Limb Embryology Limb Outgrowth • Limb bud appears ventrolaterally at day 24 (week 4) • The upper limb bud begins 1-2 days before the lower limb bud • Limb buds contain a mass of mesenchyme derived from somatic mesoderm and is covered by layer of ectoderm • Upper limb develops opposite cervical segments • Lower limb develops opposite lumbar segments Mechanism 1. Apical ectodermal ridge (AER) induced by paracrine factor (FGF-10) 2. AER then secretes FGF-8 in the proximal-distal axis 3. Other genes used to grow limb in other axes: a. Wnt7a (anterior-posterior axis) b. engrailed1 (dorsal-ventral) Note: if AER is removed, limb bud will stop developing 4. AER continues to proliferate mesenchyme in all axes 5. Chondrification begins in week 5 6. Chondrocytes within the mesenchyme differentiate and secrete molecules characteristic of ECM (collagen type II and proteoglycans). This starts proximally and ends distally 7. Entire limb skeleton is cartilaginous by week 6 8. Digit formation: Activation of Patched1 gene (Ptc1) leads to apoptosis of tissue between the cartilaginous digits. If this process is inhibited  syndactylyl birth Syndactyly • More common in foot than hand • Cutaneous syndactyly: webbing of digits with tissue  Osseus syndactyly: fusion of bones of digits  Surgical intervention: o At ~6wks of age o Skin graft taken from groin or anterior elbow Long Bone Formation • Appendicular: endochondral ossification o Clavicle + scapula  phalanges o Ilium + ischium + pubis  phalanges • Axial: intramembranous ossification o Skull, sternum, ribs and vertebrae Endochondral Ossification 1. Week 7: ossification begins at primary ossification site in diaphysis 2. The cartilaginous cells increase in size (hypertrophy) and the matrix around the cell calcifies 3. The cell dies, leaving a cavity within the cartilage 4. Blood vessels grow around the edges and deliver preosteoblastic cells which replace chondrocytes with osteoblasts which lay down a bone collar called the periosteum. 5. Nutrient artery penetrates diaphysis and delivers more osteoblasts to lay down spongey bone 6. Osteoclasts also enter via blood vessels and produce the marrow cavity 7. Osteoblasts and vessels continue to migrate into epiphysis and secdary centres develop in the first years after birth 8. At birth: diaphysis completely ossified but secondary centres slowly ossify and a layer of articular hyaline cartilage will remain at the end of long bones 9. Epiphyseal plate allows further growth. Once growth of bone has ceased (~20 years of age) the plate ossifies and closes Ossification Centres: • Humerus, radius and ulna: start of week 7 • Femur, tibia and fibula: week 8 • Scapula and ilium: week 9 • Metacarpals, metatarsals and phalanges: weeks 9-12 • Ischium and pubis: weeks 15-20