Understanding Wound Healing: A Cellular and Molecular Perspective

Posted on Aug 23, 2024 in Biology

Wound Healing

Clotting, Scarring, and Re-establishment of Function in Complex Tissues

Four Stages of Wound Healing

• HOMEOSTASIS (within minutes)

  Stops the loss of blood and sets the stage for repair.

  • Spilled blood and the fibrous clot
    • Blood vessels become sticky.
    • Sticky platelets flow to the area of the wound due to blood vessel damage.
      • Platelet: Stem cell population, a piece of a megakaryocyte filled with vesicles for secreting growth factors (regulated secretion: makes a vesicle, but it sits there waiting for a signal to fuse).
  • Platelet degranulation activates much of the ensuing response.
    • Degranulation: Signals causing regulated secretion of all the vesicles.
    • Platelets activate the blood clotting system.
      • Platelet activation in the clot makes them sticky and releases their signal storage vesicles.
        • Platelet vesicle: Filled with growth factors.
          • Stimulate cells to go from G0 to G1 and migrate to the wound.
            • Less than 5% of cells are in the cell cycle in the body pre-wound.
            • A very low amount of cells are migrating in the body pre-wound.
          • Contains everything that fibroblasts need to do their jobs.
          • Very tightly balanced.
      • Clotting factors lead to the synthesis of fibrin protein.
        • Fibrin: Cleaved fibrinogen, which strengthens the platelet plug into a stable plug.
          • Temporary ECM for all cells to migrate on since they can’t jump.
          • The clot creates an environment needed for wound healing.
    • Secretory vessels fuse with the plasma membrane.
      • Platelet activation releases growth factors by regulated secretion.
        • Secrete ligands (signaling molecules) to recruit more cells to the wound area.

• INFLAMMATION (fast)

  Disinfects and accelerates the response mediated primarily by white blood cells as part of our innate immunity.

  • Blood vessels become leaky and release plasma into the surrounding tissues.
  • Mast cells and neutrophils are the first line of defense against bacteria.
  • Fibrin is also broken down during this phase.
  • Mast cells (WBC) supply most of the visual aspects of healing.
    • Resident mast cells: Create large amounts of vesicles, and when ligands bind to their receptors, they degranulate.
    • Resident mast cells that live in different tissues degranulate once activated and release signaling molecules that contain pro-inflammatory mediators and move towards blood vessels.
      • Leads to:
        • rubor = redness, calor = heat, tumor = swelling, dolor = pain
      • Activated so the body knows it needs to repair.
  • Circulating neutrophils arrive first to clean out bacteria.
  • Circulating and local monocytes, once they are in the wound site, are activated to become macrophages (attracted to the wound site by growth factors released from platelets and other cells).
    • Resident macrophages: AKA alarm bells, perform phagocytosis; eat everything.
      • Almost all tissues have these waiting for information from platelets or bacteria, and when the signal tells them to, they secrete and set off inflammation.
    • Activated macrophages: Colony-stimulating factor.
      • Diffusion of lots of cells (every single cell responds to the wound).
    • Mast cells and macrophages clean up and activate the release of expression and synthesis of signaling molecules by secretion.

• PROLIFERATION

  Replacement of dermal/subdermal tissue.

  • Re-establishes most or all of their pre-wound functions.
    • Reconnection of the dermal connective tissues.
      • Fibroblasts, macrophages, and smooth muscle cells are recruited.
        • Fibroblasts: Make dermal connective tissue and reconnect the dermal connective tissue.
          • Growth factors diffuse out, and fibroblasts respond.
          • Go from G0 connected to ECM to activated, resulting in migrating and dividing.
      • Scars are collagen-based.
        • Fibroblasts
      • Cell migration or “crawling”
        • Actin-myosin-based movement.
        • Form attachments and then release them, stimulated by growth factors.
      • Chemotaxis: Crawling to a particular place.
        • This is how platelets diffuse out by following their concentration gradient to find the wound.
        • Rho/Rac – internal to the cell, stimulate actual actin-myosin movement.
        • PDGF – primary stimulate, causes cell division.
        • Ras is also activated – G0 to G1 by divergent crosstalk.
        • TGF-β also provides cell division by convergent cell division.
      • Formation of the scar matrix.
        • The concentration of these changes with a scar in the dermis, a shift of the ECM that the fibroblasts are making.
          • Glycosaminoglycans
          • Proteoglycans
          • Fibrous proteins
          • Elastic proteins
        • % of collagen is increased during scar making.
      • Want to reduce scar size, so the scar is chipped away slowly by macrophages, leaving an empty space that is filled with connective tissue by fibroblasts.
    • Integrity of the epidermal layers.
      • Re-establishment of the epidermal epithelium involves both mitosis (new cells) and epithelial migration.
      • Fibroblasts repair connective tissue.
      • Contraction by myofibroblasts.
      • Keratinocytes: Epithelialization and make the outer layer.
    • Re-establishment of blood flow by vascular cells.
      • Form epithelial layers, continuous flow as they develop, they close high top.

• MATURATION

  Maximizes restored function.

  • Contracture minimizes the final scar area.
  • Fibroblasts and macrophages remodel collagen for a year+.

Two Major Errors in Healing

1. Failure to heal from an extended inflammatory stage.
2. Fibrosis results from the excessive formation of scar matrix tissue.
   1. HYPERTROPHIC SCARS – Don’t have fibroblast contraction.
   2. KELOID SCARS – Associated with excessive TGF-β activity.
      1. Too much is made, and fibroblasts have too many receptors.

What We Did in Lab

• DAY 1: Protein Isolation and Quantification via BioRad RC/DC Assay

  • Flask of cells in the incubator.
  • Make complete RIPA lysis buffer, then place on ice (1 ml of RIPA buffer is not enough, should go to 1.5 ml, but then need to extract more protein since the concentration will be too small otherwise).
    • Add 1.0 ml RIPA lysis buffer.
      • SDS: Sodium dodecyl sulfate; creates a uniform negative charge.
      • Triton X-100: Detergent; solubilizes the membrane.
      • Deoxycholate: Emulsifier; lysing the cells, getting rid of the membranes, suspending the lipids, and grabbing all the proteins in the membrane.
    • Add protease inhibitor cocktail solution: Stops protease activity in the cell.
  • Remove the culture medium.
  • Wash cells with ice-cold PBS.
  • Keep the plate on ice.
  • Scrape cells (protocol expected to get more than 1 g of protein, but we failed and got less due to bubbles or just not enough).
  • Pass cell lysate to form a homogeneous lysate.
  • Refrigerate at 4 degrees C for 5 min.
  • Centrifuge to separate cell debris from the protein.
  • Prepare standard dilutions.
    • 0.25-1.25 need a very straight line if the protein is outside of the curve (r=1).
    • Machines out there can photograph and assess the density and compare the wells to each other to make it a semi-quantitative evaluation.
  • To each tube, add:
    • RC reagent 1, then vortex and incubate at room temperature.
    • RC reagent 2, vortex, then centrifuge.
    • Discard the supernatant, keep the pellet.
    • Reagent A+S.
    • Reagent B, then vortex.
  • Incubate in the dark for 10 min at room temperature.
  • Read absorbance using a reader.

• DAY 2: Polyacrylamide Gel Electrophoresis (PAGE) and Transfer to Nitrocellulose

  • Take the tape off the bottom of the ReadyGel, remove the comb, and rinse with water.
  • Clamp into place.
  • Fill with SDS running buffer: Makes everything negative and has lots of ions, positives and negatives, just add salt to change the concentration.
    • Too much = hot current, and the gel will melt into solution.
    • Too little = too little current won’t run.
    • Glycine: Neutral amino acid, runs after the protein.
  • Mix standards with PBS and 4x protein loading buffer.
  • Heat and boil samples and blanks, BUT NOT STANDARDS.
    • Boiling for protein denaturation.
  • Run the gel at 200 V for 30 min.
  • Transfer proteins from SDS-PAGE to nitrocellulose membrane: Proteins stick to it.
    • Place the gel into cold transfer buffer.
      • Transfers proteins and promotes binding.
    • Wet the membrane by incubating in cold transfer buffer.
      • Positive behind the membrane, perpendicular current, horizontally with SDS run to positive.
      • If overrun, then the proteins will run past the membrane into the sponge.
      • Need to make sure to have the right concentration of ions in the buffer, too much salt will increase the current, and it can run through the membrane.
    • Run the membrane at 100 mA for 7 hrs.
  • Analyze the gel for remaining proteins with Coomassie Blue.
    • Disassemble and cover with Coomassie Blue, cover with saran wrap and lock overnight.
      • Tells if proteins are left on the gel.
  • Rock the gel on a rocker.
  • Save the membrane in a sealed container at 4 degrees C.

• DAY 3: Antibody Staining of the Membrane for β-actin, Treatment of the Cells with GF

  Rinse the membrane with water, then with blocking solution for 30 min, then put on a lab shaker. Blocking: Milk protein casein, a protein soup. Need blocking to eliminate background on the membrane, blocks non-specific binding, lots and lots more blocking, add primary antibody: Beta-actin – housekeeping gene; positive control; false-negative results, more blocking, secondary antibody: Horseradish peroxidase, can’t add a fluorescent probe since the membrane is too thick and cannot fluoresce. Negative control, false positive, washing solution, replace with Vector Red staining solution complete: Reducing dye; gives the enzyme what it needs to turn red.

Need to load the full amount of protein.

Too much confluency = not enough cyclin B1.

Too low confluency = not enough protein.