Derm 8 - Wound Healing

Remodeling Phase: Scar Tissue

*Amount of scar tissue produced is directly proportional to tension on wound during healing* - As the wound matures, *collagen bundles become progressively thicker with greater and greater tensile strength* - Wound remains *<80% as strong* as adjacent skin *1yr* after injury - *number of fibroblasts decreases* as the wound matures suggesting that *apoptosis of fibroblasts is a normal* part of the repair process

Epithelialization: Changes in Keratinocytes (lecture)

- flatten out and form pseudopods - increase actin - shut down proliferative machinery (are moving not dividing) - increase proteases - secrete cytokines etc

Neuropeptides (KNOW)

- increase blood flow to allow important plasma proteins to leak into the wound - increase expression of adhesion molecules on endothelial cells - stimulate endothelial cell proliferation - increase macrophage chemotaxis and DNA synthesis in keratinocytes and fibroblasts

Question: After wounding, what is the most important first step for survival of the host?

...

Question: Can you name 2 clinical settings in which wound healing may be delayed?

...

Question: In what clinical setting(s) may abnormal peripheral nerves adversely influence wound healing?

...

Question: Using what you learned in prior lectures about keratinocyte maturation and their relationship with the basement membrane zone and each other, what kind of changes must the keratinocyte make in order to migrate across a wound?

...

Question: What are some of the characteristics of the wound that are likely to promote keratinocyte migration?

...

Question: What other factors may delay wound healing in a patient?

...

Question: Which form of wound healing, regenerative or scarring, would be most desirable for a wound created by an incision during surgery?

...

Question: Why are only keratinocytes from the wound edge seen to migrate in full thickness wounds?

...

Question: Why did a scarring form of wound healing develop in mammals?

...

Question: Why would we see a decrease in capillaries during the remodeling phase?

...

Question: Where on the face would you expect secondary intention healing to result in a poor cosmetic outcome for a patient?

....

Proliferation Phase: Endothelial cells

Produce molecules to *limit coagulation, initiate lysis of clots, regulate leukocyte rolling and migration, and stimulate angiogenesis and ECM production* Variety of *autocrine and paracrine GFs* are produced including: - *nerve growth factor (NGF)* - *FGF* *Nitric oxide (NO)* is an important *modulator of angiogenesis*

Questions to contemplate before and after lecture:

1. What is the timetable for the events of epidermal repair in incisional wounds? 2. What are the key cells that participate in wound healing in each of the defined phases: early and inflammatory response, proliferative, and remodeling. 3. What functions are attributed to fibroblasts in wound healing? What molecules do fibroblasts make at different phases of wound repair? 4. What functions are attributed to macrophages? 5. What are some potential strategies for treating chronic non-healing wounds? Keloids? 6. What are the characteristics of an optimal wound dressing?

Clinical Wound Healing Summary:

A.) *Depth of the Wound* - Epidermis only - recapitulates the pre-injury state - Superficial dermis: a.) Minimal contraction b.) Epithelialization from hair follicles & wound edge - Deep dermis & Subcutis: a.) Contraction a major component b.) Epithelialization from wound edge only B.) *Primary & Secondary Intention Healing* - Primary: wound edges are brought together a.) Predictable re-epithelialization time b.) Less contraction - Secondary: wound edges remain separated a.) Slower time to re-epithelialization b.) Contraction a major component

Activated Macrophages

Activated macs, *proinflammatory (M1)*, are present early in repair process while *anti-inflammatory, pro-angiogenic macs (M2)* are present in later stages of wound repair - *M1* macs release inflammatory cytokines e.g. TNF-α, NO, and IL-6 that can damage host cells as well as bacteria - *M2* macs however suppress inflammation and adaptive immune responses

What are some advantages and disadvantages to reparative wound healing?

Advantates: - protection from infection - don't bleed to death (as often) - faster than regenerative wound healing Disadvantages: - scarring - loss of function

The Sequence of Events after Skin Injury

Although the healing process is separated into phases, *wound healing is continuous and the processes of each phase have significant temporal overlap.*

Inflammatory Phase:

Characterized by *early vascular response* and an *inflammatory cellular response* - vascular response maintains hemostasis and attracts additional help - inflammatory cellular response cleans wound of potential pathogens and leads to forward progress along the healing pathway

Remodeling Phase

Characterized by *wound contraction and ECM reorganization*

Clinical Appearance of the Wound

Clinically wound appearance changes over time, corresponding to microscopic changes occurring during healing process 1.) Initially wound has *glistening and moist surface* that corresponds to *fibrin exudate and serum* in wound bed 2.) *Scab or eschar forms, then detaches* as *keratinocytes migrate to re-epithelialize* the open wound beneath 3.) *Red, indurated (swollen) scar* forms in coming weeks as a result of *changes in the dermis during proliferative and remodeling phases* 4.) *Color fades* overtime due to a *decrease in newly formed capillaries* formed in proliferative phase resulting in a scar that is lighter in pigment than the surrounding skin 5.) *Parallel orientation of collagen fibers* in remodeling phase creates the *visible scar*. If wound is deep enough, a *lack of adnexal structures* (hair follicles, sebaceous and sweat glands) is also noted.

Inflammatory Phase: Immediate Response and Inflammatory Response (Lecture - REDUNDANT INFO)

FIRST need to stop bleeding - fibrin clot - cells nearby up and down regulate various factors - Increased vascular permeability etc - Cytokines to attract inflammatory cells (n.phils, macs) - Upregulation of adhesion molecules

Excessive Wound Healing

For structure and function to be restored after injury, equilibrium between cell proliferation and apoptosis as well as matrix synthesis and degradation must be reestablished. Excessive response to wounding can result in - *hypertrophic scars*: a scar that is thicker or more raised than desired but stays w/in boundary of original wound *Keloids*: scar that is thickened and tends to overgrow boundaries of original wound - sternal region is most common location for keloid formation, but can form elsewhere on trunk such as shoulders and scapular region, ears, neck and extremities; they rarely develop on the face - Trauma is usually causative event - more common in African-Americans and in pts <30y/o

Epithelialization: Keratinocytes

Have amazing array of functions in wound environment: - release proteases to help dissect under devitalized tissue and the eschar - phagocytize wound debris - mount front-line defense in response to bacteria - release GF's such as transforming growth factor (TGF)-α, insulin-like growth factor (ILGF), PDGF and VEGF - epidermal KCs also play a key role in innate immune response important in response to bacterial invaders

Remodeling Phase: ECM Reorganization

Largely a function of fibroblasts: - *Fibroblasts make fibronectin and hyaluronic acid* early in the wound repair process --> Subsequently, *sulfated proteoglycans and collagens are synthesized* --> finally *weeks after wounding, elastic fibers* may be observed in wound bed

Depth of the Wound: Superficial vs Deep

Mechanism of wound healing differs depending on wound depth *Superficial Wound*: only epidermis is injured; repaired using a regenerative process that recapitulates the pre-injury state *Deep Wound*: BMZ disrupted and dermis injured - regeneration does not occur; repair process takes place w/ the end result looking quite different from the original - wounds involving dermis acquired by embryo in first 1/3 of gestation do not heal w/ scarring; developmental pathways are reactivated thereby recreating the original tissue

Granular Tissue: Neovascularization and Fibroplasia

Neovascularization and fibroplasia begin *~3 days after injury* - Neovascularization largely complete by day 10 - Fibroplasia and dermal remodeling continue considerably longer - *Endothelial cells, fibroblasts and macs* are the main cells participating in the granulation tissue response in the healing wound

Early Response to Injury: Neuroinflammation

Nerve fibers are severed along w/ blood vessels when skin is injured. *Neuroinflammatory response begins immediately* and is heralded by *pain* - *Small C-fibers* in epidermis and papillary dermis not only provide afferent pain input, alerting the CNS of the injury, but also have efferent functions --> Large number of *neuropeptides including substance P* are released into the wound at the time of injury

Granulation Tissue

New *stroma* that *replaces fibrin clot*; Composed of a *vascular network, fibroblasts, CT, and immune cells* - formation of new blood vessels (angiogenesis/neovascularization) is essential to restoration of cutaneous function since these vessels provide nutrition and O2 needs of cells participating in repair - Should be emphasized that *many of the events in wound healing occur simultaneously, esp neovascularization and fibroplasia*, both key events in the formation of granulation tissue - Pic: overview of the time line for wound healing process.

Remodeling Phase: Wound Contraction

Occurs as result of *fibroblasts assuming a myofibroblast phenotype w/ prominent actin fibers as part of their internal architecture* - Myofibroblasts *align along lines of contraction and attach to one another as well as to collagen and fibronectin fibers in wound* - Myofibroblasts *pull the edges of wound together* This is a major factor in *closing wounds by secondary intention* healing esp in *full thickness wounds* where *contraction peaks around 2wks* and can account for up to *40% of the decrease in size of the wound*

Secondary Intention Wound Healing

Occurs when wound is not closed mechanically but closes by natural means - often requires considerable amount of wound contraction and epithelial migration - time to complete re-epithelialization varies depending on several factors including location, depth, and size. Though we define them differently, both types of wound healing (primary vs secondary) progress through the same phases of wound healing. - Do NOT want to do this at margin areas

Early Response to Injury: Platelets

Platelets play key role in clotting process through *adhesion, aggregation*, and in *cellular signaling w/ release of GF's* - *platelet-derived growth factor (PDGF)*: important for fibroblast, macrophage, and neutrophil chemotaxis - *Transforming growth factor (TGF)-β* - *Activated platelets* also contribute to regulation of angiogenesis in the new wound.

Excessive Wound Healing: PDGF

Potent positive signal for fibroblast collagen production - receptors are up-regulated in both types of excessive scarring

What are some characteristics of an effective wound dressing/bandage?

pic - not in syllabus, skipped in lecture

Epithelialization: Eschar

When a scab or eschar (made of *cellular debris and dried serum and blood*) is present, *migrating cells move between it and granulation tissue below* - to do this, they must *up-regulate proteolytic enzymes* such as *matrix metalloproteinases (MMPs)* - tends to *slow epithelialization, deepen the wound and possibly create a more unpleasant scar* - Once a *single layer of keratinocytes* covers the wound, cells are signaled to *proliferate rapidly by EGF and TGF-α* to create a more typical stratified epidermis

Early Response to Injury: Coagulation and Wound Repair

Wound is filled w/ blood and clotting cascade is immediately set in motion. Most crucial step for survival is formation of *hemostatic fibrin clot*. Clot consists of *platelets embedded in a mesh of cross-linked fibrin* Coag process results in: 1.) Release of molecules such as *Factor XII and bradykinin* which *change vascular permeability* 2.) *Complement activation* which results in *leukocyte recruitment* 3.) *Provisional matrix for cell migration*

Epithelialization: Keratinocytes - First Changes

Regeneration of epidermis is a critical step in reestablishing barrier function of skin; Epidermis provides cutaneous barrier to prevent fluid loss and bacterial invasion - In *chronic ulcers this step is notably deficient!* - In normal wounds the *first histological changes in keratinocytes (KCs) at wound margin are seen at 8-24hrs after injury* - exact mechanisms not understood, but one *important factor in stimulating KC migration is exposure to serum during wounding* - Many molecular signals and gene expression profiles have been worked out in recent years

Clinical Examples of Abnormal Wound Healing

Repair of intentional and accidental wounds is a very complex and integrated process bringing together the coagulation cascade, platelets, inflammatory cells, fibroblasts and numerous cell-to-cell signals. As a result, there are several places along the pathway where healing can be derailed. Abnormal healing, *characterized by delayed re-epithelialization or excess response to wounding*, is observed in a number of clinical settings. - Excessive Wound Healing - Delayed Wound Healing

Wound Healing

Response to external injury of skin is a primitive response w/out which humans could not survive - Rapid reconstitution of epidermal barrier is necessary for protection from fluid loss, chemical and bacterial invasion - Dermis must be repaired to restore tissue strength and blood and nerve supply to the area - Every year in US 1.25 million people suffer burn wounds, and 6.5 million people develop chronic ulcers from venous insufficiency, pressure (e.g. bed sores), and DM - 15% of all pts w/ DM will experience a lower extremity ulcer and up to 25% of those pts will require an amputation

Primary Intention Wound Healing

Typical of a surgical wound in which *wound edges are approximated by sutures or other physical means* - repair process typically moves forward in a very predictable fashion - Normal variation occurs, based on factors such as age and anatomical location [head and face heal faster than arms>trunk>legs>feet]

Epithelialization: Keratinocytes - Epithelial Migration

Unclear which population of keratinocytes actually migrates across the wound bed *"Sliding" Model*: theory says only the *basal keratinocytes* migrate, pulling the rest of the epidermis as a sheet to close the wound (pic-a) *"Roll-Over" Model*: theory involves *suprabasal keratinocytes*; thought to "roll over" anchored basal keratinocytes to fill in wound (pic-b) Target cell (basal vs. suprabasal keratinocyte) may be an *important issue as we try to develop molecular therapies to speed epithelial migration*

Proliferation Phase: Fibroblasts

Very important for *production of granulation tissue* necessary in dermal repair - actively proliferate and migrate into new wound at ~2-3 days - Induced by macs via PDGF and TGF-β1, they make the components of granulation tissue including, hyaluronic acid, fibronectin, collagen and elastin fibers - Predominant collagen produced in early wound is type III collagen and it tends to be less well organized than collagen seen in uninjured skin - Release autocrine and paracrine GF's such as keratinocyte growth factor (KGF) and ILGF - NO

Inflammatory cells and wound repair:

W/in first few hours after injury, cells begin to appear in the wound bed. - *Neutrophils and macs* are attracted to wound site by a variety of *chemotactic signals (GFs, by-products of bacterial proteins, proteolysis of fibrin)*

Inflammatory Phase Summary: Day 1-3

*Contains the injury and prevents infection* A.) *Early Response* - Pain causes withdrawal to reduce additional injury - Platelets & coag cascade prevent exsanguination, promote vascular permeability, and attract inflammatory cells to wound - Provisional fibrin clot matrix is created to aid cell migration B.) *Cellular Response* a.) Neutrophils: - 1st inflammatory cell to arrive in the wound - Decontaminate wound bed - Degrade provisional matrix b.) Macrophages - Most important inflammatory cell in wound healing - Proinflammatory (M1) cells assist in wound debridement, bacterial phagocytosis, and matrix degradation early - Predominate inflammatory cell by Day 3 - Promote angiogenesis w/ production of VEGF - Phenotypic switch to anti-inflammatory (M2) promotes progression to proliferative phase of healing

Proliferative Phase Summary: Day 3-30

*Development of the epidermal permeability barrier and granulation tissue* A.) *Epidermis* - Keratinocytes loose adhesions, flatten and migrate; process begins w/in 24 hours - New keratinocytes come from hair bulge and basal layer stem cells - Reconstitution of epidermis a.) Basal layer: 2-3 days b.) Cornified layer: 5-6d c.) BMZ: 7-10d B.) *Granulation Tissue Formation* - Begins by Day 3 - Characterized by angiogenesis and fibroplasia stimulated by M2 macs a.) Angiogenesis: 3-10d b.) Fibroplasia: 3-30d - Endothelial cells, fibroblasts and macs are main effector cells - Fibroblasts, induced by TGF-β1, produce type III collagen, the main structural component of granulation tissue - Endothelial cells promote angiogenesis and ECM formation - Granulation tissue causes the healing wound/scar to appear red/pink C.) *Macrophages* - Stimulate angiogenesis - Stimulate collagen synthesis - Promote repithelialization

Neutrophils:

*First to arrive*; Main functions are *decontamination and fibrin matrix degradation* - *Endothelial cells up-regulate selectins* on cell surface to help control rolling and tethering of neutrophils and other leukocytes - Once attached, neutrophils produce *elastase and collagenase* to *migrate into the wound* - Neutrophils have *important cleansing role and kill invading microorganisms* through several strategies, including bursts of reactive oxygen species *(ROS)* - *Wound Contamination may prolong duration neutrophils are present in wound and slow wound healing* - Neutrophils also produce *pro-inflammatory cytokines that activate local fibroblasts and keratinocytes* - Also have a role in *resolution of fibrin clot and provisional ECM promotion of angiogenesis, and reepithelialization*

Nitric Oxide (NO) and Inducible Nitric Oxide Synthase (iNOS)

*Inducible nitric oxide synthetase (iNOS)*: in later stages of wound healing, keratinocytes and fibroblasts produce iNOS *NO*: important modulator of fibroblast function and fibroplasia - lays a critical role in angiogenesis - critical for collagen deposition - also plays role in regulating inflammation by modifying or amplifying cellular responses

Question: What are potential mechanisms for preventing or treating hypertrophic scars and keloids?

*Inhibit fibroblasts* - Decrease TGF-Beta 1&2 & PDGF - Decrease tension, avoid excess movement - Corticosteroids - Increase TGF-Beta3 *Decrease angiogenesis/neovascularization* - Laser - Decrease VEGF *Reduce inflammation* - Prevent infection, contamination - Suture material = foreign body reaction - Corticosteroids - Increase IL-10

Epithelialization: Stem Cells

*Partial Thickness Wound*: Hair bulge contains the epidermal stem cells that help to re-epithelialize the wound *Full thickness wound*: adnexal structures and their stem cells are lost --> only epidermal stem cells from basal layer exist to cover the open wound

Remodeling Phase Summary: Day 10 - 1 year

*Wound contraction and Extracellular matrix reorganization* A.) *Wound Contraction* - Begins on Day 10 - Myofibroblasts slowly contract wound over days - Important in secondary intention wound healing B.) *Extracellular Matrix Reorganization* - Matrix metaloproteinases from macrophages, fibroblasts and endothelial cells breakdown type III collegen - Fibroblasts produce type I collagen - Type I collagen parallel w/ the wound tension lines persists. The basket-weave orientation of normal skin is lost giving rise to the clinical appearance of scar tissue - Strength of scar tissue vs normal surrounding skin: a.) 10% at 1 week b.) 40% at 1 month c.) 80% at 1 year (max strength)

Strategies to help diabetic wounds heal? (lecture)

- Control diabetes - Off-weight the wound - Improve arterial supply - Treat infection - Add growth factors (PDGF, EGF, FGF) - Induce Mac type switch (M1 to M2) - Promote keratinocyte migration

Proliferation Phase

- Epithelialization - Granulation tissue - Endothelial cells - Fibroblasts - Macrophages

Epithelialization: Timing

- Narrow wounds and wounds healing by primary intention are often re-epithelialized by 72hs (pic-a,b) - Complete restoration of BMZ, (Pic-c), does not occur until ~day 7 Time frame after injury of a small incisional wound for reconstituting the epidermis: *Basal Layer*: 2-3d *Granular Layer*: 4-5d *Cornified Layer*: 5-6d *BMZ*: 7-10d

Macrophages: Key Cell in Wound Healing

Appear in wound from blood w/in *first 1-3 days*; many believe this cell is "conductor of the symphony" of wound repair and is the *most important regulatory cell* - depletion of macs during inflammatory phase causes significant delay in wound healing - macs assist w/ *wound debridement, bacterial phagocytosis and matrix degradation* in early wound and appear to play an important *cell signaling role in subsequent granulation tissue formation*, helping make *shift from inflammatory phase to the proliferative phase* - Macs *release GF's including fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), PDGF and TGF-β*

Early Response to Injury: Mechanical stresses

Earliest physical events following injury are *perception of pain* and *onset of bleeding* - Cells and tissues respond to early stress signals w/ alterations in transcription of hundreds of genes resulting in alterations in cell survival and metabolism - Cells adjacent to and w/n wound respond to: 1.) mechanical "stretch" 2.) electrical current changes 3.) exposure to external environment 4.) microorganisms

Question: Healing of ulcers in patients with diabetes is much delayed. What are some potential mechanisms?

Early Response: Nerves - no sensation, and no neuropeptide signaling Inflammatory phase: prolonged, no M1 to M2 switching Proliferative Phase: insufficient vasculature Remodeling: problems with nerves, wound is weak and lack of sensation = wound easily reopened

Macrophage Switch (M1 to M2 Switch)

Early in healing Macs are inflammatory, later switch to Anti-Inflammatory pro-proliferative = SUPER IMPORTANT - if the switch does not happen, wound will stay in inflammatory stage and not heal --> Chronic inflammation

Depth of the Wound: Superficial vs Deep *Dermal* Wound

If deep dermis and SubQ tissue are intact (*partial thickness*); wounds heal w/ minimal contraction compared to wounds that extend into subQ tissue (*full thickness*) - stem cells located in basal layer of interfollicular epidermis and bulge in hair follicles act as reservoirs for new keratinocytes; epithelium from wound edge and follicles migrate to cover the open wound *Full-thickness wounds*: intact pre-existing tissues move toward the center of the wound by contraction, which is typically inadequate to completely close wound - Only keratinocytes from the wound edge migrate for re-epithelialization.

Excessive Wound Healing: TGF-β

Iimportant cytokine in proliferative phase of wound healing w/ *3 isoforms*: *TGF-β1 & 2*: promote scar formation *TGF-β3*: reduces scarring - Fibroblasts from both hypertrophic scars and keloids *overexpress proteins involved in TGF-β signaling*

Proliferation Phase: Macrophages

Important role in wound healing long after acute inflammatory phase of wound healing has resolved - Macs that survive initial inflammatory phase have a *phenotypic shift* that is *signaled by phagocytosis of apoptotic cells* --> shift creates macs that are important in *stimulation of angiogenesis, re-epithelialization, and collagen synthesis* in this phase of wound healing

Remodeling Phase: Collagen Reorganization

In attempt to restore normal architecture, *type III collagen*, laid down in the proliferative phase, is *replaced by new type I collagen over weeks to months* in a tightly controlled process of *new collagen synthesis and old collagen breakdown* - *Type III collagen degraded by MMPs* produced by macs , fibroblasts and endothelial cells - New *type I collagen fibers are oriented parallel w/ the lines of tension in order to provide max strength* - collagen laid down parallel to the tension escapes degradation by collagenases. This parallel oriented collagen structure is quite different from the basket-weave orientation see in uninjured skin. More tension = more collagen I and scarring (e.g. chest, back)

Delayed wound healing

Seen in many clinical contexts and stems from a multitude of causes. Prototypical chronic wound is diabetic foot ulcer - the leading cause of non-traumatic lower extremity amputation - Ulcers typically arise in areas of pressure or repeated trauma and commonly on the feet Several changes occur in feet of pts w/ DM creating deformities in shape and function: - *peripheral neuropathy*: pts do not receive appropriate feedback telling them damage is occurring to deformed foot --> pt may continue to wear an inappropriately sized pair of shoes or walk w/ a pebble in their shoe, resulting in a foot ulcer - Once ulcer is formed, many *mechanisms of wound healing are impaired* creating a chronic, non-healing wound - w/ repeated pressure and trauma, wound may deepen to eventually involve bone. Infection may develop leading to amputation.