Histology SSN – October 11, 2005 – Microscopy and Cytology Stains
Tabassum Sardharwala (ts2193) and Shannon Watkins (saw2123)
Microscopy and Cytology Stains
Name
Color
Staining
Properties
Structures Stained
Nissl Stain
Dark blue or blackish
Negatively charged
RER, ribosomes and DNA
particles
Eosin (H&E)
Hematoxylin (H&E)
Pink
Blue or blackish
Positively charged
Proteins (cytoplasm and
(acidophilic) structures
membranes), collagen
Negatively-charged
DNA, RNA, ribosomes *NOTE:
(basophilic) Structures
fat, mucus, lipid & golgi do not
stain w/H&E
Periodic Acid
Pink or magenta
Carbohydrates
Glycocalyx, membranes,
Schiff &
(more vibrant than
glycoproteins, mucus,
Hematoxylin (PAS)
eosin)
proteoglycans, glycogen and
lysosomes
Acid Fuschin-
Pink (acid fuschin) &
Acid fuschin acts like
Toluidine blue
Blue (toluidine blue)
eosin, toluidine like
Just like H&E
hematoxylin
Régaud's Iron
Black
Mitochondria
Hematoxylin
Acid Phosphatase
Black
Acid hydrolases
Lysosomes
Silver &
Black (silver) & red
Reticular fibers, basal lamina
Azocarmine
(azocarmine)
(silver) & nucleus/cytoplasm
(azocarmine)
Neutral Red
Red
Stains when protonated
Lysosomal contents
Janus Green
Green
Stains when oxidated
Inside of mitochondria
Histology SSN – October 11, 2005 – Mitosis
Tabassum Sardharwala (ts2193) and Shannon Watkins (saw2123)
MITOSIS
Tabassum Sardharwala [email protected]
Shannon Watkins [email protected]
Mitosis (M phase)
Mitosis is divided into 5 stages:
1) Prophase- mitotic spindle begins to form, and chromosomes condense so that sister
chromatids are attached at the centromere.
2) Prometaphase- the nuclear envelope disassembles, and chromosomes attach to spindle
apparatus via kinetochores.
3) Metaphase- chromosomes migrate to align centrally on the metaphase plate.
4) Anaphasea) A- sister chromatids separate and move towards the pole.
b) B- two spindle poles move apart. Cytokinesis begins.
(see diagram on next page)
5) Telophase- nuclear envelope reforms, chromosomes decondense, and cytokinesis is
completed.
Histology SSN – October 11, 2005 – Mitosis
Tabassum Sardharwala (ts2193) and Shannon Watkins (saw2123)
Mitosis Summary Table
Prophase
Prometaphase
Metaphase
Nuclear
Envelope
Yes
Yes
Disassembles
No
Chromosome
Degradation
Decondensed
Condensed
Condensed
Condensed
Anaphase
No
Condensed
Telophase
Reassembles
Decondensing
Stage
Interphase
Spindle Apparatus
Not formed
Centrosomes separate
Chromosomes attach
Fully formed
K-MTs shorten;
Polar MTs elongate
Disassembling
Chromosome
Location
Nucleus
Nucleus
Nucleus/Cytoplasm
Metaphase plate
Spindle poles
Within reforming
nuclear envelope
*MTs=microtubules (K-MTs= kinetochore microtubules)
MITOSIS PRACTICE QUESTIONS
1) Put the numbered cells in sequential order.
a) 1, 3, 4, 2, 5
b) 2, 4, 5, 1, 3
c) 4, 5, 3, 2, 1
d) 4, 3, 5, 2, 1
2) This structure is part of the
a) Kinetochore
b) Centromere
c) Microtubule Organizing Center (MTOC)
d) Cleavage furrow
e) Chromatid
3) What characteristics would you expect to find in the cell at the pointer:
i) Spindle apparatus
ii) Nuclear envelope
iii) Contractile ring
iv) Equatorial plate
a) I and II only
b) I, II, and IV
c) I and IV
d) All
4) All of the following pertain to the cell labeled 1, EXCEPT:
a) Nuclear envelope has disbursed
b) Mitotic spindle has begun formation
c) Centrosomes have separated
d) DNA has been duplicated
----------------------------------------------------------------------------------------------------------------
ANSWERS
1)
2)
3)
4)
C
C, EM (slide 13)
C, #112 (slide 2)
A, #112 (slide 3) Cell labeled 1 is in prophase. Nuclear envelope will disburse during prometaphase.
Histology SSN – October 11, 2005 – Epithelium
Jen Chang (jtc2109) and Bram Welch-Horan (tbw5)
EPITHELIUM
Definition: Avascular tissue made of cells that cover exterior surfaces and line both internal closed
cavities and body tubes that communicate with the exterior. Epithelium also forms the secretory portion of
glands and ducts.
Features:
ƒ POLARITY: distinct apical, basal, and lateral surfaces
ƒ Basal Surface: attached to basal lamina (collagen Type IV, made by epithelial cells) which is
part of basement membrane
ƒ Cells adhere to each other via specialized junctions (explained below)
FUNCTIONS
Protective Layer
Absorption of water and solutes
Secretion
Containment
Excretion
EXAMPLE
Epidermis (stratified squamous)
Intestinal Epithelium (simple columnar)
Glands: salivary, pancreatic (cuboidal)
Bladder (transitional)
Kidney tubules (simple cuboidal)
TYPES OF EPITHELIA
Simple
CHARACTERISTICS
ƒ One cell layer thick
ƒ Absorption, secretion, diffusion
ƒ Ex.: simple columnar in small intestine,
simple squamous in capillaries
ƒ A simple epithelium
ƒ All cells rest on basement membrane, but not all reach
apical surface
ƒ Ex.: Lining of trachea(ciliated) and
epididymis (stereociliated)
ƒ Two or more cell layers thick
ƒ Classified based on cell type of surface cells
ƒ Protection, barrier
ƒ Ex: Stratified squamous in epidermis
(keratinized) and
esophagus (non-keratinized)
ƒ A stratified epithelium
ƒ Apical surface may appear “half-domed”
ƒ Accommodates distension by flattening
ƒ Ex.: lining of the bladder, ureters, urethra
Pseudostratified
Stratified
Transitional
TYPES OF CELLS
Squamous
(stratified is usually protective, and simple
for diffusion)
Cuboidal
(often absorptive, but sometimes secretory)
Columnar
(usually absorptive, but sometimes
secretory)
CHARACTERISTICS
ƒ Cells are flattened and irregularly shaped
ƒ Appear “scale-like” or “squashed”
ƒ Ex.: endothelium of vasculature, alveoli
ƒ Round, central nucleus
ƒ Width = height (ice cube shaped)
ƒ Ex.: pancreas-secretory, kidney-absorptive
ƒ Elongated nucleus
ƒ Width < height (cells long and tall)
ƒ Ex.: lining of small and large intestines
Histology SSN – October 11, 2005 – Epithelium
Jen Chang (jtc2109) and Bram Welch-Horan (tbw5)
SPECIALIZATIONS
Cilia
Microvilli
Stereocilia
Keratin
DESCRIPTION
ƒ Insert into basal bodies (1 cilium per 1 body)
ƒ Motile processes of microtubules move synchronously
ƒ 9 +2 microtubule arrangement
ƒ Ex.: trachea and oviduct
ƒ insert into terminal web (stains eosinophilic – pink)
ƒ actin skeleton above intermediate filaments
ƒ increase surface area for absorption
ƒ Ex.: small intestine
ƒ long microvilli – actin (NOT cilia!)
ƒ non-motile
ƒ Ex.: epididymis (pseudostratified)
ƒ Formed from dead layer of squamous cells
ƒ Protects against desiccation and abrasion
ƒ Ex.: epidermis (stains strongly eosinophilic)
Basement membrane = basal lamina & reticular lamina
ƒ Stains with PAS
Basal Lamina
ƒ Separates epithelia from connective tissue
ƒ Collagen type IV, proteoglycans, glycoproteins
ƒ Synthesized by epithelial cells
Reticular Lamina
ƒ Connective tissue below epithelium
ƒ Collagen type III
CELL-CELL CONTACTS:
Zonula Occludens (apical end)
Terminal bar = Junctional Complex = Zonula Adherens
Macula Adherens
ƒ Stains dark with Bodian silver
CELL CONTACT
DESCRIPTION
Zonula Occludens (tight junction)
ƒ Diffusion barrier
ƒ Most apical, forms band around cells
Zonula Adherens
ƒ Forms band around cell at lateral surfaces
ƒ Adds to integrity of epithelial surface
Macula Adherens (desmosome)
ƒ Spot adhesions on lateral surfaces
Hemidesmosome
ƒ Link cell to basement membrane at basal surface
IMPORTANT: Don’t confuse terminal bar (junctional complex) with terminal web
(network of actin and intermediate filaments microvilli insert into)
Questions
1.
This structure is typically found in the…
a. Trachea
b. Kidney
c. Epididymis
d. Small intestines
2.
Which of the following is FALSE regarding the structure at the pointer?
a. Its permeability determines whether the epithelia is “tight” or “leaky”
b. It occurs at small discrete sites
c. It separates the apical surface from the basolateral surface
d. It is a component of the junctional complex
Histology SSN – October 11, 2005 – Epithelium
Jen Chang (jtc2109) and Bram Welch-Horan (tbw5)
3.
What type of collagen is found in the tissue at the pointer?
a. Type II
b. Type III
c. Type IV
d. Type VII
4.
What kind of epithelium lines the secretory alveoli of this exocrine gland?
a. Simple columnar
b. Simple cuboidal
c. Squamous
d. Transitional
Questions 5-6: Figure A (Lab 3, slide 35); Figure B (Lab 3, slide 25)
5.
Select the one correct statement regarding the surface epithelium:
a. In both figures all of the cells reach the lumen.
b. In both figures the superficial cells are keratinized
c. In both figures all of the cells rest on a basal lamina
d. Only in Figure B do all the cells rest on a basal lamina
6.
The tissue or tissues that are specialized to provide a barrier to luminal absorption are shown in:
a. Figure A only
b. Figures A and B
c. Figure B only
d. Neither figure A or B
7.
This cell type is typically found in the:
a. Bladder
b. Kidney tubules
c. Intestinal epithelium
d. Epidermis
8.
What type of epithelium is this?
a. Pseudostratified
b. Stratified Columnar
c. Stratified Squamous
d. Stratified Cuboidal
Answers:
1. D; These are microvilli. They function to increase surface area for absorption and are found in the small
intestines.
2. B; This is the zona occludens (tight junction). They are located around the entire perimeter of the cell.
3. C; The basal lamina stains red on a PAS stain. It contains collagen type IV.
4. B; The small ducts of exocrine glands are lined by simple cuboidal cells.
5. D; In the trachea pseudostratified epithelium, all cells rest on the basal lamina. Bladder transitional epithelium is
stratified and therefore not all cells touch the basal lamina.
6. A; Transitional epithelium, a special type of stratified epithelium, serves as a barrier.
7. C; Simple columnar epithelium lines the small intestine and colon. The stomach lining, gastric glands, and lining
of the gall bladder are all lined by simple columnar epithelium.
8. C; The image shows epidermis (i.e., skin), which is a stratified squamous (keratinized) epithelium. More on that
next time…
Histology SSN – October 11, 2005 – Connective Tissue
Patrick McCormick (pjm2109) and Anastasia Spencer (azs2103)
CONNECTIVE TISSUE
I. COMPONENTS OF CONNECTIVE TISSUE
Most Connective Tissue (CT) is derived from mesenchyme, which is mostly mesoderm.
Connective tissue is composed of cells and their secreted ECM (fibrous proteins, ground
substance, and fluid). The combination and ratio of each determines the function & role of
various connective tissues.
A. Cells
Structure
elongated cells with
cigar-shaped nuclei
and little cytoplasm
large nucleus (often
Macrophages eccentric), many
(written MΦ) surface folds on EM,
monocyte-derived
small condensed
Adipocytes
nucleus on the side,
thin rim of cytoplasm
Fibroblasts
Function
Stain
secrete collagen,
ground substance,
carbohydrates, elastin
phagocytose bacteria
& cell debris, and
digest them in
lysosomes
plasma membrane often
obscured by surrounding
collagen
PAS positive cytoplasm due
to carbohydrate-rich
enzymes in lysosomes
FAT
appear clear; lipid
droplets lost during staining
Other cells found in CT include immune cells (lymphocytes, neutrophils, eosinophils & basophils)
that migrate out from the blood.
B. Fibers (long, slender protein polymers)
Collagen Structure
Found in
thick fibrils bone
Type I
bundled
skin
into fibers
tendon, ligament
fibrocartilage, LCT,
DCT
thin fibrils
hyaline cartilage
Type II
elastic cartilage
meshed
reticular fibers
Type III
fibrils
lymphatic tissues
(except thymus)
does not
basement membrane
Type IV form fibrils
Elastic
fibers
elastin and
fibrillin
vertebral ligaments,
larynx, elastic arteries
(aorta), often
interwoven w/collagen
Function
resists
tension
resists
pressure
organ
framework
Stain
pink w/H&E (pos chged
aa)
red-brown w/Ag stain
yellow w/Orcein
blue w/ trichrome
look at cartilage notes
brick-brown w/ Ag stain
(due to glycoproteins)
support;
filtration
barrier
resists shear
and tearing
- pink w/H&E (so hard to
distinguish)
- black w/Orecin
Histology SSN – October 11, 2005 – Connective Tissue
Patrick McCormick (pjm2109) and Anastasia Spencer (azs2103)
C. Ground substance (gel-like substance)
1) Glycosaminoglycan (GAG)
a) long, unbranched polysaccharides
composed of repeating
disaccharides
b) generally linked to a core protein
c) may be classified into different
groups based on their chemical
structure:
i) hyaluronic acid (aka
hyaluronan)
(1) very large, nonsulfated
molecule
(2) not attached directly to a
core protein
(3) attached to the core
proteins via linker proteins
ii) chondroitin sulfate
(1) attached directly to the
core protein
iii) keratan sulfate
(1) attached directly to the core protein
2) Proteoglycans
a) composed of a protein core covalently bound to many glycosaminoglycans (GAG)
b) large molecules shaped like a bottle brush (see picture!)
c) negatively-charged GAGs attract cations
i) cations draw in water, hydrating the ground substance
3) Glycoproteins
a) includes fibronectin & laminin
b) fibronectin Æ multifunctional molecule
i) mediates cell adhesion to the ECM by binding to fibronectin receptors on the cells
surface
ii) has domains for binding collagen, heparin, various cell-surface receptors, and celladhesion molecules
c) laminin Æ mediates interactions between epithelial cells and ECM by anchoring cell
surface to the basal lamina
II. TYPES OF CONNECTIVE TISSUE
1) Loose Connective Tissue (LCT or Areolar)
a) many cells, mostly fibroblasts, also macrophages and lymphocytes
b) much more ground substance than fibers
c) well vascularized
d) found beneath many epithelia (e.g. the lamina propria of the GI tract)
e) Adipose Tissue (one special type of LCT)
i) white adipose tissue – unilocular adipocytes
Histology SSN – October 11, 2005 – Connective Tissue
Patrick McCormick (pjm2109) and Anastasia Spencer (azs2103)
ii) brown adipose tissue – multilocular adipocytes and mitochondria for heat
production
f) Reticular Tissue (second special type of LCT)
i) distinctive black appearance when stained w/silver salts
ii) contains reticular fibers (type III collagen), glycoproteins & proteoglycans
iii) provides structural support to stroma of lymph nodes, spleen, liver, bone marrow
2) Dense Connective Tissue (DCT)
a) Dense Irregularly Arranged Connective Tissue (DIACT)
i) fibrous tissue with fewer cells (cells are mostly fibroblasts)
ii) little ground substance
iii) collagen fibers are bundles, without definite orientation
iv) found in dermis, prostate, mammary glands, outer capsule of many organs
b) Dense Regularly Arranged Connective Tissue (DRACT)
i) made of many fibers that run in the same direction & offer resistance to stress
ii) little ground substance
iii) forms collagenous tissue & elastic tissue
iv) found in tendons and elastic ligaments (vertebral column, true vocal cords, large
arteries)
Helpful Hint: To distinguish between CT and muscle note that CT (fibroblast) nuclei are flatter and
between fibers rather than within fibers. CT can look wavy due to the fixation process, but
muscles have striated banding patterns and stain more deeply.
3) Embryonic Connective Tissue (mesenchyme)
a) characterized by many cells and few fibers
b) found around developing notochord
c) can differentiate into all kinds of connective tissue
Histology SSN – October 11, 2005 – Connective Tissue
Patrick McCormick (pjm2109) and Anastasia Spencer (azs2103)
CONNECTIVE TISSUE
Questions:
1. A 23 year old, 6' 4" female first-year medical student presents with Marfan's syndrome, a autosomal dominant
mutation of the fibrillin gene, which is necessary for forming elastic fibers. She has just taken anatomy (but didn't
attend SSN Histology) and wants to know which organ will be most affected by her condition. What do you tell her?
A. Middle cerebral artery (MCA)
B. Hyaline cartilage
C. Bone
D. Aorta
E. Lymphatic vessels
2. Collagen Type III is used in what capacity?
A. ligaments
B. organ framework
C. fibrocartilage
D. basement membrane
E. hyaline cartilage
3. Collagen in which material is most able to resist pressure?
A. bone
B. lymph node
C. elastic cartilage
D. fibrocartilage
E. glomerulus
4. Why do the large pink cells in the slide to the right stain pink? (or
purple, or gray, depends on your point of view.)
A. Reticular fibers in the stroma
B. Enzymes in the lysosomes
C. Transfer RNA in the cytoplasm
D. Diffuse chromatin in the nucleus
E.
FAT
ANSWERS:
1. D. The aorta is the most affected because it has the most elastic fiber and is under the greatest stress. Patients
with Marfan's syndrome are at great risk for developing an aortic aneurysm leading to aortic rupture and death.
2. B. Collagen type III forms reticular fibers, and creates the organ framework for several organs including the spleen
and lymph nodes.
3. C. Elastic cartilage is composed of collagen type II, which is good at resisting pressure. Collagen type I, found in
bones and tendons, is good at resisting tension. (Think tendon = tension.)
4. B. The large pink cells are macrophages, which contain a large number of lysosomes. The lysosomal enzymes are
carbohydrate-rich. Carbohydrates stain bright pink with a PAS stain. (Note also the carbohydrate-rich brush border
at the top of the slide which also stains bright pink.)
Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
~BONE~
Throughout life, bone is in a dynamic state of growth & resorption to accommodate changing
mechanical stress & the demands of calcium homeostasis.
Bone is a specialized connective tissue consisting of cells and mineralized matrix.
Functions:
1) support & protection
2) storage site of calcium & phosphate
3) encloses hematopoietic elements of bone marrow
~THE CELLS OF BONE~
Osteoblasts
Osteocyte progenitors
Secrete collagen I & ground
substance for osteoid
(unmineralized bone
matrix)
Initiate matrix calcification
Single layer on surface of
forming bone
Basophilic*
Osteocytes
Mature, non-dividing bone
cells
Release lysosomal enzymes
to digest bone; bone
remodeling/ resorption
Enclosed in calcified bone
matrix
Lacunae
Less basophilia than
osteoblasts
Cuboidal, Polygonal
Eccentric, Euchromatic
nuclei
Prominent nucleoli & Golgi
Osteoid newly-secreted
from osteoblasts is lightstaining (not calcified)
Osteoclasts
Remodeling cells
Howship’s lacunae-region
where bone is eroded
Acidophilic*
Large cells, ruffled border
on EM (membrane foldings
around bone seal off spaces
for localized enzymatic
digestion)
Multinucleated
Canaliculi connect
osteocytes and can be wellvisualized in ground bone
sections.
Parathyroid hormone Æ
increased osteoclast activity
Calcitonin Æ decrease
osteoclast activity
(calcitonin “puts the bone
in”)
*Rather than memorize details, think about patterns: rER and other necessary synthetic
components stain basophilic to H&E, so cells producing high levels of proteins, etc., will have
basophilic cytoplasm. Cells with high levels of lysosomal enzymes, like osteoclasts, tend to be
acidophilic.
11
Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
~MATRIX~
1) type I collagen
2) ground substance w/ proteoglycans & non-collagenous glycoproteins (ALL MINERALIZED)
3) mineral = calcium phosphate in form of hydroxyapatite crystals
~CLASSIFICATIONS OF BONE~
Mature (Adult bone; lamellar)
Compact
Spongy
• Haversian
• Trabecular
systems
appearance
• Dense layer
• At bone interior
covering bone
exterior
Immature
Immature Woven Bone
• Deposited in fetal skeleton/following a
fracture
• Nonlamellar
• Irregular collage in proteoglycan matrix
(unmineralized)
• More cells & ground substance than
mature bone
• Stains more intensely w/ H&E than
mature bone
Can also classify bones as long vs. flat:
Long Bones
*Growth = endochondral ossification
*Examples: Tibia, Metacarpals
Flat Bones
*Growth = intramembranous ossification
*Examples: Skull, Sternum
Components of Long Bones:
• diaphysis = shaft; marrow cavity surrounded by compact bone
• epiphysis = expanded bone end; spongy bone surrounded by thin compact bone shell
• metaphysis = flared portion between diaphysis & epiphysis
• epiphyseal plate = cartilage separating epiphyseal & diaphyseal cavities; maintains growth
process
~BONE GROWTH~
• All growth of bone tissue is appositional, in that it grows on pre-existing surface of bone,
beneath periosteum. This results in increase in width.
• All bones increase in length by interstitial growth of cartilage bone growth via formation of
new cartilage within existing cartilage mass. This results in increase in length.
12
Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
~BONE FORMATION~
Endochondral:
• cartilage model serves as a precursor
• fetal development
• growth in young adulthood – at epiphyseal plate
1. Mesenchymal cells condense, aggregate, and differentiate into chondroblasts.
2. Chrondroblasts lay down cartilage model; cartilage model grows in length by interstitial
growth and width by appositional growth.
3. Bony collar develops around shaft of growing bone.
4. Calcification of cartilage matrix occurs in this region, causing death of chondrocytes
5. Lacunae become confluent, creating larger cavity in center of model.
6. Periosteal cells migrate in, differentiate into osteoblasts, and begin to lay down osteoid on
calcified spicules that remain in cavity.
7. Calcified cartilage that remains is basophilic. New bone is eosinophilic.
Zone
Zone of Reserve
Cartilage
Description
• Randomly arranged
chondrocytes
• No proliferation
• Source of bone-destined
chondrocytes
Hallmarks
• Cells most sparse
• Appears like “normal”
cartilage
• Closest to distal edge of
epiphyseal plate
Zone of
Proliferation
• Chondrocytes undergo
division and are organized in
distinct columns (stacks of
poker chips)
• Actively producing matrix
• Chondrocytes and lacunae are
enlarged
• Look for cells of “normal”
size that have increased in
number & appear to stack
Zone of
Hypertrophy
• Matrix begins to mineralize
• Cuts chondrocytes from
nutrients
• Chondrocyte death
Zone of
• Osteoblasts deposit osteoid on
Ossification
exposed cartilage
Zone of Resorption • Nearest diaphysis
• Osteoclasts absorb oldest
bone on spicules
Zone of
Calcification
13
Epiphysis
• Clear cytoplasm from
glycogen accumulation
• Matrix compressed between
columns of large cells
• Huge dying cells
• Empty lacunae
• Lacunae invaded by blood
vessels
• Look for layer of osteoblasts.
• Cells look irregular, warped
• Osteoclasts present
• Look for bone marrow
nearby.
Diaphysis
Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
Intramembranous:
* No cartilage model
1. Mesenchymal cells begin to condense, area becomes vascularized.
2. Mesenchymal cells become larger, rounder. Cytoplasm changes from eosinophilic to
basophilic as cells differentiate into osteoblasts.
3. Osteoblasts secrete collagen and proteoglycans of matrix (osteoid). When surrounded by
matrix, these osteoblasts become osteocytes and maintain bone.
4. Matrix is calcified and forms shape of spicules, which enlarge and interconnect, forming
trabeculae
5. Osteoblasts on surface of spicules reproduce to maintain population capable of growth.
6. Fibrous periosteum surrounds growing bone.
7. As bone continues to grow, it undergoes remodeling via resorption by osteoclasts.
~PREPARATION OF SAMPLES FOR MICROSCOPY~
GROUND BONE (MINERALIZED)
• Dried and finely ground preparations of bone that are not decalcified
• Black and tan
• Allows visualization of Haversian systems (a.k.a. osteons) = structural unit of compact bone
• Consists of Haversian canals: blood vessels, nerves, lymphatics
• Surrounded by concentric lamellae of bone. The outermost rings are oldest
• Lacunae = w/in lamellae; contain osteocytes. Lacunae and osteocytes are interconnected via
thread-like canaliculi that contain the cytoplasmic processes of osteocytes. Allow gap
junction communication between osteocytes, circulation of extracellular fluid, wastes and
metabolites.
• Volkmann’s canals run perpendicular to Haversian canals, passing through the lamellae.
They carry neurovascular bundles from endosteum and periosteum into Haversian canals.
• Interstitial lamellae are remnants of Haversian systems that have been resorbed. Lie
between osteons.
• Cementing lines delimit Haversian systems. Basophilic due to proteoglycans.
DECALCIFIED BONE
• Demineralized with acid and then stained with H&E
• Able to see cells, organic matrix & periosteum
• Periosteum – sheath of dense connective tissue surrounding outer surface of bone containing
osteoprogenitor cells
• Endosteum – lines bone cavities (marrow cavity of compact bone & the marrow spaces
between trabeculae of spongy bone). Contains endosteal cells which can differentiate into
osteoblasts
14
Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
BONE
Question 1: Panel B is a low magnification micrograph of the tissue shown in Panel A. Circle all
that apply to the projected slides.
a.
b.
c.
d.
e.
f.
normally calcified
vascularized
collagen type II
cells capable of division
matrix contains proteoglycans
lacunae present
Questions 2-3: For questions 2 and 3 select the correct combination of terms.
2.
a.
b.
c.
d.
Figure A illustrates_______________and _____________.
membrane bone formation, interstitial growth
formation of Haversian systems, appositional growth
endochondral bone formation, interstitial growth
membrane bone formation, appositional growth
3.
a.
b.
c.
d.
Figure B illustrates_______________and _________________.
intramembranous bone formation, interstitial growth
formation of Haversian systems, appositional growth
endochondral bone formation, interstitial growth
intramembranous bone formation, appositional growth
Questions 4 and 5:
4.
a.
b.
c.
d.
The progenitor that gave rise to the type of cell at the pointer in Figure A was a:
chondroblast.
osteoclast.
osteoblast.
chondrocyte.
5.
a.
b.
c.
d.
The eosinophilic component of the matrix in Figure B provides for:
tensile strength.
diffusion.
interstitial growth.
protein synthesis.
Question 6:
6. Which of the following is NOT true about the cells at the pointer?
a. they are accumulating glycogen
b. they originate from hyaline cartilage
c. the matrix surrounding them is completely mineralized
d. they are actively secreting matrix
15
Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
Bone Answers
Question #1: A. decalcified bone, tibia (high mag), B. decalcified bone (low mag)
a. Yes - the bone shown in slide B is decalcified in this preparation, but would normally be
calcified
b. Yes - bone is made up of Haversian systems through which vessels travel via the Haversian
canals
c. No - bone is made up of type I collagen
d. No - Mature chondrocytes, as seen in this image of calcified bone, cannot proliferate.
e. Yes - the matrix for both hyaline cartilage and bone is made up of proteoglycans
f. Yes - osteocytes are found in lacunae within the lamellae of the Haversian systems
Questions 2-3: Figure A. #93 fetal endochondral bone formation; Figure B #94 parietal
membrane bone with osteoblasts; Figure C same as Figure B but with pointer on osteoclasts.
Question 2: c – The tissue shown in Figure A was endochondral bone formation, which is the
formation of new bone be growth of a cartilage model via interstitial growth. A is wrong
because membrane bone does not form from a cartilage model. B is wrong because although
appositional growth is the way the bone is growing (vs. the cartilage also shown), we are not
seeing the formation of Haversion systems. D is incorrect because, once again, this is
endochondral bone, not membrane bone.
Question 3: d – Figure B shows parietal membrane bone with osteoblasts. Membrane bone
grows without cartilage model, growing via appositional growth. A is wrong because interstitial
growth applies to cartilage only. B is wrong because we are not seeing Haversian channels, and
C is wrong because this is not endochondral bone (since there’s no cartilage model) and thus
there is no interstitial growth.
Questions 4 and 5 Slides: Figure A #9 ground bone, pointer on osteocyte; Figure B #10
decalcified bone
Question 4: c – Figure A shows ground bone, pointer on osteocyte, which started out as an
osteoblast, but as it secreted bone matrix, it became embedded in bone and confined to a lacunae
where it remained as an osteocyte. Chondroblasts and chondrocytes are found in cartilage only,
and osteoclasts break down bone and do not form osteocytes.
Question 5: a – Figure B shows decalcified bone, which appears eosinophilic due to collagen
(matrix is basophilic). Collagen is important for tensile strength of bone. Bone is not capable of
diffusion (except via Haversian systems) nor of interstitial growth. Collagen cannot produce
proteins as it is itself a long polymer of proteins.
Question 6: c – Pointer on zone of hypertrophy within the epiphyseal plate. These
stacked cells are very large and actively secrete unmineralized matrix. Once cells
enter the zone of calcification, the newly-mineralized matrix isolates chondrocytes from their
nutrient supply, leading to cell death and vacant lacunae.
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Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
Cartilage
The Basics:
• specialized connective tissue
• rigid, elastic & resilient – RESISTS COMPRESSION
• AVASCULAR – necessary nutrients diffuse through matrix
The Components:
• perichondrium – dense irregularly arranged connective tissue (type I collagen); eosinophilic
due to positively charged amino acids (hydroxyproline, hydroxylysine) of collagen; ensheaths
cartilage; houses vasculature; home of chondroblast precursors (look like fibroblasts closer to
cartilage)
• chondroblast – progenitor of chondrocyte; secretes type II collagen and other extracellular
matrix components (chondroblasts build); lines the border b/t perichondrium and matrix
• chondrocyte – mature cartilage cell surrounded by matrix; reside in spaces called lacunae;
active chondrocytes secrete collagen, GAG, and proteoglycans
• matrix – provides rigidity, elasticity, and resilience; is composed of fibers (either collagenous
or elastic) and ground substance (rich in glycosaminoglycans [GAGs], especially chondroitin
sulfates)
Note: GAGs are highly negatively charged due to sulfate and carboxyl groups on their sugars;
this negative charge causes matrix to stain basophilic, and attracts water. The water creates a
hydrated gel-like matrix
Methods of Growth:
• appositional – increasing in GIRTH or WIDTH; new cartilage cells, chondroblasts, arise
from inner layer of the surrounding perichondrium and secrete extracellular matrix; forms
new cartilage at surface of preexisting cartilage
• interstitial – increasing LENGTH; forms new cartilage within the cartilage mass;
chondrocytes divide and secrete matrix from within their lacunae; This cartilage cell division
occurs in all types of cartilage and is associated with endochondral bone formation.
17
Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
Types of Cartilage
HYALINE
Hundreds of eyes staring
back at you.
ELASTIC
Similar to hyaline,
but differs in
staining (see below)
Support of tissues &
organs; bone
development
Support with
flexibility
Nasal septum, larynx,
tracheal rings, articular
surfaces of joints, sternal
margins of ribs
External ear,
external auditory
canal, epiglottis,
part of laryngeal
cartilage,
Eustachian tubes
Type II (thin fibrils)
Type II w/ elastic
fibers
APPEARANCE
FUNCTION
LOCATIONS
FIBROCARTILAGE
Layers of collagen
fibers visible;
Chondrocytes
aligned between
collagen fibers
Support with great
tensile strength
(must sustain
pressure & shear)
Intervertebral discs,
pubic symphisis,
articular disks of
sternoclavicular
joint
MATRIX
COLLAGEN
GROUND
SUBSTANCE
STAINING
3 types of GAGSs
• chondroitin sulfate
• keratin sulfate
• hyaluronic acid
(proteoglycan monomer
= GAG + core protein)
contains a lot of water
Lots of Type I
collagen layers
(oriented parallel to
stress plane)
(same as hyaline)
Matrix – basophilic due
Weigert stain –
H&E – type I
to GAGs (neg. charged
elastic fibers stain
collagen layers are
sulfate grps)
black
intense pink;
matrix is
Territorial matrix –
basophilic
surrounds lacunae;
more basophilic due to
Orcein van Giesen
high concentration of
Elastic stain –
proteoglycans secreted
Fibrocartilage is
by chondrocytes
reddish brown
(nucleus pulposus
Chondrocytes – active in
at center); NOTE:
matrix creation; dark
hyaline cartilage
blue nuclei; clear areas
is yellow
b/c Golgi apparatus
and lipid droplets
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Histology SSN – October 11, 2005 – Bone, Cartilage and Joints
Sarah Murray (sgm2106) and Yixing Xu (yx2109)
Bone vs. Cartilage
H&E STAINING PROPERTIES
FUNCTION
NUTRIENTS
GROWTH
CELLS
MATRIX
BONE
Eosinophilic
Rigid structure & support
HC, VC, & canaliculi
Appositional
Osteocytes, -blasts, -clasts
CARTILAGE
Basophilic
Shape, precursor to bone
Diffusion across matrix
Appositional & interstitial
Chondrocytes
GAGs, type II collagen
Mineralized; type I collagen (hyaline & elastic) and type
I collagen (fibrocartilage)
Joints
Types of Joints:
• immovable or slightly movable
o syndesmoses – fibrous joints; bone connected to connective tissue
o synchondroses – cartilaginous joints; bone connected to cartilage
o synostoses – osseous joints; bone connected to bone
• freely movable joints
o synovial (diarthroid) - articulating bones separated by a fluid-filled cavity; articular
surfaces covered by hyaline cartilage
Terms:
• synovial cavity
Fluid-filled space between two bones
• synovial fluid
Comprised of water and GAGs
Maintains articular cartilage (provides nutrients than enter cartilage
through diffusion)
• synovial membrane
Specialized secretory connective tissue
Consists of collagenous fibers and fibroblasts
Fibroblasts secrete synovial fluid
Highly vascular
May be attached to perichondrium at lateral regions of articular cartilage
• synovial villi
Folds of the synovial membrane
Project into the synovial cavity to allow increased secretion/absorption of
Synovial fluid
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