Bone Tissue Histology

• Bone tissue is specialized connective tissue
• Learning objectives are defining histological termination of bone tissue, learning histological structures, defining histological features of cells, and listing bone formation.

Terminology

• The matrix is densely packed with collagen fibrils, infiltrated with bone mineral as fine crystals of calcium salts.
• The densely packed collagen fibrils are primarily type I
• Small amount of non-collagenous proteins are calcium-binding osteocalcin, and bone sialoproteins
• Hydroxyapatite is calcium and phosphate that gives bone hardness
• Hydroxyapatite crystals are associated with collagen in bone to give strength and resistance.
• Bone consist of calcified bone matrix surrounding osteocytes, osteoblasts, and osteoclasts.
• Canaliculi are little tunnels called canaliculi where the osteocyte extends numerous processes.
• Spaces within the bone matrix are called lacunae
• Ligaments are fibrous tissue connecting bones to other bones
• Tendons connect muscle to bone.

Bone Matrix: General Specifications

• Bone, or osseous tissue, represents the highest differentiation among supporting tissue
• Bone is rigid tissue
• Bone is distinct for the mineralization of its matrix
• The matrix is composed of an extremely hard structure that provides support and protection
• Hydroxyapatite crystals are formed from calcium and phosphate minerals
• Bone is highly vascularized and metabolically very active.
• Organic portion makes up 1/3 of the dry weight of bone which is 35%
  • Type I collagen fibers are 90%, and also type V collagen.
  • Proteoglycans
  • GAG's chondroitin sulfate, keratan sulfate, and hyaluronic acid
  • Sialoproteins
  • Glycoproteins osteocalcin, osteonectin, and osteopontin
• Inorganic portion makes up 2/3 of the dry weight which is about 65%.
  • Hydroxyapatite is calcium phosphate which makes up 85%
  • Calcium carbonate makes up about 10%
• Other components include Mg++, K+, Na+, SO4, CO3-, OH-, and F-.

Classification of Bone

• Long bones are larger in one dimension than other bones
  • They consist of a shaft and two ends.
  • Examples include bones of the upper and lower arms and legs as well as the metacarpals and metatarsals.
• Flat bones are thin and plate-like
  • Examples sternum, ribs, and scapulae
  • These have no diaphyses, epiphyses
  • Cancellous bone lays between two compact bones
• Short and Irregular Bones are nearly equal in length and diameter
  • Carpals and tarsals are examples of short bones
  • Irregular bones shape typically does not fit
  • Vertebrae are an example of an irregular bone
  • No diaphyses and not elongated
  • Some flat and irregular bones of the skull have sinuses lined with mucous membranes

Long Bone Anatomical Architecture

• Diaphysis
  • Shaft
  • Compact bone
• Epiphysis
  • End of the bone
  • Cancellous bone
• Metaphysis
  • The flared portion of the bone between the diaphysis and the epiphysis.
• Medullary cavity:
  • Red marrow
  • Yellow marrow
• Periosteum
  • Bones covered by periosteum
  • A sheath of dense fibrous connective tissue containing osteoprogenitor cells
• Endosteum
  • Bone cavities lined by endosteum
  • Relative loose connective tissue cells that contains osteoprogenitor cells.

Long Bone Histological Architecture

• The periosteum consists of two layers
  • These are not sharply defined
• The inner layer
  • Is loosely arranged connective tissue
  • During embryonic and postnatal growth, inner layers consist of bone forming cells.
  • This layer is also called the osteogenic layer.
  • In the adult, the periosteum contains inactive connective tissue cells that remain their osteogenic potential in case of bone injury and repair
• The outer layer
  • Is a dense connective tissue
  • Rich in blood vessels
  • Some of this vessels enter Volkman's canals
  • This layer has thick anchoring collagen fibers, called Sharpey's fibers
• Sharpey fibers penetrate through the periosteum and into the bone
• Periosteum fuses with tendons and ligaments to produce an extremely strong
• They invade and fuse with the fibers of the cortical bone
• Sharpey fibers are not founded in the lamellar system or internal circumferential lamellae.
• The endosteum consists of reticular connective tissue fibers and squamous cells that resemble fibroblasts.
• The endosteum covers the spongy walls
• It is the housing for the bone marrow
• The endosteum extends into all cavities of the bone, including the haversian canals.
• The endosteum contains osteoprogenitor cells osteoblasts and osteoclasts.
• Mature bone has better organization than woven bone
• It has mineralized substance deposited
• Woven bone is observed in the developing bone.
• Mineralization of woven bone is less and loose.
• Histological Classifications of bone:
  • Spongy or Cancellous Bone
  • Compact Bone

Cancellous bone

• Trabeculae is the primary anatomical and functional unit that is interconnected with rods or plates
• Intertrabecular spaces are filled with marrow
• This space is covered with endosteum
• Trabeculae develop along lines of stress
• Mature compact bone is composed of structural units called Osteons (Haversian Systems)
• Osteons consist of concentric lamellae and osteonal canal (Haversian Canal)
• The central canal is called the osteonal canal (Haversian Canal)
• Volkmann's canal and intertitial lamellae are present
• Vascular and nerve supply of the osteon is contained in this canal
• Haversian canals run longitudinally
• Volkmann's Canals run at right angles to the central canals and perforate the osteon shaft
• These canals provide nutrition to the bone and joints that haversian canals do not
• Interstitial lamellae are remnants of previous osteonal lamellae located between osteons
• Concentric lamellae is surrounded by a central canal
• Each lamella consists of osteocyte, lacuna ve canaliculi
• Osteons consist of from 5 to 20 lamellae that surround the central Haversian canal
• Interstitial lamella are remnants of previous osteonal lamellae, now called interstitial lamellae
• Circumferential Lamellae
  • These follow the inner and outer circumferences of the shaft of a long bone
  • These lamellae appear much like the growth ring of a tree
• Outer circumferential lamellae are called perioseal circumferantial lamellae
• İnner circumferential lamellae are called endosteal circumferantial lamellae

Bone Cells

• There are four special cell types to bone
  • Osteoprogenitor Cells
  • Osteoblasts
  • Osteocytes
  • Osteoclast
• Osteoprogenitor Cells are stem cells derived from mesencyme
  • They have capacity or mitosis and further differentiation into mature bone cells.
  • They are found near bone surface, in the inner portion of the periosteum, in endosteum and within the vascular canals of compact bone
• Osteoblasts are differentiatied bone-forming cells that secrete bone matrix
  • Lie on the bone surface
  • In a one-cell thick layer
  • Most lie on the endosteum and inner periosteum
  • These cells secrete both type I collagen which is 90% of protein in bone, and bone matrix proteins (BMPs), which constitute the initial unmineralized bone, or osteoid.
• Osteocytes arise from osteoblasts trapped in lacunae
  • They are mature bone cells
  • Stellate shaped which are surrounded by matrix
  • Make small amounts of matrix to maintain.
  • Processes extending from the body down the canaliculi not visible by LM
  • Like osteoblasts, remain connected by gap junctions.
• Canaliculi containing the processes of osteocytes are generally arranged in a radial pattern with respect to the canal
• Canaliculi opens to the osteonal canal to serve the passage of substances between the osteocytes and blood vessel.
• Osteoclasts are responsible for bone resorption
  • Are large, multinucleated cells.
  • Derived from monocytes from stem cells in red bone marrow
  • They rest directly on bone tissue where is taking place
  • Lie on the surface of bone, often in an eaten-out hollow - Howship`s lacuna.
  • Osteoclast of the ruffled border agitate the resorbing - bone- destroying - materials
  • In dense bone, many osteoclasts act together to erode resorption tunnels

Bone Formation

• The two processes of bone formation or osteogenesis
  • Intramembranous Ossification
  • Endochondral Ossification
• Intramembranous Ossification
  • Takes place in connective tissue membrane formed from embryonic mesenchyme
  • Forms many flat bones, part of mandible, diaphyses of clavicles
  • When remodeled, indistinguishable from endochondral bone.
• Intramembranous ossification occurs in the following sequence:
  • Embryonic mesenchyme changes into a highly vascularized connective tissue
  • Mesenchymal cells embedded in a gelatinous extracellular matrix.
  • These cells are typical osteoblasts
  • Develop and eventually fuse, forming a network of anastomosing trabeculae resembling a sponge, the so-called spongy bone or primary spongiosa
• Collagen fibers in the newly formed trabeculae are randomly oriented to describe woven bone
• Calcium phosphate is deposited in the bone matrix, which is laid down by apposition.
• Interstitial bone growth does not occur
• Bone matrix mineralization leads to two new structures
• Osteoblasts transform into osteocytes
• Connective tissue in the intertrabecular space transforms into hematopoietic tissue.
• Increased vascularity of tissue occurs in intramembranous bone formation
• Active proliferation of mesenchymal cells mesenchymal cells give rise to osteogenic cells to develop into osteoblasts
• Osteoblasts begin to lay down osteoid, which is the organic part of bone without inorganic constituent
• Osteoblasts retreat or become entrapped as osteocytes
• Osteoid calcifies to form spicules of spongy bone
  • The spicules unite to form trabeculae.
  • The inorganic salts carried in by the blood vessels supposedly bring about calcification
  • The salts are deposited in an orderly fashion as fine crystals of hydroxyapatite crystals intimately associated with the collagenous fibers which are only visible with the electron microscope
• Bone remodeling occurs, and periosteum and compact bone are formed.
• Endochondral/Intracartilaginous Ossification involves the replacement of a cartilaginous model by bone. Best observed in long bones, such as the humerus or femur.
• The primary ossification zone establishes itself across the width of the shaft and starts extending in both directions towards the epiphyses
• This results in two transverse fronts of ossification across the diaphysis with a cartilaginous growth plate at each front.
• The first change indicative of beginning ossification takes place about the center of the future bone shaft
• Cartilage cells hypertrophy and the cartilage matrix becomes calcified
• Calcified matrix disintegrates and opens cavities that communicate with the connective tissue and vessels at the surface
• The bone collar forms concurrently with the primary ossification center
  • Cells of the perichondrium begin to form bone
  • The bone collar holds together the shaft through the disintegration of the cartilage.
  • The connective tissue about the bone collar, previously a perichondrium, is now called periosteum.
• About the time of birth, a secondary ossification center appears in each end (epiphysis) of long bones
• Cartilage between the primary and secondary ossification centers is the epiphyseal plate.
• It continues to form new cartilage, which is replaced by bone, resulting in longer bones
• Growth continues until the individual is about 21 years old or the cartilage in the plate is replaced by bone.
• Union of the primary and secondary ossification centers is called the epiphyseal line
• Process begins with the formation of a cartilage model
• A periosteal (perichondrial) collar of bone forms around the shaft (diaphysis) of the cartilage model
• Cartilaginous matrix begins to calcify
• Blood vessels and connective tissue cells erode calcified cartilage to create a primitive marrow cavity
• Endochondral bone forms on these spicules of calcified cartilage, and constitutes the metaphysis
• Blood vessels and perivascular cells invade the proximal epiphysial cartilage
• Secondary center of ossification is established in the proximal epiphysis
• A similar epiphyseal secondary ossification center forms at the distal end
• Epiphyseal cartilage is formed between each epiphysis and the diaphysis
• Continued growth of the long bone occurs, and the distal epiphyseal cartilage disappears
• Finally, with the end of growth, the proximal epiphyseal cartilage disappears and the metaphysis becomes continous with epiphysis
• Identify and study the five zones of cartilage associated with endochondral ossification:
  • Zone of reserve cartilage
  • Zone of cell proliferation (ZP)
  • Zone of cell and lacunar maturation and hypertrophy enlargement (ZH)
  • Zone of calcification (ZC)
  • Zone of cartilage removal and bone deposition.
• The zone of reserve cartilage is a site composed of primitive hyalin cartilage that is responsible for growth in length of the bones
• The zone of proliferation contains flattened chondrocytes in columns or clusters parallel to growth axis Chondrocytes are seperated by the territorial matrix.
• The zone of cell and lacunar maturation and hypertrophy enlargement (ZH)displays chondrocytes increasing in size due to fluid influx which causes thinner septa and mineralization in the longitudinal septa
• In the zone of calcification (ZC) the enlarged cells begin to degenerate matrix becomes calcified, and chondrocytes die
• In the zone of cartilage removal and bone deposition, which are very nearest the diaphysis, the calcified cartilage is in direct contact with the connective tissue of the narrow cavity
• During the development of cartilaginous joints, the interzonal mesenchyme between the developing bones differentiates into hyaline or fibrocartilage
• During the development of synovial joints, the interzonal mesenchyme between the developing bones differentiates:
  • Peripherally the interzonal mesenchyme forms the joint capsule and other ligaments.
  • Centrally the mesenchyme disappears and the resulting space becomes the joint or synovial cavity.
  • Where it lines the joint capsule and articular surfaces, it forms the synovial membrane to line the joint capsule
  • Synovial membrane helps the joint capsule
• Axial skeleton composed of the cranium, vertebral column, ribs, and sternum.
• Cells in the sclerotomes surround the neural tube and the notochord for vertebrae development during the fourth week
• Positional change of the sclerotomal cells is effected by differential growth and not by stem cell migration
• Sclerotomes are found in three main areas of notochord, neural tube, and in the body wall
• Each sclerotome consists of loosely arranged cells cranially and densely packed cells caudally.
• Some densely packed cells move cranially form the intervertebral disc
• Nerves lie in close relationship to disc and intersegmental arteries bodies, which become the intercostal arteries
• Nucleus pulposus and annulus fibrosus together form the disc.
• Two primary ossification centers ventral and dorsal for the centrum
• These centers soon fuse to form one center, and three primary centers are present for the eighth week. One is the centrum, and the others comprise of the neural arch
• Ribs develop from the costal processes of the spinal vertabrae
• Ribs become cartilaginous at some point in embryonic stage
• Union points where the costal processes meet the vertebrae are replaced by costotransverse joints

Types of Ribs

• True ribs attach to the sternum cartilage
• False ribs have cartilage for the sternum
• Floating ribs lack sternums
• sternal bars develop ventrolaterally in the body wall
• These also undergo chondrification
• By 10 weeks, they fuse craniocaudally in the median plane to form sternabrrae and xyhoid process
• The cranium develops from mesenchyme around the developing brain
• Neurocranium is initiated from ossification centers within the desmocranium mesenchyme
• Bones are classified into neurocranium and viscerocranium
• Appendicular skeleton comprises of the pelvic girdle, girdle, and bones
• During the 5th/6th week, mesenchymal bone models undego chondrification to form cartilage bone models
• Clavicles develop by membranous ossification at both ends
• Lower limb bones are done before the pectoral and limb bones
• Models appear in sequence
• Ossification begins within 8 weeks in the diaphyses
• Knee bones show secondary centers