CONNECTIVE TISSUE PROPER: CELLS AND FIBERS

Readings: Chapter 4, In: Atlas of Functional Histology by Kerr; Chapter 5, Bloom and Fawcett: A Textbook of Histology,12th Ed.
 

OBJECTIVES
1. Learn to recognize microscopically or from written or verbal descriptions, the cells and fibers of connective tissues.
2. Be able to describe the interrelationships among the components of the connective tissue.
3. Be able to differentiate among connective tissue classes and cite examples of their locations in the body.

I. INTRODUCTION

A. Types of Connective tissues:

1. Connective tissue proper

Supporting C.T.

2. Cartilage (specialized)    

3. Bone (specialized)

4. Blood (specialized,.)    

Atypical C.T


Common characteristics:

1. Composition:

a). Cells:

1). Several cell types;
2). Few cells per unit volume of tissue (i.e. compared to epithelial tissues).

b). Intercellular matrix:

1). Fibers (constitute the formed elements)
2). Ground Substance (on amorphous, semifluid substance)

2. Functions:

a). Binding and packing of tissues (connective tissue proper);
b). Connect, anchor and support the body and its organs;
c). Transport of metabolites between capillaries and tissues;
d). Defense against infection (via ground substance and cells);
e). Repair of injury (via cell proliferation and fiber formation);
f). Fat storage (as determined by age, sex, nutrition or disease)

 

II. CELLS OF THE CONNECTIVE TISSUE PROPER

SOURCE OF
CELLS

CELL TYPES

MOTI-
LITY

NUCLEUS

CYTOPLASM

FUNCTION

Mesenchymal cell
 of the embryo or those persisting in the adult.

Mesenchymal cell (persists along capillaries)

Fixed

Large, round,euchromatic (pale staining)

Stellate, scanty, little differentiation of cytoplasm

Functions for all three cell types:
In embryo:
Loose packing between organs, Secrete ground substance;

Mesenchyme is pluripotent: gives rise to CT cells, bone, cartilage, blood, endothelium, mesothelium.

In adult:
during wound healing; differentiates into other cell types;

Produce:
   1. Tropocollagen for collagen and reticular fibers;
   2. Tropoelastin and microfibrils for elastic fiber formation;
   3. Ground substance containing glycosaminoglycans;

Fat cells - function mainly in lipid storage (lipid droplet=mostly triglicerides). (Insulin to glucose to triglicerides)

Fibroblast (deployed along collagen fibers)
Fixed

active cells: euchromatic;
 inactive cells: heterochromatic; 1-2 nucleoli;

Fusiform or stellate (difficult to distinguish) Have abundant granular E.R.

Fat cell (singly or in clusters in loose CT, along small blood vessels, and in adipose tissue)
Fixed

Flattened, peripheral, deeply staining

 Thin, surrounds a single lipid droplet. "Signetring shaped" in appearance.
Bone marrow precursor

 Mast cells
(adjacent to blood vessels)

Size: 20-30µm diam.

Motile
(amoeboid)

Round, relatively small, usually central, with clumped chromatin

 Round or ovoid, filled with large granules;

Granules are:

1.Metachromatic with basic aniline dyes (i.e., methylene blue);
2. Water soluble;

Produce:
1. Histamine;
 2. Slow-reacting substance of anaphylaxis (SRS-A);
 3. Eosinophil chemotactic factor (ECF);
 4. Heparin (anticoagulant);

Mechanism of action:
1. Sensitization with antigen (Ag);
2. Antibody produced: IgE
3. Mast cell + [Ag-IgE] = Mast cell-[Ag-IgE] complex; Next:
4. Degranulation, release of histamine, SRS-A, ECF; Next:
5. Smooth muscle contraction: BP^, constriction of air ways, constriction of intestines;
  Accumulation of eosinophils (in allergic reactions) to endocytose Ag-Ab complexes (dampen rxn).

Basophils
(at sites of inflam­mation)

Size: 9-12µm diam.

Motile
(amoeboid)

Elongate, bent in a U or S shape, with 2 or more constrictions;

Stains paler than in eosinophils or neutrophils

 Round; Filled with coarse granules that stain metachromatic, are water soluble, difficult to see in routine preparations;

 

 

 

II. CELLS OF THE CONNECTIVE TISSUE PROPER (Continued)

SOURCE OF CELLS CELL TYPES MOTILITY

NUCLEUS

CYTOPLASM

FUNCTION
Eosinophils of blood

Eosinophils

[in CT under "wet epithelia" (mucosae), i.e., lung, intestine, mammary gland, omentum];
Size: 9-12µm

Motile
(amoeboid)

Bilobed in man,
(annular in rodents;)
Chromatin: heterochromatic;

Round (spherical) with acidophilic (eosinophi-lic) granules;

Granules contain a crystalloid (seen with E.M. only); Diagnostic;

Participate in allergic reactions;

1. Granules contain: hydrolytic enzymes;

2. Do not ingest bacteria;

3. Dampen allergic reaction by phagocytizing Ag-Ab compl.

Neutrophils of blood

Neutrophils

(at sites of injury and inflammations)

Size: 10-12µm diam.

Motile
(amoeboid)

2-5 interconnected lobes (segments);

Polymorphonuclear leukocyte ("polys");

The number of lobes increases with age of the cell;

Round, contains granules:

1. Numerous small  specific granules;

2. Few larger azuro philic granules --(lysosomes);

Avidly phagocytize:
  1. bacteria;
  2. particular matter (cell debris);

Produce:
 1. Specific granules with bacteriocidal proteins (phagocytins);

 2. Azurophilic granules are:
a. lysosomes with hydrolytic enzymes;
b. myeloperoxidase (combines with H2O2> O2 ^ kills bact.
Dead neutrophils = PUS
Bone marrow

Monocytes

(emigrate from blood during inflammations to CT
Size:12-17µm.

Motile
(amoeboid)

Eccentric,
oval or kidney shaped,

Heterochromat­ic,
1-2 nucleoli

Round to oval, contains:

 1. Scattered small
azurophilic granules         (primary lysosomes);
 2. Some vacuoles;

Becomes phagocytic in the connective tissue;

Differentiates into tissue macrophages;
Monocytes in blood

Macrophages

Histiocytes (outdated term);

Found along bundles of fibers;
Size: 20-80µm

Fixed or Motile
(amoeboid)

Smaller than in fibroblasts, heterochromatin stains darker than in fibroblast;

Outlines nucleus;

Variously shaped;

Contains:
1. Azurophilic granules    (lysosomes);
2. Vacuoles;
3. Phagosomes;

1. Phagocytosis of foreign materials and cell debris;

2. Integral part of the immunological defense (acts as antigen presenting cell); Important in local defense against bacterial invasion;

 

 

 

 

 

 

II. CELLS OF THE CONNECTIVE TISSUE PROPER (Continued)

SOURCE OF CELLS CELL TYPES MOTILITY NUCLEUS CYTOPLASM FUNCTION
Lymphocytes in blood Lymphocytes
(In small numbers in CT, but increase mark­edly during inflamma­tions)
Size: 7-8µm diam.		 Motile
(amoeboid)		 Round or slightly indented;
Densely heterochromatic;		 Round,
Scanty, thin basophilic rim around nucleus in small lymphocytes but more abundant in medium and large lymphocytes;		 Participate in immune responses;
1. In cell-mediated immune responses:
   a. Long lived (5-20 yrs);
   b. Cytotoxic lymphocytes;
2. In humoral immune responses:
   a. Short-lived (less than a week);
   b. Differentiate into Ab forming plasma cells.
Lymphocytes in CT. Plasma cells
Uncommon in CT; abundant in:
1. Lymphoid tissues
2. Sub-epithelial CT of:
gastrointestinal       and respiratory       tracts and mammary glands;
Size: variable
7-20µm in diam.		 Motile
(amoeboid)		 Eccentric, round;
Peripherally distributed
heterochromatin resemble: a "spoked wheel" or a "clock-face"; 		Ovoid,
basophilic,
(extensive rough endoplasmic reticulum);
Negative Golgi
(adjacent to nucleus);		 Produce antibodies
(Ab is localized in the endoplasmic cisternae);
Functional in the humoral immune response;

 

 

 

 

 

 

III. The Intercellular Matrix of theConnective Tissue Proper  
  A. Ground Substance (amorphous intercellular gel)  
   

1. Physical properties:

  
   

a. Viscous, varying from semifluid to jelly-like consistency;

b. Fills the space between cells and fibers (lubricates and cushions to
diminish friction and wear);

c. Water soluble ( difficult to demonstrate histologically gets leached out in preparation);

   

 

2. Chemical composition: Proteoglycans (a protein core with polysaccharide side chains called glycosaminoglycans; Old term: mucopolysaccharides); Protein and glycosaminoglycan composition is varied in each CT type. There are two main clas-
ses of glycosaminoglycans:

 

a. Non-sulfated glycosaminoglycans

1.  Hyaluronic acid;

2.  Aggregates proteoglycan sub­units
oo(see figure on left);

3. The aggregates form viscous barriers
oo& prevent spread of microbes;
 
b. Sulfated glycosaminoglycans

1. Chondroitin sulfate - primarily in
oocartillage;

2. Others: Keratan (in cornea),
ooooooooDermatan (in dermis);

3. Very hydrated; facilitate diffusion of
oometabolites;


B. CONNECTIVE TISSUE FIBERS (Formed, non-living elements):

COLLAGEN FIBERS

RETICULAR FIBERS

ELASTIC FIBERS

Physical characteristics: 

1. Subunits: fibrils= 1000-5000A diam.

2. Form fiber bundles (may branch)
   bound together by matrix
= 1-10 µm diam.

3. Flexible -- not elastic;  tensile srength;

4. Stain pink with H&E

Physical characteristics:

1. Subunits: fibrils= 1000-5000A diam.

2. Form network of branching fibers
   bound together by matrix
   = 0.2-1 µm diam.

3. Flexible -- not elastic;

4. Argyrophilic staining (affinity for reduced silver ions); also stain with PAS;

Physical characteristics:

1. Subunits: microfibrils= 110A diam.

2. Form branching fibers, = 0.5-1µm diam. bound together by elastin (a protein); or sheets bound together by elastin;

3. Highly elastic -- can stretch 150% of its length;

4. Refractile in light microscope, stains poorly with H&E, well stained with  special stains.

Chemical composition of both, COLLAGEN and RETICULAR fibers:
 Chemical composition of ELASTIC Fibers & Sheets:

1. Fibers are made up of : tropocollagen;

1. Microfibrils: cystine-rich protein;

2. Elastin: amorphous valine-rich protein;

2. Tropocollagen is made up of: 3 helical polypeptide chains coiled around each other (2800 A long).

3. Characteristic amino acids:hydroxyproline and hydroxylysine;

Origin: Both collagen and reticular fibers are secreted by fibroblasts of CT proper

Origin: 1. Fibers secreted by fibroblasts;
ooooooo2. Sheets produced by smooth muscle
oooooooo (e.g., aorta);

C. CLASSIFICATION OF CT based on composition of Cells, Fibers, and Ground substance:



D. EXAMPLES OF CT CLASSES:
CT Types Mesenchyme Mucoid CT Loose-FECT Reticular CT Adipose CT Dense Regular FECT Dense Irregular
FECT
Examples of the 7 CT classes: Embyonic CT Umbilical cord;
(Whartons Jelly)

Placenta;

Embryonic sucutaneous tissue;		 1. Lamina propria of mucosae;

2. Hypodermis;

3. Packing in and  around organs;

4. Stroma of muscles;

5.  Around blood  vessels and erves;

1. Bone marrow;

2. Lymphoid organs;

(Except reticular fibers around adipose cells, muscle fibers, around endocrine cells;)

1.White dipose     tissues:

a. tela subcutanea
b. mesenteries
c. retroperitoneal     fat;
d. orbital fat;
e. breasts,
hips, buttocks;
f. Palms and soles;

2. Brown adipose tissue

a. human infants
b.hibernating animals
c. Path.: (Lipomas)

1. Dermis of skin;

2. Capsules of  organs;

3. Muscle and
nerve sheets;

4. Periodontal ligament.

Tendons and ligaments

e.g. yellow
ligaments:
Ligamenta
flava of  vertebrae, vocal cords, suspensory    ligament of penis,  stylohyoid  ligament;

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

LABORATORY GUIDE FOR CONNECTIVE TISSUE PROPER 

OBJECTIVES:
1. To learn to identify CT types, fiber types, and CT cell types;
2. To learn to classify the stroma of tissues and organs;

ATLAS: Use your CD and Kerr, or Berman, on Connective tissue proper; or Wheater, Chapter 4; 

Note: It is essential that you know the difference between parenchyma and stroma before you start.

a. Parenchyma: is typically represented by the cells primarily expressing the function of a tissue or organ, for example: the lymphoid cells of spleen or lymph nodes, muscle fibers (cells) of muscles, hepatocytes of liver etc.

b. Stroma: of tissues is typically represented by the supporting connective tissue elements and it includes blood vessels, nerve fibers and lymphatic vessels, for example: the reticular CT of spleen, the CT in the liver, etc  
              

A. Thick Skin - (Slide #65)  Stained with H & E.  Shows examples of loose and dense irregular FECT.
          

Orient your slide with the aid of the 4x objective so that the epithelial surface of the skin is facing up (or "North") in the field of view.  Study the layers with the 4x and 10x objectives and for the fibers and cell types use 10x, 40x and oil.  Identify the three fibro-elastic connective tissue (FECT) layers and classify each layer; Identify fiber and cell types.

1.  Papillary layer:  Look beneath the epithelium and locate the thin, fibrous layer which stains lighter than the layer below. There are more nuclei and finer fibers than in the C.T. layer below. (This is the papillary layer, a term which you will learn when you study the histology of the skin).  

a.  C.T. type: (Relatively) loose FECT; but not typical, - does not have all the cellular elements of typical loose FECT such as the numerous wandering cell types.

b.  Fiber types: Some fine collagen fibers are continuous with layer below; also there are many thin wavy elastic fibers.

c.  Cell types: Active fibroblasts with euchromatic, round to oval nuclei. (Do not confuse with the more elongate, darker nuclei of capillary endothelial cells.)

What is the advantage of having elastic fibers in this layer?

2.  Reticular layer: This layer is beneath, and continuous with the papillary layer. A thick layer; fibers form a feltwork, a reticulum therefore the name). Most of the fibers run parallel to the surface, but many run in various  directions; Highly eosinophilic layer.

a. C.T. type:  typical dense irregular FECT (fibro-elastic connective tissue).

b. Fiber types: Primarily collagen fibers, some elastic fibers (typically around blood vessels, nerves and especially around glands.

c. Cell types:  mostly fibroblasts (Active and inactive).

Can you distinguish between active and inactive fibroblasts?

3.  Hypodermis: The thickest, lower most layer of the thick skin.  Lightly stained.

a.  C.T. type:  typical loose FECT.

b. Fiber types:  few collagen fibers, elastic fibers (continuous with fibers of the Reticular layer); Reticular fibers (this name has nothing to do with the name of the above layer) are associated with fat cells and produced by them.

c.  Cell types - Numerous; Try to identify:
    (1) Fibroblasts (5) Macrophages
  (2) Fat cells  (6) Mast cells  
(3) Small lymphocytes (7) Plasma  cells    
(4) Monocytes (8) Neutrophils   
  (9) Eosinophils   

Look also in the lumen of larger blood vessels of the hypodermis and practice the identification of these cells. If you can’t find any than practice with the mammary tissue (Slide #190). 

Can you find mast cells, macrophages, and plasma cells in the lumen of the blood vessels?

 

 




B. Spleen: (Slide #67)   Stained with silver. Shows and example of reticular fibers and reticular C.T.
 
This spleen section was impregnated with silver to show argyrophilia of fibers. Viewing the tissue with the 4x and 10x objectives you should see dense, darkly staining fibrous areas which are the trabeculae of the spleen (incomplete C.T. partitions) compose primarily of collagen fibers.  If you follow some of these wavy collagen fibers away from the trabeculae using the 40x and oil objectives you will see that the collagen fibers are continuous with the sparsely distributed, thin, black, branching reticular fibers. Where have you seen reticular fibers stained with PAS?
 

a.  C.T. type:  typical reticular C.T.

b.  Fiber types:   reticular; produced by reticular (fibroblast-like) cells (Collagen fibers only in trabeculae; trabeculae are not a reticular C.T.).

c. Cell types:     not seen in this preparation. (Would expect lots of lymphocytes, macrophages, some granulocytes, and even megakaryocytes among the reticular fibers).

What are the differences between collagen and reticular fibers? (See lecture notes.) Do all tissues associated with reticular fibers constitute a reticular connective tissue?

C. Aorta: (Slide #80) Stained with H & E.  An example of elastic laminae (Use 40x oil objective).
  Identify the elastic sheets or laminae forming fenestrated, concentric, wavy layers in the wall of the aorta (4x;10x). These elastic laminae are the product of smooth muscle cells. Between these layers of elastic sheets there are mostly smooth muscle cells and only a few elastic fibers and associated fibroblasts. Elastic fibers are produced by fibroblasts. Elastic fibers are made up of elastin+microfibrils but elastic lamellae are elastin + microfibrils in an amorphous layer.

What does fenestrated mean?  What is the function of elastic laminae in the aorta? Is this slide an example of elastic C.T.?
D. Tendon: (Slide #13) -- An example of Dense regular C.T.
  Proceed in examining the tissue from low (4x) to high (10x, oil) power.  Note: if you are looking at fibers with clearly visible cross banding, you are looking at striated muscle fibers.  Look towards the end of these fibers as they merge with their tendons.  Observe the parallel dense, sometimes wavy arrangement of fibers. Other slides will show only the tendon; these are stretched out better and the fibers are more parallel (See: tendon in CD). If you have one of these slides let your neighbors see it too. Fibroblast nuclei are mostly heterochromatic and elongated in tendons and ligaments (inactive f.b)
   

a.  C.T. type:   dense regular FECT.

b.  Fiber type:  collagenous

c.  Cell types:  only fibroblasts (mostly inactive)
  What is the function of a tendon? - Would your strength increase or decrease if tendons were made of elastic fibers?

 

 

 

 

 

 

 

 




E. Mesentery:   (Slide #9). --   An example of typical loose FECT, collagen and elastic fibers as well as cells.
 

This is a whole-mount of a rodent mesentery spread.  The tissue consists of loose FECT sandwiched between two layers of mesothelium (define mesothelium).  In addition to the thicker collagen fibers, elastic fibers can be easily visualized. Elastic fibers form a branching network of thin, eosinophilic fibers. Mesothelial cells may be identified with the following procedure on many mesentery preparations. Turn your coarse focus knob all the way toward you if the stage is away from you. Next start focusing in the opposite direction or dawn on the section. When you focus down on the tissue the first structures coming into focus are the large, plump nuclei of mesothelial cells (squamous epithelium). [Unfortunately some slide preparations are over stretched and one can't see much difference and essentially all cells come into focus at the same depth.]
   

a.  C.T. type:  typical loose FECT. 
b.  Fiber types:  collagen and elastic fibers 
c.  Cell types: all types; identify the following cell types: active and inactive fibroblasts, macrophages, mast cells and as many “wandering cell”“ types as you can. 

What are the functions of the different cells listed?

F. Mammary Tissue:  (slide #190A or B) -- Good example of loose FECT.
  Contains most of the“wandering cells;” What are the “wandering cells?”

Orient yourself at low power (4x or 10x) on the slide and examine with the 40x and oil objectives areas of connective tissue between lobules and between the glandular and ductal epithelia. Classify the C.T. and identify the following cell types:
       
   1.  Fibroblasts
2.  Fat cells  		 3.  Mast cells
4.  Lymphocytes		 5.  Plasma cells
6.  Macrophages
  Why do lymphocytes especially small lymphocytes appear to have different morphology in blood smears vs. in tissue sections?

G. Duodenum: (Slides #118 or #118A) -- The lamina propria beneath the epithelium.
 

Another good example of loose FECT, lymphocytes, plasma cells, eosinophils, and perhaps macrophages.

This tissue from the duodenum illustrates the above cell types to advantage in the lamina propria beneath the epithelium (Define lamina propria).  Orient yourself on the slide and identify in addition to the eosinophils as many of the other cell types as possible. 
 

Have you identified all 7 types of C.T.?  How many?                                        

Have you identified all 11 cell types? 

CT cell types and wandering cells?

Have you identified all 3 types of fibers?          
OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO