Cartilage, bone and

ossification (formation of bone)

Ivan VARGAInstitute of Histology and Embryology

March 12, 2020

Histology lecture

On-line documents for histology

lectures: introductory notesDear students,

until now, it hasn´t been our custom to publish any of histology lectures in freely accessible on-line form on the website of our faculty and there has been a good reason for that. The most important elements of each one of the histology lectures are high quality schematic pictures, drawings and photomicrographs. The source of these didactic materials is commonly some internationally accetedtextbooks, however, any further spread of these materials to wider public requires a consent from the publisher/copyright owner. Therefore, the lecture you will obtain is a version with significantly reduced number of figures. The only graphic materials I have been able to use come from a textbook which I co-authored, so I have the "copyright" to share them. Only other option has been to use free-access documents from the web. It is important to point out that any figure without proper commentary loses its educational value, so for the purpose of your self-study, I must refer to other textbooks we have written for you. Please feel free to contant me via an e-mail if you have any other queries. I hope, that the epidemiological situation will allow us to continue the in-person education as soon as possible, so we can get back to "classic" lectures followed by discussion, which represent the highest form of teaching and manifestation of creativity within the university education.

Best regards, prof. RNDr. Ivan Varga, PhD. ivan.varga@fmed.uniba.skwww.fmed.uniba.sk/en/science/

Connective tissue - classification

Connective tissue proper

Cartilage

Bone

CELLS

EXTRACELLULAR MATRIX

+ blood and lymph

Cartilage and bone

Cells of develop from embryonic mesenchymal cells

The amorphous extracellular matrix / ground substance is hardened to provide:

Rigidity

Support

Attachment for tissues

Cartilage (lat. cartilago, gr. chondros)

avascular (withoutvessels) and aneural(without nerves) connective tissue

firm and flexible tissue

few cells, more than 95% of the content -extracellular matrix,

surface is covered by perichondrium (a

type of connective tissue „capsule or envelope“)

Structure of cartilage

cartilage

cells

extracellular

matrix

chondrocytes

chondroblasts(in perichondrium)

Fibers

Ground substance

collagen I.

collagen II.

elastic

Perichondrium – envelope („capsule“ of a cartilage)

Fibrous layer =

Dense connective tissue

Chondrogenic layer =

chondroblasts

Cartilage

Perichondrium(gr. peri = around, chondros

= cartilage)

Cover of connective tissue proper (dense)

Nutrition and growth

2 layers: Outer: connective tissue proper

collagen I, fibroblasts, blood vessels

Inner: more cells – chondroblasts

Blood vessels

not present in fibrocartilage and articulating ends of joints (articular cartilage)

CELLS

Embryonic chondrogenic cells – derivatives of mesenchymal cells

Chondroblasts –inner part of perichondrium

Chondrocytes – embedded in extracellular matrix, spherical, deeper localized in cartilage

Ultrastructure of the chondrocytes

In lacunae (as small

chambers)

Territorial and

interterritorial matrix

Dilated cisterns of rough

endoplasmic reticulum, many cisterns of Golgiapparatus, secretoryvesicles - high activityduring production of proteins

Chemical

composition of

hyaline cartilage

IX-Interactions between fibrils and proteoglycan molecules

XI-Regulation of the size of fibrils

X-Organization of collagen fibrils into the 3-dimensional network

Hyaluronan

Chondroitin-sulfate

Keratan-sulfate

Anchorin CII

Majority of water is bound to hyaluronan aggregates – diffusion of metabolites from and into the chondrocytes

Growth of cartilage

Adult organism –growth and regeneration are limited

1) Interstitial growth –mitosis of chondrocytes

2) Appositional growth –differentiation of chondroblasts in inner part of perichondrium

1.

2.

3 types of cartilage – according

arrangement of the cells and

composition of extracellular matrix

Hyaline cartilage: chondrocytes arranged in the

isogenous groups („cell nests“), located in lacunas,

collagen fibrils – collagen II.

Elastic cartilage: chondrocytes arranged mostly

separately, elastic and collagen fibrils – collagen II

Fibrocartilage: chondrocytes arranged separately

or in rows, collagen fibrils – collagen I.

Where can we find a hyaline cartilage?

Maintains airways in respiratory passages (nose, larynx, trachea, bronchi)

Hyaline cartilage – other functions

Development of bones – fetal skeleton

Growth of bones – epiphyseal (growth) plates

Articulation of bones – articular cartilage

Costal cartilages

Articular

cartilage

Hyaline cartilage (glassy)

LACUNAS with CHONDROCYTES

ISOGENOUS GROUPS (cell nests)

NON-VISIBLE FIBERS

(has the same refractive index as

the ground substance)

collagen II.

chondrocyte

hyaluronan

proteoglycan

monomeresHyaline cartilage

Hyaline cartilage

2 regions of the Matrix:

• territorial – matrix around lacuna, few collagen fibers

but rich in chondroitin sulphate → basophilia of the

cytoplasm

• interterritorial – matrix between lacunas

Articular cartilage

Covers the articular surfaces of movable joints

No perichondrium

Exposed to pressure and friction

Tightly connected to the bone, blurred boundary line between cartilage and bone

Without vessels and nerves

Articular capsule

Connective tissue layer

Synovial membrane

Extends folds and villi into cavity, many capillaries, joint cavity is filled by lubricanct –

is derived from blood plasma, high concentration of hyaluronic acid

Synovialocytes A – macrophage-like cells –remove debris from the synovial fluid

Synovialocytes B – fibroblastic synovial cells –produce abundant hyaluronan

Clinical correlations

Degenerative diseases of joints, affecetd principally the articular cartilage

Up to 40% of people older than 70 years suffer from clinically manifested osteoarthrosis of knee joint.

High prevalence of arthritis: immune responses or infectious processes localized in joints (rheumatoid arthritis, tuberculosis)

Microscopic picture of partial damage of articular cartilage 2 months after injury. Locality of damage (D) is healing by the tissue that is not

similar to articular cartilage (DB – damage boundary)

Elastic cartilageAuricle, external auditory

meatus, some cartilages

of larynx, epiglottis

Elastic cartilage

Yellow color – elastic

fibers, collagen type II fibrils

High flexibility

More chondrocytes in

comparison with hyaline

cartilage, more separately

few isogenous groups

perichondrium

Fibrocartilage

• without perichondrium

• thick boundles of

collagenous fibers (I)

• chondrocytes in rows

or separately

Symphysis ossis pubisIntervertebral disc

Fibrocartilage Intervertebral disc

Bone

Strong, hard and

Rigid form of connective tissue

In most areas bone is covered by periosteum

and the central marrow cavity is lined with

endosteum

Bone tissue

protection, support, supply of Ca2+ a PO42-

only 7% of water, many minerals (hydroxyapatite, ions of organic acids)

proteoglycans and glycoproteins (bindingcalcium)

Collagen type I fibers - bone strength

blood vessels and nerves run in canals(Haversian and Volkmann´s canals)

Cells of bone Osteoprogenitor cells: capable of

differentiation and mitotic division

Osteoblasts: produce an osteoid = organic, unmineralized matrix (type I collagen and bone matrix)

Osteocytes: placed in lacunas, connected with each other by their processes, processes placed in canaliculi ossium

Osteoclasts: many nuclei, mononuclear origin (from monocytes), bone resorbtion

Osteoprogenitor cells Derived from mesenchymal stem cells

Located in periosteum and endosteum,

in blood (during bone formation)

Osteoblasts• Produce a bone matrix

• have an ability of mitotic division,

responsible for calcification of bone matrix

• cuboidal or polygonal cells with basophilic cytoplasm, many

rER and GA, arranged into 1 layer – epitheloid arrangement

at the surfaces of bone matrix

Osteocytes

Mature cells, without mitotic activity, are embeded in the bone

Responsible for maintaining the bone matrix

Located in lacunas – via their processes communicate with neighbouring osteocytes, also with osteoblasts and also pericytes of vessels

Are smaller than osteoblasts, less basophilic cytoplasm, connected by nexuses

Osteoclasts Multinucleated cells -

derivated from

mononuclear

precursors

Acidophilic cytoplasm

with many lysosomes

Located in Howship´s lacunas

- ruffled border with microvilli

(hydrolytic enzymes)

Parathormone ↑ activity,

calcitonin ↓ activity of

osteoclasts

Types of bone according their

maturity and structure

Primary bone (immature, woven, nonlamellar):

- during embryonal development, during repair of fractures - random arrangement of collagen fibers

and osteocytes, less minerals

- in dental alveolar bony sockets, insertions of tendons into bones

Secondary bone (mature, lamellar):

- compact bone (diaphysis of long bones - femur)

- spongy – trabecular bone (epiphysis of long bone,

diploe in flat bones of skull...)

Basic morphologic unit of

mature bone is osteon –

Haversian system.

Haversian canal with vessels and nerves

Bone lamellae

Osteocytes in lacunas

Canaliculi ossium

Volkman´s canals

Interstitial, outer and inner

circumferential lamellae

Types of mature bone:

Compact bone

Spongy-trabecular bone

Bone section

Periosteum and

endosteum

Periosteum – composed of outer layer (dense collagen connective tissue) and internal layer (loose collagen connective tissue, osteoprogenitor cells), in both layers - vessels

Endosteum – composed of 1 layer of osteoprogenitor cells, few collagen connective tissue

Function: nutrition of bone tissue and source of bony cells

Sharpey´s perforating fibers

Penetrating collagen fibers from outer layer of periosteum to Haversian system

Formation of bone – general rules

1. Undifferentiated embryonic mesenchymal cells can proliferate and differentiate to osteogenic cells

2. In a highly vascular environment, mesenchymal

cells differentiate to osteoblasts

3. In a poorly vascular environment, mesenchymal

cells form chondroblasts (and cartilage)

4. If calcification occurs around chondrocytes, they

will die leaving empty spaces in lacunae – osteoid tissue will occupy these empty spaces, cartilage models are replaced by bone cells

Bone histogenesis - ossification

Desmogenous (intramembranous)– in embryonal connective tissue -mesenchyme(flat bones of skull, clavicula)

Chondrogenous (endochondral)– in embryonal hyaline cartilage (long and short bones)

Intramembranous ossification

Mesenchymal cells are condensed together in a

highly vascular area where bone formation will

start

Mesenchymal cells proliferate and differentiate

to osteogenic cells – osteoblasts

Osteoid synthesize organic components of the

bone matrix, collagen type I. and osteoid

When osteoblasts are trapped inside lacunase

they are called osteocytes

Bony spicules fuse together as bony trabecules

Endochondral ossification

Replacement of a cartilage model by bone

NOTE! Cartilage does not change to or form bone! Because chondrocytes die and are replace with osteoblasts

Endochondral

ossification –

fundamental steps:

1. Cartilage model of future bone

2. Periosteal bony collar (intramembranous ossification of perichondrium)

3. Proliferation of chondrocytes

4. Hypertrophy of chondrocytes

5. Calcification of extracellular matrix of cartilage and subsequent die of chondrocytes

6. Origin of primary marrow cavity

7. Penetration of the vesselsthrough periosteal bud

8. Formation and storage of an osteoid and its mineralization

Periosteal bony collar (intramembranous ossific. of perichondrium)

Stop of diffusion of oxygen and nutrients into cartilage

Bone growth in width

Proliferation of chondrocytes

Hypertrophy of chondrocytes

Calcification of extracellular matrix – calcified spicules

Penetration of the vessels into periosteal bony collar

Osteoprogenitor cell -osteoblasts (matrix)

Osteoblasts -formation and storage of an osteoid and its mineralization

Osteoclasts – resorption of the matrix

Progress towards the epiphysis

Epiphysis – secondary ossification centre (2)

The same process of ossification as in diaphysis but circular direction

The result –epiphyseal plate

20 years old - epiphyseal closure –stop of the bone growth in the length

Zones in the hyaline cartilage during endochondral

ossification:

I. resting zoneII. proliferative zoneIII. hypertrophic cartilage zoneIV. calcified cartilage zone

-------- V. erosion line -------

VI. ossification zone

future bone

Mineralization of the bone

No satisfactory mechanism can be presented for calcification, two theories exist

Osteoblasts produce alkaline phosphatase,

which could locally increase the deposition of

insoluble calcium phosphate

Osteoblasts produce into bone matrix small matrix vesicules by an active transport

mechanism – formation of calcium phosphate

crystals

Repair of bone after fracture Destruction of bone and haemorrhage,

formation of blood clot

Neutrophils, macrophages – removal of debris

Proliferation of fibroblasts and capillaries into the place of injury = granulation tissue

Formation of connective tissue proper and cartilage in the place of injury – soft, temporary callus

Osteoprogenitors from periosteum and endosteum

– osteoblasts – new, immature bone

Repair of bone after fracture

Vitamins and bone

Vitamin D – promotes calcium and phosphate absorption. Deficiency in children causes the bone disease rickets

Vitamic C – collagen formation. Deficiency results in condition known as scurvy

Vitamin A – ballance between production and resorption of bone

Hormones and bone

Parathormone – activates and increases the number of osteoclasts promoting resorption of bone matrix (raising the blood calcium level)

Calcitonin – decreses the activity of osteoclasts and inhibits bone resorption

Growth hormone – stimulates proliferation of chondrocytes in epiphyseal plate of long bones

Thank you for you attention!