Connective Tissues and Blood Dr. Jack L. Haar jlhaar@vcu.edu

Learning objectives

1. Describe the light and electron microscopic appearance of connective tissue cells and explain the function(s) of each.

2. Describe the fiber types in connective tissues and define their unique physcal and chemical properties.

3. Define the components of the extracellular matrix. 4. Describe the classification of connective tissue tissues.

I. Introduction

A. Distribution and role of connective tissues B. Embryonic origins

1. Mesoderm 2. Neural crest

II. Components : Ground substance, Fibers, and Cells bathed in tissue fluid

A. Ground substance

1. Functions a. Forms a gel-like matrix of variable consistency in which cells and fibers are

embedded b. Provides a medium for passage of molecules and cells migrating through the tissue c. Contains adhesive proteins which regulate cell movements

2. Components

a. Glycosaminoglycans (GAGs)

I. Long, unbranched polysaccharides composed of repeating disaccharide units which are usually sulfated

II. Large negative charge of the sugars attracts cations, resulting in a high degree of hydration.The matrix formed ranges from a liquid passageway to a viscous shock absorber.

III. GAGs are generally attached to proteins to form proteoglycans.

IV. Proteoglycan aggregate–Many proteoglycans attached to hyaluronic acid, which is itself a glycosaminoglycan.

b. Adhesive glycoproteins–e.g., fibronectin and laminin

c. Tissue fluid -a filtrate of blood similar to blood plasma. Contains ions, diffusible substances,and a small percentage of plasma proteins. In pathologic conditions the amount of tissue fluid increases resulting in edema.

B. Fibers: Collagen, Elastic, Reticular

1. Collagen fibers a. Tropocollagen

I. Basic collagen molecule consisting of three alpha subunits intertwined in a triple helix; collagen types are distinguished by their subunit composition.

II. Produced by fibroblasts and other matrix-forming cells III. Secreted into the matrix where they spontaneously orient

themselves into fibrils with a 64nm repeating banding pattern

b. Major collagen types I. Type I–fibrils aggregate into fibers and fiber bundles; most

widespread distribution; “interstitial collagen” II. Type II–fibrils do not form fibers; present in hyaline and elastic

cartilage III. Type III–fibrils aggregate into fibers; present surrounding

smooth muscle cells and nerve fibers; forms the stroma of lymphatic tissues and organs

IV. Type IV–chemically unique form of collagen which does not form fibrils; major component of the basal lamina

2. Elastic fibers

a. Composed primarily of elastin; produced by fibroblasts b. Elastin forms the central amorphous core of the fiber which is surrounded

by microfibrils. c. Unique chemical properties of elastin provide for elasticity. d. Elastic fibers occur in nearly all connective tissues in varying amounts and

are intermixed with collagen fibers. When present exclusively, they constitute elastic connective tissue.

e. Frequently difficult to differentiate from collagen with conventional stains.

3. Reticular fibers a. Collagen Type III fibers b. Highly glycosylated and stain with silver (argyrophilic) c. When they constitute the major fiber fiber type, e.g., in the stroma of

lymphoid organs, they constitute reticular connective tissue.

C. Connective Tissue Cells Some c.t. cells are long term residents and are responsible for the formation and maintenance of the tissue. Others are motile and migrate from blood into the c.t. where they are responsible for protecting the organism

1. Fibroblasts

a. Active fibroblast

1) Nucleus is oval to spindle-shaped, very euchromatic, usually exhibiting one or two prominent nucleoli. Largest of c.t. cells 2) Cytoplasm usually is not discernible in L/M preparation. At the E/M level abundant rER and prominent Golgi complex are features. 3) Functions to synthesize and secrete all types of c.t. fiber and ground substance.

b. Inactive fibroblast (fibrocyte)

1) Nucleus is very spindle-shaped, quite heterochromatic. 2) Cytoplasm is not apparent with L/M; at E/M level is thin and attenuated. 3) Metabolic level is very low.

2. Monocytes

a. Large, spheroid cell 12-20 �m in diameter, abundant basophilic cytoplasm, indented nucleus. Golgi complex and centrioles lie in nuclear indention. b. Monocytes are progenitors of macrophages.

3. Macrophages

a. Function to ingest foreign matter (phagocytosis) and process antigen to stimulate cells of the immune system. b. Nucleus is smaller and more condensed than an active fibroblast nucleus. It may have the shape of a football or be indented similar to that of a monocyte. Chromatin is slightly condensed peripherally. c. Cytoplasm is difficult to discern at L/M level unless it contains phagocytosed material. Lysosomes and various types of phagosomes are evident at the E/M level.

4. Lymphocytes

a. Small cells 6 to 7 �m in diameter with dense spheroidal, heterochromatic nucleus. Nucleus may have a slight indentation occupied by the Golgi complex and centrioles b. The front line for the immune system. Can be divided into two groups, T- lymphocytes and B-lymphocytes.

c. B-lymphocytes are the progenitors of plasma cells

5. Plasma cells

a. Ovoid cells with well-defined basophilic cytoplasm which at the E/M level is seen to be occupied by abundant rER. b. A well-developed Golgi complex and the centrioles are located adjacent to the nucleus. c. Nucleus is eccentrically located with abundant, clumped, peripherally located heterochromatin resulting in a clock-face or spoke-wheel appearance.

6. Mast cell

a. Spheroid or oval cells having a well-defined cytoplasm packed with dense granules. b. Granules contain glycosaminoglycans, histamine, neutral proteases; they are metachromatic. c. The nucleus is small and centrally located. It may contain peripheral heterochromatin. d. Two populations of mast cells have been identified; connective tissue mast cells and mucosal mast cells. e. Connective tissue mast cells contain heparin and mucosal mast cells contain chondroitin sulfate.

7. Neutrophils

a. Distinguished by having a nucleus with 3 to 5 lobes. b. Possess two types of granules, one a lysosome containing an enzyme, the other containing a non-enzyme protein. c. Neutrophils are attracted to sites of infection. They are very phagocytic, attacking and digesting bacteria and are responsible for the characteristic signs of inflammation, viz. dolor, calor, tumor, rubor.

8. Eosinophils

a. Possess a bi-lobed nucleus, thus when sectioned for microscopic examination they may appear to have two small nuclei. b. Cytoplasm contains large distinct bright red granules in H&E preparations. c. Granules are lysosomes, but cells are not as phagocytic as neutrophils.

9. Adipocyte (fat cell)

a. This is the dominant cell type in adipose or fat tissue. b. In mature cells the cytoplasm and its organelles are restricted to a thin rim surrounding a large lipid droplet. c. The nucleus is a thin crescent structure pushed to one side by the lipid mass and may or may not be in the plane of section examined. d. These cells synthesize and store lipid for nutrition and insulation. e. May appear as small cell clusters in loose c.t. or in large accumulations at other sites. f. Yellow fat is the predominant form and has a single large lipid droplet (unilocular) . g. Brown fat, found in infants and hibernating animals, contains cells having multiple small droplets (multilocular) with numerous brown mitochondria and a central nucleus. It is thought to assist in thermoregulation.

D. Blood Cells

1. Erythrocytes (red blood cells)

a. Biconcave shape measuring 7.5 �m in diameter

b. Filled with hemoglobin for the efficient transport of oxygen

c. Actin-containing cytoskeleton which maintains cell shape

d. No cell organelles

e. 4.0 – 7.0 X 106 per �L of peripheral blood

2. Leukocytes (white blood cells) 6,000-10,000 /�Lof peripheral blood

a. Neutrophils (as described above) 40-75% of WBC’s in peripheral blood

b. Eosinophils (as described above) 5% of WBC’s in peripheral blood

c. Lymphocytes (as described above) 20-50% of WBC’s in peripheral blood

d. Monocytes (as described above) 1-5% of WBC’s in peripheral blood

e. Basophils 0.5% of WBC’s in peripheral blood. The least common wbc in blood measuring 12-15 �m in diameter. Possess large basophilic cytoplasmic granules containing heparin and histamine. Lobulated nucleus is usually obscured by granules. May assist mast cells in hypersensitivity reactions.

3. Platelets

a. 2-4 X 105 per �L of peripheral blood; 2-4 �m in diameter

b. Nonnucleated, disk-like cell fragments

c. Promote blood clotting

d. Formed by megakaryocytes in the bone marrow

Megakaryocytes are giant cells measuring 35-150 �m in diameter with an irregularly lobulated nucleus. They are found in the hematopoietic compartments of the bone marrow.

E. Hematopoiesis

1. Phases a. Mesoblastic – mesenchymal cells in the yolk sac and body stalk form

nucleated blood cells. Third week of gestation in humans b. Hepatic - blood cells form in liver, thymus, spleen between 4 – 8 weeks

of gestation

c. Myeloid – blood cells form in bone marrow beginning at 12 weeks.

2. Pluripotential hematopoietic stem cells

a. Arise from a single type of stem cell

b. Proliferate and form either lymphoid multipotential cells or myeloid multipotential cells.

3. Storma

a. A meshwork of reticular fibers

b. Rich vascular supply with multipotential cells

4. Production/Differentiation of blood elements – Terminology

a. Lymphopoiesis – lymphocytes

b. Erythropoiesis – erythrocytes

c. Thrombopoiesis – platelet formation from megakaryocytes

d. Monopoiesis – monocytes

e. Granulopoiesis – granulocytes

F. Theory of hematopoiesis

1. Pluipotent cells gives rise to lymphoid multipotent cells and myeloid multipotent

cells 2. Lymphoid multipotent cells form T and B lymphocytes

3. Myeloid multipotent cells form erythrocytes, megakaryocytes, monocytes, neutrophils,

eosinophils and basophils

III. Connective tissues can be divided into three categories

A. C.T. Proper

B. C.T .with special properties

C. Supporting C.T.

IV. Features considered when classifying connective tissues

A. Number and types of cells present

B. Number and type of fibers and/or ground substance

C. Arrangement of fiber V. C.T. Proper

A. This includes what one generally thinks of as connective tissue, i.e. the tissue holding

organs together and providing a passageway through which nerves and blood vessels travel.

B. Loose or Areolar fibroelastic C.T.

1. Contains a large number of cells and a large variety of cell types which may include, fibroblasts, macrophages, neutrophils, lymphocytes, plasma cells, eosinophils, mast cells, and adipocytes.

2. The majority of the fibers are collagen although some elastic and reticular fibers are present. 3. The collagen fibers are irregularly arranged and in small accumulations which never measure more than 7 �m diameter.

4. Ground substance is gelatinous in character. 5. Distribution – surrounds muscles and supports the epithelial tissue of all organs.

6. Lamina propria is the specific term given the loose connective tissue which supports an epithelium.

C. Dense Irregular C.T .

1. Composed primarily of bundles of collagen which measure at least 7 �m in diameter. Bundles are randomly arranged, having no particular orientation.

2. Some elastic fibers are present but few reticular fibers are present.

3. Since larger amounts of fiber are present the number of cells is fewer, although all cell types may be represented.

4. Ground substance is similar to that found in loose FECT but also is present in smaller amounts since fibers occupy a larger proportion of the space.

5. Distribution -dermis of the skin

D. Dense Regular C.T .

1. Composed of collagen bundles all arranged in parallel orientation. Few elastic or reticular fibers. 2. Few cells present and all are of one type, fibroblasts.

3. Ground substance is in short supply since most area is occupied by fibers.

4. This tissue is not very vascular

5. Distribution -tendons

IV .Connective tissue with special properties

A. Elastic C.T .

1. The primary fiber type is elastic, and these fibers are arranged into sheets. 2. The sheets have holes or fenestrations and the sheets are loosely held together by collagen fibers 3. Fibroblasts and smooth muscle cells are able to produce elastic fibers and sheets. 4. Distribution -form the walls of large arteries

B. Reticular C.T .

1. Composed primarily of collagen type III fibers which collectively form a mesh or network scaffolding. 2. The cell type present is a special population of fibroblast, a reticular cell. The reticular cells wrap cytoplasmic processes around the reticular fibers.

3. Distribution -form the framework of the blood cell forming area of bone marrow, and the framework of lymphoid organs, viz. lymphoid nodules, lymph nodes, and spleen.

C. Adipose C.T .

1. White adipose C.T .

a. Predominant cell type is white adipocytes which store fat as triglycerides b. Collagenous fibrillar septa divide the tissue into lobules. c. Reticular fibers form the supporting framework d. Distribution -throughout the body except for eyelids, penis, scrotum, and auricle of external ear. The amount of adipose deposited is determined by caloric intake, age, and sex.

2. Brown adipose C.T .

a. C.T. septa are sparse compared to white adipose C. T. A rich blood supply permeates the tissue. b. Brown multilocular fat cells predominate. c. Distribution -found in newborns in neck region and lower abdominal wall.

D. Mucous (Mucoid) C. T .

1. Cells are a primitive fibroblast

2. Abundant amorphous ground substance composed of hyaluronic acid. 3. Contains some collagen and a few elastic and reticular fibers. 4. Distribution -forms the core of the umbilical cord where it is called Wharton's jelly.

E. Blood – This tissue contains all the components of C. T., viz. cells, fibers (which are potential and form when blood clots), and ground substance (serum).

V. Supporting Connective Tissues -Cartilage and Bone These two important connective tissues will be covered in separate lectures, but are mentioned at this point to emphasize the fact that they are truly connective tissues.

Connective tissue proper laboratory

You are responsible for identifying the following structures in the images in the “Digital Histology CD package. While additional features may be mentioned in the legend or indicated on the images, you are not required to identify them at this time.

Connective tissue types: Loose or areolar connective tissue Dense, irregular connective tissue Dense, regular connective tissue Adipose connective tissue Reticular connective tissue Connective tissue fiber types: Collagen fibers/bundles Collagen fibrils Elastic fibers Reticular fibers Cell types: Active fibroblast with euchromatic nucleus Adipocyte Eosinophil Inactive fibroblast with heterochromatic nucleus Lymphocyte Macrophage Mast cell Neutrophil Plasma cell with negative Golgi

VIRTUAL SLIDE LINKS

Loose and dense irregular connective tissue: Thick SkinDense irregular connective tissue: TestisAdipose connective tissue: Small Artery and Vein