LABORATORY GUIDE FOR BONE

In this lab, questions will be asked for you to quiz yourself on as you move through the exercise; answers are provided at the end.

COMPACT BONE

Examine Slide 11 (ground bone), which is a piece of dried compact bone which has been ground thinly enough to be viewed through a microscope. As stated on your side, do not use your oil objective (due to the height of the specimen), or you will crack your slide and have to receive abuse from the Slide People. No cellular elements remain in this specimen, but the spaces previously occupied by soft tissues are easily distinguishable. Identify the following structures.

1. How are interstitial lamellae formed, i.e. why don't they form complete circles like concentric lamellae?

SPONGY BONE

The human skull, Slide 3, is provided to study compact and spongy bone. In these and all the following slides, the bone has been decalcified, fixed, cut and stained with H & E. Cells remain, but lamellae are harder to distinguish than in ground bone. Also bone, like cartilage and c.t. proper, is eosinophilic. Hold the slide against a white background. It is grossly easy to distinguish spongy bone with its clear marrow spaces from the denser cortical bone around the perimeter. This easy distinction will vanish when you look in the microscope at these same areas. Spongy bone is central and consists of a ramifying system of trabeculae, interrupted by marrow spaces. Identify the following:

In these slides, many of the Haversian systems appear to be immature and have large central canals with cells in them. You may distinguish these Haversian canals from marrow spaces by the presence of surrounding concentric lamellae, which you must look for closely.

2. The endosteum of slide 3 is poorly developed, consisting only of sparse, spindly cells. Osteoclasts are not found. Is this representative of actively growing or quiescent bone?

3. What cell type do you think you're seeing in the endosteum?

4. Is there a clear demarcation between the cortical (compact) bone of the femur (Slide 4) and the spongy bone?

5. You see a Haversian system in a trabeculum. How can this possibly happen if Haversian systems are characteristic of compact bone?

Look at Slide 6 (human finger joint) grossly and identify: two articulating bones, their marrow spaces and cortices, a thin, slightly basophilic rim of tissue on the articular surfaces representing hyaline cartilage, the joint cavity, joint capsule, and synovial membrane. It is essential to make these identifications before placing the slide under the microscope.

With the microscope now, identify again the following features:

The joint capsule forms a hood over the articular ends of the bone. It consists of two not clearly delineated parts. One is a thick outer fibrous layer, which is dense regular c.t. that can be followed and seen inserting into the bone. The collagen fibers of the capsule that continue into the bone are Sharpey's fibers, but they are subtle and optional features to identify. The innermost layer of the joint capsule is the synovial membrane. It lines the entire inner surface of the joint capsule except over the cartilage, and is composed of loose connective tissue, fat, and blood vessels. On its free inner layer, an incomplete layer of cells elaborates and resorbs synovial fluid.

6. If hyaline cartilage and bone both have abundant matrix and lacunae, how can you tell them apart?

7. Does the bone of slide 6 look active or quiescent?

ENDOCHONDRAL BONE FORMATION

Fetal hand (H-2) is an excellent example of endochondral bone formation at its earliest stage, that of primary ossification from a hyaline cartilage primordium. Look at these primordia, as in the digits, in various stages of development, and find the following:

ENDOCHONDRAL BONE GROWTH

Examine Slide 1 (human costochondral junction) and Slide 5 (human intervertebral disk). These sections illustrate all the steps seen in endochondral bone growth; the zones are much more abbreviated in slide 5 than slide 1, and somewhat easier to distinguish. Use your lecture handout to identify the following zones:

Reserve Cartilage:
Most cartilage on this slide is this type.

Cartilage Proliferation:
The cells in this zone are increased in number, slightly larger than reserve cells, and stacked like coins.

Cartilage Hypertrophy:
The lacunae attain a maximal size.

Cartilage Calcification:
The hallmark of this zone is its intense basophilia. Easy to see in slide 1, but not in slide 5.

Ossification:
This zone is well-demarcated in slide 5, and can be distinguished by its bright reddish-orange color.

Additionally, use Slide 1 to identify the following features of bone:

ANSWERS TO QUESTIONS

  1. Interstitial lamellae are the remnants of concentric lamellae; they represent old osteons that are partially removed. At one time they were concentric lamellae.
  2. Flat, sparse cells, little osteoid, no osteoclasts represents bone with little turnover, i.e. quiescent.
  3. Cells of this type of endosteum are mostly osteoprogenitor cells.
  4. There is frequently no clear demarcation between compact and spongy bone when viewed microscopically - it is very difficult to draw a line and say where one ends and the other begins. You only know for sure that trabeculae are hallmarks of spongy bone, and the outermost bone is compact, especially where you see circumferential lamellae running parallel to the surface.
  5. Two ways. One, as bone grows in width, cortical areas are partially resorbed and the marrow space expands. Bone that was once on the outside, with Haversian systems, can become a trabeculum hanging from that outer wall. Two, trabeculae that are too thick can be tunnelled through by osteoclasts for formation of a new Haversian system. Therefore, although osteons typically form compact bone, they are not found there exclusively, and may be seen in spongy bone.
  6. Hyaline cartilage will always be more basophilic than bone. Stains vary from slide to slide, but even when the color of hyaline cartilage is washed out and not very blue anymore, the bone in our sections is still orange-red and relatively more eosinophilic. At higher mags, bone has a "weave" to it due to the presence of lamellae, like rings on a tree. Hyaline cartilage is smooth. Lastly, hyaline cartilage has pronounced basophilic capsules surrounding the lacunae, while bone has no capsules.
  7. Quiescent