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
Review the four primary types of collagen and where they are typically found.
Answer: Type 1 - Bone, Ligaments, Skin, Tendons.  Type 2 - Cartilage.  Type 3 - Reticular Fibers.  Type 4 - Basement Membrane
What is the difference between white and brown adipose tissue? What makes white fat "white" and brown fat "brown" under the microscope?
Answer: White adipose tissue is composed of large cells with prominent central vacuoles. It is white because the lipid is washed away during fixation and the vacuoles appear white under the microscope. Brown adipose tissue has smaller cells with many lipid droplets and mitochondria. It is brown because of the large number of cytochromes present.
Distinguish the three kinds of cartilage. What type of collagen are they made of, and where are they found?
Answer: Hyaline cartilage – type II collagen – nose, tracheal rings, end of ribs. Fibrocartilage - type I collagen - tendons, intervertebral discs.  Elastic cartilage - type II collagen - ear and epiglottis.
How would you distinguish a fibroblast, macrophage, and mast cell under the light microscope?
Answer: Fibroblast may be very basophilic because it has a lot of RER, and you may actually be able to see it releasing collagen. Macrophages are mononuclear cells that are generally nondescript, although they are easily identifiable if they have been actively phagocytosing particles. Mast cells have a characteristic granulated appearance.
Slides
Please select whether to view the slides in study mode or quiz mode. In study mode, the images will contain labels and a description. In quiz mode, labels and description will be hidden.