Flashcards in Skin Wound healing Deck (41):
where are the stem cells of the skin found?
the basal layers
what part of the skin its is involved in wound healing and regeneration?
- the basal and supra basal layers
what is the wound healing process?
- red blood cells coagulate and release fibrin and thrombin which forma clot
- they also release factors which stimulate the inflammatory response so that neutrophils will fight infection and release ore pro inflammatory growth factors.
- they also stimulate the cells to become proliferative
- re-epithelialisation occurs and then ECm deposition and then re-repithelialisation
- then wound healing occurs
what are gap junctions and which is the most implicated in wound healing?
connexins 43- come together in a raft and content two membranes. They can be inserted or removed from the membranes very quickly.
what are the dominant connexins and where are they found?
- connexin 43 in the basal layer
- connexin 26 and 30 in the lower and upper spines layers
how do connexin expression change during wound healing?
- connexin 43: normally expressed in moderate levels n the basal layer in air follicles and blood vessels. When you wound the skin- the would edge the keratinocytes and fibroblast turn off connection 43 rapidly as they become migratory. In the blood vessels it turns on as they come leaky.
- connexin 26 is normally at low levels and turns on highly in the cell that become migratory and stays on until the epithelialisation is finished.
how did they try to investigate the role of connexin 43 at the wound edge?
- they wanted to accelerate the turning down of CD43 at the wound edge and use an oligonucleotide against the cx43 as an antisense and only effect the wound site. They deliver it as a gel. It is called nexagon. They found that the treated wounds healed much quicker than the controls- healer quicker. this works for excisions and incisions
how does hexagon affect neutrophil invasion?
- it reduces the number of neutrophils that are present
how does nexagon affect blood vessels?
when they measure leakiness they saw that the leakiness is reduced- resulting in a reduction in swelling and neutoprhils
how are cheekiness affected by nexagon?
chemokine ccl2 is reduced at day 2 post injury
- TNF-alpha is reduced d7 after injury
how is TGF-beta expression changed as a result of nexagon?
- significantly elevated eels of TGF-beta 1 mRNA on day 2 following nexagon treatment . This results in increased keratinocyte and fibroblast proliferation n the nascent epidermis and nascent granulation
how does cell migration change in response to nexagon?
- cx43 levels inversely correlates with the rate of fibroblasts migration - can't form filopodia
what does cx43 do inside the cell?
- can control the expression of tight junctions and adherens
- can bind to the cytoskeleton
- can bind to alpha beta tubular and actin- anchor the ctokseletaon to the membrane
generally what is the role of cx43 inhibition?
reduce inflammatory response
how did they investigate what genes cx43 controls the expression of ?
- they produced fibroblast cells in culture which were knock down for cx43- and they find that ZO-1 and N-cad expression is also reduced
how did they investiage which genes are involved in migration? what did this result in?
they knocked down cx43 and sa that cell migration was increased and the same was seen for N-cadherin knock down. But not ZO-1 knockdown.
- they found that cell adhesion and connexins have affects on the ability of these cels to migrate
what happens to cell stickness when you knockdown cx-43
cells normally become less tricky astray migrate- which reduce connexion 43, the stickiness is reduced. the same for n-cad knockdown
what happens to GTPases expression when there is knockdown of cx43 and n-cadherin?
- actiate RhoA and Rac1
how does connexion affect the F-actin and tubulin expression when measured by cx43 KD?
- lose the tight band of f-catin at the front and see cells putting long processes out.
how can you test the effect of cx-43 DN in a sheet of cells- when measuring the effects on a single cell within a sheet?
- you can tranfesct individual cells with cx-43 DN and see that it extends much longer processes and lamella podia compared to others
how is collagen affected in nexagon ?
There is significantly more collagen I mRNA in treated wounds a 2d and 7d
There is significantly more “good” collagen I in treated wounds at 7d which correlates with enhanced granulation tissue formation
how did nexagon treatment affect scar healing
yofiber blasts contract and pull the wound down. they find at day 10 the myofibroblasts appear earlier and lost sooner with enhanced granulation tissue maturation •Treated wounds have significantly smaller granulation tissue areas at 7 & 12 days compared to controls.
how does nexagon affect blood vessel formation?
Accelerated invasion of very fine blood vessels throughout the granulation tissue at 7dfollowing treatment - thickening 14d
what are the 7 main points of nexagon treatment?
•Enhanced proliferation and migration of both keratinocytes and fibroblasts
•Enhanced “good” collagen I expression and production
•Reduced pro-inflammatory mediators
•Reduced numbers of neutrophils and macrophages •Faster angiogenesis
•More rapid granulation tissue maturation
•Smaller granulation tissue area
how can connexion treatment be used to treat diabetic wounds?
- can be used totter chronic wounds of diabetic ulcers
what rat is used to study the diabetic wound healing?
- STZ diabetic rat
what is the STZ rat?
has chronic wound healing as it is used as a diabetic model
what is the difference between the dermis and the epidermis?
- the dermis is deeper and the epidermis is ore superficial
what did they find about the expression profile of connexin in the diabetic rat?
- cx43 was reduced in the epidermis but increased in the dermis
how did they investigate there role of cx43 in chronic wound healing in rats?
they took a square out of the back of the rat and looked at the expression of cx43-
- in the epidermal wound edge: he epidermis start to turn down cx43 and thin down to a thin layer in the control. In the diabetic, instead of thinning there is a thick bulb of cells with turn of cx43 and stops them migrating
- so think that this cd43 turning on and not turning off was how this chronic wound was occurring
- then they added the antisense and were able to turn off the abnormal turn on and you see that you can reduce the healing process
how have scaffolds mean used to help wound healing? why does this not work? how did they investigate this
- use collagen scaffolds to electrospin collagen into a matrix which could replicate the dermis and this will encourage the growth of fibroblasts and kertatinocytes into the wounds
- but they found that they didn't grow and when stained for connexion 43 you see a massive turn on rather than a natural turn off. So this actually mimics the chronic wound and represses the ability of cells to migrate and prevents the turning off of cx43.
- they then used antisense and saw it can encourage heeling of the collagen a bit.
- this is due to a foreign body reaction caused by the collagen scaffold- the cells the engineers were seeing were neutrophils not fibroblasts
how did they measure whether it was the chemical or physical properties of the alginates that was causing the foreign body response in the collagen? (*)
- they made the alginate dress, microspheres and in a hydrogel
- the found the gel was improved so it is physical
what does a diabetic ulcer look like superficially?
there is swelling around the edge of the wound and there is a bacterial biofilm which prevent the wound healing
what did a biopsy of the choleric wound ulcer show?
the epidemis becomes very thick in the wound and turns on cx43 and a loss in collagen and elastic de to elastase and MMPs from the neutrophils. there are many neutrophils
what does staining of cx43 in the chronic diabetic ulcer show?
- that there is an increase of 25 fold cx43 in the epidermis
- 100 fold increases in cx43 in the dermal fibroblasts (hugely unable to crawl out and heal the wound)
- also in venues leg ulcer
- basically every chronic wound had a massive regulation of cx43.
how have compassionate use of connexion 43 been used so far?
- 13 month after leg removal and no wound heeling - they added nexagon nd there was compete heeling- broke the inflammatory cycle
- damaged cornea- burnt epidermis off. no blood flow to stem cells to keep the eye transparent- found epidermis grown and 6 weeks later his eye was recovered.
what are the key reasons that cx43 causes chronic ulcers?
- prevent migration and proliferation and stimulates blood vessel leakiness.
-•Faster down regulation of Cx43 in keratinocytes and fibroblasts gives enhanced proliferation and migration of both keratinocytes and fibroblasts
•Reduced blood vessel leakiness, numbers of neutrophils, macrophages, pro-inflammatory mediators
hat two types of wound did nexagon show to improve nt he mouse?
incision and excision wound
how does cx43 bind to the actin?
it binds to F-actin via a ZO-1 linker
how was nexagon shown to increase wound healing in the STZ diabetic rat?
- it prevent the faulty upreglation of cx43 in the epidermis and improved wound healing- this is normally upregulated rather than down regulated in the wound edge od TSZ diabetic rats - the kertinoyctes for a thicker bulb at the wound edge and this wounds take a long time to heal- This effectively inhibited the increase in Cx43 that was seen with control (sense) gel and allowed the keratinocytes to adopt a thinner, tongue-like profile More importantly, it also improved wound-healing rates, which more than doubled, reaching untreated control levels or exceeding them