Șef lucr. dr.

Grigoraş Adriana

FACULTY OF MEDICINE

Practical work no. 7

Connective tissue (2 hours)

Compact and spongy bone (slides of ground sections and decalcified bone). Intramembranous
ossification. Endochondral ossification.

Bibliography
Junqueira LC, Carneiro J, Basic Histology. Text and Atlas, 11th. ed., McGraw-Hill
Companies, New-York, 2005
Ross MH, Pawlina W, Histology. A text and atlas With Correlated Cell and Molecular
Biology, 7th edition, Wolters Kluwer Health, 2015

Bone
Bone tissue is a mineralized connective tissue. It is formed by cells, called
osteoblasts, that deposit a matrix of type-I collagen and also release calcium, magnesium, and
phosphate ions that ultimately combine chemically within the collagenous matrix into a
crystalline mineral, known as bone mineral, in the form of hydroxyapatite. The combination
of hard mineral and flexible collagen makes bone harder and stronger than cartilage without
being brittle.

Bone cells

Osteoblasts arise from osteoprogenitor cells located in the periosteum and the bone
marrow. Osteoprogenitors are immature progenitor cells . Osteoprogenitors are induced to
differentiate under the influence of growth factors, in particular the bone morphogenetic
proteins (BMPs). Osteoblasts form a closely packed sheet on the surface of the bone, from
which cellular processes extend through the developing bone.

Osteocyte, a cell that lies within the substance of fully formed bone. It occupies a
small chamber called a lacuna, which is contained in the calcified matrix of bone. Osteocytes
derive from osteoblasts, or bone-forming cells, and are essentially osteoblasts surrounded by
the products they secreted. Cytoplasmic processes of the osteocyte extend away from the cell
toward other osteocytes in small channels called canaliculi. By means of these canaliculi,
nutrients and waste products are exchanged to maintain the viability of the osteocyte.

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Osteoclast is a large cell that contain 15-20 closely packed oval-shaped nuclei. They
are found in bits in the bone surface which are called resorption bays or Howships Lacunae.
Osteoclasts are characterized by a cytoplasm with a homogeneous, "foamy" appearance. This
appearance is due to a high concentration of vesicles and vacuoles. These vacuoles are
lysosomes filled with acid phophatase. The rough endoplasmic reticulum is sparse. The Golgi
complex is extensive. At a site of active bone resorption, the osteoclast forms a specialized
cell membrane, the "ruffled border," that touches the surface of the bone tissue. The ruffled
border, which facilitates removal of the bony matrix, is a morphologic characteristic of an
osteoclast that is actively resorbing bone. The ruffled border increases surface area interface
for bone resorption.

The osteon, or Haversian system, is the fundamental functional unit of much compact
bone. Osteons, roughly cylindrical structures that are typically several millimeters long and
around 0.2mm in diameter .Each osteon consists of concentric layers, or lamellae, of compact
bone tissue that surround a central canal, the Haversian canal. The Haversian canal contains
the bone's nerve and blood supplies.Between adjoining osteons there are angular intervals that
are occupied by interstitial lamellae.These lamellae are remnants of osteons the greater parts
of which have been destroyed. Near the surface of the compact bone the lamellae are arranged
parallel to the surface these are called circumferential lamellae. Some of the Osteoblasts
develop into osteocytes, each living within its own small space, or lacuna. Osteocytes make
contact with the cytoplasmic processes of their counterparts via a network of small canals, or
canaliculi. This network facilitates the exchange of nutrients and metabolic waste. Collagen
fibers in a particular lamella run parallel to each other but the orientation of collagen fibers
within other lamellae is oblique. The collagen fiber density is lowest at the seams between
lamellae, accounting for the distinctive microscopic appearance of a transverse section of
osteons.

Osteons are separated from each other by interstitial lamellae between systems.The
space between osteons is occupied by interstitial lamellae, which are the remnants of osteons
that were partially resorbed during the process of bone remodelling. Osteons are connected to
each other and the periosteum by oblique channels called Volkmann's canals.

Trabecular bone or spongy bone has a higher surface area but is less dense, softer, weaker,
and less stiff like compact bone. It typically occurs at the ends of long bones, proximal to
joints and within the interior of vertebrae. Spongy bone is highly vascular and frequently
contains red bone marrow where hematopoiesis, the production of blood cells, occurs. The
primary anatomical and functional unit of spongy bone is the trabecula.

Ossification (or osteogenesis) is the process of laying down new bone material by cells called
osteoblasts. It is synonymous with bone tissue formation. There are two processes resulting in
the formation of normal, healthy bone tissue. Intramembranous ossification is the direct
laying down of bone into the primitive connective tissue (mesenchyme), while endochondral
ossification involves cartilage as a precursor.

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Intramembranous ossification is one of the two essential processes during fetal

development of the mammalian skeletal system resulting in the creation of bone tissue.

Embryologic mesenchymal cells condense into layers of vascularized primitive connective

tissue. Certain mesenchymal cells group together, usually near or around blood vessels, and
differentiate into osteogenic cells which deposit bone matrix constitutively. These aggregates
of bony matrix are called bone spicules. Separate mesenchymal cells differentiate into
osteoblasts, which line up along the surface of the spicule and secrete more osteoid, which
increases the size of the spicule. As the spicules continue to grow, they fuse with adjacent
spicules and this results in the formation of trabeculae. When osteoblasts become trapped in
the matrix they secrete, they differentiate into osteocytes. Osteoblasts continue to line up on
the surface which increases the size. As growth continues, trabeculae become interconnected
and woven bone is formed. The term primary spongiosa is also used to refer to the initial
trabecular network.

Endochondral ossification - Unlike intramembranous ossification, which is the other process,

cartilage is present during endochondral ossification. It is also an essential process during the
rudimentary formation of long bones, the growth of the length of long bones and the natural
healing of bone fractures.

During endochondral ossification, four distinct zones can be seen at the light-microscope
level.

1. Zone of resting cartilage. This zone contains normal, resting hyaline cartilage.
2. Zone of proliferation. In this zone, chondrocytes undergo rapid mitosis, forming
distinctive looking stacks.
3. Zone of maturation / hypertrophy. It is during this zone that the chondrocytes undergo
hypertrophy (become enlarged). Chondrocytes contain large amounts of glycogen and
begin to secrete alkaline phosphatase.
4. Zone of calcification. In this zone, chondrocytes are either dying or dead, leaving
cavities that will later become invaded by bone-forming cells. Chondrocytes here die
when they can no longer receive nutrients or eliminate wastes via diffusion. This is
because the calcified matrix is much less hydrated than hyaline cartilage.