Bone and Bone Formation

OBJECTIVES: At the end of this laboratory, you should be able to:

  1. Identify the types of bone and the components of an osteon.
  2. Identify osteoblasts, osteocytes and osteoclasts, and describe their relationships to each other and their role in bone remodeling.
  3. Thoroughly describe the way in which bone develops and grows, including intramembranous versus endochondral ossification.
  4. Understand how this mineralized tissue is vascularized.
  5. Understand the distinction between primary, secondary, trabecular, and compact bone.


Bone:

Bone is specialized connective tissue with a calcified extracellular matrix (bone matrix) and 3 major cell types: the osteoblast, osteocyte, and osteoclast. The first type of bone formed developmentally is primary or woven bone (immature). This immature bone is later replaced by secondary (or lamellar) bone (mature). Bone is further classified as two types: trabecular bone (also called cancellous or spongy bone) and compact bone (also called dense or cortical bone) based on their anatomical appearance.

Be aware that trabecular and compact bone refers to the anatomical appearance of the bone, and not necessarily to the histological appearance, which can be either primary or secondary bone, although compact bone is usually composed of secondary (mature) bone and trabecular bone is often composed mostly of primary (immature) bone.

Developing bone
Primary bone (or woven bone) is characterized by the irregular (i.e.; woven) arrangement of collagen fibers, large cell number, and reduced mineral content. Primary bone matrix is acidophilic due to the abundance of collagen in the matrix. Lacunae containing osteocytes are arranged in a random orientation. In this specimen, note the preponderance of flat endosteal (bone-lining) cells on the surface of the bone matrix instead of osteoblasts, since there is not a high level of bone formation occurring at this stage. Be aware that endosteal cells are usually quiescent osteoblasts.

Woven bone, mammal
Primary (woven) bone is present throughout this entire specimen. Osteoblasts are cuboidal-appearing basophilic bone matrix-forming cells located on the surface of the bone matrix immediately above the osteoid (newly formed bone matrix which often stains lighter than mineralized bone matrix). Osteoblasts become entrapped in spaces within the bone matrix that they produce, and become osteocytes which are found within lacunae. Note that the orientation of the lacunae is random in woven bone. Osteoclasts and endosteal cells are not readily observed in this specimen since bone is being rapidly formed, and is not being remodeled.

Bone, femur
The trabecular (spongy) bone present in this specimen is found mostly within the epiphysis and some in the bone marrow cavity. Osteoblasts are located immediately above the osteoid (newly formed bone matrix). Osteocytes are found within lacunae. Giant multinucleated osteoclasts, which break down bone, are occasionally found in depressions in the bone surface termed Howship's lacunae. These are readily found in the ossification zone of the growth plate.
The compact bone in this specimen surrounds the marrow cavity and spongy bone. Locate the periosteum (external) and endosteum (internal) linings of the bone. Note that the separation of these linings is an artifact of specimen preparation. Bone lining cells, also sometimes referred to as endosteal cells are flattened basophilic cell located on the surface of the bone matrix, and represent quiescent osteoblasts. Observe the dense connective tissue of the periosteum which surrounds the bone tissue.
Note the presence of osteoclasts in the “cutting cone” of compact bone, in which these bone-resorbing cells are in the process of burrowing a tunnel through the bone to form a new osteon.

Bone, ground preparation (cross section)
Observe the Haversian systems (or osteons) of secondary (mature) bone in this specimen. The concentric lamellae are concentrically located around a central canal (Haversian canal) which contained blood vessels, nerves, and loose connective tissue. Volkmann's canals may be seen connecting Haversian canals. The other lamellae of compact bone are organized into inner circumferential lamellae, outer circumferential lamellae, and interstitial lamellae. Also note the empty lacunae and canaliculi that housed the osteocyte and its cell processes, respectively. Compare the ground bone specimen to H&E-stained bone sections.

Bone, ground preparation (longitudinal section)
Observe the Haversian canals (or osteons) of a longitudinally-oriented secondary ground bone specimen. Volkmann's canals may be seen connecting Haversian canals. Compare the ground bone specimen to H&E-stained bone sections.

 

Bone formation:

Developing facial bones
Intramembranous ossificationis visible in the facial bones on this specimen. Bone arises directly within mesenchymal condensations. This process can be identified by the appearance of bone spicules (islands of bone) among mesenchymal cells. Look for the eosinophilic bone matrix. The surrounding mesenchymal cells are stellate (central part with smaller parts radiating out from it) in appearance, and will differentiate into osteoblasts. Note the abundance of osteoblasts on the surface of the bone matrix, and the lighter-staining osteoid directly below the cells, which is newly-formed unmineralized bone matrix.

Bone, femur
Endochondral bone formation (ossification) is represented in this specimen. Bone arises by replacement of a small hyaline cartilage model. Locate the epiphyseal plate; it is the site for bone elongation. First, find the hyaline cartilage and move toward the diaphyseal bone marrow cavity. Identify the 5 overlapping zones:
1. Zone of Reserve or Resting Cartilage - young small cells evenly distributed, appears as typical hyaline cartilage.
2. Zone of Cell Proliferation - chondrocytes divide, forming parallel columns.
3. Zone of Cell Maturation and Hypertrophy - cells produce collagen and ground substance
4. Zone of Cartilage Calcification - septa of cartilage matrix become calcified, cells die.
5. Zone of Ossification - osteoblasts invade cavities, and deposit bone matrix.

Developing bone
This is an example of endochondral bone formation. Identify the zones in the developing epiphysis.
1. Zone of Reserve or Resting Cartilage - appears as typical hyaline cartilage.
2. Zone of Cell Proliferation - parallel columns of chondrocytes.
3. Zone of Cell Maturation and Hypertrophy - cells produce collagen and ground substance
4. Zone of Cartilage Calcification - cartilage matrix becomes calcified and the cells die.
5. Zone of Ossification - osteoblasts deposit bone matrix over the calcified cartilage