Histology Exam 1: Connective Tissue

Front 1

Connective Tissue (General)

Back 1

Connective tissue is the most abundant, widespread, and diverse tissue type. It is highly vascular and has a nerve supply.

Front 2

What makes up CT?

Back 2

Cells and extracellular matrix. CT cells are sparse in density and not attached as epithelial cells are. The ECM of the CT separates them.

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What makes up the ECM of CT?

Back 3

Protein fibers and ground substance (which can be fluid, gel, or solid and contributes to ECM consistency).

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What are the two groups of CT cells? Give examples of each.

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Cells are classified as fixed or wandering. FIxed cells include fibroblasts (the only true fixed cell), macrophages, and mast cells. Wandering cells (WBC's) include lymphocytes, plasma cells, eosinophils, and basophils. *Monocytes (and possibly basophils?) are not seen in CT.

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What is the function of CT?

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Structural support (provides form and shape and facilitates locomotion), Binds organs together (CT capsules around organs/ligaments bind bones together), Packing (areolar CT acts as soft packaging, surrounding organs), Defense and protection (provides the stage for inflammation), Communication/transport, Thermoregulation, Repair and replacement (scarring- CT fills the space where damaged tissue cannot regenerate).

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What is the origin of CT?

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It is derived from mesenchyme (embryonic connective tissue).

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Mesenchyme

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Embryonic connective tissue that has mesodermal and ectodermal (neural crest) origins. It consists of many cells and few fibers found around the developing notochord that can differentiate into all kinds of mature CT. Its matrix is a semifluid ground substance that contains reticular fibers and embedded, star-shaped mesenchymal cells (embryonic stem cells).

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What do embryonic mesenchymal cells differentiate into?

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Anything they want! All other cell types including fibroblast, chondrocyte, osteocyte, or adipocyte.

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What are fibroblasts? Where are they from and where are they found?

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Fibroblasts are the main cell type of CT and lie very close to collagen fibers. They are of mesenchymal origin.

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What do fibroblasts do?

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Synthesize collagen, elastin, and reticular fibers and carbohydrates of ground substance.

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What do fibroblasts look like?

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They are elongate cells with oval nuclei and fine cytoplasm. They usually only have one visible nucleus. When they are active, they have lots of RER for protein synthesis.

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What are myofibroblasts and what do they do?

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Myofibroblasts are specialized fibroblasts containing actin that lack a basal lamina. They have contractile functions (both fibroblast and smooth muscle properties).

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What are macrophages derived from?

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Macrophages (aka histocytes) are derived from monocytes (blood cells that arise in bone marrow).

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What do macrophages do?

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Phagocytize bacteria and cell debris and digest them in lysosomes, present antigens, and act as part of the mononuclear phagocytic system (MPS).

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What do macrophages look like?

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Macrophages are round cells with lots of cytoplasm and vacuoles that are folded in cell membrane. The cytoplasm of macrophages stain positive with PAS because of the carbohydrate-rich enzymes found in the lysosomes.

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What are mast cells?

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Mast cells are related to basophils (blood cells with similar granules) and develop in the bone marrow where they differentiate into CT. Mature mast cells do not circulate (unlike basophils). You can see mast cell granules with a routine H&E stain.

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What do mast cells do?

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Mast cells release granules in the immune response following exposure to antigen and function in allergy, hypersensitivity, and anaphlaxis.

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What do mast cells look like?

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Mast cells are large round cells with round nuclei. Their cytoplasm stains positive with PAS and T-blue due to cytoplasmic granules.

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What are the three major types of lymphoctyes?

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B, T, and NK (natural killer cells).

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Function of Lymphocytes

Back 20

Immune response and antibody production.

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What do lymphocytes look like?

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They are round cells with small, dark blue nuclei. Their cytoplasm is difficult to see.

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Function of Plasma Cells

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Immune response and antibody production.

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What are plasma cells derived from?

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B-lymphocytes.

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What do plasma cells look like?

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Plasma cells are round with pale blue cytoplasm and round nuclei that have a "clockface" or "cartwheel" appearance. They have abundant RER and a pale halo which represents the golgi body.

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Neutrophils

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The most numerous WBC. They are the first at the site of injury and function in phagocytosis. They also create chemical mediators.

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What do neutrophils look like?

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They are round cells wtih multilobated nuclei.

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Eosinophil Function

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Eosinophils are important in allergy, hypersensitivity, and anaphalyxis as well as parasitic infections.

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What do eosinophils look like?

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Eosinophils are round cells with multilobated nuclei. Their cytoplasmic granules stain bright pink/red and contain chemical mediators such s proteins and enzymes.

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Basophils

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Basophils are the least numerous WBC type and are related to mast cells (they are derived from the same stem cell).

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Function of basophils

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Basophils are important in allergy, hypersensitivity, and anaphylaxis.

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What do basophils look like?

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Basophils have round, lobed nuclei and blue granules in their cytoplasm. They are not usually seen in tissue sections.

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Monocytes

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Monocytes are WBC's that are precursors of the mononuclear phagocytic system (MPS). Their cells are round with slightly indented nuclei. They are not usually seen in tissues.

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ECM of CT (General)

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CT is 95% ECM, which is a web-like meshwork of fibers embedded in a homogenous ground substance. It is composed of ground substance and fibers.

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What are the functions of the ECM of CT?

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Mechanical and structural support, binds tissues together ("glue"), acts as a biochemical barrier, aids in extracellular communication, anchors cells in tissues, and provides a path for cell migration in wound repair.

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What produces the matrix of CT?

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The principal cell of the CT produces all elements of the matrix; each tissue has a name that indicates the principle cell type (eg fibroblast, chondroblast, osteoblast...).

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Ground Substance of CT

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The background material all other CT elements are embedded in. It occupies the space between fibers and cells, provides the medium for diffusion of O2 & nutrients, and allows for the migration of cells. It may be highly modified in specialized cell types.

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What makes up the ground substance of CT?

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Ground substance of CT is made up mostly of water (allows for communication and diffusion between tissues). This is stabilized by proteoglycans, glycosaminoglycans, and glycoproteins (which combined are a very small fraction of the ground substance)

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How is CT ground substance modified in specialized cell types?

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Blood: it lacks stabilizing macromolecules and is fluid (this free-flowing ground substance is plasma).
Skeletal tissues: it may become mineralized by deposition of calcium salts (called bone).
Cartilage: it is much more solid than in ordinary CT, but still retains more resiliency than in bone.

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Proteoglycans in the ECM of CT (General)

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Composed of GAG's (95%) attached to a protein core (5%) and found in the ground substance of all CT. Some are transmembrane. They are responsible for the highly viscous character of the ground substance.

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How are glycosaminoglycans situated within the proteoglycans of CT?

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GAG's extend perpendicularly from the protein core and are often attached by a noncovalent bond to hyaluronic acid.

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What role do transmembrane proteoglycans play in CT?

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They participate in cell-cell or cell-ECM interactions (eg versican and syndecan).

Front 42

Give examples of extra-cellular proteoglycans in CT.

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Aggrecan (hydrates the ECM of cartilage) and decorin (plays a role in fibrillogenesis- it orients fibers and regulates fibril thickness).

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Glycosaminoglycans in the ECM of CT (General)

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Negatively charged polysaccharide chains that provide the structural framework for CT cells. They are found in the ground substance (mostly) and on the surface of cells in the ECM.

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What are the glycosaminoglycans in the ECM of CT made up of?

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Repeating disaccharide units, typically of iduronic or glucuronic acid coupled to a sulfated amino sugar (except in hyaluronic acid, which is not sulfated).

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Hyaluronic Acid

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A dominant GAG that acts as the backbone for assembly of other GAGs in the CT. Hyaluronic acid is nonsulfated and is always present as a free carbohydrate chain (not bound to protein) so it doesn't form proteoglycans. It can bind proteoglycans via link proteins to form proteoglycan aggregates (as in cartilage which can subsequently absorb shock and resist compression).

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Examples of Glycosaminoglycans

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Include: Hyaluronic Acid (dominant GAG), Chondroitin sulfate, Dermatan sulfate, Keratan sulfate, and Heparan sulfate.

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How do glycosaminoglycans stain with Periodic Acid Schiff (PAS) stain?

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GAGs stain positive with PAS stain because of their carbohydrate composition.

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How do glycosaminoglycans function to provide structure in the CT?

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The carbohydrates and negative charge of GAGs attracts large amounts of water and cations and the effect is gel-forming. This gel composition allows for the diffusion of substances such as nutrients and O2.

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What are polysulfated GAGs used for clinically?

Back 49

To therapeutically treat degenerative joint disease in dogs and horses.

Front 50

What are glycoproteins and what do they do in the ECM of CT?

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Glycoproteins are a small but important group of large ECM proteins involved in cell adhesion that serve to stabilize the ECM by linking it to the cell surface (which is necessary for survival). They aid in cell movement/migration within the ECM and play a role in cell proliferation and differentiation.

Front 51

Give examples of glycoproteins found in the ECM of CT.

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Examples include: Fibronectin, Laminin, Tenascin, and Osteopontin.

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Fibronectin

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The most abundant GP in CT, it is present in the ECM of many tissues and functions in cell attachment to the ECM. It also influences deposition and orientation of collagen.

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Laminin

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Present in the basal laminae of epithelium and in the external laminae of muscle cells, adipocytes, and Schwann cells.

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Tenascin

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Important in embryogenesis, its synthesis is switched off in mature tissues but reappears during would healing. It functions in cell attachment to the ECM.

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Osteopontin

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Present in the ECM of bone, it binds to osteoclasts and attaches them to the bone surface below. It is important in sequestering calcium and promoting calcification of the ECM.

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What are the three types of protein fibers embedded in the ECM and what produces them?

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The three types are collagen fibers, elastic fibers, and reticular fibers. They are all produced by fibroblasts.

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Collagen Fibers (General)

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Made up of bundles of the fibrous protein collagen (the most abundant protein in the body). This is the dominant fiber type in most CTs and is tough and flexible, providing high tensile strength. The function of collagen is to add strength to CT.

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What do collagen fibers look like?

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Collagen fibers have a wavy appearance under the microscope and stain well with H&E (they are pink). They also show up with trichrome stain and variants such as Mallory's, Masson's, Gomori's, etc... and appear blue/green with these stains.

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What characteristics are used to classify collagen fibers?

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The genes that produce them, their properties, and the locations at which they are found.

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Collagen Type 1

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The most common type of CT, it functions to resist tension. It occurs in ordinary fibrous CT (e.g. dermis, tendon, fascia) as well as bone and fibrocartilage.

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Collagen Type 2

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Reinforces hyaline and elastic cartilage and functions to resist pressure.

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Collagen Type 3

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Forms reticular fibers and occurs in the basal laminae and in bone. It provides the framework for organs and is the first type of collagen produced during would healing (it is ultimately replaced by T1)

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Collagen Type 4

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Occurs in the basal lamina and doesn't form fibrils. It is secreted by epithelial cells and functions in support and as a filtration barrier.

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Collagen Type 5

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An interlinking collagen important in formation of basement membranes.

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What is the basic structural unit of collagen?

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A triple, right-handed helix; the helix consists of 3 alpha-chains and results from an abundance of glycine, proline, and hydroxyproline making up the motif of Gly-Pro-X.

Front 66

Type 1 Collagen Fibers (Structure)

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Longitudinal striations may be visible in Type 1 collagen. Striations reveal fibers are non-branching and composed of thinner collagen fibrils. Each fibril is, in turn, composed of microfibrils (seen with EM) which are assemblies of collagen molecules in a regular arrangement (the triple helix). In Type 1, many triple-helices pack side by side to form fibrils, which pack side-by-side in bundles to form fibers.

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Collagen Biosynthesis: Intracellular Events (in the Fibroblast)

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Collagen alpha-chains are synthesized as long precursor molecules (preprocollagen) at the polyribosomes and then transferred to the RER. At the RER, the preprocollagen chain is processed via post-translational modification (possible PTMs: clipping of the signal peptide, hydroxylation of proline and lysine, and glycosylation of hydroxylysine. Bonds are made within/between chains to align the chains for creating a helix.

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Collagen Biosynthesis: Extracellular Events

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After processing and assembly, procollagen is secreted into the extra-cellular environment. Collagen peptidases then remove the N and C-terminal propeptides to produce the mature collagen. Mature collagen can assemble into fibrils (and then fibers).

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Elastin Fiber Function

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Provides strength and elasticity to CT and resists shearing and tearing.

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Location of Elastin Fibers

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Found where greater elasticity (stretch and recoil) is needed. Examples: skin, lungs, elastic arteries (aorta), pinna, and between vertebrae.

Front 71

Describe the arrangement and composition of elastic fibers.

Back 71

They are arranged in a branching pattern and are made up of an elastin core and surrounding fibrillin (a glycoprotein) microfibrils.

Front 72

Elastin (General)

Back 72

A fiber rich in proline and glycine (with less hydroxyproline and no hydroxylysine). Its random distribution of glycines makes it hydrophobic and allows for the coiling of fibers. Desmosine and isodesmosine are unique to elastin and allows elastin molecules to bond to one another. Fibers can be stretched to 150% length.

Front 73

What stain should be used with elastin?

Back 73

Elastin doesn't stain well with H&E. Verhoeff-van Gieson stain is a good stain to use (elastic fibers will appear dark blue-black).

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Reticular Fibers (General)

Back 74

These are actually composed of a variety of collagen (mainly Type 2). The fibers are very thin, fine, and delicate. They form branching fiber networks (NOT thick bundles like Type 1 collagen in the dermis) and are produced by fibroblasts in most locations.

Front 75

Name two organs whose framework is supported by reticular fibers.

Back 75

Liver and spleen.

Front 76

Where are reticular fibers NOT produced by fibroblasts?

Back 76

In lymphoid and hematopoietic tissues, peripheral nerve endoneurium, blood vessel tunica media, the intestinal muscularis layer, reticular cells, Schwann cells, and smooth muscle cells.

Front 77

What is the function of reticular fibers?

Back 77

To provide a supporting stroma...