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PCB 4023 – Cell Biology
Lab 3: Connective Tissue - Part I
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Name: ____________________
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Preparation assignment (to be completed before lab (1/25/02):
1)
Kerr (1999) Chapter 4. Connective or Supporting Tissue (pp. 59-80).
Web resources:
N.B.
Again, you may wish to examine the modules pertaining to connective tissue at the
University of Florida College of Medicine histology tutorial. For connective tissue, there
are two review modules and one on-line quiz. As before, these modules may contain
information that is not covered in this course and you will not be held responsible for any
information presented there. However, you may wish to consider using them as
additional preparation and/or review. The URL is as follows:
http://www.medinfo.ufl.edu/year1/histo/index.html
Know of any other web sites pertaining to connective tissue that you have found helpful
or interesting? E-mail me the link at [email protected]. and let me know what and why
you found it informative and/or interesting.
I.
INTRODUCTION
This is the first of two labs devoted to connective tissue. In Part I you will examine the
general connective tissues (mesenchyme, adipose, areolar, dense) and one of the special
connective tissues, cartilage. In Part II, you will examine the remaining special connective
tissues (bone, blood and hemopoeitic tissue).
As before, the laboratory assignment will consist of microscopic observation of slides
containing sections or smears of cells, tissues, or organs. The laboratory directions include the
number of the slides in your collection that you are to observe. The slides have cells and tissue
types which correspond to many of the photographs in your texts. The text may help you locate
and identify particular structures; it also describes and correlates the cells and tissues seen under
the microscope.
II.
CONNECTIVE TISSUE
You will recall that there are four basic types of tissue and vast majority of cell types can
be classified as one of these four.
1)
2)
epithelia - continuous layers of cells with littler intercellular space which line surfaces
and form glands
connective tissue - cells embedded in intercellular substances (extra-cellular matrix);
the matrix is typically abundant and the cells less so.
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3)
4)
muscles - specialized contractile cells with electrically excitable membranes
nervous tissue - (1) cells specialized for conducting and transmitting electrochemically
mediated information (i.e., neurons) and (2) the cells which support those engaging in
this activity (e.g., neuroglia or glia ).
Connective tissues are defined as tissues primarily composed of intercellular material (or
extracellular matrix; ECM) with varying densities of cells. As such, you can think of them as the
antithesis of epithelia (all cells, no matrix). The intercellular matrix (or ECM) typically consists of
(2) fibers (either collagen or elastin) and/or (2) a relatively amorphous “ground substance” (it’s
molecular composition is better described in your text). The functional properties of the tissue
are determined primarily by the composition of the matrix, rather than the cells themselves.
Connective tissues in the body are derived from mesoderm whereas in the head it is derived from
mesoderm and neural crest (ectomesenchyme; ask Dr. Kos about neural crest).
Connective tissues are extremely variable and thus serve a variety of functions, these
include:
1) structural - posses special mechanical properties; e.g., bone, cartilage, tendons
etc.
2) transport - fluids such as blood and lymph
3) energy reserves - fat (adipocytes)
4) defensive (immunological) - cell based (e.g., mast cells, platelets, leukocytes and
their derivatives) immune responses
As your text (Kerr, 1999) point out, there are a plethora of classification schemes for
connective tissues, each with its strengths and weaknesses. For the purposes of this lab, we will
employ the rather general classification shown in the accompanying figure. Connective tissues
can be divided into two broad groups, (1) general and (2) special.
figure: 3K5-219.tif
General connective tissues are dispersed widely throughout the body. Their
predominant cell type is the fibroblast and the matrix is generally a network of protein fibers
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(collagen or elastin) in a ground substance of polysaccharide gel. If the fibers are relatively
sparse, they are considered loose connective tissues (mesenchyme, adipose, alveolar). If the
fibers are densely packed, they are considered fibrous (or dense) and further subdivided as to
regular (fibers exhibit uniform orientation) or irregular (fibers exhibit irregular orientation).
The remaining connective tissues are classified as special. Two, cartilage and bone,
form the skeleton and provide a rigid framework to which all other tissues/organs attach. Blood is
considered a fluid connective tissue. The blood stem cells, hemopoietic tissue, is the fourth
special connective tissue. Bone and blood will be examined next week (Connective Tissue - Part
II) and even board-certified hematologists cannot agree on the identification of these cells, so we
will avoid the topic.
Cartilage is a firm but flexible skeletal tissue. The matrix consists of gel-like groups
substance (proteoglycans) and varying amounts and types of protein fibrils. Two types of protein
fibrils are found in cartilage, collagen and elastin. Collage in much more common and its fibers
are relatively flexible but with limited reversible extensibility. Elastin fibers are less widely
distributed but stretch easily with almost perfect recoil. These are found in the ligaments of the
vertebral column and in elastic cartilage. The proteoglycans are carbohydrate polymers linked to
a core protein molecule which bind water. These form hydrated gels which allows for the rapid
diffusion of water soluble molecules and cells. The latter is important because cartilage is an
avascular tissue.
Cartilage is produced by chondrocytes. Within the cartilage, the chondrocytes are
housed in small cavities called lacunae (L., dim. of lacus (lake, hollow)). Surrounding the
cartilage is the perichondrium, a fibrocellular layer containing the proliferative cartilage stem
cells..
The physical properties of cartilage are determined by the ground substance and by the
type and abundance of the protein fibers. Cartilage is both aneural and avascular, the nutrients
gases and waster diffusing through the ECM to provide the chondrocytes. Cartilage can calcify
(become embedded with calcium salts) but its minerals are not as highly ordered as in bone.
figure: 3K5-20.tif
Three types of cartilage can be differentiated on the basis of their type and abundance of
protein fibers: hyaline, fibro- and elastic cartilage. Hyaline (Gr. hyalos, glass) is the most
common. It consists of sparse collagen fibrils in a “glassy” matrix. It is firm with slight elasticity
and can be found in the costal cartilages, bronchi, trachea and articular cartilages. It also forms
the cartilaginous precursor to endochondral bones.
Fibrocartilage has abundant, densely interwoven collage fibrils with correspondingly less
ground substance. It is found in areas subject to compressive loading, e.g., all symphyses
including intervertebral discs (between vertebral bodies), articular menisci (discs), and sutures
Elastic cartilage contains numerous elastin fibers. It is often associated with vibrational
functions (sound wave production and collection) and thus can in the pinna (external ear; sound
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collection) and the smaller laryngeal cartilages (sound production). It is also the most flexible
cartilage and thus is also found in the nasal septum and epiglottis.
Lab Assignment: Connective Tissue – Part I
Work through the following sections using your atlas as a guide. Make sure to answer
the questions (marked by “?”) at the end of the lab; these will be evaluated when you turn in your
handout next week. A list of structures which will form the basis for next week’s quiz is given at
the end of the handout.
To learn how to identify the structures, write down criteria which will assist you in your
identification (e.g., simple squamous epithelium: single layer, flat cells with flattened nuclei).
Your text is a good source for such material as well as your own observations. Some students
find it helpful to make rough sketches of the structure to assist in their learning.
N.B. Due to [unprogrammed] slide death, your slide box may not contain the required
slide. If this is the case, notify an instructor and they will provide a replacement or suggest an
alternative. If you end up borrowing a slide from one of your colleagues’, please don’t forget to
return it to them.
I.
Mesenchyrne
slides:
EE 4-2
92W9029
93W5569
chick embryo, 24 hours, transverse section (best; extras available)
chick embryo, 24 hours, sagittal section
embryo, mammal; faded
Mesenchyme is any non-epithelial, undifferentiated embryonic tissue whose cells are
capable of migration. Its inclusion in a classification based on features found in differentiated
cells has always been nonsensical but, for the sake of tradition, it is included here.
Examine the mesodermal layer. The mesenchymal cells have fine processes which form
a network in the pale, amorphous intercellular substance. This material supports the tissues and
organs in the embryo and later transforms into connective tissue (but also muscle and nervous
tissue. Scream!).
II.
Loose Areolar Connective Tissue
(demo?)
HK 7-24
93W4576
93W4542
93W3224
small intestine
duodenum
colon
areolar tissue; probably orcein stained mesentery smear; DEMO slide
This tissue with its relatively sparse fibers (areolar is the Latin diminutive of space, thus
little spaces occur between the fibers) is easily observed supporting the epithelial lining of hollow
organs and glands, e.g. in the submucosa of the gut. If the section is stained with orcein, then it
will be possible to distinguish the collage and elastin fibers. The major cell type is the fibroblast
which has a relatively pale and elongate nucleus. Other cells present will include macrophages,
mast cells, leukocytes, and fat cells. These will be easier to identify after next week’s lab on
blood.
III.
Adipose
small intestine: HK 7-24; look superficial to capsule but deep to mesothelium
skin: HI 1-3
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Fat cells are found in aggregates in areolar tissue. In routine tissue fixation the fat is
dissolved out, leaving empty spaces. [The fat may be preserved by fixation with osmic acid and
then stains black.] The fat cell is often referred to as a signet cell because of its thin rim of
cytoplasm and thickened region containing the nucleus.
IV.
Reticular Tissue
lymph gland: H1930; H&E, not widely available
liver: H3230 or HK 10-2 or 934567
thymus: HH 1-5, or 93W4123 or H9160
Reticular fibers are subtype of collage (type III; nauseating details in your text) and form
the supporting network for a variety of organs (lymph nodes, liver, thymus). Other than size
(reticular fibers are thinner) it is difficult to distinguish reticular tissue from alveolar tissue without
special staining.
V.
Dense Connective Tissue
Regular:
tendon: H6275; not widely available (DEMO)
ligamentum nuchae: 93W3256 – orcein stained; demo?
tail: H 800 (extras available) or 93W3321
Irregular:
[mammalian] skin: HI 1-3 or H2081
In dense connective tissue, the fibers are densely packed and thus lack the spaces
characteristic of loose or areolar. Due to tissue preparation artifacts, however, there are spaces
in dense collagenous tissue but in comparison to areolar, they are significantly decreased.
Dense connective tissue is subdivided into regular (fibrils have uniform orientation) and irregular
(fibrils have varying orientation). A piece of tendon is a good example of regular dense
connective tissue. The fibers, in longitudinal sections, are extremely straight with thin, dark,
elongated fibroblast nuclei lined up in parallel rows between the fibers. In addition to tendons this
tissue is the major component of ligaments, aponeuroses and some fascia. Some ligaments
contain elastin fibers which can be visualized if stained with orcein.
A good example of irregular dense is the dermis of terrestrial vertebrates. It differs only
from regular dense connective tissue only in the varying orientation of the fibrils. Interestingly,
the dermis of most aquatic vertebrates is regular. Ask why.
VI.
Dense Elastic Tissue
aorta: H1725 (extras available), Verhoeff stain
This tissue is found primarily in large arteries, as in the aorta. The elastic tissue forms a sheet or
membrane in concentric rings within the vessel wall and helps the vessel maintain its shape
under conditions of high arterial pressure.
VII.
Hyaline cartilage
trachea: HJ 2-1 (not widely available)
endochondral bone formation : HB 8-1, use portion distant from ossification
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Notice the nest of large cells (chondrocytes) lying within hollow lacunae surrounded by a
glassy matrix. Near the cells the matrix is often more basophilic Observe that along the edge of
the cartilage is the pink, dense collagenous perichondrium, with parallel rows of fibroblasts.
VII.
Elastic cartilage
elastic cartilage (probably pinna): H6026 (best), H 690 (OK; extras available)
epiglottis: 93W3275
The dark stained fibers coursing throughout the cartilage and forming heavy networks of
rings around the lacunae are elastic fibers. The chondrocytes and matrix are otherwise similar to
hyaline cartilage.
VIII.
Fibro-cartilage
Fibrocartilage: H710 (DEMO)
Fibrocartilage: H 2.65; bad example, looks like immature IV disk
Fibro-cartilage is composed of collagen fibers in a cartilage matrix. The chondrocytes
are scattered and small but do lie within lacunae surrounded by matrix. The fibers are distinct
and the tissue is a modified fibrous connective tissue.
IX.
Questions
As before, time has run out so you will be supplied with some questions and a list of
structures for next week’s quiz in lab tomorrow.
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