HISTOLOGY FULL-TEXT

William A Beresford MA, D Phil ©
Professor of Anatomy
Anatomy Department, West Virginia University, Morgantown, USA

Chapter 5 CONNECTIVE TISSUES

Connective tissues (including cartilage and bone: Chapters 6, 7 and 8) are derived from mesoderm or mesectoderm (for the head) of the embryo, via an intermediate stage called mesenchyme. Mesenchyme consists of pale cells, with extended processes, lying in jelly-like matrix. In later development, the cells and extracellular matrix (ECM) become specialized for various tasks, and the matrix comprises amorphous 'ground substance' reinforced to greater or lesser extent by specialized fibres. The various cells, fibres, and ground substances will be discussed, followed by a treatment of the tissues that they combine to build. Connective tissue from hereon may be abbreviated to CT. (Caution! This abbreviation usually signifies computed tomography.)Powerpoint

A CELLS OF CONNECTIVE TISSUES

l Fibroblast
l Occurs in young active, and adult quiescent/less active forms.
2 Young has abundant, basophilic cytoplasm, with a well-developed Golgi complex and GER for protein and proteoglycan synthesis.
3 Nucleus is ovoid, with weakly staining chromatin granules.
4 The cell is elongated, and often sends out processes to take on a more elongated or stellate form.
5 Adult fibroblasts (fibrocytes) have smaller, darker nuclei, and very little cytoplasm. They remain fixed and squashed into a spindle/cigar form amongst the fibres that they formed.
6 Function - forming and remodelling collagen, reticular and elastic fibres, and the ground substances. The remodelling requires the production of destructive enzymes, and inhibitors to help restrain their action. TIMPs - Tissue Inhibitors of MetalloProteinases - are an example.
In some sites, e.g., the periodontal ligament holding the teeth in place, the fibroblasts more aggressively destroy fibres, in the process of matrix turnover.
7 Young fibroblasts, aside from making fibres, may in some circumstances (e.g., wound repair) take on some smooth-muscle characteristics, and become contractile myofibroblasts, which contribute to the disabling contractures of some scar tissue.

2 Mesenchymal cell
l Has a similar appearance to a small, young fibroblast, but is far more multipotential in what cell types it can turn into.
2 In adult tissues, two views are:
...(a) a few are present and can explain such findings as the formation of ectopic (out of its expected place) bone in soft CT, otherwise difficult to account for unless differentiated cells such as fibroblasts can dedifferentiate and change their role;
...(b) mesenchymal cells all differentiate early in life and thereafter are not present, and fibroblasts or other cells can de- and redifferentiate and become osteoblasts.

3 Macrophage/histiocyte
l An ovoid or spheroid cell, which may change its shape while lying alongside fibres, or when extending pseudopodia to move and ingest materials.
2 Phagocytoses dead cells, cell debris, live and inert foreign bodies.
3 Coordinates the inflammatory response and healing by means of signalling peptides and proteins - cytokines, e.g., IL-1, TGF-b (Chapter 8.F).
4 Nucleus is smaller and more condensed than that of the active fibroblast.
5 Cytoplasm is pale with little GER, but has many lysosomes, when digesting phagocytosed material.
6 Macrophages may fuse to become foreign-body giant cells with many nuclei, when faced with a large object for digestion. More on macrophages.

4 Macrophage/reticuloendothelial/mononuclear phagocyte system (MPS)

l Comprises cells related directly to blood monocytes, or derived from the same precursor in marrow.

2 A tentative division of the macrophage-system cells recognizes:
...Phagocytic antigen-presenters (Chapter l9.B.l)
... (a) Macrophages of connective tissues and serous cavities.
... (b) Alveolar macrophages/lung dust cells.
... (c) Macrophages of lymph nodes, spleen and bone marrow.
... (d) Kupffer sinusoid-lining cells of liver.
.........................................
... Weakly phagocytic antigen-presenters
... (e) Dendritic and interdigitating reticulum cells of lymphoid tissues.
... (f) Langerhans cells of epidermis and other epithelia.
.........................................
... Specialized (Some not phagocytic? Some not antigen-presenters?)
... (g) Foreign-body giant cells.
... (h) Microglia cells of CNS.
... (i) Synovial A cells lining joints.
... (j) Osteoclasts resorbing bone.

3 The phagocytic group (i.e., the original reticulo-endothelial series) can be revealed by vital injection (into the living animal) of colloidal or particulate coloured matter, e.g., Trypan blue or India ink, which the phagocytic cells of the system preferentially accumulate in their cytoplasm, thereby identifying themselves. Nowadays, MPS cells are distinguished by their cell-surface glycoprotein profiles, e.g., CD antigens.

5 Mast cell
l A `watchdog' cell starting the inflammatory response to noxious intruders.
2 From the German verb, mästen, it meant a `fattened' cell.
3 Spheroid or ovoid with a small central nucleus, and its cytoplasm packed with dense basophilic granules.
4 Granules give a metachromatic staining reaction with thionine or toluidine blue, i.e., a reddish-purple colour, because they contain a sulphated polysaccharide - heparin.
5 Heparin is an anticoagulant for blood, first obtained from the liver (hepar), but it also inhibits vascular smooth muscle proliferation and some immune complement reactions. As a polyanion, it can complex materials, e.g., the trypsin-like enzyme, tryptase, in the granules.
6 Histamine, increasing capillary permeability, is also present in the granules. The chemokines also released can then more easily attract white blood cells out of the vessels.
7 Many stimuli (e.g., antigens and agents released by lymphocytes during an immune response) activate a release of the granule contents, from this `mobile-pharmacy' cell, with its many chemical mediators.
8 Mast cells favour positions in CT close to veins (MCt subtype), and at dermal and mucosal interfaces with the hostile environments of the skin, airway, and gut (MCtc subtype).
9 The mast cell subtypes in man differ in the proteases that they contain:
...MCt cells have mast-cell tryptase and are involved directly with defence.
...MCtc cells contain chymase, cathepsin G, and other proteases, in addition to mast-cell tryptase, and are more concerned with adaptive and remodelling responses of blood vessels and CT.

6 Fat cell/adipocyte
l A genuinely fattened cell, initially resembling a fibroblast with a few droplets in the cytoplasm.
2 For the white or yellow unilocular fat seen in adult man, the droplets (mainly glycerides of fatty acids) coalesce and more fat is added,
3 until the nucleus is bulged to one side of a spheroid cell up to 200 µm in diameter, distended by a huge droplet.
4 Cytoplasm, with a Golgi complex, ER and mitochondria, is present as an attenuated peripheral shell.
5 The cell is static, but its content is not. The stored fat is participating in the body's carbohydrate and fat metabolism.
6 Fat in the usual wax-imbedded section is dissolved out, but with osmium tetroxide fixation it remains and is black. Some dyes will colour it, if it is preserved by frozen sectioning.
7 Besides a number of adipocyte-specific enzymes for fat metabolism, fat cells secrete leptin, which helps control energy balance and body fat mass.

7 Melanophore/CT pigment cell/CT melanocyte
l A process-bearing cell with melanin pigment granules in its cytoplasm.
2 Found in the skin`s dermis, brain's pia matter and the scleral and choroid coats of the eye.

8 Plasma cell
l Many tissues, particularly those lining tracts open to outside the body, are not immunologically virgin, but have been exposed to foreign organisms that have provoked immune responses by local CT plasma cells and lymphocytes. A lamina propria may have many of both and some eosinophils, e.g., in the gut.
2 Plasma cells are ovoid, roughly l0 µm in length, with an eccentrically placed nucleus having its denser chromatin granules clumped regularly around the nuclear membrane (clock-face appearance).
3 Cytoplasm is deeply basophilic from the rich GER, except for a pale central region where the Golgi complex lies.
4 Proteins synthesized by plasma cells in lymphoid organs reach the plasma as immunoglobulins/ antibodies, inactivating foreign invaders, e.g., viruses.
5 Plasma cells in CT make antibodies for local use, e.g., in the airway or gut, to counter toxins and control microbial populations.

9 Reticular/reticulum cells
l Immunocytochemistry, EM, and enzymatic analysis distinguish at least three kinds of reticular cell: fibroblastic, and two phagocytic kinds - interdigitating (T-zone:) and dendritic (B-zone: antigen-presenting).
2 The supporting reticular fibres of lymphoid tissues and bone marrow are presumed to be produced by the fibroblastic variety.
3 Caution! The principal reticular cell in the thymus is an epithelial kind, although extending cell processes to build a reticulum.
[Any time you hear 'reticular cell', ask for the type meant.]

B FIBRES OF CONNECTIVE TISSUES

l Collagen fibres
l Fibres 2 Fibrils 3 Collagen molecule and fibril-formation 4 Collagen types: distribution and use
1 All collagen molecules are trimers of helical alpha chains (with two-number designations) intertwined together, mostly as a robust super-helix, e.g., [a-1(II)]3 in cartilage, [a1(IV)2a2(IV)] in basement membrane.
2 Collagen molecules differ in their amounts of helical versus globular shapes along the molecule. The ones (fibrillar) that are cleaved to be only helical assemble into fibrils, the others (non-fibrillar) attach to and space the fibrils, in scaffolds of various patterns, fibril-widths, densities and strengths, appropriate to the mechanics of the tissue. Some of the scaffold-glueing ones, e.g., types IX, XII, and XIV, are termed Fibril-Associated Collagens with InterrupTed helices - FACIT.
3 The types are relatively tissue-specific, but not absolutely so as once was thought.
4 Of the nineteen types, some important ones are:
Type I in bone, fibrocartilage, and established soft connective tissues
Type III in these same tissues as embryonic or reparative forerunners (and as a minor mature component)
Type II in hyaline cartilage
Type IV in basement membranes
Type VII to anchor BMs, and
Type VIII from endothelium lining vessels.

5 Collagen staining
(a) Collagen (type I) often is present in bulk, and is stained selectively by: aniline blue in Mallory's method, light green in Masson's, or red acid fuchsin in van Gieson's. (Eosin stains it orange.)
(b) Mallory's, Masson's and van Gieson's trichrome methods distinguish collagen from muscle, and also react with the nuclei and cytoplasm of other cells.

6 Caution for rat CT spreads. Preparations of rat subcutaneous tissues may be contaminated by hairs. The segmentation of the medulla of a hair gives a crossbanding effect in LM. Collagen fibril crossbanding is visible only in EM.

7 Collagen types: as classified by Prockop DJ & Kivirikko KI. Ann Rev Biochem 1995;64:403-434

Fibril-forming     I  II  III    V                   XI
Network-forming               IV          VIII     X
Beaded filament-forming            VI
Anchoring filament-forming            VII
FACIT                                          IX       XII       XIV       XVI             XIX
Nonsecreted transmembrane                                   XIII                XVII*                                                                     XV             XVIII
Basement membrane zone                                                XV             XVIII       
* transmembrane collagen XVII is a component of hemidesmosomes. An autoimmune reaction to it can cause poor epidermal adhesion and hence skin blistering in humans.

2 Reticular fibres
l Collagen fibres, running parallel to one another, do not join up with others running differently. Such an arrangement is seen, however, with reticular fibres, which form a network or reticulum.
2 Reticular fibres stain black with reduced silver methods, hence their other names - argyrophil or argentophil. H and E and some trichrome stains leave them unstained.
3 X-ray diffraction and EM show them to be like fine collagen fibres, having the same 67 nm-repeating crossbanding. Furthermore, they appear first at many sites, as in mesenchyme and healing wounds, where collagen fibres will later form. Thus reticular fibres are an immature, fine kind of collagen fibre, mostly of type III collagen.
4 They persist into the adult in several organs, where a fine fibrous support is needed that does not interfere with a close relation between fixed cells and blood or lymph, e.g., in endocrine glands.
5 Reticular fibres fasten to the underside of basal laminae of epithelia and endothelium, and bind and secure muscle and nerve fibres, using their external laminae.

3 Elastic fibres
l May be fine, single and branching in areolar CT, or thick and parallel in elastic ligaments. Walls of blood vessels have incomplete elastic membranes.
2 The elastic nature of the fibres is shown by the spiralling and kinking of their recoiled broken ends, in spread preparations.
3 Elastic fibres and membranes, if thick, stain pink with eosin, or red with Masson's method; otherwise, they remain unseen, unless elastic stains, e.g., orcein or Verhoeff's, are used.
4 In bulk, unstained, they appear yellow to the naked eye.
5 Formation and nature - fibroblasts and vascular smooth muscle cells form and release two components: (a) fine protein microfibrils thought to orient (b) tropoelastin as it polymerizes into amorphous elastin. With little structure in EM, elastin is a network of long protein chains held in a springy arrangement crosslinked by desmosines, each derived from four lysines of the protein amino-acid chains.

C GROUND SUBSTANCES

l Location - in interstitial/tissue spaces, cartilage and bone matrices, under basal laminae, on and between CT fibres. Ground substance(s) is the extracellular matrix, less the fibrous and fibrillar elements.

2 Nature - large negatively charged proteoglycan molecules (polyanionic macromolecules) bind to a varying degree water, electrolytes, and other macromolecules, such as collagen, and the glycoproteins, fibronectin and tenascin.

3 Proteoglycan chemistry - from a long protein backbone molecule, many long sugar side chains stick out, because negative charges along each chain repel adjacent chains and each other. The chains are composed of repeating pairs of sugar/saccharide units. Each pair has an hexosamine and a uronic acid. The loss of hydrogen ions from the many acids in the chain of glycosaminoglycans (GAGs) leaves negative charges, only some of which are neutralized by counterions such as Na+.

4 Nomenclature - the many linked sugars of the side-chains are polysaccharides, hence with the protein backbone the general name - 'protein-polysaccharide'. However, this also describes glyoproteins, for example, mucoproteins and mucopolysaccarides. Proteoglycans differ from glycoproteins in: their core proteins; the use of fewer species of sugar; lack of branching of the sugar chains; and usually their longer sugar chains, and more acidic/negative character

    O-LINKED GLYCOPROTEIN an example
                                             
 |Core protein
 AA                    Fu    Side chain is short (1-20 sugars) & branching
 |                     |
 AA        Ga - Gln - Ga     Wide variety of sugars
 |       /  ^
 T - Gln                     Uses one of several sugar core^ types
 |    ^   \                   for attachment to the protein
 AA        Gun - Gln - Na                 
 |          ^                Na - sialic acid,  Fu - fucose



    TYPICAL STRAIGHT PROTEOGLYCAN SUGAR SIDE-CHAIN
                                                            -------->
 |                                        Repeating disaccharide pair 
 AA                                       ___|___
 |                                       |       |
 S - Xy - Ga - Ga - Ua - Gln - Ua - Gln - Ua - Gln - Ua - Gln
 |                              -     -          - Unsatisfied negative
 AA                                                             charges
 |Core protein, with serines (S) & threonines (T)      

5 Proteoglycan varieties - dependent on the specific sugars, and the sites of sulphation, if any:
... Hyaluronate - soft connective tissues; synovial fluid; vitreous humour;
... Dermatan sulphate (chondroitin sulphate B) - skin and corneal CT;
... Keratan suphate - cartilage matrix;
... Chondroitin-4-sulphate (A) - cartilage matrix;
... Chondroitin-6-sulphate (C) - cartilage matrix:
... Heparin (also sulphated) - granules of mast cell and basophil.

6 Staining - the failure of counterions to neutralize all anions leaves regions of high negative charge density. If the proteoglycan is prevented from dissolving out, its reactions are:

7 Physical properties - the high negative charge:

8 Overview of proteoglycans (PGs) and glycoproteins in connective tissues

1 The large PG monomer molecules may be aggregated by being strung along a hyaluronate backbone, by means of a link protein for the core protein-HA attachment.
PG aggregation produces huge molecules extending over micrometres, and visible with conventional TEM. Proteoglycans amenable to such assembly are aggrecans, susceptible to breakdown by aggrecanase.
However, the chemical nature and heterogeneity of monomers and their aggregates make study of these important matrix constituents difficult.
Note that proteoglycans are also kept within some cells to work with other molecules.

2 The glycosaminoglycan side chains of proteoglycans vary in number, nature and length. Combinations of sulphated and non-sulphated hexosamines, and relatively tissue-specific core proteins, yield a diversity of PGs, crudely classifiable by molecular size into large and small:

LARGE
Chondroitin-6-sulphate, skeletal keratan sulphates - Cartilage
Versican/Fibroblast PG - Soft CTs
Cell-surface-associated, e.g., the membrane-attached PGs syndecans, with heparan-sulphate and chondroitin-sulphate chains, and the HSPGs - glypicans - on epithelial and other cells
Basement-membrane heparan-sulphate PGs - basement membranes, e.g., perlecan

SMALL
Decorin/PGII (chondroitin/dermatan sulphates) - extracellular matrix
Biglycan/PG-S1 ( " ) - associated with a variety of cells including non-CT ones
Fibromodulin (keratan sulphate)
Dermatan sulphate
Small bone proteoglycans I & II

3 Non-collagenous glycoproteins of connective tissues include: Fibronectin, Tenascin, Thrombospondin, Bone sialoprotein/BSPII, Osteopontin/BSPI, Osteonectin/Bone Gla protein, Cartilage-matrix protein, Alkaline phosphatase, Chondronectin, and Fibrillin.
They interact with other macromolecules and influence cell behaviour.

One clinical aspect is their use as urinary or serum markers of excessive turnover, e.g., Gla protein for bone disease.
Fibrillin is a crucial component of elastic fibres and other structures in CTs; and genetic defects in its formation result in the weak arterial walls, poorly suspended eye lens, lax ligaments, etc. of Marfan's syndrome.

4 Fibronectin and Tenascin

5 For more on vulnerabilities from the cellular and ECM-molecular interactions see ECM <>

D TYPES OF CONNECTIVE TISSUES

Based upon: (a) the density and order of fibre packing; and (b) the predominant cell and fibre types.

l Areolar tissue
l Loose textured with a mixture of all cell and fibre types (but seldom pigmented cells).
2 Rich in ground substances which fill the spaces or areolae, and confer physical properties and control transport.
3 Locations - the lamina propria of the gut, under the skin, around joints, muscles and some viscera, and other sites needing some freedom of movement; the eye's choroid coat serving a more nutritive role also has pigment cells.
4 Areolar tissue merges with the somewhat denser CT of D.6. Both types may be regarded as belonging in one broad loose category.
5 Serous membranes are similar to areolar tissue but also have a layer of simple squamous mesothelium (sometimes two layers).
6 Milky spots on serous membranes are dense accumulations of the macrophages and lymphocytes present to protect serous body cavities.

2 White adipose tissue
l Comprises primarily fat cells enclosed in basal lamina, and held on a framework of reticular fibres in association with many blood capillaries.
2 Fibrous CT encloses the tissue, subdividing it with septa.
3 Found subcutaneously in the hypodermis (in the child, a panniculosus adiposus), and in the mesentery, omentum, and retroperitoneal area.
4 Padding fat in palmar, plantar and intraorbital sites is not so freely available as an energy store, and can survive starvation.
5 Adipose deposits in the hips, buttocks, and breasts are especially under the control of female sex hormones, but many hormones control fat metabolism.
6 Functions - energy store; insulation; padding; steroid conversions.

3 Brown adipose tissue
l Cells have many separate (multilocular) fat droplets, relatively more cytoplasm, and are smaller than white fat cells.
2 Found around the thorax and kidneys of animals naturally exposed to severe cold, particularly hibernators.
3 Brown fat is a thermogenic organ providing a prompt and direct source of heat to maintain the temperature of vital organs. Uncoupling protein 1 lets mitochondria divert energy in this otherwise unwanted thermal way by uncoupling respiration from ATP formation.
4 Seen in the human newborn; in adults BAT is detectable after adrenergic stimulation. Brown fat might dissipate surplus energy from overeating.

4 Reticular tissue
l Has the reticular fibre as the supporting fibre, and phagocytic fixed macrophages.
2 The fibres are made by some of the stellate reticular cells acting as fibroblasts.
3 Reticular tissue also contains parenchymal cells (the main working cells) held by the fibres, e.g., hepatocytes or lymphocytes.

5 Elastic tissue
l Elastic fibres or membranes are the predominant element.
2 The fibres may be:
(a) thick or very thick (l0-l5 µm) and orderly as in the elastic ligaments, e.g., ligamentum nuchae (in the neck of heavy-headed grazing animals), vertebral ligamentum flavum, penile suspensory ligament, and in the vocal chords; or
(b) finer and mixed with membranes in elastic arteries. The lung and airway also have many elastic fibres.
3 In the ligaments, elastic fibres are formed by fibroblasts and held together by reticular fibres, proteoglycan, and glycoproteins.

6 Dense fibrous (collagenous) tissue
Two kinds:
...(a) Regular, e.g., tendon, ligament, aponeurosis, fascia, with collagen fibres oriented to take stress principally in one direction. (The dense corneal stroma has very orderly collagen for transparency as well as strength.)
...(b) Irregular, e.g., dermis, organ capsules, periosteum, perichondrium, epitendineum, with irregular, interwoven bundles of collagen.

7 Loose fibrous (collagenous) tissue
l Although 6(b) and 7 have fibroblasts and collagen fibres as the principal elements, reticular and elastic fibres and other cells are present to a lesser degree, together with blood and lymphatic vessels and nerves.
2 An example of a loose fibrous tissue is the lamina propria of the urinary bladder, looser than dermis, denser than that of the gut. Indeed, the gut's lamina propria is so given over to defence and defensive cells that it is hardly recognizable as a CT.
. However, fibrous CTs form a continuum from dense, regular to areolar, making implausible any assignment to rigid categories.

8 Mucous/mucoid/primitive connective tissue
l Very rich in proteoglycans and water, has some fine collagen fibres and widely separated young fibroblasts.
2 As Wharton's jelly of the umbilical cord it encloses and cushions the vessels; the ocular vitreous and young dental pulp also fit tolerably well in this class.

E FUNCTIONS OF CONNECTIVE TISSUES

1 Mechanical and protective - supporting, restraining, binding, separating, directing and padding.
2 Transport of nutrients, metabolites, and signalling factors.
3 Storage of energy-rich lipids, water and electrolytes.
4 Defence against pathogenic organisms.
5 Repair of damage to itself, and organs supported or enclosed, by fibrosis - the formation of irregular collagenous scar tissue.
6 Thermogenesis (brown fat) and insulation (white fat).

Physiological factors controlling connective tissues are listed in Chapter 8.E.


William A Beresford, Anatomy Department, School of Medicine, West Virginia University, Morgantown, WV 26506-9128, USA - - e-mail: -- wberesfo@wvu.edu -- wberesfo@hotmail.com -- beresfo@wvnvm.wvnet.edu -- fax: 304-293-8159