Tissue Repair & Wound Healing

What color is collage in a trichrome stain?

BLUE

What part of the ECM will provide a scaffold for cells & ECM deposition during healing?

FIBRONECTIN (found in tissue and plasma)

Which stem cells are closest to final differentiation of the stem cell?

PROGENITOR or *lineage committed* --these are stem cells that are capable of limited differentiation.

The accumulation of A1AT in hepatocytes will cause apoptosis and necrosis -- this will activate what process?

Repair processes are activated which results in: >*Development of regenerative nodules*: hyperplasia & hypertrophy of hepatocytes, formation of oval cells and continued stimulation can lead to DNA damage & neoplasia >*Scarring* (Cirrhosis) - from fibroblast proliferation and collagen deposition.

What is the composition of scar tissue? What are the advantage of this tissue?

SCAR TISSUE: pale, avascular tissue that is composed of *collagen*, *fragments of elastic tissue*, *ECM* and *inactive fibroblasts* > Advantages of this tissue are that it provides a *resilient permanent patch* and *tensile strength* for the tissue after healing.

During wound healing, fibroblasts will divide and secrete what? Then what will occur?

Secrete *Collagen Types III* -after secretion the first type of collagen in the wound will be replaced by *Type I collagen* and the eventual result is fibrosis w/ connective tissue matrix. -Replacement involve *macrophages*

What is the function of *transforming growth factor-beta* (TGF-b)? What does it work to suppress?

TGF-b has 4 main functions: > *Chemotactic* for leukocytes & fibroblasts > Stimulates *ECM protein synthesis* > *Suppresses Acute Inflammation* > Act as a growth stimulator or inhibitor.

Describe the steps of inserting new genetic material to transdifferentiate cell (with transduction and transdifferentiation).

TRANSDUCTION = genes from a host cell are incorporated into the genome of a recipient cell by vector. TRANSDIFFERENTIATION = non-stem cell transform into a different type of cell.

Growth factors bind to what type of receptor?

TYROSINE-KINASE Receptors --- activation of these receptors indicates to the cell to *replicate*

Why would there be nodules in the liver of a person with A1-antitrypsin deficiency?

There would be *nodules* of REGENERATION (*hypertrophy & hyperplasia*)

What are Labile cells? Where are these cells found?

These are *stem cells* which are continuously dividing via [M --> G1 --> S --> G2 --> M] -Found in the *epidermis*, *mucosal epithelium*, *GI tract epithelium*, & *bone marrow*

What are Stable cells? Where are these cells found?

These are *stem cells* which has a *low level of replication* via [Go --> G1 --> S --> G2 --> M] -*Hepatocytes*, *renal tubular epithelium*, *pancreatic acini*

What are the four different types of STEM Cells?

These are undifferentiated cells capable throughout life of renewing itself as well as generating one or more types of differentiated cells. > TOTIPOTENT > PLURIPOTENT (embryonic stem cell) > MULTIPOTENT > PROGENITOR or LINEAGE COMMITTED

Why is *laminin* found in the basement membrane?

This ECM component *ANCHORS basal cells* of epithelia and modulates cell growth, differentiation & motility.

What is a pyogenic granuloma?

This is a *permanent problem* that is of granulation tissue (overgrowth) -- mixture of abnormal repair & dysplasia.

What is a wound contracture?

This is the abnormal formation of scar tissue --- the tissue grows across a gap that should naturally be there (surgery is often needed to remove the access tissue in certain areas (common in burn victims) -function of area is limited.

A hematoxylin stain on liver tissue will show what components of the cell?

This stain will show: --*Nucleic Acids* --*Ribosomes* --*Calcium* --*bacterial walls*

Where is collagen *type IV* found?

This will be found in the *basement membrane*

How many different types of VEGF are there? What are their receptor and what induces the production of VEGF?

VEGF --> 5 types (*A-D, F*) *3 distinct VEGF receptors *VEGF Production*: induces by -*hypoxia* (via HIF-1) -*TGF-a* -*PDGF*

Where are Collagen Type VI and Type VII found?

VI --> found in *Microfibrils* VII --> found in anchoring fibrils in *hemidesmosomes*

Which growth factor will stimulate the proliferation of *endothelial cells*? What is this secreted by?

Vascular Endothelial Growth Factor *(VEGF)* -this is secreted by Mesenchymal cells and will also increase vascular permeability.

What are the main events which occur in the 1st, 2nd & 3rd week following a cutaneous wound?

*1st Week* = primarily INFLAMMATION *2nd Week* = primarily cell/tissue PROLIFERATION & MATRIX deposition *3rd week* = primarily MATURATION & REMODELING of tissues

Emphysema that is present in a non-smoker may be due to what condition?

*A1-antitrypsin Deficiency (A1AT)* > A1AT is a protein that is produced abnormally (genetic condition) which will cause decreased A1AT activity in the blood & *lungs* > There will also be increased A1AT accumulation in the Liver (which is why liver enzymes can be elevated in these patients)

What are the main methods (4) of cell-to-cell interactions?

*AUTOCRINE* --> note to self, target on same cell. *SYNAPTIC* --> target to directly synapsed cell(s) via specialized cell junctions. *PARACRINE* --> targets are nearby cells ("message to neighbors") *ENDOCRINE* --> Distant target cells via blood stream (public notice)

The second week after sustaining a cutaneous wound will involve what events?

*Angiogenesis* w/ *Granulation tissue* Re-epithelialization begins.

What component of the wound will influence the scarring that occurs to repair the wound?

*Background Connective Tissue* --- the presence of this tissue determines what structure is available (if it is not available then normal structure cannot be obtained and scarring occurs)

The first week after sustaining a cutaneous wound will involve what events?

*Clot* (crust) formation *Chemotaxis* of inflammatory cells

What collage in found ONLY in soft tissues?

*Collagen Type III* (fibrillar collagen)

The third week after sustaining a cutaneous wound will involve what events?

*Collagen* laid down. Wound contraction.

What aid in the debridement activity of macrophages?

*Collagenase* *Elastase*

What is signaling in angiogenesis that is used to induce *vascular remodeling*? What else occurs due to this stimulus?

*DLL4/Notch* = Vascular remodeling > Decreased sprouting > Decreased endothelial cell proliferation > Increased Vascular organization > Increased *lumen diameter*

Which growth factor will stimulate keratinocyte migration & formation of granulation tissue?

*EPIDERMAL GROWTH FACTOR* > mitogenic for keratinocytes, smooth muscle, glial cells & fibroblasts.

What provides the elasticity and recoil ability of tissues?

*Elastin*

Which growth factor will induce endothelial cell to *migrate* to injury site & release proteolytic enzymes? What are the other function of this GF?

*FIBROBLAST GROWTH FACTORs (FGFs)* --chemotactic & mitogenic to *fibroblasts* --*ECM protein synthesis*

What tissue forms in wound healing and will fill the incision or wound in the original tissue? What is the purpose of this new tissue?

*GRANULATION Tissue* > Soft, pink, & granular tissue that is *anti-infection* & protects the would surface. > Replaces necrotic tissue, effusion & other removed foreign body.

What are some abnormalities of healing that involve *granulation tissue*? what about those involving *cicatrization* (scar formation)?

*Granulation Tissue* = Proud flesh, pyogenic granuloma *Cicatrization* = contractures, hypertrophic scars, keloid

What is the function of proteoglycans (found in ECM)?

*Hold water* so they are found in tissues which require compression or lubrication. *Reservoir for growth factors* Modulate cell growth, migration, adhesion & differentiation.

How are cells able to have adhesion with the ECM?

*INTEGRINS* -these are cell-surface receptor proteins mediating adhesion of cells to ECM (laminin, fibronectin, etc.)

What is the difference between a keloid and a hypertrophic scar?

*KELOID* = patient must have a predisposition to form this large amount of scar tissue (Fibroblasts are stimulated to lay down abnormal collagen) *Hypertrophic Scar* = This is abnormal raising of scar with excessive amount of tissue, but not as large as keloid.

What cell is known as the "leader of the band" in angiogenesis? How does it signal migration and proliferation?

*Leading ("TIP") Cell* -this cell will cause migration, proliferation and remodeling via *VEGF* and *Notch signals*

Which stem cells are found in the fetus and adult?

*Multipotent* stem cells

Which cell almost NEVER divide? What are the examples of these cells in the body?

*PERMANENT cells* at Go > CNS neurons, cardiac & skeletal myocytes.

What are the different unions that can occur when healing a cutaneous wound?

*PRIMARY union* (or 1st intention): minimal scarring with short healing process. *SECONDARY union* (or 2nd intention): lots of granulation tissue and larger scar that may be indented.

What are important events that must occur to heal a open wound?

-Debridement (removal of necrotic tissue) -Angiogenesis -Myofibroblasts -Contraction of fibrin -Contraction of wounds -Wound shapes

What occurs if there are problems with the PiZZ phenotype?

*PiZZ Phenotype* --substitution of lysine to glutamic acid at position 342 (SERPINA1 gene) in protein Z, this lead to a protein that contains a large beta-pleated sheet conformation & allows *polymerization* to similar proteins. --proteins align w/ each other to cause large aggregates that cannot leave the ER (protein accumulation leads to ER stress, *Apoptosis* & *necrosis* = liver toxicity

What are the events of hemostasis stage in healing by 1st intention (primary union)?

*Platelet plug* ---> involved platelet degranulation and then PDGF which acts as mitogen and chemotaxin for fibroblasts. *Clotting* ---> collagen contract & tissue factor form injured cells will cause thrombus. *thrombin* attract macrophages and induces fibroblast replication.

What is another term for exuberant granulation tissue (overgrowth of granulation tissue)?

*Proud Flesh* -will be an overgrowth of soft, pink tissue.

What are the two different repairs which can be carried out on damage tissue? What are the results of these repairs?

*RESOLUTION* --> this is healing that will have very minimal scarring -think surgical incision *Repair by CONNECTIVE TISSUE* --> this is healing that will form a scar

Which wound repair process will have very little residual evidence of the initial injury? What are the characteristics of this process?

*Resolution* > minimal tissue damage > Neutralization of chemical mediators > Restoration of normal vascular permeability > Removal of dead tissues, edema, proteins & other materials released during inflammatory process.

What growth factor will stimulate the proliferation of *hepatocytes* & other epithelial cells?

*TRANSFORMING GROWTH FACTOR - alpha* > TGF-a is similar to EGF

What stem cells are found very early in embryonic development?

*Totipotent* stem cells.

What is the main function of VEGF in angiogenesis? What else occurs due to its stimulus?

*VEGF* = Vascular Proliferation > Increased sprouting > Increased endothelial cell proliferation > Increased endothelial cell survival > *Decreased* vascular organization

WHat are the VEGF subtypes which promote lymphatic vasculature?

*VEGF-C* *VEGF-D*

What is important about the wound healing in hepatocytes?

*hepatocyte proliferation* --induces replication in stable cells (hepatocytes) that are resting in Go phase of cell cycle.

How does A1AT deficiency cause apoptosis in hepatocytes?

--Accumulation of mutant 1ATZ in the ER results in activation of the *caspase cascade* via both the ER & mitochondrial pathways. --> Membrane dysfunction --> Ca2+ influx --> activation of phospholipases --> cell membrane damage & dysfunction --> more Ca2+ influx

What are the mediator of the phagocytic activity of macrophages?

-Reactive oxygen species (free radicals) -Nitric Oxide

Vascular endothelial growth factor (VEGF) has what functions (4)?

1. *ANGIOGENESIS* - endothelial cell proliferation & migration. 2. *Increases vascular permeability* via stimulation of NO production 3. Increases expression of plasminogen activator, plasminogen activator inhibitor & collagenase by endothelial cells. 4. *VEGF-C* & VEGF-D* promote *Lymphatic vasculature.

What are the functions (3) of *platelet-derived growth factor* (PDGF)?

1. *Chemotactic* for neutrophils, macrophages, fibroblasts & smooth muscle cells. 2. Activates & stimulates *proliferation* of *fibroblasts*, *endothelial*, smooth muscle & *monocytes* 3. Stimulates *ECM protein synthesis*

What are two important post-surgical complications of wound healing?

1. *DEHISCENCE* -- this is the re-opening of a surgical incision 2. *EVISCERATION* -- re-opening that involves intestinal contents protruding form the incision (much more concerning)

What are some important patient conditions (7) which can inhibit or affect outcome of wound healing?

1. *Nutrition*: commonly a vitamin deficiency 2. *GLucocorticoid* use 3. *Immune Deficiencies*: such as DM 4. *Infection*: either from wound or systemic 5. *Foreign bodies* in wound 6. *Decreased venous drainage* or *blood supply* 7. *wound dehiscence*: separation of the layers of a surgical wound.

What are the 5 activities of macrophages?

1. *Phagocytosis* 2. *Debridement* (removal of necrotic tissue) 3. *Cell recruitment* and activation 4. *Angiogenesis* 5. *matrix synthesis*

What are the three different events a cell with replicative potential can undergo?

1. *Proliferate* & make additional cells 2. Differentiate into terminal *non-replicating cells* 3. *Die* (apoptosis)

What are the four steps of Angiogenesis?

1. *Proteolytic degradation* of basement membrane 2. *Migration* & *proliferation* of *endothelial cells* 3. Migration & proliferation of *pericytes* & *smooth muscle cells* 4. *maturation

What are two general methods which have been used to induce Pluripotent stem cells?

1. Embed nucleus of differentiated cell into cytoplasm of oocyte (i.e. Dolly the lamb) 2. Insert new genetic material to *Transdifferentiate* a differentiated donor cell (first with transduction and then transdifferentiation)

What are three factors that influence the Repair of a tissue?

> *Cell-Cell interactions* conditioned medium and contact inhibition are parts of this factor. > *Growth Factors* > *Extracellular Matrix* laminin and fibronectin are important components.

What are the 6 components of the extracellular matrix (ECM)? Where is each component found?

> *Fibronectin*: tissue & plasma > *Laminin*: basement membrane > *Integrins*: cell surface receptor proteins > *Proteoglycans* : in tissues that require compression or lubrication > *Elastin* > *Collagen*

during the Early scar stage of healing by 1st intention (primary union) - what are the events?

> Capillaries will bridge wound and lymphatic channels appear > *Macrophages scavenge* > Type I collagen appears

What are the main processes that the body will utilize to restore the structure & function of a tissue after a wound?

> Induction of *acute inflammatory response* by an initial injury. > *Angiogenesis* > *Fibroblast* & parenchymal cell regeneration. > Migration & proliferation of parenchymal & CT cells. > Synthesis of *extracellular (ECM) proteins* > *Scar* formation > Remodeling of parenchymal elements to restore tissue function. > Remodeling of CT to achieve wound strength.

When healing by 2nd intention (secondary union), what are the different reasons for keeping a wound OPEN?

> Margins are *too far apart* > Wound is *infected*-penetrating intestinal injury is common example > Tissue if *devitalized* (such as gangrenous bowel and necrotic ulcers)

What are the stages of healing by 1st intention (primary union)?

> Minimal disruption: surgical incision or laceration w/ sutures or surgical glue. > *Hemostasis*: platelet plug, clotting (thrombus) and thrombin attract macrophages for fibroblast replication > Scab formation (dried exudate) > *Inflammation* (migration of cells in 24 hrs.) > *Regeneration* = 3 days > *early Scar* = 7-10 days > *Scar maturation*

Stem cells can be what two type of cells?

LABILE Cells = continuously dividing (M--> G1 --> S --> G2 --> M) STABLE Cells = low level of replication (G0 --> G1 --> S --> G2 --> M)

Where are hematopoetic blast cells main located?

Located within the BONE MARROW.

What is true of the strength of tissue following an injury vs. the strength of normal uninjured skin?

After a wound the tissue is NEVER as strong as normal, uninjured skin. *< or = 70-80% of strength*

What is the collagen found in Cartilage of the body?

Collagen *Type II* --This collagen is also found in vitreous and intervertebral disks. (is a fibrillary collagen)

Which collage is found in both soft tissues and blood vessels?

Collagen *Type V* (fibrillary collagen)

What are the important growth factors (3) and cytokine for the process of angiogenesis (by macrophages)?

GFs = *FGF, VEGF, & PDGF* Cytokines = *TNF* -*Nitric oxide* is also important for angiogenesis.

What are the growth factors and cytokines which aid in cell recruitment & activation (for macrophage activity)?

GFs = *PDGF, TGF-b, EGF, IGF* Cytokines = *TNF, IL-1, IL-6* -Fibronectin (ECM component) also is a mediator for this function.

In wound healing, what provides the strength and nutrients to the *granulation tissue*?

Granulation tissue will contain *fibroblasts* & thin-walled *capillaries* (angiogenesis) which provide conduit for nutrients. > Inflammatory cells w/n the loose edematous ECM, and fibroblasts (*collagen type III*) will aid in building strength in the tissue.

Where is collagen type I found?

Hard & soft tissues -This is a fibrillary collagen.

The enzymes *arginase* and *collagenase* are important for what macrophage function? What other mediators are important to this function?

Important for *MATRIX (ECM) Synthesis* --GFs: *TGF-b, EGF, PDGF* --Cytokines: *TNF, IL-1, IFN* --*Prostaglandins* --*Nitric oxide*

Alpha 1 antitrypsin inhibits the action of what? What occurs if A1AT is defective?

Inhibits the *Neutrophil elastase* enzyme. > w/o this inhibition, areas w/ inflammation have *increased tissue destruction* > in lung, unchecked neutrophil elastase secreted secondary to inflammation leads to destruction of alveolar septa leading to *emphysema*