BONE
Bone is an exceptionally important tissue for dentistry. It also is complex because we must consider the dynamic aspects of this tissue instead of just its static structure. Therefore today's laboratory is long and has seven objectives.
Objectives:
1. To recognize bone in ground and decalcified sections:
2. To visually distinguish:
| Adult from woven bone | |
| Spongy from compact bone. |
3. To identify on your slides:
| Osteocyte | |
| Osteoblast | |
| Osteoclast | |
| Bone matrix | |
| Osteoid | |
| Lacuna | |
| Canaliculi | |
| Haversian system = osteon | |
| Haversian canal | |
| Circumferential, concentric and interstitial lamellae | |
| Trabeculum (= spicule) of bone | |
| Periosteum | |
| Endosteum | |
| Epiphysis, diaphysis | |
| Epiphyseal plate composed of epiphyseal cartilage |
4. To understand the processes of intramembranous bone formation
5. To understand the processes
of endochondral bone formation
and recognize :
| Primary and secondary ossification centers | |
| Reserve cartilage | |
| Zone of proliferation | |
| Zone of hypertrophy = cell enlargement | |
| Zone of calcification | |
| Zone of chondrocyte death | |
| Zone of resorption | |
| Zone of ossification | |
| Spicules of mixed calcified cartilage and bone |
6. To understand the histology of bone remodeling
7. Observe the structure of joints:
| Diarthrosis | ||
| articular cartilage | ||
| synovial space | ||
| synovial organ | ||
| Synarthrosis | ||
* * * * *
 | Blue Histology |
Slides
| D-87 | Fetal palate: woven and lamellar bone (H&E) | |
| D-142 | Suture of a newborn: synarthrosis (H&E) | |
| D-143 | Fetal skull: intramembranous bond formation (H&E) | |
| D-145 | Adult finger joint: diarthrosis (H&E) | |
| D-146 | Newborn knee: endochondral osteogenesis (PASH) | |
| D-147 | Decalcified femur: lamellar bone (H&E) | |
| D-149 | Compact bone (Ground) |
Optional slides
| D-8 | Intervertebral disk (H&E) | |
| D-89 | neonatal palate (H&E) | |
| D-144 | Toe, monkey, as a second example of a diarthrotic joint | |
| D-148 | Lamellar bone, newborn (Bodian) | |
| Bone associated with teeth: D-152, S-153, D-158, D-158 | |||
| Adult hard palate, to compare with D-87: D-90, D-91 | |||
Prologue
A word about examining bone histologically
Bone is a hard, calcified tissue and cannot be sectioned like the other soft tissues that you have seen. The two ways around this problem produce two sorts of preparations. Your slide set has examples of both.
To make a ground section, a small chip of dried bone is attached to a backing and then ground down to a very thin section, essentially with fine sand paper. Your section on slide D-149 is about 50 microns thick, much deeper than cut sections but thin enough to be transparent. As you focus up and down different planes of the tissue become visible. Ground sections are not stained. You look directly at the constituents of bone themselves (i.e. hydroxyapatite crystals and collagen). Obviously, no soft structures survive the grinding process. Instead of cells and blood vessels, all you can see are the holes (lacunae) where they had been.
The alternative approach is decalcification. The tissue is soaked in dilute acid or EDTA solution until all of the calcium crystals are dissolved. The tissue then can be sectioned in the ordinary way and stained. Decalcified sections show all of the soft tissues in and around the bone; such as bone cells, blood vessels, periosteum, endosteum and bone marrow. Decalcified bone matrix is highly visible because of the considerable amount of Type I collagen that remains.
* * * * *
Slide descriptions
D-147, Lamellar bone in a decalcified femur
This tissue has been decalcified and sectioned so you can see its soft components - cells, protein and connective tissue. Scan the slide with low power to pick out the bright orange ring of bone, the periosteum on the outside and the shrunken bone marrow within.
The outer part of the bone is formed by circumferential lamellae that extend around the entire circumference (illustration). These were laid down, one after another, under the periosteum. There are bluish lines between some of the lamellae, making them particularly obvious. These lines represent sites (or times during bone deposition) at which extra amounts of ground substance were secreted when the bone was being laid down. Further explanation
The larger white spaces you see in the bone tissue are spaces around blood vessels. These Haversian canals run longitudinally in a long bone such as a femur so you see them here in transverse section. In the outer part of the bone the blood vessels lie between circumferential lamellae and were incorporated from the periosteum as new lamellae were deposited. There are no Haversian systems around these vessels, showing that no remodeling has yet taken place in this part of the bone. Remodeling has occurred only in the inner (older) portion of the bone. There, the vascular canals are surrounded by series of concentric lamellae as Haversian systems. Obviously this bone came from a young monkey. Almost all of the bone in your femurs have been remodeled into Haversian systems.
Pick out a Haversian system to focus on. Lower your condenser until the field starts to darken. You should then see the circular lamellae arranged around the Haversian canal. The tissue within the canals is generally poorly preserved. However, if you look around carefully at 430X you can find examples which contain a tiny arteriole and venule and are lined by flattened osteoblasts (illustration). The many smaller blank spaces around the Haversian canals are lacunae for osteocytes. In most cases the cells themselves were washed out when the section was prepared.
This section was taken from near the end of the femur where large ligaments were inserted. The ends of their collagen fibers extend deeply into the bone for secure attachment. Such embedded fibers from a ligament or tendon are called Sharpey's fibers. In parts of the bone you can see the cross sections of individual collagen bundles as small, red circular structures, about the size of the osteocyte lacunae (illustration). Being dense bundles of collagen they are discernibly less basophilic than the bone matrix. The bundles run through holes in the bone, of course, and have shrunk during fixation so that one sees a lacuna around them.
Now, turn your attention to the layer of connective tissue on the outside of the bone (between it and some skeletal muscle). This is the periosteum, consisting of two sub-layers. The obvious outer fibrous layer is really no more than a typical adventitia of dense irregular connective tissue, and in this case quite vascular. The inner osteogenic layer is composed of a single layer of osteoblasts (with a scattering of indistinguishable osteoprogenitor cells external to them). Look for the osteoblasts at high power as scattered, very flat nuclei on the surface of the bone, underneath the coarse collagen bundles of the fibrous layer (illustration).
The osteogenic layer is crucial for the growth of the bone because bone, unlike cartilage, can grow only by apposition at a surface. That surface may be on the outside of the bone, an inner surface or even the surface inside of a Haversian canal, but bone can only grow on a surface. When osteoblasts actively deposit bone they are numerous, plump and easily seen (as in some of the later slides). Here bone growth has greatly slowed or stopped. The quiescent osteoblasts have reverted to a very flattened condition. (What is their function in this state? Answer)
Finally, glance at the shrunken remains of the marrow inside the bone. Its edge is smooth because it is lined by the endosteum that has peeled away from the inner surface of the bone. The abundant marrow cells obscure your view of the very flattened osteoblasts of the endosteum.
D-149 Ground bone
This is a small dried slab of compact, lamellar bone. It was cut from a cross-section of a femur. Therefore the lamellae have been cut perpendicular to their surfaces and show up well. The bone is compact and lamellar. Lower the condenser to make the lamellae more visible. (What does lowering the condenser do?) Most of the bone is made up of osteons. This is a sign of extensive remodeling. Remains of older lamellae survive between the osteons as "interstitial lamellae". In some cases you may be able to see where a new osteon has partially obliterated an older one, clear evidence of remodeling (illustration).
At least three of the edges of your bone chip are artificial breaks, but most of your slides have one natural edge that had been covered with periosteum. Along this edge the outermost lamellae run parallel to the surface of the bone and are called circumferential lamellae. See if you can find them on your slide (illustration).
Between lamellae are dark lacunae where osteocytes used to be. (Why are lacunae dark?) Focus up and down with your 43X objective and note the spidery canaliculi traversing the lamellae. They really are little canals running through the calcified tissue from one lacuna to the next (illustration).
Here and there on some slides, it is possible to observe somewhat larger vascular canals running at an angle to the Haversian systems. These are Volkmann's canals, which carry larger blood vessels to the Haversian canals. Look for them as channels that are cut obliquely instead of in good cross section.
D-87, Fetal palate, woven and lamellar bone (H&E)
Focus on the bone and
ignore the mucosae, the developing tooth at the anterior end of the palate and
other soft tissues. Scan the spongy bone to find areas of woven bone juxtaposed
to lamellar bone. This provides an excellent opportunity to compare these two
forms of bone (illustration).
List the ways that they differ.
| Ways to distinguish woven from lamellar
bone | |
| ______________________________________________________ | |
| ______________________________________________________ | |
| ______________________________________________________ | |
| ______________________________________________________ | |
| ______________________________________________________ | |
| ______________________________________________________ | |
(Only after you fill in this table check here for confirmation). Often a bluish reversal line runs along the junction of lamellar and woven bone. Eventually all of the woven bone will be remodeled and replaced by lamellar bone. Slides D-90 and D-91 show the bone of adult palates. All or most of their bone is lamellar, although very irregular in staining (illustration). This is partly because there are many Sharpey's fibers from ligaments and also because there are series of reversal lines in some places. (By way of orientation, D-87 is a sagittal section, going from the front to the back. D-90 and 91 are frontal sections going from left to right. In all cases the palate separates the oral from the nasal cavities.
D-143, fetal skull (intramembranous bone formation) (H&E)
Scan this slide with your low power objective. The scalp forms the upper half of the tissue, recognizable by numerous oblique sections through hair follicles. The developing bone is in the lower half, represented by irregular purple spicules (illustration). Look at the bone-forming area with intermediate magnification. Note how the area between the bone spicules is filled with loose, undifferentiated mesenchyme. Also see how highly vascular this area is (illustration).
Now, study one of the bone spicules. It has no embedded blood vessels, Haversian systems, nor lamellae. The osteocytes are embedded in a very haphazard way. This is fast growing embryonic = woven bone. Find the lighter staining band on the surface of the spicule. This is matrix that has not yet calcified and is called osteoid or prebone (illustration). The osteoblasts lined up on its surface have been busy laying down this organic matrix and soon will calcify it (consult your lecture notes for how they do it). Osteoblasts will continually become trapped in the bone as they lay it down. Replacements will differentiate from osteoprogenitor cells of the indistinct developing periosteum.
This woven bone forms a scaffold for lamellar bone to be deposited on. After the sheet of osteoblasts has laid down a sufficient amount of woven bone the cells abruptly change to synthesize lamellar = adult form of bone. Some of your slides show mixed spicules of woven and lamellar bone. The others of you will just have to be content knowing that your section was cut at an earlier stage, with woven bone only.
Eventually adult bone will replace all of the woven bone through remodeling. Remodeling begins even on spicules that are still growing. You should be able to find some multinucleate giant osteoclasts on your spicules (illustration). They usually lie in depressions that they have hollowed out (called Howship's lacunae [careful how you say this] or resorption cavities), usually on the opposite surface from the lined-up osteoblasts.
D-146, Newborn knee, endochondral osteogenesis (PASH)
This section was stained with PAS (for carbohydrate) to emphasize the matrix of cartilage and bone. Cartilage appears an intense magenta. That which has calcified is even darker. Bone tissue stains more palely because it has much less carbohydrate. Cells, of course, stain poorly.
Find the two opposed masses of hyaline cartilage, first by eye and then with your low power objective. At this stage these bones have only a single ossification center. Later they would develop a secondary calcification center in the middle of the masses of reserve cartilage. Since these cartilaginous masses constitute the epiphyseal ends of the bones, scanning along them will show all the stages of endochondral bone formation. You have two bones on your slide so choose the one which is the best to study (without tears, cut more medially and so forth).
Flip over to 100 X and find, the extensive zone of reserve cartilage at the ends of the bone where the growth begins (illustration). The chondrocytes here are randomly scattered. Because they have been stained with PASH they show the glycogen in them. Glycogen stores are a characteristic feature of hyaline cartilage mentioned earlier.
Proceeding down towards the bone shaft, find the zone of cell proliferation where the chondrocytes reproduce by specialized cell division (illustration). The division planes are all in the same orientation resulting in flattened cells arranged like a stack of coins. One cell in the reserve cartilage divided to produce all of the cells in one stack. Next, in the zone of enlargement, the cells swell up to a spherical shape. After this is the zone of calcification where the matrix begins to calcify as shown by its deeper reddish-purple color (illustration). The chondrocytes die as they calcify the matrix around them and you can see empty lacunae. A bit lower the amount of calcified cartilage matrix suddenly decreases. A front of chondroclasts covers the exposed surface of the calcified cartilage matrix in this zone of resorption. These cells remove all of the matrix except for a few bits which remain as elongate spicules extending into the marrow cavity. (Chondroclasts are the same cell type as osteoclasts, just eating a different dinner). These chondro/osteoclasts are difficult to recognize here, in part because the slide was stained with PASH instead of H+E and in part because their cytoplasm is filled with unstained vesicles, making the cells look like balls of foam .
The surviving spicules of calcified cartilage form a firm substrate for osteoblasts to attach to. Flip down to 430X and find these cells lined up on the thin, almost black calcified matrix. Osteoblasts are flattened so that they appear large when viewed face on but narrow when seen edgewise. The cells have a triangular shape with the nucleus pushed into the narrow end and a large Golgi apparatus in the center. Their cytoplasm is basophilic, but this is not revealed by the PASH staining of this slide. Some of the nearby spindle shaped cells may be osteoprogenitor cells but they cannot be distinguished with certainty from the reticular cells supporting the bone marrow between the spicules (illustration).
Question: If osteoblasts always are right on the surface of bone why do you see some without bone around? Explanation
The osteoblasts form a thin layer of pink woven bone over the cores of dark purple, calcified cartilage matrix. As you might expect the bone layer gets progressively thicker towards the lower ends of the spicules. Finally, osteoclasts sit at the ends of the spicules and chew their way upwards (illustration). They keep the length of the spicules more or less constant.
To understand what is going on at the spicules it is important to realize that the cartilage matrix is polarized and that cells can distinguish the longitudinal direction from lateral directions. This polarity is obvious up in the zone of proliferation where the cells divide only longitudinally to form stacks of disk-shaped cells. It persists in the spiculesOsteoblasts are attracted to the lateral sides of spicules (by signals that are unknown). Osteoclasts are attracted to the ends of spicules (and to the surface of newly calcified cartilage in the zone of resorption) where they can chew their way upwards towards the articular end of the bone.
The diaphysis of the bone also is growing, under the periosteum. The osteogenic layer here is made up of large, easily visible osteoblasts (illustration). These cells deposit bone to increase the diameter of the bone. Meanwhile, osteoclasts on the inner surface of the bone increase the size of the marrow cavity. The diaphysis is considered to grow intramembranously because new bone is deposited directly onto existing bone under a periosteum without any involvement of cartilage, Thus, a long bone, such as a femur, is formed through both endochondral and intramembranous processes. Flat bones are formed by only intramembranous osteogenesis.
Return to low power and review the entire process to get the "flavor" of endochondral bone formation. Consult your text if necessary to understand the significance of each step in the process.
D-145 Adult finger joint (H&E)
This section shows: 1) later stages in the chondrogenic formation of an endochondral bone, 2) the histological structure of a diarthrotic joint and 3) a variety of tissues. Let's begin with the first of these.
By eye, find the bone on this slide with a secondary ossification center in its epiphysis. Then find the entrapped plate of blue hyaline cartilage between the two ossification centers. The other bone on the slide is more mature and has lost its cartilaginous epiphyseal plate altogether. It is even impossible to say exactly where the plate had been because the primary and secondary ossification centers have fused completely into a single marrow cavity.
Examine the epiphyseal plate under low magnification (illustration). Endochondral bone formation takes place from both of its faces. However, this finger is from a young adult and its growth has dwindled to a snail pace. The individual developmental zones in the epiphyseal cartilage have regressed to a minimum (illustration). Growth on the epiphyseal side is even slower than of the diaphyseal side and the stages there more abbreviated. Nevertheless, you can still see some longitudinally oriented lines of chondrocytes. Columns of (calcified) cartilage extend down into some of the bony spicules. Can you distinguish any woven bone here? The thin layer of endosteum can be seen over most of the spicules and around the cavity in the diaphysis. See how vascular it is. The marrow in the spongy areas of this bone is all yellow.
The articular surfaces of the two adjacent bone ends are covered with articular cartilage. (These cartilages tend to stain redder than most of the other hyaline cartilages that you looked at in the last laboratory session.) The distinctive feature of articular cartilage is that it has a free surface not covered by a perichondrium (illustration). The articulation involves two very smooth cartilage surfaces sliding across one another. The basal surfaces of these cartilages are directly attached to the underlying bone. Locate the junction between the cartilage and bone to verify that there is neither perichondrium nor periosteum here on the bone surface (illustration).
Compare the structure of the joint itself with the figure of a joint in your textbook. Also, read the short section of text. The space between two articular cartilages is filled with synovial fluid. A collar of ligament holding the two bones together encloses this joint cavity. A triangle of tissue extends from the inner side of the ligament part way into the space between the two articular cartilages. This synovial membrane is specialized to secrete synovial fluid. In these areas the epithelium is cuboidal to low cuboidal and underlain by a rich plexus of small blood vessels (illustration).
Finally, how many types of connective tissue can you identify on the slide?
| 1-4 | Wimp, she/he loves you not | |
| 5-7 | Good but no cigar | |
| 8 | Awesome! | |
| 9 | BIG DUDE, even if you must be splitting hairs | |
| 10 | Honorary Australian |
D-142, Suture of a newborn (synarthrosis) (H&E)
A synarthrosis is a junction where two flat bones (of the skull) meet. A ligament of very dense fibrous connective tissue holds the two bones together. Obviously this is not a movable joint, like in a finger. Its purpose is to attach the bones instead of encouraging movement. The opposing edges of the bones are very irregular with rows of interdigitating projections and cavities. The narrow ribbon-like ligament runs between.
It is essential to understand the orientation on this slide. This suture has been sectioned along its length and perpendicular to the surface of the skull. One bone would lie mainly in front of the plane of section and the other mainly behind it. Most of the tissue on your slide is from one bone. In places the section cuts into the ligament of the suture between the two bones. This gives roundish islands of dense connective tissue (illustration). In a few places the section has dipped all of the way through the ligament to graze the tip of protrusions of the other bone. These bits of the other bone shows up as a bluish blob in the middle of islands of ligament (illustration). They look strange for bone because they are riddled with Sharpey's fibers. Also, they are sectioned parallel to and just below the surface of the bone. For some reason the lamellae near the surface have excessive amounts of ground substance and therefore a variegated bluish appearance. You can find some nice views of Sharpey's fibers cut transversely and longitudinally in these blobs of bone if you are patient.
It is worth while examining the ligament with some care as it has been cut in various orientations across the slide. In particular, with a bit of perseverance you can find some transverse sections. Here you can see how large the bundles of fibers are. They form a mosaic pattern with the fibroblasts (fibrocytes) squeezed in between. By the way, this is what a tendon looks like in cross section, a view that our slides, unfortunately, do not show.
Now drop back to 100X and examine the bone. What type is it? Dr. Bernard thinks that some of the bone is woven. I think that it is almost all lamellar and that its sloppy bluish look just comes from the extra chondroitin sulfate and the fact that the orientation of the lamellae varies from place to place (so that lamellae are not seen everywhere). Bone often seems to have extra amounts of ground substance at its surface where Sharpey's fibers go in. What do you think about Dr. Bernard's claim? In any case much of the bone is lamellar and has beautiful, that's right, beautiful, osteons.
* * * * *
Optional exercises
D-148 Lamellar bone in a newborn (Bodian)
This slide has been subjected to Bodian staining to emphasize the canaliculi. Osteoblasts and osteocytes extend cell processes (filapodia) through these small passageways. Use your low power objective to find the circular bone amid the skeletal muscle on this slide. You can't really see the lamellae of bone in this preparation, but never mind, it is lamellar. Flip over to an intermediate power and note that the vascular canals are large in this immature specimen. The small "spiders" are the lacunae of the osteocytes. Change to high power and observe that the legs of the spiders are canaliculi.
Scan around to verify that canaliculi connect one lacuna to its neighbor. Now, find canaliculi extending right to a vascular space. An osteoblast on the bone surface sends filapodia down these canaliculi to make contact with osteocytes closest to the endosteum. This is how the living portion of bone is nourished. You cannot see the osteoblasts in this preparation but if they were not here, the bone under the bare surface would starve to death.
D-89 Neonatal palate (H&E)
This section has some mighty fine woven bone. If you look around, there are also several sheets of lamellar bone for comparison. The lamellar bone has a more even distribution of more uniform osteocytes and a paler pink color. There are a few osteoclasts, especially near the areas of lamellar bone, but you will have to look diligently to find them and probably shouldn't bother.
The glands, mostly mucous, are well developed. On the other hand the stratified squamous epithelium lining the hard palate has not yet become keratinized. You can see nuclei extending up to the top of the epithelium.
D-144 Toe joint of Monkey
Your slide box has a section through an additional joint on slide D-144. Examine it as you did D-145. There is no epiphyseal cartilage left in either bone of this joint from a mature individual. The slide is a good one for reviewing a variety of types of connective tissues and for examining the synovial organ and chamber.
Can you find Sharpey's fibers?
Do you see any muscle tissue on this slide? (Really? What type is it?)
Recall that Haversian systems run longitudinally in long and short bones. Therefore you will see them sectioned lengthwise here. Do they look the way you expected them to for this orientation? In several cases, the section goes through the Haversian canal. Can you see the layer of osteoblasts lining those canals?
D-8 Intervertebral disk (H&E)
Return to this slide which you examined earlier for fibrocartilage. Look carefully and see if you tell whether vertebrae are formed by intramembranous or endochondral osteogenesis? How would this tissue have looked when bone formation had just begun?
Now compare this
situation with the septum of the nose in D-83.
Hold this slide up to the light. Running up the midline is a long thin bar of
purple-blue staining cartilage. This is the cartilaginous portion of the nasal
septum (which divides the nasal cavity into right and left chambers). Twist your
own nose from right to left. The flexibility is present because the most anterior
portion of the nasal septum is composed of hyaline cartilage. This cartilage abuts
the more posterior bony portion of the nasal septum (illustration).
Did the septum form endochondrally from this cartilage?
* * * * *
Other slides
with bone on them
| D-90, D-91 | Adult hard palate, to compare with D-87 | ||
| D-152, D-153 | Slides showing maxillary and mandibular bone: | ||
| D-157, D-158 | |||
| D-159 | |||
* * * * *
Teasers
Look at D-146. How many microns of calcified cartilage will a single reserve cartilage cell produce as it goes through the special steps leading up to calcification during endochondral bone formation, in the baby rat femur?
What is the function of depositing bone on the calcified cartilage spicules at the epiphysis? What would a cross section of an epiphyseal plate look like sectioned just below the zone of resorption?
Think about a blood vessel running in the inner part of periosteum of a femur and getting incorporated into the growing diaphysis. Draw what you think the pattern of lamellae would look like around the incorporated blood vessel. Now look at the blood channels in the outer part of the femur on slide D-147. Draw what you actually see and discuss the matter with your lab mates.
Estimate the average distance in microns between blood vessels in the compact bone of slides D-147 and D-149. Would you be able to make this estimate for the finger bone in D-145 cut longitudinally?
What are the shapes of: an osteocyte in lamellar bone, an osteoblast actively laying down bone, an inactive osteoblast, an osteoprogenitor cell and an osteoclast?
Osteoblasts form a sheet of cells over bone but this is not considered to be an epithelium. In what ways, do you think, such a sheet of osteoblasts differs from an epithelium?