Session 5

Question 0

Discuss Stem Cells

Answer 0

Potentially limitless proliferation giving rise to two new cells.

Daughter cells either remain as a stem cell to maintain the stem cell pool OR differentiate to a specialised cell type (e.g. Myoblast or fibroblast).

Stem cells are part of ‘internal repair system’ to replace lost or damaged cells in tissue.

They may have therapeutic utility in degenerative or cardiac disease to replace cells that cannot normally be replaced.

Question 1

What is regeneration?

Answer 1

The replacement of dead or damaged cells by functional, differentiated cells (which are derived from stem cells).

Question 2

Define unipotent, multipotent and totipotent stem cells

Answer 2

Unipotent: can only produce one type of differentiated cell e.g.epithelia

Multipotent: can produce several types of differentiated cell e.g. haematopoietic stem cells in bone marrow can give rise to a limited variety of cells

Totipotent: can produce any type of cell e.g. Embryonic stem cells

Question 3

The propensity to regenerate varies between cell types (different rates of proliferation). What are Labile cells?

Answer 3

E.g. Epithelial or haematopoietic cells (found in areas where cells are constantly being lost and replaced) are constantly dividing.

Normal state is active cell division - continuously cycling between G1-S-G2-M-G1) Usually rapid proliferation

Question 4

The propensity to regenerate varies between cell types (different rates of proliferation). What are Stable cells?

Answer 4

E.g. Hepatocytes, osteoblasts, fibroblasts

Cells are normally in the resting state (G0) but can enter active cell division cycle when requested/stimulated.

Speed of regeneration is variable. E.g. When renal tubules are damaged, repair requires epithelial cells to leave G0 and enter active cell division.

Question 5

The propensity to regenerate varies between cell types (different rates of proliferation). What are Permanent cells?

Answer 5

E.g. Neurones, cardiac myocytes

Unable to divide (permanently in G0)

Unable to regenerate

Question 6

Describe hepatocytes in alcoholic cirrhosis in the liver

Answer 6

Proliferating nodules of new hepatocytes can be seen separated by areas of fibrous tissue,

This can impair flow in the portal system and biliary tree - impair function of the liver.

Question 7

What are the factors controlling regeneration?

Answer 7

Complex and poorly misunderstood.

1. Growth factors
2. Contact between basement membranes and adjacent cells

Question 8

Discuss the role of Growth Factors in regeneration

Answer 8

Promote proliferation in the stem cell population

Extracellular signals are transduced into the cell which promotes expression of genes controlling the cell cycle.

Growth factors are quite heterogeneous:

• proteins e.g. EGF and PDGF;
• hormones e.g. Oestegrogen, Testosterone and Growth Hormone

Location can be autocrine, paracrine and endocrine

signals from many cell types: inflammatory, mesenchymal, extracellular matrix

Signalling pathway might involve the receptor dimerising and becoming kinase-like, phosphorylating other molecules causing a phosphorylation cascade.

Question 9

Discuss how contact between basement membranes and adjacent cells controls regeneration

Answer 9

Anti-proliferative signalling through adhesion molecules inhibits proliferation in intact tissue - ‘contact inhibition’

Loss of contact promotes proliferation

E-Cadherin dimerises across extracellular space.

Accumulation of beta catenin when there is loss of contact, drives proliferation.

These mechanisms are deranged in cancer (proliferation is out of control)

Question 10

What is fibrous repair?

Answer 10

The replacement of functional tissue by scar tissue

Question 11

What are the three outcomes after injury and inflammation?

Answer 11

Resolution

Fibrous repair and scarring

Death

Question 12

What are the key components of fibrous repair?

Answer 12

1. Cell migration
2. Blood vessels - angiogenesis
3. Extracellular matrix production and remodelling

Initiate fibrous repair by combining to form granulation tissue.

Question 13

What are the cell types involved in cell migration?

Answer 13

Inflammatory cells

Chemical mediators

Endothelial cells

Fibroblasts/myofibroblasts

Question 14

Discuss the roles of inflammatory cells and chemical mediators in healing and repair

Answer 14

Inflammatory cells such as neutrophils and macrophages) are involved in phagocytosis of debris.

Chemical mediators such as lymphocytes and macrophages orchestrate processes in inflammation, healing and repair

Question 15

Discuss the role of endothelial cells and fibroblasts/myofibroblasts in healing and repair

Answer 15

Endothelial cells: angiogenesis

Fibroblasts/myofibroblasts produce: extracellular matrix proteins e.g. Collagen and are involved in wound contraction - myoblasts pull the edges of a wound together.

Question 16

Describe Angiogenesis

Answer 16

The development of a blood supply is vital to wound healing as it provides access to the wound for inflammatory cells and fibroblasts and the delivery of oxygen and other nutrients.

Endothelial proliferation is induced by proangiogenic growth factors such as VEGF (vascular endothelial growth factor)

Pre-existing blood vessels sprout new vessels

Mechanisms are exploited by malignant cells (in cancer)

Question 17

What is the mechanism for angiogenesis?

Answer 17

Endothelial proteolysis of basement membrane.

Migration of endothelial cells via chemotaxis - transported to sites of hypoxia

Endothelial proliferation

Endothelial maturation and tubular remodelling

Recruitment of periendothelial cells (cells that reside next to and support endothelial cells)

Over time new blood vessels are produced in the area of healing and repair.

Question 18

List the functions of the Extracellular Matrix

Answer 18

Supports and anchors cell

Separates tissue compartments e.g. basement membrane

Sequesters growth factors (which can be released when required)

Allows communication between cells

Facilitates cell migration

Question 19

Discuss collagen (key component of EM)

Answer 19

Provides extracellular framework

Composed of triple helices of various polypeptide alpha chains (different combinations give rise to different types of collagen)

Remodelled by specific collagenases.

Question 20

Discuss the Synthesis of Collagen (Fibrillar collagens - Types I-III)

Answer 20

Polypeptide alpha chains synthesised in ER

Enzymatic modification steps post transcription include vitamin C dependent hydroxylation

Alpha chains align and cross link to form Procollagen triple helix

Soluble Procollagen is secreted

After secretion Procollagen is cleaved enzymatically to give tropocollagen

Bundles of fibrils form fibres

Slow remodelling by specific collagenases. See MGD notes

Question 21

Discuss Vitamin C Deficiency - Scurvy

Answer 21

Inadequate Vit C dependent hydroxylation of alpha chains leads to defective helix formation

Lacks strength, vulnerable to enzymatic degradation

Particularly affects collagens supporting blood vessels #

Haemorrhage, skeletal changes in infants

Question 22

What is wrong in Ehlers-Danlos Syndrome?

Answer 22

Defective conversation of Procollagen to tropocollagen

Question 23

What is Alport Syndrome?

Answer 23

Inherited defective Type 4 collagen (basement membrane)

Question 24

Apart from collagen, what are the other key components of the Extracellular Matrix?

Answer 24

Matrix glycoproteins (e.g. fibronectin, Iaminin, Tenscin) organise and orientate cells, support cell migration

Proteoglycans (e.g. Heparan sulphate proteoglycan) are involved in matrix organisation, cell support, regulate availability of growth factors

Elastin provides tissue elasticity

Question 25

List the steps involved in the mechanism of Fibrous Repair

Answer 25

1. Inflammatory cell infiltrate
2. Clot replaced by granulation tissue
3. Maturation

Question 26

Discuss the Inflammatory Cell Infiltrate step of Fibrous Repair

Answer 26

Blood vessels get damaged and blood clot forms.

Acute inflammation around the edges - neutrophils infiltrate and digest clot

Chronic inflammation - macrophages and lymphocytes migrate into the clot.

Question 27

Discuss the Clot Replaced by Granulation Tissue step of Fibrous Repair

Answer 27

Angiogenesis: capillaries and lymphatics sprout and infiltrate (ingress of new cells into the site of injury)

Myo/fibroblasts migrate and differentiate, Extracellular matrix such as glycoproteins are produced by myo/fibroblasts.

There is now a vascular network; collagen synthesised; macrophages reduced

Question 28

Describe the Maturation step of Fibrous Repair

Answer 28

Comparatively long lasting

Cell population falls

Collagen increases, matures, contracts and remodels

Myofibroblasts contract - reduces volume of defect (pulls the edges close together)

Vessels differentiate and are reduced

Left with a fibrous scar.

Question 29

Discuss the Control of Fibrous repair

Answer 29

Complex and poorly understood:

Inflammatory cells recruited by chemotaxis

Angiogenesis is due to angiogenic cytokines produced by platelets, extracellular matrix and others in response to hypoxia e,g, VEGF, bFGF

Fibrosis is due to macrophages releasing pro-fibrotic cytokines e.g. IL1, TNF alpha, TNF beta) causing fibroblast proliferation and production of extracellular matrix.

Question 30

Describe and discuss the healing of a clean incised skin wound

Answer 30

Healing by primary intention: Incised wound (very localised tissue damage)

Apposed edges (as close together as possible)

Minimal clot and granulation tissue formation

Epidermis regenerates

Dermis undergoes fibrous repair

Sutures out at 5-10 days: approximately 10% normal strength.

A clean sutured wound - transition from granulation tissue to scar tissue.

Maturation of scar continues up to 2 years, minimal contraction and scarring, good strength

Risk of trapping infection - abscess

Question 31

Describe and discuss the healing of a large skin defect

Answer 31

Healing by Secondary Infection - infarct, ulcer, abscess or any large wound (including most burns).

Quantitative differences:

Unapposed wound edges

Large clot dries to form a scab (ESHAR)

Epidermis regenerates from the base up

Repair process produces much more GRANULATION TISSUE

Question 32

Compare healing with secondary intention with healing with primary intention

Answer 32

Produces much more contraction to reduce volume of defect

Produces a larger scar; not necessarily weaker

Takes longer

Question 33

Describe the healing of bone fractures

Answer 33

Haematoma forms from ruptured vessels within marrow cavity and periosteum.

Organising haematoma provides a framework for ingress of macrophages, endothelial cells, fibroblasts and osteoblasts.

Endothelial cells start to produce new blood vessels.

Necrotic tissue removed

Capillaries develop

Angiogenesis occurs

Callus formation - initially soft

Bone is laid down in an irregular woven pattern, sometimes with Islands of cartilage

External callus provides sprint-like support

Woven bone gradually replaced by more organised lamellar bone.

Lamellar bone gradually remodelled to the direction of mechanical stress.

Question 34

What are the Local Factors influencing Wound Healing?

Answer 34

Type, size, location of wound

Apposition, lack of movement - skin wounds, bone fractures, severed nerves

Blood supply: arterial, venous

Infection: suppurations (pus areas), gangrene, systemic

Foreign material: dirt, glass, sutures, necrotic tissue

Radiation damage: cancer patients undergoing radiotherapy which could impact their endothelial regeneration and cause inactivation of fibroblasts.

Question 35

What are the General Factors influencing Wound Healing?

Answer 35

Age (elderly may have impaired ability)

Drugs (steroids can impair) and hormones

General dietary deficiencies e.g. Protein Specific dietary deficiencies e.g. Vitamin C (alpha chain hydroxylation) and essential amino acids

General state of health - chronic diseases e.g. Diabetes, rheumatoid arthritis

Chronic cardiovascular status

Question 36

What are the complications of repair?

Answer 36

Insufficient fibrosis: wound dehiscence; hernia, ulceration. High risk in obesity (particularly with abdominal wounds), elderly, malnutrition, steroids etc

Excessive fibrosis: cosmetic scarring, keloid (abnormal collagen in skin); cirrhosis; lung fibrosis (affects gas exchange)

Excessive contraction: obstruction of tubes and channels (strictures)

Excessive contraction: limitation of joint movement (contractures).

Question 37

Describe the healing process in liver

Answer 37

Hepatocytes have excellent regenerative capacity.

In some circumstances, hepatic regeneration comes from liver progenitor cells rather than hepatocytes; bone marrow-derived stem cells are a third option.

The hepatic architecture however cannot be satisfactorily reconstructed if severely damaged.

Consequently, conditions that result only in hepatocytes loss may be followed by complete restitution, whereas damage destroying both the hepatocytes and architecture may not.

In the latter situation the imbalance between hepatocytes regeneration and failure to reconstruct the architecture may proceed to cirrhosis.

However following partial surgical resection of the liver, there can be substantial regeneration of functioning liver

Loss of only scattered liver cells or even small groups can be stored without architectural disturbance.

However if there is confluent loss of liver cells and architectural damage the liver heals by scarring (fibrosis - chronic) and nodular regenerations of liver cells, resulting in cirrhosis

Question 38

Describe the healing process in kidney

Answer 38

Similar to the liver with respect to tissue injury, in that it has an epithelium that can be regenerated and an architecture that cannot.

Loss of tubular epithelium following an ischaemic episode or exposure to toxins may result in renal failures but in general there is sufficient surviving epithelium to repopulate the tubules and enable normal renal function to return.

Inflammatory or other damaging resulting in destruction of the glomerulus is likely to be permanent or result in glomerular scarring, with loss of filtration capacity.

Similarly, interstitial inflammation is likely to proceed to fibrosis and thus, impaired reabsorption from tubules

Question 39

Describe the healing process in muscle

Answer 39

Cardiac muscle fibres and smooth muscle cells are permanent cells (fibrosis - scarring will occur); vascular smooth muscle may be different in that new vessels can be formed so it has some limited regeneration capability.

This means that damaged muscle is replaced by scar tissue.

However if contractile proteins only are lost, then it is possible to synthesise new ones within the old endomysium.

Voluntary skeletal muscle has a limited capacity for regeneration from satellite cells

Question 40

Describe the healing process in neural tissue

Answer 40

There is no effective regeneration of neurones in the CNSbut glial cells however may proliferate in response to injury, a process referred to as gliosis.

Peripheral nerve damage affects axons and their supporting structures such as Schwann cells.

If there is transection of the nerve, axons degenerate proximally for a distance of about one or two nodes; distal of there is Wallerian degeneration followed by proliferation of Schwann cells in anticipation of Axonal regrowth.

If there is good realignment of the cut ends, the axons may regrow down their previous channels (now occupied by proliferated Schwann cells); however full functional recovery is unusual.

When there is poor alignment or amputation of the nerve, the cut ends of the axons still proliferate but in a disordered manner to produce a tangled mass of axons and stroma called an amputation neuroma.

Sometimes these are painful and require removal

Question 41

What is Wallerian degeneration?

Answer 41

Degeneration of nerve fibres that occurs following injury or disease and that progresses from the place of injury along the axon away from the cell body while the part of the axon between the place of injury and the cell body remains intact

Question 42

Describe the healing process in the heart

Answer 42

Myocardial cells are permanent cells so cannot divide in a regenerative response to tissue injury.

In myocardial infarction, a segment of muscle dies and if the pstient survives, it is replaced by scar tissue (fibrosis).

As the remainder of the myocardium must work harder for a given cardiac output, it undergoes compensatory hypertrophy (without cell division).

Occasionally there may be right ventricular hypertrophy as a result of left ventricular failure and consequent pulmonary hypertension

Question 43

Describe healing in cartilage

Answer 43

Cartilage is avascular.

Articular cartilage does not usually regenerate after injury or disease leading to loss of tissue and formation of a defect.

Small damage does not repair itself, as cartilage is also aneural (lack of nerve supply) so shallow damage often does not trigger pain.

When the damage increases and the chondral defect reaches the subchondral bone, the blood supply in the bone starts a healing process in the defect.

Scar tissue made up of a type of cartilage called Fibrocartilage is then formed. although Fibrocartilage is able to fill in articular cartilage defects, its structure is slightly different to hyaline cartilage.

It is much denser and doesn’t withstand the demands of everyday activites as much as hyaline cartilages. It is therefore at a higher risk of breaking down.

Defects may also progress to osteoarthritis over time if left untreated.

An articular cartilage defect that initially may be small still has the potential to have a physical and chemical domino effect in the surrounding normal articular cartilage

Question 44

Explain about pressure sores

Answer 44

• Also known as decubitus ulcers or bedsores.
• This occurs when tissues are compressed by either exogenous agents (atrophy of skin and soft tissues overlying the sacrum in bedriiden patients) or endogenous factors (atrophy of a blood vessel wall compressed by a tumour).
• In both of these circumstances, a major facotr is actually local tissue hypoxia - pressure applied to soft tissue resulting in completely or partially obstructed tissue, usually over a bony promience. Shear is also a cause as it can pull on blood vessels feeding the skin.
• Most commonly develop in people who are not moving about or confined to wheelchairs.

Question 45

How would you prevent or treat pressure sores?

Answer 45

Although often prevented and treatable if detected early, pressure ulcers can be very difficult to prevent in critically ill patients, frail elders, wheelchair users (especially where spinal injury is involved) and terminally ill patients.

Primary prevention is to redistribute pressure by turning the patient regularly.

In addition to turning and repositioning the patient in bed or wheelchair, eating a balnced diet with adequate protein and keeping the skin free from exposure to urine and stool is very important.

Question 46

Throughout the 4 phases of wound healing, collagen performs what following functions?

Answer 46

Guiding: collagen fibres serve to guide fibroblasts. Fibroblasts migrate along a connective tissue matrix.

Chemotactic properties: the large surface area available on collagen fibres can attract fibrogenic cells which help in healing

Nucleation: collagen in the presence of certain neutral salt molecules can act as a nucleation agent, causing formation of fibrillar structure. A collagen wound dressing might serve as a guide for orienting new collagen deposition and capillary growth.

Haemostatic properties: blood platelets interact with the collagen to make a haemostatic plug.