TISSUE REPAIR: REGENERATION, HEALING AND FIBROSIS

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TISSUE REPAIR REGENERATION, HEALING AND FIBROSIS

• Tissue repair is the response of organisms to
  overcome the damage caused by toxic insults,
  inflammation and trauma.
• The inflammatory response is not only the first
  to deal with any type of tissue injury, but also
  initiates the process of repair which consists of
  the restoration of tissue architecture and
  function.
• Some tissues are able to replace the damaged
  cells and tissues in its entirety (regeneration).
• If the tissue injury is too severe and
  regeneration is not possible, repair occurs by
  laying down fibrous tissue (healing) that results
  in scar formation.
• Injured tissue can usually function despite the
  presence of scars.

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• Fibrosis refers to the heavy deposition of
  collagen that occurs in organs such as lungs,
  liver and kidney following chronic inflammatory
  processes or in the myocardium after extensive
  ischemic necrosis (infarction).
• Fibrosis replacing purulent exudates is called
  organization.
• Repair involves the proliferation of different
  types of cells and their interaction with the ECM
  (extracellular matrix).

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CELL PROLIFERATION AND REGENERATION

• Tissue repair involves the proliferation of
  cells from
• a) the remnants of the injured tissue
• b) vascular endothelial cells to form new blood
  vessels
• c) fibroblasts which provide fibrous tissue for
  the formation of scars.
• The normal size of the cell population is
  determined by the balance between cell
  proliferation and cell death by apoptosis and the
  appearance of new differentiated cells produced
  by tissue stem cells.

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• The main steps in the proliferation of cells are
  DNA replication and mitosis and this sequence of
  events is known as the cell cycle.
• The cell cycle consists of several steps in
  order to check the accuracy of cell division.
• Non-dividing cells are either in cell cycle
  arrest in G1 or they exit the cycle (G0).
• Checkpoint controls prevent DNA replication or
  mitosis of damaged cells or eliminate damaged
  cells by apoptosis.

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• The major action of growth factors is to
  overcome these checkpoint controls by releasing
  suppressors of CDK activity (cyclin dependent
  kinases).

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• A. Tissue Repair
• The ability of tissues to repair themselves
  depends on their intrinsic proliferative
  capacity.
• Based on this principle, the tissues of the body
  can be divided into three groups
• a) continuously dividing tissues (labile
  tissues) such as hematopoietic cells of the bone
  marrow, stratified squamous epithelium, cuboidal
  epithelium of excretory ducts, and
  gastrointestinal tract.
• These tissues can easily regenerate after injury
  as long as stem cells are intact.

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• b) stable tissues cells of these tissues are
  quiescent and have minimal replicate activity.
  However, cells are able to replicate in response
  to injury or loss of tissue mass.
• Stable tissues constitute the parenchyma of most
  solid organs such as the liver, kidney, and
  pancreas, as well as endothelial cells,
  fibroblasts, and smooth muscle cells.
• With the exception of the liver, stable tissues
  have a limited capacity to regenerate.

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• c) Permanent tissues. The cells of these tissues
  are terminally differentiated in post-natal life.
  Cardiac muscle cells and most neurons are in this
  category. Injury to brain and heart muscle
  results in liquefaction, necrosis and scar
  formation.
• The liver has a great regenerative capacity that
  occurs after surgical removal or injury of
  hepatic tissue.
• As much as 40 to 60 of the liver may be
  removed in a procedure called living-donor
  transplantation. In this situation, replication
  after partial hepatectomy is initiated by the
  cytokines TNF and IL6 that trigger the transition
  of hepatocytes from stages g0 to g1 in the cell
  cycle.

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• HGF and EGF provide the progression of
  hepatocytes along the cell cycle.
• HGF is produced by fibroblasts, endothelial
  cells and liver non-paremchymal cells.
• HGF also stimulate the proliferation of most
  epithelial cells including those of the skin,
  breast, and lungs.

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• Extensive regeneration or compensatory
  hyperplasia can take place only if the residual
  tissue of the affected organ is structurally or
  functionally intact.
• Skeletal muscle is considered permanent tissue.
  However, cells attached to the endomysial sheet
  provide some regenerative capacity.

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• In continuously dividing tissues, mature cells
  are terminally differentiated and short-lived
  (for example, epidermal cells). The dead cells
  are replaced by cells produced by stem cells
  located at the basal layer of the epithelial
  lining. Cells differentiate progressively as they
  migrate to the upper layers of the mucosa.

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• Stem cells with the capacity to generate
  multiple cells lineages (pluripotent stem cells)
  can be isolated from embryos (ES cells).
• Stem cells with similar capacity have also been
  identified in the bone marrow with the capacity
  to generate fat, cartilage, bone, endothelium,
  muscle, as well as all myeloid and lymphoid
  hematopoietic elements.
• The new field of regenerative medicine deals
  with the regeneration and repopulation of injured
  organs using embryonic or adult stem cells.

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• B. Growth Factors
• Cell proliferation is triggered by many chemical
  mediators such as growth factors, hormones and
  cytokines.
• Growth factors are polypeptide molecules causing
  an expansion of cell populations which include an
  increase in cell size, mitotic activity and
  protection from apoptotic death (survival).
• In addition to stimulating cellular
  proliferation, they promote cellular migration,
  differentiation, contractibility, as well as
  enhancing the synthesis of special proteins such
  as collagen by fibroblasts.

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• C. Repair by Connective Tissue
• If extensive tissue surgery is performed or if a
  chronic inflammatory process causes damage to
  parenchymal cells, epithelia and stromal network,
  repair cannot take place by regeneration alone.
  The same thing happens when non-dividing cells
  are injured.
• In this situation, repair occurs by replacing
  the necrotic tissue with connective tissue or by
  the combination of regeneration of some cells and
  scar formation.
• The extracellular matrix (ECM) is an essential
  participant of the repair process.

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• D. Structure and Function of the ECM
• Tissue repair depends not only on growth factor
  activity, but also on interactions between cells
  and ECM components.
• The ECM is a dynamic, constantly remodeling
  macromolecular complex creating a network that
  surrounds every cell.

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• The ECM can be divided in two basic forms
  interstitial matrix, and basement membrane.
• The interstitial matrix is present in connective
  tissue between epithelium and supportive vascular
  and smooth muscle structures.
• It is synthesized by mesenchymal cells
  (fibroblasts) and tends to forms a
  three-dimensional amorphous gel.
• The basement membrane lies beneath the
  epithelium and is synthesized by overlying
  epithelium and underlying mesenchymal cells. It
  is a highly organized ECM around epithelial,
  endothelial, and smooth muscle cells.

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• The ECM has three basic components
• a) fibrous structural proteins such as collagens
  and elastins
• b) water hydrated gels, hyaluronan and
  proteoglycans
• c) adhesive glycoproteins and adhesion receptors

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• a) Collagens and elastins
• There are basically two types of collagen the
  fibrillar types (I, II, and V) and the
  non-fibrillar types (IV and IX).
• The fibrillar collagens form a major portion of
  the connective tissue of healing wounds and scars
  as well as the interstitial matrix. The synthesis
  of these collagens are Vitamin C dependent.
• The nonfibrillar collagen (Type IV) is a
  component of the basement membrane.

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• Elastin consists of a core of elastin surrounded
  by a mesh-like network of fibrillin glycoprotein
  and is the main component of elastic tissue.
• It has the ability to recoil and return to a
  baseline structure after physical stress.
• Elastin is also part of the interstitial matrix.

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• b) Proteoglycans and Hyaluronan
• Proteoglycans are highly hydrated, compressible
  proteins providing resilience and lubrication
  (cartilage and joints).
• It consists of long polysaccharides called
  glycosaminoglycans (heparan sulfates) linked to a
  protein backbone.
• Proteoglycans may be components of the cell
  membrane and have roles in cell proliferation,
  migration, and adhesion.
• Hyaluronan is composed of many disaccharide
  repeats without a protein core.
• It binds water, forming a viscous, gelatin-like
  matrix.

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• c) Adhesive glycoproteins and adhesion receptors
• These compounds are involved in cell to cell
  adhesion, the linkage between cells and ECM and
  binding between ECM components.
• The adhesive glycoproteins are fibronectin,
  which is a major component of the interstitial
  ECM and laminin which is part of the basement
  membrane.
• These are synthesized by fibroblasts, monocytes
  and epithelium.
• Fibronectin binds to many components of the ECM
  (collagen, proteoglycans) and can also attach to
  cell integrins.

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• There are two types of fibronectin tissue and
  plasma.
• Tissue fibronectin forms fibrillary aggregates
  at wound healing sites, while plasma fibronectin
  binds to fibrin to form a provisional clot of a
  wound that serves as the base for ECM deposition
  and reepithelization.
• Laminin is the most abundant glycoprotein of the
  basement membrane.
• It mediates attachment to the BM and modulates
  cell proliferation, differentiation, and motility.

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• Adhesion receptors, also known as CAMs (cell
  adhesion molecules), are divided in four groups,
  namely immunoglobulins, cadherins, selectins,
  and integrins.
• Integrins are present in most animal cells
  except red cells.
• These are present in the surface of leukocytes
  that mediate cell adhesion and transmigration and
  are the main cellular receptors for the ECM
  components.
• By linking to intracellular domains (actin)
  integrins initiate signaling cascades affecting
  cell locomotion, proliferation, and
  differentiation.

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• E. Summary of the roles of the ECM
• In addition to filling spaces around cells, the
  ECM does the following
• Provide mechanical support for cell anchorage,
  migration and maintenance of cell polarity.
• b) Control of cell growth by signaling through
  link with intracellular integrins.
• c) Affect the degree of differentiation of the
  cells in a given tissue via
  cell surface integrins.
• d) Provide scaffolding for the basement
  membrane and interstitial cellular matrix. The
  integrity of the ECM is critical for the
  organized regeneration of parenchymal cells.

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• e) Establishment of tissue microenvironments. The
  basement membrane acts as a boundary between
  epithelium and underlying connective tissue.
• f) Storage and presentation of regulatory
  molecules like growth factors FGF and HGF,
  synthesize by epithelial and stromal cells,
  allowing for rapid deployment of growth factors
  after local injury or during regeneration.

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Repair by Connective Tissue

• It involves the replacement of dead cells and
  tissues with connective tissue leading to scar
  formation. The sequence of this process is as
  follows
• 1) Repair begins within 24 hours of the time
  of injury by the emigration of fibroblasts and
  the induction of fibroblast and endothelial cell
  proliferation.
• 2) After 3 to 5 days, it begins the formation
  of granulation tissue which is pink and soft,
  with a granular appearance such as seen
  underneath the scab of an injured skin.
  Histologically, it is composed of proliferating
  fibroblasts, newly formed thin capillaries and
  loose ECM.

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Granulation Tissue
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• Angiogenesis.
• Blood vessels are formed by vasculogenesis
  originated by angioblasts (endothelial precursor
  cells (EPCs)) present in the bone marrow or by
  neovascularization, where preexisting blood
  vessels send out capillary sprouts to produce new
  vessels.
• Angiogenesis is involved in the development of
  collateral circulation at sites of ischemia and
  allowing tumors to increase in size. EPCs may
  migrate from the bone marrow to areas of injury
  but the homing mechanism is not known.

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Vasculogenesis
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• Capillaries present in granulation tissue are
  leaky due to defective endothelial junctions and
  because VEGF increases vascular permeability.
  This leakiness explains why granulation tissue is
  edematous, and the persistent presence of edema
  in healing wounds.
• ECM proteins participate in vessel sprouting
  through interaction with integrin receptors of
  endothelial cells. They also interfere with ECM
  cell interactions which facilitate the migration
  of endothelial cells.
• The most important growth factors involved in
  angiogenesis are VEGF and FGF2.

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• 3) Migration of fibroblasts and ECM deposition
  (scar formation).
• Scar formation takes place on the network of the
  newly formed granulation tissue and loose ECM.
  The scar develops in two steps
• Migration and proliferation of fibroblasts at the
  injury site, and
• b) deposition of ECM by fibroblasts.

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• The migration and proliferation of fibroblasts
  is triggered by several growth factors such as
  PDGF, FGF, and TGF2. These are synthesized by
  activated endothelial and inflammatory cells,
  especially macrophages which also clear
  extracellular debris and elaborate mediators that
  induce fibroblast proliferation and ECM
  components.
• The fibroblast migration starts early in
  wound healing, and continues for several weeks,
  depending on the size of the wound.
• As the healing progresses, there is a decrease
  of the number of proliferating fibroblasts and
  newly formed blood cells but there is an increase
  in the deposition of ECM, particularly collagen.

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• Eventually, the granulation tissue becomes a
  scar composed of inactive spindle-shaped
  fibroblasts, dense collagen, fragments of elastic
  tissue, and other ECM components.
• There is also a progressive vascular regression
  resulting in a pale avascular scar.

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Early Scar Formation
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Fully Developed Scar
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Normal Myocardium
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Acute Myocardial Infarction
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Healing Myocardial Infarct
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Healed Myocardial Infarct
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Healed Myocardial Infarct
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• TGFB is a potent fibrogenic agent. It simulates
  production of collagen, fibronectin, and
  proteoglycans, and it inhibits collagen
  degradation.
• It also inhibits lymphocyte proliferation and
  has a strong anti-inflammatory effect.
• PDGF is produced by endothelial cells, activated
  macrophages, smooth muscle cells and many tumor
  cells. It is stored in platelets and released on
  platelet activation. PDGF causes migration and
  proliferation of fibroblasts, smooth muscle cells
  and macrophages.
• Cytokines like 1L, and TNF may also function as
  growth factors by inducing fibroblastic
  proliferation and fibrogenic effects. They are
  also chemotactic for fibroblasts and stimulate
  the synthesis of collagen and collagenase.

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• 4) ECM tissue remodeling.
• After scar deposition, the ECM continues to be
  modified and remodeled. The outcome of the repair
  process depends on the balance between ECM
  synthesis and degradation.
• The degradation of collagen and ECM is done by
  matrix metalloproteinases (MMPs) which are zinc
  dependent. ECM can also be degraded by neutrophil
  elastase, cathepsin, plasmin, and other serine
  proteases.

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• MMPs are produced by fibroblasts, macrophages,
  synovial cells and some epithelial cells. Their
  synthesis and secretion are regulated by growth
  factors, cytokines and other agents. Their
  synthesis is inhibited by steroids, TGF, and TIMs
  (tissue inhibitors metalloproteinases). The
  activity of MMPs is tightly controlled.
• CUTANEOUS WOUND HEALING
• This process involves epithelial regeneration
  and scar formation.
• Reepithelialization of the wound surface happens
  by cell migration from the edges of the wound,
  triggered by the interaction between growth
  factors and the ECM.
• Cutaneous wound healing ends with scar formation
  following the steps already described. It may
  occur by first or second intention.

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• Healing by first intention or primary union
  takes place on wounds with focal damage of the
  basement membrane and the death of few epithelial
  and connective tissue cells.
• In this instance, epithelial regeneration
  predominates over fibrosis. A small scar is
  formed with minimal wound contraction. The
  incisional space is filled with fibrin clotted
  blood, rapidly invaded by granulation tissue and
  covered by new epithelium.
• The whole process is usually completed in two
  weeks, with the formation of a thin scar which is
  covered by normal epidermis in approximately one
  month.

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Normal Skin
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• Healing by second intention takes place when
  cell and tissue loss is extensive, such as large
  wounds, abscesses, ulcerations, or ischemic
  lesions of solid organs. This healing process is
  also called secondary union.
• In this type of healing, a large clot or scab
  rich in fibrin and plasma fibronectin forms at
  the surface of the wound. The inflammatory
  response is severe, because large tissue defects
  have a great volume of necrotic debris that must
  be removed.
• A large amount of granulation tissue is formed
  in order to fill the gap and to allow regrowth of
  epithelium and formation of a usually large scar.
• At the end of the process, there is contraction
  of the wound (5 to 10 of the original size,
  brought about by myofibroblasts).

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• Eventually there is a recovery of the tensile
  strength of the wound is brought about by
  collagen synthesis that exceeds degradation.
• After three months, wound strength reaches 70 to
  80 of the normal unwounded site.
• Pathologic aspects of repair
• Several conditions may alter the process of
  repair, causing either inadequate scar formation,
  or an exuberant deposition of collagen and ECM,
  leading to the formation of a prominent raised
  scar.

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• Production of an inadequate scar or delay in
  scar formation may be due to the following
• 1) Infection is the most common cause of delay in
  healing, by prolonging the inflammatory response
  and by increasing local tissue injury.
• 2) Poor nutrition and specially Vitamin C
  deficiency inhibits collagen synthesis and
  retards healing.
• 3) Glucocorticoids have a known anti-inflammatory
  effect and their administration results in poor
  wound strength due to diminished fibrosis.
• 4) Mechanical variables such as local pressure or
  torsion may cause wounds to pull apart or dehisce.

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• 5) Poor perfusion secondary to arteriosclerosis,
  diabetes, or obstructive venous drainage also
  impairs healing.
• 6) Foreign bodies like glass fragments, steel
  fragments, or bone may disturb healing.
• Aberrations of cell growth may be due to a
  genetic predisposition (Keloid formation). This
  condition is more common in African American
  patients.

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Review of Repair Responses