Limb development and bone formation

Question 1

Skull origin

Answer 1

Mesenchyme surrounding the brain

Question 2

Viscerocranium

Answer 2

facial bones surrounding neck viscera
forms through endochondral and intramembranous ossification
forms from mesenchyme of head (neural crest ectoderm - ectosoderm)

Question 3

Neurocranium

Answer 3

skull bones

Question 4

Base of skull

Answer 4

endochondral ossification

formed from somites

Question 5

Flat bones of skull

Answer 5

Intramembranous ossification
Forms from mixture of head mesenchyme and somites
Induced by neural tissue
No brain formation - lack of skull formation

Question 6

Limb formation

Answer 6

begins in 4th week, vulnerable period: 5.5-7 wks
Position controlled by homeobox genes
1)Two paddle-shaped limbs grow outwards on either side
Core of dense mesenchyme surrounded by epithelium
2) Differentiation begins with condensation of mesenchyme into cartilage, then eventually bone - occurs simultaneously with muscle differentiation
3) Tips of limbs become paddle-shaped in week 6

Question 7

Mesenchyme of limbs

Answer 7

primarily from myotomes of somites, some lateral plate cells, some neural crest cells

• neural crest cells: pigment and Schwann cells
• lateral plate cells - cartilage and bone
• myotomal cells - muscles

Question 8

Epithelium of limbs

Answer 8

called apical ectodermal ridge (AER)
AER interacts with mesenchyme and causes it to continue growing, mediated through the release of several fibroblast GF’s

Loss or damage to AER: cause amelia/meromelia

Question 9

Digit formation

Answer 9

Week 6 - apoptosis between digits to form fingers and toes
Controlled by activity of retinoic acid on zone of polarizing activity (ZPA) to release Sonic hedgehog and bone morphogenetic proteins

Improper gradient of ZPA/sonic hedgehog —> polydactyly or syndactyly

Question 10

AP axis of limbs

Answer 10

present from time of limb bud formation

determined by sonic hedgehog genes

Question 11

DV axis of limbs

Answer 11

determined later in development

Determined by Wnt7

Question 12

PD axis of limbs

Answer 12

maintained by Wnt7

Question 13

Limb rotation

Answer 13

elbow rotates so that it points posteriorly

Hind limb rotates so that knee joint points anteriorly

Question 14

Limb innervation

Answer 14

Forelimbs grow out from cervical region of spinal cord
Hind limbs grow out from lumbar region of spinal cord
Week 5
Only neurons that find the appropriate target survive

Question 15

Limb blood supply

Answer 15

Each somite has segmental artery
Arm - brachial`
Leg - profunda femoris

Question 16

Somites

Answer 16

42-44 in total
4 occipital
8 cervical
12 thoracic
5 lumbar
5 sacral
8-10 coccygeal

Question 17

Parts of somites

Answer 17

Dermatome
Myotome
Sclerotome - becomes bone

Question 18

Vertebrae formation

Answer 18

Each forms from fusion of sclerotome of 4 somites
Myotome connects vertebral discs
Mesoderm left between vertebrae forms annulus fibrosus
Notochord forms nucleus pulposus of vertebral disc and centrum of vertebrae

Question 19

Myotome division

Answer 19

Myotome splits into two groups in thorax and abdomen
Epaxial divison - back and neck muscles, innervated by dorsal rami of spinal nerves
Hypaxial division - trunk and limb muscles, innervated by ventral rami of spinal nerves

Question 20

Muscle development

Answer 20

Most use mesenchymal cell precursors from somites

1) begins in week 4-5
- muscle cell precursors form and undergo migration to face, septum transversum, trunk, developing limb buds
2) week 5-6
- muscle cell begins differentiation
- fusion of myoblasts –> formation of multinucleated myotubes
- begin to synthesize actin and myosin
3) week 9 - month 5
- nuclei migrate to outside of myotube
- actin and myosin organized into contractile elements
- primary myotubes form without nerve cell involvement
- secondary myotubes require nerve cell involvement

Question 21

Formation of synovial joints

Answer 21

Mesenchyme in centre of developing limb condenses and releases bone morphogenetic protein –> causes mesenchyme to develop into cartilage, then bone

Noggin: secreted by regions that form synovial joints, antagonizes BMP
- apoptosis –> formation of fluid-filled space, becomes the synovial joint

Surrounding mesenchyme condenses into ligaments of joint capsule and tendons

Question 22

Intramembranous ossification

Answer 22

Develops directly from mesenchyme
does not use cartilage model
For flat bones in skull and face, mandible, clavicle

Question 23

Stages of Intramembranous Ossification

Answer 23

1) Aggregation
2) Osteoblast trapping
3) 3D network of spongy bone
4) remodelling

Question 24

Aggregation - IM ossification

Answer 24

1) mesenchymal cells migrate and aggregate
2) differentiate into osteoprogenitor cellsa nd osteoblasts
3) osteoblasts produce osteoid
- proteoglycans and type I collagen
- also produce alkaline phosphatase, which induces mineralization by causing ppt of calcium and phosphate salts

Question 25

Osteoblast trapping - IM ossification

Answer 25

1) Osteoblasts get trapped in mineralized matrix they produce 2) Calcification of matrix and formation of spicules

Question 26

3D network of spongy bone - IM ossification

Answer 26

1) mesenchyme condense to form periosteum 2) initially - osteoid is laid down in random arrangement --> woven bone, will be remodelled later 3) Osteoblast produce more bony matrix - generate a lattice network - appositional growth (growth outwards) - vascularization of spongy bone brings in bone marrow and osteoclasts

Question 27

Remodelling - IM ossification

Answer 27

Osteoclasts remodel bone - trabecular just deep to periosteum gets remodelled to compact bone collar - trabeculae on inside of bone gets remodelled to spongy bone - formation of collar of woven bone, later replaced by lamellar (compact) bone

Question 28

Endochondral ossification

Answer 28

cartilage model precursor | used by bones of extremities, axial skeleton that bear weight

Question 29

Stages of endochondral ossification

Answer 29

1) development of hyaline cartilage model 2) bony collar development 3) chondrocyte death, blood vessel invasion 4) formation of primary ossification centre 5) growth of endochondral bone 6) secondary ossification centres form 7) skeletal maturity

Question 30

Development of hyaline cartilage model - EC ossification

Answer 30

1) aggregation and differentiation of mesenchymal cells into chondrocytes 2) Chondrocytes produce cartilage matrix - forms model of bone 3) Two types of growth will occur: - interstitial: growth in length due to chondrocyte division - appositional: growth in width due to chondrocyte differentiation from perichondrium, resulting in more cartilage deposition

Question 31

Bony collar development - EC ossification

Answer 31

1) perichondral region starts to produce osteoblasts instead of chondrocytes 2) Perichondrium becomes periosteum 3) Collar becomes an osteogenic area, developing a collar of bone around the diaphysis and transitioning from a perichondrium to periosteum Formed through intramembranous ossification

Question 32

Chondrocyte death/BV invasion - EC ossification

Answer 32

1) formation of bony collar cuts of blood supply to cartilage in diaphysis --> hypertrophy 2) Hypertrophic chondrocytes produce AP - induce cartilage ECM to calcify and mineralize 3) calcification results in nutrient cut off to chondrocytes - death 4) Blood vessels then grow through bony collar into diaphysis of bone

Question 33

Formation of primary ossification centre - EC ossification

Answer 33

1) mesenchymal cells migrate along blood vessel and differentiate into osteoprogenitor cells in marrow cavity 2) Osteoprogenitor cells contact calcified cartilage plates, become osteoblasts and lay down osteoid 3) get mixed spicules of bone and cartilage (differentiate using Mallory stain)

Question 34

Growth of endochondral bone - EC ossification

Answer 34

1) Diaphyseal marrow cavity enlarges, pushes up toward epiphyses 2) get distinct zone of cartilage at each end = epiphyseal cartilage 3) Epiphyseal growth plate - division between diaphyseal cavity and cartilage - site where avascular cartilage is converted to vascularized bnoe - continues until skeletal maturity - responsible for growth in length of bones

Question 35

Formation of 2ndary ossification centre - EC ossification

Answer 35

1) develop in the same way as primary ossification centre 2) Bone laid down on calcified spicules, leaving primary spongy bone - cartilage is resorbed 3) Happens shortly after birth - 2ndary ossification centres appear in proximal eiphysis - same process as in diaphysis - formation delayed after primary

Question 36

SKeletal maturity - EC ossification

Answer 36

1) proliferation of cartilage in epiphyseal plate continues until skeletal maturity achieved 2) deposition of bone until cartilage is gone 3) epiphysela and diaphysela marrow cavities become confluent 4) epiphyseal closure/elimination of epiphyseal growth plate 5) only remaining cartilage is bone found on articular surfaces

Question 37

Growth plate layers

Answer 37

1) zone of reserve cartilage 2) zone of proliferation 3) zone of hypertrophy 4) zone of calcified cartilage 5) zone of resorption

Question 38

Zone of reserve cartilage

Answer 38

1 Closest to epiphysis normal, resting chondrocytes no proliferation/active matrix production

Question 39

Zone of proliferation

Answer 39

``` 2 Actively dividing chondrocytes cells get larger organize into columns cells actively produce collagen and matrix proteins actual lengthening ```

Question 40

Zone of hypertrophy

Answer 40

3 Accumulate glycogen granules in cytoplasm secrete type 1 and type X collagen synthesize AP

Question 41

Zone of calcified cartilage

Answer 41

4 hypertrophied chondrocytes degenerate cartilage matrix becomes calcified calcified cartilage --> scaffold for new bone formation

Question 42

Zone of resorption

Answer 42

5 Closest to diaphysis in contact with marrow cavity small blood vessel invasion - brings osteoprogenitor cells Undergo differentiation to osteoblasts Begin deposition of bone onto calcified cartilage get mixed spicules, eventually replaced by spongy bone

Question 43

Bone modeling

Answer 43

How bone attains its adult shape Growth in length: endochondral ossification - proliferation of cartilage at growth plates - occurs at long bone epiphyses Growth in girth: periosteal growth, at long bone diaphysis Metaphyseal inwaisting - adding length and manipulating circumference to maintain bone shape Modified by envelopes and attachments - tendons, capsules

Question 44

Bone metabolizing unit

Answer 44

Unit bone where bone formation is coupled to bone resorption Consists of osteoblasts, osteoclasts and osteocytes arranged in: - osteons in cortical bone - trabeculae in spongy bone

Question 45

Formation of osteons

Answer 45

Form when capillaries invade cortical bone/woven bone Cutting cone model: - osteoclasts move through dense bone down axis of diaphysis, degrading bone and producing a tube of empty space - osteoblasts follow and lay down concentric lamellae in immature woven bone - primary osteon in matural cortical bone - secondary osteon

Question 46

Wolff's law

Answer 46

bone will adapt to the load it is placed under stress causes strain on bone - mixture of tensile and compressive stress - tensile: osteoclast activity - compressive: osteoblast activity

Question 47

Types of bone healing

Answer 47

Primary: no motion at fracture site Secondary: motion at fracture site

Question 48

Primary bone healing

Answer 48

Due to stress fractures, reduction of fractures surgically - 20% direct contact between fragments - no fracture callus - gap filled with woven bone - cutting cones then cross fracture site to create new osteons

Question 49

Secondary bone healing

Answer 49

1) Inflammation 2) Repair 3) Remodelling

Question 50

Inflammation - 2ndary bone healing

Answer 50

1) begins immediately, continues ofr a couple weeks 2) Hematoma develops 3) Necrotic tissue is absorbed 4) Bleeding becomes source of progenitor cells 5) granulation tissue forms at site of fracture 6) proliferation of osteoblasts and fibroblasts

Question 51

Repair - 2ndary bone healing

Answer 51

begins within 2 weeks, for weeks-months 1) formation of bridging soft callus: non-ossified cartilaginous and fibrous tissu 2) replaced by a hard callus via endochondral ossification

Question 52

Remodelling - 2ndary bone healing

Answer 52

1) starts part way through repair phase, continues for years 2) bone assumes a more normal shape 3) woven bone replaced by laminar bone through Haversian remodelling 4) based on stress

Question 53

Influences on fracture healing

Answer 53

General: age, comorbidities (diabetes, vascular disease), nutrition, function, smoking Mechanical: amount of energy/trauma, anatomical location, fracture stability, bone type (scaphoid and clavicle take a long time), bone loss Biologic: soft tissue envelope health, infection, drugs (osteoporosis drugs, NSAIDs) Biochemical/hormonal

Question 54

Metaphyseal healing

Answer 54

Cortex is thing - minimal external callus laid down Tissue goes through similar staging but callus is formed within bone Radiographs: sclerotic fracture line Large external callus would interfere with joint function

Question 55

Diaphyseal healing

Answer 55

Cortex is thick - large external callus visible on radiographs

Question 56

Remodelling of childhood bone deformities

Answer 56

Potental greater with: - young children - fracture close to growth plate - deformity in the plane of mortion rotational deformities do not remodel well

Question 57

Osteoporosis

Answer 57

Loss of bone density/imbalance in osteoclast and osteoblast activity Role of estrogen - inhibits osteoblast apoptosis and increases lifespan --> increased osteoblast deposition - mediates oxiative stress on osteoblast - mediates RANKL-induced differentiation of osteoclasts, ensures there isn't too much differentiation that would increase osteoclast activity

Question 58

Calcium-regulating hormones

Answer 58

Low Ca in blood: - PTH stimulates osteoclasts to resorb bone and liberate Ca - bind osteoblasts, stimulates osteoblasts to express RANKL and downregulate OPG expression High Ca: - calcitonin stimulates osteoblasts to take calcium out of blood for deposition - binds osteoclasts, disrupts cytoskeleton and inhibits its resorptive ability

Question 59

Bone components

Answer 59

``` inorganic (65%) hydroxyapatite calcium phosphorus magenesium citrate potassium sodium ``` ``` organic (35%) type I collagen PGs multi-adhesive glycoproteins bone-specific vitaminK dependent proteins GHs and cytokines ```

Question 60

Periosteum structure

Answer 60

Outer layer: Fibroblasts, type I collagen, nerve, blood vessels Inner: blood vessels, osteoprogenitor cells, osteoblasts anchored to the bone by collagen fibers

Question 61

Endosteum

Answer 61

membranous lining covering the inner surface of compact bone and spongy bone often 1 layer thick - osteoprogenitor cells, bone-matrix secreting cells, bone-lining cells Osteoprogenitor + bone-lining cells = endosteal cells

Question 62

Fracture fixation

Answer 62

intramedullary nail fixation | doesn't stabilize it completely - callus still forms

Question 63

Healing time

Answer 63

``` fracture in metaphyseal: 6-8 weeks for a reasonable heal 2x in elderly 1/2 children 2x cortical bone 2x open fracture 2x smoker 2x non-compliant patient ```

Question 64

TGF beta

Answer 64

transforming growth factor | induce mesenchymal cells to produce type II collagen

Question 65

IGFII

Answer 65

stimulates type I collagen cartilage matrix synthesis bone formation

Question 66

PDGF

Answer 66

platelet-derived growth factor released from platelets attracts inflammatory cells to fracture site

Question 67

Hormonal influences on bone healing

Answer 67

TH/PTH - increase callus, affect remodelling Cortisone - decreases callus proliferation GH - increases callus volume Calcitonin - effects unclear