Embryo Simple & Bone - Fundamentals

Early Embryology & Stem Cells Lecture Sessions 6 & 7
Problems for Self-Study

(annotated answers follow)
1. In the context of embryonic development, "mesenchyme" refers to cells which:

A. form cohesive sheets
B. exist in a non-aggregated state
C. are derived from embryonic endoderm only
D. will give rise to extraembryonic structures only
E. are restricted to becoming just few different cell types
2. All adult epithelium is derived from the embryonic:

A. ectoderm
B. endoderm
C. mesoderm
D. neuroectoderm
E. epiblast
3. The neural crest:

A. is derived from the notochord
B. is differentiated mesoderm
C. only give rise to connective tissue structures of the head and neck
D. comes the spinal cord
E. gives rise to the dermis of the face and cranial nerves
4. A correct description of stem cells is that:

A. all daughter cells form more differentiated cell types
B. daughter cells form at least 2 differentiated cell types
C. they are only found in morula or blastocyst
D. only some of the daughter cells will form more differentiated cell types
E. their presences is well document in brain and heart tissue
5. The drawing below represents a blastocyst prior to implantation in the endometrium of the uterus.
The cell labeled "C" can develop into:
A. the hypoblast only

B. endoderm only
C. the endoderm or mesoderm, but not ectoderm
D. any tissue of the embryo
E. the morula
6. Hematopoietic stem cells:

A. are equivalent to embryonic stem cells
B. can form all of the cells found in circulating blood except lymphocytes (a type of white blood
cell)
C. are derived from hypoblast cells
D. can form resident cell types in certain other tissues in addition to blood
E. are totipotent
7. During gastrulation:
A. mesenchymal cells form tightly adhering sheets of cells between the endoderm and ectoderm
B. paracrine signaling molecules are produced by cells of the primitive streak
C. epiblast cells migrate to the lateral edges of the embryo
D. the ectoderm is formed from the hypoblast layer
E. neurulation, somite formation, and cardiovascular formation are two weeks in the future
8. A skin graft is a section of skin, often removed from the injured person, and placed over a large
skin wound. In order for the graft to be capable of generating new epidermis, which cells must
maintain their viability and be included in the graft?
9. An example of apoptosis in adult tissue is:

A. death of intestinal cells at the luminal surface of the intestine
B. loss of cell adhesion molecules by epidermal basal cells
C. cell death due to irreversible chemical, mechanical or genetic injury
D. the formation skeletal muscle precursor cells in the dermomyotome due to notochord SHH
E. elimination of tissues between fingers or toes
10. Wilms tumor is the most common primary tumor in the pediatric kidney and one of the top five
common pediatric malignancies. Biopsy and inspection of a typical Wilms tumor will reveal
incomplete differentiation of embryonic tissue into postnatal renal tissue derived from the:
A. ectoderm
B. endoderm
C. paraxial mesoderm
D. intermediate mesoderm
E. lateral plate
Wilms tumor in the lower pole of a pediatric kidney.

Robbins, Pathological Basis of Disease, 7/e, Fig 10.31
11. The concept of a stem cell niche refers to stem cells that are:
[NOTE: More than one answer may be correct.]
A. capable of dividing only under condition requiring compensatory reconstruction or scar
formation
B. found in a specific tissue microenvironment
C. capable of responding to changing extracellular conditions or injury
D. found only during the embryological period of development
E. capable of forming only a relatively small number of differentiated cells
12. Apoptosis:
A. is a cellular mechanism by which longitudinal limb growth is achieved
B. is the process by which cancerous cells undergo rapid hyperplasia
C. is a process of programmed cell death that eliminates transitory tissues
D. usually involved cell volume expansion leading to lysis
E. is a secondary, non-essential cellular process during normal embryological development
13. The embryonic cells forming the outer cell mass (a.k.a. trophoblast) will develop into:
A. the embryonic contribution to the placenta
B. skeletal muscle tissue of the body wall
C. embryonic ectoderm
D. the neural crest
E. epiblast
14. Five pairs of embryonic structures are listed below. In which pair are the structures listed in the
correct temporal order of their appearance during normal development?
A. blastocyst morula
B. neural crest notochord
C. embryonic mesoderm embryonic endoderm
D. hypoblast inner cell mass
E. epiblast primitive streak
15. Which of the following statements regarding the primitive streak is most clearly FALSE?
A. The primitive streak is a linear, midline thickening of epiblast cells on the dorsal epiblast disk.
B. During invagination of the primitive streak, epiblast cells increase their cell-to-cell adhesions
forming a single layer of mesoderm.
C. The notochord develops from a collection of cranial primitive streak cells called the primitive
node.
D. The primitive streak is involved in initiating the development of the cranial-caudal body axis.
E. The presence of the primitive streak is taken as the first observable sign that gastrulation is
occurring.
16. In adult tissues, transit amplifying cells (TACs):

A. are found only in tissues lacking stem cells
B. have a greater differentiation potential than stem cells in the same stem cell niche
C. typically cannot divide
D. have a higher capacity for self-renewal than stem cells
E. typically divide rapidly to form a large pool of committed cells
17. Which of the following correctly describes stem cells?

A. Only some of their daughter cells will form more differentiated cell types.
B. Adult stem cells are only found in red bone marrow and epidermis.
C. Stem cells have high replication activity in quiescent cell populations found in the liver or
pancreas.
D. Osteoprogenitor TACs become adult mesenchymal stem cells.
E. Stem cells are evenly distributed throughout all cell layers of the skin and mucosal linings.
18. Somites:
A. are derived from mesenchyme and become intervertebral disks
B. develop as a single column of "blocks" anterior to the notochord beginning in Week 3
C. differentiate into vertebrae and muscles of the back and body wall
D. contribute to the parietal mesothelium, which are derived from the lateral plate mesoderm
E. undergo rapid apoptosis, which renders them useless in assessing embryonic age
19. Which of the following is true regarding differentiation of somite cells?

A. WNT and SHH paracrine factors are released by the dermomyotome.
B. SHH causes differentiation of the somite cells into cartilage precursors.
C. The neural crest cells act as a Responder Group to the dermomyotome.
D. The notochord, but not the neural tube, releases paracrine signalling factors to the somite.
E. SHH comes from cells adjacent to neural crest cells.
20. Teratomas are an encapsulated tumors comprised of normal tissues derived from the endoderm,
mesoderm, and ectoderm. Sacrococcygeal teratomas are located in the tailbone and are the most
common tumor of newborns and upon biopsy bone, nerve, and hair are often found within the
teratoma. A sacrococcygeal teratoma is a derivative of which of the following embryonic
structures?
A. caudal notochord
B. trophoblast
C. caudal somites
D. caudal neural tube
E. primitive streak
Sacrococcygeal Teratoma
Robbins, Pathological Basis of Disease, 7/e, Fig 10.26
Answers to Self-Study Problems
1. B. Although most of the mesenchymal cells arise from mesoderm migrations, the only consistent
description of these cells is that they are not aggregated into sheets. This is because they have lost
the cell adhesion molecules (including E-cadherin), and do not adhere to each other. Embryonic
and adult (post-natal) mesenchyme is usually considered to be multipotent in that it can become
many different cell types found in the tissue derivatives of endoderm, mesoderm, or ectoderm.
2. E. Epiblast. The epiblast cells will give rise to all three embryonic layers, and all three embryonic
layers contribute to the epithelium of the adult. Epithelium is typically found at the boundary
between the external and internal environment. The mucosal linings of the trachea, gut tube (oral
cavity to anus) and urogenital tract are examples of epithelium. The skin epidermis and anterior
pituitary gland are also examples of epithelial tissue.
3. E. Neural crest cells have greater potency than neural tube cells.. The neural crest will give rise to
a wide range of tissues, including connective tissue found in the head, neck and face. Neural crest
cells also give rise to all PNS structures such as peripheral nerves, autonomic ganglia, and cranial
nerves. Neural crest cells undergo significant migration to embed themselves into the walls of the
gut tube forming the enteric nervous system "brain in your belly".
4. D. Stem cell populations divide by mitosis to form daughter cells. Some of these daughter cells
become committed to developing into mature differentiated cells while other daughter cells remain
'behind' in the stem cell niche as 'stem cells'. In some tissues an intermediate pool of Transit
Amplifying Cells (TACs) is found near the stem cell niche. Embryonic stem cells are found in the
morula and blastocyst. Adult stem cells have been characterized in many tissues including bone
marrow, epithelial tissue (skin, mucosa of gut tube, trachea), liver, kidney, pancreas, endothelium,
and resting lymphocytes. However, with a few exceptions, current research efforts have not well
characterized stem cell populations that support baseline populations of CNS neurons, cardiac m.
fibers, or skeletal m. fibers.
5. D. This is a cell of the inner cell mass (a.k.a. embryoblast), which is pluripotent, that is, capable of
differentiating into any of the embryonic ectoderm, mesoderm, or endoderm tissues (but not
extraembryonic tissues such as the placenta). The morula is the stage which precedes the
blastocyst.
6. D. Hematopoietic stem cells can form all types of red and white blood cells as well as bone cells
(osteoclasts), nervous system cells (neurons, microglia), liver cells and primitive cardiac myocytes.
HSCs are multipotent and exhibit more multipotency than other types of adult tissue stem cells
(TSCs) located outside of bone marrow. The epiblast is the layer of the bilaminar disk adjacent to
the nascent amnionic cavity from which the ectoderm, mesoderm, and endoderm are derived.
Hence embryonic and adult stem cells are derived from the epiblast and not hypoblast since they
differentiate from the mesoderm layer of the gastrula.
7. B. The appearance of the primitive streak in midline of epiblast layer signifies the onset of
gastrulation and grows in the caudal-to-cranial direction. Gastrulation occurs in week 3 in which
the three layers of the embryo are formed (ectoderm, mesoderm, endoderm) and the body plan and
form begin to take shape: Recall- "3s in Week 3". Cells undergo hyperplasia and self-organize
along the cranial-caudal axis once the primitive streak appears. Primitive streak cells elaborate
paracrine signalling molecules which induce nearby epiblast cells to invaginate and migrate to
become either embryonic endoderm or mesoderm. Some of the mesodermal cells begin to
differentiate into embryonic connective tissue called mesenchyme. Within the third week
neurulation, somite formation, and cardiovascular development commences.
8. Adult tissue stem cells (TSCs) located in the most basal layers of the epidermis just superficial to
the dermis. A graft consisting of only the superficial layers of the epidermis would not work
because these cells are mature differentiated cells that can no longer divide. In general, tissue grafts
are most successful only if the section of healthy tissue excised contains the relevant adult stem
cells.
9. A. The finite lifespan of intestinal cells is an example of apoptosis, programmed or non-pathologic

cell death. The loss of cell adhesion molecules is not apoptosis, but a change in the status of
intercellular linkages due to intercellular junctions. Necrosis is cell death due to irreversible injury.
The formation of the skeletal muscle precursor cells in the dermomyotome from sonic hedgehog
(SHH) paracrine signals released by the notochord is an example of inductive signalling.
Elimination of tissues between fingers and toes is an example of apoptosis in fetal tissue, not adult
tissue.
10. D. Urogenital system tissues (kidneys, ureters, bladder, gonad stroma) are derived from
intermediate mesoderm. Paraxial mesoderm becomes skeletal muscle, CT and dermis of trunk,
limbs and portions of the head; lateral plate mesoderm becomes mesothelium that lines the ventral
body cavity as well as the most of the gut tube wall thickness (except mucosa which is derived
from endoderm), the heart, blood and lymph vessels and the spleen. Ectoderm becomes the
epidermis, anterior pituitary gland, cornea & lens of eye and nervous tissue. Endoderm becomes
the epithelial inner lining of the respiratory tract, urinary bladder, most of digestive tract, accessory
digestive tract organs (liver, pancreas) and contributes to the parathyroid gland, thyroid gland,
thymus and tonsils
11. B. and C (two answers are correct): A stem cell niche is both an anatomical and a functional
concept. Anatomically the niche is the specific geographical location of stem cells found in a
tissue. Functionally, it is a collection of multipotential cells that can respond to changes in
extracellular chemical or mechanical signals or injury. Continuously dividing cell populations (e.g.
epithelia, most glands, bone marrow) require a continuous input of cells derived from local stem
cell niches. Quiescent cell populations (found in liver, kidneys, pancreas; also smooth m. fibers,
fibroblasts, chondrocytes, osteocytes, endothelium, lymphocytes) require an stem cells usually in
times of compensatory reconstruction of tissue due to reversible injury or when scar formation is
warranted by fibroblasts. Stem cells are found in both the embryo and in the postnatal being ('adult'
stem cells). Adult stem cells come in two broad categories: Hematopoietic Stem Cells and Tissue
Stem Cells. Adult Mesenchymal Stem Cells are one type of Tissue Stem Cell.
12. C. Cell death due to apoptosis is a normal part of development as compared to cell injury due to
trauma or disease. Apoptosis eliminates unwanted or unneeded regions of tissues during
development. Lack of proper apoptosis can lead to disfigurement or fusion of postnatal structures.
13. A. The outer cell mass (a.k.a. trophoblast) is a collection of pluripotent cells that differentiate into
extraembryonic tissues i.e. various placental structures and membranes that will be detailed in PSL
535. The trophoblast surrounds the fluid filled blastocyst cavity and physically touches the
epithelium of the uterine endometrium. Body wall skeletal muscle is derived from paraxial
mesoderm. Embryonic ectoderm is derived from the epiblast layer derived from the inner cell mass
(embryoblast). Ectoderm eventually differentiates into epidermis of the skin and its derivatives
(hair, sweat glands), the anterior pituitary gland, cornea & lens of eye and CNS/PNS tissue. The
neural crest is derived from neuroectoderm and hence ultimately comes from the inner cell mass
(embryoblast as well). The neural crest cells lose their cell-cell attachments and migrate to become
PNS tissue. The epiblast is one of the two cell layers of the bilaminar disk and is adjacent to the
amniotic cavity. Epiblast cells are precursors to ectodermal cells from which all embryonic tissues
eventually differentiate from
14. E. Overall: zygote > morula > blastocyst > inner cell mass (embryoblast) > bilaminar disk
(epiblast + hypoblast) > trilaminar disk (ectoderm + mesoderm + endoderm) > embryological
folding and formation of ventral body cavities > organogenetic period > fetal period > birth. The
primitive streak forms in the epiblast midline and signifies the onset of gastrulation (trilaminar
disk). Several waves of mesoderm migration occur due to invagination of epiblast cells adjacent to
primitive streak: paraxial mesoderm, then intermediate mesoderm then lateral plate mesoderm.
Both endoderm and mesoderm cells are simultaneously derived from the invaginating midline
epiblast cells- neither precedes the other. At the cranial end of the primitive streak the primitive
node forms and some cells from the primitive node migrate and invaginate to form the notochordal
plate among the cells of the embryonic endoderm. The notochordal plate cells detach and fold
length-wise into a cranial-caudal midline tube called the notochord. Paracrine signalling molecules
from the notochord induce overlying ectoderm cells to form the neural plate. As the neural plate
forms the neural tube, some cells, called the neural crest cells, detach, migrate and form PNS
structures.
15. B. is a false statement; A, C, D, and E are all true statements. Epiblast cells adjacent to the
primitive streak invaginate, lose their cell-to-cell adhesions and either become endoderm cells that
displace some of the hypoblast cells or migrate in three sequential waves of mesoderm: paraxial
then intermediate then lateral plate mesoderm. Regardless of the migration wave, some of the
mesodermal cells become embryonic connective tissue called mesenchyme that specializes to
become fetal cartilage, bone, fascia, and stroma of glands.
16. E. In some types of tissues (e.g. skin), a population of Transit Amplifying Cells (TACs) are found
adjacent to the stem cell niche. TACs do not exist without their parent stem cell population nearby.
TACs are daughter cells of stem cells and have less potency and less capacity to for self-renewal
(asymmetric replication). Under the appropriate stimulation, TACs are capable of rapid mitosis
resulting in hyperplasia and thus can form a larger population of cells that can further differentiate
into mature cells. For example weight lifting will cause callus formation in the epidermis of the
hands. The external mechanical stress results in expansion of the TAC population and increases the
thickness of the epidermis.
17. A. Self-renewal (asymmetric replication) means that only some of the daughter cells in a stem cell
niche will form more differentiated mature cell types. The remaining daughter cells remain part of
the anatomical and functional collective of stem cells. Adult stem cells are found in many types of
tissue including red bone marrow, skin epidermis and the epithelial mucosal lining of the gut tube,
trachea and urogenital tract. The mitotic activity of adult stem cells is the greatest in continuously
dividing tissue such as epidermis and the lining of the gut tube. Quiescent cell populations of the
liver, pancreas, endothelium, fibroblasts, resting lymphocytes have stem cells with low mitotic
activity. Non-dividing cell populations found in the CNS, heart and skeletal muscle have no
therapeutically significant stem cell populations (current state of science) and are composed of
cells that have left the cell cycle. Adult mesenchymal stem cells can become osteoprogenitor TACs
or chondroprogenitor TACs but not the other way around. Stem cells are located in just the basal
regions of the epidermis near the dermis or the basal regions of the intestinal mucosa layer away
from the lumen.
18. C. Somites are derived from the paraxial mesoderm and thus become bilateral (Left and Right)
structures found adjacent to the neural tube. Nearly 40 pairs of somites form by the end of Week 4
and can be used to asses embryonic age. Somite cells organize themselves into first a
dermomyotome and sclerotomes. The dermomyotome further segregates into a dermotome and
myotome. All three cell regions (dermatome, myotome, sclerotome) co-develop to form the
dermis, skeletal muscle, and bone of the body wall and limbs and certain neck regions. Tissues
derived from the dermatomes, myotomes, and sclerotomes are all are innervated by general
somatic afferents and efferents. Lateral Plate Mesoderm, the third wave of mesoderm, gives rise to
parietal and visceral layers. The partial layer of the LPM contributes to the parietal mesothelium
lining of the ventral body cavities (parietal pleura, parietal pericardium, parietal peritoneum).
19. B. Both the neural tube and notochord contain Inducer Groups of cells that release paracrine
signalling factors that induce the differentiation of Responder Groups in the somites. SHH is
released from the dorsal regions of the neural tube (i.e. not near the neural crest cells located in the
ventral neural crest) as well as the notochord. WNT is released from cells adjacent to the ventral
neural crest cells. WNT promotes the development of precursor dermis cells from the dermatome
and precursor muscle cells from the myotome. SHH also promotes the development of precursor
cells from the myotome as well as development of precursor cartilage cells in the sclerotome
20. D. The only embryological tissue in the list that can produce all three germ cell layers is the
primitive streak. The primitive streak is a midline thickening of epiblast cells that gives rise to first
ectoderm. Shortly after the ectoderm forms some of these nascent ectoderm cells near the
primitive streak invaginate and migrate to form both mesoderm and endoderm. Somites
differentiate from the paraxial mesoderm and have no ectoderm nor endoderm cells. Notochord
cells are derived from mesodermal cells from the primitive streak and helps form the vertebral
column. The notochord has no ectoderm nor endoderm. The trophoblast is the outer cell layer of
the blastocyst and does not contribute to embryonic tissue. The neural tube is derived from
ectoderm and has no mesoderm nor endoderm.
Instructional Objectives
A. Gestational Overview & Cell Potency
1. List the periods of human development and the titles of key events in Weeks 1, 2, 3 and 4. When does
the embryological period occur versus the organogenetic period verus the fetal period occur?
2. Define totipotent, pluripotent, multipotent and oligopotent, and give some examples of the
developmental stages when these potency stages present. (remember that, in reality, there is a
continuum of potency from totipotent to oligopotent).
a) What is the last totipotent and pluripotent stage of embryological development?
b) In the early embryo, how would the potency of a daughter cell typically compare to its
parent cell (more or less or about the same?)
B. Weeks 1 & 2 Overview

1. Consider the zygote, morula, blastocyst, and bilaminar disk; the embryoblast and trophoblast; the
epiblast and hypoblast. You should know the approximate temporal order in which these
embryological structures arise relative to each other.
2. What will the cells that form the outer cell mass and inner cell mass develop into? How is the
blastocyst cavity related to the inner and outer cell mass?
3. What does the epiblast and hypoblast give rise to?
4. What does '2s in Week 2'?
C. Week 3: Gastrulation and Body Tissue Correlates

1. Describe a few salient characteristics of epithelial tissue, connective tissue, muscle tissue, and nervous
tissue to the extent introduced in these embryology lectures. You will learn many (!) more
details later this semester. For example, which tissue covers exposed surfaces? fills internal
spaces?, produces active movement?, carries information?, lines internal passageways?, stores
energy?
2. Define hypertrophy, atrophy, hyperplasia, and neoplasia. Which of these four processes is necessary to
produce an overall increase in the size of embryo to grow into a fetus and hence baby?
3. What is the first visible sign of gastrulation? (3-layer formation?)
a) What is significance of the primitive streak and primitive node? Describe the events of
gastrulation in terms of inductive signalling by the primitive streak, cell
migration and changes in cell adhesion
b) What direction (cranial ->caudal?, vice versa?; medial -> lateral or vice versa?) is the
primitive streak and where is the primitive node formed? Which germ layer is
largely responsible for developing the medial-lateral body axis?
4. What does "3s in Week 3' mean?
5. Describe the development of the ectoderm, endoderm and mesoderm with regard to when it occurs
during embryological development and where the cells come from in the blastocyst. Use the
terms "invagination" and "migration" in your explanation.
6. Which embryonic germ layers (ecto, meso, endo) become which of the 4 tissue types (muscle,
nervous, CT, epithelial)?
D. Fates of the 3 Germ Layers- a closer look

1. What is the fate of the surface ectoderm and neuroectoderm?
2. Which of the 3 germ layers becomes the following structures: CNS, PNS, skin, skeletal muscle of
head/neck, skeletal muscle of body wall/trunk, skeletal muscle of the limbs, blood vessels,
heart, lungs, stomach, kidneys, limb bones, ribs, inner lining of the intestines, lymphatic vessels,
dermis, cornea of eye, posterior pituitary, anterior pituitary gland
3. What are the three mesodermal tissues and what is the order in which they form/migrate?
4. Which of the three mesodermal tissues are the following structures derived from: somites, urogenital
system, parietal mesothelium (e.g. parietal pleura or visceral peritoneum); visceral mesothelium
(e.g. visceral pleura or visceral peritoneum), smooth muscle of the gut tube, connective tissue of
the gut tube; blood vessels of the gut tube, skeletal muscle of the body wall, connective tissue of
the limbs, skeletal muscle of the head/neck; ribs
5. Besides the innermost lining (mucosa) of the gut tube what other structures related to the gut tube are
derived from endoderm?
6. What is mesenchyme, where does it come from and what does it eventually specialize into? Is
mesenchyme a tightly or loosely bound cell mass? Is mesenchyme fixed or can it migrate? Is
mesenchyme totipotent, pluripotent, multipotent or oligopotent?
E. Notochord Development and Significance

1. where structure of the epiblast does the notochord come from?
2. is the notochord longer, shorter or about the same length as the primitive streak? Is the notochord more
dorsal or more ventral or in the same tissue layer as the primitive streak?
3. what is the significance of the notochord and what does it become in the post-natal human?
F. Neurulation Overview
1. Describe neurulation- that is, what is the sequence of event in which the neural tube and neural crest
cells are formed from the neuroectoderm?
2. What embryonic tissue becomes the PNS and what embryonic tissue becomes the CNS? How are the
following PNS structures formed: posterior (dorsal) root ganglia, sympathetic trunks,
prevertebral ganglia, adrenal medulla, enteric ganglia
3. True/False: Neural crest cells develop into connective tissue structures of the face, skull and neck?
G. Somite Development and Significance

1. What is a somite and where to they develop in relation to the superior-inferior axis of the body?
2. What is the relationship (anatomical/spatial or functional or both) between the notochord and somites?
How are somites used to asses embryonic age?
3. what do sclerotomes, myotomes, and dermatomes become? What innervation do they receive?
4. How do the limbs develop? Do the UL and LL develop at the same time, if not, which develops first?
Which limb (UL or LL) undergoes a significant rotation during development? That is, can you
visualize why the palms of the hand face anterior and the soles of the feet face inferior in the
anatomic position?
H. Some Universal Mechanisms of Development

1. Define cell proliferation, specialization, interaction, movement and memory
2. What does it mean for a cell to have 'positional value' during early development?
3. Is inductive signalling an endocrine, paracrine, juxtacrine or autocrine process? (two may apply). What
is the difference between the Inducer Group and Responder Group?
4. Be able to recognize the names of molecular families of paracrine signalling proteins discussed in
lecture
5. Give an example of inductive signalling by the primitive streak, neural tube and notochord at the level
of detail in these lectures
6. Define apoptosis and describe its importance/significance. Are apoptosis and necrosis the same or
different?
I. Adult Stem Cell Basics

1. Distinguish between adult and embryonic stem cells in terms of origin and potential. Why are adult
stem cells important or significant?
2. Define what a stem cell is including its two basic properties.
3. What is a stem cell niche and transit amplifying cells? Give examples of niches that contain TACs.
Comment on the differentiation potential of TAC and their relationship to committed cells.
a) To the extent discussed in this lecture outline give examples of post-natal stem cell niches
and describe the purpose of these stem cells. What tissues do NOT have well
characterized stem cell niches?
b) What are the three general categories of the proliferative activity of tissues? Give examples
of each cell population in this tissue category and the relative importance of
adult stem cells in maintaining a competent tissue
4. What types of cells or tissues do adult mesenchyma stem cells (a.k.a. multipotent stromal cells)
differentiate into?
5. Where are hematopoietic stem cells found and what kinds of cells can they differentiate into?
PSL 534
STUDY GUIDE
(Fall Semester, 2011)
Connective Tissue
Including Connective Tissue Proper
and Cartilage & Bone *
Lecture Sessions 8-11

Lab Sessions 3-4
* Blood, another connective tissue, will be dealt with separately.
CONNECTIVE TISSUE: THE EXTRACELLULAR MATRIX
Chemical Components of Connective Tissue
PSL 534 Lecture Session 8
Dr. John Wang Tuesday, 9/13/11
BRIEF OVERVIEW
If living cells didnt have a fondness for sticking together, we would all be colorful globs of jelly
oozing all over the floor. Fortunately, cells hold to a basic biological premise that stickiness is
desirable for FORM and essential for FUNCTION. (Time, 9/28/92). Stickiness is, in fact central to
many aspects of biological processes. When cells become too sticky, arteries get clogged. When cells
become too slippery, tumor cells skate around the body looking for sites to metastasize. Inflammation
(which, normally, is a healing process) can turn subversive to become arthritis and multiple sclerosis.
Cells are able to form organs and function as a unit, thanks to a fascinating category of complex glues,
collectively known as the extracellular matrix. We now consider the components of the extracellular
matrix, their biochemical and physical properties, and how these properties serve their biological
function(s). This description will serve as the lead-in to our consideration of the various types of
connective tissue.
PREREQUISITE MATERIAL
Readings:
1) Ross and Pawlina Chap. 6, pp. 158-178
Embryonic Adult
Epithelial tissue forms sheets which line the
Endoderm, internal organs, the external surface, and
Mesoderm, Epithelium the blood and lymph vessels. It comprises
Ectoderm the barrier which determines what will enter
and leave the interior of the body.
Neurons, cells specialized for signaling,

Ectoderm Nervous Tissue and glia (support cells) of the brain,
spinal cord, and peripheral nerves
Mesoderm Specialized for motility and includes

(except some Muscle skeletal, cardiac, and smooth muscle
regions of head) (muscle of the viscera)
Mesoderm Connective Scaffold upon which everything else hangs;

(except some Tissue (CT) it includes many different cell types and
regions of head) large amounts of extracellular material.
Connective Tissue: Extracellular Matrix Lecture Session 8
LECTURE MATERIAL
EPITHELIA NERVOUS MUSCLE CONNECTIVE
TISSUE TISSUE (CT)
A continuum
blood adipose mucous loose dense dense cartilage bone

CT irregular regular
(body CT CT
cavity) (fascia) (tendon
ligament)
(hematopoietic)
(lymphatic)
SPECIALIZED CT PROPER SPECIALIZED
CELLS EXTRACELLULAR MATRIX
ADHESIVE GLYCOPROTEIN GROUND SUBSTANCE FIBERS
I) Extracellular Matrix (ECM): definition and general considerations

a) ECM is a collection of macromolecules that surround the plasma
membrane and comprises the SUBSTRATUM on which the cells
are attached.
b) Serves as biological glue, to maintain association with surrounding
molecules and other cells.
c) A hydrated gel-like ground substance with embedded fibers
large resistance
the gel --- resists forces of compression
little resistance
the fibers --- withstands tensile forces enormous tensile strength
(streching)
little tensile strength
II) Chemical composition
Fibrous CT (e.g. tendon) Cartilage Basement Membrane

Tissue_________ Some Basement Membrane Vitreous Body __________________
Cell Type Fibroblasts; Hepatocytes Chondrocytes Epithelial and
Myoblasts Endothelial Cells
COLLAGENS Type I Type II Type IV
(forms bundles) (forms fibers) (forms sheets)
GLYCOSAMINO- Hyaluronic acid (HA) HA CS
GLYCANS (GAGS) Chondroitin sulfate (CS) CS HS
Heparan sulfate (HS) Keratan sulfate (KS) Dermatan sulfate (DS)
ADHESIVE Fibronectin Chondronectin Laminin
GLYCOPROTEINS
[Type III collagen forms reticular fibers (loose fibrils) and is found in smooth muscle, marrow, lung
vessels and lymph organs.]
Difference from one kind of ECM to another is mainly due to differences in the
identity and/or proportion of these three classes of molecules.
III) Histological staining of ECM
(a) cells, as
indicated by the
dark staining of
(b) white space,

corresponding to
the ground
substance
(does not pick up
stain)
(c) pinkish fibers

(eosinophilic) ---
these are bundles
of
OUTSIDE of cells
In this micrograph, the Type I collagen bundles are irregular in direction and in
size: some large bundles, others small bundles.
IV) Collagens Collagen

A) Abundance and diversity (as % dry weight)
Tissue of organic molecules
1) Collagen is an important component bone 88
of the protein scaffold of the body; tendon 86
it constitutes ~25% of total body skin 74
protein and is a major organic cornea 65
(C,H, N) component of many cartilage 50
tissues. liver 4
2) There are >40 distinct genes/polypeptides (with different amino acid

sequences), classified into >20 main types based on their amino acid
sequence and higher order organization (e.g. bundles, fibers, sheets).
Different types predominate in different tissues. (see table, p. )
B) Structural features
all of the different collagen polypeptides share important general features
1) Primary Structure
a)
b) Also high content of Pro and Lys, some of which are
4-OH Pro 5-OH Lys

Serves as the point of attachment of sugars to the collagen polypeptide
Enzymes for hydroxylation ( )

require, as co-factors, ascorbate (vitamin C) and deficiency in the vitamin
affects collagen processing.
2) Secondary Structure ---entirely helical but NOT -helical

-helix collagen helix
residue/turn 3.6 3.3
rise/residue 0.15 nm 0.29 nm
pitch (height 0.54 nm 0.96 nm
of complete turn)
3) Tertiary Structure (There is no tertiary structure.)

4) Quaternary Structure
The collagen polypeptide is translated on ribosomes of the RER.
As it passes through the ER- Golgi, it is post-translationally modified
(e.g. hydroxylation of Pro and Lys) and then assembled into a triple helix.
The three collagen polypeptides can fit closely

together because every third residue, facing the
interior of the triple helix, is a Gly (small, little
steric hindrance). Mutation(s) of Gly to another aa with bigger
side chain gives rise to problems in collagen assembly.
C) Assembly to form fibril

Alignment of collagen important:
Tendon all aligned in one

direction --->resist tensile forces
in one longitudinal direction.
Skin less well aligned --->

resist tensile forces in many
different directions.
(collagen is weak when forces
applied perpendicular to the direction of the fibril)
Fibrils of collagen molecules are stabilized by repetitive H-bonds, both

perpendicular and parallel to axis of the helix (lots of OH for H-bonds from
OH-Pro, OH-Lys, and the saccharides attached via these OH-Pro and OH-Lys).
They are also covalently cross-linked (x-linked)
rxn with NH2-groups
Lys and OH-Lys ----------------------->aldehydes ---------------------> covalent x-links
on another collagen molecule
Extraordinary length extraordinary strength
(Stiff skin/tough meat --- due to spontaneous oxidation and cross-linking)

D) COLLAGEN DEFECTS and DISEASED STATES

1) A 5-year-old white male is brought to the emergency room with a fracture of his right
forearm that he sustained after falling off a couch. This is the fifth bone fracture in the past
two years. Physical exam revealed right leg and right arm slightly deformed from poor
healing of past fractures. Abnormal softening of tooth enamel. Mild scoliosis (spine curved)
and hypotonia (low muscle tone). Additional complications of osteopenia and hearing loss.
(Physical child abuse? Idiopathic juvenile osteoporosis?)
a group of connective tissue disorders
e.g. defective type I collagen: 1 amino acid residue changed
:disrupts the triple helix cannot form ordered
fibrillar arrays;
brittle bones; multiple fractures; some skeletal deformities
Fig. 4.11 Harvey and Ferrier, Biochemistry Fig. 4.10 Harvey and Ferrier, Biochemistry
2011, LWW. Reproduced with permission. 2011, LWW. Reproduced with permission.
2) A 9-year-old boy is brought to the emergency room with pain, inability to move his left
shoulder, and flattening of the normal rounded shoulder contour. Patient history revealed
many prior shoulder dislocations in the past: 9 times right shoulder and 3 times left shoulder.
The boy also has a history of easy bruising. Laboratory data indicated that the blood clotting
profile is normal.
another group of genetically inherited disease,
defective post-translational steps of fiber formation
e.g. deficiency in
hypermobility of
poor cross-linking -----> loss of fiber strength ------> joints
Note on nomenclature: Ehlers-Danlos Type IV refers to type IV of the group of Ehlers-
Danlos diseases. The specific collagen affected is Type III collagen, resulting in rupture
of vessels and internal organs!
3) (vitamin C deficiency)
Vitamin C, required as a cofactor of prolyl hydroxylase and lysyl hydroxylase
less H-bonding parallel to the length of helix ---> loss of strength/stability fibers
Problems of deficiency show up in tissues with high turnover of collagen
e.g., Gums weakened and teeth fall out!
V) RETICULAR FIBERS
(1) Type III collagen.
(2) narrow diameter (~20 nm); do not bundle but forms meshwork.
(3) prominent in initial stages of wound healing and scar tissue formation;
also in embryonic, hemopoietic and lymphatic tissues (lymph node).
(4) best revealed by special silver staining procedures.
VI) ELASTIC FIBERS ---

(1) composed of three key proteins
(a) a collagen-like protein
rich in Gly and Pro, as well as two unusual amino acids,
desmosine and isodesmosine, which are easily cross-linked,
providing a high degree of elasticity to the fibers.
(b)
forms microfibrils upon which

elastin is deposited and organized
(c) MAGP (microfibril-associated

glycoprotein) links elastin to fibrillin
(2) elastic fibers allow tissues to respond to stretch and distension;

- are arranged in a branching pattern to form a network;
- are interwoven with coexisting collagen fibers to limit distensibility
of tissue and prevent tearing from excessive stretching.
- are revealed and distinguished from collagen fibers by special stains.
(3) elaborated by fibroblasts of loose CT,

by smooth muscle cells of blood vessels, and
by chondroblasts and chondrocytes of elastic cartilage
(4) degree of assembly varies with tissue ---

- thin fibers, interspersed among collagen, in loose CT;
- thick fibers in elastic ligaments (e.g. nuchal ligament of neck);
- perforated sheets (lamellae) in blood vessels (especially aorta)
VII) Matrix Metalloproteinases (MMPs)

- collagenases (degrades various types of collagens);
- gelatinases (degrades collagens, elastin, fibronectin, laminin, etc.)
- stromelysins (degrades proteoglycans, fibronectin, collagens)
- excessive collagen degradation in cartilage (rheumatoid arthritis), bone
(osteoporosis), various tissues (tumor metastatsis).
VIII) Glycosaminoglycans (GAGs)

1) All are polysaccharides consisting of repeating disaccharide units
monosaccharide A + monosaccharide B
2) All are negatively charged, due to COO- and SO42- groups.
Repeat unit of hyaluronic acid Repeat unit of chondroitin 4-sulfate

Repeating Disaccharide (A-B)
Mol. Sulfates Linked
Glycosaminoglycan Weight Monsaccharide A Monosaccharide B per to Tissue Distribution
Disaccha- Protein
ride Unit
Hyaluronic acid 1 to 10 D-glucuronic acid N-acetyl-D-glucosamine 0 - various connective tissues,
5 skin, vitreous body,
x 10
cartilage, synovial fluid
Chondroitin 4-sulfate 5,000- D-glucuronic acid N-acetyl-D-galactosamine 0.2-1.0 + cartilage, cornea, bone, skin,
50,000 arteries
Chondroitin 6-sulfate 5,000- D-glucuronic acid N-acetyl-D-galactosamine 0.2-2.3 + cornea, bone, skin, arteries
50,000
Dermatan sulfate 15,000- D-glucuronic acid or L- N-acetyl-D-galactosamin 1.0-2.0 + skin, blood, vessels, heart,
40,000 iduronic acid heart valves
Heparan sulfate 5,000- D-glucuronic acid or L- N-acetyl-D-glucosamine 0.2-3.0 + lung, arteries, cell surfaces
12,000 iduronic acid
Heparin 6, 000- D-glucuronic acid or N-SO4-D-glucosamine 2.0-3.0 + lung, liver, skin, mast cells
25,000 *L-iduronic acid
Keratan sulfate 4,000- D-galactose N-acetyl-D-glucosamine 0.9-1.8 + Cartilage, cornea,
19,000 intervertebral disc
*L-iduronic acid is produced by the epimerization of D-glucuronic acid at the position where the carboxyl group is located.
3) PROTEOGLYCANS---Most GAGs exist in the form of proteoglycans, in
which the polysaccharide chain of GAG is COVALENTLY attached to a
polypeptide, called the
domains (exemplified here by the C4S-KS proteoglycan of cartilage)
COOH
CS region: ~100 C4S chains
covalently attached to core protein
KS region:~60 KS chains
covalently attached to core protein
HA BINDING region: devoid of GAGs in the

NH2 PROTEOGLYCAN
but is used to bind non-covalently to HA in forming
a PROTEOGLYCAN
Proteoglycan Aggregate (e.g. aggrecan of cartilage)

many PROTEOGLYCAN MOLECULES
links individual
PROTEOGLYCAN MOLECULES
(through the HA-binding region) to the HA
backbone to form the
PROTEOGLYCAN AGGREGATE
actual EM of proteoglycan aggregate from cartilage

4) Physical properties of proteoglycans in relation to their structure

- (-COO & SO42)
- adjacent GAGs and proteoglycans repel each other
- proteoglycans occupy largest possible space,
encompassing a large volume of water
- can regulate fluid balance through
when pressure is applied, structures deform by squeezing

out H2O; when pressure is removed, repulsion drives the
structure to expand, water reenters. This represents a
mechanism for fluid exchange, which is important for an
avascular tissue to obtain nutrients.
- SO42 & COO- groups can bind

providing physical shield to prevent growth of
calcium crystals (calcification).
IX) ADHESIVE GLYCOPROTEINS

Adhesive glycoproteins link ECM components to the cell
e.g. (others: laminin; chondronectin)
FN has two polypeptides (each Mr ~220,000), linked by disulfide bonds

Each polypeptide has discrete DOMAINS for binding different things.
FN binds to ECM FN can bind to cells

cell-binding domain has the RGD
collagen binding domain (Arg-Gly-Asp) sequence motif,
heparin-binding domain which binds to
a generic name for a family of

receptors, all of which share the
recognition of RGD.
Thus, FN can serve as a glue to maintain the association of cells with the matrix
of a tissue.
Integrins also link the outside of the cell

to the inside of the cell
Binding of ligands (e.g. heparin) to
FN on the outside
signals to the inside
Alters the actin cytoskeleton
Modulates the response of the cells
to other signals
(e.g. heparin-dependent growth factors: fibroblast growth factor)
(mechanical linkage, followed by a biochemical signal --- juxtacrine signaling)
X) Interface between connective tissue and other tissues

A) Basal Lamina --- interface between a sheet of epithelial cells and the
underlying CT, to which it is anchored;
synthesized by the epithelial cells. Schematic diagram of connective
tissue underlying an epithelium
1) collagen: primarily type IV
2) proteoglycans: percalan, a
heparan sulfate proteoglycan
3) adhesive glycoproteins:
laminin and entactin
B) Lamina reticularis---
synthesized by the CT cells,
with primarily type III collagen
and FN as adhesive glycoprotein.
(Basement membrane is a light
microscopy designation corresponding to
basal lamina + lamina reticularis; Fig. 19-31 from Alberts. Molecular Biology of
basal lamina is observable by electron microscopy.) the Cell, 3rd edition.
C) External lamina --- structure corresponding to the basal lamina for

1) smooth and skeletal muscle;
2) adipose tissue;
3) Schwann cells (glial cells of peripheral nervous system)
D) Functions --- all of the types of basal lamina serve the purpose of:
(1) attachment (of epithelial cells); (2) barrier of passage for cells and molecules
(e.g. filtration in kidney); and (3) restricts/regulate epithelial migration.
SELF-INSTRUCTIONAL PROBLEMS
1) The diagram shown below schematically illustrates the structure of a proteoglycan molecule
isolated from cartilage. Match the names (Arabic numerals) with the parts shown on the
diagram (Roman numerals).
1. keratin sulfate region

2. chondroitin sulfate region
3. core protein
4. hyaluronic acid binding region
2) The diagram below schematically illustrates a transmembrane protein embedded in the lipid
bilayer of a plasma membrane. Identify the parts (domains) of the molecules labeled A, B, and
C. Use terms that apply, in general, to any old garden-variety plasma membrane protein.
3) Use the same diagram shown above. Now suppose the molecule depicted is the cell surface
receptor for fibronectin (one member of the integrin receptor family on fibroblasts). Identify
the parts (domains) of the molecules labeled A, B, and C. In this case, indicate specifically
what molecule(s) each part (domain) will bind. Discuss (very briefly) the possible significance
of this integrin with respect to the extracellular matrix and the cytoskeleton.
4) The extracellular matrix consists of several distinct parts. Below, each part of the extracellular
matrix is matched with a specific example of that component. Which of the following pairs
represents an incorrect match?
A. cells --- fibroblasts

B. fibers --- collagen
C. ground substance --- elastin
D. ground substance --- proteoglycan aggrecan
5) Which of the following pairs of molecules are glycosaminoglycans?
A. collagen and elastin

B. hyaluronic acid and heparan sulfate
C. fibronectin and chondronectin
6) For which of the following reactions is ascorbic acid directly required in the synthesis of the
correct form of collagen?
A. polypeptide synthesis
B. carbohydrate attachment
C. hydroxylation of proline
D. proteolytic cleavage
E. formation of lysine-lysine cross-links
7) Lysyl oxidase is an enzyme involved in post-translational modification of collagen. Which of

the following statements describes it correctly?
A. It requires ascorbic acid for activity.

B. It converts lysine to hydroxylysine.
C. It is involved in cross-linking collagen fibers
D. Its activity is markedly diminished with aging.
E. Its activity is diminished in the disease state scurvy.
8) The genetic code provides codons for 20 amino acids. These are known as the primary amino
acids. Secondary amino acids are not coded at the level of the gene but are derived through
post-translational modification of certain primary amino acids. Of the secondary amino acids
listed below, which one is found on collagen and whose post-translational modification
depends on vitamin C?
A. phosphoserine
B. phosphothreonine
C. phosphotyrosine
D. hydroxyproline
E. -carboxyglutamic acid
9) The extracellular matrix of most connective tissue proper is composed of a hydrated gel-like
ground substance with fibers embedded in it. Ground substance is composed of
glycosaminoglycans, proteoglycans, and adhesive glycoproteins. The most prominent protein
that makes up the fibers of the extracellular matrix is _______________________.
10) Look at Figure 6.19 of the Ross and Pawlina text, which shows a representative histological
stain of connective tissue. In this micrograph, what is the color of the collagen fibers?
11) Look at Figure 6.2, panel b of Ross and Pawlina, which shows a representative histological
stain of mucous connective tissue (Whartons jelly from umbilical cord). Does the ground
substance yield any staining? Is so, what is the color of the stain?
ANSWERS TO SELF-INSTRUCTIONAL PROBLEMS
1) 1. III
2. II
3. I
4 IV
2) A. extracellular domain
B. transmembrane domain
C. cytoplasmic domain
3) A. ligand binding domain (binds RGD sequence of fibronectin)

B. transmembrane domain (binds lipids of membrane)
C. cytoskeletal binding domain (binds to cytoskeletal components, e.g., actin)
Binding of ligand (fibronectin) on the outside of the cell can alter the organization of the
cytoskeleton on the inside mechanical signal transduction, rather than biochemical.
4) C (elastin is the protein component of elastic fibers)
5) B
6) C
7) C (lysyl oxidase should not be confused with lysyl hydroxylase; the latter is responsible for
hydroxylation of lysine and is the enzyme that requires vitamin C/ascorbate as a cofactor)
8) D (hydroxylation of proline and lysine residues require vitamin C/ascorbate)
9) collagen
10) pink
11) white (the ground substance fails to stain with H & E)

INSTRUCTIONAL OBJECTIVES
What you should be able to do:
I) Extracellular matrix
A. List the cellular and non-cellular components of connective tissue.
B. Categorize and compare the three major classes of molecules constituting the
extracellular matrix.
II) Structural features of collagen polypeptide

A. Describe the primary structure and the special features of glycine, lysine, and proline
residues.
B. Describe the secondary structure and report the lack of typical tertiary structure.
C. Diagram the quaternary structure in terms of a triple helix and rationalize in terms of the
forces stabilizing such a helix.
D. Identify hydroxylation and glycosylation as examples of post-translational modification
of proteins, particularly of collagen.
E. Identify proteolytic cleavage as another example of post-translational modification and
illustrate its effect on collagen.
F. Identify covalent cross-linking as still another example of post-translational
modification and illustrate its effect on collagen.
G. Categorize the basis of genetically inherited collagen disorders and predict their
consequences.
H. Identify the basis of acquired collagen defects in ascorbate deficiency.
III) Glycosaminoglycans (GAGs) and adhesive glycoproteins

A. Recognize the structure and nomenclature of the GAG molecules and identify their
common characteristics: repeating structures of disaccharides with negative charges.
B. Sketch and distinguish GAGs versus proteoglycans and proteoglycan molecule versus
proteoglycan aggregate. Identify their components.
C. Rationalize the physical properties of the proteoglycans in relation to their structure.
D. Describe how an adhesive glycoprotein (e.g. fibronectin) can link a cell to its
extracellular matrix.
IV) Elastic fibers and reticular fibers

A. List the three major proteins of elastic fibers.
B. Identify the three most common forms of elastic fibers as exemplified in loose CT,
in elastic ligaments, and in blood vessels of the aorta.
C. Distinguish collagen fibers, reticular fibers, and elastic fibers in terms of their principal
protein composition, morphological appearance, and function.
V) Distinguish basal lamina and lamina reticularis in the basement membrane.

Cellular Components of Connective Tissue
PSL 534 Lecture Session 9
Dr. John Wang Tuesday, 9/13/11
BRIEF OVERVIEW
Having now described the chemical components and the physical properties of the extracellular matrix,
we can catalog and distinguish the cells of the connective tissue. There is actually a continuum of
connective tissues: blood at one extreme and bone at the other. Because of this heterogeneity, many
functions can be attributed to connective tissue. In general, however, the connective tissue serves as
the supporting tissue (stroma) of an organ, helping the parenchyma of the organ to carry out its
function.
Because of the wide range of this continuum of connective tissues, there are many cell types, each with
its distinctive function(s) and histological staining characteristics. The best way to learn these is by
repeated exposure to micrographs, using both a hard copy atlas and the electronic images available at
the recommended website(s).
PREREQUISITE MATERIAL
Readings:
1) Ross and Pawlina Chap. 6, pp. 178-197

Chap. 9, pp. 254-267
Connective Tissue: Cellular Components Lecture Session 9
LECTURE MATERIAL
I. General Considerations
A) Continuum of extracellular fiber density and their orientation
Type
Loose Low density Body cavity; surrounding blood vessels & glands;
(areolar) of fibers CT subjacent to basal lamina and external lamina
Dense Dense, Fascia, capsules of organs, underlying epidermis

irregular irregularly
arranged
fibers
Dense Dense, Tendons and ligaments
regular regularly
arranged
fibers
Other designations:
Adipose: adipocytes (fat storing cells)
Reticular: loose CT of primarily Type III collagen; meshlike organization
support for certain organs (e.g. spleen)
Elastic: CT in which elastic fibers predominate; elastic laminae (sheet)
of blood vessels (aorta) or elastic ligaments (nuchal ligaments)
B) Functions
1) structural support dense CT loose CT
2) medium exchange loose CT dense irregular dense regular
3) defense/protection: (i) physical barrier to microorganisms
(ii) immune response (chemical and cellular)
(Loose CT is usually just underneath the epithelium, which separates the
outside from the inside of the body; thus, it represents the first barrier.
Loose CT also has high permeability to fluids and cells and thus is usually
the site of edema (water entry) and inflammatory response.)
4) fat storage
In general, the CT represents the stroma or the supporting tissues of an organ,

helping the parenchyma of the organ to function (e.g. the CT forms the routes
by which blood vessels can gain access into the organs)
C) Components 1) ECM: (i) ground substance; (ii) fibers

2) cells
(a) ---resident in CT; stable, long-lived
(fibroblasts, adipocytes, mast cells, [macrophages], adult stem cells)
(b) ---circulating in blood stream;

short-lived and replenished
(neutrophils, eosinophils, basophils, monocytes, macrophages,
lymphocytes and plasma cells)
II. Fixed CT cells: (mostly) derived from mesenchymal stem cell

A) Fibroblast
1) note on general nomenclature:
= precursor (fibroblast; erythroblast);
= final form (fibrocyte; erythrocyte)
Here, the parts of fibroblast mean

blast---gives rise to something
fibro---fibers (collagen)
produces the major part of the ECM
2) fusiform shaped; sometimes elongated

In H & E stain of tissues, dark nuclei appear flattened
Cytoplasm pale staining under H & E
(many more mitos and vesicles under EM in fibroblast than in fibrocyte)
B) Myofibroblast
fibroblast-like in appearance but includes contractile proteins (myosin
and -smooth muscle actin) like a smooth muscle cell;
prominent proliferation during wound healing;
contractile property responsible for closing the wound --- tissue scars
C) Pericyte (perivascular cells) and adult stem cells

Surrounds the endothelial cells of capillaries (with contractile properties);
Most likely the multipotential mesenchymal stem cell
D) Adipocytes (adipose cells; fat cells)
1) white space under H & E---most of the lipids are extracted from tissue during
the fixing-staining protocols.
2) cells synthesize and store triglycerides (fat)
3) (white adipose tissue; yellow fat)
one large fat droplet; crowds the nucleus to edge of plasma membrane
the white adipose cell stores triglycerides for energy (BMB 514)
4) adipose tissue is a secretor of a wide variety of signaling proteins!!!

a) regulation of (decreases) appetite (satiety factor);
stimulate metabolic rate;
regulation of vascular tone (blood pressure control);
b) interferes with insulin receptor signaling (possible
cause of development of insulin resistance in obesity);
c) increases insulin resistance, linking obesity to type 2
diabetes.
5) (brown fat)
many small fat droplets; nucleus not crowded to edge; numerous
mitochondria, whose cytochromes contain heme Fe (brown color)
prominent in fetus/newborn; brown fat important in thermogenesis (heat
generation) via regulated uncoupling of oxidative phosphorylation (BMB 514)
From Histology: A Text and Atlas by M.H. Ross and W. Pawlina, Fifth Edition
Copyright 2006 by Lippincott Williams &Wilkins. Reproduced with permission.
III) Transient CT cells: (mostly) derived from bone marrow
From Histology: A Text and Atlas by M.H. Ross and W. Pawlina, Sixth Edition
Copyright 2011 by Lippincott Williams &Wilkins. Reproduced with permission.
A) Plasma Cells
1) large, ovoid cells (20 m diameter), eccentrically placed nucleus
heterochromatin radiating from center of nucleus (clockface)
cytoplasm intensely basophlic, with well-developed rough ER.
2) antibody-producing cell, derived from differentiation of B-lymphocytes
upon antigenic stimulation (MMG 522)
3) scattered throughout the connective tissue but numbers are greatly
increased in areas of chronic inflammation
B) Mast Cells
1) similar in origin and related to basophils but mast cells usually in CT of
skin and mucous membranes
. Comparison of Features Characteristic of Mast Cells and Basophils
Characteristic Features Mast Cells Basophils
Origin Hemopoietic stem cell Hemopoietic stem cell
Site of differentiation Connective tissue Bone marrow
Cell divisions Yes (occasionally) No
Life span Weeks to months Days
Size 20-30 m 7-10 m
Shape of nucleus Round Segmented (usually bilobed)
Granules Many, large, metachromatic Few, small basophilic
Surface Fc receptors for IgE Present Present
2) surface receptors bind immunoglobulin E (IgE) and mediate inflammatory

immune response
a) cytoplasm contains different granules:
Primary mediators (preformed and packaged)
(i) histamine - vasodilation and permeability of small blood vessels,
causing edema and skin reaction such as itching.
It increases mucous production in the bronchial tree of

the lung and prompts contraction of the smooth muscle
in the pulmonary airways.
(ii) chemotactic factors to attract neutrophils and eosinophils to
site of inflammation\
(iii) proteases: tryptase and chymase; markers of mast cells and used to
distinguish subclasses of mast cells (e.g. skin versus lung)
(iv) heparin (for mitogenic action of heparin-dependent growth factors)
[Heparin, a GAG, is a polyanion --- stains purple with toluidine blue,
indicative of polyanions and referred to as metachromatic.]
b) secondary mediators (synthesized on stimulus)

(i) leukotrienes - vasodilation and vascular permeability;
Triggers prolonged constriction of smooth muscle in the pulmonary
airways, causing bronchospasm.
[These are Slow Reacting Substances of Anaphylaxis observed during
immediate hypersensitivity (anaphylactic rxn) to bee venom, pollen, etc.]
(ii) prostaglandin D2 - increase in mucous secretion
(iii) cytokines - interleukins (IL-3, IL-4, etc.), tumor necrosis factor (TNF),
Colony Stimulating Factors (GM-CSF) that affect growth
proliferation, and differentiation.
C) Macrophages
1) monocytes (which are derived from the bone marrow) that have left the
circulatory system and taken up residence in CT.
previously designated by names peculiar to the organ---
Name Location
Macrophage (histocyte) Connective Tissue
Macrophage Bone Marrow, spleen, thymus, lymph nodes
Kupfer cell Liver
Dust cell (alveolar macrophage) Lung
Microglia Central nervous system
Osteoclast Bone
Langerhans cell ?(dendritic cell) Skin
Dendritic cell Lymph node; spleen
2) 10-30 m diameter; irregular shape

Typically identified under light microscopy by pretreating
with a dye, which the macrophage ingests (phagocytosis)
ovoid shaped nucleus, usually indented on one side
3) three general functions
destroy foreign microorganisms

and old cells and cell debris
produce cytokines, proteases, etc.

(e.g. interleukins; plasminogen activators)
MMG 522
D) Leukocytes (white blood cells)

1) travel in bloodstream but function in body parts other than the
circulatory system (e.g. function after infiltration of CT spaces)
2) cell numbers (in units of cells/mm3):
<5 X 103 6 10 X 103 >12 X 103
3) classification:
(granular leukocytes) (mononuclear leukocytes)
Neutrophils eosinophils basophils monocytes lymphocytes
staining characteristics of granules gives rise to the names of the cells (will be
covered in Lecture Session #12, under blood cells)
SELF-INSTRUCTIONAL PROBLEMS
Please go to the Web site: www.pathguy.com/histo/000.htm
There you will find Eds Basic Histology Gallery
Scroll down to Start Learning
Look at the following images and read Eds commentary for each:
#1 Cells and Fibers
#2 Cells in Relationship
#4 More epithelium and Fibrous Tissue
#5 Nuclei and Cytoplasm
#11 Heterochromatin and Euchromatin
#51 Lymphocytes and Plasma Cells
#52 Dense Irregular Connective Tissue
#64 White Fat
#70 Brown Fat
#97 Macrophages
#99 Neutrophils in Connective Tissue
________________________________________________________________________________
The questions below refer to images found at the Web site www.pathguy.com/histo/000.htm
1) In image #51, Ed tells you that plasma cells are differentiated from .
2) From what tissue was image #52 prepared? Does this belong to the category of dense irregular
connective tissue?
3) In image #64 (White Fat), what does Ed say immediately below the image?
4) With respect to image #70, why is multilocular fat brown?
5) In image #97 (Macrophages), Ed tells us that the macrophages are loaded with a chemical.
Describe the person from whom these macrophages are derived. List three functions for
macrophages. Of these, which function does image #97 best illustrate?
6) In image #99 (neutrophils in Connective Tissue), what cell (right of center in the image) is
highlighted by Ed?
ANSWERS TO SELF-INSTRUCTIONAL PROBLEMS
1) B-lymphocytes
2) Dermis is indeed classified as dense irregular connective tissue.
3) Ed says, This is common, yellow fat.
4) Multilocular fat is brown because it is loaded with mitochondria, which contain the cytochrome
proteins. These proteins carry the prosthetic group heme, which contains Fe, giving rise to the
brown coloration.
5) The macrophages shown in the image are derived from a smoker. These cells have
phagocytosed lots of carbon particles.
The three major functions of macrophages: (a) phagocytosis; (b) secretion of cytokines, etc.;
and (c) processing and presentation of antigen.
6) Plasma cell.
INSTRUCTIONAL OBJECTIVES
What you should be able to do:
1) List in order the continuum of connective tissues

a) distinguish connective proper and specialized connective tissues
b) distinguish loose versus dense connective tissues
c) distinguish regular versus irregular dense connective tissues
d) classify the different connective tissues (e.g. fascia, tendon) into each category
2) Describe the general functions of connective tissues and relate the predominant fiber type,
fiber density, and fiber orientation to the function of the tissue:
a) structural support
b) medium exchange
c) defense
d) fat storage
3) Describe the functions of individual types of connective tissue cells

a) fixed connective tissue cells (fibroblasts, adipocytes, mast cells)
b) transient connective tissue cells (plasma cells and macrophages)
4) Distinguish the various cell types in histological stains

Lab Session #3: Connective Tissue Proper
9/14/11 or 9/15/11
Introduction
General properties and components of connective tissue
Connective tissue cells are not linked to one another, and in some cases (wandering cells) they are able
to move through the extracellular matrix.
The type and arrangement of extracellular fibers in a connective tissue will largely dictate its strength,
permeability and function. In H&E stained sections, one type of extracellular fiber, type I collagen,
stains well with eosin, so that the extracellular bundles can be seen easily. We will evaluate the density
of these type I collagen bundles and their orientation in order to categorize connective tissues as either
loose or dense, irregular or regular.
Ground substance in most preparations will be washed away. Even though it is not stained, however,
you should recognize the locations of ground substance, and understand the composition and function of
this component of connective tissue. (As you progress through this and future Lab Sessions, you will see
how special stains may be used to specifically highlight various components of connective tissue.)
Types of connective tissue named for the dominant extracellular fiber type and its arrangement.
Loose (areolar) connective tissue (Plate 4, top, pp. 192-193)
o As compared to other types of connective tissue, loose connective tissue has relatively few
extracellular fibers (primarily type I collagen) and relatively more ground substance. Extracellular
fiber bundles in this type of tissue are thin compared to the thick bundles seen in dense connective
tissue.
o It usually has relatively more cells and less extracellular matrix than does dense connective tissue.
o It surrounds blood vessels, lymph vessels and glands, and lies immediately subjacent to the
epithelium of the viscera, where it is called lamina propria.
o Properties of loose connective tissue:
Highly permeable to oxygen, water and other molecules
Usually contains many fixed and transient cell types, including cells of the immune system.
These immune cells interact with foreign material that has succeeded in breaching the epithelial
barrier, so that loose connective tissue may be a site of inflammatory and allergic reactions.
Dense irregular connective tissue (Plate 4, top, pp. 192-193)

o Composed mostly of type I collagen.
o Extracellular fibers form thick, irregular eosinophilic bundles.
o Cells are sparse.
o Properties of dense irregular connective tissue:
Can withstand mechanical stresses from varying directions
Examples include the dermis of the skin, capsules of organs and the submucosa (a layer in the
walls of hollow organs, such as in the gastrointestinal tract).
Dense regular connective tissue (Plate 5, pp. 194-195)

o Extracellular fibers are predominantly type I collagen.
o Fibers are arranged in densely packed, parallel, eosinophilic bundles.
o The fibroblasts tend to be aligned in rows, and the cytoplasm of the fibroblasts is often difficult to
distinguish from the extracellular fibers.
o Properties of dense regular connective tissue:
Strong when mechanical stresses are applied parallel to the direction of the fibers.
Found in ligments, tendons, and aponeuroses.
Connective Tissue Proper Lab Session 3
Reticular tissue (Fig. 6.12, pg. 171)

o In this tissue, the extracellular fibers are type III collagen (reticular fibers). These fibers are much
too thin to be seen in routine H&E stained light microscopic sections. Silver stains are used to make
these fibers visible as brown or black.
o Reticular tissue is mesh-like in its arrangement.
o Properties of reticular tissue:
Provides structural support in the stroma of many non-tubular organs, such as the liver,
kidneys and lymph nodes.
Its mesh-like arrangement facilitates passage of cells and other material through the organ.
*** Please note, we will discuss type II collagen in Lab Session 4, Cartilage and Bone. ***
Elastic tissue
o Elastic tissue allows stretch followed by a return to its original shape.
o Elastic tissue may be in the form of elastic fibers or elastic lamellae.
Elastic fibers are found in ligaments which need to stretch and return to their original length,
such as the nuchal ligament. In these ligaments, thick elastic fibers are interspersed among
collagen bundles. In routine H&E stained sections, elastic fibers stain with eosin, but not as well
as collagen fibers stain. Special stains can be used to preferentially highlight elastic fibers and
distinguish them from other extracellular fibers.
Elastic lamellae (Plate 6, lower right, pp. 196-197) are present in muscular and elastic arteries,
such as the aorta. They are arranged as thin, perforated sheets with intervening smooth muscle.
Unlike elastic fibers, which are synthesized by fibroblasts, elastic lamellae are synthesized by
smooth muscle cells. Elastic lamellae appear refractile (white and glassy) in H&E stained
sections.
Adipose tissue (Fig. 9.2, pg. 258)

o Large aggregations of adipocytes are referred to as adipose tissue.
o In adipocytes, a thin rim of cytoplasm surrounds a large, unstained lipid droplet. The small amount of
extracellular matrix between adipocytes may be difficult to identify.
o The nuclei of adipocytes are pushed peripherally by the lipid droplet, giving the cell an appearance
similar to that of a signet ring.
Resident cells are relatively stable components of connective tissue.
Fibroblasts (Fig. 6.20, pg. 181)

o Synthesize the fibers (type I collagen, type III collagen, elastic fibers) and ground substance of
connective tissue.
o Thin, elongated cells, they have sparse cytoplasm, which may not be visible in LM.
o Usually the nucleus is dark and flattened.
o Fibroblasts which are actively producing extracellular fibers are plump, with relatively more
euchromatin in their nuclei compared to inactive fibroblasts.
Adipocytes (Fig. 9.2, pg. 258)

o Store neutral fat and produce hormones.
o Unilocular (one cytoplasmic lipid droplet) adipocyte in white adipose tissue.
o Multilocular (numerous cytoplasmic lipid droplets) in brown adipose tissue.
Wandering (transient) cells generally migrate into the connective tissue in response to a
specific stimulus, such as an infectious process or an allergic reaction.
Mast cell (Fig. 6.23, pg. 186)

o 25 m diameter ovoid cell
o Small spherical nucleus
o Cytoplasm filled with granules containing mediators (such as histamine), which participate in
hypersensitivity reactions, allergies and anaphylaxis
Macrophage (Fig. 6.22b, pg. 184)

o 20-30 m diameter irregularly shaped cell
o Ovoid, slightly indented nucleus
o Cytoplasm has inclusions with a variety of shapes and sizes. Most of these inclusions represent
material which has undergone phagocytosis
o Often requires special preparation to be identified in LM section
o Functions in phagocytosis and antigen presentation
Lymphocyte (Plate 18, top, pp. 304-305)

o Slightly larger than an erythrocyte (>7.5 m diameter)
o Nucleus appears round, oval or slightly indented
o Cytoplasm is scant and basophilic
o Participates in cell mediated and humoral immunity
Plasma cell (Fig. 6.25, pg. 190)

o 20m diameter ovoid cell
o Nucleus is eccentric (off-center) within the cell, and its chromatin is condensed in a pattern similar to
the numbers of an analog clock (you will hear the term clock-face nuclei).
o Cytoplasm generally basophilic. A large Golgi apparatus near the nucleus is sometimes apparent as a
large unstained region in the cytoplasm.
o Produces immunoglobulins (proteins), which are secreted constitutively.
Also included in this category would be white blood cells, such as neutrophils and eosinophils, which
infiltrate as a part of inflammatory processes. We will study these cells in more detail in Lab Session 5,
Blood and Hematopoiesis.
Preparation
Review your lecture notes and readings regarding connective tissue and adipose tissue. In Chapters 6 and 9 of
Ross and Pawlina, pay particular attention to the figures and tables. These should help clarify the characteristics
of connective tissue proper. If necessary, review your lecture notes and readings regarding staining procedures.
Complete the Pre-Lab Problem Set on LON-CAPA (deadline is Noon on the day of your assigned lab section).
Remember to bring your i>clicker, Study Guide, and Ross and Pawlina textbook with you to your assigned lab
section.
LABORATORY OBJECTIVES
Objective 1: Identify cells, extracellular fibers and ground substance in loose and dense irregular
connective tissue.
1.1 Mammary gland (Plate 4, top, pp. 192-193)
Using the labeled image provided, identify:
(A) Glands
(B) Loose connective tissue
(C) Dense irregular connective tissue
Increase magnification to 20X as you examine
the glands and loose connective tissue.
o Glands are composed of epithelial cells.
These are cells which are similar in staining
characteristics and which are joined
together. In this tissue, the epithelial cells
form tubes, which appear as circles in
cross-section.
o In loose connective tissue, cells are
separated from each other by extracellular fibers, which take up stain in H&E preparations, and ground
substance, which is washed away in H&E preparations.
Decrease magnification to 5X and center the dense irregular connective tissue. Increase magnification to
20X and identify the following:
o Extracellular fibers, which are abundant and well stained.
o Cells, which are sparse, as indicated by the relatively few cell nuclei visible in this tissue.
o Unstained ground substance, which is relatively sparse when compared to loose connective tissue.
Slide note: The cells labeled A are epithelial cells, which form the ducts and inactive secretory regions of
this mammary gland. The connective tissue immediately surrounding these epithelial cells is loose
connective tissue, and is populated by fibroblasts and many immune cells. The small ducts seen in this view
are continuous with major duct branches, which cannot be seen in this view. The major duct branches are
surrounded by dense irregular connective tissue. What is the name given to the supportive structures
formed by this dense connective tissue in the mammary gland (from ANTR 551)?
1.2 Fibroblast, EM (Fig. 6.8, pg. 167)

The region enclosed by the box in the image on the top is shown at higher magnification in the image on the
bottom.
Locate the cell in the middle of the image on the top. This is a fibroblast.
Examine the extracellular matrix around the fibroblast, and increase magnification to examine the
extracellular matrix more closely. Locate bundles of collagen fibrils (banded or striped in longitudinal
section) embedded in more amorphous (lacking a definite structure) ground substance.
Examine the detail of the fibroblast in the image on the bottom. Locate rough endoplasmic reticulum and
vesicles undergoing exocytosis (see the labels on the cited R&P figure). At what stage in the synthesis
of collagen does it undergo exocytosis from the fibroblast? Notice the fuzzy gray material that lies just
outside the cell. This includes collagen precursor material, which will be assembled into its final form by
enzymes in the extracellular matrix.
Objective 2: Identify characteristics of loose, dense irregular and dense regular connective tissue.
Connective tissue classification is based upon the predominant type of extracellular fiber present as well as the
relative density and arrangement of these fibers. Type I collagen is the primary extracellular fiber in loose, dense
irregular and dense regular connective tissue.
2.1 Loose (areolar) connective tissue, colon (Plate 4, bottom, pp. 192-193)
Without moving the field of view, increase magnification one step at a time while keeping centered on the
lamina propria (loose connective tissue lying just deep to the epithelial lining). This is a good specimen
to study one of the basic differences between epithelium and connective tissue.
o Epithelial cells form a continuous sheet of cells. In the tissue shown in this Objective, the
epithelial sheet lines a lumen, and folds inward to form glands.
o Loose connective tissue (lamina propria) cells are separated from each other by extracellular
matrix, and do not form sheets.
Use nuclear shape and cytoplasmic staining characteristics to identify at least two different cell types in
the lamina propria. (You dont need to name them yet).
Locate extracellular fibers (eosinophilic).
Identify unstained areas, which represent areas where ground substance was located prior to tissue
processing. You will recall that ground substance is one of the materials that is washed away during
routine tissue processing.
Slide note: In many tissues, loose connective tissue forms an important interface between epithelium and the
underlying tissues. Because of its sparse extracellular fiber content, loose connective tissue lacks the strength
of dense irregular connective tissue. However, the cells and matrix in loose connective tissue allow nutrient
and oxygen diffusion and help provide protection against pathogen invasion. Many of the cells in the lamina
propria are part of the diffuse system of immune cells, called the mucosa associated lymphoid tissue
(MALT), which will be discussed during Spring semester.
2.2 Dense irregular connective tissue
2.2a Skin (Plate 42, top right, pp. 514-515)
Skin consists of a surface epithelium (epidermis) and an

underlying thick layer of dense irregular connective
tissue (dermis). Underlying the dermis is adipose tissue
(hypodermis; superficial fascia). In this slide, the
epidermis is the thin, basophilic layer at the top of the
section. The dermis is the underlying thick, eosinophilic
region, and the hypodermis is very lightly stained.
Center the dermis and increase magnification.

Note the thick eosinophilic bundles of collagen in the dermis. The dominant fiber here is type I collagen.
As you will see in Objective 2.2b, other fiber types which are present require special stains to be seen.
Note the varying orientations of the fiber bundles. Gross anatomists refer to a pattern in the orientation
of these fiber bundles in the dermis, which is important when a surgeon is deciding the direction of an
incision through the skin. This pattern is called ____________________ lines.
Identify the unstained areas representing ground substance. There is relatively less ground substance in
dense irregular connective tissue than there is in loose connective tissue.
Locate the nuclei of fibroblasts. Because these fibroblasts are inactive, their nuclei are small and
flattened, with dark basophilic, condensed chromatin.
Slide note: The dermis consists primarily of dense irregular connective tissue. The extracellular fibers of the
dermis, primarily type I collagen, form thick bundles arranged in many different orientations. This
arrangement of dermal collagen gives this tissue strength in a variety of directions. This is important,
because skin is typically subjected to mechanical stresses applied in a variety of directions. Dense irregular
connective tissue is also present in the capsules surrounding many organs and in the septa which divide
organs and tissues.
2.2b Skin, H&E and elastin stain
In this slide you can see that, in addition to type I collagen, other extracellular fibers can be found in
dense irregular connective tissue. Center the dermis and increase magnification. This section has the
same orientation as the section seen in Objective 2.2a. There is a thin, darkly stained epidermis at the
top. The dermis is thick and eosinophilic. The hypodermis is relatively unstained.
When an elastin stain is used with H&E stain, collagen bundles in the dermis will still be eosinophilic.
With the addition of the elastin stain, however, you can see strands of elastic fibers, which stain black,
among the dermal collagen bundles. You should also be able to identify areas of ground substance in the
dermis. You will learn more about elastic tissue in Objectives 4.1 and 4.2.
2.3 Dense regular connective tissue, tendon (Plate 5, pp. 194-195)

Most of this section is skeletal muscle. Center the right portion of the section. This is the tendon. You can
see an abrupt transition from skeletal muscle (left) to the dense regular connective tissue of the tendon on the
right.
Increase magnification until you can distinguish nuclei of fibroblasts.
At 5X and 10X, examine the collagen bundles. These bundles are wavy in this tissue, because the tendon
is not stretched, but relaxed. Are these bundles of collagen randomly oriented, or do they follow an
ordered pattern?
Also at 10X, identify fibroblast nuclei. Based upon the location of these nuclei, you should be able to
determine the organization of fibroblasts in this tissue. Do these fibroblasts appear to be randomly
distributed, or are they following an ordered pattern?
Increase magnification and locate the ground substance separating fibroblasts and collagen bundles.
Ground substance appears relatively sparse in dense regular connective tissue when compared to dense
irregular and loose connective tissue.
Slide note: Tendons and ligaments are typically subjected to great stress, but only in one direction. The
orientation of the fiber bundles gives the tendon great strength to withstand these unidirectional stresses.
However, the tissue is relatively vulnerable to stresses applied in other directions. Tendons can stretch
slightly when the attached muscles contract. When relaxed, the collagen fibers are wavy, as is seen in this
section. When the tendon is stretched, the collagen bundles are straight.
Objective 3: Type III collagen fibers (reticular fibers), lymph node, silver stain (top) and H&E (bottom)
(Fig. 14.18, pg. 462)
Type III collagen fibers, also called reticular fibers, are thin and branched. They do not form bundles as type
I collagen fibers do, and do not provide much resistance to mechanical stresses. Type III collagen fibers
intersect each other, forming a loose meshwork. They are always present in loose connective tissue, and
provide the primary support for some tissues.
Type III collagen fibers are difficult to identify unless special stains are used. In the silver stain preparation
used in the top virtual slide for this Objective, reticular fibers are black and the cell components are brown.
The silver stain clearly delineates these thin fibers. The H&E stained section of a comparable area of lymph
node (bottom) shows how reticular fibers are easily obscured by cells in routine H&E tissue preparations.
Slide note: The primary support tissue (stroma) of some organs, such as these lymph nodes, is reticular
fibers. While type I collagen provides strong structural support, the loose reticular meshwork of type III
collagen provides relatively weak structural support while permitting passage of cells and other material
though the organ. Normal function of a lymph node requires that fluid, solutes and cells be allowed to pass
through the organ. However, rather than expediting this passage, the intersecting fibers of reticular stroma
impede flow enough to allow the maximum number of interactions among the cells, solutes and fluid.
Objective 4: Elastic Tissue
4.1 Elastic fibers, nuchal ligament

There are two sections on this slide: longitudinal section (top) and cross section (bottom).
Using the Crossman trichrome stain:
Nuclei are black
Collagen is blue
Elastic fibers are pink
Move the cursor so that it is within the green rectangle in the top (longitudinal) section of the thumbnail
image.
Increase magnification until you can identify fibroblast nuclei. These are not very numerous in this
section. They appear to be embedded in the areas of collagen.
Identify the elastic fibers. How would you describe their orientation relative to each other?
Locate collagen fibers between the elastic fibers. How does the orientation of the collagen fibers
compare to the orientation of the elastic fibers?
Repeat this examination for the cross section (bottom section), by first moving your field of view within
the outlined region of the bottom section on the thumbnail image, and then increasing magnification.
Slide note: Ligaments are composed of dense regular connective tissue. In most ligaments, the primary
extracellular fiber is type I collagen. However, in some ligaments (ligamentum flavum, nuchal ligament) and
the true vocal folds (vocal cords), the primary extracellular fiber is the elastic fiber. In these latter
ligaments, type I collagen surrounds each elastic fiber, providing the ligament with increased strength.
4.2 Elastic lamellae, aorta, H&E (top) and toluidine blue (bottom) (Plate 33, top, pp. 434-435)
Each of the slides used for this Objective shows a section of aorta, which is an elastic artery.
Top slide: In this H&E stained section, elastic lamellae are pale eosinophilic and appear glassy. Increase
magnification so you can see the layering of cells and extracellular material in the wall of each aorta. The
cells layered between elastic lamellae in an elastic artery are smooth muscle cells. It is these smooth
muscle cells which synthesize the elastic lamellae of the arterial wall.
Bottom slide: In this toluidine blue stained section of aorta, everything is blue except for the unstained
(white) elastic lamellae. This stain allows you to delineate the elastic lamellae more clearly. In this
section you can see that, when the aorta has lost its luminal pressure, the elastic lamellae appear folded.
Slide note: In elastic arteries (aorta, pulmonary arteries) elastin is arranged in perforated sheets, called
lamellae, rather than in fibers. The perforations in these sheets cannot be seen in light microscopic sections.
There are intervening layers of smooth muscle between the elastic lamellae. Smaller arteries may have single
elastic lamellae near their lumina (internal elastic laminae), with elastic fibers dispersed throughout the rest of
their walls. The elasticity of an arterial wall provided by these elastic lamellae helps maintain blood pressure
during diastole. You will learn more about this in the cardiovascular lectures later in this semester.
Objective 5: Unilocular adipose tissue, hypodermis (Plate 16, top, pp. 266-267)
Center the boundary between the dermis and the

hypodermis and increase magnification.
Adipocytes are large, polygonal cells
o Single large lipid droplet, which appears as
an unstained area in the cytoplasmic space.
o Thin rim of stained cytoplasm surrounding
the lipid droplet.
o Flattened peripheral nucleus. Because of the
large size of the adipocyte, its nucleus is
often out of the plane of section, and so
likely will not be visible histologically.
There is a thin region of extracellular matrix between adipocytes. However, this extracellular matrix
cannot be distinguished from the thin rim of adipocyte cytoplasm in LM.
Slide note: You will remember that lipid is lost during routine histological preparation, and so will not be
stained. For this reason, regions that contained lipid in living tissue will appear empty (little or no staining)
after routine preparation.
Objective 6: Resident cells of connective tissue

6.1 Fibroblasts
Top: Fibroblast in EM (Fig. 6.20, pg. 181 and Fig. 6.4, pg. 162)
Note the overall shape of the fibroblast and the shape of its nucleus.
Find the long cellular processes extending into the extracellular matrix.
Find examples of extracellular collagen in longitudinal section and in cross section.
Image note: The fibroblast in this EM is quiescent, meaning it is producing relatively little extracellular
material. It has limited numbers of organelles, and it has long, thin cytoplasmic processes. This cell can
become activated if there is a need to modify the extracellular fibers and matrix, such as in the case of tissue
injury.
Bottom: Fibroblasts in LM (Fig. 6.19, pg. 181)

Click on the links provided to review the light microscopic appearance of fibroblasts in LM section. In these
sections, fibroblasts have elongate nuclei, with such small amounts of cytoplasm that it usually is not visible
in routine H&E sections. The links will take you to slides of:
Dense regular connective tissue (Tendon, Objective 2.3)
Dense irregular connective tissue (Dermis of the skin, Objective 2.2a)
Loose connective tissue (Colon lamina propria, Objective 2.1)
6.2 Adipocyte
Adipocytes store lipid, provide support for other tissues and secrete hormones.
Top: Adipose tissue in LM (Fig. 9.2, pg. 258)

Large, polygonal or round cell.
Flattened nucleus often not visible in LM sections, just internal to the cell membrane.
Single large lipid droplet in cytoplasmic space. Lipid is washed away in routine H&E preparations.
.
Bottom: Adipocyte in EM (Fig. 9.3, pg. 259)
Identify the large lipid droplet. Lipids are retained during preparation for EM. What is the diameter of
this lipid droplet? What is the diameter of this adipocyte?
In this image you can identify the nucleus, flattened and peripheral to the lipid droplet.
What cytoplasmic organelles can you identify in the unlabeled image? Use the labeled version of this
image to confirm your identification.
Note also the extracellular fibers and extracellular matrix peripheral to this adipocyte.
Objective 7: Wandering cells of connective tissue
7.1 Mast cells

Top: Mast cells in intestinal lamina propria, toluidine blue stain
Without moving the field of view, examine the cells scattered within the loose connective tissue of
the lamina propria.
Mast cells are ovoid. In this preparation, their numerous cytoplasmic granules stained dark blue. You
can identify the nucleus of the mast cell as the lightly stained region without granules. There are
several mast cells in this field of view.
Bottom: In this section, the loose arrangement of the collagen fibers allows you to more easily identify
cell boundaries. In addition to fibroblasts, which can be identified by their oval nuclei and thin
cytoplasmic extensions, plump oval cells with round to oval nuclei and abundant cytoplasm can be seen
among the loose connective tissue collagen fibers. The cytoplasmic spaces of these cells are filled with
granules, which stain dark red to purple in this preparation. As you scan through this tissue, you will
occasionally find mast cells which have undergone degranulation. Free mast cell granules can be seen
peripheral to such cells. This change most likely occurred during tissue preparation, so would not be
indicative of pathology.
Slide note: Mast cells are found primarily in connective tissue of the skin and underlying the epithelium of
visceral organs, such as the intestine. Secretory granules fill the cytoplasm of mast cells, except for the region
immediately surrounding the nucleus. These granules are typically lost during normal histological
preparation. Therefore, mast cells will often require special preparation to be identified in LM section. See
also Fig. 6.23, pg. 186 for an EM image of a mast cell. In EM mast cell granules are electron dense, regular in
shape and quite large relative to the size of the cell. A mast cell was included in the EM view used in
Objective 6.2.
7.2 Macrophages
Top: Macrophage, spleen, LM
The pointer indicates a large cell with an irregular outline. This cell contains brown-to-red material in its
cytoplasm. By identifying the location of the nucleus, you can see that this material is within the cell.
This is a macrophage.
Note that there are many other examples of macrophages with cytoplasmic pigment in this image. The
other cell types (those without the brown cytoplasmic pigment) are primarily lymphocytes.
Bottom: Macrophage in connective tissue, EM (Fig. 6.22b, pg. 184)

Large cell, irregular shape.
Indented nucleus.
Numerous cytoplasmic inclusions of varying size, shape and density. These inclusions represent material
which has undergone phagocytosis by the macrophage.
Note also the fibers in the extracellular matrix surrounding the macrophage.
Slide note: In most tissues macrophages are difficult to distinguish without special tissue preparation. In the
LM image described above, the macrophages are engaged in destroying old or damaged erythrocytes, which
is one of the functions of the spleen. The brownish-red material in the macrophage cytoplasm is a product of
the breakdown of hemoglobin from these erythrocytes. The results of this process have allowed us to see the
macrophages in LM without special staining.
7.3 Lymphocytes
Top: Lymphocytes in colon, LM (Plate 4, bottom pp. 192-193; Plate 18, top, pp. 304-305)
Small, round cell (6-15m).
Nucleus fills most of the cell.
Thin rim of basophilic cytoplasm. What cytoplasmic organelle is most likely responsible for this
basophilic staining?
Commonly found in loose connective tissue, particularly in the lamina propria of viscera.
Bottom: Lymphocyte, EM
Refer to Fig. 10.11, pg. 284.
Note the size of the nucleus of this cell as well as its heterochromatin and euchromatin.
Note the relative volume of the cytoplasmic space occupied by its nucleus.
Slide note: Most of the lymphocytes in tissue are B lymphocytes. B and T lymphocytes cannot be
differentiated in routine H&E preparations. Inactive lymphocytes are small. Activated lymphocytes are
slightly larger, and activated B lymphocytes can differentiate into plasma cells.
7.4 Plasma cells

Top: Plasma cells in dermis of skin, dermatitis (Fig. 6.25a, pg. 190)
This slide opens in a region which contains numerous plasma cells.
Ovoid cell.
Eccentric nucleus with a clockface heterochromatin arrangement.
Prominent perinuclear Golgi apparatus (pale region next to nucleus).
Basophilic cytoplasm.
Slide note: This is a section of skin. Using the 20X objective, you should recognize the dense irregular
connective tissue of the dermis. Using the 5X objective, you should be able to see that this dermis is more
cellular than the dermis you examined in Objective 2.2a. The presence of large numbers of immune cells in
the slide of this Objective is due to an inflammatory process (dermatitis) in this individual.
Bottom: Plasma cell, EM (Fig. 6.25b, pg. 190)

Eccentric nucleus.
Chromatin condensed in a regular pattern along the periphery (clockface).
Large, perinuclear Golgi apparatus (more obvious in the higher magnification EM on the right)
Extensive RER. The number of RER profiles in this EM helps explain why the plasma cell cytoplasm is
so basophilic in LM section.
Image note: Although plasma cells are active in protein synthesis and secretion, their chromatin is condensed.
This suggests that these are relatively inactive cells. However, the plasma cell is a specialist, secreting large
amounts of a single antibody, a glycoprotein. Consequently, only relatively small segments of its DNA need
to be transcribed. The organelles needed for antibody synthesis and secretion, the RER and Golgi apparatus,
are prominent. The secretion of antibody is constitutive, meaning the secretory products do not remain stored
within secretory vesicles, but are continuously released from the cell.
Objective 8: Embryonic connective tissue (mesenchyme) (Fig. 6.2, pg. 160)
This is a section through the head of a fetal mammal. The dark eosinophilic regions are developing bone.
Between these regions is the mesenchyme, which is composed of mesenchymal cells and mesenchymal matrix.
Mesenchymal cells are spindle shaped, and have sparse cytoplasm. They have oval nuclei with prominent
nucleoli.
The mesenchymal matrix includes very fine collagen fibers.
What material is most likely responsible for the wide, unstained spaces between mesenchymal cells and
mesenchymal fibers seen in this slide?
.
Low power High power
Slide note: Mesenchymal cells are stem cells for the fixed connective tissue cells, and are present in both the
embryo and the adult. In adults mesenchymal cells are found in the bone marrow space, and may also be present
in connective tissue elsewhere. These cells proliferate and migrate as dissociated cells; however, under the
influence of specific signals, they can differentiate into a variety of cell types.
Integrative Questions
1a. What is the dominant form of the elastic material in the tissue in the top slide?
A. fenestrated lamellae
B. thick fibers
C. fine, branching fibers
b. What is the dominant form of the elastic material in the tissue in the bottom slide?
A. fenestrated lamellae
B. thick fibers
C. fine, branching fibers
c. What effect does the form of elastic material have on the function of these two tissues?
2a. Examine the sections of adrenal gland (left) and spleen (right). Both of these organs are surrounded by a
connective tissue capsule. What type of extracellular fiber predominates in both capsules?
A. Type I collagen
B. Type II collagen
C. Type III collagen
D. Elastic fibers
E. Elastic lamellae
b. In the virtual slide below, examine the splenic trabeculae and capsule. Can you find an extracellular fiber
type other than collagen in these locations?
c. Which of these two organs is most likely to have to expand and contract under normal physiological
conditions?
3. In this slide type I collagen fibers are dark pink to purple, reticular fibers are black and cell nuclei are dark
gray to black. What differences would you predict in cell and fluid movement through region "A" vs. region
"B"?
Self-study Review
1a. Find regions of loose connective tissue and dense irregular connective tissue.
b. Identify adipocytes and fibroblasts.
c. Find an example of an elastic lamella in a blood vessel wall
d. Find lymphocytes within a vessel.
2a. Identify a region of loose connective tissue.

b. Identify a region of dense irregular connective tissue
c. Sometimes it is possible to distinguish elastic fibers in H&E preparations. These fibers are often branched,
do not form bundles and may look "kinked" in preparation. Try to find some of these fibers?
d. Find a fibroblast.
e. Find a cell that has lipid within its cytoplasm, but is not an adipocyte.
3a. Identify the connective tissue within this window and identify its type.
b. Identify a fibroblast, increasing magnification to confirm your identification.
c. Identify extracellular fibers.
d. Identify cells with mucin in their cytoplasmic spaces.
4. Which of the labeled arrows indicates a region representative of ground substance?
5a. Find examples of antibody secreting cells.

b. How do these cells differ from lymphocytes in their histologic appearance?
c. Find examples of lymphocytes.
d. What type of connective tissue is this?
e. Find cells with eosinophilic cytoplasmic granules.
6a. In this EM, find an example of a heparin secreting cell.

b. Identify collagen and a portion of the cell that synthesizes the collagen.
c. What type of connective tissue is this: loose, adipose or dense irregular?
d. How is the appearance of the adipocyte cytoplasm different in this EM as compared to the LM you
examined in Objective 6.2? Why is this difference present?
7. The left image is from a section through a keloid in the dermis; the right section is normal dermis. Compare
the extracellular material in the keloid to that in the normal dermis. What differences do you see? Identify the
fibroblasts in the keloid and in the normal dermis
For more information about keloids, click on the following link:
http://www.aocd.org/skin/dermatologic_diseases/keloids_and_hypert.html
8. This is a section of the stomach. An asterisk is located in an eosinophilic band of material (smooth muscle).
a. What type of connective tissue do you see in the area to the left of the smooth muscle?
b. What type of connective tissue do you see in the area to the right of the smooth muscle?
c. How do the cell types in the two connective tissue regions differ?
In addition to answering the bolded questions in the lab manual:
Identify connective tissue, and specify what type of connective tissue it is based upon its histologic
appearance. This is an important part of analyzing the structure and function not only of the connective tissue
itself, but of the organs supported by this connective tissue.
Understand the relationship between the function of these connective tissues and their structure in terms of the
extracellular fiber type, density and orientation, as well as cellular constituents.
Relate the different types of connective tissue to the function of the regions of the body in which the
connective tissues are found.
Identify areas representative of ground substance, and know the composition and function of ground
substance.
Identify the different types of extracellular fibers in connective tissue, and know which of these needs special
staining for LM visualization.
Identify the resident and wandering cells of connective tissue covered in this lab, and be able to describe their
primary function.
Be able to include the following terms in your basic medical vocabulary:

o stroma
o lamina propria
o submucosa
o basal lamina
CARTILAGE AND BONE
PSL 534 Lecture Sessions 10 & 11

Dr.Laura McCabe Wednesday, 9/14/11
Thursday, 9/15/11
Brief Overview
Cartilage and bone are both specialized forms of connective tissue, and follow the same
general pattern in structure as the other connective tissues: cells elaborate an extracellular
matrix of fibers and ground substance. The distinct biomechanical properties of cartilage and
bone reflect primarily the differences in the extracellular matrix.
Cartilage is a confined gel, both rigid and resilient. The articular cartilage (hyaline cartilage)
provides a lubricated and low friction surface for joint movement, while at the same time
absorbing the energy of impact. These properties require the normal complement of cells and
normal extracellular matrix. Any alterations in cells or matrix can change the biomechanical
properties of the tissue as a whole and influence its function. Cartilage is avascular and has
limited capacity for repair. Its importance in normal movement is evidenced by the pain and
immobility suffered by adults with damaged cartilage
Bone, in addition to its obvious role of biomechanical support, has many functions including
calcium/phosphate homeostasis and the production of blood cells and stem cells. Like
cartilage, bone has a population of cells which elaborate an extracellular matrix, but this
matrix is mineralized primarily by crystal of calcium and phosphate (hydroxyapatite). Unlike
cartilage, bone is highly vascular, and dynamically responds to applied stress and metabolic
needs for calcium and phosphate homeostasis. Understanding the complex interactions of
biomechanical and metabolic controls on the bone cells is the goal for the many researches
concerned with disease of bone, including the excessive bone loss seen in many older adults
(osteoporosis). The dead, rigid bone you study in Gross Anatomy is nothing like the
continuously changing living bone.
Reading Assignment
Chapters 7 & 8, Ross
Review :Composition of extracellular matrix and extracellular fibers in connective tissue
Specific differences of ECM in cartilage and bone
Connective tissue stem cells and their derivatives
Cartilage and Bone Lecture Sessions 10, 11
Lecture Outline
Cartilage
I. Cartilage Types
II. Cartilage Structure hyaline cartilage
A. Extracellular matrix
B. Cells
III. Biomechanical properties
A. Collagen fibers in cartilage
B. Role of fluid in cartilage
C. Structure of the synovial joint
IV. Cartilage over a lifetime
A. Embryonic development
B. Growth
C. Modulation of cartilage (disease, trauma)
D. Aging
V. Cartilage repair
Bone
I. General considerations
II. Bone matrix
III. Bone structure
A. cortical
B. trabecular
IV. Bone cells
A. Bone formation
1. mesenchymal stem cells
2. osteoblasts
3. osteocytes
B. Bone resorption osteoclasts
V. Bone during a lifetime
A. Embryonic growth and development
B. Bone growth
C. Bone repair
D. Bone balance
1. Remodeling
2. Biomechanical control of BMU
3. Factors regulating bone resorption
4. Factors regulating bone formation
E. Aging Bone
CARTILAGE
I. Cartilage Types
Connective tissue
Unique in that its sole purpose is biomechanical
Three types:
Elastic: ear, epiglottis, tip of nose
Fibrocartilage: intervertebral disks,

pubic symphysis, menisci
Hyaline: joints, trachea, costal cartilage, nose

septum, it is also the
template for developing bone
Elastic Cartilage
ECM
o type II cartilage
o proteoglycans (like hyaline cartilage)
o ELASTIN
Perichondrium. Chondrocytes are often in isogenic pairs, similar to hyaline

cartilage.
Located in: epiglottis, pinna, vocal folds (true vocal cords), external auditory canal,
eustachian tube, tip of nose
Flexible.
Does not calcifiy with age, unlike hyaline cartilage.
Fibrocartilage
A composite material of chondrocytes
and ECM embedded in dense
connective tissue.
ECM
o Type I and II collagen
o proteoglycans
NO perichondrium
Found in regions subjected to greater
shear force, as in the intervertebral Cormack, Clinically Integrated Histology,
Lippincott-Raven 1998
discs, and in menisci in some joints
(knee, e.g.)
greater tensile strength than hyaline cartilage
No tendency to calcify with age. However with aging there are changes in the types
of aggregates formed in the ECM, changing the permeability of the matrix, and
therefore, changing the response to loading.
Hyaline cartilage
ECM
o Type II collagen
o proteoglycans
Periochondrium
Often found where joints meet, costal cartilage, trachea, nose (ie; septum),
developing bone
Allows smooth movement between joints
Can calcify with age
II. Cartilage Structure: (using hyaline cartilage as the exemplar)

A. Extracellular matrix (ECM)
Proteoglycan: primarily Aggrecan which contains:
chondroitin sulfates (~100) and keratan sulfates (~60)
attached to protein core
Proteoglycan aggregates: Aggrecans (~300) attached to

a hyalurinic acid core by linking proteins
GAG Disaccharide
Hyaluronic D-Glucuronic acid +
acid N-acetyl-D-glucosamine
Chondroitin D-Glucuronic acid +
4-sulfate N-acetyl-D-galactosamine 4-
sulfate
Chondroitin D-Glucuronic acid +
6-sulfate N-acetyl-D-galactosamine 6-
sulfate
Keratan Galactose or galactose 6-sulfate
sulfate + N-acetylglucosamine 6-sulfate
Safranin staining
of GAGs
Hyaluronic acid
Chondroitin sulfate
Keratin sulfate
Hyaline
cartilage
Proteoglycan concentration is greatest in

matrix immediately surrounding chondrocytes.
Capsular dense staining around

chondrocyte
Territorial matrix surrounds isogenous

groups of cells
Interterritorial matrix space between cell

groups
Figure 7.4. Ross & Pawlina, 6th edition. P. 201
Collagen Fibers in Matrix: primarily type II collagen (fibrous, not bundles), but do
see some type 6 (and a little 9) near chondrocytes (this is what the cells bind to)
Hyaline cartilage everything together (the rest is water, which accounts for 65-
80% weight).
Proteoglycan/aggrecan
Collagen II
Fibers Figure 7.3. Ross & Pawlina,
(not bundles) 6th edition. P. 200
Hyaluronic acid
B. Cells
Chondrocytes and Chondroblasts synthesize and degrade ECM
Due to a fixation artifact, it appears the chondrocytes are in a lacuna, but in fact
in life the chondrocytes tightly adhere to the ECM.
Consistent with synthetic activities, chondrocytes have abundant RER, large
Golgi, and lipid and glycogen deposits (deposits increase with age). Glycogen
and calcium content increases when cells hypertrophy.
Isogenous groups: pairs of closely apposed chondrocytes, representing the two

daughter cells of a previous mitotic division. The daughters move farther
apart as each synthesizes matrix.
Maturation of stem cells into chondrocytes

Chondro- chondrocyte
Pre- blast
chondroblast
Sox 9
MESENCHYMAL pre-
osteoblast osteocyte
STEM CELL osteoblast
RUNX2
pre-
PPAR2 lipoblast lipoblast adipocyte
Perichondrium: See perichodrium on hyaline

(except for articulating surfaces) and elastic
cartilage. It contains some fibroblasts, vessels,
connective tissue and progenitor cells.
Chondroblasts: Differentiate from chondrogenic

cells; produce and modify ECM.
There are no blood vessels within the cartilage itself
(internal to the perichondrium). This means that
chondrocytes inhabit an anaerobic /low O2
environment. Low O2 levels are one of the signals to
progenitor cells to select the chondroblast lineage
over other lineages. Because of the avascularity,
the cells are not very metabolically active.
Chondrocytes: Once the chondroblasts are

completely surrounded by ECM they differentiate
into chondrocytes. Chondrocytes produce and
modify ECM.
III. Biomechanical properties of cartilage

A. Collagen fibers in the cartilage are oriented for shear and tensile strength
collagen nearest to joint surface is aligned parallel to the surface, to withstand

shear forces
deepest layers: fibers and chondrocytes are oriented vertically, with respect to
the joint hydration of the proteoglycans in the matrix is limited by the cage of
collagen fibers
synovium
Collagen Articular
fibers cartilage
bone
B. Fluid is the key for both nutritional and mechanical support
Nutritional Function:
Cartilage is avascular. So nutrients, intercellular signals, ions, etc.
must all reach chondroctyes by diffusion in fluid phase of matrix.
Diffusion is very slow (up to hours for some molecules), luckily

chondrocytes are not very metabolically active.
Any interference in the diffusion properties of the cartilage matrix

will have an impact on chondrocyte viability.
Compression induced fluid flow: compression (i.e., standing) squeezes

cartilage, and exerts pressure on the fluid in matrix, which can force some
fluid out into the synovial fluid of the joint. With release from compression,
fluid flows back into cartilage from the synovial fluid. This is an
opportunity for exchange of molecules and ion with the synovial fluid.
---
--- ---
---
Mechanical support
Fluid flows out of the cartilage matrix during compression. As the

load increases, the fluid permeability in the matrix decreases
because 1) matrix pores are decreased and 2) there is a negative
charge repulsion of proteoglycans which are pushed together this
helps keep some sodium and water so all the fluid is not squeezed
out. With increasing compression, the fluid phase actually begins to
support some of the applied weight.
C. Structure of the synovial joint: To fully understand the biomechanical properties

of hyaline cartilage, we need to look at its environment within the synovial joint (there are
actually 3 types of joints: fibrous, cartilaginous and synovial).
The articular surfaces of bone are covered with hyaline cartilage (note: there is
NO perichondrium in articular cartilage)
The cartilage is separated by a fluid synovial fluid, which acts both as a

lubricant and nutrient source for the cartilage
Synovial fluid includes hyaluronic acid.
The non-articulating surfaces of the synovial cavity are lined by the synovial
membrane
The synovial membrane= 1-4 layers of synoviocytes, plus the underlying loose
connective tissue. Synoviocytes include fibroblast like cells (Type B),
which produce the fluid, and macrophage-like cells (type A).
Inflammation of the synovial membrane produces degeneration of the joint,

and is a hallmark of rheumatoid arthritis
Externally, the synovial joint is encapsulated by an outer layer of fibrous

connective tissue
Synovial
Articular membrane
cartilage
Netter
IV. Cartilage over a lifetime

A. Embryonic development
mesenchymal cells (star-like)

round up, and aggregate
differentiate into chondroblasts

(this process requires suitable
environment, and specific
signals/factors)
B. Growth: There are two forms of growth, appositional and interstitial
Appositional growth:
Chondrogenic cells in the perichondrium differentiate into chondroblasts, which

then lay down ECM, differentiating into chondrocytes
growth at the surface/edge of cartilage
Interstitial growth:
Chondrocytes divide into daughter cells=isogenic group(in mature cartilage,

pairs of adjacent chondrocytes are remnants of these isogenic groups).
Cells spread apart by secreting ECM in between; matrix is distensible.
growth from within the cartilage
Cartilage formation is also important for endochondrial bone growth because it

serves as the early template. We will discuss further when we talk about bone.
Endochondrial bone formation:
Secondary ossification
center ADULT BONE
Epiphyseal growth
plate
Primary ossification
center
Hyaline cartilage
Cartilage mineralized in process of being resorbed
Bone
C. Modulation of cartilage matrix by loading and disease

Changes in the fluid permeability of the matrix alter normal function and cartilage
health.
Loading:
Use it or lose it! Cartilage health requires normal
loading. Since fluid movement is important for
nutritional support, immobility (and lack of load applied
to cartilage) can lead to further cartilage damage
Disease:
Cartilage can degenerate and calcify as a consequence of arthritis or joint damage.
This can lead to bone-on-bone rubbing and joint inflammation.
Osteoarthritis
Cartilage is ideally suited to absorb impact and provide a smooth gliding surface for articulating
bones. However, when the cartilage is damaged, the tissue has limited capabilities to repair
itself, and the use of the joint may be severely impaired. An example is in osteoarthritis, in which
the cartilage becomes damaged and eventually deteriorates, until the bone surfaces may come
into direct contact. The cartilage in osteoarthritis is marked by both structural and biochemical
changes. Deep fissures form in the cartilage, as well as isolated tears and other lesions.
Crystals may be deposited within the matrix, and the matrix itself is less dense in proteoglycan.
The decrease in proteoglycan levels -- results in a reduction in the intercellular water content of
the cartilage matrix. The initial causes of changes in the cells and ECM may be related to the
effect of stress on the joint, although not all joints are equally susceptible, and there are clearly
familial tendencies in developing the disease. Chondrocyte and synoviocyte (cells at the
synovial membrane) contribute to increased cytokine levels (i.e., IL-1, TNF-) which in turn
increase local levels of prostaglandins, nitric oxide, matrix metalloproteases (MMPs) and
aggrecnases. The latter enzymes work to degrade the cartilage matrix.
D. Aging-induced changes in cartilage

Proteoglcyans:
Young cartilage has lots of proteoglycans and sulfates, so lots of negative
charge and lots of water (65-80%). When loaded the high charge retains some of
the water as a biomechanical support.
Old cartilage has less proteoglycan, less sulfates, so less negative charge and
less water retained. When loaded, the charge does not retain much water in the
matrix so more of the load is put on the cartilage on bone.
aged
Matrix (GAG, collagen)
+
Chondrocytes Matrix metalloproteases More or Less
Produce Collagenases Matrix
+
Inhibitors of degradation
(TIMP)
synovial
membrane
synovial
fluid
degradation
PGE2
products
ROS
synoviocytes
MMPs
MMPs
PGE2 IL-1
aggrecanse iNOS
Hyaluronic
acid
Collagen II ch
on
dro
cy
tes
cartilage
Calcification:
During normal aging, hyaline cartilage tends to calcify, especially in larynx,
trachea and costal cartilage. Calcification reduces diffusion of solutes through
matrix, and adjacent chondrocytes die, increasing the damage to the cartilage.
Collagen II
Pre- chondroblast chondrocyte

chondroblast
LOW OXYGEN
Osteocalcin
MESENCHYMAL
pre- osteocyte
STEM CELL osteoblast osteoblast
OXYGEN
V. Cartilage Repair: limited capability in adults

Poor repair: cartilage isnt vascular, chondrocytes arent mobile, and
chondrocytes are not mitotically active in mature individuals, so no interstitial
growth is possible, and damaged areas are not replaced.
Appositional growth is limited in adults
Chondrocytes cannot substantially increase production of ECM to make major
repairs
Calcified cartilage tends to be replaced by bone
Avascular tissue - usual mechanisms for repair are not available
For example, when skin is damaged, the dead cells and debris are effectively
removed. In cartilage, there are no blood vessels, and the damaged regions are left
in place. Some injuries extend deep into the subchondrial bone, and can promote
the growth of blood vessels from the bone into the cartilage. These vessels bring in
stem cells, but the high O2 levels produced by the blood vessels, as well as other
chemical signals, promotes bone formation, not cartilage formation. This bone
formation interferes with both the mechanical and diffusion properties of the matrix.
Any cartilage which does form is typically fibrocartilage (see below), which cannot
withstand the normal compressive forces of joints, and breaks down.
BONE
I. General considerations
Bone is a connective tissue with a calcified extracellular matrix to provide greater support
and protection and a rigid platform for muscle action. In addition to providing mechanical
support, bone also serve as a reservoir for calcium and other minerals, and houses the
marrow, the blood forming tissue. A dynamic tissue, it continuously responds to changing
support and metabolic needs of the body. Recent studies also suggest that bone is
involved in body metabolism regulation through its release of osteocalcin in the blood
stream which enhance insulin secretion by pancreatic beta cells!
II. Bone Matrix

As with all connective tissues, bone has an extensive extracellular matrix of ground
substance and fibers
ECM COMPONENTS
1. Organic matrix -- OSTEOID
Proteoglycans: lower proteoglycan content than in cartilage
Water= 5% by weight
Adhesive glycoproteins:
osteoclacin, osteonectin, oestopontin
bone sialoprotein
Ca++ binding
Extracellular fibers : primarily collagen Type I
Osteogenesis Imperfecta
- mutations in collagen I
- can have dozens of fractures at birth
- can have more than 140 fractures by
20 y/o (some caused by sneezing)
- can have short stature (i.e., only 3 feet
tall)
2. Inorganic -- MINERAL
65% by weight, primarily Ca++ and PO4- as hydroxyapatite crystals
mineralization: hydroxyapatite binds to both collagen and ground
substance.
In bone formation, the osteoid is synthesized first, followed by mineralization.

Osteoid formation and mineralization can be affected independently by various
pathologies: vitamin C deficit, versus Ca++ deficit (due to factors such as low Ca
uptake or low vitamin D status.
NOTE: Bone is 6 times as strong as steel of similar weight.

Bone is more flexible than steel.
Bone can take 10-15X body weight without breaking.
III. Bone Structure

Bones have an outer dense region (called compact or cortical
bone) and an inner, spongy (trabecular, cancellous) bone. The
interior cavity is the medullary cavity, and contains active bone
marrow (depending on the bone and the age) and fat.
A. Compact or cortical bone:

OSTEONS (Haversian systems): cylindrical units of cells and matrix.
The long axis of the osteon cylinder is parallel to the direction of normal external
force, such as the line of gravity or normal muscle action
Each osteon consists of 4-20 concentric cylinders of bone ECM and cells, in
cross section these appear as concentric layers, or lamellae
CHANNELS within the bone link cells and osteons.

o Lacunae (with osteocytes) are
concentrically arrayed with the lamellae.
o Canaliculi link adjacent lacunae, and

provide channels for osteocyte cytoplasmic
processes.
o Haversian canal: central canal within

each osteon, containing blood vessels and
nerves, and lined by endosteum.
Extracellular fluid is confluent with that of
the canaliculi.
o Volkmanns canal: transverse or oblique

connections between Haversian canals of
adjacent osteons. (also lined by endosteum)
Volkmanns canal, Haversian canal and
canaliculi all created by osteoclasts.
o Osteons are continuously rebuilt

(remodeled; see below) for appropriate
alignment under changing conditions: i.e.
change in overall posture, or muscle mass.
Normal loads on bone are compressive: Bone is

strongest (least likely to fracture) under compression,
but weaker to tension (as in bending)
Gartner & Hiatt, Color Atlas of Histology,
3rd ed., LWW 2000
o In between adjacent osteons is bone

called interstitial lamellae, the remnants of
partially resorbed osteons.
B. Spongy, cancellous, trabecular bone
Trabeculae or spicules of bone, separated by marrow or fat, having a spongy

appearance
Lamellar organization of cells and matrix, but lamellae are less regular. Very thick
trabeculae may have osteons
Trabeculi
Trabeculae have a lot of surface area compared to cortical bone. Therefore they are
resorbed to a greater degree than cortical bone due to increased surface that bone
resorbing cells (osteoclasts) can attach to. Thus, this region is most affected by
osteoporosis (excessive bone loss).
IV. Bone Cells Collagen II
A. Bone forming cells chondroblast chondrocyte

Pre-
chondroblast
Osteocalcin
Sox 9
pre-
osteoblast
RUNX2
MESENCHYMAL
apoptosis
STEM CELL
PPAR 2
pre- lipoblast adipocyte

lipoblast
aP2
1. Mesenchymal Stem Cells

Periosteum . Bone is surrounded by an external sheath, analogous
to the perichondrium (except at articular edges).
o Outer periosteum : fibrous, dense connective tissue
o Inner periosteum: osteoprogenitor cells, derived from
mesenchymal stem cells
Clinically, periosteum is used for grafts to replace bone
Endosteum: The interior surface of bone is also lined with

progenitor cells, called bone lining cells. These are flattened in the
quiescent state, rounded when active.
2. Osteoblasts cells involved in bone formation
o Synthesize ECM (collagen I)
o Osteoid (collagen, organic matrix) first, followed by mineralization

(secretion of phosphatases and calcium binding proteins -
OSTEOCALCIN)
o Found on the surface of bone, either flattened (relatively inactive;

sometimes referred to as bone-lining cells), or plump (cuboidal) and
quite basophilic (more active).
o Once they are surrounded by a mineralized matrix, they differentiate

into osteocytes or die (the majority die via apoptosis).
osteocalcin
FORMATION
OSTEOBLASTS
Osteoid
Mineralized
bone
Osteocalcin Total serum levels are used in clinic as a marker of bone

formation. Its expression is regulated by vitamin D, insulin, and other
factors. Osteocalcin (undercarboxylated form) is now thought to participate
in the regulation of pancreatic insulin secretion and adiponectin secretion.
Thus, it increases insulin release and sensitivity and be involved in a bone-
pancreas signaling loop!!
3. Osteocytes
Derived from osteoblasts, these mature cells synthesize and maintain the
fibers and ground substance of the matrix (both organic and inorganic
components)
Gartner & Hiatt, Color Textbook of Histology 2nd ed., W.B. Saunders
Osteocytes occupy lacuna in the matrix. The fluid within the lacuna is
extracellular fluid. Fluid movement may be sensed by osteocytes, and act
as a signal for osteocyte activity.
Osteocytes send cytoplasmic processes through very fine, fluid filled

channels within the bone extracellular matrix called canaliculi and contact
the processes of adjacent osteocytes
Osteocytes communicate with osteoblasts and other osteocytes via gap

junctions: specialized communication channel between cells
B. Bone resorbing cell lineage
Osteoclasts: phagocytic cells which arise from bone marrow precursor (probably
monocytes); large multinucleated cells, well-stained eosiniphilic cytoplasm. m-CSF
(macrophage-colony stimulating factor) and gm-CSF needed to promote hematopoetic
stem cells toward the monocyte lineage. Secreted by osteoblasts, and stromal cells in
the marrow. Also need activation of RANK, a receptor on preosteoclasts.
active osteoclast
RANK
GM-CSF RANKL
Hematopoetic Monocyte/ Cathepsin K
Stem cell macrophage
H-ATPase
H Cl
multinucleated
phagaocytic cells
integrins
Tartrate resistant
acid phosphatase (TRAP)
Acid pump (H-ATPase)
Often identified by staining for Tartratrate resistant acid phosphatase

(TRAP) activity. This enzyme is also increased in the serum of patients
with active bone resorption (so it is a good way to monitor bone
resorption)
These cells are responsible for most of the bone resorption by dissolving
organic and inorganic matrix (H+, phosphatase and metalloproteinase
secretion) and phagocytosing the organic components (lysosomes).
The cell margin near the bone surface = ruffled border, created by
extensive membrane folds to increase surface area. Cells adhere to
matrix by integrin binding.
The bone underneath the osteoclast = Howships lacuna

Tartrate resistant
Deoxypyridinoline
RESORPTION
acid phosphatase (TRAP5b)
(DPD)
OSTEOCLAST
Erosion pit
Mineralized
bone
V. Bone During a Lifetime

A LIFETIME OF BONE ACTIVITY
BF - BONE FORMATION
BR BONE RESORPTION
Bone
YOUNG 20-50 OLD Mineral
Density
osteoporosis
(BMD)
BF>BR BF=BR BF<BR
formation remodeling
Birth 20-30 50 70
years old
A. Embryonic growth and development
There are two METHODS by which bone is formed: intramembranous or
endochondral.
1. Intramembranous bone formation:

This process is analogous to the formation of cartilage: mesenchymal cells
aggregate and differentiate into osteoblasts; osteoblasts synthesize bone
matrix, become surrounded by it, and differentiate into osteocytes.
The initial bone is in form of trabeculae or spicules, which enlarge, coalesce

and form osteons; the final form of bone may be either compact or
cancellous.
Intramembranous formation occurs in most of the skull bones and in the

clavicle.
2. Endochondral bone formation

Instead of developing directly from mesenchyme, the bone develops from a
cartilage model, which is then replaced by bone.
Chondrocytes differentiate from mesenchymal cells. The cartilage formed is

in the shape of the intended bone (cartilage model or template)
Primary ossification center: fetal period

First a collar of bone forms around region of diaphysis or shaft
The extracellular matrix of the cartilage in the soon to be marrow then

calcifies, and the chondrocytes of the cartilage lose nutrients and die.
Blood vessels penetrate into the interior, bringing osteogenic cells and
hemopoietic cells. Osteoblasts differentiate, deposit bone, while
hemopoietic cells occupy the future marrow.
This ossification of the diaphysis is the primary ossification center. The

bone will be continuously remodeled into the typical osteon structure of
compact and trabecular bone.
Endochondrial bone formation:

Secondary ossification
center ADULT BONE
Epiphyseal growth
plate
Primary ossification
center
Hyaline cartilage
Cartilage mineralized in process of being resorbed
Bone
Secondary ossification centers develop in each epiphysis

At about birth, blood vessels and osteoprogenitor cells invade each end of
the bone (epiphysis).
This is the secondary ossification center, and ossification proceeds as

in the primary center, but not all cartilage is replaced:
Cartilage remains at the articular surface throughout life, and at the

epiphyseal plate (the junction of diaphysis and epiphysis) until growth has
ended (20yr. approx for some bones)
B. Bone Growth
Length: chondrocytes at the epiphyseal plate divide. The plate is a region of hyaline
cartilage in between the primary and secondary ossification centers. The cartilage
within the plate forms distinct layers, reflecting the process of bone formation:
Zone of reserve or resting cartilage: Region of cartilage,

providing a pool of dividing cells for the stages below.
Bone growth will cease once the resting zone
chondrocytes cease to divide
Zone of proliferation: Chondrocytes undergoing rapid

mitotic activity, and are aligned into rows parallel to the
long axis of the bone.
Zone of hypertrophy: Chondroctyes accumulate

glycogen and fluid and begin to swell in size; the matrix
between cells becomes thinned. Cells accumulate large
stores of intracellular Ca++ (in mitochondria)
Zone of calcified cartilage: Calcium is released and the

matrix calcifies; diffusion is impaired and chondrocytes
die. The empty lacunae become confluent.
Zone of resorption and ossification: bone is laid down on

calcified cartilage by osteoblasts; primary bone first,
then secondary; bone is then remodeled by osteoclasts
and osteoblasts as needed to maintain the appropriate
shape and size
Epiphyseal closure: chondrocytes lose ability to divide,

ossification continues until only the articular cartilage
remains.
Width: Differentiation of osteoprogenitor cells in the periosteum to osteoblasts build

bone on the outside of bone thereby increasing width. This is also called
appositional bone growth.
As the bone grows in length and width, it is also remodeled by osteoclasts and
osteoblasts to retain normal proportions.
Bone development and growth
+++
resorption
+ +
+ +
Immature bone vs. Mature bone

Immature WOVEN bone = woven or primary bone, bone matrix with lower
mineral content, and more irregular arrangement of collagen than in mature
form. This is the initial type of bone regardless of the method of bone formation
(intramembranous or endochondral) during early periods of growth in embryonic
life and childhood, and during the initial stages of repair throughout life
Mature COMPACT bone = secondary bone, with high mineral content, regular
collagen in matrix, and organized in lamellae
Stephen E. Fish.
Joan C. Edwards School
of Medicine, Marshall
University, with
permission
C. Bone repair
For many fractures, repair processes are similar to embryonic bone formation
1. Clot formation -- hematoma
2. Callus formation - fibrous, cartilage, vessels
3. Callus ossification - vessels, woven bone
4. Remodeling - vessels, resorption
Blood vessels bring in osteogenic cells, as well as numerous signaling molecules
Callus: A periosteal callus forms at the periphery of break and leads to intramembranous
ossification. Intramedullary callus forms inside the marrow at the center of the fractures
and endochondrial ossification occurs here.
1. Hematoma 2. Soft Callus 3. Hard Callus 4. Bone

/inflammation Formation (Woven bone) remodeling
formation
D. Bone balance (middle age)

1. Bone Remodeling Unit (Bone Multicellular Unit; BMU):
groups of osteoclasts and osteoblasts which participate in remodeling.
(1) Osteoclasts resorb bone in a cutting cone, creating the elements of new
osteons: canals and canaliculi and resorbing trabecular bone. This cutting
cone is followed by capillaries, bring osteoprogenitor cells, which differentiate
into osteoblasts (2). The osteoblasts deposit bone (3) to form new osteons (4).
As always the initial ECM is osteoid, followed by mineralization.
The bone remodeling units remodel bone to fit changing mechanical or

metabolic needs. The activity is not quite balanced: after final growth is reached,
each BMU deposits slightly less bone than it resorbs, so there is a normal, slow
decline in bone mass with age.
Bone loss tends to be greatest for the trabecular bone than for cortical
(compact) bone
In normal bone there are millions of ongoing BMUs

2. Biomechanical control of BMUs

LOAD:
Bone responds to changes in external load or stress;
A reduction of the external load produces bone resorption, while an
increase in load elicits formation
(sometimes referred to as Wolfes Law -- or Use it or lose it)
Mechanical load Wolffs law =

Bone is laid down where it is needed, and resorbed
where it is not needed.
ie: use it or lose it!
Reductions in load cause bone loss Examples include bed rest, limb
immobilization, and some metal pins and plates used to fix broken bones (can
shield the load onto bone and cause local bone loss).
Bone responses are local
BONE RESPONDS AT THE SITE OF LOADING

and bone is built to be strongest with regard to the angle of load (the trabeculi
of trabecular bone orient in the direction of the load). Think of tennis player
example who has more bone density in the arm that hits the ball.
Thoughts on mechanisms of load-induced bone formation:
1. Peak stresses may be most effective

-> High amplitude stress ie: gymnastics
2. Low amplitude, high frequency

Load/vibration alter cell-ECM contacts that lead to stimulation of BMU.
3. Shear stress/nutrient delivery

to osteocytes
Load/vibration (caused by gravity or muscles attached to bones)
causes extracellular fluid movement in the canaliuculi. This is like a
mechanostat which amplifies the signal of a very small bend in the
bone. The osteocytes then sense the fluid and they respond by
signaling through gap junctions and secreting factors to promote
bone growth at that site.
4. Micro-fracture stimulation
of remodeling
Small microfractures are made in bone with loading and help to
release signals from the bone matrix for the loaded area to be
remodeled and strengthened.
3. Regulation of bone remodeling

Factors that promote osteoclast activity (bone resorption):
M-CSF or GM-CSF increase hematopoetic stem cells toward monocyte lineage

(osteoclast precursor).
Chronic PTH Parathyroid hormone (PTH) is produced by an endocrine gland, the

parathyroid gland (small glands near the thyroid) in response to low Ca++ levels.
PTH stimulates bone resorption indirectly by binding to receptors on the osteoblast,
stimulating the production of RANKL, and decreasing the production of OPG,
leading to an increased activation of osteoclasts to raise blood calcium to normal
levels.
RANK, RANKL, OPG signaling mechanism:
RANKL (receptor activator of NF B ligand ). RANKL is both a membrane

bound and secreted protein produced by osteoblasts. It binds the RANK
receptor on osteoclast precursors. Because RANKL is on the membrane of
the osteoblasts or stromal cells (or is locally secreted), osteoclast precursors
must be in the local area in order for RANKL to bind to RANK (a receptor
located on osteoclast precursors surface) and activate the osteoclasts (and
promote their differentiation into resorptive cells).
Osteoblast-osteoclast signals
RANK receptor on osteoclasts RANKL osteoclast
when bound it activates
osteoclasts
RANKL ligand on osteoblasts
that binds and activates RANK
Osteoprotegrin (OPG) binds

RANKL and prevents osteoclast
activation osteoblast
OPG RANK
RANK
RANKL
RANKL
Secreted
RANKL
osteoblast
osteoblast
Factors that suppress resorption:

Osteoprotegerin (OPG, also known as a decoy receptor). Secreted by
osteoblasts (and some other cells), it binds to RANKL (This is competitive binding,
meaning that if OPG is bound to the RANKL, then RANK cannot bind, and vice
versa). When OPG binds to the RANKL then local osteoclast precursors are NOT
induced to differentiate into an osteoclast (see cartoon above).
This is a common scenario in physiological control, in which

OPG RANK the same cell (here, the osteoblast or stromal cell) produces
both an inhibitory and a stimulatory control. The final outcome,
the number of osteoclasts maturing, will depend on the
balance of these two proteins (osteoprotegrin and RANKL),
RANKL
each of which can be activated by a wide variety of local and
systemic signals.
osteoblast Calcitonin - endocrine hormone, released in response to high Ca++ levels. It

binds to receptors on osteoclasts, and inhibits osteoclast activity. Its action is
therefore a direct effect on bone resorption, with no effect on osteoblast activity
Bisphosphonates: These compounds incorporate into newly

synthesized bone and can stay there for more than a year. Bisphosphonates
Osteoclasts die or have reduced function when they come into OH OH
contact with bone containing bisphosphonates. There are a R
variety of bisphosphonates on the market. Those containing
nitrogen in their side groups are most effective. Some stay in O=P - C P=O
the body longer than others. There may be a link with jaw R
necrosis, but only at high doses (only used to treat some OH OH
cancers). They may, in fact, stimulate bone healing this is a
current topic of research in the bone field.
4. Selected factors simulating bone formation

Vitamin D and calcium: mineralization requires availability of Ca++ and PO4-;
Vitamin D (either ingested or synthesized from UV exposure), and a normal
response to vitamin D, is required for normal Ca++ levels. Osteomalacia (rickets
in children) results from low calcium levels (osteoid is set down but not
mineralized.
A patient can do all the right things to stimulate bone formation, but without
mineral the bone cant be made.
Bone Mass
500 1000
mg calcium
Vitamin C: important for osteoid (collagen fiber formation). Also, genetic or

nutritional deficits can compromise collagen synthesis, and therefore the
mechanical properties of the ECM.
Intermittent PTH In contrast to chronic PTH, intermittent increases in PTH

actually increase bone formation and bone density. It is thought that this occurs
because osteoblasts do not die in response to intermittent PTH but do with
chronic PTH. The net result is bone formation with intermittent PTH vs. bone
resorption with chronic PTH. Intermittent PTH is the only anabolic bone
treatment on the market!
BMP (bone morphogenetic protein; a member of the TGF super-family of growth
factors) is synthesized by osteoblasts, and binds to the bone matrix. It is
released from the bone matrix during bone resorption, and activates osteoblast
differentiation, and bone deposition. Bone resorption itself therefore stimulates
bone formation. BMP is used on bone implants to enhance healing.
Estrogen. Effects are complex, but ESTROGEN
several mechanisms are known (Ive noted
only a few here). Estrogen can directly OPG
enhance osteoblast maturation, but it is
RANKL
also known to inhibit pro-inflammatory
RANK
cytokine release by osteoblasts and bone
marrow stromal cells. It also increases Estrogen
OPG production by osteoblasts, decreases ESTROGEN
RANKL secretion by osteoblasts and
RANK expression in osteoclasts
IL-6, IL-1 osteoclast
osteoblast
E. Aging Bone
EXERCISE, Ca++
Bone 1- 2.5 SD BMD
Mineral > 2.5 SD BMD
Density
osteoporosis
DISEASE
(BMD)
Birth 20-30 50 70
years old
Definition of osteoporosis: Normal bone density is within 1 standard deviation (SD)
below average age bone mineral density (of a 25 year old female). Osteopenia is
between 1 and 2.5 SD below the mean, while osteoporosis is greater than 2.5 SD.
Osteoporosis increases fracture risk.
8
relative 6
fracture 4
risk
2
0
-3 -2 -1 0 1 SD BMD
Vertebral compressions/fractures and hip fractures are often seen in the elderly.
These sites contain a large proportion of trabecular bone.
Osteoporosis can occur with aging, disuse and is also associated with some
diseases. Some (aging, disuse , type I diabetes), but not all cases of bone loss are
associated with increased marrow adiposity suggesting that 1) mesenchymal
stem cells are actively choosing to be adipocytes over osteoblasts or 2) that
adipocytes fill in the marrow space that no longer contains bone.
Collagen II, X
chondroblast chondrocyte
Pre-
chondroblast
Osteocalcin
Sox 9
adiposity
Marrow
pre-
osteoblast
RUNX2
MESENCHYMAL
STEM CELL
apoptosis
PPAR2
pre- lipoblast adipocyte

lipoblast
aP2
Additional Therapeutics:
PREVENTION OF OSTEOPOROSIS:
o Increasing bone formation. The best current way to
Obtain and maintain increase bone formation is by the use of appropriate
mechanical stress exercise. The optimal time to
maximal bone mineral density increase bone mass is up to early adulthood, but bone
and strength mass can be increased somewhat even in the elderly.
PTH is the only anabolic therapeutic on the market.
Prostaglandin E2 is also being studied for its possible
effects on promoting osteoblast differentiation from
B o n e M in e r a l
progenitor cells.
D e n s it y
o Optimizing the quality of the extracellular matrix, both

the extracellular fiber formation (vitamin C), and
10 20 30 40 50 60 70 80
years
mineralization (vitamin D and Ca++ )
In summary: Systemic and local factors can act at any of the steps involved in bone
remodeling and may act at several of these steps.
Differentiation of osteoblasts
Synthesis of osteoid by osteoblasts
Mineralization of osteoid
Differentiation of osteoclasts
Bone resorption by osteoclast
Questions for Self-study

1. Which of the following comparisons and contrasts between the three types of cartilage is
CORRECT?
A. Hyaline cartilage is the only form of cartilage which is avascular.
B. Elastic and fibrocartilage are characterized by bundles of Type I collagen in the ECM.
C. Hyaline cartilage tends to become calcified with age, but elastic cartilage does not.
D. Fluid permeability in the ECM is important for hyaline cartilage, but not for
fibrocartilage.
E. All three types of cartilage have an external perichondrium.
2. Sulfate and carboxylate groups GAGs bind calcium, and can prevent the formation of
calcium crystals. Why is this important for cartilage? What are the consequences to the
health of the cartilage if calcium crystals form?
3. In hyaline cartilage, interstitial growth:

A. occurs throughout life
B. is stimulated by parathyroid hormone
C. occurs at the epiphyseal plate during endochondral bone formation
D. occurs only at the perimeter of the cartilage
E. results from the differentiation of chondroblasts from progenitor cells
4. Which of the following cells is not derived from the mesenchymal stem cell?
A. osteoblast
B. adipocyte
C. osteoclast
D. chondrocyte
E. fibroblast
5. At a synovial joint, the extracellular fluid of the cartilage ECM:

A. can flow out of the matrix and exchange with the synovial fluid, but only at very high
loads
B. is tightly bound to the GAGs of the matrix, except under pathological conditions
C. is a source of exchange for solutes from the internal capillaries of the cartilage to the
capillaries of the perichondrium
D. is restricted to the interior of the cartilage
E. can help support the load applied to the joint, even in normal circumstances
6. The figure below depicts a sagittal section through lumbar vertebrae in an adult. Which of
the following statements is correct?
Netter
A. The regions labeled X are compact bone.

B. There are no canaliculi in the regions labeled Y.
C. These bones are formed by intramembranous bone formation.
D. There are no blood vessels in the regions labeled X.
E. The regions labeled Y are woven bone.
7. Which of the following would increase bone resorption?

A. differentiation of osteoblasts to osteocytes
B. decreased binding of RANKL to RANK receptor on macrophages
C. increased binding of calcitonin by osteoclasts
D. decreased production of osteoprotegrin by osteoblasts
E. decreased binding of PTH to osteoblasts
8. In the figure of bone below, which of following descriptions of Regions I, II, III, and IV is
correct?
A. Osteoblasts are found lining the space indicated by "I".

B. Regions I and III are linked by small channels containing extracellular fluid.
C. Regions I and III are channels filled with blood vessels.
D. Region IV is the source of progenitor cells.
E. Region IV is the endosteal region.
9. A defect in the ability of chondrocyte to divide would affect which of the following?
A. formation of the bones of the skull
B. longitudinal growth of long bones in children
C. growth in the diameter of long bones in children
D. normal repair processes of articular cartilage in adults
E. adaptation of bone in adults to changes in stress
10. Look at the middle image in Plate 13 (p. 249) in Ross and Pawlina. What is the direction of
growth at the epiphyseal plate? Which cells are responsible for growth in the width of the
bone? What is this type of growth called? Examine the medullary cavity. Does this cavity
stay the same size as the bone grows? Why or why not?
Answers to Self-Study Problems

1. C. All cartilage is avascular, and all forms have similar problems with responding to
damage. The bundles of Type I collagen are found only in the ECM of fibrocartilage. These
bundles give the cartilage its fibrous quality, and resistance to shear. Fluid permeability is an
important property of all cartilage, regulating both nutritional status, and mechanical
properties of the cartilage. Fibrocartilage lacks a perichondrium
2. There would be two consequences. The first relates to the nutritional requirements of the
chondrocytes. Chondrocytes depend on diffusion and fluid movement through the matrix for
the delivery and exchange of nutrients. Calcium crystals change the diffusion characteristics,
and large deposits can even produce cell death in nearby chondrocytes. The second
consequence has to do with the permeability relative to external load. Changes in the
composition change this relationship, and could limit the load bearing properties of cartilage.
3. C. Interstitial growth refers to cell division of chondrocytes, not the differentiation of

chondroblasts. That latter process underlies appositional growth, which occurs at the
perimeter, as progenenitor cells in the perichondrium differentiate. Interstitial growth
continues at the growth plate until around puberty, or a little later, depending on the bone.
Parathyroid affects the differentiation of osteoclasts.
4. C, osteoclast. These cells are derived from the macrophage/stromal cells. Knowing the
cells derived from mesenchyme makes it easier to understand the seemingly odd changes
which can occur in bone and cartilage, such as bone deposition in cartilage, and fat deposition
in bone.
5. E. The fluid of the ECM is part of the support for external loads applied to the joint. The fluid
of the ECM flows even with gentle movements of the joints. The rate of flow is related to the
load, but the rate is actually lower for higher loads than for small loads (this is the reason fluid
is not simply squeezed out of the matrix). The fluid is an important source of nutrient
excahnage, but not with internal capillaries. There are no internal capillaries in cartilage.
Blood supply is in the perichondrium, or other external tissues.
6. A. The outer region of any bone is compact, even if this region is very thin. The inner region
is cancellous or spongy bone (region Y). Spongy bone and compact bone both have canaliculi,
the small channels used for intercellular communication by the osteocytes. Both types of bone
are vascularized. Woven bone refers to the composition of the extracellular matrix in
immature bone. Woven bone has a lower mineral content, and more disorganized
arrangement of collagen. As the bone matures, the matrix acquires its normal mineral content
and assumes a lamellar organization. Since the question referred to an adult, one would
expect that overall the bone would be mature, and not woven.
7. D. Osteocytes do not resorb bone, so increasing differentiation from osteoblast to

osteocyte would not be expected to increase the bone resorption. In fact, this increase in
osteocyte formation would most likely be the result of increased bone formation, as more
osteoblasts become surrounded by more matrix. Calcitonin does bind to osteoclast receptors,
but this binding results in the inhibition of osteoclast activity. Regular activity, especially
weight training, is used to increase the overall deposition of bone. The RANKL signalling
molecule and its receptor, RANK, are required for the promotion of osteoclast formation.
Therefore, diminished binding of the RANKL would result in fewer osteoclasts, and less bone
resorption. The correct answer, a decrease in the production of osteoprotegrin, would result
in an increase in osteoclast formation. (Note that your textbook does not correctly describe
this process).
8. B. This is a diagram of several osteons. (in which direction would you expect the normal
applied stress to this bone?) I & III indicate lacunae in the same osteon, II a Haversian canal,
and IV a lacuna in a remodeled osteon. Lacunae are the locations of osteocytes, not
osteoblasts. They are linked by the fine channels or canaliculi, through which the osteocytes
send cytoplasmic processes. These canaliculi include extracellular fluid, and it is the
movement of this fluid which may provide the signal for osteocytes concerning local variations
in applied load to the bone. Progenitor cells would be found on the internal and external
surfaces of bone, including the Haversian canals, but not within the lacunae.
9. B. This is the defect in achondroplasia, a type of dwarfism. Chondrocyte cell division is the
process occurring in the proliferation zone of endochondral bone formation, and defects in
this process affect the longitudinal growth of bones formed in this way. Skull bones are formed
by intramembranous bone formation, and are not affected by this defect. Appositional growth
of bone (increased diameter) is also not affected, since this is due to the activity of
periosteum. Similarly, bone remodeling does not specifically involve chondrocyte
proliferation, and would also be unaffected. Normal adult repair would be no worse than usual,
that is to say, still very limited.
10. Epiphyseal plate: growth in length (to the left and to the right, in this figure). Width:
differentiation of osteoblasts from progenitors in the periosteum; appositional growth; the
medullary cavity will increase in size during growth, in order to maintain the proper proportion
of bone andcavity while the entire bone enlarges.
You should be able to
1. Compare cartilage and bone with respect to: GAGs, extracellular fibers, developmental
growth, function, source of nutrition for osteocytes and chondrocytes.
2. Know the differences between hyaline cartilage, elastic cartilage and fibrocartilage in
terms of fiber types, features (ie: flexible) and location in the body
3. Understand the biomechanical properties of cartilage, understand the importance of fluid

and fluid movement, and extracellular fiber type and orientation on cartilage biomechanics.
Where are chondrocytes derived from?
4. Know the relationship between osteon orientation, bone remodeling and external load in
bone.
5. Be able to describe the relationship between osteoblasts, macrophage or stromal cell, and
osteoclast; role of RANK, RANKL, OPG, PTH (intermittent versus chronic), calcitonin and
BMP.
5. Define intramembranous and endochondral bone formation, and understand how these
relate to mesenchyme, hyaline cartilage, epiphyseal plate. Which bones are formed only by
intramembranous means?
6. Understand the effects of disuse on both cartilage and bone. How does this relate to
Wolffs Law? What region of bone is particularly prone to bone loss?
7. Identify the features in the osteon which relate to: communication between osteocytes;
blood vessels; communication between osteons
8. Define the terms: osteoid, woven bone, mature bone, cancellous bone, trabecular bone,
compact bone, epiphysis, diaphysis, medullary cavity.
9. Understand the difference between the organic and inorganic components of bone matrix,
and the effects of vitamin C, vitamin D and Calcium on bone matrix and mechanical
properties.
10. Understand the difference between osteoporosis and osteopenia. Why do we care about
bone loss? What are some things that can cause bone loss?
11. What are some potential therapeutics for bone loss? How do they work?
12. Understand how changes in the following processes would affect bone formation or
resorption:
Differentiation of osteoblasts
Synthesis of osteoid by osteoblasts
Mineralization of osteoid
Differentiation of osteoclasts
Bone resorption by osteoclasts
13. How do cartilage and bone change with age?
14. Discuss a potential mechanism for the pathology of osteoarthritis.
15. Why do osteoporotic women and men have curved backs and not bowing legs?
16. What is osteocalcin?
17. What are markers of bone formation? What are markers of bone resorption? Why do
these markers work?
18. Name 3 factors that activate osteoclasts.
19. Name 3 factors that stimulate bone formation.
20. Name 3 things that suppress osteoclast activity.

Lab Session #4: Cartilage and Bone
9/21/11 or 9/22/11
Introduction
Cartilage and bone have some of the same general characteristics that you studied for connective tissue proper.
They are composed of specialized cells and an extracellular matrix (ECM), which contains fibers and ground
substance. In this Lab Session you will learn how to distinguish cartilage and bone from other tissues. You will
also learn to distinguish differences between the three types of cartilage. These differences, which primarily
involve variation in fiber content of the extracellular matrix, can be seen histologically, and reflect the different
mechanical properties of these cartilage types.
Overview of Cartilage
General properties of cartilage (Plate 7, top, pp. 210-211)
The first thing you will notice about cartilage is that it is avascular. You should already have some idea as to
how this characteristic limits the function of cartilage and limits its reaction to injury.
The specialized cells of cartilage, chondrocytes, are ovoid, and are often found in pairs, a remnant of
interstitial growth. Numerous small cytoplasmic processes protrude from the chondrocyte into the ECM (Fig
7.6, pg. 202). The cytoplasm of chondrocytes has pale or vacuolated areas, due to the presence of a large
Golgi apparatus, as well as glycogen and lipid, which are washed away during tissue preparation (Fig. 7.5, pg.
202). Because chondrocytes lose glycogen and lipid during LM tissue preparation, they will shrink, leaving a
surrounding space which separates the chondrocyte from the ECM. This space resembles the lacuna (small
lake) which you will examine in bone. Unlike bone, however, the lacunae of cartilage are artifacts of LM
tissue processing, and will not be seen in EM preparations.
The ECM of hyaline cartilage appears homogeneous in H&E sections. However, it does contain fibers,
which consist primarily of Type II collagen. Type II collagen is not discernible in routine H&E preparations.
Unlike the ground substance of connective tissue proper, the ground substance of cartilage contains a high
density of sulfated proteoglycans. These sulfate groups bind hematoxylin, making the cartilage matrix
basophilic. Sulfate groups are present in highest concentration around the lacunae. Therefore, there will be
rings of more intense basophilia around these spaces, with intervening more eosinophilic ECM. There are
also changes in the sulfated proteoglycans during the course of normal aging, so that the staining of cartilage
from older individuals may be less intensely basophilic than that from younger individuals. Sulfate groups
can also be lost during fixation. As a result, you should expect much variability in the intensity and hue of
staining of the ECM of cartilage.
Key features of the three types of cartilage (Table 7.1, pg. 206)
Hyaline cartilage: (Plate 7, pg. 210-211)
Perichondrium containing blood vessels surrounds non-articular hyaline cartilage.
Chondrocytes are often found in isogenous pairs.
ECM tends to be basophilic (see explanation above) with no fibers visible in the matrix on H&E stained
section.
Elastic cartilage: (Plate 9, pp. 214-215)
Perichondrium present.
Chondrocytes tend to be closer together (less ECM) than in hyaline cartilage.
ECM includes fine elastic fibers and lamellae, which can be specifically stained with elastin stains.
As a person ages, there may be accumulations of adipocytes within elastic cartilage.
Fibrocartilage: (Plate 10, pp. 216-217)
Perichondrium NOT present.
There are fewer chondrocytes than are present in either hyaline or elastic cartilage. Chondrocytes are often
arranged linearly in isogenous groups, which are widely separated by thick bundles of type I collagen in the ECM.
These collagen bundles stain eosinophilic on H&E stained section.
Cartilage and Bone Lab Session 4
Overview of Bone
Preparation of bone specimens for histologic examination
The mineralized matrix of bone presents a special challenge when preparing sections for histological examination.
The matrix is too hard to be cut with routinely used microtomes. Therefore, bone is usually prepared in one of the
following two ways:
Ground bone: this method is used to examine matrix organization into osteons (Plate 11, pp. 244-245).
After removal of the soft tissue, the bone is dried. This drying process destroys cells in the bone, so that they
cannot be examined histologically. The dried bone is then cut into thin sections using a saw. This procedure
preserves the details of the mineralized matrix. Dyes, such as India ink, may then be applied to highlight
the fine structure of the matrix.
Decalcified bone: this method is used to examine the cells of bone (Plate 14, pp. 250-251).
Most of the processing of decalcified bone is the same as that used for other tissues. The bone is fixed to
preserve the cellular structures. However, in order to dissolve the hard, inorganic components of the
matrix before sectioning, the fixed tissue is treated with acids or other agents. Processing then proceeds as
with soft tissues, with the tissue being embedded, sectioned and stained routinely. This technique is used to
examine the cellular components of bone. The lamellar organization of the cells within the osteon is
preserved, although the fine cytoplasmic processes which enter the canaliculi usually cannot be seen using
this technique. The bone cells, blood vessels and periosteum can be examined in this type of preparation.
Composition of bone:
Osteoblast: secretes both the collagen (primarily type I) and ground substance of bone ECM
o Cuboidal or polygonal if active, flat if inactive
o Basophilic cytoplasm (What organelle is most likely responsible for this basophilia?)
o Located on the surface of forming bone
Osteocyte: name given the osteoblast after it is completely surrounded by bone ECM
o Ovoid cell
o Often shrinks during preparation
o Located in a lacuna
Osteoclast: removes bone during the process of bone development (see Fig. 8.21, pg. 240)
o Very large cell with multiple nuclei
o Present in a small indentation (Howships lacuna) on the surface of forming/resorbing bone
Bone matrix
o Collagen (primarily type I) and ground substance
o Eosinophilic, homogeneous in staining
Ultrastructure: osteocyte and osteoblast
o Ovoid cells
o Long, thin cytoplasmic processes extend beyond the lacunae and into the matrix
o Osteoblasts have a large Golgi apparatus and large amounts of RER (Why are these organelles more
extensive in osteoblasts than in osteocytes?)
Bone development: You will examine sections of embryonic bone in order to understand both intramembranous
and endochondral bone formation in developing bone. Because the mineral content of embryonic bone varies as
it develops, the matrix of developing bone may contain both basophilic (mineralized) and eosinophilic
(non-mineralized) regions.
Intramembranous bone formation (Plate 15, pp. 252-253)

o Trabeculae of bone with osteoblasts, osteocytes and osteoclasts.
o Mesenchymal precursors between trabeculae.
Endochondral bone formation (Plate 14, pp. 250-251 and Figs. 8.17, pg. 236 and 8.19, pg. 238) is
characterized by the following zones, which correspond to progressive changes as bone develops:
o Zone of reserve cartilage: chondrocytes have relatively little intervening matrix, as compared to
hyaline cartilage in other locations.
o Zone of proliferation: parallel stacks of chondrocytes (daughter cells) resulting from rapid mitosis.
o Zone of maturation/hypertrophy: enlarged chondrocytes with pale cytoplasm, due to the presence
of cytoplasmic glycogen.
o Zone of calcified cartilage: matrix calcifies (basophilic), chondrocytes die, lacunae are empty.
o Zone of resorption: blood vessels and connective tissue invade area of dying chondrocytes.
o Zone of ossification: osteoblasts and osteocytes are present, ossified matrix is eosinophilic.
Synovial Joints
(Plate 13, bottom, pp. 248-249)
The structure of synovial joints will be examined microscopically. As you study their histology, you should be
able to relate your knowledge of the microscopic structure of bone, cartilage and connective tissue to what you
have previously learned about the gross structure of synovial joints.
Preparation:
Review your lecture notes on bone, cartilage and extracellular matrix. The figures and plates in Ross and Pawlina
Chapters 7 and 8, as well as Table 7.1, pg. 206 will be helpful as you try to identify the similarities and
differences between cartilage and bone and among the different types of cartilage.
Complete the Pre-Lab Problem Set on LON-CAPA.
Remember to bring your i>Clicker, Study Guide, and Histology Text with you to your assigned lab section.
LABORATORY OBJECTIVES
Objective 1: Cartilage in light microscopic section

As you work through Objectives 1.1 through 1.3, use the following grid to help you understand the similarities
and differences among hyaline cartilage, elastic cartilage and fibrocartilage.
Location of Perichondrium
Type of ECM Dominant Chondrocyte
cartilage type in present or
cartilage characteristics ECM fiber arrangement
the body absent
Hyaline
cartilage
Elastic
cartilage
Fibrocartilage
1.1 Hyaline Cartilage
1.1a Hyaline cartilage, trachea (Plate 7, pp. 210-211; Fig. 19.6, pg. 673)
In this preparation, the extracellular matrix of cartilage is prominently stained and the cytoplasm of most of
the chondrocytes is pale.
Center the tracheal cartilage using the image provided above or your text images as a guide. Increase
magnification.
Locate the perichondrium.
o Outer layer of elongate cells. These are the precursors of chondroblasts.
o Inner layer of less flattened to ovoid cells. These are the chondroblasts.
o Note that when we say outer and inner we mean layers on the outside of the cartilage, not
the outside and inside of the trachea. Perichondrium is on both the luminal and abluminal
surfaces of the tracheal ring.
Locate chondrocytes surrounded by the ECM of cartilage.
o Ovoid cells, sometimes in isogenous pairs.
Isogenous (isogenic) pairs are remnants of prior growth is this growth interstitial or
appositional?
o Sometimes chondrocytes appear to be within empty spaces (lacunae). These are artifacts due to
cell shrinkage during preparation. Loss of what cytoplasmic components during preparation
results in this shrinkage?
o Chondrocytes are surrounded by matrix. Note that the matrix nearest the chondrocytes is more
basophilic than that farther from the chondrocytes. This reflects variation in the GAG
composition of the matrix. Variation in which component of GAG results in this staining
variability?
o The cytoplasm of chondrocytes is often pale staining, with this staining varying depending upon
the level of chondrocyte activity.
***Note the complete absence of blood vessels within the cartilage.***
1.1b Hyaline cartilage, trachea, alcian blue and PAS stain

Using this staining procedure, everything is blue except for complex carbohydrates, such as intracellular
glycogen and extracellular glycoproteins of the cartilage matrix. The trachea is the bottom structure in this
slide.
Center the tracheal cartilage and increase magnification.
Identify the perichondrium. This is stained blue in this preparation, and its cells are flattened.
Examine the chondrocytes in the cartilage.
o The nuclei of these chondrocytes are pale blue. Lipids within the chondrocyte cytoplasm are
unstained, because they were lost during preparation. Glycogen has been preserved in this
preparation, and stains dark magenta with this special stain.
o Glycogen storage varies with the maturity of the cartilage. Older, less active chondrocytes have
greater glycogen stores. Therefore, in a routine H&E preparation, because of the loss of
glycogen, older chondrocytes have unstained regions in their cytoplasm.
1.2 Elastic cartilage

1.2a Elastic cartilage, elastin stain (Plate 9, pg. 214-215)
We will use this special stain first, so you can see the distribution of elastic fibers in the extracellular matrix
of this cartilage. This is a section of external ear, and the cartilage is the dark band running longitudinally in
the middle of the section. Elastin (and therefore elastic fibers) is black in this preparation.
Locate the perichondrium. The perichondrium also has some elastic fibers.
Identify the chondrocytes.
o They are sometimes present in isogenous pairs.
o Some are within lacunae. Would these lacunae be seen in an EM of this tissue?
Examine the ECM of the cartilage. Find the elastic fibers in this matrix.
In this slide, do the elastic fibers appear to be more densely arranged immediately around the
chondrocytes or farther from the chondrocytes?
***How does the flexibility of the external ear compare to that of the nasal septum? What type of cartilage
is found in the nasal septum?***
1.2b Elastic cartilage (Plate 9, pp. 214-215)

Now we will look at elastic cartilage in a routine H&E preparation.
Center the cartilage. At low magnification, the cartilage can be seen as the dark eosinophilic band
running longitudinally near the top of the section. Increase magnification.
Locate the perichondrium using the same criteria you used when examining hyaline cartilage in
Objectives 1.1a and 1.1b.
Identify chondrocytes. Most have large unstained regions in their cytoplasm. This indicates that this is
older cartilage. Cartilage in mature individuals is less active. The chondrocyte in the cartilage of mature
individuals will have increased cytoplasmic stores of glycogen and lipid, which will cause them to have
areas of unstained cytoplasm. The Golgi apparatus will be smaller in these less active chondrocytes.
The extracellular matrix has fine streaks, due to the presence of elastic fibers. Elastic fibers stain
eosinophilic in routine H&E preparations. Although they are thin, it is easier to see individual elastic
fibers between chondrocytes, where they are more loosely arranged, than immediately around
chondrocytes, where they are more densely packed.
1.3 Fibrocartilage (Plate 10, pg. 216-217)

Increase magnification until you can examine the chondrocytes. These are ovoid cells with spherical
nuclei. You can see lacunae around most of these chondrocytes. In fibrocartilage, chondrocytes are
sometimes arranged in rows or clusters, called isogenous groups.
Examine the extracellular matrix. Identify eosinophilic streaks in the extracellular matrix. These streaks
are composed of bundles of what fiber type?
Slide note: The eosinophilic streaks in the ECM of fibrocartilage are composed of bundles of Type I
collagen fibers. Because of its arrangement into bundles and its staining characteristics, it can be
distinguished from the surrounding cartilage ground substance. You will remember that Type II collagen,
which is present in the ECM of all three types of cartilage, cannot be distinguished from the surrounding
ground substance in LM section. Fibrocartilage is resistant to both compression and shear, and is found
in intervertebral discs, articular menisci and the pubic symphysis.
Objective 2: Ultrastructure of chondrocytes
2.1 Chondrocyte, EM (Figs. 7.5 and 7.6, pg. 202)

Which organelles identifiable in this chondrocyte are important in the synthesis of collagen and other
components of the ECM?
In the ECM surrounding the chondrocyte, note the absence of banded fibrils of Type I collagen. Only
loosely arranged, thin fibers and dark particles containing proteoglycans can be seen in the ECM
surrounding this chondrocyte.
Note that the extracellular matrix extends to the cell surface. In tissue prepared for EM examination, the
cytoplasmic components which are lost during routine H&E preparation (glycogen, lipid) remain in the
cytoplasm. Therefore, in EM images you will not see a lacuna surrounding the chondrocyte.
2.2 Chondrocyte isogenous pair, EM

Depending upon their level of activity, chondrocytes will have variably prominent organelles, such as RER and
Golgi apparatus, which are associated with the process of secreting the proteinaceous fibers and complex
carbohydrates of the ECM. A large Golgi apparatus will appear as an unstained area near the nucleus in H&E
preparations (see R&P Fig. 7.5, pg. 202). In this image, you can identify numerous small cytoplasmic
processes extending slightly from the chondrocyte into the ECM. Do you see a lacuna in this EM?
Objective 3: Bone in LM
3.1 Organization of bone extracellular matrix, ground bone (Plate 11, pp. 244-245)
Ground bone is the best preparation for review of the structure of the Haversian system. It will not allow,
however, visualization of any of the cellular elements of bone.
Identify the following features of the Haversian system:

o Haversian canal
o Lamellae and interstitial lamellae
o Canaliculi
o Lacunae
o Volkmanns canal
Although you cannot see cellular elements in this specimen, identify locations where you would
normally find the following:
o Osteocytes
o Osteoblasts
o Osteoclasts
o Blood vessels
3.2 Cells and sheaths of bone, decalcified bone, calvaria (Plate 12, pp. 246-247)
This specimen of bone underwent decalcification, a technique which preserves the cellular components
of bone for examination. Use this slide to identify the cells of bone as well as the sheaths of bone, the
periosteum and endosteum.
Osteoblasts, when active, are rounded or plump with basophilic cytoplasm. They are flattened when
inactive. Osteoblasts lie along the surface of forming bone. In this preparation, look for osteoblasts in
the endosteum on the surfaces of trabeculae and in Haversian canals.
Osteocyte is the term used for an osteoblast which has become surrounded by bone matrix. Osteocytes
are the small, dark, angular cells within lacunae of the bone. How does the appearance of an osteocyte
in LM section differ from that of a chondrocyte in LM section?
Osteoclasts (see Fig. 8.21, pg. 240) are very large cells which have multiple nuclei. They can be found
in the endosteum along the surface of trabeculae and in Haversian canals. Osteoclasts often are present
within depressions, called Howships lacunae, on the surfaces of forming bone. A good spot to begin
looking for osteoclasts in this slide is in the region indicated by the arrow in the figure provided below.
Periosteum is the external covering of bone. In this section of calvaria it covers both the upper concave
surface of the bone and the lower, convex surface of the bone. It consists of an outer layer of dense
fibrous connective tissue and a layer of progenitor cells immediately adjacent to the bone. These
progenitor cells may be flat, like fibroblasts, or more rounded, like osteoblasts.
Endosteum lines the inner surface of bone, including all of the Haversian canals and Volkmann's canals.
Endosteum is composed of spindled cells, which resemble fibroblasts. These are osteoprogenitor cells.
As their name implies, they have the capability to form bone.
Examine the extracellular matrix of this specimen of decalcified bone. Bone extracellular matrix is
homogeneous and eosinophilic in LM preparations.
Although it will be more difficult than when examining ground bone, locate a Haversian canal with its
blood vessels in this specimen of decalcified bone.
Slide note: The calvaria is composed of the frontal, parietal, and temporal bones. It is also known as the
skullcap. As this slide is oriented on your computer screen, the internal surface of the calvaria is at the top and
its external surface is at the bottom. In life the brain would have been located at the top and the scalp at the
bottom of this specimen. As is seen in this section, the periosteum, the endosteum, osteoclasts and other cells of
bone may be pulled away from the surface of the bone during sectioning. This occurs because of the difference
in density between the cellular components of bone and the bone matrix.
Objective 4: Osteocytes, EM
Refer to R&P Fig. 8.10, pg. 228
Before reading the caption for these three figures, try to determine, based upon cell shape and organelle
content, which cell is least active and which cell is most active in producing bone matrix.
Use these EMs to identify the ultrastructural features of osteocytes at different functional stages. The
position of the nucleus may be altered by changes in organelle content. In Fig. 8.10a, you can see
cytoplasmic processes of the least active osteocyte extending into canaliculi.
Objective 5: Bone formation, intramembranous and endochondral
5.1 Intramembranous bone formation (Plate 15, pg. 252-253)

Use the image below to help orient yourself as you examine regions of developing bone in this section
through the head of a fetal mammal.
Eosinophilic trabeculae of forming bone are separated by large spaces, which will eventually become
marrow spaces. Osteoblasts and osteoclasts are on the surfaces of forming bone, with osteocytes
embedded in matrix. In this specimen, osteoclasts are very difficult to find. Therefore, use Objectives 3.2
and 5.2 for your examination of osteoclasts.
Mesenchymal cells are between bone trabeculae. Mesenchymal cells are spindled or stellate shaped.
Does intramembranous bone formation occur in long bones or flat bones?
5.2 Endochondral bone formation, finger

(Plates 13 and 14, pp. 248-249; Fig. 8.17, pg. 236; Fig. 8.19, pg. 238; Fig. 8.20, pg. 239)
As you work through Objective 5.2, use the following chart to help organize (by drawing or listing) the
histologic differences between the stages of endochondral bone formation. Correlate the histologic changes
with the processes the cells are undergoing at each stage to accomplish overall bone growth. Also, be able to
answer the following questions:
Where are osteoblast precursors located?
Where does appositional bone growth occur?
Where do you expect to find osteoclasts?
Chondrocytes typical of
Zone of reserve hyaline cartilage, although
cartilage there is less matrix
separating chondrocytes
than would be found in
mature hyaline cartilage.
There is frequent chondrocyte
Zone of mitosis and the chondrocytes are
proliferation arranged in columns parallel to
the long axis of long bones.
Cells still in columns become

Zone of enlarged due to intracellular
hypertrophy glycogen accumulation.
The intervening matrix is
narrowed.
Chondrocytes die and the

Zone of calcified matrix is calcified. In this
cartilage zone the matrix is dark
basophilic.
Blood vessels and connective

Zone of tissue invade calcified cartilage.
resorption Blood vessels source of
and osteoprogenitor cells
ossification Osteoblasts synthesize osteoid matrix.
When surrounded by matrix
osteocytes. The bone initially formed
is primary, cancellous bone.
Locate the bone collar along
Bone collar and the shaft (diaphysis) of the bone
periosteum (see Fig. 8.17 and Fig. 8.20).
Identify the periosteum.
Objective 6: Identify the following components of the synovial joint in this section of finger
(Plate 13, pp. 248-249)
Articular surface
What type of cell is
present here?
Joint cavity/
synovial cavity
In life, what is present
in this cavity?
Synovial membrane
synoviocytes
blood vessels
Synoviocytes are the cells lining the joint cavity, except for the surfaces of the articular cartilage.
Examine the portion of the synovial membrane that protrudes into the joint cavity.
Note the difference in vascularity between the synovial membrane and the cartilage. (You will remember
that cartilage is always avascular); also, note that where the two bones of this joint oppose each other, the
synovial membrane does NOT extend to cover the surfaces of the articular cartilage of either bone. If a
synovial membrane were to proliferate due to pathology, what do you think would be the patients
presenting clinical sign or symptom?
Integrative Questions
1. Using the virtual slide at the bottom, find examples of each of the features labeled A-D in the still image on
top. (Note: this virtual slide has an additional "Focus" option available in the magnification objective menu.)
2. Compare the cells and extracellular matrix in these two electron micrographs.
Which of the cells is more actively synthesizing extracellular matrix?
Which matrix appears more orderly in its arrangement of extracellular fibers?
Which matrix would be more efficient at withstanding unidirectional shear?
Do you see a lacuna surrounding either of these cells?
What is the most likely cell type shown in each EM?
3. Locate regions of appositional bone growth and bone remodeling. What do you think would be the effect of a
reduced function of osteoclasts during bone development?
4. This image is a sagittal section of a knee from Netters Atlas, showing the femur, tibia and patella. The blue
material is cartilage. The lateral meniscus is visible as two wedges of fibrocartilage, which protrude into the
joint space. Where would you expect to find synovial membrane?
Self-study Review
1. Part 1: Identify regions of bone, bone marrow and cartilage.
Part 2: Identify the type of cartilage. What criteria did you use to identify this cartilage?
2. Compare the two tissues. Identify each, and give the criteria you used to identify them.
3. Compare the two tissues

Identify each, and give the criteria you used to identify them.

5. Find examples of endosteum and periosteum.

7. Locate the zones of reserve cartilage, proliferation, hypertrophy, calcified cartilage, resorption and
ossification.
In addition to answering the bolded questions in the lab manual:
For Bone and Cartilage:

Be able to distinguish the different types of cartilage from bone and from all of the types of connective
tissue studied so far.
Describe the primary types of extracellular fibers found in bone vs. cartilage, and in the different types of
cartilage.
Identify the cells of cartilage and bone, and understand the ultrastructural differences between bone and
cartilage cells.
Identify the periosteal and perichondrial regions of bone and cartilage.
Understand the difference between interstitial and appositional growth. Be able to identify regions of
appositional growth, and the cells responsible for this growth.
For Cartilage:
Be able to distinguish the three types of cartilage, and understand their difference in structure, typical
location and normal function.
Relate the organization of cells and perichondrium (if present) to the mechanism of cartilage growth.
For Bone:
Understand the difference between ground bone and decalcified bone preparations, and the limitations on
what you can observe with each preparation.
Identify compact bone, cancellous bone and the medullary cavity in gross specimens of bone and in
microscopic images
Identify the components of the Haversian system in LM sections.
Relate the different cell types found in bone to the function and control of BMU as described in lecture.
Understand the difference between endochondral and intramembranous bone formation, and recognize
these in section.
Be able to identify the different functional regions of endochondral bone formation.
For Joints:
Identify the components of the synovial joint in light microscopic section or in diagrams.

Embryo Simple & Bone - Fundamentals

Încărcat de

Informații document

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Embryo Simple & Bone - Fundamentals

Încărcat de

Drepturi de autor:

Formate disponibile

Early Embryology & Stem Cells Lecture Sessions 6 & 7

Problems for Self-Study

1. In the context of embryonic development, "mesenchyme" refers to cells which:

2. All adult epithelium is derived from the embryonic:

3. The neural crest:

4. A correct description of stem cells is that:

A. the hypoblast only

6. Hematopoietic stem cells:

9. An example of apoptosis in adult tissue is:

Wilms tumor in the lower pole of a pediatric kidney.

16. In adult tissues, transit amplifying cells (TACs):

17. Which of the following correctly describes stem cells?

19. Which of the following is true regarding differentiation of somite cells?

Answers to Self-Study Problems

9. A. The finite lifespan of intestinal cells is an example of apoptosis, programmed or non-pathologic

B. Weeks 1 & 2 Overview

C. Week 3: Gastrulation and Body Tissue Correlates

D. Fates of the 3 Germ Layers- a closer look

E. Notochord Development and Significance

G. Somite Development and Significance

H. Some Universal Mechanisms of Development

I. Adult Stem Cell Basics

Lecture Sessions 8-11

1) Ross and Pawlina Chap. 6, pp. 158-178

Neurons, cells specialized for signaling,

Mesoderm Specialized for motility and includes

Mesoderm Connective Scaffold upon which everything else hangs;

blood adipose mucous loose dense dense cartilage bone

SPECIALIZED CT PROPER SPECIALIZED

CELLS EXTRACELLULAR MATRIX

ADHESIVE GLYCOPROTEIN GROUND SUBSTANCE FIBERS

I) Extracellular Matrix (ECM): definition and general considerations

II) Chemical composition

Fibrous CT (e.g. tendon) Cartilage Basement Membrane

III) Histological staining of ECM

(b) white space,

(c) pinkish fibers

IV) Collagens Collagen

2) There are >40 distinct genes/polypeptides (with different amino acid

b) Also high content of Pro and Lys, some of which are

4-OH Pro 5-OH Lys

Enzymes for hydroxylation ( )

2) Secondary Structure ---entirely helical but NOT -helical

3) Tertiary Structure (There is no tertiary structure.)

The three collagen polypeptides can fit closely

C) Assembly to form fibril

Tendon all aligned in one

Skin less well aligned --->

Fibrils of collagen molecules are stabilized by repetitive H-bonds, both

(Stiff skin/tough meat --- due to spontaneous oxidation and cross-linking)

D) COLLAGEN DEFECTS and DISEASED STATES

VI) ELASTIC FIBERS ---

forms microfibrils upon which

(c) MAGP (microfibril-associated

(2) elastic fibers allow tissues to respond to stretch and distension;

(3) elaborated by fibroblasts of loose CT,

(4) degree of assembly varies with tissue ---

VII) Matrix Metalloproteinases (MMPs)

VIII) Glycosaminoglycans (GAGs)

Repeat unit of hyaluronic acid Repeat unit of chondroitin 4-sulfate

HA BINDING region: devoid of GAGs in the

Proteoglycan Aggregate (e.g. aggrecan of cartilage)

actual EM of proteoglycan aggregate from cartilage