Musculoskeletal System and Dermatology Module

HISTOLOGY LAB MANUAL PART II

(Histology lab is scheduled in week 2 of MSKD module)

CONTENT OUTLINE
Part I: Integumentary System (Skin)
Part II: Cartilage, Bone and Bone Formation

Histology Guide Virtual Slide Box: http://www.histologyguide.com/

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PART II: CARTILAGE, BONE AND BONE FORMATION

http://www.histologyguide.com/slidebox/05-cartilage-and-bone.html

Learning Objectives
1. Describe the organization of cartilage
2. Identify chondrocytes, lacunae, perichondrium and isogenous groups
3. Compare hyaline, fibrocartilage, and elastic cartilage
4. Correlate the types of cartilage to the function.
5. Identify compact bone and spongy bone.
6. Identify the components of the Haversian system including: lamellae, Haversian canals, Volkmann’s
canal, interstitial lamellae, lacunae and canaliculi.
7. Identify osteoblasts, osteocytes, and osteoclasts and Howship’s Lacunae
8. Describe the zones of the epiphyseal plate, and the periosteum.
9. Compare intramembranous ossification to endochondral ossification

Identification Checklist Key Distinguishing Features/Notes

Chondroblasts
Chondrocytes
Chondrogenic Perichondrium
Fibrous Perichondrium
Isogenous Group
Chondrocyte Lacunae
Territorial Matrix
Interterritorial Matrix
Hyaline Cartilage
Articular Cartilage
Elastic Cartilage
Fibrocartilage
Intramembranous Ossification
Osteoprogenitor Cells
Osteoblasts
Osteoid
Osteocytes
Osteoclasts
Howship’s Lacunae
Osteocyte Lacunae
 Canaliculi
Spongy Bone
Bone Trabeculae
Bone Marrow
Compact Bone
Osteon
Haversian Canal
Volkmann’s Canal
Concentric Lamellae
Interstitial Lamellae
Fibrous Periosteum
Osteogenic Periosteum
Endosteum
Epiphysis
Diaphysis
Endochondral Ossification
Primary Center of Ossification
Epiphyseal Growth Plate
Zone of Reserve/Resting
Zone of Proliferation
Zone of Hypertrophy
Zone of Calcification
Zone of Resorption

*Structures not listed in this checklist will not be assessed during lab quizzes but can be assessed during
unit exams if content was covered during OLMs. Please note that quizzes will consist of mostly
identification questions, and some structure-function questions (see histology lab practice questions for
an example) *

Introduction
Cartilage and bone will be examined here. They are regarded as connective tissue derivatives (supporting
CT) modified for weight bearing. They contain cells and a large amount of intercellular material also
referred to as matrix.

Cartilage

Hyaline Cartilage
Histology Guide Slide MH136 – Trachea
http://www.histologyguide.com/slideview/MH-136-trachea/05-slide-1.html?x=0&y=0&z=-1&page=1
The most common type of cartilage is hyaline cartilage. Observe this cartilage (the basophilic portion of
the tissue) in the trachea, and using the interactive sidebar observe the main tissue features. There is dense
irregular connective tissue associated with the surface of the cartilage; this is the fibrous perichondrium,
which consists of fibroblasts and the eosinophilic type I collagen fibers that it secretes, what is the
functional significance of this CT layer? Adjacent to this CT layer is the more basophilic inner
chondrogenic perichondrium identified by its elongated chondroprogenitor cells and lightly basophilic
matrix. These stem cells are a source of new chondroblasts at the surface of the cartilage for which type
of cartilage growth? What does the suffix -blasts usually indicate about the function of cells?
Study the cartilage in high magnification and notice that the matrix is basophilic, homogeneous and glass-
like, what ECM component gives rise to the basophilia? Why is this component functionally important?
In the matrix, note the presence of small partially filled white spaces, the so-called lacunae. The lacunae
contain chondrocytes, their appearance is usually distorted after to preparation. The matrix contains type
II collagen fibers which are masked by the ground substance solidified during the histological procedure.
Chemically, the main components of the ground substance are sulfated glycosaminoglycans, which are
basophilic and therefore stain with hematoxylin. More stain is attracted just around the lacunae,
territorial matrix, due to a higher concentration of GAGs. The lighter staining matrix further away from
cells is the interterritorial matrix. Several lacunae show two to four chondrocytes, they are daughter
cells which stayed together after division(s) of the chondrocytes, called isogenous groups. These dividing
chondrocytes account for which type of cartilage growth? In the articulating surface of joints where
hyaline cartilage is devoid of perichondrium, the cartilage is referred to as articular cartilage, why is this
clinically important?

Elastic Cartilage
Histology Guide Slide MH038-039 – Epiglottis
http://134.84.138.9/slideview/MH-038-039-epiglottis/05-slide-1.html?x=0&y=0&z=-1&page=1
Examine the second type of cartilage, the elastic cartilage, in two sections of the epiglottis, one stained
with H&E, the other with Verhoeff's Stain (stains elastic fibers black). Review the structures listed for
hyaline cartilage as they are also present in elastic cartilage. As in hyaline cartilage, lacunae,
chondrocytes, and basophilic matrix are also characteristic, but the matrix also contains elastic fibers,
which are best visualized when stained brownish-black in the Verhoeff’s stain slide. In the H&E stained
slide, the pink, thin branching elastic fibres are more difficult to see.

Fibrocartilage
Histology Guide Slide MH040 – Intervertebral Disk
http://134.84.138.9/slideview/MH-040-intervertebral-disk/05-slide-1.html?x=0&y=0&z=-1&page=1
The third type of cartilage is fibrocartilage. It is a mixture of dense regular connective tissue and hyaline
cartilage usually present in regions where extra pressure has to be cushioned, for example, in between the
vertebrae (i.e., in the intervertebral disks). A structure similar to that in hyaline cartilage can be discerned
but the Type II collagen (weak H&E stain) matrix is mixed in with bundles of Type I collagen fibers
(bright eosinophilic); hence the name Fibrocartilage. When using H&E, the pink stain of the type I
collagen fibres made by fibroblasts is more prominent than that of the blue sulfated glycosaminoglycans
made by chondrocytes; thus, the matrix assumes a pink colour. Study the tissue in high magnification and
try to differentiate between the elongated nuclei of fibroblasts and the round chondrocytes. Notice also
that chondrocytes of an isogenous group are often arranged in a line, or row, as opposed to a cluster,
between collagen fibres. A typical perichondrium cannot be observed, what are the clinical consequences
of this?

Bone

Bone Formation: Intramembranous Ossification

Histology Guide Slide MH 046 – Bone Development
http://134.84.138.9/slideview/MH-046-bone-development/05-slide-2.html?x=0&y=0&z=-1&page=1
During intramembranous ossification, compact and spongy bone develop directly from sheets of
mesenchymal (undifferentiated) connective tissue and not from a cartilage template. The flat bones of the
face, most of the cranial bones, and the clavicles (collarbones) are formed via intramembranous
ossification. Using the interactive sidebar, locate the skull cap, formed through intramembranous
ossification, and in high magnification observe how mesenchymal stem cells condense and differentiate
into osteoprogenitor cells and then into osteoblasts. When active, osteoblasts appear cuboidal in shape,
with a round nucleus and basophilic cytoplasm. They can be seen on the surface of bones laying down
osteoid (unmineralized bone matrix, eosinophilic due to type I collagen) and becoming osteocytes
(appears as dark nuclei trapped in non-staining lacunae completely surrounded by pink bone matrix). As
the bone forms, spicules of immature bone are laid down, which can then be remodelled into spongy bone
or compact bone. Note the developing osteons (described in detail later), which can be recognized by
blood vessels at the center of a bony area lined internally by osteoblasts (some osteogenic cells will
remain lining the canal as the endosteum).

Bone Formation: Endochondral Ossification

Histology Guide Slide MHS 287 – Bone
http://134.84.138.9/slideview/MHS-287-bone-development/05-slide-1.html?x=0&y=0&z=-1&page=1
During endochondral ossification, bone develops by replacing hyaline cartilage. Cartilage does not
become bone. Instead, cartilage serves as a template for the region that needs to be completely replaced
by new bone. Bones at the base of the skull and long bones, like the femur, form via endochondral
ossification. The various stages of endochondral ossification can be seen in these front and rear legs of a
fetal rat. Use the interactive sidebar to study the overall structure of cartilage models and locate the
primary centres of ossification at the diaphysis of the bone. Study this site in high magnification and
note the presence of hypertrophic chondrocytes (large clear cytoplasm, round and dark nuclei), and
osteoblasts beginning to lay down osteoid, forming woven bone. Sometimes basophilic calcified cartilage
matrix can still be seen around the developing bone, this will soon be resorbed by osteoclasts – large,
multinucleated cells. Use the hyperlink in the second page of the sidebar to locate the secondary centers
of ossification at the epiphysis of the bone and the epiphyseal plate between the diaphysis and epiphysis.
The epiphyseal plate is the area of growth in a long bone. It is a layer of hyaline cartilage where
ossification occurs in immature bones. On the epiphyseal side of the epiphyseal plate, cartilage is formed.
On the diaphyseal side, cartilage is ossified, and the diaphysis grows in length. Study this slide in both
low and high magnification and note that the epiphyseal plate is composed of different zones of cells and
activity. Furthest away from the diaphysis filled with bone marrow is the zone of reserve cartilage (blue
box), followed by the zone of proliferating cartilage (red box), zone of hypertrophic cartilage (yellow
box), zone of calcified matrix (green box), and the zone of resorption (pink box), which is closest to the
marrow in the diaphysis.
Note the hypertrophic chondrocytes (brown arrow) enlarged lacunae (black box), osteoblasts laying
down osteoid (red arrow), and becoming osteocytes trapped in the bone matrix (blue arrow).
Multinucleated osteoclasts and their Howship’s lacunae (resorption bays) are present but cannot be seen
in this section; they are responsible for the resorption of bone in the process of bone-remodeling.

Histology Guide MHS 203 Bone

http://www.histologyguide.com/slideview/MHS-203-bone/05-slide-1.html?x=0&y=0&z=-1&page=1
In this slide the periosteum, where long bones grow in width through appositional growth, is labelled. In
the diaphysis of the bone, note the outer fibrous layer of the periosteum(dark blue box), consisting of
DICT, and the inner osteogenic layer (dark green box), consisting of osteoprogenitor cells and
osteoblasts. Observe also the spongy bone cavities in the inner core of the diaphysis becoming
increasingly filled with bone marrow cells.

Histology Guide Slide MHS 046 - Bone Development

http://134.84.138.9/slideview/MH-046-bone-development/05-slide-3.html?x=0&y=0&z=-1&page=1
Both a flat bone formed through intramembranous ossification is seen at the top of this slide, and a long
bone below formed through endochondral ossification are pictured in this slide. Briefly observe the main
features in each bone and compare and contrast the two types of bone formation.

Histology Guide Slide MH029B – Bone

http://134.84.138.9/slideview/MH-029b-bone/05-slide-1.html?x=0&y=0&z=-1&page=1
The epiphyseal plate zones are better appreciated in this slide of a growing long bone. Use the interactive
sidebar to find the different zones and note the differences in cell types, matrix staining and organization.
Note that this epiphyseal plate of a growing bone is a lot smaller than the previous slide of a bone during
embryological growth. Identify osteoblasts, osteocytes, and osteoclasts - large, multinucleated cells that
remove bone tissue (both mineralized matrix and type I collagen).

Mature Bone
Regardless of the type of bone formation, primary woven bone can be remodelled into spongy
(cancellous) bone, or compact bone. Most bones (long, flat, etc) contain both compact and spongy bone.
Compact bone is a dense layer that forms the outer shell or cortex of bones, and spongy bone is an
anastomosing network of spicules found in the interior of bones.

Compact Bone
MHS 233 Ground Bone
http://www.histologyguide.com/slideview/MHS-233-ground-bone/05-slide-1.html?x=0&y=0&z=-
1&page=1

Study this section of specially prepared ground bone stained with india ink to reveal small, open spaces in
the bone. Compact bone is the denser, stronger of the two types of bone tissue. It can be found under the
periosteum and in the diaphyses of long bones, where it provides support and protection. The microscopic
structural unit of compact bone is called an osteon (blue circle), or Haversian system. Each osteon is
composed of concentric rings (red arrows) of calcified matrix called lamellae. Note also interstitial
lamellae (purple square), lightly stained remnants from remodeling of bone between osteons. Running
down the center of each osteon is the central canal, or Haversian canal (green circle), which contains
blood vessels, nerves, and lymphatic vessels. These vessels and nerves branch off at right angles through
a perforating canal, also known as Volkmann’s canals (brown oval), to extend to the periosteum and
endosteum.

The osteocytes are located inside spaces called lacunae (pink arrow), found at the borders of adjacent
lamellae. They extend narrow cytoplasmic processes through Canaliculi (yellow arrow) that connect with
the canaliculi of other lacunae and eventually with the central canal. Adjacent osteocytes form gap
junctions with each other at the tips of these processes. This system allows nutrients to be transported to
the osteocytes and wastes to be removed from them. It also serves to monitor and communicate
mechanical forces that then regulate bone maintenance, formation, or resorption.

Histology Guide Slide MH045 – Bone

http://134.84.138.9/slideview/MH-045-bone/05-slide-1.html?x=0&y=0&z=-1&page=1
Explore the features of compact bone stained with H&E, noting the vessels in the Haversian canals, and
osteoblasts lining some of these canals (some osteoprogenitor cells remain following bone remodelling
and become the endosteum).

Spongy Bone
MH 047 Spinal Cord
http://www.histologyguide.com/slideview/MH-047-spinal-cord/05-slide-1.html?
x=17584&y=12818&z=26.5&page=1#

Like compact bone, spongy bone, also known as cancellous bone, contains osteocytes housed in lacunae,
but they are not arranged in concentric circles. Instead, the lacunae and osteocytes (orange arrow) are
found in a lattice-like network of matrix spikes called trabeculae (blue arrows). The trabeculae may
appear to be a random network, but each trabecula forms along lines of stress to provide strength to the
bone. The spaces of the trabeculated network provide balance to the dense and heavy compact bone by
making bones lighter so that muscles can move them more easily. In addition, the spaces in some spongy
bones contain bone marrow (green box, dark staining clusters of cells) protected by the trabeculae,
where formation of blood cells, hematopoiesis, occurs.