Bone

THE SKELETAL SYSTEM Cartilage and Bone Prof Raymond Coleman Department of Anatomy & Cell Biology Rappaport Faculty of Medicine Technion-Israel Institute of Technology Haifa, Israel

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The Skeletal System

The skeletal tissues • Skeletal tissues consist mainly of cartilage and bone • These skeletal tissues are specialized forms of connective tissue • Like all connective tissue they develop in embryos from embryonic mesenchyme

Cartilage • Cartilage is mainly a tissue of embryos and fetuses • In postnatal life and in adults cartilage has a very limited distribution, but serves very important functions • The slow-turnover of cartilage, limited growth and repair processes in adults is problematic

Functions of cartilage • Skeletal support in embryos prior to development of bony skeleton • Long bone elongation (endochondral ossification) • Articulating joints • Flexible support and protection (ears, trachea, bronchi)

Cartilage • Cells (chondrocytes) constitute only 2-5% of tissue volume • The main bulk of cartilage is the matrix (95-98%) • Cartilage cells are located in lacunae in the matrix • Cartilage is avascular and lacks intrinsic nerves • Cartilage receives nutrients from blood vessels of the perichondrium • Metabolites and nutrients diffuse via the matrix (low metabolic activity)

Types of cartilage There are three morphological types of cartilage: Hyaline cartilage Elastic cartilage Fibrocartilage The differences depend on the matrix especially the type and amount of the embedded fibers

Hyaline cartilage • This is the most common type of cartilage • The name is derived from the Greek “hyalos”=glass • This is mainly found in developing embryos where it forms the model for developing bones • In adults hyaline cartilage is found in the respiratory tract, ventral part of ribs, and articulating joints (articular cartilage)

Hyaline cartilage

Hyaline cartilage of the trachea

Hyaline cartilage

Hyaline cartilage

Cartilage cells • Chondroprogenitor cells (found in the perichondrium). These develop into chondroblasts • Chondroblasts (actively secrete matrix) • Chondrocytes (mature cells derived from the chondroblasts).

Chondrogenesis There are two different types of cartilage growth • Appositional growth (adding new cells from the perichondrium) • Interstitial growth (division of older cells to form isogenous or nest cells located in the same lacuna deeper in the matrix)

)Nest cells )isogenous cells

Interstitial growth of matrix occurs in deeper areas of cartilage and involves division of chrondrocytes within a single lacuna

Hyaline cartilage cells Chondroprogenitor cells

Chondroblasts

Chondrocyte Nest or isogenous cells

Hyaline cartilage matrix The main components of the matrix are: • Water (“solvation water”) (72-75%) • Proteoglycans (10%) • Collagen )type II) (16%) • Glycoproteins (e.g.chondronectin) 1.6% • Minerals (0.5%)

Proteoglycans of matrix The proteoglycans are a complex of protein and sulfated glycosaminoglycans (GAGs) and in particular: • chondroitin-4-sulfate • chondroitin-6-sulfate • keratan sulfate • (non-sulfated) hyaluronic acid

Cartilage matrix

HA, hyaluronic acid LP, link protein PC, core protein CS, chondroitin sulfate

Matrix staining properties • Basophilic after H & E staining (mainly due to sulfated glycosaminoglycans) • Periodic acid-Schiff (PAS) positive • Metachromatic The matrix surrounding lacunae stains a deeper color (territorial matrix) owing to more GAGs than in the interterritorial matrix

Territorial matrix

The territorial matrix surrounding lacunae stains deeper than the interterritorial matrix (owing to the greater amount of glycosaminoglycans (GAGs

Hyaline cartilage metachromasia If stained with toluidine blue or Azur II the GAGs stain a purple )color )metachromasia

Metachromasia of matrix

Elastic cartilage • Elastic cartilage has a very limited distribution. It is present in the external ear and epiglottis • It contains large quantities of elastic fibers in the matrix providing flexibility and elasticity • It has a yellowish color in the fresh state • Elastic fibers can be stained with orcein • The numbers and quantity of elastic fibers are greater in older matrix

Elastic cartilage Perichondrium

.Elastic cartilage stained with Weigert’s elastic stain The elastic fibers are present in greater numbers in older matrix rather than in the newer matrix below the perichondrium

Fibrocartilage Fibrocartilage is found in areas subject to high mechanical stress or weightbearing such as: • intervertebral disks • pubic symphysis • temporo-mandibular joints • ligament connections to bones (e.g. Ligamentum teres femoris) • tendon insertions

Fibrocartilage • high tensile inelastic properties • possesses large amounts of collagen fibers (little amorphous matrix) • white appearance because of large quantities of collagen fibers • acidophilic in H&E staining • lacks perichondrium

Fibrocartilage

The main feature of fibrocartilage is the dominance of large bundles of orderlyarranged collagen fibers that provide great tensile strength to the tissue

Intervertebral disks Nucleus pulposus

Anulus fibrosus

Theanulus fibrosus is a form of fibrocartilage. Thenucleus pulposus . represents remains of the embryonic notochord

Intervertebral disk

Secondary cartilage • This refers to cartilage that develops in association with specific bones formed by intramembranous ossification after the bones are already present (unlike cartilage associated with endochondral ossification) • The temporo-mandibular joint (TMJ), for example, has secondary cartilage

Cartilage regeneration • Cartilage in adults has very limited regenerative ability if injured or after wearand-tear of aging • This is a result of limited numbers of cartilage cells with low metabolic activity and minimal cell turnover • Absence of an integral blood supply

Bone Tissue • Bone tissue is the main skeletal tissue of the body and is the main component of bones

(Cartilage-ligament skeleton (van Hagen

Bone tissue • Bone is a specialized form of connective tissue and the main element of skeletal tissues • It is composed of cells and extracellular matrix • Unlike other connective tissues the extracellular matrix becomes hard and calcified

Bone

Functions of bone • Skeletal support • Site of attachment of tendons and muscles • Protection for vital organs (skull protects brain; rib cage protects heart and lungs) • Hematopoietic tissue (bone marrow) is enclosed and protected by bone • Calcium homeostasis (main store of calcium and phosphate)

Muscle man and his skeleton (Gunther van Hagen) The skeleton provides the support system for skeletal muscle

Bone characteristics • Hard, brittle, light-weight • Dynamic tissue, continuous formation and resorption throughout life • Remodelling is result of several factors including: mechanical stimuli, metabolic causes (diet, illness, aging), endocrine changes, use of drugs

Macroscopic structure Compact bone Spongy bone

There are two main categories of bone: •

Spongy bone (trabecular, cancellous bone)

•

Compact bone (cortical bone)

Compact bone

Compact bone appears as a mass of bony tissue lacking spaces visible to the unaided eye. All bones (long bones, flat bones and irregular bones) are composed of both .compact and spongy bone

Spongy bone

Spongy bone is composed of a lattice or network of branching bone spicules or trabeculae. The spaces between the bone .spicules contain the bone marrow

Spongy bone )Osteoid )prebone

Bone trabeculae have a lamellar structure. Osteoblasts are active at sites of bone formation.Osteoclasts are active at sites of bone resorption ((Howship’s lacunae

Endosteum is a thin layer of cells between the trabecula and bone marrow and is asite of osteoprogenitor .cells

Preparation of histological sections of bone tissue • • • •

Decalcification (acids or chelating agents such as EDTA) Ground sections Embed in hard resins and cut with tungsten-carbide knives For electron microscopy small blocks (under 1mm3) of undecalcified bone can be cut with diamond knives

Microscopic structure •

Cells form only a small part of bone tissue • The bulk of the tissue is the calcified bone matrix • The matrix has two main components: )d) organic matrix )e) inorganic matrix

Bone matrix Organic matrix • Type 1 collagen fibers (95%) • Amorphous ground substance: sulfated GAG’s (chondroitin-4-sulfate, chondroitin6-sulfate, keratan sulfate); bone proteins (sialoprotein, osteocalcin) Inorganic matrix • Mainly hydroxyapatite Ca10(PO4)6.(OH)2

Bone matrix )Organic matrix )30% Cells )2%) )Matrix )98% )Inorganic matrix )70% )Hydroxyapatite )95%

Bone cells

Osteoprogenitor cell

Osteoblast

in osteogenic tissue) on surfaces of) of periosteum and (developing bone (endosteum

Osteocyte in lacuna of bone) (tissue

Osteoclast in Howship’s lacuna of) (bone undergoing resorption

Bone cells • Osteoprogenitor cells are found in the periosteum and endosteum and possess “osteogenic potential” • Osteoblasts are found on surfaces of newly forming bone • Osteocytes are present in lacunae of bone matrix • Osteoclasts are found in matrix being resorbed (Howship’s lacunae)

Osteoblasts

Osteoblasts

Osteoblasts are present on surfaces of new bone development. Osteoblasts are characterized byhigh alkaline phosphatase activity. They secrete the components .of osteoid (prebone) which undergoes calcification to form the calcified matrix of bone

Osteocytes

Osteocytes in lamellar bone are located in lacunae. Lacunae are connected to each other by means ofbone canaliculi. Cell processes from osteocytes penetrate the bone canaliculi. Because the matrix is calcified nutrients can .only reach osteocytes by diffusion via these bone canaliculi

Osteocytes

Osteocytesare located in lacunae of bone matrix and communicate with each other by means of processes via bone canaliculi

Osteoclasts Osteoclasts are found inHowship’s lacunae at areas of .bone resorption. Osteoclasts originate from monocytes

Osteoclasts are multinuclear, highly acidophilic cells. They secrete lysosomal enzymes, which are acidic and lead to the breakdown and .erosion of bone matrix

Osteoclasts

Osteogenesis: woven bone • The first bone to develop is woven bone )immature bone, primary bone). This a temporary form of bone in which the lacunae are disordered and the matrix has thick, irregularly-arranged collagen fibers • Over the age of 14 woven bone is rare and only found in sutures of flat bones, tooth sockets, some tendon insertions and temporarily during fracture repair.

Woven bone • Woven bone (b) is temporary and is rapidly remodelled into lamellar bone (a)

IB , immature boneMB, mature bone

Lamellar bone There are 3 lamellar arrangements in compact bone: • Osteons (Haversian Systems) • Circumferential lamella (outer, inner) • Interstitial systems

The periosteum is attached to compact bone by Sharpey’s fibers ((perforating fibers

)Osteons )Haversian systems Osteons constitute the morphofunctional units of compact bone. They consist of 4-20 concentric lamellae surrounding a central vascular Haversian canal. Cement lines surround each osteon. The interstitial systems (2) are remains of earlier osteons after remodelling.

Interstitial System

(Osteons (Haversian systems

Osteons in compact bone

Lamellar arrangements in compact bone

Osteons Inner circumferential lamellae

Endosteum

Outer circumferential lamellae

(Lamellar arrangements (diaphysis of tibia

Bone lamellae: polarization microscopy

Alternate lamella are seen as bright when bone is examined using polarization microscopy. This anisotropy )birefringence) results from the orderly arrangement of the collagen fibers in secondary bone and their alternating direction in adjacent lamella.

Ansiotropy of lamella

Anisotropy of lamella

Labelling bone growth

Tetracycline is incorporated into developing bone matrix and can be used as a marker to determine rate of lamellar formation. Tetracycline-labelling can be .seen using fluorescence microscopy

Remodelling of bone

During remodelling of bone there is erosion of osteons by osteoclasts that results in connecting resorption cavities from adjacent osteons. When sufficient resorption has occurred, osteoblasts appear in the resorption cavities and start building a new generation of osteons. The remnants of earlier osteons are seen as interstitial systems.

Interstitial systems

Interstitial systems represent remains of osteons following remodelling

Blood supply of compact bone Blood supply to compact bone is from vessels in the perichondrium leading to transverse vessels (Volkmann’s canals) between the osteons that connect with the longitudinal Haversian canals.

Blood supply to osteons of compact bone

Haversian canal in center of osteon

Volkmann canal )transverse) connects periosteal blood with osteons

Trabecular bone Trabecular )spongy) bone is also lamellar. Unlike compact bone the lamellae do not show a concentric arrangement.

Ossification Ossification is the process of bone formation. There are two types of ossification: • Intramembranous ossification (e.g. in flat bones) • Endochondral ossification (e.g. in long bones)

Intramembranous ossification

Flat bones, such as those of the calvarium, develop by intramembranous ossification directly from connective tissue

Intramembranous ossification

Early stages

Advanced stages

Intramembranous ossification

mesenchyme ,2 blood vessel ,3 bone matrix ,4 osteoblasts ,5 osteoclast ,6

Developing calvarium in embryo

Osteoblasts, osteoid and matrix vesicles

osteoblast

Matrix vesicles

osteoid

Calcified matrix

Thematrix secreted by osteoblasts is at first non-calcified (osteoid or prebone). Processes of osteoblasts are nipped off to form membrane-bound matrix vesicles. Calcification processes occur on these matrix vesicles resulting in deposition of hydroxyapatite crystals . andmineralization of the matrix

Endochondral Ossification Endochondral ossification involves an initial model of cartilage which provides the basis for subsequent development of bone tissue. This process is best illustrated in long bones. Initially endochondral ossification occurs at the growth plate and primary center of ossification of the diaphysis. At later stages the process also occurs in the epiphyses.

Endochondral ossification

Endochondral ossification

Endochondral ossification

Growth plate of long bone Resting zone Zone of proliferation Zone of hypertrophy Zone of calcification and primary spongiosa formation

The cartilage matrix is stained with alcian blue. The bone develops on the remains of the calcified cartilage.

Growth processes in long bones

Elongation in long bones

)Synovial joints )diarthroses

Diarthrosis

Synovial joint

Synovial joint

a , capsular ligamentb

, adipose tissuec, synovial membrane

Synovial membrane

Two types of cells are found lining synovial joints: fibroblast-like cells and macrophages

Dynamic processes in bone

Activation Resorption Reversal Formation

Bone multicellular unit (BMU)

Bone is continually undergoing change and the dynamic remodelling processes involve coordination between several bone types. These are sometimes described as bone multicellular units. These are regulated by interaction of several endocrine factors

Fracture repair

Repair of a fractured bone by formation of new bone through proliferation of periosteal and endosteal cells

Fracture repair Bone heals more rapidly than cartilage because its blood supply is more plentiful and there is rapid activation and turnover of bone cell types

Growth factors and bone • All stages of bone development and growth are tightly regulated by a large complex system of systemic and local growth factors. • Homeostatic mechanisms of bone physiology differ according age, gender and growth phase. It is convenient to classify these stages as: • Fetal • Neonatal • Juvenile • Adult • Senile

Endocrine regulation of bone Major systemic hormones involved in skeletal growth and homeostasis include: •

• • • • • • •

Growth Hormone Insulin IGFs Glucocorticoids Thyroid Hormones )T3, T4) Androgens Estrogens Calcium-regulating hormones )Parathyroid hormone, Calcitonin, Vitamin D3)

Published March 2000 pp, 110 Illustrations 512

Systemic and local growth factors • Normal skeletal growth results from a balance between the processes of bone matrix synthesis and resorption. These activities are regulated by both systemic and local factors. • Bone turnover is dynamic, and skeletal growth must be maintained throughout life. • Although many growth promoters are associated with bone matrix, it is enriched particularly with transforming growth factor beta (TGF-beta) activity.

TGF-beta as a skeletal growth factor • Experimental evidence indicates that TGF-beta regulates replication and differentiation of mesenchymal precursor cells, chondrocytes, osteoblasts, and osteoclasts. • Recent studies further suggest that TGF-beta activity in skeletal tissue may be controlled at multiple levels by other local and systemic agents. • The intricate mechanisms by which TGF-beta regulates bone formation are likely to be fundamental to understanding the processes of skeletal growth during development, maintenance of bone mass in adult life, and healing subsequent to bone fracture.

Growth factors and bone Recent studies have indicated a wide range of bone regulatory local growth factors including:

• Insulin-like growth factors (IGFs) • • • •

Fibroblast growth factors (FGFs) Transforming growth factors (TGFs) Bone morphogenetic protein (BNP) IGF-binding proteins (IGFBPs)

Possible regulation and effects of bone-related growth factors