Skin Wound healing

Question 1

where are the stem cells of the skin found?

Answer 1

the basal layers

Question 2

what part of the skin its is involved in wound healing and regeneration?

Answer 2

• the basal and supra basal layers

Question 3

what is the wound healing process?

Answer 3

• 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

Question 4

what are gap junctions and which is the most implicated in wound healing?

Answer 4

connexins 43- come together in a raft and content two membranes. They can be inserted or removed from the membranes very quickly.

Question 5

what are the dominant connexins and where are they found?

Answer 5

• connexin 43 in the basal layer

- connexin 26 and 30 in the lower and upper spines layers

Question 6

how do connexin expression change during wound healing?

Answer 6

• 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.

Question 7

how did they try to investigate the role of connexin 43 at the wound edge?

Answer 7

• 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

Question 8

how does hexagon affect neutrophil invasion?

Answer 8

• it reduces the number of neutrophils that are present

Question 9

how does nexagon affect blood vessels?

Answer 9

when they measure leakiness they saw that the leakiness is reduced- resulting in a reduction in swelling and neutoprhils

Question 10

how are cheekiness affected by nexagon?

Answer 10

chemokine ccl2 is reduced at day 2 post injury

- TNF-alpha is reduced d7 after injury

Question 11

how is TGF-beta expression changed as a result of nexagon?

Answer 11

• 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

Question 12

how does cell migration change in response to nexagon?

Answer 12

• cx43 levels inversely correlates with the rate of fibroblasts migration - can’t form filopodia

Question 13

what does cx43 do inside the cell?

Answer 13

• 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

Question 14

generally what is the role of cx43 inhibition?

Answer 14

reduce inflammatory response

Question 15

how did they investigate what genes cx43 controls the expression of ?

Answer 15

• 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

Question 16

how did they investiage which genes are involved in migration? what did this result in?

Answer 16

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

Question 17

what happens to cell stickness when you knockdown cx-43

Answer 17

cells normally become less tricky astray migrate- which reduce connexion 43, the stickiness is reduced. the same for n-cad knockdown

Question 18

what happens to GTPases expression when there is knockdown of cx43 and n-cadherin?

Answer 18

• actiate RhoA and Rac1

Question 19

how does connexion affect the F-actin and tubulin expression when measured by cx43 KD?

Answer 19

• lose the tight band of f-catin at the front and see cells putting long processes out.

Question 20

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?

Answer 20

• you can tranfesct individual cells with cx-43 DN and see that it extends much longer processes and lamella podia compared to others

Question 21

how is collagen affected in nexagon ?

Answer 21

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

Question 22

how did nexagon treatment affect scar healing

Answer 22

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.

Question 23

how does nexagon affect blood vessel formation?

Answer 23

Accelerated invasion of very fine blood vessels throughout the granulation tissue at 7dfollowing treatment - thickening 14d

Question 24

what are the 7 main points of nexagon treatment?

Answer 24

• 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

Question 25

how can connexion treatment be used to treat diabetic wounds?

Answer 25

• can be used totter chronic wounds of diabetic ulcers

Question 26

what rat is used to study the diabetic wound healing?

Answer 26

• STZ diabetic rat

Question 27

what is the STZ rat?

Answer 27

has chronic wound healing as it is used as a diabetic model

Question 28

what is the difference between the dermis and the epidermis?

Answer 28

• the dermis is deeper and the epidermis is ore superficial

Question 29

what did they find about the expression profile of connexin in the diabetic rat?

Answer 29

• cx43 was reduced in the epidermis but increased in the dermis

Question 30

how did they investigate there role of cx43 in chronic wound healing in rats?

Answer 30

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

Question 31

how have scaffolds mean used to help wound healing? why does this not work? how did they investigate this

Answer 31

• 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

Question 32

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? (*)

Answer 32

• they made the alginate dress, microspheres and in a hydrogel
• the found the gel was improved so it is physical

Question 33

what does a diabetic ulcer look like superficially?

Answer 33

there is swelling around the edge of the wound and there is a bacterial biofilm which prevent the wound healing

Question 34

what did a biopsy of the choleric wound ulcer show?

Answer 34

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

Question 35

what does staining of cx43 in the chronic diabetic ulcer show?

Answer 35

• 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.

Question 36

how have compassionate use of connexion 43 been used so far?

Answer 36

• 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.

Question 37

what are the key reasons that cx43 causes chronic ulcers?

Answer 37

• 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

Question 38

hat two types of wound did nexagon show to improve nt he mouse?

Answer 38

incision and excision wound

Question 39

how does cx43 bind to the actin?

Answer 39

it binds to F-actin via a ZO-1 linker

Question 40

how was nexagon shown to increase wound healing in the STZ diabetic rat?

Answer 40

• 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

Question 41

how have stem cells been used for wound healing?

Answer 41

you can add MSCs to the wound sie to help the reepilethilisation - has been shown by labelled injection that they travel to the wound site.