(C28-31) Slide 16: Decalcified bone, H&E

Because bone is so hard and difficult to section, two special methods were developed to permit its study. In the first method, the mineral content is left intact. After fixation and a thorough rinsing, the bone is ground on a grinding wheel until it is transparent. These sections are referred to as "ground bone".

In the other method, the calcium salts, which make bone so rigid, are removed by treating the bone with acid and/or EDTA, a chelating agent. Besides removing the calcium salts, the acid also causes swelling of the type I collagen fibers, so individual fibers are difficult to discern. These sections are referred to as "decalcified bone". These terms refer to the techniques, and not to the condition of the living bone.

This slide shows a transverse section through a long bone of a small, growing mammal. There are two different series of sections in the collection, one with a more purple staining marrow (C28 M) than the other.

WARNING: The sections on this slide did not lay flat on the glass. They are very wavy. So, cleaning your glasses and blinking your eyes will not help. Drivers, please try to find a flat spot, and move slowly when using higher powers, to prevent having sick passengers. When looking at this slide, it is best to work with the 4x and 10x objectives as much as possible.

Identify the following (C29): Endosteum, periosteum, inner and outer circumferential lamellae, interstitial lamellae, and the concentric lamellae of Haversian systems (osteons). See figures on the next pages.

The endosteum is found on the marrow side of the compact bone and the periosteum is on the outside. Both layers are composed of osteoprogenitor cells and a little bit of loose irregular connective tissue. The inner and outer circumferential lamellae are arranged around the inner and outer regions of the bone, like growth rings of a tree. These rings are layers of appositional growth. Interstitial lamellae are lost souls, the bits and pieces of old Haversian systems that remain after remodeling (See figures below). Most of the Haversian systems on this slide have only 2 to 3 concentric lamellae around them. About the only visible evidence of the osteocytes is their nuclei, which lie in concentric circles around the Haversian canal. The osteocyte’s lacunae have little points with fine hairlike projections extending from them. These are the canaliculi. At higher magnifications, focus slowly through the hairlike projections. You should see them connecting one osteocyte to the next.

The Haversian canal includes vessels, nerves and resting osteoblasts. Within the larger canals, there are many more cells, as evidence of primary (new) bone formation. Can you find differences in staining of the different osteons? The lighter stained osteons are more recently developed, and less well calcified.

Occasionally, one can find a Volkmann’s canal (C30) connecting the endosteum with an Haversian systems. Look for the Volkmann’s canals on the endosteal surface of the bone. They are the means by which blood vessels enter and leave the bone substance as they supply the osteocytes via vessels in Haversian canals.

Look for osteoclasts on the endosteum (C31 arrow). They are monocyte derived, multinucleated cells which destroy bone.