Wound Healing Flashcards
Name 4 phases of wound healing (human articles + Dong, 2023, Lux 21)
1.Hemostasis/coagulation
2. inflammatory
3. Repair/proliferation/granulation and contraction
4. Remodeling and scar formation
How damaged keratinocytes end endothelium respond to an injury ?
-release vasoactive compounds- catecholamines, histamine, serotonin and PG
-create transient constriction= decrease blood flow from wound
+ and then vasodilatation=fluids and cells could pass from vessel and lymphatics to fill and clean the site of injury
Intrinsic blood clot results from?
1.surface activation of Hageman factor XII
2. Tissue procoagulant released from damaged cells
3. Coagulation factors expressed on activated platelets and endothelial cells
What does the fibrin clot contains?
-cross-linked fibronectin, activated factor XIII, fibrin
What is the function of fibronectin?
- allows binding of fibroblasts to ECM
-enhances fibroblast activity
When is the clot formation terminated ?
When the stimuli for the formation are removed / diluted from the system
What does the clot formation provides in wound healing process?
-scaffold for movement and organization of cells at the injury site
=fibrin, fibronectin, vitronectin =provisional Matrix (1. ECM that fills the wound )
-provides a pathway for the influx of monocytes, fibroblasts , and newly forming vessels
+ hemostasis
+barrier to micro-organisms
How is classical and alternative complement pathway activated?
By the initiation of coagulation cascade and activation of Hageman factor that create fragments of bradykinin (vasoactive agent)
What will activation of complement cascade result in ?
-release of C3a and C5a
-they will increase the blood vessel permeability
+ will attract neutrophils and monocytes at cite of injury
+they will release other vasoactive amines - histamine, LTC4, LTD4 (MC) and active oxygen products (from neutrophils and macrophages)
Function of platelets in wound healing
-platelets degranulate and release growth factor and cytokines
-They initiate wound healing process by leukocyte recruitment
-signal cellular movement for re-epithelialization, wound contraction and the angiogenic response
-cause vasoconstriction = by releasing serotonin, and forming thromboxane A2 (TXA2)
What substances does the platelet release?
-serotonin, thromboxane A2, adhesive proteins like fibrinogen, fibronectin, von Willebrand-factor VIII
What are the functions of thromboxane A2 (released from dense granules)?
-vasoconstriction
-amplifies platelet activation
+ additional platelet recruitment
What does the alpha granules of platelets release ?
-adhesive proteins like fibrinogen, von Willebrand’s factor, factor V
-it stimulates more platelet aggregation= platelet plug
Platelet activation, adhesion and aggregation are triggered by
Exposure at the wound site to thrombin and fibrillar collagen
Who limits the size of the clot to the size of defect?
Intact adjacent endothelial cells who provide factors: prostacyclin (PC) and anti-thrombin III
What does the prostacyclin do?
Inhibits platelet aggregation
How is clot lysis initiated?
By the release of plasminogen activator and convert plasminogen to plasmin
What are the chemoattractants for neutrophils in wound healing?
IL-8, Gro, kallikrein, fibrinogen degradation products (FDP), fibrinopeptides (from fibrin degradation) , cytokines (from activated platelets) and FORMYL METHIONYL PEPTIDES (cleaved from bacterial proteins)
Another name for apoptotic neutrophils
Effete
Which cells play a pivotal role in the transition between inflammation and repair
Monocytes/ macrophages
Name hallmark of the inflammatory phase (clinically)
Exudate, which can be septic or non septic
Acute- contains growth factors, leukocytes, chemotactic factors
Chronic- higher levels of damaging proteases
Purulent debris is composed of what ?
-wound fluid
-products by neutrophils-proteases, reactive and toxic oxygen species (degrade injured cells, denatured ECM, bacterial products)
-reflection of inflammatory process
Name 2 types of macrophages and their function?
M1-traditional, kill bacteria and scavenge debris
M2-suppress the immune system and aid in tissue repair
Name role of the macrophages during wound repair
- Phagocytosis and antimicrobial function
- Wound debridement
- Cell recruitment and activation
- Angiogenesis
- Matrix synthesis regulation
Phagocytosis and antimicrobial function is archived by macrophages by which substances?
-oxygen radicals
-nitric oxide
How macrophages contribute to the wound debridement?
- by phagocytosis
-enzymes: MMPs, proteases, elastases
Name factors that help macrophages for cell recruitment and activation
GF: PDGF, TGF-beta, EGF, IGF
-cytokines: TNF-alpha, IL-1 , IL-6
-fibronectin
Name factors released from macrophages important for angiogenesis
-GF: bFGF, VEGF
-TNF-alpha
Name factors important for matrix synthesis regulation released by macrophages
-GF:TGF-beta, EGF, PDGF
-TNF-alpha, IL-1, IFN-gamma
-enzyme:MMPs
-prostaglandins: PGE2
What role does the mast cells have in wound healing ?
-they become activated by C3a,C5a
-release mediators, especially TGB-beta 1(essential for triggering the inflammatory reaction , and important in all phases of wound healing)
-fibrin clot is formed-MC exposed to FIBRONECTIN and TYPE III COLLAGEN
-produce chymase and tryptase-break down the ECM
-it will allow the influx of new cells and collagen I
-NGF released will decrease the nociceptive threshold
+in the transition from proliferating to remodeling phase-MC interact with FIBROBLAST - become MYOFIBROBLAST (wound contraction)
What are the predominant cell types in repair phase?
M2 macrophages, fibroblasts, endothelial cells and keratinocytes
Function of the fibroblasts?
-protein syntesis - collagen, elastin, proteoglycans
-secretion of LYSYL OXIDASE to cross link collagen
-some are converted to myofibroblasts- wound contraction
-produce proteases (MMPs)- remove damaged matrix proteins
In tissue proliferation phase, what are the factors needed?
-tissue hypoxia
-presence of adhesion proteins
-presence of ECM components
-action of previously described inflammatory components
=this will allow the proliferation of resident FIBROBLAST, ENDOTHELIAL CELLS and KERATINOCYTES
How does low oxygen affect wound repair (blood vessels severed in a injury)?
-it has a STIMULATORY ROLE in early repair
-activates FIBROBLAST & ENDOTHELIAL CELLS
-stimulate MACROPHAGES to release angiogenic factors
+macrophages, fibroblast enhance TGF-beta1 release
+fibroblast peptide synthesis is ENHANCED
What is fibroplasia
=formation of granulation tissue and subsequent re-collagenation of dermal matrix
Formation of what allows movement of the fibroblasts into wound tissue
Formation of actino-myosin cytoskeleton
What is the hallmark of repair phase
-decrease in inflammatory cells
-appearance of granulation tissue, re-epithelialisation, wound contraction
-major cells: fibroblasts, endothelial cells, KC
Name processes that reinforce the injured dermal tissues
-fibroplasia (reinforcement)
-angiogenesis (from endothelial cells)
-re-epithelialisation (creation of permeability barrier)
-wound contraction
In what time frame does the provisional ECM start to turn into granulation tissue ?
72 h
-typical time for completion of haemostasis and inflammatory phase
+wound contamination-prolonged neutrophil presence and delay in wound healing
Name members of the matrix metalloproteinases and who secrets them?
-fibroblasts
1.collagenases
2. gelatinases
3. stromelysins
Which collagen is firstly produced to form granulation tissue and when is the maximal secretion ?
Collagen III
-max secretion 5-7 days
What replaces the collagen III wound healing?
-collagen I
+ collagen VI- regulates dermal matrix composition and assembly
Who initiates angiogenesis (“angiogenic sprouting”)?
-endothelial cells near the damaged tissue
-macrophages
-platelets
-keratinocytes
-fibroblasts
-low oxygen
-lactic acid
-growth factors- VEGF
+inhibitors of proliferation and migration- angiostatin, endostatin, antithrombin III
Does the granulation tissue has nerve endings ?
NO
What happens to blood vessels in granulation phase ?
-in the beginning bright red appearance- they are intensively forming
-at the end-will regress with remodelling and apoptosis= have pale, less fleshy appearance
In what time frame is granulation tissue formed?
3-5 days in a healthy wound
How do the keratinocytes move in process of re-epithelialization
-they change by forming LAMELLIPODIA (pseudopod-like projection)
and they leap frog over each other through the centre of the wound
-loss cell to cell & cell to matrix contact, formation of actin filaments within cytoplasm
-until they meet another KC or CONTACT INHIBITION
-in paper by Lux, 21 lamellOpodia ?
What is “epithelial tongue” ?
-form how KC advance over the wound
-by lamellopodia that allow KC to crawl of the fibronectin-rich matrix
-KC reach the centre of the wound -migration stops (by contact inhibition)
Name enzyme that degrades the KC basement membrane and tight bonds with collagen I?
MMP-1 (collagenase)
When is the basement membrane usually formed and with what collagen?
-usually 7-9 days
-with a 3D lattice-work of collagen IV
Which GF stimulates transition of dermal fibroblasts into myofibroblasts
TGF-beta
When are usually myofibroblast formed ?
-4 days after wounding
In what direction wound contraction occurs?
In the direction of the skin tension lines
How does the wound contraction happens?
-myofibroblast extends - allow intracellular actin to interact with fibronectin and collagen in wound matrix via INTEGRINS
-myofibroblast retracts- pulling the collagen towards the cell and causing contraction
What are the mediators for myofibroblast extension and retraction?
-5-hydroxytryptamine
-angiotensin
-vasopresin
-bradykinins
-epinephrine/norepinephrine
When does the the wound contraction peaks
-after 2 week following wounding
-important in full thickness wound healing!
What is angiogenesis and how does it occur?
=process of neovascularisation performed by endothelial cells that are near damaged tissue in a wound
-by destabilisation of mature blood vessels by MMP and heparinase
What inhibits wound contraction?
- excessive tension on the wound edges
2.mechanical obstruction - eg necrotic tissue
What is the hallmark of phase of remodelling and scar formation?
-apoptosis of endothelial cells and myobiroblasts -collagen remodeling
When does the tissue remodelling occur in wound healing?
-throughout all phases
-fibrin clot remodels into acellular scar
Final scar will have how much of its normal tissue strength?
-only 70-80%
In partial thickness wounds how does the epithelialisation occur ?
-migration of KCs from skin appendages- HF, nails, sebaceous glands
In full-thickness wound how does the re-epithelialisation occur?
-it cannot start unless the BED OF GRANULATION TISSUE is present and filling the wound (second intention healing)
When KCs stars migrate and when the appearance of epithelialisation occur (time frame)
- they begin as early as 24 h
-epithelialisation occurs -5 days following injury
What does the TIME acronym stands for ?
Tissue
Inflammation/infection
Moisture
Edge of wound/epithelial advancement
Prolonged inflammatory phase often results in increased what products
MMPs, elastases, leukocytes
-normal 3 days
What does excessive moisture and lack of moisture does?
Excessive= tissue maceration
Lacks= desiccation
Acute inflammatory phase and repair phase- how much moisture do they have
Inflammatory phase- abundance , and repair phase needs moisture protection
Name methods for epithelial advancement
-negative pressure wound therapy
-platelet- rich plasma
-laser therapy
-ultrasonic therapy
-hyperbaric oxygen therapy
-autologous skin harvesting
How NSAIDs affect wound healing?
theoretically decrease wound strength of healing soft tissues and interferes with endothelial cell permeability & signaling (PG deficiency)
but needed in pain management
-supress bone healing in rodents, rabbits and dogs
How does nutrition affect wound healing?
-Glucose is main energy- leukocytes and fibroblasts.
-Insufficient protein (<2g/dL serum) is linked to delayed wound healing, dehiscence, and
increased wound infection rates.
-Magnesium is needed for protein formation, collagen synthesis and tissue growth.
-Vitamin A helps with inflammatory response, epidermal growth factor receptors and collagen synthesis
-vitamin C
-copper and zinc
Why horses have predisposition for granulation tissue “proud flesh”?
-delayed wound healing, particularly in distal extremities
-myofibroblasts are organised haphazardly /unorganised
-heal more by repithelialization(than ponies)
-weak inflammatory response (deficiency in leukocyte production of inflammatory mediators and chemoattractants), and is prolonged
-extended presence of fibroblasts and TGF-beta
-exacerbated angiogenesis
-dysregulation of ECM degradation due to lower MMP-1 concentrations
-greater content of disorganised collagen
Where does granulation tissue begins in cats and where in dogs?
Cats-at periphery and progress to the centre with paler colour
Dogs-at bottom and centre with deep red colour
Why do we have different healing in cats and dogs?
-different pattern of granulation tissue formation
-different cutaneous angiosomes
-dogs produce more early inflammatory response
What is cutaneous angiosome?
=defined tissue area with specified vascular supply
How is vascular supply different in cats and dogs ?
-dog- vasculature supplying the cutaneous tissues is composed of subnormal plexus supplied by direct cutaneous vessels
-when compared to cat, dogs have GREATER DENSITY OF TERTIARY and HIGHER order VESSELS
What are hallmark of cats healing
-decreased cutaneous perfusion
- decreased granulation tissue production and epithelialisation
- reduced tissue concentration
- weaker healed tissue strength
What does “psuedohealing” in cats refer to?
delayed collagen production (incision looks healed, but normal physiological stress can lead to dehiscence )
-recommended that sutures stay longer in cats than in dogs
Is removal of SC tissue recommended during procedures in cats and dogs?
-NO in both
-negatively affect perfusion, granulation tissue production, contraction and epithelialisation
Which species in prone of indolent pocket wounds, b) what are they, c) recommended treatment ?
a) Cats
b)wound associated pockets lined by granulation tissue
-fail to contract or epithelialise (even after surgical closure)
c)introduction of a new vascular supply - omental pedicle graft or other vascularised graft
Granulation tissue forms in dogs and cats (days)
Dogs-7.5 days
Cats-19 days
What are the characteristics of chronic wounds ?
-high level of bacteria
-high levels of inflammatory cytokines, proteases and ROS
-degraded and nonfunctional ECM
-aged cells with low mitogenic activity
How does Manuka honey work?
-enhances wound debridement, reduces edema, promotes phase III formation
-accelerate collagen maturation
-maintain optimal pH for for fibroblast activity
-nutrients stimulates wound healing
-antimicrobial effect- due to Manuka factor and osmotic effect
When is the best to use Manuka honey?
-inflammatory and repair phase
-needs frequent replacing due to exudate
How sugar work on wound healing?
-hypertonic effect (like Manuka, but not the same anti-inflammatory)
How does fish skin dressing/tilapia/cod work
-tilapia induces EGF, FGF , peptides have antimicrobial activity, excellent adherence
How does platelet derived product work in wound healing ?
-enhance fibroblast proliferation, migration
-accelerate epithelial differentiation
How do mesenchymal stem cells work in wound healing?
-have ability to proliferate and differentiate into different cell lines
-anti-inflammatory
-pain-modulating properties
-immunomodulation
How does negative pressure wound therapy work ?
(1) withdraw wound exudate and necrotic debris aiding in debridement
(2) improve skin’s elasticity by applying mechanical strain to bring edges together
(3) increase perfusion to the wound as far as 3cm away from the foam
(4) reduce interstitial edema
(5) decrease inflammatory response by removing detrimental cytokines (i.e. MMP
6.increases granulation tissue
What are contraindications for NPWT?
exposed vascular or organ surfaces for risk of exsanguination, neoplasia, necrotic tissue or eschar
Why are chronic non-healing wounds hypoxic ?
due to poor perfusion
How does the hyperbaric oxygen therapy work
-Oxygen is essential for cell growth, wound healing, and resistant infections
-Increased oxygen delivery to tissues results in angiogenesis and improved immune function
=increases the amount of dissolved oxygen in plasma, resulting in increased tissue oxygen tension.
Name 4 zones of granulation tissue beds:
- Zone of mature connective tissue
- Zone of capillary proliferation
3.Zone of capillary sprouts and arches
4.Zone of necrotic debris- the “scab” or crust
What is granulation tissue ?
=new vascular tissue in granular form on an ulcer or the healing surface of a wound
How are lamellipodia formed?
-KC retract monofilaments, dissolve intracellular desmosomes and hemidesmosomes, increase their gap junctions, and form peripheral actin filaments
-these actions provide detachment, enhanced cellular communication and motor apparatus called Lamellipodia
In which phases are inflammatory phase can be divided
-vascular responses leading to haemostasis
-cellular responses resulting in influx of leukocytes
How do mice and rat predominately heal?
by contraction of the wound bed
How pigs predominantly heal?
By re-epithelialisation (skin is tightly adherent to underlying structures)
What are the reasons for exuberant granulation tissue formation in horses?
-foreign body
-bone sequestrum
-neoplasia
-environmental contamination
-deficiency of local soft tissues
-location in a high motion area
What is the difference in healing wounds in horses and ponies
-wound contraction is more significant in healing of ponies
-horses heal predominantly by re-epithelialisation
What is the clinical definition of infection?
-signs associated with pain, erythema, oedema, purulent exudate
-density of bacteria > 1 million CFU per mm3 tissue
How bacteria affect wound healing?
-release: virulence, adhesion and antiphatocytic factors
-causing neutrophils lysis -release of cytotoxic substances into the wound bed, contributes to tissue necrosis and impairs normal function
-prolong inflammatory response due to bacterial presence-poor wound healing and high levels of pro inflammatory cytokines
-imbalance between MMPs and tissue inhibitors of MMP
Name risk factors for surgical site infections?
- hypothermia
- hypotension
- poor tissue oxygenation
When we suspect biofilm formation in the wound?
-delay in wound healing (no more than 3 weeks)
-granulation tissue appears friable
-foul odour with minimal signs of local infection
Which drugs are implicated in negative effects on wound healing?
-corticosteroids
-immunosuppressive drugs
-chemotherapuetiucs
-NSAID
How chemotherapy affects wound healing ? What is recommended?
-affects rapidly dividing cells like fibroblasts
-AVOID during wound healing
How does methotrexate affect wound healing?
-prevents adherence of neutrophils to endothelial cells
-decreases neutrophil chemotaxis
-causes dehiscence + infection
How does leflunamide affect wound healing?
-stops lymphocyte proliferation
-inhibits adherence of leukocytes to endothelial cells
How does azathioprine affect wound healing?
-has minimal effect
How do corticosteroids affect wound healing ?
-decreased expression of cytokines - reduced mitogen stimuli and chemotaxis
-reduced neutrophil adhesion and migration
-less macrophage infiltration into a wound
-inhibition of fibroblast migration and deposition of collagen
=wound dehiscence, delayed second intention healing, infection
-dose and duration dependant
-5-10 days has min effect (human)
How diabetes mellitus affects wound healing ?
-hyperG- interferes with ascorbic acid transport of fibroblast and leukocytes
-potentiate renal dysfunction and malnutrition = decreased healing + infection
-VIT C- for synthesis of collagen, cell mitosis and monocyte migration
How uraemia affects wound healing?
-AKI- inhibition of capillary and fibroblast proliferation
-CKI-delayed granulation due to diminished angiogenesis and cellular proliferation, ch inflammation
Name bandages recommended for pawpads?
“clam shell” or splint bandage
What does the radiation therapy do
-induce wounds
-2nd dehiscence after surgery
-lower tissue resistance to infection
-detrimental to rapidly dividing cells-epithelial, endothelial, fibroblasts, myofibroblasts
-late effects: decrease oxygen to wound (due to fibrotic microangiopathy)
