Bone Development

BONE DEVELOPMENT: OSTEOGENESIS (OSSIFICATION)

Endochondral ossification

  • An INDIRECT form of ossification, wherein a hyaline cartilaginous model (template) is replaced with bone, such as occurs with long bones (eg, the femur).

Intramembranous ossification

  • A DIRECT form of ossification mesenchymal cells directly differentiate to osteoblasts (no cartilaginous model is first formed), such as occurs with flat bones (the skull bones).

INTRAMEMBRANOUS OSSIFICATION

  • Embryologically, skeletal tissues typically derive from mesoderm: the midline (axial) skeleton derives from the somites and the appendicular (the limb) skeleton derives from the lateral plate.
  • Mesenchymal cells migrate to vascularized gelatinous extracellular collagen fiber matrix (a primary spongiosa).
  • They differentiate directly into osteoblasts.
  • Osteoblasts form bone in a loosely arranged (disorganized), immature initial form of bone, called woven bone.
  • Osteoblasts become trapped within their own bony matrix and become osteocytes; these bony matrices are referred to as trabeculae (aka fused spicules).
  • Woven bone later matures to form lamellar bone, a much tougher form of bone that constitutes both compact bone and spongy bone.

Compact Bone

  • The outer layer: the periosteum.
    -Comprises columns of compact bone, called osteons.
    -Centrally, within each osteon, lies a longitudinally-oriented canal, the Haversian (aka central) canal.
    -Each osteon comprises concentric rings of lamellae.
    -Osteocytes are a mature form of osteoblast (the bone-producing cells) within the bony matrix.
    -Internal to the compact bone, lies the endosteum, which comprises in inner circumferential lamellae and the osteoprogenitor cells, internal to it.

Spongy bone

Lies internal to the endosteum and comprises a network of lamellae that do NOT form the Haversian channels and osteons found in compact bone.

ENDOCHONDRAL OSSIFICATION

Origins

  • Mesenchymal cells migrate and differentiate to form a hyaline cartilage model, which comprises basophilic collagen and ground substance.
  • Chondrocytes are part of a cartilaginous model that are hyaline cartilage cells, which are purplish (basophilic).
    -Key histological features: their nucleus and lipid droplets.
    -Chondrocytes produce the structural components of cartilage: collagen, proteoglycans and glycosaminoglycans, and are usually found in clusters (isogenic groups) of recently divided cells.
    -Chondrocytes hypertrophy, which signals (via vascular endothelial cell growth factor) the sprouting of blood vessels, which we’ll draw next.

Bone Regions

  • The diaphysis (the shaft)
  • The epiphyses (the articulating ends of the long bone)
  • The metaphyses that separate them.

Periosteal Buds

  • Periosteal buds invade the center of the diaphysis. Vascularization occurs via the periosteal bud, which brings forth osteoprogenitor cells and forms the primary ossification center, which forms within the cavities that are created when the hypertrophic chondrocytes starve and apoptose (die).
  • Vasculature further invades the primary ossification center and the osteoprogenitor cells remodel bony matrix as the ossification center grows linearly (via interstitial growth).
  • The outer cartilage is perichondrium, which forms a periosteal bone collar of compact bone that grows in opposite orientation, increasing the bone thickness (via appositional growth) – here, the osteoblasts secrete bone matrix directly via intramembranous ossification. Periosteum distributes blood vessels to bone and is not found in synovial articulations or muscle attachment sites.

Medullary cavity

  • Forms as the primary ossification center degenerates cavitates and remodels via interstitial growth.
  • Endochondrium delineates it, which forms a layer of lamellar bone and osteoprogenitor cells.
  • The marrow cavity is filled with hematopoetic marrow (which comprises red and white blood cell precursors)
  • Vasculature invades the cavity to fill it with marrow.

Secondary ossification centers.

  • Unlike the primary ossification center, they never grow large enough to create marrow cavities but instead remain constituted with spongy bone.

Bone Growth

  • Further appositional growth (widening) of the periosteal bone collar occurs along the diaphysis (again which forms compact bone via intramembranous ossification).
  • There is an epiphyseal (growth) plate at the border of the metaphysis and the epiphysis: elsewhere we learn about the zones and processes of interstitial growth, but pay attention that Indian hedgehog (Ihh) was discovered to be important for the stimulation of chondrocyte growth with delay of chondrocyte hypertrophy (thus delaying a key step in endochondral ossification).

MAJOR BONES & THEIR DEVELOPMENT

Intramembranous Ossification

  • Cranial Vault
  • Maxilla/Mandible
  • Clavicles

Consider that the skull bones must ossify prior to delivery of the fetus, so the brain isn’t squashed during childbirth to help us remember that intramembranous ossification is a more direct form of ossification.

Endochondral ossification

  • Skull base
  • Vertebrae
  • Pelvis
  • Long Bones

They grow extensively throughout pediatric development and require an amount of pliability via their cartilaginous template prior to committing to ossification too soon. Consider that for a time-period, children fall often and thus must bounce and not break!

Muscle Stretch Reflex

Synonyms

  • Monosynaptic reflex, myotactic reflex, deep tendon reflex, tendon jerk

Definition

  • It is an automatic, monosynaptic reflex that involves a muscle and tendon, and produces a jerk.

Most commonly tested

  • Biceps (C5, C6)
    • Elbow flexion
  • Triceps (C7,C8)
    • Elbow extension
  • Patella (L2 – L4)
    • Knee extension
  • Achilles (S1,S2)
    • Foot plantarflexion

KEY MEDIATORS

  • Muscle spindles, which activate via muscle stretch.
  • Spinal neurons, which receive sensory input and generate motor output.
  • Muscle fibers, which contract.
  • Interneurons, which modulate neuronal firing.
  • Golgi tendon organs, which activate via muscle contraction to terminate the reflex.

ACTIVATION

  • When the patellar tendon is activated,
  • the muscle spindle sends an excitatory volley along the Type 1a sensory afferent,
  • which excites the extensor motor neuron.
  • It activates the muscle extensors, which extend the knee.

INTERNEURONAL INHIBITION

  • Renshaw cells are interneurons that lie in the anterior horn of the gray matter of the spinal cord.
  • When Renshaw cells are activated, they inhibit flexor motor neurons using the inhibitory neurotransmitter glycine.

TERMINATION

  • Golgi tendon organs are situated where the quadriceps tendon inserts into the patella.
  • Type 1b fibers project from the Golgi tendon organs to the Renshaw interneurons.
  • Inhibitory fibers project from the the Renshaw interneurons to the extensor motor neurons.
  • The Type 1a and 1b fibers fire at the same rate, but the muscle spindle fibers have a much lower threshold to fire than Golgi tendon organs, thus, the muscle spindle fibers fire first, and then later the Golgi tendon organs fire, which terminates the muscle stretch reflex.
  • Neurobiological influences, such as myosin ATPase and calcium re-accumulation into the endoplasmic reticulum aid in muscle contraction.

CLINICAL CORRELATION

  • In comatose patients, presence of the triple flexor reflex to plantar stimulations a sign of disinhibition, similar to the Babinski sign.

Myofibrils

Thick filaments.

  • Form from myosin
  • The A band refers to the length of the thick filaments, “think “A” for d-a-rk – they are aniosotropic (or birefringent) in polarized light.
  • H Zone is a zone of only thick filaments.
  • M line bisects the A band.

Thin filaments

  • Form from actin
  • The I band is the region along the thin filaments (between the thick filaments).
  • Think “I” for L-i-ght – they are “isotropic” (do not alter polarized light).

Z disks

  • Transverse bands at the ends of the thin filaments.

Sarcomere

  • The contractile unit of the myofibril.
  • Comprises the area between the Z-disks.

THIN MYOFILAMENT: DETAILS

  • The thin filament slides towards the H zone.

Actin

  • Spherical molecules joined in pairs of strands (like beads on a string). It is referred to as F-actin for filamentous actin, and comprises a polymer of G-actin monomers that are arranged in a double helix.

Tropomyosin

  • Threadlike strands

Troponin

  • Protein complexes that bind tropomyosin, actin, and also calcium (show their calcium-binding sites).

Cap Z

  • Binds the Z disk to the thin filaments.
  • This is (+) end stabilizer.

Tropomodulin

  • The (-) end stabilizer.

Troponin subunits

  • TnT binds to tropomyosin
  • TnI, which inhibits actin interaction with myosin.
  • TnC, which binds calcium ions.

Nebulin

  • Attaches to the Z disk, passes along the thin filament and binds to tropomodulin to help stabilize the thin filament and determines its length.
  • It is commonly stated that two nebulin molecules wrap around each thin filament to anchor it to the Z disk.

THICK FILAMENTS: DETAILS

  • Comprise myosin molecules (technically myson II), which form a golfclub shape, and comprise two heavy chains and two light chains.
  • The head forms from the heavy chain.
  • Unlike the thin filaments, the thick filaments are not directly connected to the Z disc. Instead, show that they are anchored to the ends of the sarcomere via titin molecules, which extend from the Z disks to the M line, and regulates the sarcomere’s elasticity, in addition to managing assembly of myosin.

MUSCLE CELL IN A CYLINDRICAL, 3-DIMENSIONAL VIEW

Here, we draw a muscle cell in a cylindrical, 3-dimensional view.

Sarcoplasmic reticulum (SR)

  • Form web-like rows.
  • Store calcium.
  • Are a key component to coupling muscle cell excitation to myofibril contraction.

Terminal cisternae (aka lateral cisternae) of the sarcoplasmic reticulum.

  • Flank the transverse tubules (T-Tubules)

Transverse tubules (T-Tubules)

  • Are tubular invaginations of sarcolemma.
  • Elsewhere, we see that T-tubules and terminal cisternae connect the terminal synapse firing (its depolarization) to the sarcoplasmic reticulum, again, ultimately coupling muscle cell excitation and myofibril contraction.

Triads of terminal cisternae

  • Triads of terminal cisternae with intervening T-tubules occur at the A-I junctions.

Desmin

  • Filaments encircle the Z disks.
  • Desmin-related myopathy (DRM) is an inherited disease in which, and results in disorganized and weak skeletal muscle fibers. DRM can be fatal, as it also affects cardiac and smooth muscles.

Plectin

  • Links the desmin filaments.

Alpha-beta-crystallin

  • A heat shock protein, which protects desmin from stress-induced damange.

Thus, together, desmin, plectin, and alpha-beta-crystallin constitute a Z-disk protection network.

Alpha-actinin

  • A Z disk component that binds to actin.

Costameres

  • Specialized regions of the sarcolemma, which bind to key protein complexes.

Dystrophin-associated glycoprotein complex (DAGC), which comprises:

  • The dystroglycan subcomplex, which links dystrophin (discussed soon) to laminin, a key external lamina protein (called laminin-2 in skeletal muscle).
  • The sarcoglycan subcomplex, which, when defective can cause sarcoglycanopathies – a similar manifestation of weakness as those from dystrophinopathies – and are a common cause of limb-girdle muscular dystrophy.
  • Dystrophin, which stabilizes the sarcolemma during muscle contraction.

DUCHENNE MUSCULAR DYSTROPHY (DMD)

Pathological slide

  • Muscle from an individual with Duchenne muscular dystrophy (DMD).
  • X-linked form of muscular dystrophy affects boys and occurs from a genetic mutation that prevents the synthesis of dystrophin.
  • We see a sparse group of muscle cells and see that much of the muscle is replaced with fatty and fibrous connective tissue, which presents with pseudohypertrophic muscles: muscles that are enlarged from fat and connect tissue (not muscle).
  • Eventually patients with this illness succumb to their weakness.

Syntrophins

  • Are recruited to the sarcolemma and manage the assembly of other proteins.

Dystrobrevins

  • Link desmin to dystrophin and syntrophin.

Skeletal Muscle Organization

SKELETAL MUSCLE HIERARCHY

  • Skeletal muscles divide into fascicles.
  • Fascicles are units of muscle cells (aka skeletal myocytes, skeletal muscle fibers).
  • Skeletal muscle cells comprise myofibrils and other organelles (notably, mitochondria).
  • Myofibrils comprise proteins, notably thick and thin myofilaments.
  • Myofilaments arrange into functional contractile units, called sarcomeres.

CONNECTIVE TISSUE HIERARCHY

  • Skeletal muscle is covered in epimysium.
  • Fascicles are covered in perimysium.
  • Muscle cells are covered in endomysium.

MUSCLE ORGANIZATION

Epimysium

  • Envelopes the muscle (epi = upon, my = muscle).
  • Dense irregular connective tissue.

Perimysium

  • Divides the muscle into multiple wedges (peri = around); it covers each fascicle.
  • Each fascicle comprises skeletal muscle cells (aka skeletal myocytes or muscle fibers).

Endomysium

  • Surrounds the muscle cells.
  • Loose areolar connective tissue that maintains the extracellular environment for proper muscle cell functioning.

MUSCLE FASCICLE

Muscle cells

  • They are multinucleated (meaning, each cell contains many nuclei).
    • This reality reflects the actual process of muscle cell formation. Mature muscle cells form from fused myoblast cells (embryonic cells) and each myoblast contributes its nucleus to the adult muscle cell.
  • Each skeletal myoctye comprises numerous myofibrils.

Myofibrils contain Myofilaments

  • Thick, myosin filaments
  • Numerous thin, actin filaments.
    • Thin filaments form a hexagonal shape around the thick filaments.

Sarcolemma

  • The plasma membrane of the muscle cell.

Sarcoplasm

  • The muscle cell cytoplasm.
  • Muscle cell nuclei lie within the periphery of the cell.
    • During development, the nuclei transition from a central location to a peripheral one.
  • Muscle cells comprise numerous mitochrondria.

Muscle fibers divide into 3 types based on their: myoglobincontent and contraction speed, and, in related fashion, their number of mitochondria.

  • Type 1 (slow, red)
  • Type 2a (fast, intermediate)
  • Type 2b (fast, white)

External lamina (sometimes referred to as the basal lamina)

  • Lies external to the muscle cell.
  • Within it, lie satellite cells, which are skeletal muscle stem cells: inactive myoblasts, lying in-wait: think: Army Reserves.
    • Upon muscle injury, they enter mitosis, fuse with other satellite cells to form differentiated muscle fibers. Centrally located nuclei are a hallmark of regenerating muscle cells; whereas mature muscle cells contain peripherally located nuclei.

Satellite cell biology:

  • Myostatin inhibits satellite cells and promotes protein degradation, which regulates the formation of mature skeletal muscle cells.
  • Testosterone, instead, encourages the synthesis of proteins, thus athletes abuse anabolic steroids that mimic testosterone to promote muscle growth.

MYOFIBRIL HISTOLOGY: EXTERNAL

Sarcoplasmic reticulum (SR)

  • Form web-like rows.
  • Store calcium.
  • Are a key component to coupling muscle cell excitation to myofibril contraction.

Terminal cisternae (aka lateral cisternae) of the sarcoplasmic reticulum.

  • Flank the transverse tubules (T-Tubules)

Transverse tubules (T-Tubules)

  • Are tubular invaginations of sarcolemma.
  • Elsewhere, we see that T-tubules and terminal cisternae connect the terminal synapse firing (its depolarization) to the sarcoplasmic reticulum, again, ultimately coupling muscle cell excitation and myofibril contraction.

MYOFIBRIL HISTOLOGY: INTERNAL

Thick filaments.

  • Form from myosin
  • The A band refers to the length of the thick filaments, “think “A” for d-a-rk – they are aniosotropic (or birefringent) in polarized light.
  • H Zone is a zone of only thick filaments.
  • M line bisects the A band.

Thin filaments

  • Form from actin
  • The I band is the region along the thin filaments (between the thick filaments).
  • Think “I” for L-i-ght – they are “isotropic” (do not alter polarized light).

Z disks

  • Transverse bands at the ends of the thin filaments.

Sarcomere

  • The contractile unit of the myofibril.
  • Comprises the area between the Z-disks.

SPECIAL CHARACTERISTICS OF SKELETAL MUSCLE:

  • Rich in glycosomes, which are used for energy creation.
  • Rich in myoglobin, which binds oxygen.
  • Sarcoplasmic reticulum (SR) instead of smooth endoplasmic reticulum.
  • Have specialized plasma membrane called sarcolemma.
  • Contain myofibrils, which are the contractile elements of muscle cells.