Introduction The slides for this lab are located in the “Musculoskeletal” folder on the Virtual Microscope. You will briefly review the two types of growth: endochondral and intramembranous ossification.

These types of development occur over a long period of time and as such each image is but a snapshot of that event at that moment. Be able to interpret what you see and fit it into the overall process of bone development.

Learning objectives and activities Using the Virtual Microscope:

A Use histology to demonstrate how bone grows by intramembranous and endochondral ossification and identify the major features and steps in these processes B Complete the self-quiz to test your understanding and master your learning. 1. Intramembranous ossification During intramembranous ossification, bone is formed by the differentiation of mesenchymal cells directly into which then lay down osteoid that is mineralized to form immature woven bone. It is a type of ossification that occurs in the flat of the skull and the mandible.

Examine the fetal head again in Slide 16 – the bones here are developing intramembranously

A primary ossification center of Identify osteoblasts forms from mesenchyme mesenchyme in and osteoblasts start to secrete osteoid Slide 16a

The osteoid calcifies around the osteoblasts and they become Identify this stage trapped in lacunae. More osteoblasts and its make osteoid around the outside. This is components in the stage shown in Slide 16 Slide 16b

The process continues until numerous trabeculae of immature woven bone are formed

The immature bone is remodeled to form a mature, lamellar 2. Endochondral ossification During endochondral ossification, a hyaline scaffold is first deposited and this is then transformed into bone by a series of ossification events commencing at the primary site of ossification () and then finally at the secondary sites of ossification (epiphyses). It is not possible to observe each stage but you can see the different cellular components derived from the developmental event.

Examine the fetal in Slide 17 – it is developing by endochondral ossification

a. Mesenchyme Identify the -Endochondral ossification commences by the formation of mesenchymal and from mesenchymal cells. tissue in -Find evidence of mesenchymal tissue in this slide Slide 17a

b. Developing skeletal muscle Identify the - Note that in some regions the mesenchymal tissue has begun to developing skeletal form thin muscle fibers and the outline of whole skeletal muscles muscle in can be made out. Slide 17b

Identify the 1o c. Primary ossification center ossification center - Locate the primary ossification center in the diaphysis of the long in bone Slide 17c d. Immature woven bone trabeculae Find examples of - In the primary ossification center identify thin trabeculae/spicules immature woven of newly laid down woven bone bone and its - Note that they are surrounded by active osteoblasts that have cellular secreted a thin layer of osteoid. The trabeculae are thickening by components in appositional growth. - Note the osteoblasts trapped in lacunae within the trabeculae Slide 17d e. - Examine the periosteum and identify fibrous and osteogenic layers Find the - Note that the osteogenic layer is composed of osteoblasts that are periosteum and its secreting osteoid as the periphery of the bone continues to grow osteogenic layer in appositionally. Slide 17e Find examples of f. Osteoid osteoid in - Demonstrate a region of osteoid deposited by osteoblasts and Slide 17f observe how it is different from surrounding ossified woven bone. g. Calcified cartilage - Recall that the long bone began as a cartilage scaffold and as the chondrocytes died the cartilage matrix calcified. Differentiate - Osteoid was deposited on the calcified cartilage matrix by between the osteoblasts and this began to mineralize to form woven bone. components of - The difference in staining due all of these components gives calcified cartilage calcified cartilage an almost watercolor like appearance in - The paler region is calcified cartilage Slide 17g - The darker region around it is woven bone - The outer periphery beneath the lining of osteoblasts is osteoid. - The cells trapped within the region are osteocytes in lacunae (remember the chondrocytes already died) h. Marrow spaces Find marrow - The spaces between newly developed trabeculae are beginning to spaces in resemble marrow and are highly vascular Slide 17h i. - A layer of endosteum composed of very active osteoblasts lines Locate the the newly developed trabeculae endosteum in - Observe the newly secreted osteoid beneath them Slide 17i - j. Secondary ossification center - Observe the secondary ossification center in the of the Observe the stage bone. of development of - It is composed of the hyaline cartilage scaffold and has not yet the epiphysis in begun to ossify Slide 17j - Locate the chondrocytes in their lacunae and the extracellular matrix k. Observe the extent - the epiphyseal plate is the site where the long bone is growing and location of the in length. epiphyseal plate in - it has the following regions that represent the development of Slide 17k bone from the cartilage scaffold

Differentiate between the following regions of the epiphyseal plate l. Zone of resting cartilage Zone of resting - blends with the hyaline cartilage of the undeveloped epiphysis cartilage in Slide 17l

m. Zone of proliferation Zone of - chondrocytes are proliferating and form stacks of cells as they proliferation in quickly divide Slide 17m

n. Zone of hypertrophy Zone of - the chondrocytes get larger as their cytoplasm fills with hypertorphy in glycogen Slide 17n

o. Zone of calcification Zone of - as the chondrocytes die the cartilage matrix becomes calcified calcification in - the staining is different to that of hyaline cartilage Slide 17o

p. Zone of ossification -The calcified cartilage becomes lined with osteoblasts that Zone of ossification begin to lay down osteoid that begins to mineralize to form in woven bone Slide 17p In addition to all the basic tissues you must be able to identify the following structures and their features:

Bone Development Intramembranous ossification Endochondral ossification Mesenchyme/Mesenchymal tissue Osteoid Mineralized bone Calcified cartilage Epiphyseal plate Zone of resting cartilage Zone of proliferating cartilage Zone of hypertrophy Zone of calcification Zone of ossification Diaphysis Epiphysis