OBJECTIVES:

Mature Bone

1. Be able to use standard nomenclature to describe the microscopic structure of bone (e.g. lamella, osteon, osteocytes, canaliculi, periosteum, endosteum).
   
   
2. Be able to recognize mature bone (dense and cancellous) in conventional or ground section. Be able to identify the component parts of mature bone (e.g. osteon, lamella, lacuna, osteocyte) in appropriate sections.
   
   
3. Be able to describe the anatomical route that nutrients and metabolic products use between the vascular system and osteocyte.

Intramembranous Bone Formation

1. Be able to describe, as well as recognize in section, the process of bone matrix deposition and mineralization including the inclusion of cells within lacunae.
   
   
2. Be able to describe, as well as recognize, the process whereby cancellous bone is converted into compact bone including the process of remodeling.
   
   
3. Be able to recognize osteoblasts, osteocytes and osteoclasts in appropriate sections.
   
   
4. Be able to make an educated judgment about whether an area of bone is being formed or resorbed based on the morphology present in the section.

Endochondral Bone Formation

1. Understand the process whereby a cartilage model is broken down and replaced by bone (e.g. formation of a bony collar; chondrocyte death, and incursion of an osteogenic bud from the periosteum).
   
   
2. Be able to describe how the epiphyseal growth mechanism results in elongation of the bone.
   
   
3. Be able to recognize the different regions of a cartilage growth plate (e.g. zones of reserve, proliferation, hypertrophy, calcification).
   
   
4. Be able to describe the different functions served by an articular cartilage compared with an epiphyseal growth cartilage (growth plate).
   
   
5. Be able to describe how the shaft of a long bone increases in diameter with growth.
   
   
6. Be able to describe the morphological features of insertion of a tendon or ligament into bone.
   
   
7. Be able, in a simple way, to describe how fracture repair, resembles the process of endochondral bone formation.

There is no single perfect section in which to study bone and its formation. The process involves transitions, and the various stages are simply too many to be encompassed in any one section. Even the many slides available in this session may not have all the structures you need for study, so try to look as many different slides as you can. With the exception of slide #51 and #93, all the slides you study have been decalcified by the action of acid or a chelating agent, so you will be looking at the remaining organic matrix. Bone is difficult to cut in paraffin so there are distortions and differential shrinkages, common events that lead to an almost universal separation of the bone from the periosteum. If you look long enough, however, you'll find intact areas; these areas are the ones with which to start your study.

I. Intramembranous Bone Formation (W pg. 190, 10.5-6 and pg. 197, 10.17)
Slide 46-HE (embryonic face, H&E) [WinLab] [Mac] [WinHome]
Slide 46-M (embryonic face, Masson trichrome) [WinLab] [Mac] [WinHome]
Slide 46-M-N (new scan of slide 46M) [WinLab] [Mac] [WinHome]
Slide 120 (head, 66mm embryo, H&E) [WinLab] [Mac] [WinHome]
Slide 120-N (new scan of slide 120) [WinLab] [Mac] [WinHome]

These sections of human faces exhibits all stages of intramembranous bone formation. Use figures 10.5 and 10.6 in the Wheater's atlas (pg 190) for cell identification. Wheater's covers long bone formation exclusively, but some of the images can be useful in membrane bone formation. These frontal sections of a fetal human face are stained with either H&E or H & Masson. You should be able to find:

1. deposition of osteoid, (difficult to see but you can find examples in slides #46-HE [see example] and #120 [see example]).
2. a shift in matrix color (darker) with mineral deposition,
3. osteoblasts (active and inactive) and osteoclasts [see example],
4. osteocytes , and
5. forming Haversian systems or osteons (evident in slide #46-HE [see example] and #46-M [see example]). In order to find all the structures listed above, you will have to look at many different spicules or trabeculae of bone.

Slide 115-N (palate, H&E) [WinLab] [Mac] [WinHome]
Slide 115-M (palate, Masson) [WinLab] [Mac] [WinHome] (note: this slide is a bit dark; in ImageScope, go to the Image menu, then select Adjustments to adjust the brightness and contast if necessary)

Further stages of developing a compact bone from a spongy one are illustrated in this parasagittal section of the palate. Note the increased numbers of osteocytes, in some places forming one or two rings of an osteon. In the H&E section, the rapidly formed, immature bone (aka "woven" bone) [see example] exhibits a greater affinity for hematoxylin and higher cell density compared to mature bone. An outer fibrous layer and loose inner layer of periosteum can be seen. Sharpey's fibers made primarily of type I collagen spanning the cellular layer of periosteum and inserting into the bone are well illustrated in the both the H&E [see example] and Masson-stained [see example] sections.

Slide 48 (leg, 154mm embryo, H&E) [WinLab] [Mac] [WinHome]
Slide 48b (leg, 154mm embryo, Masson) [WinLab] [Mac] [WinHome]

One slide is stained with H&E, while the other is stained with trichrome. Only long bones grow in length by proliferative activity at the epiphyseal plate and subsequent endochondral ossification. How does the diameter of the bone shaft increase with growth? (BO1) Appositional growth increases their circumference. New bone is laid down around the shaft of a long bone by a mechanism that is essentially the same as that of intramembranous ossification (many authors use this term to describe the process--which can confuse you!). In this cross section of the developing tibial and fibular shafts (two stains again) note particularly the osteoblastic activity, and the forming osteons at the outer edge of the shafts in the H&E [see example] and trichrome sections [see example]. What will fill the cavity of these bones when they are fully developed? (BO2) Where is the endosteum? (BO3) Can you see fibrous and cellular layers of the periosteum? (BO4) With the Masson stain you may see collagen fibers [see example] running from the fibrous periosteum to the bone --what are they? (hint: refer to slide 115 above) The purple area in the Masson stained section is calcified cartilage - see endochondral section that follows.

II. Endochondral Bone Formation
Slide 108 (finger, H&E) [WinLab] [Mac] [WinHome]
Slide 110 (finger, H&E) [WinLab] [Mac] [WinHome]
Slide 47 (knee, sheep embryo, H&E) [WinLab] [Mac] [WinHome]

These slides are useful for learning about the process of endochondral ossification because you can see the entire forming bone and adjacent joint cavities. Look at the cartilage ends (epiphyses) and the forming bony shaft. The phalanges do not develop an epiphyseal plate, but the same process of growth and calcification of cartilage takes place, a process that can be seen more easily in slide #108 [see example] than in #110 [see example]. Examine the periosteum and forming marrow cavity. Slide #47 of a developing knee joint also illustrates endochondral bone formation quite well [see example].

Epiphyseal cartilages (W pgs 199-200)
Slide 49_20x (humerus, H&E) [WinLab] [Mac] [WinHome]
Slide 49_40x (humerus, H&E) [WinLab] [Mac] [WinHome]

There are two different magnifications (20X and 40X) of the epiphysis of a human long bone (those of you with even locker numbers may have a canine specimen on slide #49 that is much better). We require you to recognize 5 zones (W pg 200, 10.21): 1) resting or reserve (R); 2) proliferative (P); 3) hypertrophy (H); 4) calcification (D); and 5) ossification (O). These specimens do not permit an actual distinction between hypertrophic and calcification, but make sure you understand the sequence! Note the persistence of the cartilage cores well into the marrow cavity (W pg 201, 10.22) What will happen to these eventually? (BO5). Note that slide 49 also has a secondary center of ossification [see example] that is just stating to form in the head of the bone (there are blood vessels present in the cartilage, but it hasn't yet started to ossify); the primary center of ossification is in the shaft of the bone. Note that hyaline cartilage in the head of the bone, while avascular, certainly contains spaces for traversing large blood vessels indicative of bone formation. What is the correlation between woven, spongy and compact bone and intramembranous and endochondral bone formation? (BO6). An example in which bone can actually be seen in the secondary ossification center may be found in slide 61 in the UCSF collection [WinHome] [Mac] [WinHome].

III. Mature Bone --Note: this section is repeated from the Cartilage/Mature Bone lab session last week. However, NOW you know what "interstitial lamellae" are and how they form, so be sure you can identify them.

Slide 50 (compact bone, decalicified, H&E) [WinLab] [Mac] [WinHome] (W pg 194, 10.11)

Even though this section is distorted, you should be able to find osteons in various stages of development, lacunae, and canaliculi (to see canaliculi you will need to use your microscope and the glass slide from your collection --cut down the light by closing down the iris diaphragm to see them). Incremental deposition similar to that which would be present in inner and outer circumferential lamellae can be seen. What distinguishes between compact and spongy bone? (BO7)

Ground sections (W pgs 192-193, 10.9-10.10):
Slide 51 (cross section) [WinLab] [Mac] [WinHome]
Slide 93B (cross section) [WinLab] [Mac] [WinHome]
Slide 93A (long section, thin) [WinLab] [Mac] [WinHome]
Slide 51 (long section, 20x) [WinLab] [Mac] [WinHome]
Slide 51L-EX (long section, 40x) [WinLab] [Mac] [WinHome]
Slide 93C (long section) [WinLab] [Mac] [WinHome]

There are both longitudinal and cross sections. First, study cross sections (#51, #93B). In these sections, the trapped air bends the light giving a dark image; the mineral and matrix generally transmit the light. You should be able to identify osteons and their subdivisions (as in slide 50), interstitial lamellae, Haversian canals and nutrient canals (Volkmann). Note that the latter canals penetrate osteons without causing new lamellae to be laid down around them. Study the thinnest ground section (#93A) to identify lacunae and canaliculi. (W pg 193, 10.10a; in this figure the tissue is "unstained" but a red dye has been used to illustrate canals, lacunae and canaliculi). Now, look at the longitudinal sections (#51-20x, #51-40x, or #93C) of compact bone and try identifying the various structures mentioned above, especially Haversian and Volkmann's canals.

Electron Micrograph Wall Charts

#33 INTRAMEMBRANOUS BONE FORMATION [WinLab] [Mac] [WinHome]

This is sometimes called "direct" bone formation because it does not involve cartilage. Hence, chrondocytes would not be found in this section. The term "membrane" is used here because the periosteum around forming bone appears like a membrane. Find the capillaries and understand their significance. Make sure you know the structural and functional differences between an osteoblast and osteocyte.

#34 INTRAMEMBRANOUS BONE FORMATION [WinLab] [Mac] [WinHome]

Observe (and remember) that the bone formation which occurs from the periosteum of the diaphysis of long bones is identical to the process of intramembranous bone formation. In this unique micrograph, study the differentiation of osteoprogenitor cells to osteoblasts and subsequently to osteocytes. What is osteoid? (BO8) Note the formation of long cell processes as the osteoblast (lower right corner) prepares for the transformation into an osteocyte. Find the cell process which is already located in a canaliculus.

#35 OSTEOCYTE [WinLab] [Mac] [WinHome]

The calcium crystals of the bone matrix were removed in this preparation by a decalcification process. Note how coarse the collagenous fibrils are, and the difficulty in visualizing the periodicity of the fibrils (probably due to the process of mineralization).

#36 ENDOCHONDRAL BONE FORMATION [WinLab] [Mac] [WinHome]

Note that the bony collar is formed from the cells of the periosteum/perichondrium. Central chondrocytes of this cartilage model break down and die because the cartilage matrix becomes calcified. Subsequently, periosteal blood vessels break (erode) the bony collar and invade the lacunae of the dying chondrocytes. Find these invading blood vessels.

#37 ENDOCHONDRAL BONE FORMATION [WinLab] [Mac] [WinHome]

In the epiphyseal growth plate, continuous growth of hyaline cartilage, breakdown of calcified cartilage, ingrowth of blood vessels, bone formation on the calcified cartilage matrix, and finally resorption of this bone and formation of bone marrow occurs. Make sure that you identify each of these steps and that you understand what happens.

#38 ENDOCHONDRAL BONE FORMATION [WinLab] [Mac] [WinHome]

In this enlargement of rectangular area in chart #37, study the transition that occurs in the chondrocytes as they change from very active to hypertrophied and dying. Why do the chondrocytes die? (BO9)

#39 OSTEOCLAST [WinLab] [Mac] [WinHome]

The osteoclast is a very large cell (multinucleated) that sits on the surface of bone matrix. Note the many lysosomes and phagocytic vacuoles. What is the functional significance of these structures? (BO10) Note the proximity of the osteoblast. Remember that bone deposition can take place very near bone resorption. This explains the juxtaposition of osteoclast and osteoblast. Most osteoclasts are thought to arise by fusion of monocyte-macrophages.

#40 HAVERSIAN CANAL [WinLab] [Mac] [WinHome]

Note the "inactive" appearance of endosteal cells. The presence of a macrophage in the Haversian canal indicates the potential eroding function of the endosteal lining of the canal. Why are blood vessels so important in bone? (BO11)

Review Question Answers

BO1: How does the diameter of the bone shaft increase with growth?

answer

BO2: What will fill the cavity of long bones when they are fully developed?

answer

BO3: Where is the endosteum?

answer

BO4: Can you see fibrous and cellular layers of the periosteum?

answer

BO5: What will happen to the cartilage cores that persists in bony spicules (trabeculae) in the marrow cavity?

answer

BO6: What is the correlation between woven, spongy and compact bone and intramembranous and endochondrial bone formation?

answer

BO7: What distinguishes between compact and spongy bone?

answer

BO8: What is osteoid?

answer

BO9: Why do chondrocytes die after they hypertrophy in the epiphyseal plate of the metaphysis?

answer

BO10: What is the functional significance of lysosomes and phagocytic vacuoles present in osteoclasts?

answer

BO11: Why are blood vessels important in bone?

answer

Practice Questions

Click here to view image

1. Which of the following statements regarding the cells indicated is INCORRECT?

1. They are derived from hematopoietic progenitor cells.
2. They secrete collagenase.
3. They are involved in remodeling ALL bones, whether formed via endochondral OR intramembranous ossification.
4. Excess activation of these cells may result in hypocalcemia (low blood calcium levels).
5. NONE of the above (i.e. ALL of the above statements are CORRECT).

Click on the MAC link or PC link to view image

2. The brackets indicate:

1. interstitial lamellae
2. inner circumferential lamellae
3. outer circumferential lamellae
4. periosteum
5. endosteum

Click on the MAC link or PC link to view image

3. The bracketed area:

1. contains proliferating chondrocytes.
2. contains type II collagen fibers in its matrix.
3. is NOT present in adult bone (i.e. no longer growing lengthwise).
4. NONE of the above
5. ALL of the above

Click here to view image

4. The indented regions indicated by the yellow arrows are:

1. lacunae
2. canaliculi
3. Haversian canals
4. Volkmann's canals
5. Howship's lacunae

Click on the MAC link or PC link to view image

5. The structure indicated:

1. arose via intramembranous ossification.
2. arose via endochondral ossification.
3. is comprised primarily of calcified cartilage.
4. is undergoing extensive remodeling as evidenced by numerous osteoclasts on its surface.
5. is in a secondary center of ossification.

Produced and supported by:
The Learning Resource Center - Office of Medical Education
Department of Pathology, Virtual Microscopy Facility
Department of Cell and Developmental Biology

© copyright 2007 The Regents Of The University Of Michigan. All rights reserved.

Questions or comments? E-mail Dr. J. Matthew Velkey (jvelkey@med.umich.edu)