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PART I

human mouse206 bones > 200 bones

The Skeleton

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Bones of the inner ear

Human femur

Bones can differ dramatically in their size and shape

What is bone?

• Specialized form of connective tissue: mineralized collagen matrix, therefore very rigid and strong while still retaining some degree of flexibility

• Other types of connective tissue: - Cartilage: semi-rigid form, glycoprotein rich - Ligaments: flexible bands, rich in collagen fibers, contribute to stability of the joint - Tendons: strong flexible bands, rich in collagen fibers, connect muscles with bone

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Diverse functions of bone

• Support

• Protection (Skull)

• Mineral storage (e.g. calcium homeostasis)

• Hematopoiesis (bone marrow - postnatal)

• Locomotion - muscular-skeletal system

• Hearing

Mechanisms of bone formation

• Membranous ossification intramembranous bones: flat bones of the skull, clavicle, periosteum how: direct differentiation of cells within mesenchymal condensations into bone forming cells (osteoblasts)

• Endochondral ossification endochondral bones:axial and appendicular skeleton, some bones in the skull how: replacement of a cartilagenous template by bone

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Membranous bone formation

osteoid

compact bone

Hartmann, TCB, 2006

Endochondral Ossification

Hartmann, TCB, 2006

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Different types of bone cellsinvolved in bone homeostasis

• osteoblasts bone forming cells

• osteocytes terminal differentiated osteoblast

• osteoclasts bone resorbing cells

How do these cells look?

Osteoblastmononucleatedpositive for AP(AP= alkaline phosphatase)

Osteocytemononucleatedtrapped within lacunaeserve as mechanosensors

Osteoclastmultinucleatedpositive for TRAP(TRAP =

tatrate-resistent alkaline phosphatase)

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Origin of bone cells

• osteoblasts: mesenchymal e.g. the first ob differentiate within the periosteum and

form the bone collar postnatal: bone marrow

• osteocytes: mesenchymal, terminal differentiated osteoblasts

• osteoclasts: hematopoietic lineage; bone marrow

Postnatal bone cell differentiation

Hartmann, 2006Encyclopedic Ref.

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Bone architecture changes with age

Vertebral body ofa young person

Vertebral body ofan ederly person

Outside factors affecting bone mass

• Exercise: muscle contractions stimulate osteoblast function - increased production of bone peak bone mass reached at the age of 30 • Body weight: obesity can protect from osteoporotic bone loss • Diet affects bone: minerals and vitamins

• Menopause in women: decrease in hormone level can lead to osteoporosis (treatment: HRT)

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Terminology of changes in bone

• osteopenia: decreased calcification or density of bone

• osteoporosis: progressive reduction in quantity of bone

• osteopetrosis: excessive formation of dense trabecular bone

• osteosclerosis: abnormal hardening or eburnation of bone

• osteohypertrophy: overgrowth of bone

• osteosarcoma: tumor of the bone

• osteochondrodysplasia: extreme bending of long bones

• osteochondroma (exostosis): benign cartilaginous neoplasma

• osteoblastoma: benign tumor of osteoblasts

Changes in bones

• osteopenia: osteoporosis: osteopetrosis: (mild) (severe)

Cause: osteoclast deficiency or no active OC

Cause: osteoblasts are not active enough

Cause: osteoblasts are not active enough and/or osteoclasts are too active

(associated with bone cells)

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wtwt

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• Osteochondro- matosis (multiple exostoses)

Congenital bone disorders

• Achondroplasia (Fgfr3 act. mutations) Dwarfism

• Osteogenesis Imperfecta (brittle bones;

prenatal form of osteoporosis)

• Marfan syndrome (skeletal overgrowth,lengthening of long bones mutations in Fibrillin gene)

Skeleton formation begins duringembryogenesis

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Cartilage

Types of cartilage (avascular tissue):

• hyaline cartilage: (e.g. trachea, nose, articular ends of bones, embryonic skeleton)

• fibro-cartilage:(e.g. intervertebral disc)

• elastic cartilage: (e.g. ext. ear, epiglottis)

The skeleton is derived from three

different compartments

Ventral

Dorsal

Medial Lateral

Somite

Notochord

Lateral plate mesoderm

Intermediatemesoderm

Neural tube

Neural crest

Axial skeleton

Appendicular skeleton

Craniofacial skeleton

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Different origins of membranousbone in the mouse skull

Cranial neural crest

mesoderm

frontal

pariental

IPnasal

According to: Jiang et al. 2002

Axial skeleton

Origin: somites

young somite intermediate somite differentiated somite

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Limb skeleton

Origin: lateral plate mesoderm

Dermomyotome

Sclerotome

Starting to be distinguishable aroundday 4 in chick and day 11 in mouse

Condensing cartilageanlagen in the limb

Formation of the cartilagenoustemplate in the limb

naivemesenchyme

signals initiatingcondensation

condensation &lineage specification

pre-chondroblast chondroblast chondrocyte

perichodrialcell

cartilage templatelineagecommitment &differentiation

growth

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Growth of the cartilage template

• Appositional growth: new cartilage is added on the surface by recruiting chondroblasts from the inner layer of the perichondrium

• Interstitial growth: new cartilage is formed within the cartilagenous template by chondrocytes dividing and producing additional matrix

Two mechanisms:

After initiation and formationof the cartilage template

chondrocytes within thetemplate undergo a controlled

maturation program

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PCPHC

HTC MHTC

Round proliferating chondrocytes

flattenedproliferatingchondrocytes

Pre-hypertrophicchondrocytes

hypertrophicchondrocytes

Bone collar

periosteum

Mitotically active postmitotic

transforming region

PC: proliferative chondrocytes

PHC: pre-hypertrophic chondrocytes

HTC: hypertrophic chondrocytes

MHTC: mature hypertrophic chondrocytes

Cilia are found on chondrocytes (uni-ciliated)

Possible functions:

•“Anker” for oriented secretion of extracellular matrix

• Mechanosensor

• Signaling center, as some receptors have been shown to localize to the cilium (for example smoothened the receptor required for transmitting the hedgehog signal)

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Continuous growth of skeletal element is ensured by the growth plate

Bloodvesselinvasion

The Growth Plate

osteogenic front

hypertrophicprehypertrophic

flattened, stackedproliferatingchondrocytes

round,proliferatingchondrocytes

newly formedbone

transformation zoneWt β1 integrin -/-

- defective cytokinesis- decreased proliferation- decreased cell adhesion- increased apoptosis

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Integrin-(outside-in) signaling

Replacement of cartilage by bone

Bloodvesselinvasion

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Histology of a juvenile long bone

articular cartilage

growth plate

endosteum

periosteum

cortical bone

primary spongiosa

epiphysis

secondaryossificationcenter

secondary spongiosa

trabecular bone

periosteum

endosteumcortical bone

bone marrow

epiphysis

Replacement of HTC by Bone

• Mineralization of cartilage matrix

• Apoptosis of hypertropic chondrocytes

• Phagocytosis of old cartilage matrix by osteo/chrondroclasts

• Metalloproteases

• Invasion of vascular system

Changes happening in the transformation zone:

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Segmentation of the skeleton

Skeletal elements are separated from each other by so-called joints

Different types of joints (arthrosis):

• synovial joints (diarthrosis): has a joint cavity that is enclosed by a fibrous capsule, which is lined by the synovial membrane. e.g. joints separating skeletal elements in limbs - free movement!

• Non-synovial joints (synarthroses):• fibrous joints: skeletal elements are directly linked by fibrous tissue e.g. sutures between the skull bone - don’t allow movement!

• cartilaginous joints: two skeletal elements are linked by cartilage e.g. joints between vertebral bodies - limited movement!

Patterning of appendicular skeletonduring embryonic development

Sequential process, proceeds from proximal to distal

Alcian blue stained chicken hindlimbs

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The skeletal elements of the limb form by aprocess of branching and segmentation

prechondrogenic(Sox9+, Col2a1+)

chondrogenic(Sox9++, Col2a1++)

joint(Sox9-, Col2a1-)

Development of the synovial joint

Modified after P. Francis-West

byapoptosis

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Adult mouse knee joint

Secondary ossification center

bursa

Ligamentcruciatum

Growth plateof tibia

Jointcavity

meniscus

Articularcartilage

synovium

Ligamentsof jointcapsule

subchondralbone

Pathological changes of the joints

• arthrosis: general term for degenerative affection of a joint

• rheumatoid arthritis: systemic disease affecting connective tissue of joint, accompanied by inflammation and erosion of cartilage and bone due to synovial overgrowth

• osteoarthritis: destruction of joints due to erosions of articular cartilage, accompanied by inflammation, eburnation of subchondral bone

• gout: inflammation of the joint

• synovitis: inflammation of synovial membrane

• bursitis: inflammation of bursa (german: Schleimbeutel)

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Histological changes in RA and OA

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Rheumatoid arthritis (RA) Osteoarthritis (OA)

Part II

1. Steps involved in the initiation of cartilage formation2. Signals regulating maturation of chondrocytes within a cartilage template3. Factors controlling osteobastogenesis4. Factors involved in osteoclastogenesis5. Bone homeostasis6. Bone repair - fractures7. Joint formation