HISTO ● HISTOLOGY SHIFT

M4: CONNECTIVE TISSUES

#1
CHARLES A.C. VILLAMIN, MD, FPOA SEPTEMBER 10, 2021

■ Hematopoietic – myeloid cells

LECTURE OUTLINE
■ Immunologic (defense) – mast cells
I Origin/Function/Properties ■ Tissue repair – myofibroblast
II. Composition
A. Extracellular Matrix
a. Fibers
b. Ground Substance
B. Cell
III. Embryonic Connective Tissue
IV Clinical Significance

I. ORIGIN/ FUNCTION/PROPERTIES

A. ORIGIN
● Connective tissue
○ Basic tissue characterized by an abundance of extracellular
material and paucity of cells

Figure 2. Connective tissue classification

● Connective Tissue Repair

○ Areolar connective tissue
■ Widely distributed; found under skin, around organs,
between muscles
Figure 1. Loose areolar connective tissue (LM)
■ Wraps and cushions organs
The cells and fibers are well spaced in between the ground substance.
○ Adipose (fat) tissue
■ Found under skin, around kidneys and heart
EMBRYOLOGY
■ Functions in energy storage and insulation, cushioning for
○ Developed from the Mesoderm layer of the embryo
organs
○ Mesoderm layer of the embryo can develop into different
○ Dense connective tissue
specialized tissue such as:
■ Found in tendons and ligaments
■ Bone
■ Forms strong bonds that attach bone to muscle or bone to
■ Cartilage
bone
■ Muscle
■ Blood
● Specialized Connective tissue
○ Cartilage
● Mesenchyme
■ Found in the rings of respiratory air tubes, external ear, tip of
○ Lies within the mesoderm
nose
○ Composed of mesenchymal stem cells
■ Provides flexible support cushions
■ loosely organized, undifferentiated stem cells therefore they
○ Bone
are pluripotent, capable of differentiating into the different
■ Found in the skeleton
types of connective tissue
■ Functions in support, protection (by enclosing organs), and
movement
B. FUNCTION ○ Blood
● In general, connective tissues provide: ■ Found within blood vessel
○ Support – general structure ■ Transports nutrients, gases, hormones, wastes; fights
○ Binds & protect tissues and body organs – structural infection
framework, strength
○ Specialized functions such as:
■ Insulation – adipose tissue
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 Figure 4. Collagen fibers viewed by light microscopy
Figure 3. Different cells and fibers of the connective tissue
NOTE: (Left) Hematoxylin and Eosin (H&E) stains the extracellular matrix
● Different types of fibers found in the connective tissue usually RED or PINK; (Right) Trichrome stains the collagen fibers BLUE.
○ Collagen
○ Elastic
○ Reticular Collagen Formation
● Different cells found in the CT (mostly) ● Secreted as tropocollagen monomers into the extracellular matrix
○ Fibroblast by fibroblasts.
○ Adipocytes ● Tropocollagen monomers are called alpha chains.
○ Different blood components ● Polymerize longitudinally and side to side forming collagen fibrils
● Ground substance — space in between that are cross-linked by the enzyme lysyl oxidase.

C. PROPERTIES

Table 1 Properties of Connective Tissues

PROPERTY DESCRIPTION
Tensile Strength ● To resist pulling, stretching, and tearing
● Provided by the Collagen family

Elasticity ● To facilitate return to original shape after

mechanical distortion
● Provided by Elastin fibers (functions
like rubber)

Volume ● Due to glycoproteins and complex

(i.e. bulk/substance) carbohydrates
● Has water-binding ability
● Forms a wet gel called ground
substance
Wheater’s Functional Histology, p.65 Figure 5. Collagen Formation

II. COMPOSITION

A. EXTRACELLULAR MATRIX

1. FIBERS

● Collagen
● Main fiber type found in most supporting tissues.
● Most abundant protein in the body
● Function: Tensile strength
● Stains BLUE in Masson Trichrome stain in light microscopy

Figure 6. Collagen Fibers under Electron Microscope. Fibroblasts in

active state, intimately linked with collagen fibers seen as dots in cross
section and as black lines in the longitudinal section.

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 Table 2. Types of Collagen Fibers
TYPE DESCRIPTION
Type I Main structural collagen found in fibrous supporting
tissue, skin (dermis), tendons, ligaments and bone
Type II Hyaline Cartilage
Type III Reticulin Cartilage
Type IV Meshwork and Basement membrane
Type VII Forms anchoring fibrils, links ECM to basement
membrane
Villamin, Charles M.D., (2021). Connective Tissue.
Connective Tissue Histology [Pre-recorded lecture file]

NOTE: There are at least 28 types of collagen fibers.

Figure 7. Type I Collagen Fibers under Electron Microscope at three

different magnifications. Middle picture contains different collagen fibrils. I. TYPES OF COLLAGEN
Rightmost photo shows the cross-banding of collagen fibrils. ● Type I collagen
○ Loose connective tissue
■ Widely distributed
■ Found in Superficial and deep fascia
■ Few collagen fibers

Figure 8. EM type I collagen cross-banding.

NOTE: Cross-banding is a result of polymerization of tropocollagen Figure 10. Loose areolar connective tissue
molecules in collagen.

Figure 11. Loose Areolar Connective Tissue. The pink bands are the
collagen fibers, the dark bands are the elastic fibers. Collagen fibers are
far apart from each other.

Figure 9. Collagen under Light Microscope. Collagen intimately-linked ○ Dense connective tissue - close packing of collagen fibers
with fibroblasts. a. Dense irregular connective tissue
● Collagen fibers in bundle randomly oriented
● Found in dermis capsule of organs,
periosteum/perichondrium

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Figure 12. Skin. D - Dermis. E - Epidermis. Pink staining fibers are collagen Figure 15. Reticular connective tissue of the liver. Reticular fibers are black
fibers closely packed and randomly oriented. surrounding different cells in the liver.

b. Dense regular connective tissue

● Fibers oriented in one direction and parallel to each
other
● Tendons, ligaments, aponeurosis

Figure 13. Tendon tissue. Collagen fibers Figure 16. Reticular connective tissue of the spleen. Reticular fibers
oriented parallel to each other and are densely packed. staining black.

● Type III Collagen ● Type IV Collagen

○ Reticulin - distinct type of fiber which is part of collagen. ○ Network/mesh-forming collagen
■ Used to be a separate classification ○ Important component of basement membrane
■ Supporting framework of cellular organs (i.e. liver, lymph ○ Basement membrane
nodes, endocrine glands) ■ Sheet-like arrangement
■ Contain more sugar groups ■ Acts as an interface between support tissue and epithelium or
■ High affinity for silver salts parenchymal cells
● Argyrophilic: stains BLACK with silver salts ■ Main components:
● Type IV Collagen
● Heparan sulfate
● Structural glycoproteins - fibronectin, laminins,
nidogen-1
■ Functions:
● Binds epithelium to underlying tissue
● Barrier to epithelial growth
● Manage flow of nutrients and metabolites in and out of
the organs
● Form a selective barrier for the passage of molecules

Figure 14. Reticulin fibers of the liver organ

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 Figure 19. Elastic fibers as seen in distensible organ such as the urinary
Figure 17. Basement membrane bladder

Figure 20. Elastin Formation

NOTE: Similar to collagen, elastin is a protein synthesized by fibroblast

in the form of a precursor monomer, tropoelastin. Tropoelastin is
polymerized in the cellular matrix by the enzyme lysyl oxidase, with
extensive cross-linking of lysine amino acid side chains. These elastic
fibers require glycoprotein, fibrolin, incorporated around and within the
elastic core.

Figure 18. Basement Membrane

● Elastin
○ Arranged as fibers or discontinuous sheets -> provides stretch and
recoil of organs
○ Has less tensile strength than collagen
○ Abundant in structures with frequent stretching (i.e. arteries, lungs,
skin, urinary bladder)
○ Refractile; need special stain to be recognized
■ Usually not seen in light microscopy without use of stain
Figure 21. (Left) Electron microscopy of Elastin; (Right) Elastic Fiber

NOTE: Fibrillin is surrounded by an amorphous elastic core in the EM of

elastin.

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 2. GROUND SUBSTANCE
● Amorphous transparent material
● Semi-solid gel
● Contents:
○ Water and salts (sodium)
○ Glycoproteins
■ Fibrillin: incorporated in elastic fibers
■ Fibronectin: acts as binding site for many CT components
○ Proteoglycans/Glycosaminoglycans (GAGs)
■ Hyaluronic acid: predominant
■ Chondroitin-4-sulphate
■ Chondroitin-6-sulphate
■ Dermatan sulphate
■ Keratan sulphate
■ Heparan sulphate
■ Heparin sulphate

Figure 22. Loose Areolar Connective Tissue.

NOTE: Elastic fibers are dark purple thin bands forming a network in the
connective tissue.

Figure 25. Loose areolar connective tissue

NOTE: Pink fibers are collagen fibers. Purple thin fibers are elastic fibers.
White portion is the ground substance. Dark circles are the fibroblasts.

Figure 23. Arterial Wall

NOTE: Elastic fibers are darkly-stained.

Figure 26. Dense Irregular connective tissue

NOTE: White area is the ground substance. Anything white in the connective
tissue is termed as the ground substance.

Figure 24. Epiglottis B. CELLS

FIXED CELLS
A. Fibroblasts
● Principal cells of the connective tissue
● Most abundant cell in your connective tissue
● Main function: synthesizes proteins, glycoproteins and
glycosaminoglycans

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 ● Other function: to secrete, maintain, and recycle the matrix in
most tissues
● Produces precursor of fibers such as tropocollagen and
tropoelastin
● In tissue repair it can transform into:
○ Myofibroblasts
■ Activated fibroblasts
■ Associated with repair, contractile function
■ Generates repair/healing with scars
Types of Adipocytes:
Figure 27. Fibroblasts
NOTE: Dark stained portion of the fibroblast nucleus in (L/M), seen as
condensed and elongated in the direction of the collagen fibers. The
cytoplasm is barely visible and scanty. The cell is long and thin with fine
cytoplasmic processes extending into the matrix.
Figure 28. Dense irregular connective tissue. Fibroblasts show large active
nuclei and eosinophilic cytoplasm tapering off in both directions along the
axis of the nucleus, which makes it have a “spindle shaped” appearance.
The nuclei are clearly seen, but the cytoplasmic processes resemble the
collagen bundles that fill the ECM.
B. Adipocytes
● Fat cells
● Specialized for fat storage (triglycerides)
● Heat production, storage of fat and thermoregulation
● Distinctly see them as “signet ring”
● Fat droplets are removed in H&E stain
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Figure 28. Adipocytes
● Yellow/White Adipocytes (Unilocular)
○ Distributed throughout the body particularly the deep layers of
the skin
○ Specialized tissue for storage of triglycerides for heat under the
control of nervous system
○ Less dense in light microscopy
○ Triglyceride storage and mobilization
○ Acts as structural fillers especially the pelvis, perirectal area
and axilla
○ Contributes in the sculpting of the body shape and outline
○ Acts as thermal insulator under the skin and forms part of the
shock-absorbing padding (e.g. around the kidneys)
Figure 29. White adipocyte. The cell membrane, a thin rim of peripheral
cytoplasm and the external lamina collectively give a “chicken wire”
appearance.
● Brown Adipocytes (Multilocular)
○ Arranged in lobules separated by fibrous septa
○ Involved in non-shivering thermogenesis (increase in metabolic
activity that produces heat due to cold stress)
○ Nuclei is eccentrically located and enlarged
○ Produce heat under the control of the nervous system
○ Metabolizes triglycerides for heat under the control of the
nervous system
○ More cellular and tightly packed
○ Undergo involution in early infancy
○ Specialized form of adipose tissue most prominent in babies
■ Infants: Between the shoulder blades, surrounding the
kidneys, neck, supraclavicular area and along the spinal
cord
■ Adults: Upper chest and neck especially in the
paravertebral area
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 Figure 30. Where you can find your brown adipocyte

Figure 31. Macrophage in the spleen. Usually seen embedded especially in

the supporting structure of organs easily exposed to pathogens.

Figure 31. Contrast between brown adipose tissue and white adipose tissue

NOTE: Brown adipose tissues are more cellular and tightly packed, while the
white adipose tissues are less dense in light microscopy.
Figure 32. Macrophages in Airway

Figure 32. Brown adipocyte

Figure 33. Macrophages in electron microscope
C. Macrophages
● Function: phagocytose particulate matter (ingest bacteria, dead
and drying cells) NOTE: The nucleus is irregular, lysosomes are also seen which are
● Participate in body’s immune defense responsible for digesting the phagocytized material, a few mitochondria and 3
● Secrete a variety of cytokines to enhance immune response lysosomes. The active macrophages usually have pseudopods.
● Seen embedded in the supporting structures of organs that are
easily exposed in pathogens such as spleen D. Mast Cells
● With granules containing heparin and histamine
● Involved in anaphylactic reactions

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 ● Abundant in the skin, GI lining, serosal lining of the peritoneal
and pleural cavities and around blood vessels B. Eosinophils
● Toluidine blue stain - granules become deep purple ● Main function: Defense against helminth parasites and allergic
response
● Appearance: Eccentric, bilobed nucleus with a thin nuclear
bridge connecting both lobes

Figure 34. Mast cells mixed in connective tissue

Figure 37. Eosinophils

III. EMBRYONIC CONNECTIVE TISSUE

● Composed of mesenchymal stem cells
● Mesodermal in origin in the embryo
● Pluripotent stem cells
○ Differentiate into different specialized cells
● Common functions: synthesis, maintenance and recycling of
extracellular matrix materials

Figure 35. Mast cells in electron microscope. Cell is granular and these
granules contain Heparin and Histamine (responsible for the anaphylactic
reaction)

TRANSIENT CELLS
● Cells that come and go into your connective tissue

A. Plasma Cells
● Infrequent
● Usually seen in areas of inflammation Figure 38. Mesenchymal cell seen in light microscope. Cells are more
● Main function: Produces antibodies spindle in shape, and some are stellate in shape.
● Appearance: “Cartwheel” appearance of nucleus

Figure 36. Plasma Cells

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 Figure 39. Mesenchymal cell and loose connective tissue. Mesenchymal
cells mature into adult CT. The difference is in the shape and amount of
fibers in ground substance. More fibers in between the ground substance in
adult CT.
NOTE: Loose connective tissue is the adult form of mesenchymal cells.

I. MUCOUS CONNECTIVE TISSUE

● Abundance of amorphous and jelly-like ground substance
● Distinctive cells are the primitive fibroblast
○ Stellate or spindle (fusiform) shaped in origin Figure 42. Scurvy - Vitamin C deficiency
● Seen in Wharton’s jelly of the umbilical cord

Figure 43. Different clinical manifestations of Systemic Lupus

Erythematosus (SLE)
Figure 40. Mucous connective tissue (Wharton’s jelly, human umbilical
cord) under high magnification. This shows the typical spindle shaped or
stellate shaped fibroblast in a ground substance.

IV. CLINICAL SIGNIFICANCE

● Scurvy
○ Connective tissue is defectively formed
○ Defective collagen formation from lack of vitamin C which results
in loose teeth, skin hemorrhages and death
● Marfan and Ehlers-Danlos syndrome
○ Genes encoded in connective tissue are mutated
○ Mutation in genes coding for type I and III collagen
● Autoimmune connective tissue disease
○ Immune system attacks the connective tissue
○ Systemic Lupus Erythematosus (SLE), Mixed Connective Tissue
Disease (MCTD)
● Cancer of connective tissue
○ Connective tissue degenerate into malignancies Figure 44. Soft tissue sarcoma or Liposarcoma of the thigh. Tumor is
○ Sarcoma (either soft tissue or bone)
encasing the anterior thigh.

Figure 45. Osteosarcoma of distal femur. Primary bone sarcoma.

’
Figure 41. Marfan syndrome

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 VII. RATIONALIZATION

No. RATIONALIZATION
1 The embryonic connective tissue is mesodermal in origin.
2 Protection of tissues is not a common function of the embryonic
connective tissue but a general function of epithelial tissues.
3. Hyaluronic acid is the predominant GAG in the ground substance
4. Scurvy is a defective collagen formation from lack of vitamin C.

VIII. FREEDOM WALL

Figure 46. Wide Resection - Osteosarcoma Distal Femur.

V. APA REFERENCES
(1) Villamin, C. (2021). Connective Tissues. [Pre-recorded lecture]
(2) Rodak Young, B., O’dowd G., & Woodford P. (2014). Wheater's
Functional Histology: A Text and Colour Atlas. Elsevier: USA.
(3) Batch 2024 Transcript

VI. REVIEW QUESTIONS

No. QUESTION
1 What is the embryonic origin of the connective tissue?
a. Mesenchyme
b. Mesoderm
c. Ectoderm
d. Endoderm
2 Which is not a common function of the embryonic connective
tissue?
a. synthesis
b. maintenance
c. protection of tissues
d. recycling of extracellular matrix materials
3. What is the predominant Glycosaminoglycan (GAG) in ground
substances?
a.Keratan sulphate
b.Hyaluronic acid
c.Chondroitin-4-sulphate
d.Heparin sulphate

4 What disease is caused by a dietary deficiency of vitamin C

(ascorbic acid)?
a. Sarcoma
b. Marfan and Ehlers-Danlos syndrome
c. Systemic Lupus Erythematosus (SLE)
d. Scurvy

SUMMARY

CELLULAR COMPONENTS

FIXED CELLS

Fibroblasts ● Create, maintain and recycle the matrix in

most tissues

Fibroblasts

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 Myofibroblasts ● Activated fibroblasts specialized for tissue
repair with contractile ability
● Generates repair/healing with scar
● Role in secretion of matrix

Myofibroblasts with the presence of eosinophils (Eo)

and Lymphocytes (L)

White or yellow ● Specialized tissue for storage of

adipocyte triglycerides for heat under the control of
the nervous system

Typical appearance of the white (unilocular)

adipocyte

Extra info from book: Adipose tissue is pale staining

because virtually all the cells are occupied by lipids,
which are dissolved out in paraffin-embedded tissue
preparations.

Brown adipocyte ● Specialized form of adipose tissue most

prominent in babies
● Metabolizes triglycerides for heat under
the control of the nervous system

Brown (multilocular) adipocyte

Macrophages ● Tissue resident immune cells with

prominent phagocytic abilities and major
roles in the immune system

Macrophage in the spleen

Mast cells ● Tissue resident immune cells involved in

immediate hypersensitivity and allergy

Mast cells

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 TRANSIENT CELLS

Plasma cells ● Infrequent

● Seen in areas of inflammation
● Produce antibodies
● “Cartwheel” appearance of nucleus

Plasma cells

Eosinophils ● Defense against helminth parasite and

allergic response
● Eccentric, bilobed nucleus with a thin
nuclear bridge connecting both lobes

Eosinophils

FIBERS

Type I Collagen ● Main structural collagen found in fibrous

supporting tissue, skin (dermis), tendons,
ligaments, and aponeurosis
● Loose CT: widely distributed, superficial,
and deep fascia, few collagen fibers
● Dense regular: oriented in one direction,
and parallel to each other
● Dense irregular: in bundle; randomly
oriented, found in dermis capsule of
organs, periosteum/perichondrium

Loose Areolar Connective Tissue

Pink bands = collagen fibers; Dark bands = elastic

fibers. Collagen fibers are far apart from each other

Dense Irregular Connective Tissue

D = Dermis; E = Epidermis

Pink staining fibers are collagen fibers closely

packed and randomly oriented.

Dense Regular Connective Tissue

Collagen fibers oriented parallel to each other and

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 densely packed.

Type II Collagen ● Main structural collagen of hyaline

cartilage

Hyaline Cartilage

NOTE: No images of the examples of this type were

shown in the lecture.

Type III Collagen ● Reticulin fibers

● Used to separate classification
● Supporting framework of cellular organs
(i.e. liver, lymph nodes, endocrine glands)
● Contains more sugar groups
● Argyrophilic

Reticular Connective Tissue in the Liver

Reticular fibers are black surrounding different cells

in the liver.

Type IV Collagen ● Meshwork and basement membrane

Basement membrane

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 Elastin ● Stretch and recoil
● Refractile

Left: E/M of Elastin

Right: Elastic Fiber

CONNECTIVE TISSUE

Embryonic ● Composed of mesenchymal cells

Connective Tissue
● Mesodermal in origin
● Pluripotent stem cells
● Synthesis, maintenance and recycling of
extracellular matrix materials
● Mesenchymal cell matures into adult CT
● Differ in shape, fibers, and ground
substance

Mesenchymal cells seen in a light microscope.

Cells are more spindle-shaped, and some are
stellate-shaped.

Mucous Connective ● Abundance of amorphous jelly-like ground

Tissue substance
● Distinctive cells are primitive fibroblast
(stellate, spindle or fusiform-shaped)
● Seen in Wharton’s jelly of the umbilical
cord

Mucous connective tissue seen in Wharton’s jelly of

the umbilical cord under high magnification

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