Bone and Bone Formation Laboratory 8

Histology of Bone and Bone Formation
Laboratory 8

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Required Reading: Ross & Romrell: Chapter 8, pp. 150-160

Gartner & Hiatt: Chapter 4, pp. 60-81

YOUR JOB DURING THIS LAB: After completing this assignment you should be able to (a) identify all of the major structural features of the Haversian system, or osteon, in lamellar bone; (b) identify regions of bone, vs. bone marrow vs. calcifying cartilage etc.; and (c) identify and understand the differences in location and function between osteoblasts, osteocytes, and osteoclasts.

STRUCTURE OF BONE
First, concentrate on learning the histology of Haversian, lamellar bone. I have included an extensive series of images that should help you when you look at the glass slides.

Images for overall bone structure.

D8: A good cross-section of a single Haversian system, or osteon in compact bone. This bone has been decalcified. The Haversian canal in the center contains the blood vessel, with associated nerve and CT. Note the concentric layers of extracellular matrix (a, for example), and the osteocytes fully encapsulated by the matrix. Careful observation at the edges of the image should reveal the cement line where one osteon meets another.

D9: Compact bone in cross-section. This sample is not decalcified. Concentric lamellae (*) surround the Haversian canals (one shown at a). Some of the lacunae are empty (arrowhead), but would normally contain an osteocyte each. A Volkmann's canal is shown at (b), connecting two Haversian systems. Do you remember what is in one of these transverse canals? Interstitial lamellae (arrow) are the remnants of previously existing osteons. Faint radial lines in the lamellae are the intercellular canaliculi allowing one osteocyte to communicate and interchange material with its neighbor.

D10: Compact bone in cross-section (decalcified). Here the bone was fixed in fresh condition and decalcified before sectioning. Many osteocytes have not fallen out, but remain in their lacunae (pointer). The boundaries of a single small osteon are indicated (a), as is an Haversian canal (b), and interstitial lamellae(*). The canal marked (b) shows nice preservation of the blood vessel and associated CT.

D11: Compact bone in longitudinal section (decalcified). Compare this with image D9 where the same structures can be found. Here, concentric lamellae (*) appear as long, parallel layers because the osteons are oriented with the bone long axis. The Haversian canals (a) also appear elongated due to the direction of the section. Volkmann's canals (b) now appear clearly transverse, but interstitial lamellae are hard to detect in this type of section orientation.

D12: This image shows a sample of ground bone (not a section) and illustrates nicely the intercellular canaliculi radiating from each of the osteocytes (arrow). Here the Haversian canal is empty, but this is artifactitious.

D13: Another ground bone image showing the canaliculi nicely, along with Haversian canals (b) and Volkmann's canals (a). Is this shown in cross-sectional or longitudinal orientation?

D14: Compact and decalcified bone in longitudinal section illustrating Haversian canals (a) and (b, slightly oblique).

D15: Spongy bone and bone marrow. Spongy bone is also termed cancellous bone. It allows for light weight, without the sacrifice of much support. Spongy bone is composed of thin interconnecting trabeculae (arrowhead) with much intervening space. This space is filled by bone marrow and fat cells(*). Here, some spicules or trabeculae are also cut in cross-section; they stain the darkest. Note the lamellae within each trabeculum.

Glass slides for the study of bone structure.

Slide #21 - ground bone: Observe the relative placement of the Haversian canals, osteocytes, and canaliculi. On some slides interstitial lamellae can be seen as well.

Slide #22 - decalcified bone: here you should identify compact bone, the marrow cavity with red marrow, inner and outer circumferential lamellae, all the features of osteons, the periosteum and endosteum, and calcified cartilage. For those of you with higher numbered slide boxes, look for resorption canals which are larger than most osteons and will contain osteoclasts (one or a few). These slides may also show newly developing osteons with osteoblasts.

Slide #90 - nasal cavity: Find the area of spongy bone and identify the woven bone, the osteocytes, and osteoblasts. A few osteoclasts may also be present on some slides. Remember that this is primary bone, woven, with a non-lamellar organization of its cells.

BONE DEVELOPMENT AND REMODELING

Required Reading: Ross and Romrell: Chapter 8,
pp. 150-187

Gartner and Hiatt: Chapter 4, pp. 60-81

YOUR OBJECTIVES FOR THIS AND THE PRECEDING LAB ON BONE: You should be able to (a) identify all of the major structural features of the Haversian system, or osteon, in lamellar bone; (b) identify regions of bone, vs. bone marrow vs. calcifying cartilage etc.; (c) identify and understand the differences in location and function between osteoblasts, osteocytes, and osteoclasts; (d) identify and label all of the important features of growing or remodeling bone, with emphasis on the epiphyseal plate region of a growing long bone and the remodeling of the metaphysis of a long bone; and (e) understand the difference between intramembranous ossification and endochondral ossification.

First, we will examine the general processes of bone formation, including intramembranous ossification and then we will look at developing long bones and the process of endochondral ossification.

Images for intramembranous bone formation and bone growth.

D16: A low power frontal section of developing monkey face. OBSERVE THIS SLIDE CAREFULLY FOR ORIENTATION. You should find the tongue dominating the center of the slide, with the eye socket lower edges just barely visible at the top of the view. Towards the bottom, the arrow indicates a region of hyaline cartilage which will develop into parts of the jaw. The blue stained material here is developing bone, and the (t) indicates a developing tooth. There is another developing tooth located similarly on the other lateral side of the slide. Note the overall appearance of the forming bone, contrast it with the hyaline cartilage.

D17: The developing tooth at higher magnification. Spongy bone is also seen. The arrows indicate osteoclasts. Why are they here? How do you distinguish an osteoclast from an osteoblast?

D18: Osteoclasts are indicated by the arrows, remodeling the bone which is forming by intramembranous ossification. Osteoblasts situated on the opposite side of the bone trabeculae are visible. Make sure that you can tell the difference.

D19: Intramembranous bone formation in a section preserving nicely the osteogenic layer of osteoblasts (arrow) actually making new bone. Note how these cells line up directly on the bone spicules. Find other examples of this on this slide. Question: How are osteoblasts different from osteocytes? Find osteocytes here. The arrowhead indicates a blood vessel (use the included blood cells as your clue.)

D20: The upper surface of this bone spicule shows all or parts of at least 5 osteoclasts, some of which lie in the depressions formed in the bone by their degradative secretions. These depressions or cavities are termed Howship's lacunae. Note the osteoblasts on the bottom edge of this same bone. Question: Over time, in which direction (U/D/L/R) will this spicule of bone move and how do you know?

Glass slides to study intramembranous ossification.

Slide #90 - nasal cavity: Look at this again to make sure you can find the osteoblasts in the region of forming spongy bone.

Slide #89/89B - nasal tissue: On this slide you should identify the bone trabeculae, osteocytes, the periosteum with its fibrous layer and osteogenic layer, osteoclasts in Howship's cavities, and some calcified cartilage. Slides numbered 89B will not exhibit the osteogenic layer or calcified cartilage.

Slide #19/19A - developing tooth: Here you should find the forming and remodeling bone spicules. Then look for the nicely defined osteoblasts and the more rare osteoclasts. On some slides (those numbered 19) you may see osteoid, the unmineralized bone just subjacent to the layers of osteoblasts. Use this slide to compare osteo - clasts, blasts, and cytes.

Endochondral Ossification and Bone Remodeling

The long bones of the body are initially formed using a pre-existing cartilage model. Next in the lab, we will examine this process histologically so that you can appreciate the interplay of cartilage matrix and associated chondrocells, osteoblasts, osteocytes, osteoclasts and calcification in bone growth.

Images for the study of endochondral ossification.

D21: This is a low magnification view of the hand from a 12cm human fetus. Careful observation should reveal the hyaline cartilage "models" in each of the developing fingers. Compare these structures; which have already begun to change to bone?

D22: A higher magnification view of a similar area, stained so that hyaline cartilage is pink, formed bone is green (little of this here) as is dense connective tissue. The arrow indicates a primary site of ossification, in the middle of what will become the bone diaphysis. Examine the relationship of the cartilage model and the surrounding CT. Look at the hypertrophy of the chondrocytes in the immediate region of the ossification center.

D23: This is again an embryonic finger. Beneath the periosteum(*) near the center of the developing bone shaft is a deep red-stained collar of newly formed bone. Beneath this collar of bone, chondrocytes are aging and dying as the cartilage degenerates and changes into spicules of calcified cartilage (arrowhead). The different regions of cartilage are all shown here as follows: (1) embryonic hyaline cartilage forming the heads (epiphyses) of the bone, (2) zone of maturing cartilage, (3) zone of hypertrophy, and (4) a zone where calcification and spicule formation is occurring.

D24: A similar view of a developing monkey toe. With the stain used here, cartilage is red, and bone and CT are green. Note the more advanced primary ossification center, with the cartilage relegated only to the forming epiphyseal areas. The developing bone to the right, however, is slightly behind in its formation, with more hyaline cartilage remaining.

D25: Here, the primary ossification center is very well developed, with a substantial amount of formed bone spicules present. Examine the appearance of those chondrocytes immediately adjacent to the forming bone trabeculae.

D26: A great view of a secondary site of ossification showing the blood vessel channel penetrating from the outside. Examine the structures seen in the bone diaphysis where extensive bone marrow is seen. Only a portion of the entire longitudinal section of the bone is shown on this slide; remember that both epiphyses do not develop in total synchrony.

D27: The epiphyseal plate is shown here in red. Examine this carefully and observe the differences in chondrocyte histology from one side of the plate to the other. Note also how the expanding secondary site has left only remnants of hyaline cartilage along the inner surfaces of the outer curvature of the epiphysis.

D28: A higher magnification view of the epiphyseal plate. The arrow points to a developing bone spicule. Identify the different zones of chondrocytes in the epiphyseal plate (use Figure 8.13 on p. 165 of Ross & Romrell for reference).

D29: A section of developing endochondral bone showing mainly the zones of cell hypertrophy and the area of spicule formation and cartilage calcification.The osteoblast layer covering the spicules has been mostly lost during sample preparation, but the deep red stained osteoid containing entrapped osteocytes can be seen coating the calcified cartilage spicules.

Glass slides on which to look at endochondral ossification.

Slide #23 - developing long bones in the foot: This slide will repay very close scrutiny at LOW POWER. You should identify all of the cartilage cells, the osteo cytes/blasts/clasts, the areas of cartilage matrix and of forming bone, and the different regions of the epiphyseal plate. These are again: the resting zone, the proliferation zone, the maturation and hypertrophy zone, the calcification and ossification zone and the resorption zone. Find the synovial cavity - the space between the epiphyses of two neighboring bones. You should see that the hyaline cartilage lining this space lacks a perichondrium.

Find primary ossification centers; you will not see a secondary site on this slide. Find regions of intramembranous ossification and compare them with sites of endochondral ossification. Find developing bone marrow.

Slide #18 - This slide is of developing bone in the symphysis pubis and was used before to find fibrocartilage. NOTE THAT SLIDE #18 IS NOT THE SAME AS SLIDE #18A. Use slide #18 to study endochondral ossification (BORROW ONE FROM A NEIGHBOR IF NECESSARY).. The forming bones here are not long bones so you will not find an epiphysis, synovial cavity, epiphyseal plate or diaphysis. You will, however, find excellent views of cartilage maturation, hypertrophy, death and calcification. Osteoblasts are shown to good advantage, as are the maturing spicules of calcified cartilage/osteoid. Find some spicules which still contain a cartilage core; compare them with spicules of formed bone. Looking at the marrow spaces, what are those big empty holes?

Slide #19 - developing tooth: Review this slide in the region of bone formation. Is this intramembranous or endochondral ossification? Why?