Tissue Repair: Regeneration and Scarring

Tissue Repair Overview

• Tissue repair involves restoring tissue architecture and function after injury.
• It is often referred to as healing.
• Tissue repair occurs through regeneration and connective tissue deposition (scarring).

Regeneration

• Regeneration is cell proliferation, resulting in complete restitution of lost or damaged tissue components.
• It involves proliferation of residual uninjured cells, and maturation of stem cells.
• True regeneration does not occur in mammals, and usually applies to processes like liver growth after partial resection or necrosis.

Connective Tissue Deposition (Scarring)

• Connective tissue deposition occurs if tissues are incapable of complete restitution or experience severe damage to supporting structures.
• The resulting fibrous scar is not normal tissue, but provides enough structural stability for the injured tissue to function.
• The type of injured tissue and extent of the injury determine the relative contributions of regeneration and scarring in tissue repair.
• In inflammation of tissue spaces, exudate is often digested and resorbed.
• If fibrosis develops it is called organization.

Mechanism of Tissue Repair

• Tissue repair occurs through regeneration following mild injury.
• Tissue repair also occurs through scar formation after more severe injury with damage to the connective tissue.

Cell and Tissue Regeneration: The Cell Cycle & Tissue Types

• Tissues are divided into labile, stable and permanent groups based on their intrinsic proliferative capacity.

Labile Tissues

• Labile tissues' cells are continuously being lost and replaced through maturation of tissue stem cells and proliferation of mature cells.
• Labile tissues can readily regenerate as long as the pool of stem cells is preserved.
• Labile tissues include hematopoietic cells of the bone marrow and majority of surface epithelia.
• Examples include squamous cells in the skin, oral cavity, esophagus, and cervix, cuboidal cells in excretory glands and bile ducts, and columnar cells in the endometrium and GIT mucosa.

Stable Tissues

• Stable tissues' cells are quiescent (in G0), and proliferation is normally minimal.
• Stable tissues' cells divide in response to injury or loss of tissue mass.
• They include parenchyma of solid organs like the liver, kidney, and pancreas, endothelial cells, fibroblasts, and smooth muscle cells.
• With the exception of the liver, stable tissues have a limited capacity to regenerate after injury.

Permanent Tissues

• Permanent tissues' cells are terminally differentiated and nonproliferative.
• Permanent tissues include the majority of neurons, cardiac muscle cells, and skeletal muscle (with satellite cells providing some regenerative capacity).
• Injury in permanent tissues is irreversible.
• Repair in permanent tissues is dominated by scar formation.

Cell Cycle Checkpoints

• Cell cycle checkpoints occur at the G1/S and G2/M transitions
• The cell cycle checkpoints are tightly regulated by a balance of growth-promoting and growth-suppressing proteins and sensors of DNA damage.
• Activated DNA-damage sensors transmit signals that arrest cell cycle and initiate apoptosis if the damage cannot be repaired.
• Defects in the G1/S checkpoint are more important in cancer, leading to dysregulated growth and impaired DNA repair, resulting in a "mutator" phenotype

G1 Restriction Point

• The G1 restriction point is the stage in G1 when the cell is committed to advance further into the cell cycle without needing more growth signals.
• Cells from labile tissues cycle continuously.
• Stable cells are quiescent but can enter the cell cycle.
• Permanent cells have lost their proliferative capacity and have left the cell cycle.

Signals and Control Mechanisms

• Cell proliferation is driven by signals provided by growth factors (GFs) and the extracellular matrix (EC).
• Many different GFs have been described, with some acting on multiple cell types and others being cell-selective.
• Growth factors are produced by cells near the site of damage, especially macrophages, epithelial, and stromal cells.
• GFs activate signaling pathways and induce the production of proteins involved in driving cells through the cell cycle and releasing blocks on the cell cycle.
• Cells also use integrins to bind to ECM proteins and signals from the integrins stimulate cell proliferation.
• Tissue stem cells, which live in specialized niches, are the most important stem cells for regeneration in adults.
• Injury can trigger signals in these niches that activate stem cells to proliferate and differentiate.

Mechanisms of Tissue Regeneration in Labile Tissues

• Injured cells are rapidly replaced by proliferation of residual cells and differentiation of tissue stem cells if the underlying BM is intact.
• Growth factors (GFs) involved are not defined.
• Loss of blood cells is corrected by the proliferation of hematopoietic stem cells in the bone marrow and other tissues.
• This proliferation is driven by GFs called CSFs.
• Lifespan of blood cells: RBCs is around 120 days, platelets is about 9 to 12 days, and WBCs is 13 to 20 days.

Mechanisms of Tissue Regeneration in Parenchymal Organs with Stable Cell Populations

• Except for the liver, regeneration is usually a limited process.
• The pancreas, adrenal, thyroid, and lungs have some regenerative capacity.
• Surgical removal of a kidney elicits hypertrophy and hyperplasia of proximal duct cells in the remaining kidney.
• Mechanisms underlying this response are not understood but likely involve local production of GFs and interactions of cells with ECM.

Liver Regeneration

• Liver regeneration occurs by proliferation of remaining hepatocytes and repopulation from progenitor cells.
• The nature of the injury determines which mechanism plays the dominant role.
• Extensive destruction with collapse of the reticulin framework leads to scar formation.
• Resection of up to 90% of the liver can be corrected by proliferation of residual cells.

Proliferation of Hepatocytes Following Partial Hepatectomy

• Proliferation is triggered by the combined action of cytokines and polypeptide GFs.
• The process occurs in two distinct stages.
• Priming (1st) phase: cytokines like IL-6 from Kupffer cells make liver cells ready to receive and respond to GF signals.
• Proliferation (2nd) phase: GFs (HGF and TGF-α) produced by many cell types stimulate primed cell metabolism and entry into the cell cycle.
• Hepatocytes are quiescent cells, so it takes them several hours to enter the cell cycle (G0 to G1) and reach the S phase of DNA replication.
• Almost all hepatocytes replicate, followed by replication of nonparenchymal cells like Kupffer cells, endothelial cells, and stellate cells (termination (final) phase).
• Hepatocytes return to quiescence.
• The nature of the stop signals is poorly understood, but antiproliferative cytokines of the TGF-β family are likely to be involved.

Liver Regeneration from Progenitor Cells

• The proliferative capacity of hepatocytes is impaired due to chronic liver injury or inflammation.
• Progenitor cells in the liver contribute to repopulation.

Repair by Connective Tissue Deposition

• The repair cannot be accomplished by regeneration alone.
• It occurs if repair occurs by scar formation, which replaces the tissue with connective tissue.
• Repair also occurs by a combination of regeneration and scar formation.
• Scarring may happen if the injury is severe or chronic, or if there is damage to parenchymal cells, epithelia, or connective tissue framework.
• Scarring also happens if non-dividing cells are injured.
• In contrast to regeneration, scar formation "patches" rather than restores the tissue.

Steps in Scar Formation

• Consists of sequential processes that follow tissue injury and the inflammatory response.
• Steps include angiogenesis, granulation tissue formation, and remodeling.

Angiogenesis

• Angiogenesis involves sprouting of new vessels from existing ones.
• Critical in healing at sites of injury, it supplies nutrients and oxygen that are needed.
• Angiogenesis is involved in the development of collateral circulation at sites of ischemia and allows tumors to increase in size.
• Newly formed vessels are leaky because of incomplete inter-endothelial junctions and vascular endothelial growth factor (VEGF) induced permeability.
• Steps include: vasodilation in response to NO, increased permeability induced by VEGF, separation of pericytes from the abluminal surface, breakdown of the basement membrane to allow formation of a vessel sprout, migration of endothelial cells toward the area of injury, proliferation of endothelial cells just behind the leading front ("tip") of migrating cells, remodeling into capillary tubes and recruitment of periendothelial cells to form the mature vessel and, finally, suppression of endothelial proliferation & migration and deposition of the basement membrane.
• Angiogenesis involves several signaling pathways, cell-cell interactions, ECM proteins, tissue enzymes, and growth factors.
  • VEGFs, especially VEGF-A, stimulate both migration and proliferation of endothelial cells, thus initiating the process of capillary sprouting, and promotes vasodilation by stimulating the production of NO.
  • FGFs, especially FGF-2, stimulate the proliferation of endothelial cells, promote the migration of macrophages and fibroblasts, and stimulate epithelial cell migration to cover the wounds.
  • Angiopoietins 1 and 2 play a role in angiogenesis and maturation of new vessels and stabilize the newly formed vessels by recruiting pericytes and smooth muscle cells and depositing connective tissue.
  • PDGF and TGF-β also participate in the stabilization process: PDGF recruits smooth muscle cells, and TGF-β suppresses endothelial proliferation and migration and enhances the production of ECM proteins.
  • Notch signaling regulates the sprouting and branching of new vessels with proper spacing to effectively supply the healing tissue, ECM proteins participate in the process of angiogenesis through interactions with integrin receptors in endothelial cells and providing the scaffold for vessel growth.
  • Enzymes in the ECM, notably the matrix metalloproteinases (MMPs), degrade the ECM to permit remodeling and extension of the vascular tube.

Formation of Granulation Tissue

• Granulation tissue involves migration and proliferation of fibroblasts and deposition of loose connective tissue, together with vessels and leukocytes, to form granulation tissue.
• Histology shows proliferation of fibroblasts, new thin-walled capillaries, loose ECM, and mixed inflammatory cells (mainly macrophages).
• Granulation tissue progressively invades the site of injury.

Deposition of Connective Tissue

• Occurs in two steps: migration and proliferation of fibroblasts into the site of injury, and deposition of ECM proteins produced by these cells.
• These processes are orchestrated by locally produced cytokines and GFs, particularly PDGF, FGF-2, and TGF-β.
• Major sources of these factors are inflammatory cells, especially alternatively activated (M2) macrophages, at the sites of injury & in granulation tissue.
• Transforming growth factor-β (TGF-β) is most important for synthesis and deposition of connective tissue proteins and is produced by most of the cells in granulation tissue.
• Trasforming growth factor -β (TGF-β) stimulates migration and proliferation of fibroblasts, increases synthesis of collagen and fibronectin, decreases degradation of extracellular matrix, and is involved in fibrosis that follows chronic inflammation in the lung, liver, and kidneys and is also antiinflammatory, limiting and terminating inflammation by inhibiting lymphocyte proliferation and other leukocytes.

Collagen Synthesis

• Collagen synthesis begins early (days 3-5) and continues for several weeks.
• Net accumulation depends on both increased synthesis & diminished collagen degradation.
• Finally, granulation tissue evolves into a scar composed of inactive, spindle-shaped fibroblasts, dense collagen, fragments of elastic tissue, and other extracellular matrix components.
• As the scar matures, some fibroblasts acquire features of smooth muscle cells (myofibroblasts) and contribute to contraction of the scar over time.

Remodeling of Connective Tissue

• The outcome of the repair is a balance between synthesis and degradation of ECM proteins.
• The deposited connective tissue continues to be modified and remodeled.
• The amount increases with maturation and reorganization, eventually producing the stable fibrous scar.
• Degradation of collagens and other ECM components is accomplished by a family of MMPs.
  • MMPs include interstitial collagenases (cleave fibrillar collagen), gelatinases (degrade amorphous collagen and fibronectin), and stromelysins (degrade ECM constituents), and are produced by fibroblasts, macrophages, neutrophils, synovial cells, and some epithelial cells.
• MMP synthesis and secretion are regulated by GFs, cytokines, and other agents.
• Activity of the MMPs is tightly controlled.
• MMPs are produced as inactive precursors (zymogens) that must be first activated via proteases (e.g. plasmin) present only at sites of injury.
• Activated collagenases can be rapidly inhibited by specific tissue inhibitors of MMPs (TIMPs) produced by most mesenchymal cells.
• During scar formation, MMPs are activated to remodel the deposited ECM, and then their activity is shut down by TIMPs.
• A family of enzymes related to MMPs is called ADAM (a disintegrin metalloproteinase ), which are anchored to the plasma membrane, and cleave and release EC domains of cell-associated cytokines and GFs like TNF, TGF-β, & members of the EGF family.

Phases of Repair by Scar Formation

• The four phases repair by scar formation: Hemostasis phase, inflammatory phase, proliferative phase, and maturation phase.

Factors Influencing Tissue Repair

• Tissue repair may be altered by a variety of influences, frequently reducing the quality or adequacy of the reparative process.

Variables that Modify Healing

• Extrinsic or intrinsic factors to the injured tissue & systemic or local factors.

Infection

• One of the most important causes of delay in healing, and prolongs inflammation & increases local tissue injury.

Diabetes

• Compromises repair and is one of the most important systemic causes of abnormal wound healing.

Nutritional Status

• Protein deficiency, & mainly vitamin C deficiency, inhibits collagen synthesis & retards healing.

Glucocorticoids

• Have anti-inflammatory effects that inhibit TGF-ẞ production & diminished fibrosis.
• Reduce the transcription of genes encoding COX-2, phospholipase A2, and Proinflammatory cytokines (e.g., IL-1).

Mechanical Factors

• Increased pressure or torsion may cause wound dehiscence.

Poor Perfusion

• Arteriosclerosis or obstructed venous drainage (varicose veins) impairs healing.

Foreign Bodies

• Impede healing.

Type and Extent of Tissue Injury

• Stable & labile tissues: Complete restoration; but extensive injury leads to incomplete regeneration.
• Permanent tissue: Result in scarring.

Location of the Injury and the Character of the Tissue.

• In tissue space = exudation > digestion > resolution
• If a great deal of tissue is damaged it results in organization & scarring.

Examples of Tissue Repair and Fibrosis

• Healing of skin wounds, can occur by First, Second, or Third intention.

Healing of Skin Wounds by First Intention: Healing by Primary Union

• This is often called primary union.
• It occurs if the wound is clean and the tissue loss is minimal.
• There is a focal disruption of BM continuity and minimal death of epithelial and connective tissue cells.
• Wound edges are closely approached, and the main mechanism of repair is epithelial regeneration.
• Example - Clean surgical incision approximated by sutures.

Phases of Repair by Healing by First Intention:

• The four connected processes are hemostasis, inflammatory phase, proliferative phase, and maturation phase. Hemostasis phase: occurs immediately after injury. Blood vessels constrict, which restricts blood flow
• Formation of platelet plug seals the break & coagulation via fibrin reinforce the platelet plug
• The Clot
  • Contains Fibrin, Fibronectin, Complement proteins & Entrapped RBCS
  • Stops the bleeding by acting as a scaffold for migrating cells.
  • A Scab is formed as dehydration occurs at the external surface, prevents dehydration and facilitates rapid healing
• Inflammatory phase Releases VEGF, leading to increased permeability & edema. Within 24 hours neutrophils are seen at the incision margin.
  • Neutrophils release proteolytic enzymes, beginning to clear the debris, beginning to clear the debris
  • Basal cells of the epidermis show increased mitosis.
  • By day 3 neutrophils are replaced by macrophages. Macrophages clear extracellular debris, Fibrin & other foreign material
• Proliferative phase After 24 to 48 hours:
  • Epithelial cells begin to migrate & proliferate along the dermis, and deposit basement membrane as they progress.
  • Macrophages promote angiogenesis, and deposition of the ground substance (ECM):Type III collagin & fibronetin
  • Granulation tissue progressively invades the incision space.
  • Epithelial cell proliferation continues forming a covering approaching the normal thickness of the epidermis.
• Proliferatin and migration by Day 5
  • Neovascularization reaches its peak and As granulation tissue fills the incisional space.
  • Fibroblasts produce ECM proteins & collagen fibrils that become more abundant & begin to bridge the incision.
  • The Epidermis matures of normal thickness with keratinization. Migration is driven by chemokines, TNF, PDGF, TGF-ß, & FGF.
  • Subsequent proliferation is triggered by multiple GFs, including (PDGF, EGF, TGF-ß, & FGF, and the cytokines IL-1 & TNF).
  • Macrophages are the main source for these factors, followed by inflammatory cells & platelets that may also produce them.
• Maturation phase,During the second week
  • Collagen deposition & fibroblast proliferation are continued. Leukocytes, edema, & vascularity are markedly reduced.
  • By the end of the first month the scar comprises aceullular connective tissue, with no or minimal inflammatory cells and normal covering of epidermis -Dermal appendages are permanently lost, and the tensile strength of the wound increases with time overtime

Healing by Second Intention: Healing by Secondary Union

• Also known as healing by secondary union. It involves the healing of large wounds.
• Here - Tissue loss is more extensive, there may be an infection, and Wound edges can not be approximated.
• Repair involves the four stages of wound healing, A combination of regeneration & scarring, and Abundant ECM proteins, & collagen fibrils deposition.
• The inflammatory reaction is more intense
  • Greater volume of necrotic debris, exudate & fibrin; & hence secondary inflammation-mediated injury.
  • Abundant Granulation tissue to fill in the gaps, it results in a greater mass of scar tissue.
• At first a provisional matrix is formed
  • Containing fibrin, plasma fibronectin, & type III collagen that is changed in about 2 weeks to type I collagen.
    • Dense collagen, fragments of elastic tissue, & Other ECM components, is converted into a pale, avascular scar, with spindle-shaped fibroblasts.
  • Destroyed dermal appendages in the line of incision are permanently lost and epidermis recovers its normal thickness & architecture.
• By the end of the first month the scar is made up of Acellular connective tissue devoid of inflammatory infiltrate
  • Covered by intact epidermis.