth of elastic cartilage occurs by both interstitial and appositional growth, in the same manner as for hyaline cartilage.

FIG. 10.4 Chondrocyte
EM ×16 000
This electron micrograph illustrates a single chondrocyte C lying within its lacuna and surrounded by abundant cartilaginous matrix material M. As is typical of cells active in protein synthesis (in this case matrix turnover), chondrocytes have very prominent rough endoplasmic reticulum rER which is distended with secretory material. A well-developed Golgi apparatus G is present. Some glycogen granules are scattered in the cytoplasm. Note that the chondrocyte completely fills its lacuna within the matrix. Small cytoplasmic extensions mediate the constant interaction between chondrocytes and the surrounding extracellular matrix material.
At this magnification, the fibrous elements of the extracellular matrix can just be discerned. The deposits of electron-dense material which can be seen lying adjacent to the deep aspect of the cell represent some recently secreted extracellular matrix material MM.

C chondrocyte G Golgi apparatus H Howship lacuna M matrix material MM extracellular matrix material O osteoclast Ob osteoblast Oc osteocyte P perichondrium rER rough endoplasmic reticulum

Bone

Bone is composed of cells and a predominantly collagenous extracellular matrix (type I collagen) called osteoid which becomes mineralised by the deposition of calcium hydroxyapatite, thus giving the bone considerable rigidity and strength. The cells of bone are:

• Osteoblasts which synthesise osteoid and mediate its mineralization. These are found lined up along bone surfaces.

• Osteocytes which represent largely inactive osteoblasts trapped within formed bone. These may assist in the nutrition of bone.

• Osteoclasts are phagocytic cells which are capable of eroding bone. These are important, along with osteoblasts, in the constant turnover and refashioning of bone.

Osteoblasts and osteocytes are derived from a primitive mesenchymal (stem) cell called the osteoprogenitor cell. Osteoclasts are multinucleate phagocytic cells derived from the macrophage-monocyte cell line.

Bone forms the strong and rigid endoskeleton to which skeletal muscles are attached to permit movement. It also acts as a calcium reservoir and is important in calcium homeostasis. Bone is heavy and its architecture is optimally arranged to provide maximum strength for the least weight. Most bones have a dense, rigid outer shell of compact bone, the cortex, and a central medullary or cancellous zone of thin, interconnecting narrow bone trabeculae. The number, thickness and orientation of these bone trabeculae is dependent upon the stresses to which the particular bone is exposed. For example, there are many thick intersecting trabeculae in the constantly weight-bearing vertebrae, but very few in the centre of the ribs, which are not subjected to constant stress. The space in the medullary bone between trabeculae is occupied by haematopoietic bone marrow (see Fig. 3.3).

FIG. 10.5 Active osteoblasts and osteoid
(a) H&E (HP) (b) Undecalcified resin section, Goldner trichrome stain (HP)
These micrographs illustrate osteoblasts actively depositing new osteoid on a bone surface. When active, the osteoblasts Ob are large, broad, spindle-shaped or cuboidal cells with abundant basophilic cytoplasm containing much rough endoplasmic reticulum and a large Golgi apparatus. These features reflect a high rate of protein (type I collagen) and proteoglycan synthesis.
In micrograph (a), the tissue has been decalcified before sectioning and staining, so the distinction between mineralised bone and the newly formed unmineralised osteoid cannot be seen. In micrograph (b), which has not been decalcified, the mineralised bone (blue) can easily be distinguished from the new osteoid (red) which is being produced by the row of cuboidal osteoblasts. There is always a short delay between osteoid production and its mineralisation.
When inactive, osteoblasts are narrow, attenuated, spindle-shaped cells lying on the bone surface. In (a) the burst of new bone formation is nearly over, and the osteoblasts are becoming spindle-shaped again and will soon become virtually undetectable, only the long, narrow nucleus being visible histologically. A few cells are being incorporated in the newly formed bone as osteocytes Oc.

FIG. 10.6 Osteoclasts
(a) H&E (HP) (b) Undecalcified resin section, Goldner trichrome (HP)
Resorption of bone is performed by large multinucleate cells called osteoclasts O, which are often seen lying in depressions resorbed from the bone surface called Howship lacunae H. The aspect of the osteoclast in apposition to bone is characterised by fine microvilli which form a ruffled border that is readily visible with the electron microscope. The ruffled border secretes several organic acids which dissolve the mineral component, while lysosomal proteolytic enzymes are employed to destroy the organic osteoid matrix.
Osteoclastic resorption contributes to bone remodelling in response to growth or due to changing mechanical stresses upon the skeleton. Osteoclasts also participate in the long-term maintenance of blood calcium homeostasis by their response to parathyroid hormone and calcitonin (see Ch. 17). Parathyroid hormone stimulates osteoclastic resorption and so increases the release of calcium ions from bone, whereas calcitonin inhibits osteoclastic activity.
Micrographs (a) and (b) are taken from bone showing excessive osteoclastic activity due to the effects of Paget disease of bone, a disorder characterised by continuous disorganised bone resorption and associated new bone formation (see textbox). Micrograph (b) shows uncoordinated new osteoid formation by a row of cuboidal osteoblasts Ob.

Disorders of osteoblasts and osteoclasts

The normal maintenance and refashioning of bone is the result of coordinated activity of osteoblasts depositing new bone and osteoclasts eroding redundant bone. Some diseases are the result of excessive unbalanced activity of one or other of the cell types, commonly osteoclasts.

In hyperparathyroidism, excessive uncontrolled secretion of parathyroid hormone by the parathyroid gland (see Fig. 17.11) stimulates an increase in numbers and erosive activity of osteoclasts. This leads to diffuse destruction of bone, producing radiological areas of lucency (‘brown tumours’) and predisposition to fracture. A serious side effect of excessive bone erosion is the release of large amounts of ionic calcium into the bloodstream, producing severe symptoms of hypercalcaemia.

Paget disease is a disease of unknown cause in which there is random and haphazard excessive osteoclastic erosion of bone occurring in waves, followed by increased osteoblastic activity attempting to replace eroded bone (see Fig. 10.6). However, the new osteoid and bone formation does not always occur where bone has previously been eroded, so the architecture of the bone is grossly distorted (usually with woven bone, see Fig. 10.7) and the bone is structurally weak.

FIG. 10.7 Woven and lamellar bone, polarising microscopy
H&E (MP)
Bone exists in two main forms, woven bone W and lamellar bone L. Woven bone is an immature form with randomly arranged collagen fibres in the osteoid. Lamellar bone is composed of regular parallel bands of collagen arranged in sheets.
Woven bone is produced when osteoblasts synthesise osteoid rapidly, as in fetal bone development. It can also occur in adults when there is pathological rapid new bone formation e.g. at the site of a healing fracture and in Paget disease (see textbox).
The rapidly formed woven bone is eventually remodelled to form lamellar bone, which is physically stronger and more resilient. Virtually all bone in a healthy adult is lamellar.

FIG. 10.8 Long bone
This diagram illustrates the general structure of long bones in the mature skeleton and the gross morphological appearance of the two types of lamellar bone found in the mature skeleton, i.e. compact (cortical) bone and cancellous (medullary) bone.
Compact bone forms the dense walls of the shaft or diaphysis, while cancellous bone occupies part of the large central medullary cavity. Cancellous bone consists of a network of fine, irregular plates called trabeculae separated by intercommunicating spaces.
The articular (joint) surfaces of the expanded ends, or epiphyses, of long bones are protected by a layer of specialised hyaline cartilage called articular cartilage. The external surface of the bone is invested in a dense fibrous layer called the periosteum, into which are inserted muscles, tendons and ligaments. The inner surfaces of the bone, including the trabeculae of cancellous bone, are invested by a delicate layer called the endosteum. The endosteum and periosteum contain cells of the osteogenic series which are responsible for growth, continuous remodelling and repair of bone fractures (see textbox).
Prior to the attainment of skeletal maturity, the long bones grow in length by the process of endochondral ossification which occurs at a growth or epiphysial plate situated at each end of the bone at the junction of the diaphysis (shaft) and epiphysis.

Fractures

Bone fractures (broken bones) are common and usually occur due to trauma. Sometimes fractures occur in abnormal or weakened bone, termed pathological fractures, and may be due to a wide range of underlying disorders, including osteoporosis, Paget disease and metastatic cancer.

Fractures can be described as closed or simple (with no damage to the overlying skin) versus open or compound (where there is an overlying skin wound exposing the broken bone). In comminuted fractures, the bone is broken into a number of pieces.

Healing and repair of fractures is a specialised process which involves the formation of callus between the damaged bone ends (see Figs 10.9 and 10.23). Bleeding occurs immediately at the fracture site to form a haematoma. Fibroblasts and blood vessels then grow into this haematoma to form granulation tissue. The fibroblasts and other cells then develop into osteoblasts and chondroblasts, which produce woven bone and hyaline cartilage. This callus acts to temporarily unite the bone ends, and over time there is remodelling into mature lamellar bone.