ANAT D502 - Basic Histology

Cartilage, Bone & Joints, Bone Formation Pre-Lab

revised 9.23.12

Objectives:

1. Identify the three types of cartilage:  hyaline, elastic and fibrocartilage.

2. Define the lacuna(e), chondrocyte, territorial and inter-territorial matrix in hyaline cartilage.

3. Distinguish between primary (immature, woven) and secondary (mature, lamellar) bone as well as compact and trabecular bone.

4. Identify the three types of lamellae (circumferential, concentric and interstitial), osteons, vascular channels (osteonic and transverse), lacunae and canaliculi found in mineralized sections of compact secondary bone.

5. Distinguish the periosteum and endosteum and the path of blood vessels through compact bone.

6. Identify the cells (true and otherwise) of bone (osteoblast, osteocyte, and osteoclast).

7. Distinguish between intramembranous and endochondral ossification and [appositional] bone growth.

8. Identify the different zones observed in cartilage during endochondral bone formation.

Slides:

s2 fibrocartilage (intervertebral disc)
s4 fetal limb
s5 fetal finger (developing long bone)
m7 adult finger (monkey), demonstration slide
s7 decalcified bone, longitudinal section
s11 mineralized bone
s21 bone marrow
s23 demineralized bone, cross section
s33 external ear (H&E)
s34 external ear (orcein)
s39 fetal mandible (jaw)
s48 trachea

Index of images

Cartilage and bone are specialized connective tissues consisting of cells embedded in an extracellular matrix.  The matrix is rich in glycosaminoglycans, glycoproteins and collagen [primarily types I (bone) and II (cartilage)]. Cartilage and bone differ from dense collagenous connective tissue primarily in the differences in the type and amount of glycosaminoglycans and glycoproteins rather than the type of collagen. In the case of cartilage, the matrix is typically highly hydrated (a lot of filler or ground substance) giving the tissue resiliency (pliability). There are three types of cartilage: hyaline, elastic and fibrocartilage. In the adult, hyaline cartilage is found at the junctions between bones (joints); elastic cartilage is found in the external ear, nose, epiglottis and a few other places where elasticity and pliability are needed; and fibrocartilage is found in the spine and a few other places. In bone, the matrix is mineralized and this results in the bone having little resiliency (i.e., greater stiffness), aiding in its functions of resisting and transmitting forces.  Thus, the bones of the skeleton allow us to both stand and move. The two different forms of mature bone, compact (cortical) and trabecular (spongy) originate by different mechanisms and possess different histological as well as anatomical appearances.

Cartilage

We begin our study of cartilage by viewing the hyaline cartilage found within the wall of the trachea (s48). View the section with the naked eye and observe the transverse section of the trachea and esophagus. The trachea is denoted by its more circular shape and a basophilic (purple) ring within the wall of the trachea. This ring is not complete but rather forms a C. Further study the trachea at low power and investigate the structure of the basophilic region within the wall of the trachea. This basophilic region is hyaline cartilage. Investigate the cartilage at higher magnification and you will observe the collagenous perichondrium on each side of the cartilage wall. The perichondrium may be detached from the cartilage but this is not so in living tissue (artifact). Just beneath the perichondrium (between the perichondrium and the cartilage) are flattened cells which are the chondroblasts that can give rise to chondrocytes. Within the cartilage, you will observe the lacunae in which the chondrocyte(s) reside. You will note single cells in the lacunae near the perichondrium and multiple cells in the center of the cartilage wall as would be expected from appositional growth in the former and interstitial growth in the latter.  In these central lacunae, you will observe a subtle staining difference just around the lacunae relative to the matrix between the lacunae; this is due to a difference in the matrix composition between these two regions. The matrix nearest the lacuna is called territorial matrix whereas that between the lacunae is called the inter-territorial matrix. The cells within the lacunae are poorly preserved in that the cell cytoplasm is poorly stained and the cells have detached from the walls of the lacunae. However, the nuclei of the chondrocyte are readily observed within the lacunae. You should not observe any vasculature in the hyaline cartilage as it is typically avascular. This is a feature common to all types of cartilage. Additional examples of hyaline cartilage can be found in s5 (fetal finger, in the cartilage template from which bone will form) and in m7 (finger) at the joint (articular cartilage) that we will study later in this lab.

Next, we will study the elastic cartilage found in the external ear (pinna; s34). Fold or bend your external ear and note how it returns readily to its original shape; this is because of the elastic cartilage within it. This section has been stained with orcein to highlight the elastic connective tissue found in elastic cartilage. Examination of this section at low magnification shows an island of elastic cartilage that is more cellular than that observed in hyaline cartilage. The lacunae are more numerous as well as the chondrocytes that reside within the lacunae. The matrix has darker (rose-colored to brown) staining than the adjacent collagenous tissue because of the elastic fibers that are present. The elastin fibers are not as readily observable as those we have observed in the blood vessels. A perichondrium is present but it is not as well defined as observed in the hyaline cartilage of the trachea. As in hylaine cartilage, the cells near the perichondrium are flatter than those in the center of the cartilage island as expected from appositional growth of the cartilage in this region. The lacunae are more often filled with one cell rather than the multiple cells (isogenous groups) seen in hyaline cartilage. Territorial matrix and inter-territorial matrix are difficult to distinguish because of the high cellular density of this cartilage. This form of cartilage can be also be observed in s33 of the human external ear. This slide was stained with H&E and thus the elastin is not highlighted. The elastic cartilage is observed as an island of tightly packed cells enclosed within a thin perichondrium which has a central location within the ear tissue. The lacunae are visible at high magnification.

N.B.  In examining the next slide (s2, fibrocartilage; intervertebral disc) be aware the intervertebral disc (comprised of an inner nucleus pulposis and outer anulus fibrosus) forms an intervertebral symphysis with the hyaline cartilages covering the adjacent vertebral body surfaces.  Thus, depending upon the plane of section of your slide, you may (a) observe some hyaline cartilage in addition to fibrocartilage, and (b) not observe the nucleus pulposis.  Use this schematic of the intervertebral symphysis to determine the plane of section of your slide.

The last form of cartilage that we will study is the fibrocartilage found within the intervertebral disc (s2) and in some tendons (m7; demonstration slide). In the vertebral column, between adjoining vertebrae, is the intervertebral disk which is made of two structures. The structure in the middle of the disk is called the nucleus pulposus and is liquid filled with hyaluronic acid and a few cells. Surrounding this structure is the anulus fibrosis which encloses the nucleus pulposus. The anulus fibrosis is made of dense connective tissue along its periphery and of fibrocartilage near the nucleus pulposus. This section is poorly preserved, but, with a little work, you can observe the fibrocartilage found in the anulus fibrosis. Look for rows of cells between eosinophilic collagen fibers. The lacunae are not as distinct as that found in the other forms of cartilage but isogenous groups are present within the lacunae and these are usually arranged as rows of cells. Some ligaments and tendons also contain fibrocartilage and this can be observed in the tendons of the finger in a demonstration slide (m7). Locate the synovial joint proximal to the finger nail and study the connective tissue along the dorsal margins of the joint (these are tendons of the extensor muscles)  Note the rows of chondrocytes between the dense collagen type I fibers. This tendon continues along the finger joining with the distal phalanx (most distal bone of the digit) and attaches to the bone via Sharpey's fibers that we will view later.

Careful observation of the matrix, lacunar arrangement and density, and perichondrium should permit you to distinguish the three types of cartilage, as summarized in the chart below. 

 

Type / Character

 

Matrix

Lacunar

arrangement / density

 

Perichondrium

 

 

 

 

hyaline cartilage

clear

random; < 50% SA

distinct

elastic cartilage

fibrous

random; > 50% SA

less distinct

fibrocartilage

not observable

linear

not observable

 

Bone/Joints
Bone

For the histological study of bone, we will examine both mineralized (s11) and de-mineralized (s23 and m7) sections. Because of the rock-like nature of mineralized bone, the section of mineralized bone (s11) was prepared by cutting the bone with a jeweler's saw followed by grinding it into a thin section. The tissue is not stained but different structures are observable because they refract light differently.  This particular section is from a transverse section of compact bone from the diaphysis (shaft) of a long bone. The entire transverse section was further divided into smaller sections by cutting the round section into pie-like pieces. This unstained, mineralized section of bone is the best to study the lamellar structure of bone. Begin your microscopic examination at low power and you should observe a rectangle-like structure with two flat sides and a concave and convex side. The concave side is the endosteal side of the bone whereas the convex side is the periosteal side of the bone. Examination of the endosteal side at higher magnification shows almond-shaped lacunae arranged in a concentric fashion for several layers around the inner circumference of the bone. This is the inner circumferential lamella. On the opposite side of the section, a similar organization is noted, and this is the outer circumferential lamella on the periosteal side of bone. It is usually thinner (fewer lamella) than the inner circumferential lamella. Between the inner and outer lamella are numerous osteons (also eponymously called Haversian systems). These are noted by their central, dark appearing dot with concentric rings or lamella around this dot. The central dot is the central or osteonic canal  (also eponymously called the Haversian canal) in which blood vessels and nerves pass in the living tissue. You may observe canals which pass between or connect the central canals. These lateral canals are called transverse canals (also eponymously called Volkmann's canals) and they do not have a lamellar arrangement of lacunae around them. In living tissue these canals are also filled with blood vessels and nerves that connect to larger vessels outside the bone allowing for nourishment of the bone. Upon closer examination of the osteon, you should observe 3 or more concentric layers of lacunae around each central (osteonic) canal. Between the lacunae is mineralized bone. Upon examination of the almond-shaped lacunae, you will observe thin processes which emanate from the lacunae and connect to adjacent lacunae. These are called canaliculi and in the living tissue the lacunae are filled with osteocytes and the canaliculi are filled with osteocyte processes. The processes from different osteocytes are joined by gap junctions forming a functional syncytium of cells within the compact bone. Between the osteons, you will observe incomplete lamellae and these are called the interstitial lamella. These lamina are the remnants of older bone which have not been removed during bone remodeling.

Next we will study sections of de-mineralized bone which affords a greater understanding of the organic matrix and cells found in bone. Slide 23 is a transverse section of the diaphysis (shaft) of a long bone. Examine it at low power and in the center you will observe bone marrow with its constituent developing blood cells encased within compact bone. Lining the wall of the bone marrow cavity at the inner wall of the bone, you will observe a single layer of flattened to cuboidal basophilic cells. This layer is called the endosteum and is made of osteoprogenitor cells and osteoblasts. Beneath regions of cuboidal osteoblasts (which may be absent in your slide), you will observe a subtle difference in the staining properties of the bone matrix with that nearest the osteoblast being less eosinophilic than regions deeper within the bone matrix. This is called osteoid and is newly synthesized organic matrix which has not fully mineralized. The difference in the composition of this newly synthesized matrix is responsible for its different staining properties. Moving further into the bone, you will observe the inner concentric lamella and the concentric and layered organization of the osteocytes within their lacunae. The almond shape nuclei of the osteocytes are readily observed but the canaliculi between the lacunae are just barely visible. Linear-running transverse canals are readily observed within the compact bone. You may be able to find canals which open into the bone marrow cavity. You will also observe osteons with their central canal but their circumferential lamellae are more difficult to distinguish than in the mineralized bone. Both the central (osteonal) and transverse canals contain blood vessels but these cells are not easily distinguished. An occasional endothelial cell may be noted in the canals. The outer layer of the bone is covered by the periosteum and this can be subdivided into two layers, an inner cellular layer and outer (superficial) fibrous layer.  The inner layer contains osteoblasts or osteoprojenitor cells along the bone surface.  If osteoblasts are present you may observe a seam of osteoid deep to these cells.  The outer layer is fibrous and consists of a dense connective tissue interspersed with fibroblasts and lies adjacent to the skeletal muscle.

Slide s7.  allows for observation of compact long bone in longitudinal section. Examine the section at low power and you will observe skeletal muscle on one side of the section followed by compact bone then bone marrow on the other side. Compare and contrast the appearance of the endosteum and periosteum in this longitudinal section relative to the transverse section. The central (osteonic) canals of the compact bone are shown to good advantage in longitudinal section because of their linear orientation parallel to the long axis of the bone.  Similarly, transverse canals can be observed running perpendicular to the central canals.  Occasionally you may find an osteon undergoing active re-modeling.  Here the cells apposed to the surface of the bone are osteoblasts while those in interior are likely endothelial cells. One may also find occasional red blood cells within the endothelial-lined capillaries. The lamella are difficult to distinguish in this longitudinal section but you will observe lacunae with their resident ostoecytes.  In the periosteum, you will observe skeletal muscle fibers attaching to the fibrous layer. On the endosteal side of the bone, you may find osteoblasts and osteoclasts. Active osteoblasts are cuboidal while inactive (resting) cells are squamous with both having one side apposed to the bone and the other to the bone marrow cavity. The active osteoblasts have a basophilic cytoplasm as would be expected for a cell involved in active protein synthesis and secretion. The osteoclasts are large, multinucleated "cells"  (actually syncytiums) that are typically attached to the bone. The cytoplasm of these masses is eosinophilic as would be expected from a cytoplasm full of mitochondria to supply ATP for the numerous ion pumps involved in the function of these cells. Although not easily observed in this section, the region of bone removed by the osteoclast is called the resorption or osteoclast lacuna (also eponymously called Howship's lacuna).   Finally, if you look in the marrow cavity of this section you can observe abundant megakaryocytes.  Examine these cells closely and make sure you can distinguish them from osteoclasts.

Trabecular (also called cancellous or spongy) bone is the bone that is found in the marrow (medullary) cavity, especially in long bones. An example of trabecular bone can be seen in s21. The bone within the marrow cavity is similar to compact bone in that it is organized into lamella. However, the bone is consists of trabeculae (L, little beams) instead of a thick wall of bone. Interspersed between these bony spicules are the cells involved in blood cell synthesis. If the marrow cavity is active in blood cell synthesis, it appears red and thus is called red bone marrow. The next time you satisfy your carnivorous cravings for a chicken wing, after you have consumed the muscle tissue, break open the diaphysis and observe the interior of the bone. Most likely it will be red marrow (fryers are fairly young critters).  However, if the marrow cavity is not active, fat cells replace the progenitor cells and the marrow is said to be yellow bone marrow,  based on its yellowish appearance.  The next time you satisfy your cravings for beef with a bone in chuck roast (especially from an older critter), observe the marrow cavity and it will likely be yellow marrow.

While the histological structure of bone can be successfully studied using H&E stained slides of demineralized tissue embedded in paraffin, such as those found in your slide box, alternative embedding and staining methods can be employed to ease the identification of osteological tissues.  Osteoclasts can be readily demonstrated using enzyme histochemistry, specifically the tartrate-resistant acid phosphatase (TRAPase) histochemical method as seen in this demonstration slide from a rat proximal tibia.  Similarly, osteoid (unmineralized bone matrix), osteoblasts and osteoclasts can be shown to good advantage by using mineralized (resin-embedded) thin sections histochemically stained with the von Kossa method for minerals (which turns mineralized tissue black) and counterstained with MacNeal's tetrachrome, as seen in this demonstration slide from a fetal babboon tibia.  Examine the endosteal surface of the tibia to find osteoclasts.  Abundant osteoid and osteoblasts can be observed on both the periosteal and endocortical surfaces in this rapidly growing animal.

Synovial joint

A demonstration slide of a finger is also available for study. First, examine the section at low power and you will observe the skin covering the digit. At one end, you will observe a yellow plate of tissue and this is the finger nail. Within the finger, you will observe the two articulating bones (phalanges) forming a synovial joint. You can review the cells and structures of compact bone in the shafts of the phalanges. Of more interest is the synovial joint between the middle and distal phalanges. Upon examination of the joint at low power, you will note an empty space between the articulating surfaces. In life, this was filled with hyaluronic-acid-rich synovial fluid. At the articulating surfaces, you will observe the more eosinophilic hyaline cartilage with the chondrocytes being more flattened at the surface than deeper into the cartilage. There is no perichondrium covering this cartilage and it is called articular cartilage. There is a transition in the matrix from cartilage to bone in this region with the cartilage being defined by the larger lacunae. Following along the joint cavity to the non articulating surfaces one can observe the cells of the synovial membrane and the fibrous capsule of the joint. The former lines the margins of the joint cavity, and the cuboidal to low cuboidal cells that line the internal side of the joint cavity are called the lining cells. These synthesize and condition the synovial fluid. Beneath these cells is irregular connective tissue with fibroblasts and capillaries. The fibrous capsule is on the outer side of the joint cavity and is made of dense connective tissue or fibrocartilage. In the periosteum of the distal phalange, just beyond the joint, you will observe that the dense regular connective tissue forms fibers which fuse into the tendon. The fibers attaching to the bone are called Sharpey's fibers and link bone to muscle via the tendon.


Bone formation

For the last part of the laboratory, we will study slides representing the initial stages of bone formation (intramembranous and endochondral) and the subsequent appositional bone growth.  Initial bone formation occurs by two different processes, these being intramembranous and endochondral bone formation (ossification). With intramembranous bone formation, the anlage (precursor) of the bone is formed by a dense mat of mesenchymal cells.  Within this cell mass, differentiation of mesenchymal cells into osteoprogenitor cells then osteoblasts occurs in clusters of cells (islands). Secretion of bone-specific organic matrix (osteoid) then occurs followed by ossification of this matrix. With continued ossification, the osteoblasts are encased within mineral matrix and become osteocytes within lacunae. These islands of mineralized tissue grow and fuse with other islands. This type of bone formation occurs in flat bones such as the skull and mandible. Examine s39 of the fetal jaw (mandible) as an example of intramembranous bone formation.  Intramembranous ossification is nearly finished in most of these slides but look for regions of dense mesenchyme on the ends of the developing bone.  In these regions adjacent to thepreviously formed bone one can observe randomly arranged, large, cuboidal basophilic cells which are the osteoblasts.  These osteoblasts are secreting osteoid into the surrounding matrix but it is difficult to discern.  As the osteoid accumulates and mineralizes, the osteoblasts are trapped and become osteocytes within lacunae, as seen in the adjacent bone.  This type of bone formation yields bone called primary, immature or woven bone.  

In the other mechanism of bone formation (ossification) the anlage (precursor) of the bone formed in hyaline cartilage and this is termed endochondral bone formation. This mechanism is responsible for the formation of long bones. There are two slides within the Histology collection which we will use to learn about endochondral bone formation. The first slide is taken from the fetal limb and it shows, to good advantage, the cartilage template of long bones prior to bone formation although the section is lightly stained. Examine s4 at low power and you will observe, in the center of the section, developing joints with their attached developing long bones. The nascent epiphysis appears as hyaline cartilage with chondrocytes in lacunae but not as isogenous groups, rather as single cells within the lacunae. As you progress from the nascent epiphysis into the nascent diaphysis, you will observe a change in the cartilage from distant lacunae to closely packed, small lacuna with isogenous groups and lastly into large but closely packed lacunae. The first transition (small lacunae) is the result of proliferation (increase in cell number) of chondrocytes whereas the second (large lacunae) is the result of chondrocyte hypertrophy (increase in cell size). The hyaline cartilage may be calcified in the central region of the diaphysis.

The process of ossification of cartilage can be understood by the study of s5 which is a longitudinal section of a developing finger. Begin your study as with slide 5, at the joint. The developing epiphysis does not yet contain a secondary ossification center but a primary ossification center is present (in the diaphysis). The primary ossification center is divided into 5 zones (starting from the center of the epiphysis and moving inward): zone of resting cartilage, zone of proliferation, zone of hypertrophy, zone of cartilage calcification, and zone of ossification. Within the cartilaginous epiphysis and nearest the joint is the zone of resting cartilage. This is noted by the single chondrocytes within their respective lacunae. Following this resting zone, is the zone of proliferation in which there are multiple chondrocytes within lacunae. The lacunae are relatively small and the chondrocytes are tightly packed within the lacunae. The proliferative zone is followed by the zone of hypertrophy in which the chondrocytes within the lacunae increase in size rather than number. As such, you observe large lacunae usually in rows aligned with the long axis of the bone. The matrix in this zone may be more basophilic than the earlier zones because of the difference in the organic matrix composition. The zone of hypertrophy is followed by the zone of cartilage calcification in which the chondrocytes have died and the cartilage becomes calcified. This zone is noted by its basophilia relative to the other regions. Adjacent to these zones and in the marrow cavity is the zone of ossification which is orange (in the web image). It is noted by its eosinophilic staining relative to the adjacent basophilic calcified cartilage. You will also note small lacunae with their resident osteocytes. This zone is formed by the concerted action of osteoclasts which remove the calcified cartilage followed by osteoblast activity; the latter resulting in the synthesis of bone matrix and its subsequent calcification. The subsequent remodeling of this tissue results in the formation of bone termed secondary, lamellar or mature bone.

While bones are initially preformed either in cartilage or membrane (mesenchyme), endochondral and intramembranous, respectively do not account for the majority of bone growth to adult size.  That is, for the most part, endochondral ossification and intramembranous ossification do not substantially increase the size of the fetal / juvenile “bones”, they merely replace the precursor material (membrane or cartilage) with bone.  The vast majority of increase in bone size results from appositional bone growth as part of the process known as bone modeling.  While it is true that increases in long bone length results from continued cartilagenous proliferation followed by endochondral ossification, increases in long bone diameter are due to appositional bone growth and all growth (increase in size) in membranous bone results from appositional bone growth.  

Return to slide 39  and look for regions of appositional bone growth along the periosteal and endosteal surfaces of the growing mandible (anatomically speaking, this is the dentary bone).  In contrast to the scattered arrangement of osteoblasts and poorly discernable osteoid in the adjacent areas of intramembranous ossification, here the cuboidal osteoblasts are arranged in long rows (“string of pearls”) and thick seams of osteoid can be observed to cover the bone surface.  This type of bone deposition results in the formation of bone termed secondary, lamellar or mature bone.

 


Lab:  Bone & Cartilage