Exercise 3 Connective Tissue

Objectives : After completing the laboratory exercise, you should be able to: § § § § § § § § § § § §

Identify the following connective tissues in light microscopic preparations: dense irregular, dense regular; white and brown adipose; mucous; elastic. Identify each of the following connective tissue cell types in light microscopic preparations, and describe the known functions of each in general terms: fibroblasts, macrophages, mast cells, plasma cells, fat cells, neutrophils, lymphocytes, and eosinophils. Identify irregular and regular connective tissues in light microscopic preparations. Identify the three types of cartilage and the cells found in them. Identify the major non-cellular components of bone, and the cell types normally present (other than in bone marrow). Identify osteocytes, osteoblasts, and osteoclasts in microscopic sections. Identify the components of a synovial joint in microscopic preparations. Identify the perichondrium. Identify all the cell types of bone and the process of ossification. Identify periosteum Identify the non-cellular components and structural features of compact and spongy cancellous bone. Identify different zones in the developing endochondral bone, including: the light microscopic appearance of the growth plate.

Introduction The human body is supported and held together by a diverse range of tissues that are traditionally called connective tissue. This name implies a structural role, but the function of connective tissue extends far beyond a simple role as a framework. Connective tissue has three elements: fibers, cells, and non-cellular matrix material. The proportion of the three varies widely in different connective tissues, and it is the proportion among them that give any connective tissue its characteristic properties.

Embryonic Connective Tissue The mesoderm gives rise to almost all of the connective tissues of the body. Through proliferation and migration of the mesodermal and specific neural crest cells, a primitive connective tissue referred to as mesenchyme is established in the early embryo. Mesenchyme contains relatively uniform appearing, small spindle shaped cells. Processes of these cells extend and contact similar processes of neighboring cells forming a three-dimensional cellular network. The extracellular space is occupied by a viscous, almost jelly-like ground substance. Collagen (reticular) fibers are present, but they are very fine and relatively sparse in number List of Slides – Embryonic connective tissue § § § § §

CT-1 CT-2 CT-3 CT-4 CT-5

Mesenchyme, chick embryo Mesenchyme, chick embryo, developing limb Umbilical cord, Wharton’s jelly, fetus Umbilical cord, human, t. s. Amphibian skin (also melanocytes)

REVIEW THE FOLLOWING BOX SLIDES 16, 28, 29, 72

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Loose Connective Tissue Loose connective tissue, also called areolar tissue, is a cellular connective tissue. The fibers are thin and relatively sparse, the ground substance is abundant and small blood vessels run through it. Spaces or gaps in the matrix of this tissue are also formed during preparation. In a slide preparation the collagen fibers can be sectioned in various planes and transverse ends may be seen. Collagen fibers have various diameters and appear longitudinally striated because of their fibrillar structure. The fibers are acidophilic and stain with eosin. Thin elastic fibers are also present but are difficult to distinguish when stained with H & E. Also present in loose connective tissue are blood cells, neutrophils, lymphocytes and blood vessels sectioned in several planes. The primary location of loose connective tissue is beneath those epithelia that cover the body surfaces and line the internal surfaces of the body. It is also present in association with the epithelium of glands and surrounds the smallest blood vessels . List of slides – Loose connective tissue CT-6

Areolar

Dense Connective Tissue Irregular dense connective tissue : In dense irregular connective tissue the collagen fibers are larger, more numerous and more concentrated. Elastic fibers, which can be seen if selectively stained (orcein), are also larger and more numerous. In this tissue, the collagen fibers show very random and irregular orientation. The cell population is sparse and is typically of a single, the fibroblast. Dense irregular tissue is present in hollow organs, like the intestinal tract, allowing the organ to exhibit resistance to excessive stretching and distension. Similarly skin contains a relatively thick layer of irregular connective tissue in the dermis. List of slides – Irregular dense connective tissue CT-11 Irregular, around blood vessels CT-12 Irregular, loose and dense, skin

Regular dense connective tissue : Dense regular connective tissue is present in ligaments and tendons. The collagen fibers are arranged in compact, parallel bundles. Between these bundles are thin partitions of looser connective tissue that contain parallel rows of fibroblasts. These cells have short processes (not visible in most preparations and the nuclei appear ovoid when seen in surface view or rod-like in lateral view. List of slides – Regular dense connective tissue CT-19 Dense regular tissue, tendon CT-20 Tendon, monkey, t. s.

Reticular dense connective tissue : Reticular connective tissue has fine reticular fibers in it. These fibers form very delicate stranded networks, rather than thick bundles, and they provide the supporting internal stroma for cells in the spleen and other lymphatic organs. They are also present in the outside of small blood vessels and form part of the basal lamina of epithelial sheets. Reticular fibers are simply a very fine form of collagen. Reticular fibers can be clearly seen when stained by a silver 19

impregnation method (Wilder’s reticulum method). They can also be visualized by using the PAS reaction because they are relatively rich in sugar groups. After silver treatment, the fibers appear black. List of slides – Reticular dense connective tissue CT-13 Reticular tissue, lymph node, silver CT-14 Reticular tissue, spleen, silver

Elastic dense connective tissue: Elastic connective tissue, as you might expect, is noted for its springiness. It has some collagen fibers in it, but its fibers are principally made from elastin, which is chemically different from collagen. Elastin fibers stain with eosin, but not well, thus, they cannot always be distinguished from collagen fibers in routine H & E preparations. However, elastic fibers can be selectively stained with special dyes like orcein or resorcin-fuchsin. List of slides – Elastic dense connective tissue CT-15 CT-16 CT-17 CT-18

Yellow elastic Yellow elastic, fibers stained black Yellow elastic, teased White fibrous tissue

REVIEW THE FOLLOWING BOX SLIDES 7, 66, 69, 70, 74, 75 and almost any other slide in the collection.

Adipose Connective Tissue Adipose connective tissue is very cellular and has little if any matrix and few fibers. There are two types of adipose connective tissue, white and brown. White adipose tissue or “white fat” is very widely distributed and quite easy to find. It has the appearance of “chicken wire”. At high magnification you should be able to make out the “signet ring” appearance of individual cells, with the nucleus pushed to one side and the cytoplasm squeezed into a thin ring by the single large fat droplet in the cell. Individual adipose cells appear as empty cells because the fat was dissolved by chemicals used during routine histologic preparation of the tissue. Because adipose cell nuclei are compressed in the peripheral rim of the cytoplasm it is difficult to distinguish between fibroblast nuclei and adipose cell nuclei. Brown fat is more restrictive in its distribution. It is found in the interscapular region of most mammals. Note that in this tissue there is not one single fat droplet inside the cell, rather there are several smaller ones. List of slides – Adipose connective tissue CT-7 CT-8 CT-9 CT-10

Adipose, white, mammal Adipose, white, monkey, t. s. Adipose, white, human, t. s. Adipose, brown, mammal

Specialized Connective Tissue Cartilage Cartilage is a special form of connective tissue that has the main function of supporting soft tissue. It consists of cells (chondrocytes and chondroblasts) and the matrix (fibers and ground substances). The matrix contains collagen and elastic fibers which give the cartilage its firmness and resilience. There are three types of cartilage in the body: hyaline, elastic, and fibrocartilage, with hyaline cartilage being the most common type.

Hyaline cartilage: Sections of hyaline cartilage show ovoid spaces (the lacunae) with mature cartilage cells, the chondrocytes, inside. These spaces are distributed throughout the homogeneous ground substance, the matrix. Each cell has a granular cytoplasm and a nucleus. During histologic preparations the chondrocytes shrink and the lacunae appear as clear 20

spaces. Cartilage cells in the matrix are observed either singly or in isogenic groups. Lacunae and chondrocytes are larger in the middle, but become progressively smaller and flatter in the periphery. List of slides – Hyaline cartilage CT-21 CT-22 CT-23 CT-24 CT-25 CT-26 CT-27 CT-28 CT-29

Hyaline, trachea Hyaline, mammal Hyaline, monkey, t. s. Hyaline, trachea, mammal Hyaline, trachea, mammal Hyaline, trachea, bird Hyaline, frog Hyaline, lamprey Hyaline, shark

Elastic cartilage: Elastic cartilage differs from hyaline cartilage principally by the presence of elastic fibers in its matrix. If stained with orcein, these are visible as deep purple fibers. The density of fibers in the matrix varies among elastic cartilages as well as among different areas of the same cartilage. As in the hyaline cartilage, larger chondrocytes in lacunae are seen in the interior of the plate. The smaller ones are more peripheral. List of slides – Elastic cartilage CT-30 CT-31 CT-32 CT-33 CT-34 CT-35

Hyaline and elastic Elastic Elastic Elastic with adipose cells Elastic, monkey Elastic, human

Fibrous cartilage: In sections of fibrous cartilage the matrix is permeated with collagen fibers which frequently exhibit parallel fiber arrangement as seen in tendons. Small chondrocytes in lacunae are distributed in rows within the fibrous matrix rather than at random or in isogenous groups as is normally seen in hyaline or elastic cartilage. All chondrocytes and lacunae are of similar size. Fibrous cartilage usually forms a transitional area between hyaline cartilage and tendon or ligament. List of slides – Fibrous cartilage CT-36 Fibrocartilage

Bone Bone is also a special form of connective tissue. Like other connective tissues, bones consist of cells, fibers and matrix. Bone development begins in the embryo by two distinct processes: intramembranous ossification and endochondral ossification. Bones produced by the two different methods, however, have the same histologic structure. Review the processes of endochondral and intramembranous ossification when you study the following slides.

Intramembranous ossification: In intramembranous ossification, bone develops not from a cartilage model but from the connective tissue mesenchyme. Some of the mesenchyme cells differentiate directly into osteoblasts and produce the bony matrix. The mandible, maxilla, clavicles and most of the flat bones of the skull are formed by the intramembranous ossification method. 21

When looking at the MB slides, be sure to identify the following ele ments: 1. 2. 3. 4. 5.

Trabeculae Osteocytes – located inside the lacunae in the bone trabeculae Primitive narrow cavity – tissue located between the formed bony trabeculae having blood vessels, blood cells and hematopoietic tissue Osteoclasts – multinucleated giant cells associated with bone resorption and remodeling Osteoblasts – cells developed from mesenchymal cells and are seen in linear arrangement along the osteoclasts

List of slides – Intramembranous ossification MB -1 MB -2 MB -3 MB -4 EB-5 EB-6 EB-7 EB-8

Mesenchyme, chick embryo Membrane bone formation, fetus Membrane bone, human Mandible, chick embryo Chick, limbs Digit, mammal Long bone, l. s. Long bone, c. s.

Endochondral ossification: Endochondral ossification forms most of the bones of the skeleton. Each bone is first preceded by a temporary hyaline cartilage model. This model first continues to grow, then the chondrocytes hypertrophy and mature, finally the growing cartilage model calcifies. As a result, the chondrocytes die and the fragmented calcified matrix then begins to serve as a structural framework for the deposition of the bony material. When looking at slides of endochondral ossification try to identify the following: 1. 2. 3. 4. 5. 6. 7. 8.

Hyaline cartilage matrix Proliferating chondrocytes Degenerating chondrocytes Osteocytes Osteoclasts Osteoblasts Marrow cavity Osteoids

Study the following slides of adult endochondral bones and identify the histologic elements. Cancellous bone: 1. 2. 3. 4. 5.

Periosteum Trabeculae Marrow cavity Osteocytes (in lacunae) Osteoclasts

Compact bone: 1. 2. 3. 4. 5.

Haversian canals Canaliculi Lacunae Perforating (Volkmann’s) canals Cement line

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List of slides – Endochondral ossification EB-9 EB-10 EB-11 EB-12

Cancellous bone Compact bone, decalcified, l. s. Compact bone, decalcified, c. s. Bone, ground section

Joints Joints may be classified into two main functional groups, synovial and non-synovial. Synovial joints allow for extensive movement of the bones upon one another at the articular surfaces. List of slides – Synovial joint EB-13

Synovial joint

Identify the following structures: 1. 2. 3.

The two bones at the joint Synovium – a layer of specialized cartilage. Can you identify the type of cartilage? Tendon (s)

List of slides – Non-synovial joint EB-14

Tendon bone joint

General Slides These slides allow you to review what you have learned in this exercise. EB-19 and 20 are an example of one histopathology of bone. Try to identify the pathological changes in the histology of bone. List of slides - General EB-15 EB-16 EB-17 EB-18 EB-19 EB-20

Review slide – identify all tissues Head, Amia calva Femur, fish Vertebrae, fish Ostegenic sarcoma Ostegenic sarcoma

REVIEW THE FOLLOWING BOX SLIDES FOR EXAMPLES OF CARTILAGE AND BONE 10 – 16, 26 – 29, 48, 49, 52, 87, 90, 91

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