Overview:

The main goal of this lab is to learn how to identify key cells and structural features of cartilaginous tissues, mature bone, and developing bone.  The student should appreciate the differences in structure between cartilage and bone, and how the specific cells and matrices work together to perform the critical functions of these tissues.

Part I. Cartilage

In Part I , there are four slides to examine, showing hyaline cartilage (Webslide 26), elastic cartilage (Webslide 12 and UMich 44H), and fibrocartilage (Webslides 45 and 74). 

Hyaline cartilage

Webslide 0026_R: Trachea, human, H&E
[DigitalScope]

Note the general organization of hyaline cartilage. Observe: perichondrium,  connective tissue, fibroblasts, differentiating chondroblasts, development of chondrocytes, and mature chondrocytes, usually found in clusters of 2-4 cells each.

Note that while there is some vesiculation of the chondrocytes (especially near the middle of the cartilage -probably artifactual), the prominent "empty" lacunae with shriveled chondrocytes seen in paraffin sections are absent. Your thin section, which shows each lacuna filled with a healthy chondrocyte, is a closer approximation to the living structure.

The TB stains the cartilage matrix intensely yielding a color variation from blue to pink, an effect known as metachromasia. Note the more intense staining of the territorial matrix around each lacuna reflective of the relatively higher proportion of sulfated proteoglycans compared to the lighter staining inter-territorial matrix found between the cell clusters.

Elastic cartilage

Webslide 0012_A: Ear, monkey, 2 µm TB-AF.
[DigitalScope]

Slide UMich_044H_20x: Epiglottis, human, 7 µm, H&E
[DigitalScope]

These slides both contain central bars of elastic cartilage.  Note the same structural features present in the hyaline cartilage of Webslide 8, and the addition of clearly visible elastic fibers (the dark, interlacing amorphous elements of varying sizes and shapes) within the matrix. In Webslide 12, the fibers appear dark-purple/magenta and in Slide UMich_044H_20x the fibers are intensely red.

Fibrocartilage

Slide UMich #45: Vertebrae and intervertebral disc, human, H&E
[DigitalScope]

This slide shows an intervertebral disc (in the center) sandwiched between two vertebral bones. Locate the nucleus pulposus of the intervertebral disc (in the very center of the slide), then move either up or down to find fibrocartilage.  Note the fibrous texture of the matrix due to the presence of type I collagen fibers in the matrix. Note also the distinct chondrocyte lacunae. A distinct perichondrium is also absent; instead, the fibrocartilage merges with surrounding connective tissue (at the top and bottom) or inserts directly into the bone of the vertebrae on either side.

 

Webslide 0074_A: Developing elbow, guinea pig, Masson trichrome
[DigitalScope]

Examine the slide on low power. The section shows the front limb flexed (click here to see an orientation image).  At the peak of the flexure, the end of the ulna should be evident. Find the cartilage at the end of the bone at the bottom of the slide. Note the absence of perichondrium on what will become the articular cartilage surface. Observe the change in morphology of the chondrocytes from the surface layer (very flattened) to the deeper layers. In the deep layers the cartilage is involved in the complex process of endochondral bone formation. Fibrocartilage can be observed where the tendon joins with the cartilage at the very bottom of the slide. Examine this area for the characteristics of fibrocartilage: relatively scanty chondrocytes within lacunae surrounded a small amount of territorial matrix, and a predominant amount of fibrous material (collagen).

Part II. Bone

The slides for the bone portion of the lab have been prepared in one of two ways. In ground sections such as Webslides #70 and 71, the organic material has not been preserved which allows observation of the mineral aspect of the bone. In the other slides, the specimen has been decalcified to allow examination of the cells and the extracellular matrix portion of the bone. It is useful to combine information from both kinds of preparations to get a feel for the tissue.

A. Mature Bone.
            There are two slides to examine.

Webslide 0070_A: Bone, ground thin, human, cross-section
[DigitalScope]

Webslide 0071_A: Bone, ground thin, human, longitudinal section
[DigitalScope]
 
Compare these two slides with each other and with Webslide 74 
Identify in each slide:

• Osteon (Haversian system)
• Interstitial lamellae (remnants of old osteons between new osteons)
• Haversian lamellae
• Haversian canal - what does (did) it contain?
• Volkmann's canal - what does (did) it contain?
• Lacunae - what do (did) they contain?
• Radiating canaliculi - what do they represent?

B. Bone Formation and Growth.
Flat bones form without a cartilage precursor (intramembranous ossification) while load bearing bones develop from a cartilage model (endochondral ossification). There is an example of each for examination.

Intramembranous ossification

Webslide 0300_A: Developing Membrane Bone, mammal
[DigitalScope]

Scan the slide first with the 10X objective setting. Observe the islands of bone (trabeculae) which stain dark pink. Note the blood vessels occupying the spaces between the developing trabeculae.

At higher power, the islands of bone are seen to be covered with either osteoblasts, a palisade of cuboidal cells, or very flattened bone lining cells. Note the lacunae with poorly preserved osteocytes in the islands of bone matrix.

This is also an excellent slide for finding osteoclasts, larger multi-nucleate cells.  There are many osteoclasts in this webslide. Make sure you find at least three of them.

Endochondral ossification

Webslide 0301_A: Finger, fetal, human, H&E
[DigitalScope]

Examine the forming bone on the left hand side of the slide. Start by examining the diaphysis of the bone which is stained bright red. Notice the uniform distribution of osteocytes as well as the blood vessels in the bone. Next observe the epiphyseal cartilage which is stained light gray. An epiphyseal plate is not present. Note the following transitions in chondrocyte and matrix morphology:

1. Resting (reserve) chondrocytes
2. Zone of multiplication (an occasional mitotic figure may be found).What causes elongation in this region? What is the direction of interstitial growth?
3. Hypertrophying cartilage
4. Region of calcifying matrix; note destruction of chondrocytes. This zone appears
   to overlap c.
5. Zone of resorption and bone deposition. Note:
   • Osteoblasts along the branching trabeculae of calcified cartilage
   • Osteoid is laid down over calcified cartilage matrix (it tends to stain more darkly).  In a decalcified section such as this one, only uncalcified extracellular matrix remains, and it is impossible to tell where ossification is complete.
   • Large, multinucleated osteoclasts involved in bone resorption that are sometimes separated from the bone during processing of the specimens. Surface erosions due to osteoclastic activity are called Howship's lacunae.
   • Marrow cavity, filled with blood forming and vascular elements that extend into the ossifying region.

 

 

Webslide 0074_A: Developing elbow, guinea pig, Masson trichrome
[DigitalScope]

With a low power objective setting find the epiphyseal plate towards the center of the slide (click here for orientation image). Note the same transitions in chondrocyte and matrix morphology (a – e, above) proceeding in the direction opposite to that of growth (i.e., toward the diaphysis).

Extra Slides

Intramembranous Ossification

Webslide 0308_A: (courtesy of U. of Mich.): Developing Membrane Bone, embryonic human face, Masson trichrome
[DigitalScope]

Scan the slide first with a low objective setting. Observe the islands of bone (trabeculae) which stain dark blue. Note the blood vessels occupying the spaces between the developing trabeculae.

At higher power the bone contains lacunae with osteocytes in the islands of bone matrix. At the edge of the islands of bone are osteoblasts (a palisade of cuboidal cells), bone lining cells (flattened cells), as well as osteoclasts (larger, multi-nucleate cells).

 

Slide UMich #115-N: Developing bone (palate), human, H&E
[DigitalScope]

This section shows the bony palate in the roof of the mouth and is also good for seeing osteoblasts that build bone as well as osteoclasts that resorb bone. You may notice areas of bone that are more basophilic and mottled in appearance and with a higher density of osteocytes compared to other regions; this is so-called "woven" or "immature" bone which will eventually be remodeled and replaced with mature or "lamellar" bone.

Endochondral Ossification

Slide UCSF 61: Fetal finger, human, H&E
[DigitalScope]

This slide is similar to WebSlide 0301 above and offers yet another opportunity to identify all the features evident in the process of endochondral ossification. In addition, this slide features a readily recognizable secondary center of ossification in the head of the bone. As proliferation slows in the cartilage of the epiphyseal plate, the processes of ossification from the secondary center in the head of the bone and from the primary center in the diaphysis eventually replace the epiphyseal cartilage with bone. Once this happens, the bone can no longer grow appreciably in length and the epiphyseal plate is said to be "closed." In this particular sample, bone deposition can be seen within the zone of resting cartilage of the epiphyseal plate indicating that the process of epiphyseal closure has begun.