Zoo115 Animal Histology Laboratory

Exercise 4
SPECIALIZED CONNECTIVE TISSUE: CARTILAGE AND BONE

Connective tissues are those tissues with supportive functions. In no case is this truer than that
of cartilage and bone, which are truly "supportive" in a physical (as well as physiological) sense.
Cartilage forms the skeleton of mammalian embryos; in the Chondrichthyes (sharks and rays) and a
few other groups a cartilaginous skeleton persists throughout life. In mammals, however, the cartilage
model is found only in the earlier stages of development, for the most part and it is later replaced by
bone.
It has to be emphasized that cartilage is not "turned into" bone, it is replaced by bone in a
complicated process referred to as endochondral ossification. Not all of the cartilage of mammals is
replaced, however. Some of it persists in areas which require resilient but flexible stiffening. Examples
include the walls of larger respiratory passageways, the tip of the nose, and the ears. Cartilage is also
present at the articular surfaces of joints.
Bone, like other connective tissues, has cells, fibers, and a matrix. In bone, however, the
extracellular matrix is calcified, and the fibers (which are collagen) are very highly ordered. The
cellular component is vital to bone function, although it is a comparatively small proportion in terms of
total volume.

OBJECTIVES: After completing the exercises on cartilage and bone, you should be able to:
 recognize the three major cartilage types in typical light microscopic sections.
 use standard nomenclature to describe a section of cartilage (e.g. chondrocyte, lacuna, matrix,
perichondrium).
 use standard nomenclature to describe the microscopic structure of bone (e.g. lamella, osteon,
osteocytes, canaliculi, periosteum, endosteum).
 recognize mature bone (dense and cancellous) in conventional or ground section.
 identify the component parts of mature bone (e.g. osteon, lamella, lacuna, osteocyte) in
appropriate sections.

CARTILAGE

Cartilage is a specialized type of connective tissue whose unique combination of rigidity,

elasticity and resilience is due primarily to the special properties of its matrix. As in other connective
tissues, its matrix is composed of fibers (collagenous or elastic) and a ground substance that is rich in
extracellular glycosaminoglycans (particularly the chondroitin sulfates). The fibrillar components
provide for shape retention and tensile strength, while the viscous, hydrated matrix absorbs
compression forces. Cartilage is avascular, but its matrix is permeable to tissue fluids carrying nutrients
and waste products. Cartilage can grow from the outside (appositional growth) and from within
(interstitial growth).
Cartilage is the primary skeletal tissue of the fetus, and it serves as a model for the development
of endochrondral bone. In the adult, cartilage is more restricted in its distribution, but it forms the
articular surfaces of diarthrodial joints, the skeleton of the external ear and the septum of the nose,
supporting rings and plates of the trachea and bronchi, and intervertebral discs. Three types of cartilage
are found in the adult; these are classified according to the predominant component of their
extracellular matrix. As in other connective tissue classifications, there are gradations between these

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basic types.
The three basic forms of cartilage are: the hyaline cartilage, the elastic cartilage, and the
fibrocartilage. The hyaline cartilage serves as the "type" and the other two forms are described with
reference to it. Many authorities regard the fibrocartilage as a transitional form between cartilage and
connective tissue proper. In the case of hyaline cartilage, the matrix material is the predominant
element.

Draw, locate, and label structures mentioned in bold face.

A. Hyaline Cartilage
Hyaline cartilage is the most common type, and serves as the structural archetype; the other
forms are described in comparison to it. Hyaline cartilage is found in several places.

1. Trachea, H&E; Hyaline Cartilage, H&E

These are good examples of mature hyaline cartilage with its abundant matrix and spaces,
lacunae, occupied by cells, chrondocytes, which usually shrink extensively during fixation. For the
prepared slide Trachea in H&E, locate the incomplete C-shaped rings in the wall. This is the hyaline
cartilage. Notice the variably staining matrix. Remember that there are abundant type II collagen
fibrils in the matrix. However, they are too small to be resolved in the light microscope, so the matrix
has an amorphous, glassy (or "hyaline") appearance. Note that the cartilage matrix is relatively
homogeneous and basophilic. This is due to the masking of the collagen fibers by the high
concentration of the glycosaminoglycans in the ground substance. In H&E, the cartilage ECM will
stain a very strong purple (which you should be able to see by simply holding the slide up to the light).
Note that the basophilia and that the matrix immediately surrounding the lacunae is more intensely
basophilic. This zone is the territorial matrix. The interterritorial matrix is less basophilic, with
even some eosinophilic reflecting loss (or minimal content) of negative charges. The differential
staining of the territorial matrix compared to the interterritorial matrix is perhaps best shown in the
prepared slide Hyaline Cartilage in H&E.
Also note that the outside of the cartilage is covered with a two-layered connective tissue sheath
called perichondrium. At higher magnification, observe that the perichondrium surrounds the
cartilage; this merges with the cartilage on one side and with the surrounding connective tissue of the
other side. Blood vessels within the perichondrium provide the blood supply for the avascular cartilage.
Chondroblasts are cells found in the inner layers of the perichondrium and recently derived from it.
They are not yet completely embedded in the matrix. They give rise to new chondrocytes as they
secrete matrix around themselves. Mature cartilage cells or chondrocytes are surrounded by matrix and
lie within spaces called lacunae. In life the chondrocytes completely fill the lacunae. Look for cell
clusters called, isogenous groups; these are likely daughter cells of the same mitotic division.

Question: Considering the absence of blood vessels in cartilage, why do you think the content and
organization of the matrix is so important?

B. Elastic cartilage
The second form of cartilage, which resembles hyaline cartilage fairly closely in its structure, is
elastic cartilage. Elastic cartilage provides support with flexibility. The general organization of this type
of cartilage is similar to that of hyaline cartilage, except that elastic fibers predominate over collagen

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fibers in the matrix. Elastic fibers are stained specifically (black) by the van Gieson's stain or dark
purple by the aldehyde fuchsin and Masson's trichrome.

1. Epiglottis; Elastic cartilage Section

In H&E, look for plates of elastic cartilage found just under the glands deep to the respiratory
epithelium.
View the slide under the scanner and take note of the more darkly-staining predominant area.
Focus on this area then shift to a higher magnification and observe that there are chondrocytes within
lacunae just as in hyaline cartilage, but note the eosinophilic, fibrillar matrix due to the presence of
elastic fibers. As with hyaline cartilage, fibrils of type II collagen are present, but they cannot be seen
in the light microscope. You may also notice that elastic cartilage tends to be more cellular than
hyaline cartilage and in most histological preparations elastic cartilage tends to be more heavily
stained, because of the decreased proportion of matrix material. You can only convincingly identify the
elastic cartilage when the section is specifically stained for elastin.
With specific stains, preparations of the epiglottis are usually dominated by the cartilage
surrounded by varying amounts of connective tissue and epithelia. The appearance of the cartilage (in
this preparation a blue-green colour) will depend on the method used to show tissue components other
than elastic fibres. Although the matrix appears blue-green, the typical organisation of cartilage is
readily visible. Within the green matrix you can see the fine elastic fibres which give this cartilage its
elastic properties. The elastic fibres may form dense masses in which individual fibres are difficult to
distinguish. The staining of these masses of fibres may appear more reddish than dark-violet.
Notice the connective tissue, perichondrium, (pale-staining) on the outside of the cartilage. In
elastic cartilage, chondrocytes, in isogenous groups are not so well defined as in hyaline cartilage.

2. External Ear
In elastic cartilage isogenous groups are not so well defined as in hyaline cartilage. The inter-
group matrix material is considerably lessened in volume, hence the groups are closer together. The
nature of the fibrillar component also is different, in that it consists of elastic fibers, not exclusively
collagen. Identify in this slide the same structures mentioned (i.e. matrix, chondrocytes in lacuna,
elastic fibers, isogenous groups, perichondrium) in bold face in Epiglottis.

C. Fibrocartilage
The last form of cartilage is fibrocartilage. You will find it on a section from an intervertebral
disk. This cartilage is named for its textured matrix; it looks fibrous, and in addition lacunae can be
seen. Fibrocartilage has only very limited distribution in the body. In addition to the intervertebral
disks, it's found in the pubic symphysis, and a few other locations.

1. Intervertebral disc; H&E

Locate the nucleus pulposus (clear area) of the intervertebral disc, then move out to the edge of
the section to see fibrocartilage. Note the fibrous texture of the matrix due to the presence of type I
collagen fibers in addition to the type II collagen present in all cartilage tissue (type II fibrils are not
bundled into fibers large enough to be visible in the light microscope), but note also the distinct
chondrocyte lacunae. Also, note that there is no perichondrium in this cartilage. Fibrocartilage, seen
here, is much more similar in appearance to dense collagenous CT than to traditional cartilage, and it
shares several structural similarities to both. In this type the fibrous component (which is collagen) is

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predominant, and the matrix is minimal, but the cells are in lacunae. Some of the lacunae may be
incomplete. The presence of lacunae is the tip-off that this is, in reality, a cartilage and not dense
fibrous CT. The lacunae are pockets in the scanty matrix material that fills the spaces between the
fibers.

BONE
Bone is a calcified connective tissue, and like other connective tissues, it consists of cells, fibers
and ground substance. The deposition of inorganic calcium phosphate salts as hydroxyapatite crystals
within its matrix is a distinguishing characteristic of bone. This renders it structurally rigid. Both the
macroscopic and microscopic structure of bone reflects the response of this tissue to its mechanical
function. In addition, bone functions as a homeostatic reservoir of calcium and phosphate ions, and it
encloses the hematopoietic elements of the bone marrow.

The two processes of bone formation or osteogenesis observed in the embryo are: 1.
Endochondral ossification (EO), where bone and marrow replace a preexisting hyaline cartilage
template or anlagen of future bone; and 2. Intramembranous ossification (IO), where bone is
deposited directly within primitive connective tissue or mesenchyme. In both cases, a primary or
immature bone is first laid down, and is then transformed (“remodeled”) into mature bone. By these
two processes, two types of bone are formed. Cancellous (also called spongy or trabecular) bone
results from EO; this tissue is web-like and porous, is surrounded by bone marrow, and likely provides
appropriate niches for blood cell development in the marrow cavity. Compact (also called dense or
cortical) bone results from IO; this tissue is dense, and is designed for a supportive and weight-bearing
role.

Mature bone: There are two types: compact (lamellar) and spongy (trabecular or cancellous).
Compact bone is characterized by the regularity of its collagen fibers. Spongy bone consists of a lattice
of branching bony spicules, known as trabeculae, which in some regions are surrounded by bone
marrow. When the trabeculae are sufficiently thick, they may contain osteons (see description below).

Immature (woven) bone: (see in "bone development") It is the first bone laid down in prenatal
life or in the repair of bone fractures. In this type of bone, the matrix immediately surrounding the
osteoblast is called osteoid and is not mineralized. Immature bone is characterized by irregularly
arranged, interwoven collagenous fibers within a matrix containing proteoglycans.

Note: Do not confuse the term spongy (a type of mature bone) and woven (immature bone).

Because of its calcified matrix, bone presents difficulties in its preparation for microscopic
study. There are two basic techniques for studying bone with the light microscope, and both of these
types of preparations must be studied to appreciate the organic and inorganic components of bone. (1)
Bone may be decalcified by acid solutions prior to embedding and sectioning. This permits study of the
cells and organic matrix of the bone. (2) To study the lamellar and canalicular pattern of the calcified
matrix, it is necessary to grind dried bone that has not been decalcified to a thickness that permits the
microscope light to be transmitted ("ground bone").

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A. Compact Bone

1. Bone decalcified section

Look at a ground bone in cross section. Examine it first at low power. This is a piece of dried
bone cut into a wafer with a diamond saw. It's from the shaft of a long bone, and it's histology is pretty
typical. You will note there appear to be circular areas, and between these there are less regularly
shaped regions. These are respectively the osteons and interstitial systems of compact bone. You
should be able to identify osteons and their subdivisions, interstitial lamellae, Haversian canals and
nutrient canals (Volkmann).
The basic unit is the osteon (Haversian system). In three dimensional view, an osteon is a
cylinder composed of 4 to 20 concentric lamellae arranged around a central opening, the
osteonal/Haversian canal. A useful structural analogy is a roll of paper towels: layers around a
relatively open central core. In cross section, as you see them in your slide, there are discrete areas.
Some are organized as concentric rings around a small opening. These circular profiles are the
osteons/Haversian systems; compact bone showing this organization is referred to as osteonal bone.
The central osteonal canals/haversian Canals are visible here as dark holes, more or less round in the
middle of the osteon. They are the route for the blood vessels and nerves serving the osteons. They run
along the length of the long bone and provide major vessel supply to the osteocytes, the mature bone
cells. The branches that run between adjacent osteons are passed through lateral canals.
Adjacent osteons are linked together for nutritive purposes by lateral canals or Volkmann's
canals which connect the osteonal canals. These canals run perpendicularly or obliquely to the course
of the Haversian canals. Thus the osteonal (Haversian) and lateral (Volkmann's) canals form a
complicated and ramified set of channels through the dense material of the bone.
Now examine an osteon at higher magnification. The cementing lines between osteons should
be visible. You will also be able to make out small spaces, lacunae (which you see as dark "spots"), in
which the "maintenance" cells of the bone, the osteocytes, would be located in living tissue. Since bone
is lamellar (unlike cartilage, which has an amorphous structure) the lacunae are located between
adjacent lamellae; hence they're neatly arranged into rows, which coincide with the division between
one lamella of the osteon and the next.
Between any given osteocyte and the next, there's a lamella of hard, calcified bone matrix, and
quite a few collagen fibers. Obviously, unlike cartilage, there can be no diffusion of materials through
this stuff. But the living cells of the bone have to have facilities for nutrient and waste exchange. This is
provided for by the osteocytes themselves, and their relationship to each other. Look at the layers of
bone and osteocytes running around the Haversian canal. Focus up and down and you can see tiny
channels, like spider legs, extending from oblong lacunae. The osteocytes are sitting in the lacunae and
the canals are canaliculi, which interconnect the lacunae with the major vessels. This series of channels
allows the osteocyte to be nourished and regulated.

B. Spongy Bone

1. Articular cartilage/surface; Trabecular/Cancellous Bone, H&E

Let us examine the cancellous bone located inside the round articular end. Cancellous bone is
lamellar, but it doesn't form the orderly osteons and interstitial systems of compact bone. Instead it
forms sheets and plates, which in sections are seen as anastomosing trabeculae and spicules.
The spaces between the trabeculae are continuous with the main marrow cavity in the

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diaphysis. Both the marrow cavity and the spaces among the trabeculae are lined with a very thin layer
of mixed cell types. This is the endosteum. Some of these cells are osteogenic, and some osteolytic.
We'll look at them in detail below. The trabeculae can be thought of as a mosaic of angular pieces, each
covered with endosteum.
Similarly, the outer surface of the shaft is covered with a tough collagenous CT, the
periosteum. Some of the cells in it have osteogenic capabilities, and are derived from the same stem
line as the fibroblasts. The periosteum is analogous to the perichondrium, and it's tightly adherent to the
surface of the bone.
If you examine the trabeculae at medium magnification, you can verify that they have
osteocytes enclosed within lacunae, and these lacunae are arranged in orderly rows. Osteons do not
form here, however. Since the trabeculae are quite thin, the osteocytes are nourished by diffusion from
the bone marrow via the canaliculi, and there is no need for the osteonal system, with its tortuous
channels to carry nutrient vessels. Nor is there much need for compressive strength, as in long bones.
The osteons of compact bone are a structural adaptation to provide mechanical strength under vertical
loads, and the spongy portions of the bone aren't subjected to this kind of stress at all.

C. Developing Bone
The developing bone will in sections usually be associated with a number of other tissues which
develop in close association with it. In case of the mandible, there can be developing teeth, the tongue,
skin and salivary glands.

1. Membrane bone developing

The first job - best done at low magnification - is to find the developing bone. It should look
like a coarse meshwork (trabecular bone) of pink tissue surrounding patches of much lighter or
unstained tissue. Lamellae are not visible (woven bone) and the lacunae are larger than lacunae in
mature bone. Ossification centres appear as areas of a gradual transition from connective tissue to bone.
Light, pinkish bone matrix is found between the osteoblasts.
Depending on the state of development of the bone, it is occasionally possible to find bone
trabeculae which are lined by a layer of osteoblasts. These osteblasts are depositing the first lamellae
on the already existing trabeculae. The trabeculae will therefore have a core of woven bone, which is
surrounded by lamellar bone. Compare the shapes, sizes and frequencies of lacunae in lamellar and
woven bone if both types of bone are present.

Question: Why are blood vessels important in bone?

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