CONNECTIVE TISSUE

Question 1

Connective tissue - general structure and classification

Answer 1

Classification of Connective Tissue

• Connective tissue develops from mesenchyme, an embryonic type of tissue.
  
  • Embryonic connective tissue is present in the umbilical cord and in the pulp of the developing teeth.
• Like all tissue types, it consists of cells surrounded by a compartment of fluid called the extracellular matrix (ECM).
  
  • With the exceptions of blood and lymph, connective tissue consists of cells and extracellular material called matrix.
• Based on the cells present and the ECM structure, we differ two types of connective tissue:
  
  • Connective tissue proper;
    
    • further divided into loose and dense connective tissues
      
      • The connective tissue is classified as either loose connective tissue or dense connective tissue, depending on the amount, type, arrangement, and abundance of cells, fibers, and ground substance.
  • Specialised connective tissue;
    
    • reticular, blood, bone, cartilage and adipose tissues
• Structure:
  
  • The extracelluar matrix consists
    
    • of connective tissue fluid,
    • ground substance
    • Protein fibers (collagen, reticular, and elastic).
• Function:
  
  • The connective tissue binds, anchors, and supports various cells, tissues, and organs of the body.

Key facts

• Dense connective tissue
  
  • Cells: fibroblasts
  • Fibers: collagen fibers heavily packed in the ECM either in parallel order (dense regular), or randomly interlaced (dense irregular)
• Loose connective tissue
  
  • Cells: fibroblasts
  • Fibers: collagen fibers loosely scattered in the ECM
• Reticular connective tissue
  
  • Cells: reticular cells
  • Fibers: reticular fibers organized in delicate networks
• Cartilage
  
  • Cells: chondrocytes
  • ECM: collagen II (hyaline cartilage), elastic fibers (elastic cartilage), collagen I (fibrocartilage)
• Bone
  
  • Cells: osteoblasts, osteocytes, osteoclasts
  • ECM: calcified lamellae
• Blood
  
  • Cells: erythrocytes, leukocytes, platelets
  • ECM: blood plasma
• Adipose tissue
  
  • Cells: white and brown adipocytes
  • ECM: no ECM
• Embryonic connective tissue
  
  • Mesenchyme: mesenchymal cells in reticular fibers rich ECM
  • Mucoid tissue: mesenchymal cells in collagen rich ECM

Structure overview

• Cells
  
  • Resident cells (e.g., fibroblasts, fibrocytes), which synthesize extracellular matrix
  • Transient cells (e.g., macrophages, neutrophils)
• Extracellular matrix (ECM)
  
  • Three types of fibers
    
    • Collagen fibers
    • Reticular fibers
    • Elastic fibers
  • Glycosaminoglycans
  • Proteoglycans
  • Glycoproteins
  • Water

Cells of the connective tissue

• Resident cells
  
  • Fibroblasts (the most common cell type in connective tissue)
    
    • Origin: derived from mesenchymal stem cells
    • Function: synthesis and organization of the ECM
    • Histological features:
      
      • spindle-shaped cells arranged in a branching pattern
  • Fibrocytes: fibroblast with low metabolic activity, inactive or resting fibroblasts
  • Myofibroblasts: contractile hybrid cells with features of both fibroblasts and smooth muscle cells
    
    • Function: synthesize ECM components and are involved in the proliferative phase of wound healing
      
      • Histological features: indistinguishable from fibroblasts under the light microscope without immunohistochemical staining for, e.g., actin or desmin
  • Adipose (fat) cells,
    
    • which may occur singly or in groups, are seen frequently in the connectivetissue; these cells store fat.
    • When adipose cells predominate, the connective tissue is called an adipose tissue.
• Transient immune cells
  
  • Immune cells: lymphocytes, plasma cells, macrophages, and mast cells
    
    • Macrophages or histiocytes
      
      • phagocytic cells and are most numerous in loose connective tissue.
      • They are difficult to distinguish from fibroblasts, unless they are performing phagocytic activity and contain ingested material in their cytoplasm.
    • Mast cells,
      
      • usually closely associated with blood vessels, are widely distributed in the connective tissue of the skin and in the digestive and respiratory organs.
      • Spherical cells filled with fine, regular dark-staining and basophilic granules
    • Plasma cells
      
      • arise from the lymphocytes that migrate into the connective tissue.
      • These cells are found in great abundance in loose connective tissue and lymphatic tissue of the respiratory and digestive tracts.
    • Leukocytes,
      
      • migrate into the connective tissue from the blood vessels.
      • Their main function is to defend the organism against bacterial invasion or foreign matter.

Extracellular matrix

• The extracellular matrix (ECM) is composed of various macromolecules arranged in a three-dimensional structure.
• Its specific composition determines the biochemical properties of the connective tissue.

Extracellular matrix fibers (connective tissue fibers)

• There are three types of connective tissue fibers:
  
  • collagen, elastic, and reticular.
• The amount and arrangement of these fibers depend on the function of the tissues or organs in which they are found.
• Fibroblasts synthesize all of the collagen, elastic, and reticular fibers.

Type of Collagen Fibers

• Collagen fibers are tough, thick, fibrous proteins that do not branch.
• They are the most abundant fibers and are found in almost all connective tissue of all organs.
• The most frequently recognized fibers in histologic slides are the following:
  
  • • Type I collagen fibers.
    
    • These are found in the dermis of skin, tendons, ligaments, and bone.
    • They are very strong and offer great resistance to tensil stresses.
  • • Type II collagen fibers.
    
    • These are present in hyaline cartilage and elastic cartilage. The fibers provide resistance to pressure.
  • • Type III collagen fibers.
    
    • These are the thin, branching reticular fibers that form the delicate supporting meshwork in such organs as the lymph nodes, spleen, and bone marrow.
  • • Type IV collagen fibers.
    
    • These are present in the basal lamina of the basement membrane, to which the basal regions of the cells attach.

Reticular Fibers

• Reticular fibers, consist maily of type III collagen, are thin and form a delicate netlike framework
• in the liver, lymph nodes, spleen, hemopoietic organs, and other locations where blood and lymph are filtered.
• Reticular fibers also support capillaries, nerves, and muscle cells. These fibers become visible only when the tissue or organ is stained with silver stain.

Elastic Fibers

• Elastic fibers are thin, small, branching fibers that allow stretch. They have less tensile strength than collagen fibers, and are composed of microfibrils and the protein elastin.
• When stretched, elastic fibers return to their original size (recoil) without deformation.
• Elastic fibers are found in abundance in the lungs, bladder, and skin.
  
  • In the walls of the aorta and pulmonary trunk, the presence of elastic fibers allows for stretching and recoiling of these vessels during powerful blood ejections from the heart ventricles. In the walls of the large vessels, the smooth muscle cells synthesize the elastic fibers.

GROUND SUBSTANCE

• Glycosaminoglycans (GAGs)
  
  • Definition:
    
    • a family of unbranched polysaccharide chains of repeating disaccharide units with multiple negative charges that constitute a large volume fraction of the ECM
  • Structure:
    
    • polymer of repeating disaccharide units
    • First sugar = a derivative of uronic acid (e.g., glucuronic acid),
    • second sugar = a hexosamine (e.g., the amino sugar N-acetylglucosamine)
  • Four main groups
    
    • Hyaluronic acid
    • Chondroitin sulfate and dermatan sulfate
    • Heparan sulfate
    • Keratan sulfate
  • Function
    
    • Bind H2O in connective tissue due to its negative charges → act as a cushion
  • Component of proteoglycans
• Proteoglycans
  
  • Definition:
    
    • proteins with numerous covalently linked GAG side chains
  • Function
    
    • Bind H2O → shock absorption and a supportive function (e.g., in cartilage → resistance to compression of articular cartilage)
    • Formation of cell-cell or cell-matrix junction
    • Further signaling and regulatory functions (e.g., by binding signaling molecules)
  • Examples
    
    • Aggrecan:
      
      • large proteoglycan found in cartilage as an aggregate with hyaluronic acid → shock absorption withstands compression
    • Decorin:
      
      • small proteoglycan that binds collagen fibrils → regulates fibril assembly
• Glycoproteins of the ECM
  
  • Definition:
    
    • proteins with short carbohydrate side chains that contribute to the organization of the extracellular matrix by offering specific binding sites for cells and other matrix molecules
  • Examples
    
    • Fibronectin: glycoprotein important for cell-matrix interactions
      
      • Function:
        
        • binds to collagen and integrins and plays an important role in embryogenesis (regulates cell migration) and hemostasis (cross-linking of fibrin molecules with thrombocytes and fibroblasts)
    • Laminin:
      
      • major component and organizer of the basal lamina (besides type IV collagen)
    • Function:
      
      • binds to collagens, integrins, and proteoglycans

• Loose Connective Tissue
• Loose connective tissue is more prevalent in the body than dense connective tissue. It is characterized
• by a loose, irregular arrangement of connective tissue fibers and abundant ground substance.
• Numerous connective tissue cells and fibers are found in the matrix. Collagen fibers,
• fibroblasts, adipose cells, mast cells, and macrophages predominate in loose connective tissue,
• with fibroblasts being the most common cell types. The overview figure shows the various types
• of cells and fibers that are present in the loose connective tissue.
• Dense Connective Tissue
• In contrast, dense connective tissue contains thicker and more densely packed collagen fibers,
• with fewer cell types and less ground substance. The collagen fibers in dense irregular connective
• tissue exhibit a random and irregular orientation. Dense connective tissue is present in the dermis
• of skin, in capsules of different organs, and in areas that need strong support. In contrast,
• dense regular connective tissue contains densely packed collagen fibers that exhibit a regular and
• parallel arrangement. This type of tissue is found in the tendons and ligaments. In both connective
• tissue types, fibroblasts are the most abundant cells, which are located between the dense collagen
• bundles.

CONNECTIVE TISSUE

• Diverse group of cells within a tissue specific extrcellular matrix
• Classification of CT is based on composition and organizatio nof its cellular and extracellular components and function

Classification of CT: NEED TO KNOW EXAMPLES OF EACH!

1- Embryonic CT

• Mesenchyme:
  
  • non diffrentiated embryonic tissue
  • Embryology:
    
    • middle germ layer: mesoderm- gies rise to almost oll of the CT of the body
    • exception: head region: specific proginator cells which are dirivef from ectoderm- neural crest cells
    • through prliferation and migartion mesodermal and neural crest cells- become primitive connective tissue: mesenchyme: in head region- ectomesenchyme o
    • mesenchyme also gives rise to:
      
      • smooth muscle, vascular and urogenital systemsm, serous membrane of body cavities, smooth, Endothelial cells(inner part of blood vessel)
      • It takes part in the hemopoiesis (mesoblastic period) and in the vasculogenesis.
  • Contents:
    
    • It contains small, satellite-shaped with processes extend forming a three-dimensional cellular network
      
      • (Gap junctions)
    • extracellular space contains reticular fibers
• Wharton’s Jelly/Mucous connective tissue:
  
  • Umbilical cord (Wharton´s jelly), dental pulp
  • Composition:
    
    • fibroblasts, fine collagen and reticular fibrils, high proportion of the amorphous matrix, rich in hyaluronan (water attraction???)
  • Wharton’s jelly occupies large intercellular spaces located between thin, wispy collagen fibers.
    
    • High amount of GAG (negative charged! ATTRACTS water, diamers of saccharides, one is sulfated the other has an amino group. Stores water!)

CT proper: פירוט בשאלה ממוקדת לזה

ig CT proper: general structure and classification

• loose CT
• Dense CT
  
  • regular
  • irregular

Specilized CT

• cartilage
• bone
• adipose tissue
• blood
• lymph

DIRRFIENCIATED- SPECILIZED

Elastic connective tissue

• Elastic tissue is composed of bundles of thick parallel elastic fibers
• Can be found in:
  
  • lig. Flava – They connect the laminae of adjacent vertebrae all the way from the second vertebra
  • supensory lig. Of pinnes
• basicly connective tissue proper with high amount of elastin fibers!

Reticular Connective tissue :

• form delicate 3D network that support cells in reticular tissue.
• Because of their affinity for silver salts, these fibers are called argyrophilic.
• Composed of:
  
  • Cellular aspect: reticular fibers of type III collagen produced by reticular cells
  • for hematopoietic organs and lymphoid organs
• Location:
  
  • stroma of lymphnodes,
  • spleen and
  • bone marrow
  • Thymus, reticular epithelium! Not reticular connective!

Adipose tissue

• white (unilocular adipocytes),
• brown (multilocular adipocytes)
• recieve postganglionic sympathetic fibers
• Adipose tissue is the largest repository of energy (in the form of tryglycerids)
• Can be found in:
  
  • Subcutanos layers that help to shape the surface of body + form pads act as shock absorber(sole,palm)
• Thermal insulation – since fat is poor thermal conductor

Connective Tissue is composed of:

1. Cells -

• The cells inside the connective tissue produce the fibers (function)in every connective tissue there are fibroblasts that produce the three types of fibers.
• In addition, the cells (not just the fibroblast) produce the ground substance in the tissue.
• Blast – active! Working, Cyt – “Sleeping” not working!

2. Extracellular matrix

• Produced by the Cells,
• fill in spaces, affect mechanucak consistincy of tissues (effectivness of diffusion, metabolism and such)
• The ECM has 2 basic parts:
  
  • Fiber part: Composed out of fibers 🡪 Fibrils

1. Thick (composed of bundle of fibrils 20-300nm): Collagen
2. Thin unbranched (0.3-0.5µM) Reticular fibers
3. Thin and branched Elastic (elastin) fibers

● Ground Substances

1. Glycoaminoglycans (GAG’s)

hyaluric acid, Proteoglycans

3. Multi adhesive glycoproteins

(laminin, fibronectin and others) that stabilizes the ECM by binding to receptor proteins (integrins) on the surface of cells and to the other matrix components.

Question 2

Extracellular matrix – structure and function

Answer 2

Function EMC

• support
• segregating tissues from one another
• Regulating intercellular communication.
• Hold wide range of cellular growth factors and acts as a local store for them.
• Essential for processes like growth, wound healing, and fibrosis.
• The stiffness and elasticity of the ECM has important implications in:
  
  • cell migration
  • gene expression
  • Differentiation

1. Extracellular matrix 🡪 Produced by the Cells, which become surrounded by the EM.

The ECM has 2 basic parts:

• Fiber part:
• Ground Substances

——————————————————–

CT fibers are of 3 principle types

• Each type of fiber is produced by fibroblasts and is composed of protien consiting of long peptide chains
  
  • Collagen
  • Reticular
  • Elastic fibers

1-Collagen Fibers – Molecules of Tropocollagen -

• Collagen is the most abundant protein in the human body – 30% of its dry weight.
• Stain with eosin and other acidic dyes
  
  • Also: aniline blue: mallory’s CT stain
  • Light green: Masson’s stain

***Fibrils: subunits of collagen fibers

• Strength of collagen fibril: due to covalent bonds of adjacent rows
• Fibrillar collagens: I, II, III, V, XI
  
  • characterized by uniterupted glycine-proline- hydroxyproline repeates, 68 nm banded fibril
  • Type I:
    
    • loose and dense CT
    • heterotrimeric:
      
      • 2 chains of alfa I and 1 chaind of alfa II
    • for bone and proper connective tissue (Found in skin, tendons, bone, ligaments and cornea)
  • Type II:
    
    • hyaline and elastic cartilage
    • homotrimeric: onyl alf II
    • cartilage (and in intervertebral discs (form collagen fibrils)
  • Type III:
    
    • Reticular fibers, (found in blood vessels and hematopoetic organs: liver, bone marrow, lymph nodes, and spleen).
• Fibril assosiated collagens with interupted triple helix
  
  • interuptions provide flexability to molecules
  • (Type IX, XII, XIV)
  • IX: collagen binfs and interacts with type II collagen in cartilage at intersections of fibrils
    
    • Stabalizing tissue by binding type II collagen fibrils with proteoglycans of ECM
• Hexagonal network forming collagens: short chain (VIII, X)
• Transmembrane collagens
  
  • XIII: focal adhesions
  • XVII: hemidesmosomes
  • XXIII: cancer
• ​Basment membrane forming collagens
  
  • Type IV: found in the basement membrane (“four on the floor”)
    
    • responsible for the collagen suprastructure in the basment membrane of epithlial cells
    • the ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen
  • VII: forms ancoring fibrils that attach the basment membrane to the ECM
• Other…

***Formation of collagen

• 3 micro fibril collagen form collagen fibrils, and few fibrils create a fiber
• Can be colored in acid dyes
• They are Eosinophilic! Meaning acid dyes will paint them! Will be stained by Eosin,In bone, dentin and cementum 🡪 always fibrils, never fibers.

Production of Collagen:

• Production of fibrillar collagen
  
  • Series of event in fibroblast that lead to the production of procollagen: precusor of collagen molecule
  • Events take place in membrane bound organells within the cell
    
    • Pro-alfa chains synthesized in rER
    • intracellular processing and modification in cisternae of rER
      
      • Ascorbic acid (vitamin C) is essential
    • Tripple helix of alfa chains is formed, except at terminal ends where they remain uncoliied
    • Stabilization of tripple helix molecules
    • Result: procollagen
    • Transfered to golgi apperatus and beggin to assosiate in small bundles that are packaged into secratory vesicles and transported to cell surface
  • Assemblly of the fibril is in the ECM, under gudlines of cell
    
    • ​cleavlage of uncoilled ends of procollagen forms a mature collagen molecule: tropocollagen
    • aggrigated collagen molecules align and form fibrils: fibrillogenesis
    • Collagen fibrils can consist of more than 1 type of collagen molecule
• secretion of collagen collecules:
  
  • fibroblasts: tissues
  • chondrocytes: cartilage
  • oxteoblasts: bone
  • pericytes: blood vessels
  • collagen molecules of basment membrane are produced by epithelial cells

Other

• every 3rd amino acid is glycine- except and ends of alfa chains- known as a glycogprtien
  
  • hydroxyproline: frequently preceeds glycine in chain
  • proline: frequently follows in chain
• ​many variations for alfa chains: classified by roman numerals

2- Reticular Fibers:

• provide supporting framwork fot the cellular constitunts of tissues and organs
• diameter – 0.5-2um (extrimly thin)
• Compostition: collagen type III fibrils
  
  • (diameter of 20-45 nm), narrow diameter
  • 68 nm banding pattern as well
  • which are embedded into the glycoprotein matrix
• Contain more polysacharides then normal collagen
  
  • Stained by:
    
    • silver impregnation and PAS reaction is used for their visualization​
      
      • Argyrophilic – impregnation with salts of silver (AgNO3)which gives dark black colour
• Location:
  
  • In loose CT reticular ribers are found:
    
    • at the boundry of the CT and epithelium, as well as surrounding adipocytes, small blood vessels, nerves and muscle cells
  • Found in embryonic tissue
  • Prominet in te initial stage of woulnd healing and scar tissue fomation
    
    • provide mechanical strength to the newly synthesyized ECM
    • as healing progresses the reticular fibers and gradually replaced by stronger collagen type I fibers
  • Supporting stroma in hemopoietic and lymph tissue
    
    • BUT NOT THYMUS
    • In these tissues reticular cells produce the collagen of the reticular riber
  • In most other locations: reticular fibers are produced by fiberblasts
    
    • Important exceptions:
      
      • Endometrium of peripheral nerves: schwann cells secrete reticular fibers
      • Tunica media of blood vessels and muscularis of alimentary canal: smooth muscle cells secrete reticular and other collagen fibers

***RETICULAR TISSUE

• Is composed of retiular cells (modified fibroblasts) and reticular fibers.
• Reticular tissue creates stroma of the hemopoietic organs
  
  • (the red bone marrow, spleen and lymph nodes)
• ​The reticular cells surrounds the fiber with its cytoplasm, thus isolating the fiber from other tissue components

3- Elastic Fibers:

• Allows tissues to respond to stretch and distention
• Typaiclly thinner than collagen fibers and are arranged in a branching pattern to form 3D network
  
  • The fibers are intervoven with collagen fibers to limit the disrensibility of the tissues and preent tearing from exsessive stretching
• Gives elasticity to tissues, allowing them to stretch when needed and then return to their original state.
• (diameter 0.5 - 4 μm)
  • EFs are composed of fibrillin microfibrils (10 -12 nm in diameter) and elastin
  • Elastin is highly insoluble (hydrophobic)
    
    • random distrubution of glycine, maked the molecule hydrophobic and allows for random coliing of fibers
    • missing hyproxylysine and less hydroxyproine
    • contains amino acids
      
      • desmosine** and **isodesmosine
        form covalent vonds between elastin molecules
  • Elastin is synthesized by fibroblasts and smooth muscle cells.
• This is useful in:
  
  • blood vessels(as fibrils), elastic arteries
    
    • in the form of fernestrated lamellae:m sheets of elastic amterial with gaps/openings
      
      • arranged in concentric layers between layers of smooth emsucle cells
  • the lungs
  • in skin
  • Ligaments:
    
    • ​ ligament flava of spinal cord, ligamen nuchae of neck
    • larynx: thinner eleastic ligaments of volcal cords
• ​Staining methoods:
  
  • Orcein – brown colour
  • Aldehyde – fuchsin – black colour
  • Resorcin – fuchsin – black colour

Ground substances:

• The ground substance occupies the space between the cells and the fibers of the connective tissue
• viscous colorless matter, can’t be stained well. Fills the spaces between cells and fibers PAS will work, but HE for example won’t work.

1-Glycosaminoglycan (GAG):

• Linear polysaccharides composed from repeating disaccharides units:
  
  • composed of N/acetylgalactosamine or N-acetylglucosamine + uronic acid
  • synthesized in the Golgi complex.
  • Hydrophilic: Attract water- nutrition by diffusion
• Staining:
  
  • The GAG’s are negatively charged due to carboxylic and sulfate groups, thus it is stained with basic dyes (hematoxylin)
• Function:
  
  • The negatively charged GAGs attract water forming a gel like substance which allows rapid diffusion of nutrients, lubricant or as a shock absorber.
  • Rigid enough to provide structural support for the tissue.
• Types of GAG’s: Hyaluronic acid. Sulfated GAGS

***Hyaluronic acid

• Long and large molecule, non sulfated
• Dissosiated form: hyaluronan
• Can attract large amount of water- forms viscous gel like substance
• Not covalently bond to proteins, but indirectly bound with ptoteoglycans (forming proteoglycan aggregates)
• Mostly found in cartilage. Frequent in mucous connective tissue, vitreous and synovial fluid.

***Sulfated glycoaminoglycans:

• Synthesized in Golgi complex
• Covalently attached to proteins (as a part of proteoglycans)
• Function:
  
  • Bound to cell surfaces, membrane receptors and growth factors
• Types:
  
  • chondroitin sulfate(cartilage, bone, dentin),
  • keratan sulfate (bone, cartilage, cornea),
  • dermatan sulfate (skin, tendon),
  • heparan sulfate (basal lamina, aorta) and
  • heparin (mast cells)

* Proteoglycans:

• Protein cores
  
  • ​Made in the RER are post- translationally modified in the Golgi complex
• GAGs are added to protein cores are extend perpendicularly from the core in a brush-like structure.
• Function:
  
  • Derives from the physicochemical characteristics of the glycosaminoglycan component of the molecule, which provides hydration and swelling pressure to the tissue, enabling it to withstand compressional forces.
• The types parts of proteoglycans are:
• Aggrecan –
  
  • found in cartilage, responsible for the hydration of the ECM of cartilage.
• Decorin –
  
  • found in CT proper, cartilage and bone.
  • Plays a role in fibrilogenesis where it binds to neighboring collagen molecules and help in orienting the fibers
• Versican –
  
  • Found in fibroblasts, skin, smooth muscle, brain and kidney mesangial cells
    
    • (Mesangial cells are specialized cells around blood vessels in the kidneys).
  • Participates in cell-cell and cell-ECM interactions
• Syndecan –
  
  • links cells to the ECM.

2-Multi adhesive glycoproteins:

• Function:
  
  • Stabilizing and linking ECM to cell surface
  • Regulate cell movement and migration, and stimulate cell proliferation and differentiation.
• ​The most common multiadhesive glycoproteins are:
  
  • Fibronectin
    
    • (cell adhesion and cell migration), It has binding sites for integrins and collagen type IV.
  • Laminin
    
    • (present in basal lamina),
    • It anchors the cell surface to the basal lamina.
    • Together with Collagen type IV.
  • Nidogen/entactin
    
    • (links laminin and type IV collagen in basal lamina),
  • Chondrodronectin
    
    • (binds chondrocytes to ECM),
  • Osteopontin
    
    • (attachment cells to the bone matrix)

Question 3

Connective tissue proper – characteristics and classification

Answer 3

CT classification

• non diffrentiated
  
  • Mesenchyme
  • Wharton’s Jelly/Mucous connective tissue:
• DIRRFIENCIATED- SPECILIZED
  
  • Elastic connective tissue
  • Reticular Connective tissue
  • Adipose tissue

Connective tissue Proper

divided into 2 sub categories

• loose CT: aka areolar tissue
• dense CT
  
  • regular
  • irregular

Dense CT

• Regular
  
  • high fibrous component, little ECM
    
    • fibers arranged in parallel array and are densly paack for maximum strenth
    • cells that produce and maintain fibers are paced and aligned between fiber bundles
    • Tendons:
      
      • muscle to bone, fibroblasts alligned between collagen type 1 fibers (NEED TO CHECK IF ITS TRUE!)
      • contains collagen type 2 fibrils as well (CHECK!!!)
      • adapted to offer resistance and protection
    • Ligaments
      
      • less regullarly arranged that tendons
      • bone to bone
      • in some locations require elasticity: like in the spinal cord
        
        • collagen is the major componet but some contain elastic fibers- yellow
    • Aponeurosis
      
      • resemble a broad flat tendon
      • arranged in multiple layers
      • arranged usually in 90 degree andgle to neighboring fibers, but in each sheet arranged regularrly
        
        • orthogonal array
        • also in the cornea of the eye: reponsible for transperancy
• Irregular
  
  • Contains mostly collagen fibers, arranged in various directions
  • Cells are sparse and of a single type: fibroblasts
  • Relativly little gound substance
  • Function: privide strength and withstand stress
  • Location:
    
    • hollow organs in intestinal tract ??
    • skin: reticular layer of dermis

Loose connective tissue

• General characteristic:
  
  • Cellular CT with thin and relativly sparse collagen fibers and abundent ground substace
• Cellular Aspect:
  
  • Most cell types in loose CT are transient wandering cells that migrate from local blood vessels in response to specific stimuli.
    
    • site of inflammatory and immune reactions.
• Can be found in:
  
  • Hypodermis
    
    • located beneath the epithelia that covers the body surfaces and line internal surfaces of body
  • Papillary layer of the dermis
  • Associated with the epithelium of glands and surrounds the smallest blood vessels
• Function:
  
  • role in diffusion
  • It is flexible, well vascularized and not very resistant to stress.
  • Most dominant cells are fibroblast and macrophages(histocytes)
  • High amount of cells in contract to the extra cellular matrix:
    
    • relatively sparse collagen fibers, thus loose connective tissue has a delicate consistency.

MOVE TO RELEVANT CARD

3. Elastic connective tissue

• Elastic tissue is composed of bundles of thick parallel elastic fibers
• Can be found in:
  
  • lig. Flava – They connect the laminae of adjacent vertebrae all the way from the second vertebra
  • supensory lig. Of pinnes
• basicly connective tissue proper with high amount of elastin fibers!

4. Reticular Connective tissue :

• form delicate 3D network that support cells in reticular tissue.
• Because of their affinity for silver salts, these fibers are called argyrophilic.
• Composed of:
  
  • Cellular aspect: reticular fibers of type III collagen produced by reticular cells
  • for hematopoietic organs and lymphoid organs
• Location:
  
  • stroma of lymphnodes,
  • spleen and
  • bone marrow
• Thymus, reticular epithelium! Not reticular connective!

5. Adipose tissue

• white (unilocular adipocytes),
• brown (multilocular adipocytes)
  
  • recieve postganglionic sympathetic fibers
• Adipose tissue is the largest repository of energy (in the form of tryglycerids)
• Can be found in:
  
  • Subcutanos layers that help to shape the surface of body + form pads act as shock absorber(sole,palm)
  • Thermal insulation – since fat is poor thermal conductor

Question 4

Cells in the connective Tissue – and their function.

Answer 4

Can be divided into 2 groups:

• Resident:
  
  • Fibroblast (fibrocytes)
  • Myofibroblast -fibroblast that located in the epimysium
  • Adipocyte
  • Melanocyte
  • Adult stem cell
• Free (wandering)
  
  • Macrophages (histocyte)
  • Granulocytes
  • Lymphocytes
  • Monocytes (procurers of macrophages)
  • Plasma cells
  • Mast cells (heparinocyte)

Fibroblast/Fibrocyte: Most common cell in the connective tissue

• Function:
  
  • main: produce ECM
    
    • including proteins: Collagen and Elastin (which upon secretion will form collagen, reticular and elastic fibers),
    • production of GAG, proteoglycans and multiadhesive glycoproteins
    • Ground substance.
    • • rich in rER, well developed Golgi complex, many mitochondria, basophilic cytoplasm.
• Shape of cells:
  
  • Nucleus – large, egg shaped and appears pale, light nucleolus,
  • rER, Golgi apparatus, Basophilic cytoplasm: Proteosynthesis.
• Fibrocyte: Resting form, elongated, spindle shaped, predominatly heterochromatin

Myofibroblast:

• Varient of fibroblast
• Some propeties of smooth muscle cells
• Wound healing, they secret ECM during wound healing
• Myofibroblast can contract by using smooth muscle type actin-myosin complex, rich in a form of actin called alpha-smooth muscle actin. These cells are then capable of speeding wound repair by contracting the edges of the wound.

Reticular Cells

• Not reticular epithelium! We are in a connective tissue, they are modificated fibroblasts- with more processes, light nucleus, nucleolus.
• They form the basic net in some organs, such as: Lymph bodes, Spleen, Bone marrow.

Pigment Cells

• not from mesenchyme cells!
• miagrates from the neuroectodermal neural crest and colonises in target organs (in the “from” of mesenchyme cell)
• melanine production, protection against UV
• Location:
  
  • Sclera – outer layer in the eye
  • Choroidea – lamina suprachoroidea = Iris
  • Pia matter around the brain

Adipocytes

• Mainly intercellular storage of Fat/lipids in vacuoles in cytoplasm. Two types of adipocytes:
• Unilocular Adipocytes:
  
  • Size50-100um, one big vacuole in the cytoplasm, White adipose tissue.
  • We won’t see the lipid droplet, but a white hollow cell, without anything inside (because of paraffin and ethanol etc).
  • The nucleus is flattened and located on the periphery. Different embedding! Sudan black.
• Multilocular adipocytes:
  
  • more vacuoles, nucleus in the center, Brown adipose tissue!
  • Newborn baby up to 2-3 years.
  • Hibernation.
  • The nucleus is round, and, although eccentrically located, it is not in the periphery of the cell. The brown color comes from the large quantity of mitochondria. They are heat-energy generator

WANDERING

Mast Cells/Heparinocytes:

• Originate from progenitor cells in the bone marrow
• shape:
  
  • 10-13 um in diameter
  • Oval shape
  • Nucleus - oval or round
  • Small microvilli
  • Contain basophilic granules in the cytoplasm.
  • Create histamine and heparin
• dying:
  
  • PAS – reaction
  • alcian blue
  • Metachromatic dyes
  • (Heparin – blue, histamine – violet) dyes.
• Histamine:
  
  • increase vascular permeability and contract smooth muscles
  • when IgE connects with mast cells the response is secreting of histamine
• Heparin is anti-coagulant
  
  • chemically : It is a sulfated and contain glycosaminoglycan
• Can be found in :
  
  • Smooth muscle cells in capillaries and bronchioles
  • Skin and peritoneal cavity
  • Connective tissue of the intestinal mucosa and lungs (mucosal mast cells)

Appears as spread blue cytoplasm with magenta nucleus

Plasma Cells - Note B Cells!

• Shape:
  
  • Round or slightly pyramidal shape
  • round small nucleus on the cell wall
    
    • typical sign : “clock – face chromatin”
  • contain many RER
• basophilic cytoplasm
• Function :
  
  • Proteosynthesis
    
    • Immunoglobulins antibodies
    • immunoblast
    • created from a B-lymphocyte

Macrophages 🡪 Histiocytes🡪 Phagocytosis, Monocyte 🡪

• Macrophages:
  
  • Can be also called:
    
    • Histiocytes – a macrophage in the CT
    • Fagocytosis – a eating cell
    • Monocyte –when it is in the blood stream
      
      • The process of changing the name from monocyte to macrophage is due to increase in protein synthesis and cell size(maturing)
  • Monocytes leave blood circulation and migrate into connective tissue where they differentiate into tissue macrophages/ Histiocytes.
  • Structure of macrophages depends on their state of activity and localization.
    
    • In resting state they are similar to fibrocytes.
    • Active macrophages enlarge, have irregular ovoid shape, contain abundant lysosomes, distinct Golgi complex, variable amount of free ribosomes and rough endoplasmic reticulum.
  • Although the main function of the macrophage is phagocytosis (bacteria and other foreign particles, cell debris), it plays an important role in immune antibody presenting cell (MHC 2)
  • Contain oval/kidney nucleus and can get up to 30 um body size
• Leukocytes
  
  • lymfocytes, monocytes,
  • contain eosinophilic granulocytes
    
    • function for
  • leukocyets are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders

Leukocytes 🡪 Lymphocytes, monocytes, Eosinophilic Granulocytes.

The rules are always the same! First describe the ROLE/Function of the cell, than follow the same steps 🡪 producing proteins? Light nucleus, rER, Golgi apparatus, a lot of ribosomes etc. By this we can understand the plasma membrane is basophilic.

Question 5

Cartilage - structure and function, description of the individual types

Answer 5

Intro:

• Is a specific avascular connective tissue composed from cells (Chondrocytes) and ECM.
• The nutrition of the Chondrocytes depends on the ECM (result of diffusion through the ECM).
• Cartilage is a specialized form of connective tissue in which the firm consistency of the ECM allows the tissue to bear mechanical stresses without permanent distortion.

Cartilage provides:

1. Shock absorbing,
2. Sliding area for joints
3. Facilitates bone movements.
4. It is essential for the development and growth of long bones (endochondral ossification).

Cellular aspect:

• The chondrocytes synthesize and secrete the ECM and are located in matrix cavities called lacunae.

ECM is rich in:

• Collagen type II, Hyaluronic acid, Proteoglycans - Aggrecan, and other.
• The Hydrophilic ground substance enables diffusion of nutrients.

3 types of cartilage:

1. Hyaline Cartilage – most common, collagen type 2 is the main fibrils here.
2. Elastic Cartilage – in addition to collagen type 2, there is abundance in Elastic fibers within its matrix.
3. Fibrocartilage – can be found in places around the body subjected to pulling forces, matrix is rich with collagen type I.

Hyaline Cartilage – “Glass in Greek”

Structure

• Outer fibrous layer of perichondrium:
  
  • Dense CT: protection, nourishment(vasuclar and neural support
• Inner endochondral layer: flat proliferating chondroblasts. allow for appositional growth- thickening
• Growing chondroblast layer
• Interteritorial matrix
  
  • Cells: chondrocytes in lacunae, producing ECM
    
    • isogenies groups of chondrocytes: tend to stay together after mitosis. May appear in groups of up to eight cells originating from mitotic division of a single chondrocytes.
    • The territorial matrix is the area surrounding each chondrocyte and characterized by an ECM rich in GAGs and poor in collagen. This area usually stains more basophilic from the rest of the inter-territorial matrix which is richer in collagen.
  • ECM:
    
    • type II collagen fibrils
      
      • ​other cartilage-specific collagen molecules are types VI, IX, X and XI
    • ground substance: glassy apperance, homogenous in living state
      • proteoglycan aggrecan: fromed from Chondroitin sulfates and keratan sulfates
      • Agreggan is liked to hyaluric acid with a connecting/linking protien (because they cant covalently bond)- forming proteoglycan aggregate
      • PG aggregates are bound to the collagen fibrils
        
        • Water bounds with GAG and occupyes up to 60% of whight of the cartlige

Staining:

• Cartilage matrix is generally basophilic,
• stained by basic dyes blue, due to the high concentration of sulfated GAGs.
• stained with alcian blue and nuclear red

ECM is rich in sulfated glycosaminoglycans which are stained with alcian blue. Capsules of chondrocytes and teritorial matrix are intensivly stained (arrows).

Location:

1. Respiratory passages: Larynx rings, Trachea, Bronchi
2. Connections between the ribs and the sternum
3. Articular surface of Joints (as there is no perichondrium) – it is important to note that Endochondral ossification is based on the blue print which is Hyaline cartilage.

Growth:

• The mesenchyme: the precursor of all types of cartilage.
• Mitotic proliferation-> prochondral blastema
• Appositional growth – on the surface of cartilage: differentiation of chondroblasts in the inner chondrogenic layer of perichondrium.
• (Differentiated chondroblasts produce ECM and are separated from one another by great amount of matrix)
• Multiplication of chondrocytes (interstitial growth) gives rise to isogenous groups surrounded by a condensation of teritorial matrix.

Elastic Cartilage

Elastic properties in addition to the resilience and pliability that are characteristic of hyaline cartilage

Structure

• Chondrocytes: small isogenous groups or single cells
• ECM: abundant network of elastic fibers in addition to collagen type II fibrils, ground substance (PG, multiadhesive glycoproteins)
• Its Perichondrium is responsible for blood supply – as vessels course through it.

Staining:

• will be stained by Orecin (Elastin fibers, not nucleus),
• HE: the fibrils are Eosinophilic.

Location of Elastic Cartilage:

1. Cartilage of the ear, auricle
2. wall of external acoustic meatus and auditory tube
3. Small cartilages in the larynx
4. Epiglottis of the larynx - in its center there is a plate of elastic cartilage.

Fibrocartilage:

a combination of dense regular CT and hyaline cartilage

• There is no perichondrium.
• Cellular:
  
  • Chondrocytes, Oval Shaped, dispersed among the collagen fibers singularly, in rows, and in small isogenous groups.
  • Also present are Fibroblasts – they will produce a typical proteoglycan: Verscian (similar to aggrecan)
• ECM
  
  • Fibrilar component predominates,
  • mainly type I collagen fibers (determined eosinophilia of ECM),
  • and type II collagen fibrils.

Location:

• Intervertebral discs- annulus fibrosus of the intervertebral disc.
• Pubic symphysis
• Articular discs of the sternoclavicular and temporomandibular joints,
• Menisci of the knee joint.
• Junctions between flat bones

Staining: Blue Trichrome is possible, Alinin blue (Acid dye), Eosin (collagen fibers/fibrils – Eosinophilic)

Question 6

Microscopical structure of the bone tissue, description of the individual types

Answer 6

Bone Intro:

• hard CT composed of cells and ECM,
  
  • mineralization of its matrix: the mineral is calcium phosphate in the form of hydroxyapatite crystals
    
    • ​Hydroxyapatite from 97%
    • 3% salts (Calcium carbonate, Calcium Phosphate, Calcium Fluride).
• Function:
  
  • • hard tissue providing support, structure and protection to tissues and body in general
• Bone has
  
  • organic component (33% collagen 1,3,5,9, type 1 is dominant in bone)
  • inorganic component (67%).

Function:

1. Movement system
2. Supports fleshy structures
3. Protects vital organ
4. Harbors the bone marrow
5. Reservoir of calcium and phosphate:
   
   • ​​Regulation of blood calcium levels (controlled by parathyroid hormone and calcitonin)
   • Our bones store Calcium (1 KG in all bones)

Cells: osteoprogenitor cells, osteoblasts, osteocytes, osteoclasts

• Osteoprogenitor cells (osteoblast precursor cells) - derived from mesenchymal cells, have a potential to differentiate into osteoblasts.
  
  • They reside on external and internal bone surface (in the innermost layer of periosteum and endosteum) and in bone vasculature.
• Osteoclasts – involved in the resorption and remodeling of bone tissue
  
  • large (50-100 μm)
  • multinucleated (up to 10 nuclei) cells resulting from the fusion of granulocyte/macrophage precursor cells
  • Location: Howship’s Lacunae.
  • The active osteoclast exhibits:
    
    • rufflet border (deep plasma membrane infoldings, exocytosis of hydrolytic enzymes and protons, endocytosis of degradation products and bone debris)
    • clear zone (demarcates and seals bone area being resorbed - actin filaments)
    • basolateral region (exocytosis of digested material)
• Osteocytes – which are found in cavities called lacunae, between layers (called lamellae) of bone matrix.
  
  • It has thin processes running in canaliculi (blood supply through canaliculi)
  • Flat nucleus (not seen well, due to its lack of function)
  • On the end of each process there are Gap junctions, proving support and communication to one another.
• Osteoblasts – Synthesize the organic components of the matrix (basophilic cytoplasm at this time)
  
  • Surface of bone matrix- side by side, look similar to simple epithelium (But they are cells in the connective Tissue!).
  • Stimulated by parathyroid hormone.
  • (osteoid) = collagen I, proteoglycans, multiadhesive glycoproteins, calcium - binding proteins, alkaline phosphatase (strong ALP reaction on the cell membrane), matrix vesicles (calcification)

ECM:

• inorganic component 65%
  
  • (hydroxyapatite): A mineral, natural form of calcium, Color white grey yellow green and brown, Hardness 5, Dissolved in HCL
  • As well as bicarbonate, citrate, magnesium, potassium and sodium.
• organic component 35%:
  
  • both collagen and gorund substance become minerlized to form the bone tissue
  • Main component- collagenous componets: (collagen 90% of weight bone matric protiens)
    
    • collagen fibrils- type 1
    • lesser extent: type V collagen
    • also: minor collagens (III, V, XI, XIII)
  • Non collagenous components: constitute the gorund substance of the bone
    
    • minor component: onyl 10% of weight of bone matrix protien
    • esential for development, growth, remoddling and repair
    • Types:
    • Proteoglycan marcomolecules
      
      • with covlently attached side chains of GAG
        
        • GAGs (chondroitin sulfate, keratan sulfate), hyaluronan, proteoglycans
      • strength of bone and responsible for bining growth factors
    • Multiadhesive glycoproteins
      
      • ​(osteonectin, sialoproteins I and II, osteopontin) -
      • responsible for the attachment of cells and fibrils to the mineralized ground (amorphous) substance
      • osteocalcin - captures calcium from the circulation and stimulates osteoclasts
    • Growth factors and cytokines –
      
      • e.g. bone morphogenic proteins
      • (BMPs - they induce the differentiation of mesenchymal cells to osteoblasts – used also clinically)

Types:

• Woven/Fibrillar – Bone AKA non lamellar bone
  
  • Composed of:
    
    • Irregular/random net of thin Collagen Fibrils.
    • Less mineralized, higher proportion of osteocytes (Primary bone tissues are characterized by lower mineral content =less inorganic components (easily penetrated by x-ray).
    • the matrix stains more intensly with hematoxyline where as the matrix of mature boen stains more inteslly with eosin
    • • Function:
    • The primary bone is the first bone tissue to appear in embryonic development and in fracture repair.
    • It is usually temporary and replaced by secondary bone (except in the skull sutures).
• Lamellar Bone - Compact and Spongy Bone:
  
  • Found in Long bones, short bones, flat bones – specific bones according to shape
  • We can divide into: Diaphysis, Epiphysis and Metaphysis.
  • Periosteum >Compact bone >spongy

1- Compact bone:

• Composed of:
  
  • Osteons (also called Haversian systems) – in its center, blood vessels flow, around concentric arrangement of lamella 4-20. Interstitial lamellae are the region between osteons.
  • Lamellas are concentric (in a circle) 3 – 10 Micrometer Thick per one Lamella.
  • The FIBRILS are running parallel but in different opposition.
• Lamella:
  
  • Can be as thick as 3-7um.
  • Lamella is a thin layer of extracellular matrix.
  • Surface is covered by Periosteum (dense regular connective tissue)
  • Interstitial lamellae (remnants of previous concentric lamellae between osteons),
  • Outer (are under the periosteum and follow the outer circumference of the bone)
  • inner (are located around the bone marrow cavity and are covered by the Endosteum) circumferential lamellae.

Volkmann´s canals– bring vessels - from the periosteum inside the compact bone.

Staining: Eosinophilic

Periosteum:

• Shape: is a dense regular connective tissue, on the outer surface of the bone, inside it we can find Vessels and nerves,
• Consists of two layers:

1. Outer fibrous layer: Rich in fibers
2. Inner cellular layer: contains mesenchymal stem cells called osteoprogenitor cells.

• Sharpey’s Fibers: thick and short collagen fibers holding the periosteum to the outer lamella of the compact bone.

Endosteum:

• The large internal marrow cavities of bone are lined by it.
• It is a single thin layer of connective tissue, containing as well osteoprogenitor cells and osteoblasts.

Function: The principal functions of Periosteum and Endosteum are nurishment of bone tissue and provision of a continuous supply of new osteoblasts for repair or growth of bone.

2- Spongy Bone:

• Location:
  
  • In the center of Diaphysis and Epiphysis
  • At the ends of long bones
  • As well as in the pelvic bones, ribs, skull, and vertebrae.
• Composed of:
  
  • Sponge like meshwork consisting of numerus interconnecting bone trabecules (which are composed out of bone lamellae).
  • The spaces are filled with the bone marrow.
  • No haversions system! Reticular connective tissue! The lamella are seen, many osteocytes are present. Lamella forming bone trebcules
• Cellular Aspect:
  
  • Between the lamellae lying osteocytes.
  • In the spaces around the trebcules we can find Bone marrow – which sits on reticular connective tissue (net like formation), responsible for hemopoiesis/ developing blood elements. Bone marrow can be Yellow or Red.
• Function:
  
  • Spongy bone plays a crucial role in producing blood cells and stem cells and it’s the primary tissue affected by osteoporosis

Question 7

Endochondral and intramembranous ossification

Endochondral Ossification

Endochondral ossification is the mechanism responsible for the formation of all long bones of the axial skeleton (vertebrae and ribs) and the appendicular skeleton (limbs). Most of the axial skeleton is derived from cells of the paraxial mesoderm that condense early in embryogenesis on both sides of the neural tube and the notochord. Some cells of this mesoderm form segmented structures called somites, portions of which later become the sclerotomes that will give rise to the vertebral bodies. The appendicular skeleton arises from the lateral plate mesoderm. The mechanisms underlying the early condensation, segmentation, differentiation, and patterning events define the precise arrangement of the individual anatomic elements and their patterning along the proximal-distal, dorsal-ventral, and posterior-anterior body axes. These mechanisms involve actions and cross-talk of several morphogens, including fibroblast growth factors(FGFs), sonic hedgehog (Shh), bone morphogenetic proteins (BMPs), and Wnts, as well as control by Notch signaling and by transcription factors encoded by HOX, PAX1, and TBX genes.9

As in intramembranous ossification, the development of the long bones proper starts with mesenchymal progenitor cells forming condensations at the sites where the bones will form.10 Yet, in the mesenchymal condensations of endochondral bones, cells do not differentiate into osteoblasts but instead differentiate into chondrocytes that synthesize a characteristic extracellular matrix(ECM) rich in type 2 collagen and specific proteoglycans. As such, a cartilaginous model or anlage is established that prefigures the future bone. In mice, these differentiated cartilage structures appear around embryonic day 12, with the limb elements emerging in sequence along the proximodistal axis (i.e., hip to toes, shoulder to fingers). The sequential steps of the endochondral ossificationprocess starting from this stage are illustrated in Figure 60-1. Initially, the cartilage further enlarges through chondrocyteproliferation and matrix production. Chondrocytes in the midportion of the bone model then stop proliferating, undergo further maturation, and ultimately become hypertrophic. These large hypertrophic chondrocytes secrete a distinct matrix, containing type X collagen, and then rapidly direct the calcification of the matrix. Concomitantly, the hypertrophic chondrocytes direct the cells surrounding the cartilage element called the perichondrium to differentiate into osteoblasts that deposit mineralized bone matrix—the “bone collar”—around the cartilage template. This bone collar forms the initiation site of the cortical bone, the dense outer envelope of compact, lamellar bone that provides the long bone with most of its strength and rigidity (see Fig. 60-1).

At this time in development, the cartilage model starts to become replaced by bone, vascular, and marrow elements: the primary ossification center (see Fig. 60-1). The transformation is initiated by the invasion of the hypertrophic cartilage core by blood vessels(around embryonic day 14 to 15 in mice). This process is accompanied by apoptosis of terminally differentiated hypertrophic chondrocytes, resorption of the calcified cartilage matrix by invading osteoclasts or related “chondroclasts,” and deposition of mineralized bone matrix on the remnants of calcified cartilage by perichondrium/periosteum-derived osteoblasts. Recent studies have visualized the entry of osteoblast lineage cells into the primary ossification center at these early stages, showing a close temporal and spatial association between osteoprogenitors and blood vessels co-invading the developing long bones.11

With the disappearance of the diaphyseal cartilage (see Fig. 60-1), the remaining chondrocytes, restricted to the opposing ends of the long bone, provide the engine for subsequent bone lengthening. This process is typified by precise temporal and spatial regulation of chondrocyte proliferation and differentiation, with the chondrocytes first flattening out and forming longitudinal columns of rapidly proliferating cells, and next, as they reach the ends of the columns closest to the center of the bones, maturing further to hypertrophic chondrocytes (Fig. 60-2). Finally, at the border with the metaphysis(see Fig. 60-1), the terminally differentiated chondrocytes are thought to mostly disappear through apoptosis, and the calcified hypertrophic cartilage matrix is progressively replaced with cancellous or trabecular bone (forming the primary spongiosa). This process of cartilage turnover and replacement by bone requires adequate neovascularization of the chondro-osseous junction by metaphyseal capillaries (see Fig. 60-1 and cellular details in Fig. 60-2). Thus, similar to the initial formation of the primary ossification center, endochondral bone formation at the growth cartilage involves rigorous coupling of vascular invasion with maturation and activity of chondrocytes, osteoclasts, and osteoblasts (for review, see reference 12).

At a certain time (around postnatal day 5 in mice), epiphyseal vessels (see Fig. 60-1), derived from the vascular network that overlays the cartilage tissue, invade the growth cartilage and initiate the formation of the secondary center of ossification. As a result, discrete layers of residual chondrocytes form true growth plates between the epiphyseal and metaphyseal ossification centers, mediating further postnatal longitudinal bone growth. Ultimately, at least in humans, the growth plates completely disappear (close) at the end of adolescence in a process that actively requires the action of estrogen in both boys and girls, and growth stops. Remodeling of existing bone, replacing the primary spongiosa with lamellar bone in the secondary spongiosa and renewing the cortical bone, takes place throughout adult life, ensuring optimal mechanical properties of the skeleton and contributing to mineral ion homeostasis. This continual bone turnover is accomplished through the balanced action of osteoclasts and osteoblasts (see later) and results in a dynamic organization of honeycomb platelike structures or trabeculae in the interior of the bone that are surrounded by blood vessels and bone marrow and housed within the cortical bone.

The mechanisms of embryonic bone development described here are largely recapitulated in the adult upon repair of bone defects. In contrast to soft tissues, which repair predominantly through the production of fibrous scar tissue at the site of injury, the skeleton possesses an astounding capacity to regenerate upon damage. As such, bone defects heal by forming new bone that is indistinguishable from adjacent, uninjured bone tissue. It has been appreciated for a long time that fracture repair in the adult bears close resemblance to fetal skeletal tissue development, with both intramembranous and/or endochondral bone formation processes occurring depending on the type of fracture. This close resemblance has been supported by genetic and molecular studies showing that similar cellular interactions and signaling pathways (see later) are at work in both settings,8,13-15 although additionally, some molecules that are dispensable for development have been found to play essential roles in fracture repair.16,17

https://opentextbc.ca/anatomyandphysiology/chapter/6-4-bone-formation-and-development/

Figure 2. Endochondral Ossification. Endochondral ossification follows five steps. (a) Mesenchymal cells differentiate into chondrocytes. (b) The cartilage model of the future bony skeleton and the perichondrium form. (c) Capillaries penetrate cartilage. Perichondrium transforms into periosteum. Periosteal collar develops. Primary ossification center develops. (d) Cartilage and chondrocytes continue to grow at ends of the bone. (e) Secondary ossification centers develop. (f) Cartilage remains at epiphyseal (growth) plate and at joint surface as articular cartilage.

As more matrix is produced, the chondrocytes in the center of the cartilaginous model grow in size. As the matrix calcifies, nutrients can no longer reach the chondrocytes. This results in their death and the disintegration of the surrounding cartilage. Blood vessels invade the resulting spaces, not only enlarging the cavities but also carrying osteogenic cells with them, many of which will become osteoblasts. These enlarging spaces eventually combine to become the medullary cavity.

As the cartilage grows, capillaries penetrate it. This penetration initiates the transformation of the perichondrium into the bone-producing periosteum. Here, the osteoblasts form a periosteal collar of compact bone around the cartilage of the diaphysis. By the second or third month of fetal life, bone cell development and ossification ramps up and creates the primary ossification center, a region deep in the periosteal collar where ossification begins (Figure 2c).

While these deep changes are occurring, chondrocytes and cartilage continue to grow at the ends of the bone (the future epiphyses), which increases the bone’s length at the same time bone is replacing cartilage in the diaphyses. By the time the fetal skeleton is fully formed, cartilage only remains at the joint surface as articular cartilage and between the diaphysis and epiphysis as the epiphyseal plate, the latter of which is responsible for the longitudinal growth of bones. After birth, this same sequence of events (matrix mineralization, death of chondrocytes, invasion of blood vessels from the periosteum, and seeding with osteogenic cells that become osteoblasts) occurs in the epiphyseal regions, and each of these centers of activity is referred to as a secondary ossification center (Figure 2e).

Endochondral Ossification

Endochondral ossification occurs in several steps. Initially, endochondral ossification begins with a pre-existing cartilage template. The cartilage template begins to be calcified. As the cartilage template calcifies, the chondrocytes in the cartilage become hypertrophic and undergo apoptosis (Bilezikian et al., 2002; Tuan, 2004; Franz-Odendaal et al., 2006). Then, mesenchymal stem cells in the membrane surrounding the calcifying cartilage, periosteum, differentiate to osteoblasts. These osteoblasts lay down an osteoidmatrix around the exterior of the cartilage template. At the same time, a bud of cells originating from the periosteum invades the interior of the partially calcified cartilage template. This periosteal bud leads to vascularization and innervation of the developing bone. The periosteal bud also supplies mesenchymal and hematopoietic stem cells to the center of the cartilage template. The mesenchymal stem cells differentiate to osteoblasts, and the hematopoietic stem cells differentiate to osteoclasts. These osteoblasts and osteoclasts remodel the partially calcified cartilage into woven bone, which is ultimately remodeled to become lamellar bone. Lamellar bone contains collagen fibrils that are arranged in parallel areas, and exhibits greater strength compared to woven bone (Shapiro, 2008). As the bone tissue created from cells originating from the periosteal bud increases, it radiates outward and eventually joins the bone tissue created by the osteoblasts on the surface of the cartilage template

Answer 7

Intramembranous ossification - flat bones of the skull and face, mandible,clavicle

Endochondral ossification - vertebrae, bones of the extremities

Osteogenesis

• Bone can be formed initially by two ways
• In both processes, the bone tissue that appears first is primary (woven) bone.

Intramembranous ossification:

• The development of bone from embryonic mesenchymal tissue, occurs before week 8
• Most of the flat bones are being developed by intramembranous ossification.

1. A condensed layer of mesenchymal cells differentiate into osteoprogenitor

Condensation of mesenchyme - blastema - primary ossificationcenter

2. The cells proliferate and form incomplete layers of osteoblast around a network of developing capillaries: Formation of ossification center
3. Polarized osteoblasts secrete osteoid components
4. The components calcify and form tubercle of woven bone
5. The osteoblasts becomes trapped inside the space, they remain trapped there and differentiate into osteocytes (responsible for bone maintenance) after a few days the osteon calcifies and harden
6. Osteoid continues to be deposited,assembles in a random manner around embryonic blood vessels and form more woven trabeculae
7. Continued growth leads to the fusion of neighboring ossification centers and gradually lamellar bone (compact and spongy) replaces woven bone that broadly encloses a region of bone marrow.

Endochondral Ossification:

• Responsible for most types of bones
• Primary ossification center: starting point for endochondral ossification
• Embryonic model of the skeleton is made of hyaline cartilage (Blueprints)
• Begins in second trimester of intrauterine development

Process:

• With aggregation of mesenchymal cells which differentiate into chondroblasts→ due to: FGF (fibroblastic growth factors), BMP (bone morphogenic proteins)→produce cartilage matrix:
  
  • hyaline cartilage model surrounded by perichondrium
• Cartilage model gows by interstinal and appositional growth→
  
  • increase in length: interstinal growth
  • increase in width: appositional growth: addition of cartilage matrix produced by new chondrocytes that diffrentiate from the chondrogenic layer of the perichondrium surrounding the cartilage mass
• First signs of ossification:
  
  • perichondrial cells in the midregion of the cartilage model no longer give rise to chondrocytes→ instead: bone forming cells are produced: OSTEOBLASTS→ secreting bone matrix
  • →the CT surrounding this portion of the cartilage in this area is known as PERIOSTEUM
    
    • =apperance of a cuff of bone arround the cartilage model= BONY COLOR FORMATION
    • an osteopgenetic layer can be identified within the newlly “formed” periostium
• Osteoblasts enlarge →

1. Collar of bone tissue is formed around the perichondrium (outer layer of the cartilage) by osteoblasts (mesenchymal that diffrenciated)
   - prevents oxygen and nutrients from the cartilage tissue.
   - causes enhance degeneration: chondrocytes begin to secreting alkaline phosphate swell and, enlarging their lacunae (“pockets”).
   - these changes compress the matrix and cause calcification and death of chondrocytes.
2. The posterior bud invades the periosteum (used to be perichondrium) and penetrate the bone collar.
   - osteoblast which adhere to the remnants of calcified cartilage matrix and produce woven bone- this process forms the primary ossification center

*calcified cartilage appears basophilic and the new bone is acidophilic

Primary Ossification center continues to grow

3. Around birth Secondary ossification centers appear in the epiphysis and develop similarly

During childhood the first and second ossification centers are separated only by the epiphyseal disk that helps in bone elongation- epiphyseal disk disappear.

Cavities are formed, during primary and secondary ossification, that are gradually filled with bone marrow and trabeculae of ossification bone.

Epiphyseal disk – necessary for growth in length. Disappears in adulthood (around age 20) causing the bone growth to stop. The disk helps to detect the age of the bone.

The area adjacent to the epiphyseal disk is called the metaphysis; this area will first develop by endochondral ossification.

In the secondary ossification centers, cartilage remains in two regions: Articular cartilage persists through adult life and doesn’t contribute to bone growth in length and the epiphyseal cartilage (epiphyseal plate or growth plate) is responsible for the growth in length of the bone and disappears in adults, which is why bone growth ceases in adulthood.

Repair of Bone fractures: Fracture is interruption of the integrity of the bone with the interruption of the blood supply of the bone. Fractures in Pathology: Many types, Physiological or pathological fractures. During Fracture: Interruption of the blood supply (Blood will flow!) rapture in almost all blood vessels in the bone, around the bone, inside the bone etc.

• 1. Interruption of blood vessels – in periosteum, havarsian canal, volkmann’s canals- large hematoma, in 24 hours, markophages leukocytes etc
• 2. New vessels grow inside the hematoma, nutrituion, fibroclasts extra cellular matrix, up to collagen fibers, 6-8 weeks, organization of a connective callus.

3. in the callus differentiate first chondroblast, we see the first small islands of hyaline cartilage

Zones of ossification (from epiphysis to diaphysis) –
Elongation process of the bone from the epiphysial disk.

1. Resting zone: consist of hyaline cartilage with typical chondrocytes
2. Proliferation zone: chondrocytes begin to divide rapidly and form columns of stacked cells parallel to the long axis of the bone
3. Hypertrophic cartilage zone: contains swollen degenerative chondrocytes whose cytoplasm has acclimated glycogen (this compresses the matrix between the chondrocytes)
4. Calcified cartilage zone: loss of the chondrocytes by apoptosis is accompanied by calcification of the cartilage matrix by formation of hydroxyapatite crystals
5. Ossification zone: bone tissue first appears. Capillaries and osteoprogenitor cells (originally from the periosteum) invade the cavities left by the chondrocytes. Many of these cavities will be merged and become marrow cavities. Osteoblast settle in a layer over the septa of calcified cartilage matrix and secrete osteoid over these structures- forming woven bone.