Histology laboratory manual

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HISTOLOGY LABORATORY MANUAL 2016-2017 College of Physicians and Surgeons Columbia University Patrice F Spitalnik MD Histology Director pfs2101Columbia.edu 3 OVERVIEW OF THE COURSE As the structural and functional relationships of various cells, tissues, and organs are considered throughout the course, always be aware of one simple concept: All of the tissues and organs of the body are composed of cells and the extracellular products of cells (the matrix). It is for this reason that we emphasize the basic components of cells and their matrices during the early portion of the course. With an understanding of the nature of the relationship between cells and their matrices, we can proceed to the study of the organization of these two components into the basic tissues of the body. There are only four basic tissues in the body, although each category can be subdivided. In turn, the four basic tissues are organized into the various organs of the body, and these generally exist as interrelated functional units termed organ systems. The four basic tissues of the body are: 1) Epithelium 2) Connective tissue 3) Muscle 4) Nervous tissue Again, we emphasize: All of the organs of the body are composed of varying proportions of the four basic tissues, and each of the four basic tissues consists of cells and extracellular matrices. This simple concept is fundamental to the study of histology. MANUAL This manual is a guide to work in the histology laboratory. For each topic there is a brief introduction. This is followed by a list of images, with commentary. Note: The images were scanned from the Histology Slide Collection, which is listed at the end of this manual. Sets of these slides and microscopes are available for those who wish to use them. A few slides were not scanned (indicated by an asterisk). The online version of this manual is available at: http://www.columbia.edu/itc/hs/medical/sbpm_histology/index.html. In the online version, there are low power thumbnail images of the microscopic slides that have been scanned. These can be viewed from low to high magnification. There are also selected electron micrographs. The text of the online lab manual is an abbreviated version of this manual. TEXTBOOK Any of following textbooks are recommended: th Histology, A Text and Atlas, 7 ed., Michael H. Ross and Wojciech Pawlina, Lippincott Williams & Wilkins, 2016 th Junquiera’s Basic Histology, Text and Atlas, 13 ed., Anthony L. Mescher, McGraw Hill, NY, 2013. Histology and Cell Biology, an Introduction to Pathology, 4th ed., Abraham L. Kierszenbaum, Mosby, 2016. Whichever of these you choose, it is advisable to read the appropriate material in preparation for lab and bring the histology text to lab. 5 MICROSCOPY, CELLS, ORGANELLES, MITOSIS Learning objectives: 1. Understand and be able to describe how the most common dye combination, hematoxylin and eosin (H&E), stains various components of cells and tissues. 2. Identify cells and subcellular organelles. 3. Relate the appearance of a cell as seen with the light microscope (LM) with that at with the electron microscope (TEM, transmission electron microscope). 4. Recognize and understand the stages in mitosis. CELLS, ORGANELLES Slide Preparation: The first step in preparing a tissue or organ for microscopic examination is fixation, or preservation, of the specimen. Formalin is a commonly used fixative. Many other fixatives are available and are used in the study of specific structures. Note: There is a more complete description of methods for preparation of histological samples at the end of this laboratory manual (p. 92) under the heading “Histological Techniques”. The specimen on the microscope slide is a thin section (usually 5 micrometers) of the fixed tissue or organ. The section is stained by one or more dyes. Without staining the section would be nearly invisible with the microscope. Components of the specimen generally stain selectively and, on this basis, various regions of the specimen may be differentiated from each other. Most dyes are neutral salts. In some stains, the dye moiety is a cation and such dyes are called cationic or basic dyes. These form salts with tissue anions, especially the phosphate groups of the nucleic acids and the sulfate groups of the glycosaminoglycans. When the dye moiety is an anion, the dye is called anionic or acid dye and salt formation occurs with tissue cations including the lysine and arginine groups of tissue proteins. Tissue components that recognize basic dyes are "basophilic" and those that recognize acid dyes are "acidophilic". A common combination of stains is hematoxylin and eosin (H&E), which are commonly referred to as basic and acid dyes, respectively. 39 Colon H&E This slide illustrates different kinds of cells; do not be concerned at this time with the structure of the colon. Large numbers of cells are seen. Nuclei are basophilic and are stained blue. At lower magnifications they appear as blue dots and at higher magnifications chromatin and nucleoli may be identified within the nucleus. Surrounding the nucleus is the acidophilic cytoplasm stained pink (due to the positive charges on arginine and lysine). The luminal surface (center of the 40x view of colonic mucosa slide) is smooth and consists of pale cells (called Goblet cells), absorptive cells, and enteroendocrine cells that make up the mucosa. These cells are forming columnar structures called intestinal glands. The free surface of the cell, facing a lumen, is referred to as the cell apex and the opposite surface is the cell base. The lateral borders should be seen and contain structures that connect the cells together. Note that (luminal) mucosa is densely packed with cells arranged in rows. 6 HISTOCHEMISTRY 101 Small Intestine Periodic acid-Schiff (PAS) reaction and hematoxylin Carbohydrates, e.g., glycogen, proteoglycans, glycoproteins, glycosaminoglycans, are demonstrated by this histochemical reaction (see description PAS on p. 97 and in textbooks). Their presence is indicated by a red reaction product. Such structures are termed PAS positive. Note the intense reaction at the apical surface of the epithelial cells and within scattered goblet cells (containing mucin) at the luminal surface. Nuclei are stained blue because the section was counterstained with hematoxylin. POLARITY AND CELLULAR SPECIALIZATIONS Epithelial Cells in Glands In the diagram below identify the base and apex of the cells of a secretory unit, the acinus or gland. Note the basophilia in the basal compartment and the acidophilia in the apical (luminal) compartment of the cytoplasm. What subcellular organelle is responsible for attracting the basic stain? Pancreatic Acinus FYI: The pancreatic acini are apical surface the exocrine glands of the lumen pancreas. These cells contain secretory granules, which basal surface release digestive enzymes into nucleus the lumen of the acinus. The lumens of the acini converge into interlobular ducts, eventually merging to become secretory granules the pancreatic duct. 115 Gall bladder H&E - Microvilli Examine the cells lining the lumen of this organ, the gall bladder. The apical border of these cells faces the lumen. A border may be identified at the apex of the cells, which has slightly different optical properties from the remainder of the cell. Under optimum conditions faint striations, oriented parallel to the long axis of the cell, are seen in the border. These are difficult to resolve at the light microscopic level, but with electron microscopy, these striations are seen to be precisely arranged microvilli, containing cores of actin filaments. Why are the microvilli not visible on all cells lining the lumen? 40x view of gallbladder mucosa 117 Small intestine, H&E - Microvilli Study the cells lining the lumen of the small intestine as another example of a microvillous border. The lining cells of the small intestine will be studied in more detail at a later time. 7 68 Uterine, Fallopian tube H&E – Cilia and basal bodies Locate the cells lining the lumen of the oviduct. Using the 20-40X, study the free surface of the cells. The majority have fine, hair-like projections called cilia. At the apex of these cells note the pink line, which indicates the presence of the basal bodies that give rise to the cilia. Consult electron micrographs for the content and morphology of cilia and their basal bodies. There are also secretory cells along this epithelium. These have elongated nuclei and sometimes project above the epithelial surface Uterine Tube Epithelium cilia secretory cell basal bodies nuclei basement membrane MITOSIS Mitosis is divided into four distinct stages: prophase, metaphase, anaphase, and telophase. During prophase, the nuclear envelope disperses, replicated chromosomes condense, and the two sister chromatids become attached at a site called the centromere. At metaphase, duplicated chromosomes become aligned in a single plane. At anaphase A, the sister chromatids separate and begin to migrate to the poles. At anaphase B, the sister chromatids continue to migrate toward the poles and the microtubules of the spindle elongate. During telophase, the sister chromatids reach the poles, the nuclear envelope re-forms and the chromosomes decondense. Cytoplasmic division usually begins in anaphase and is complete by the end of telophase. Slide 112: Whitefish Mitosis The whitefish embryo has been stained with hematoxylin and eosin (H&E). There are examples of cells at all stanges of the cell cycle since the cells are dividing asynchronously. Assess nuclear envelope breakdown, chromosome condensation, mitotic spindle development, and location of condensed chromosomes in the whitefish mitotic cells. On the basis of these parameters, identify and determine the distinguishing features of cells in prophase, metaphase, anaphase (A and B) and telophase. 8 ELECTRON MICROGRAPHS Examine the listed electron micrographs so that you understand the ultrastructural equivalents of the structures you have seen at the LM level on the slides. Also use the electron micrographs to examine organelles that are not visible at the LM level. QUESTIONS Answers to questions for all laboratories are given at the end of the laboratory manual, p. 86. 1. What are serial sections and why are they important? 2. What is the relationship between heterochromatin and the synthetic activity of DNA? 3. What is the site of synthesis of ribosomal RNA? 4. What is the function of mitochondrial cristae? 5. What is the functional significance of the Golgi complex? 6. How many membranes comprise the nuclear envelope? The wall of a mitochondrion? 7. What are the cytological and functional differences between cilia and microvilli? 8. Know the structural characteristics and functional significance of the following organelles and inclusions: nucleus, nucleolus, ribosomes, endoplasmic reticulum (two types), mitochondria, Golgi apparatus, lysosomes, microtubules, cilia, microvilli, glycogen, lipid, peroxisomes. Which of these require more than pure light microscopy for their identification? 9 EPITHELIUM Learning objectives: 1. Recognize the different types of epithelia. 2. Relate characteristics of particular epithelia to their function, keeping in mind their essential features including junctions, apical modifications, and polarity. An epithelium is a layer or sheet of cells that covers a surface or lines a cavity. Functions of epithelia include formation of a protective layer (epidermis), absorption of water and solutes (intestine), secretion (intestine, various glands) and excretion (kidney tubules). Classification of epithelia is generally based upon two criteria: number of cell layers and cell shape. Simple epithelia are one cell layer thick and stratified epithelia are two or more cell layers thick. Pseudostratified epithelium is an intermediate type that appears stratified but really is one cell layer thick. The shape of epithelial cells may be squamous, cuboidal, or columnar; intermediate forms are often encountered. Stratified epithelia are classified according to the shape of the cells at the free surface and can be squamous, cuboidal, columnar, or transitional. Transitional epithelia line cavities in the urinary tract, which may be distended, and the thickness of the epithelium varies with the degree of distention. Beneath the layer of epithelial cells is an underlying non-cellular structure known as the basal lamina, which is secreted by the epithelial cells. The basal lamina is often associated with an additional layer secreted by other cells. Together the basal lamina and the underlying layer make up the basement membrane, which can usually be seen with light microscopy. Higher magnification (e.g., electron microscopy) is usually required to resolve the basal lamina. Epithelial Tissue Types simple columnar simple cuboidal pseudostratified stratified cuboidal stratified squamous transitional 10 SIMPLE CUBOIDAL AND SIMPLE COLUMNAR EPITHELIA Simple cuboidal epithelia 50 Kidney, H&E Examine the cuboidal epithelium that makes up the kidney tubules. Find the basement membrane and lumen of the tubules to help you determine the basal and apical membranes, respectively. Note that in some cases the lateral borders of cells are distinct while in many they are not. This is because they are highly interdigitated, a configuration that increases the surface area for transport across the cell membranes. This can be seen in electron micrographs of kidney tubules. Remember that each nucleus corresponds to one cell. 107 Pancreas, Acid fuchsin and toluidine blue Examine the epithelial cells. Note the basophilic structures that at the base of the cells are rough endoplasmic reticulum. At the apex of the cell, secretory granules appear as acidophilic structures. The contents of these granules are proteins, which are the precursors of digestive enzymes. Review the subcellular structures involved in protein synthesis. Simple columnar epithelia 115 Gall bladder. H&E Examine the lining of the gall bladder as an example of simple columnar epithelium. Be sure you locate regions where the epithelium is cut longitudinally to observe the simple columnar epithelium. Note the microvillous border that you identified in the previous lab. In tangential sections portions of cells in various planes of section may give the impression that the epithelium is stratified. Why do the nuclei appear at different levels in tangential sections? 101 Small Intestine (PAS and hematoxylin) Locate the epithelium with its microvillous border and PAS positive glycocalyx. The basal lamina is also PAS positive, but is not intensely stained. What is the basis for PAS stain? 102 Small intestine (Bodian/silver) This slide illustrates modifications found in the apical region of the columnar epithelium: the striated (microvillous) border and the junctional complex. In longitudinally sectioned cells, the junctional complex is seen as a dark dot of silver deposit at the apical lateral borders of the cells. In regions where the epithelium has been cut in cross or oblique section, the junctional complex has a belt-like appearance and can be seen to encircle the cells (hexagonal Junctional complex in cross-section shape). What types of intercellular junctions are commonly found in epithelia? Review the appearance of junctional complexes at the EM level in the electron micrograph in the lab. In addition the Bodian silver stains secretory granules within enteroendocrine cells in the epithelium and the basal lamina. 11 PSEUDOSTRATIFIED EPITHELIUM 5 Trachea, H&E Pseudostratified epithelium appears to be stratified as nuclei are seen at various levels. In fact all of the cells rest on the basal lamina, but not all of the cells have apices that reach the lumen. The cells that are confined to the base are stem cells that are the sources of the cells whose apices do reach the lumen. Identify the two major types of cell that reach the lumen. What are their characteristics? What is responsible for the eosinophilic line at the apex of the majority of the cells? Note the position of the nuclei. STRATIFIED EPITHELIUM In this type of epithelium, no cells on the basal lamina reach the lumen. The layer of cells that rests on the basal lamina is the source of the upper layers of cells. Stratified squamous keratinized epithelium 4 Skin, H&E The epithelium of the skin is known as the epidermis. Its superficial layer is comprised of keratinized (cornified) squamous cells. Note the multiple layers of the stratified squamous epithelial layer of the skin. As cells are displaced toward the skin surface their characteristics change. They ultimately die and are sloughed. This topmost keratinized layer stains dark pink and has no nuclei because the cells are dead. Stratified squamous non-keratinized epithelium 33 Esophagus, H&E. The stratified squamous epithelium lining the esophagus is non-keratinized in humans, but keratinized in some other species. Which part of the slide corresponds to the esophageal lumen? TRANSITIONAL EPITHELIUM (UROTHELIUM) 54 Urinary Bladder, H&E This is a special, highly impermeable, type of stratified epithelium found only in the urinary tract. The surface cells change their shape depending upon the volume of the lumen. In this slide they are dome-shaped because the bladder is empty and is in the contracted state. Consult electron micrographs to understand the morphological changes that accompany expansion and contraction of the lumen. ELECTRON MICROGRAPHS Examine the electron micrographs so that you understand the ultrastructural equivalents of the structures you have seen on the slides. 12 REVIEW Reviewing what you have learned in the first labs, be sure that you know the structural characteristics and functional significance of the following organelles and inclusions, and be able to identify them in light and/or electron micrographs: nucleus, nucleolus, mitochondria, Golgi apparatus, lysosomes, ribosomes, endoplasmic reticulum (two types), microtubules, cilia, microvilli. apical surface microvilli zonula occludens lysosome zonula adherens ribosomes gap junction golgi body endoplasmic reticulum nucleolus mitochondria nucleus basal lamina basal surface 13 CONNECTIVE TISSUE Learning objectives: 1. Know that cells, fibers and ground substance constitute connective tissue. 2. Be able to describe the relationship of these constituents, their structures and functions. 3. Learn the distribution of collagen types (Types I, II, III and IV) in the connective tissue types. Connective tissue is comprised of cells, formed fibers, and amorphous extracellular matrix (ground substance). Both the fibers and ground substance are secreted by the connective tissue cells that are interspersed and embedded in the matrix. Functions of the connective tissue include support and binding together of the other tissues; providing a medium for the passage of metabolites; serving as a storage site for lipids, water and electrolytes; aiding in protection against infection by an inflammatory reaction mediated by cells that have migrated into the connective tissue from the blood; and repair by the formation of scar tissue. Connective tissues are derived from the embryonic connective tissue or mesenchyme. Mesenchyme is derived primarily from the mesodermal germ layer of the developing embryo, but the ectodermal neural crest is known to give rise to some mesenchymal cells (ecto- mesenchyme). See examples in subsequent lab (Cartilage, Bone, Bone Development). CONNECTIVE TISSUE A CLASSIFICATION OF CONNECTIVE TISSUE A. Loose Connective Tissue 1. Subcutaneous tissue, lamina propria. B. Specialized Connective Tissue 1. Reticular connective tissue - forms a supporting framework for spleen, lymph nodes, bone marrow, liver, glands, and striated muscle fibers. 2. Adipose connective tissue - a modification of reticular connective tissue, characterized by an extensive intracellular accumulation of lipid droplets. C. Dense, Irregular Connective Tissue 1. Predominantly collagenous - dermis, capsule of spleen and other organs, such as the prostate gland. 2. Predominantly elastic - for example, the elastic membrane of large arteries. D. Dense, Regular Connective Tissue 1. Collagenous - tendons, most ligaments, cornea 2. Elastic - elastic ligaments (ligamentum nuchae flavate and interspinous ligaments), true vocal cords E. Mesenchyme (Embryonic Connective Tissue) Primitive connective tissue that contains precursors for connective tissue, as well as other tissue types. LOOSE CONNECTIVE TISSUE This form of connective tissue has the largest number of cells per unit volume of extracellular matrix. The large number of cells frequently makes it difficult to distinguish the fibrous component without the use of special stains. The fibers in the matrix have a loose and irregular arrangement, and they consist of collagenous, elastic, or reticular fibers. Fibroblasts and macrophages are the most common cells in loose connective tissue, but mast cells, plasma cells, neutrophils and fat cells may also be found. 14 39 Colon, H&E. Examine the scanned image at low power, and note that one surface is indented by pits that are lined by columnar epithelial cells. Immediately beneath these cells is the loose connective tissue called lamina propria. Mucosa of the colon epithelium FYI: The mucosa of the colon is comprised of the epithelium and lamina propria. It is lamina propria separated from the submucosa by the muscularis mucosae muscularis mucosae RETICULAR CONNECTIVE TISSUE Reticular tissue, a type of loose connective tissue in which reticular fibers are the most prominent fibrous component, forms the supporting framework of the lymphoid organs (lymph nodes, spleen, tonsils), bone marrow and liver. Reticular fibers (type III collagen) are too thin to stain in ordinary histological preparations, but they are readily demonstrated by techniques involving the reduction of silver from silver nitrate by the glycosaminoglycan surface coat. 22 Lymph Node, ( Silver Stain) Identify the outer, collagenous connective tissue capsule surrounding the lymph node and the trabeculae, which are projections of the capsule that penetrate into the interior of the node. These fibers (type I collagen) are colored red-brown by the counterstain Azo-carmine. The black, silver-stained reticular fibers form the supporting framework for the cortical nodules. The lymphocytes, which are located within the interstices of this framework, are not well seen in this slide. The organization of lymph nodes will be studied in a future laboratory. ADIPOSE CONNECTIVE TISSUE Trabecula surrounded by reticular fibers 46 Skin, scalp, H&E Lying deep to the dermis is the loose subcutaneous connective tissue layer (superficial fascia). The subcutaneous connective tissue may be composed largely of adipose tissue. The epithelium (epidermis) and dense, irregularly arranged connective tissue appear deeply stained. The adipose connective is the palely stained region. At higher magnification observe that the intracytoplasmic lipid has been extracted from the fat cells during the histological preparation of the tissue. The thin peripheral ring of cytoplasm and the flattened peripheral nucleus, coupled with the large central vacuole results in the "signet ring" appearance of fat cells. In white fat each cell contains a single fat droplet (unilocular). 15 16 Aorta, Cross Section In the connective tissue surrounding the aorta, note the presence of both white and brown adipose cells. At higher magnification observe the white fat in which each cell contains a single fat droplet (unilocular). In brown fat cells the lipid is accumulated in droplets, giving the cells a multilocular appearance. Where is the majority of brown fat found in humans? DENSE, IRREGULAR CONNECTIVE TISSUE In this connective tissue collagenous fibers predominate. 4 Skin, (H&E) Under the stratified squamous epithelium examined earlier is the dense irregular connective tissue of the dermis. Its thick collagenous (type I) bundles stain intensely with eosin and can be seen to course in various directions. 70 Breast, Inactive (H&E) Find the regions that contain ductal tissue at low power. Immediately surrounding the lining cells is a very small zone of pale-staining loose areolar connective tissue. Outside of this, there is a large zone of acidophilic dense irregularly arranged connective tissue. Clumps of white adipose tissue are scattered throughout the stroma of the mammary gland. From left to right: duct surrounded by loose DENSE, REGULAR CONNECTIVE connective tissue, dense connective tissue, TISSUE adipose tissue Collagenous 3 Muscle and Tendon, (H&E) The thick, collagenous bundles of the tendon run parallel to each other, slight waviness of the tissue is due to fixation. Rows of fibroblasts with heterochromatic nuclei are aligned between the collagenous bundles. Compare the appearance of the collagen bundles (Type I collagen) and fibroblasts with that of the skeletal muscle fibers on the same section. Tendon top, skeletal muscle bottom 11 Bone, rib (H&E) Find the regions of the dense fibrous regularly arranged connective tissue (tendon). Collagen is stained pink and can be distinguished from skeletal muscle that is stained purple. Note the fibroblasts aligned along the collagen fibers in the tendon. These are flattened cells with heterochromatic nuclei. 16 Elastic 20 Aorta, (Elastin stain) The fibers are predominantly elastic rather than collagenous. Elastic fibers stain reddish-brown to black and form prominent fenestrated, elastic sheets in the aorta. Most Common Types of Collagen ELECTRON MICROGRAPHS Examine the electron micrographs. QUESTIONS 1. Are reticular fibers distinguishable in tissue stained with H&E? 2. Why do adipocytes appear empty? 17 CARTILAGE, BONE, BONE DEVELOPMENT Learning objectives: 1. Describe the components and organization of cartilage and bone. 2. Relate the structure of bone and cartilage to their function. 3. Understand the differences between the development and growth of cartilage and bone. 4. Describe the processes of intramembranous bone development and endochondral ossification. CARTILAGE Cartilage is a specialized type of connective tissue whose unique combination of rigidity, elasticity, and resilience is due primarily to the special properties of its matrix. As in other connective tissues, its matrix is composed of fibers (collagenous or elastic) and a ground substance that is rich in extracellular glycosaminoglycans (particularly the chondroitin sulfates). Cartilage is avascular, but its matrix is permeable to nutrients and waste products. Cartilage is the primary skeletal tissue of the fetus, and it serves as a model for the development of endochondral bone. In the adult, cartilage forms the articular surfaces of joints, the skeleton of the external ear, the septum of the nose, supporting rings and plates of the trachea and bronchi, and intervertebral discs. Three types of cartilage are found in the adult: hyaline, elastic, and fibrocartilage. These are classified according to the predominant component of their extracellular matrix. As in other connective tissue classifications, there are gradations between these basic types. HYALINE CARTILAGE 5 Trachea (H&E) At low power, locate the incomplete rings of hyaline cartilage in the wall of the trachea. At higher magnification observe that a perichondrium surrounds the cartilage; this merges with the cartilage on one side and with the surrounding connective tissue of the other side. Blood vessels within the perichondrium provide the blood supply for the avascular cartilage. Chondroblasts are cells adjacent to the perichondrium and recently derived from it. They are not yet completely embedded in the matrix. Mature cartilage cells or chondrocytes are surrounded by matrix and lie within spaces called lacunae. In life the chondrocytes completely fill the lacunae. Note that the cartilage matrix is relatively homogeneous and basophilic. This is due to the From top to bottom: masking of the collagen fibers by the high concentration of cartilage, pericardium, the glycosaminoglycans in the ground substance. connective tissue ELASTIC CARTILAGE 6 External Ear (Elastin Stain) Elastic cartilage provides support with flexibility. The general organization of this type of cartilage is similar to that of hyaline cartilage, except that elastic fibers predominate over collagen fibers in the matrix. Elastic fibers are stained specifically (black) by the Weigert's stain. Where else does elastic cartilage occur in the body? Elastic cartilage 18 FIBROCARTILAGE Fibrocartilage can be considered as a transitional type of tissue, between hyaline cartilage and dense collagenous connective tissue, and it occurs in regions where support and great tensile strength are desirable. QUESTIONS: 1. What are the mechanisms of cartilage growth? 2. Are blood vessels found in cartilage and how does this relate to the nutrition of cartilage? BONE Bone is a calcified connective tissue, and like other connective tissues, it consists of cells, fibers, and ground substance. The deposition of inorganic calcium phosphate salts as hydroxyapatite crystals within its matrix is a distinguishing characteristic of bone. This renders it structurally rigid. In addition, bone functions as a homeostatic reservoir of calcium and phosphate ions and it encloses the hematopoietic elements of the bone marrow. There are two types of mature bone, compact (lamellar) and spongy (trabecular or cancellous). Compact bone is characterized by the regularity of its collagen fibers. Spongy bone consists of a lattice of branching bony spicules, known as trabeculae, which are surrounded by bone marrow in some regions. When the trabeculae are sufficiently thick, they may contain osteons (see description below). Immature (woven) bone (see below in "bone development") is the first bone laid down in prenatal life or in the repair of bone fractures. In this type of bone, the matrix immediately surrounding the osteoblast is called osteoid and is not mineralized. Immature bone is characterized by irregularly arranged, interwoven collagenous fibers within a matrix containing proteoglycans. Because of its calcified matrix, bone presents difficulties in its preparation for microscopic study. There are two basic techniques for studying bone with the light microscope, and both of these types of preparations must be studied to appreciate the organic and inorganic components of bone. (1) Bone may be decalcified by acid solutions prior to embedding and sectioning. This permits study of the cells and organic matrix of the bone. (2) To study the lamellar and canalicular pattern of the calcified matrix, it is necessary to grind down dried bone that has not been decalcified to a thickness that permits the microscope light to be transmitted (“ground bone”). GROUND BONE 9 Dried Bone, Shaft of Tibia Cross and longitudinal sections (unstained). Use the illustrations in your textbook as a guide and identify the following structures. Haversian Systems (osteons) are distinctive structural units of compact bone that reflect the developmental and nutritive pattern of its lamellar configuration. Haversian systems consist of Haversian canals containing blood vessels and nerves surrounded by concentric lamellae of bone. Lacunae lie between or within the lamellae. In life these lacunae are occupied by osteocytes. Lacunae are connected with each other, and ultimately with the perivascular spaces of the Haversian canal, by canaliculi. This communicating system of canaliculi is essential for exchange of gases and metabolites between the osteocytes and the perivascular spaces of the Haversian canal. Volkmann's canals, which also contain vessels and nerves, are larger in diameter than Haversian canals and run perpendicularly to them. 19 Interstitial lamellae lie between the more distinct Haversian systems; these are the remnants of earlier Haversian systems that have been partially resorbed during bone remodeling. C B A A: four Haversian systems and interstitial lamellae. B: Haversian system. C: Haversian systems at left, Volkmann’s canal at right Depiction of development of an osteon (Haversian system) DECALCIFIED BONE 11 Rib, Cross Section, (H&E). Surrounding the sectioned rib are bundles of skeletal muscle, tendon, nerves and blood vessels. Note the gradation of the surrounding connective tissue with the periosteum and the increased cellularity of the periosteum. The reversal lines (also known as cementing lines) that delimit the Haversian systems may appear refractile or slightly basophilic. What accounts for this basophilia? Trabeculae of bone extend into and partially subdivide the marrow cavity, which contains hematopoietic bone marrow. Bone marrow will be studied in more detail in a later lab. 8 Rib and Cartilage, (H&E) This slide demonstrates periosteum, which has dense cortical bone on the surface (better illustrated in the preceding slide) and spongy bone centrally. Osteoblasts are prominent on the surface of the bony trabeculae. Osteoclasts (multinucleated giant cells with acidophilic cytoplasm, related to the process of bone resorption) may also be seen near the osteochondral junction. Calcifying cartilage and rows of hyaline cartilage cells are present and 20 extend into the cartilage of the proximal end of rib. Around the rib section, skeletal muscle and tendon are present. Osteoblasts Osteoclast QUESTIONS 1. What structures are found within Haversian canals? 2. Is the osseous lamella adjacent to the Haversian canal the youngest or the oldest lamella of a particular osteon? Be sure you know how cartilage and bone differ morphologically, functionally, and with respect to blood supply. BONE DEVELOPMENT The process by which bone is formed is termed osteogenesis or ossification. Bone is never formed as a primary tissue, it always replaces a preexisting support tissue. There are two types of bone formation: intramembranous and endochondral ossification. INTRAMEMBRANOUS OSSIFICATION In intramembranous ossification, bone develops directly on or within membrane formed by mesenchyme. This, like all bone growth, is appositional. This process can be studied best in the irregular bones of the face and the flat bones of the skull. 94 Parietal bones, Human fetuses, 3.5 and 6.5 mos. –Decalcified At low power note the appearance of the developing bone as well as the total absence of cartilage. In the bone from the older fetus, scalp is present in which numerous hair follicles can be seen. Note the connective tissue has begun to condense as a fibrous periosteum on either side of the anastomosing trabeculae of the growing bone. The trabeculae surround large spaces (primitive marrow cavities) containing embryonic connective tissue, thin-walled blood vessels, and nerves. In active regions, a unicellular row of osteoblasts (each with an eccentric nucleus and strongly basophilic cytoplasm) lines the surface of the trabeculae. Osteoclasts may be seen to occupy shallow pits in the bone (Howship's lacunae). Within the trabeculae, notice osteocytes in their lacunae and the woven bone matrix, which, unlike that of mature bone, is unevenly stained pink and exhibits a patchy basophilia. The acidophilic collagenous fibers embedded in the matrix tend to be obscured by the matrix. At these stages the matrix is not calcified (i.e., contains no calcium phosphate salts). This uncalcified early bone is termed the osteoid. Later, minerals are deposited as minute hydroxyapatite crystals (calcium phosphate salts) in close association with the collagenous fibers to form a solid rigid matrix. 21 ENDOCHONDRAL OSSIFICATION Endochondral ossification involves the replacement of a cartilage model by bone. This ossification process can be studied in the long bones, such as the bones of the limbs. In a typical adult long bone, one can distinguish grossly a cylindrical shaft or diaphysis of compact bone (with a central marrow cavity) and, at the ends of the shaft, the epiphysis, each consisting of spongy bone covered by a thin peripheral shell of compact bone. In the growing long bone, the epiphysis and the diaphysis are united by a transitional zone called the metaphysis. diaphysis metaphysis epiphysis Events in development of long bone: 1. A hollow cylinder called the periosteal collar forms through intramembranous ossification around the middle of the cartilage model. The periosteal collar causes the underlying cartilage cells to begin to degenerate and die. 2. The primary center of ossification begins with calcification of matrix at the diaphysis and eroding by blood vessels. These blood vessels bring osteoprogenitor cells with them when they penetrate the bone collar. 3. The osteoprogenitor cells differentiate into osteoblasts and begin depositing matrix, forming spicules. 4. Secondary centers of ossification begin in the epiphysis at each end with invasion by blood vessels 22