PBL Unit 11

• What are the 4 phases of wound healing?

  Haemostasis (scab formation)

  
  

  Inflammation

  
  

  Proliferation

  
  

  Maturation
• What occurs during the haemostasis phase of wound healing?

  Blood loss is minimisedandepithelial cells begin to crawl over wound

  Initial trauma causes broken blood vessels to constrict and platelets to be recruited --> aggregation at wound site

  Platelets become activated --> clotting cascade -->scab formation- minimises blood loss

  Meanwhile resident macrophages release EGF and TFGa which both induce epithelial cell prolif - begin to crawl over wound (only occurs if small wound - larger wounds are aided by myofibroblasts BUT if too large or deep, a scar will eventually form)

  
• How is the inflammatory phase initiated?

  Via DAMP release
• What occurs in the early phase of inflammation?

  Release of PIC and prescence of DAMPs and/or PAMPs --> acute inflammatory response:

  
  

  - dilated and congested bvs

  - neutrophil infiltration via release of chemokines

  - oedema = big gaps between CT fibres
• What is the role of neutrophils during the early inflammatory response in wound healing?

  Prevent infection and phagocytose any extracellular pathogens present as well as tissue debris
• Along with recruiting neutrophils via chemokine release, how else does the body minmise the risk of infection during wound healing?

  PIC --> inc complement production

  
  

  Activated directly by pathogen or by antibody-bound pathogen to C3b - opsinises pathogens
• What occurs in the late phase of inflammation?

  Infiltration of macrophages, derived from circulating monocytes

  
  

  Converted along with resident macrophages to M1 state (inflammatory)

  
  

  These phagacytose any extracellular pathogens present as well as tissue debris

  
  

  Persit until wound heals (not always in M1 state)
• What occurs in the final stage of the inflammatory phase of wound healing?

  Accumilation of lymphocytes due to release of PIC and chemokines

  
  

  Activated CD4+ cells upregulate healing whereas CD8+ cells downregulate it
• At what point do the n.o lymphocytes peak?


  Day 7
• What occurs as debris begins to clear?

  Conversion of M1 to M2 (anti-inflammatory) macrophages - promote tissue repair
• How are M1 macrophages converted to M2?

  1) Innate and adaptive IR has cleared PAMPs and DAMPs --> less present to be able to bind to TLRs on macrophages and epithelial cells = less pro-inflammatory cytokines released - epithelial cells release TGF-B instead - alters recruited Th cell's phenotype to Treg which secrete IL-10 and TGF-B which inhibits other effector Th cells

  
  

  2) Less pro-inflammatory cytokine release causes neutrophils previously recruited to apoptose (no more survival pro-inflammatory signals)

  
  

  3) Apoptotic neutrophils release "find me, eat me" signals which attract macrophages which phagacytoses them and inhibits further neutrophil recruitment

  
  

  4) The combination of IL-10 released by iTregs and the phagocytosis of neutrophils causes the macrophages to become anti-inflammatory

  
  

  5) These promote tissue repair by secreting growth factors, MMPs and TIMPs, IL-10 and IL-IRA

  
• What permits the progression from inflammation to proliferation of a wound?

  M2 macrophages
• What determines how long the inflammatory phase of wound healing is?

  Infection
• What are the 3 possible outcomes of acute inflammation?

  Tissue repair:-regeneration(when cause is eliminated and surviving tissue able to proliferate)

  -scarringvia fibrosis (when extensive damage has been caused)

  Most cases involve both of these e.g. deep skin laceration

  -chronic inflammation(when cause cannot be eliminated)
• What are the roles of the anti-inflammatory macrophages?

  Phagacytose debris, fibrin and foreign material within ECM of intersitual matrix

  
  

  Release collagenases, MMPs and TIMPs to clear ECM to allow for new tissue to be formed (debride the wound)

  
  

  Release growth factors (TGF-B, PDGF, VEGF, EGF and FGF), collagenases, MMPs and TIMPs, IL-10

  
  

  --> formation of granulation tissue

  
• What are the functions of the growth factors secreted by anti-inflammatory macrophages in the proliferation phase of tissue repair? (TGF-B, PDGF, VEGF, EGF and FGF)

  - FGF, PDGF and TGFB induce fibroblast migration to wound site and causes them to proliferate

  
  

  - TGFB cause the fibroblasts to rapidly produce type 3 "quick" collagen to immediately repair ECM

  
  

  - VEGF and PDGF cause endothelial cell proliferation = angiogenesis --> supplies wound site with O2 and nutrients (currently hypoxic due to sealing of wound by epitehlial cells and platelets in inflammatory phase)

  
  

  = granulation tissue
• What makes up granulation tissue?

  Fibroblasts, collagen, proteoglycans, macrophages (M2) and new capillaries

  
• What do fibroblasts produce during the proliferative phase of tissue repair?

  Release of gfs by M2 macrophages --> production of ECM components:

  
  

  - Collagen - type 3 to begin with - forms foundation to new tissue and gives wound strength

  
  

  - Proteoglycans
• Why is angiogenesis important during wound healing?


  Damage to bvs caused by injury results in wound site being hypoxic - need O2 for repair to occur
• Describe the process of angiogenesis during tissue repair

  M2 macrophages release VEGF and PDGF

  
  

  These bind to receptors on the endothelial cells of surrounding blood vessels

  
  

  Causes them to proliferate

  
  

  MMPs also released by these macrophages degrade the bm and allow for "sprouting" of the tip cells which proliferate in the direction of the these growth factors

  
  

  Pericytes migrate back over the vessels and the bm reforms

  
  

  = new vessel

  
• What occurs after the formation of granulation tissue during the proliferative phase of wound healing?

  Epithelialisation- continued release of TGF-a and EGF results in epithelial cell proliferation and migration from the wound edges over the wound

  
• Does epithelialisation occur in all tissues wounds?

  No - regeneration of the epithelial tissue can only occur in labile and stable tissues, not permanent tissues such as cardiac muscle or nervous tissue
• What is regeration/epithelialisation dependent on?

  Intact ECM (as surviving cells use integrins to bind to ECM and replicate) and whether surviving cells areable to proliferate(depends on tissue type)

  If not, scarring occurs

  In most cases, both occurs e.g. deep skin laceration, scar formation occurs in dermis and epithelium of epidermis regenrates due to replicative nature of epithelium UNLESS damage is too extensive
• What occurs if epithelialisation cannot occur?

  Ascar- collagen deposition

  Also occurs in wound is very large or deep and the epithelial cells cant close together
• What occurs after the proliferative phase of wound healing?

  Maturation phase (remodelling/scarring)
• What occurs during the maturation phase of wound healing?

  Tissue is remodelled - type 3 "quick" collagen is replaced by stronger cross-linkedtype 1 collagen

  How?

  1) Fibroblasts begin to secrete type 1 collagen instead and continue to proliferate

  2) MMPs released by the anti-inflammatory macrophages slowly degrade the type 3 collagen ready to be replaced (balanced by TIMPs)

  Meanwhile, as the scar tissue matures,additional bvs are lostand some fibroblasts acquire smooth muscle features and become myofibroblasts -->wound contraction

  
• What factors affect how well a wound heals?

  Tissue type- is it able to regenerate?

  Wound size- is ECM in tact?if epithelial cells cant grow over wound a scar will form (collagen depositted instead) - flattens as tissue matures

  Location- highly vascularised areas heal much quicker

  Infection
• What results in abnormal wound healing? What does it result in?

  Interruption of normal reparative process --> chronic wounds and ulcers
• What is ulceration caused by?

  Caused by the loss of inflamed, necrotic tissue near the surface of a tissue due to inadequate formation of granulation tissue

  
• What can prolong the inflammatory phase of wound healing? What can this lead to?

  Wound infection --> chronic open wounds
• Why would a wound take longer to heal in patients with diabetes? What can this result in?

  Diabetes affects the inflammatory, proliferative and maturation phase of wound healing

  Patients have dysfunctional neutrophils and macrophages --> inc susceptibility to infection

  Patients have impaired proliferation due to less collagen deposition, defects in collagen maturation and poorer perfusion of the area due to formation of athersclerotic plaques caused by prolonged hyperglycaemia

  Advanced Glycation End Products(AGE) also result in thickeneing of the ECM --> less remodelling capacity

  This can result in diabetic foot ulcers (more likely to injure foot due to peripheral neuropathy, more susciptable to infection and less able to repair tissue --> loss of inflamed, necrotic tissue...)

  
• How does sustained hyperglycemia lead to the formation of atherosclerotic plaques?

  Sustained hyperglycaemia leads to the production of glycosylated proteins and LDL referred to asAdvanced Glycation End Products(AGE) --> thickening of the bm

  Glycosylated proteins also stick to RAGE on endothelial cells - makes them sticky - LDL sticks --> athersclerotic plaque --> narrowing and/or blockage

  Diabetics have increased levels of LDL (bad chlesterol) in the blood due to inc levels of de novo lipogenesis causing inc VLDL production which is then broken into FFAs, LDLs and glycerol

  --> macrovascular disturbances
• Why would a wound take longer to heal in patients with low vitamin C?

  Vit C needed for collagen synthesis low levels can result in unstable poorly linked collagen and slower synthesis = slower proliferation phase of wound healing
• Why would a wound take longer to heal in patients with arteosclerosis?

  = comprised blood flow this causes longer to heal as decreased blood flow to the wound means decreased oxygen for tissue repair per minute.
• How can glucocorticoids increase the time taken for a wound to heal?

  Glucocorticoids are glucocorticoid Tf agonists --> reduced inflammation and inhibition of gfs involved in proliferation

  
  

  Also inhibit collagen synthesis = slower proliferation phase of wound healing
• What can abnormalities in the maturation phase of wound healing lead to?

  Abnormal scarring
• What are hypertrophic scars?

  Scar which forms due tomyofibroblastssecreting too much TGF-B- stimulates fibroblasts to secrete to much collagen before epithelial cells have regrown over

  Raised, itchy, red and inflammed

  
• What are keloids?


  Mass of fibrous tissue formed when scars grow outside of wound boundary due to patients having pathologically high levels of VEGF (causes fibroblasts to secrete collagen as well as stimulates angiogensis)

  
  

  Often have to be surgically removed

  
• What type of scar can chicken pox result in? Why?

  Indented scars - due to degragation of collagen due to production of MMPs>TIMPs

  
• What type of scar can severe burns result in? Why?

  Contracture scars - due to extensive inflammation