Connective Tissue Lab

Learning Objectives

  • Describe the structural organization of the fibers in the extracellular matrix and the cells residing within connective tissue
  • Distinguish loose and dense connective tissue using the light microscope
  • Contrast white and brown adipose tissue
  • Describe the structure and function of cartilage
  • Identify a few key pathological examples involving connective tissue

Keywords

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Pre-Lab Reading

Introduction

Connective tissue is a term used to describe the tissue of mesodermal origin that that forms a matrix beneath the epithelial layer and is a connecting or supporting framework for most of the organs of the body. This lab will focus on the so-called connective tissue proper and cartilage; the next lab will focus on bone.

Overview of Connective Tissue

In contrast to epithelia, connective tissue is sparsely populated by cells and contains an extensive extracellular matrix consisting of protein fibers, glycoproteins, and proteoglycans. The function of this type of tissue is to provide structural and mechanical support for other tissues, and to mediate the exchange of nutrients and waste between the circulation and other tissues. These tissues have two principal components, an extracellular matrix and a variety of support cells. These two components will be the focus of this lab.

Most frequently, the different types of connective tissues are specified by their content of three distinguishing types of extracellular fibers: collagenous fibers, elastic fibers, and reticular fibers.

Ground Substance

The ground substance is an aqueous gel of glycoproteins and proteoglycans that occupies the space between cellular and fibrillar elements of the connective tissue. It is characterized by a gel-like viscous consistency and is polyanionic. The characteristics of the ground substance determine the permeability of the connective tissue layer to solutes and proteins.

Collagenous Fibers

Collagenous fibers consist of types I, II, or III collagen and are present in all types of connective tissue. Collagenous connective tissue is divided into two types, based upon the ratio of collagen fibers to ground substance:

  • Loose (areolar connective tissue) is the most abundant form of collagenous connective tissue. It occurs in small, elongated bundles separated by regions that contain ground substance.
  • Dense connective tissue is enriched in collagen fibers with little ground substance. If the closely packed bundles of fibers are located in one direction, it is called regular; if oriented in multiple directions, it is referred to as irregular. An example of regular dense connective tissue is that of tendons; an example of irregular dense connective tissue is that of the dermis.

Reticular Fibers

Reticular fibers are composed of type III collagen. Unlike the thick and coarse collagenous fibers, reticular fibers form a thin reticular network. Such networks are widespread among different tissues and form supporting frameworks in the liver, lymphoid organs, capillary endothelia, and muscle fibers.

Elastic Fibers

Elastic fibers contain the protein elastin, which co-polymerizes with the protein fibrillin. These fibers are often organized into lamellar sheets, as in the walls of arteries. Dense, regular, elastic tissue characterizes ligaments. Elastic fibers are stretchable because they are normally disorganized – stretching these fibers makes them take on an organized structure.

Cells of the Connective Tissue Proper

Although the connective tissue has a lower density of cells than the other tissues you will study this year, the cells of these tissues are extremely important.

Fibroblasts are by far the most common native cell type of connective tissue. The fibroblast synthesizes the collagen and ground substance of the extracellular matrix. These cells make a large amount of protein that they secrete to build the connective tissue layer. Some fibroblasts have a contractile function; these are called myofibroblasts.

Chondrocytes and osteocytes form the extracellular matrix of cartilage and bone. More details and chondrocytes can be found later in this laboratory; osteocytes will be covered in the Laboratory on Bone.

The macrophage is the connective tissue representative of the reticuloendothelial, or mononuclear phagocyte, system. This system consists of a number of tissue-specific, mobile, phagocytic cells that descend from monocytes - these include the Kupffer cells of the liver, the alveolar macrophages of the lung, the microglia of the central nervous system, and the reticular cells of the spleen. You will encounter each of these later in the course; for now, make sure you recognize that they all descend from monocytes, and that the macrophage is the connective tissue version. Macrophages are indistinguishable from fibroblasts, but can be recognized when they internalize large amounts of visible tracer substances like dyes or carbon particles. Macrophages phagocytose foreign material in the connective tissue layer and also play an important role as antigen presenting cells, a function that you will learn more about in Immunobiology.

Mast cells are granulated cells typically found in connective tissue. These cells mediate immune responses to foreign particles. In particular, they release large amounts of histamine and enzymes in response to antigen recognition. This degranulation process is protective when foreign organisms invade the body, but is also the cause of many allergic reactions.

White fat cells are specialized for the storage of triglyceride, and occur singly or in small groups scattered throughout the loose connective tissue. They are especially common along smaller blood vessels. When fat cells have accumulated in such abundance that they crowd out or replace cellular and fibrous elements, the accumulation is termed adipose tissue. These cells can grow up to 100 microns and usually contain once centrally located vacuole of lipid - the cytoplasm forms a circular ring around this vacuole, and the nucleus is compressed and displaced to the side. The function of white fat is to serve as an energy source and thermal insulator.

Brown fat cells are highly specialized for temperature regulation. These cells are abundant in newborns and hibernating mammals, but are rare in adults. They have numerous, smaller lipid droplets and a large number of mitochondria, whose cytochromes impart the brown color of the tissue. The electron transport chain of these mitochondria is disrupted by an uncoupling protein, which causes the dissipation of the mitochondrial hydrogen ion gradient without ATP production. This generates heat.

Cartilage

Cartilage is a specialized form of connective tissue produced by differentiated fibroblast-like cells called chondrocytes. It is characterized by a prominent extracellular matrix consisting of various proportions of connective tissue fibers embedded in a gel-like matrix. Chondrocytes are located within lacunae in the matrix that they have built around themselves. Individual lacunae may contain multiple cells deriving from a common progenitor. Lacunae are separated from one another as a result of the secretory activity of the chondrocytes.

A highly fibrous, organized, dense connective tissue capsule known as the perichondrium surrounds cartilage. The fibroblast-like cells of this layer have chondrogenic potentiality, and are responsible for the enlargement of cartilage plates by appositional growth. Appositional growth involves cell division, differentiation, and secretion of new extracellular matrix, thereby contributing mass and new cells at the cartilage surface. It is in contrast to interstitial growth, in which new matrix is deposited within mature cartilage.

Three kinds of cartilage are classified according to the abundance of certain fibers and the characteristics of their matrix:

  • Hyaline cartilage has a matrix composed of type II collagen and chondromucoprotein, a copolymer of chondroitin sulfates A and C with protein. Its high concentration of negatively-charged sulfate groups makes it appear intensely basophilic under H&E. This cartilage is found in the nose, tracheal rings, and where the ribs join the sternum.
  • Fibrocartilage is distinguished by its high content and orderly arrangement of type I collagen fibers. It is typically located in regions where tendons attach to bones, the intervertebral discs, and the pubic symphysis.
  • Elastic cartilage is characterized by the presence of abundant elastic fibers and is quite cellular. It is made up of type II collagen and is located in the auricle of the ear and the epiglottis.

Pre-Lab Quiz

  1. Review the four primary types of collagen and where they are typically found.
  2. Answer:
  3. What is the difference between white and brown adipose tissue? What makes white fat "white" and brown fat "brown" under the microscope?
  4. Answer:
  5. Distinguish the three kinds of cartilage. What type of collagen are they made of, and where are they found?
  6. Answer:
  7. How would you distinguish a fibroblast, macrophage, and mast cell under the light microscope?
  8. Answer:

Slides

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  1. Loose Connective Tissue
  2. Dense Regular Connective Tissue
  3. Dense Irregular Connective Tissue
  4. Collagen Fibers EM
  5. Reticular Fibers
  6. Elastic Fibers
  7. Elastic Fibers EM
  8. Fibroblast EM
  9. Mast Cell EM
  10. Macrophage EM
  11. White Adipocytes
  12. Brown Adipocytes
  13. Chondrocytes
  14. Hyaline Cartilage
  15. Fibrocartilage
  16. Elastic Cartilage

Virtual Microscope Slides

  1. Skin
  2. Identify two different types of connective tissue that are prominent in this slide. What are the functions of each type?
  3. Trachea
  4. This is a section of trachea. Begin by recalling the pertinent information from the Laboratory on Epithelia. What type of epithelium is present? Next, move down and locate the cartilage ring that surrounds the trachea.
  5. Bone
  6. This image shows a developing bone. Focus on where the tendon attaches to the bone and locate the fibrocartilage. Describe the orientation of the collagen fibers at high magnification.
  7. Elastic Cartilage
  8. This is a section of epiglottis that has been treated with a special stain for elastic fibers, so the cells and tissues will have different color than in an H&E-stained sample. Locate the cartilage in this section of the epiglottis. The elastic fibers are stained black.

Pathology

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  1. Ehlers-Danlos Syndrome
  2. Osteoarthritis

Quiz

  1. Locate and classify the connective tissue in this image of the intestine. What functions does this type of connective tissue play to support the epithelium?
  2. Answer: This is loose connective tissue. It provides metabolic support (blood vessels), immune support (white blood cells) and structural support.
  3. Classify the connective tissue and describe its function.
  4. Answer: Dense, regular collagenous. Resists tension in one direction.
  5. Name the cell type.
  6. Answer: Fibroblast
  7. Name the cell type and its major product.
  8. Answer: Mast Cell - Histamine and Heparin
  9. In this image of an artery, What type of fibers are stained darkly? What is their function?
  10. Answer: Elastic fibers. Allow wall of artery to expand and contract.
  11. Classify the connective tissue.
  12. Answer: Hyaline Cartilage. Note glass appearance and relatively fewer cells than in elastic cartilage.
  13. Identify two cells in this image.
  14. Answer: Macrophage engulfing a red blood cell
  15. Losartan is drug primarily used to treat high blood pressure. Recent work found that losartan also decreases production of TGF-β. Why would losartan be used in clinical trials for treatment of Marfan’s Syndrome?
  16. Answer: When mutations in fibrillin were discovered to lead to Marfan’s Syndrome, the explanation was that loss of or defects in fibrillin weaken elastic fibers leading to many of the symptoms of Marfan’s including aortic aneurysms. Recently, fibrillin mutations were found to increase levels of TGF-β and antibodies to TFG-β were shown to reverse the effects of fibrillin mutations in mice. Finally, fibirillin has been shown to lock TGF-β in a protein complex that keeps it inactive. Based on these results, our current thinking is that fibrillin mutations lead to increased TGF-β signaling which leads to the symptoms of Marfan’s Syndrome.