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REGENERATION
HEALING
(repair)
LEARNING OBJECTIVES
• Review the normal physiology and concepts
of cell proliferation, cell growth, cell “cycle”,
and cell differentiation
• Understand the basic factors of tissue
regeneration
• Understand the relationships between cells
and their ExtraCellular Matrix (ECM)
• Understand the roles of the major players of
healing---angiogenesis, growth factors
(GFs), and fibrosis
• Differentiate 1st & 2nd intention healing
DEFINITIONS:

• REGENERATION: Growth of
cells to replace lost tissues

• HEALING: A reparative tissue
response to a wound, inflammation or necrosis,
often leads to fibrosis

• GRANULATION TISSUE
• “ORGANIZING” INFLAMATION
REGENERATION
• Replacement of lost structures
• Is dependent on the type of
normal turnover the original
tissue has
• Can be differentiated from
“compensatory” growth
HEALING (repair)
• Needs a wound, inflammatory process, or
necrosis
• Many disease appearances anatomically are
the result of “healing” such as
atherosclerosis
• Often ends with a scar
• Fibrosis, as one of the 3 possible outcomes
of inflammation, follows “healing”
• Requires a connective tissue “scaffold”
• Fibrosis occurs in proportion to the damage
of the ECM
Minarcik robbins 2013_ch3-regen
Cell Population Fates
• PROLIFERATION
– Hormonal, especially steroid hormones
– eg., EPO, CSF

• DIFFERENTIATION

*

– UNIDIRECTIONAL, GAIN (specialization)
and LOSS (versatility)

• APOPTOSIS

*One of the most KEY concepts in neoplasia
ECTODERM
MESODERM
ENTODERM
Minarcik robbins 2013_ch3-regen
CELL CYCLE
• G0
– Quiescent (not a very long or dominent phase)

• G1
– PRE-synthetic, but cell GROWTH taking place

• S
– Cells which have continuous “turnover” have
longer, or larger S-phases, i.e., DNA synthesis
– S-phase of TUMOR CELLS can be prognostic

• G2
– PRE-mitotic

• M (Mitotic:, P,M,A,T, Cytokinesis)
CELL TYPES
• Labile: eg., marrow, GI
• Quiescent: liver, kidney
• NON-mitotic: neuron,
striated muscle
STEM CELLS
(TOTIPOTENTIAL*)

• EMBRYONIC
• ADULT
EMBRYONIC
STEM CELLS
• DIFFERENTIATION
• KNOCKOUT MICE (mice raised
with specific gene defects)
• REPOPULATION OF DAMAGED
TISSUES, in research
ADULT
STEM CELLS
• MARROW
(HEMOCYTOBLAST)
(hematopoetic stem cells)

• NON-MARROW
(RESERVE)
MARROW STROMAL CELL
ADULT TISSUE DIFFERENTIATION and
REGENERATION PARALLELS EMBRYONIC
DEVELOPMENT
Growth Factors (GFs)
• Polypeptides
• Cytokines

• LOCOMOTION
• CONTRACTILITY
• DIFFERENTIATION
• ANGIOGENESIS
Growth Factors (GFs)
•
•
•
•
•
•
•
•

Epidermal
Transforming (alpha, beta)
Hepatocyte
Vascular Endothelial
Platelet Derived
Fibroblast
Keratinocyte
Cytokines (TNF, IL-1, Interferons)
Minarcik robbins 2013_ch3-regen
CELL PLAYERS
(source AND targets)
•
•
•
•
•
•
•
•

Lymphocytes, especially T-cells
Macrophages
Platelets
Endothelial cells
Fibroblasts
Keratinocytes
“Mesenchymal” cells
Smooth muscle cells
E (Epidermal) GF
• Made in platelets, macrophages
• Present in saliva, milk, urine, plasma
• Acts on keratinocytes to migrate,
divide
• Acts on fibroblasts to produce
“granulation” tissue
T (Transforming) GF-alpha
• Made in macrophages, T-cells,
keratinocytes
• Similar to EGF, also effect on
hepatocytes
H (Hepatocyte) GF
• Made in “mesenchymal” cells
• Proliferation of epithelium,
endothelium, hepatocytes
• Effect on cell “motility”
VE (Vascular Endothelial) GF
•
•
•
•
•

Made in mesenchymal cells
Triggered by HYPOXIA
Increases vascular permeability
Mitogenic for endothelial cells
KEY substance in promoting
“granulation” tissue
PD (Platelet Derived) GF
• Made in platelets, but also MANY
other cell types
• Chemotactic for MANY cells
• Mitogen for fibroblasts
• Angiogenesis
• Another KEY player in granulation
tissue
F (Fibroblast) GF
• Made in MANY cells
• Chemotactic and mitogenic, for
fibroblasts and keratinocytes
• Re-epithelialization
• Angiogenesis, wound contraction
• Hematopoesis
• Cardiac/Skeletal (striated) muscle
T (Transforming) GF-beta
• Made in MANY CELLS
• Chemotactic for PMNs and MANY
other types of cells
• Inhibits epithelial cells
• Fibrogenic
• Anti-Inflammatory
K (Keratinocyte) GF
• Made in fibroblasts
• Stimulates
keratinocytes:
– Migration
– Proliferation
– Differentiation
I (Insulin-like) GF-1
• Made in macrophages, fibroblasts
• Stimulates:
– Sulfated proteoglycans
– Collagen
– Keratinocyte migration
– Fibroblast proliferation

• Action similar to GH (Pituitary
Growth Hormone)
TNF (Tumor Necrosis Factor)
• Made in macrophages, mast cells,
T-cells
• Activates macrophages (cachexin)
• KEY influence on other cytokines
• The MAJOR TNF is TNF-alpha
Interleukins
• Made in macrophages, mast cells,
T-cells, but also MANY other cells
• MANY functions:
– Chemotaxis
– Angiogenesis
– REGULATION of other cytokines
INTERFERONS
• Made by lymphocytes,
fibroblasts
• Activates MACROPHAGES
• Inhibits FIBROBLASTS
• REGULATES other cytokines
SIGNALING

• Autocrine (same cell)

• Paracrine (next door neighbor)
(many GFs)

• Endocrine (far away, delivered
by blood, steroid hormones)
Minarcik robbins 2013_ch3-regen
TRANSCRIPTION FACTORS
HEPATIC
REGENERATION
TNF
IL6
HGF
ExtraCellular Matrix (ECM)
•
•
•
•

Collagen(s) I-XXVII
Elastin
Fibrillin
CAMs (Cell Adhesion Molecules)
– Immunoglobulins, cadherins, integrins,
selectins

• Proteoglycans
• Hyaluronic Acid
ECM
•
•
•
•

Maintain cell differentiation
“Scaffolding”
Establish microenvironment
Storage of GF’s
Collagen One - b

ONE (main component of bone)

Collagen Two - car

TWOlage (main component of cartilage)

THREEculate (main component of reticular fibers)
Collagen Four - FLOOR - forms the basement membrane
Collagen Three - re
GENETIC COLLAGEN DISORDERS
•
•
•
•
•
•
•
•
•
•
•

I
OSTEOGENESIS IMPERFECTA, E-D
II
ACHONDROGENESIS TYPE II
III
VASCULAR EHLERS-DANLOS
V
CLASSICAL E-D
IX
STICKLER SYNDROME
IV
ALPORT SYNDROME
VI
BETHLEM MYOPATHY
VII
DYSTROPHIC EPIDERMOLYSIS BULLOS.
IX
EPIPHYSEAL DYSPLASIAS
XVII
GEN. EPIDERMOLYSYS BULLOSA
XV, XVIII KNOBLOCH SYNDROME
DEFINITIONS:
• REGENERATION:
Growth of cells to replace lost tissues

• HEALING: A reparative tissue
response to a wound, inflammation or
necrosis
HEALING

• FOLLOWS INFLAMMATION
• PROLIFERATION and MIGRATION of
connective tissue cells
• ANGIOGENESIS (Neovascularization)
• Collagen, other ECM protein synthesis
• Tissue Remodeling
• Wound contraction
• Increase in wound strength (scar = fibrosis)
ANGIOGENESIS
(NEOVASCULARIZATION)

• From endothelial precursor cells
• From PRE-existing vessels
• Stimulated/Regulated by GF’s,
especially VEGF
• Also regulated by ECM proteins
• aka, “GRANULATION”, “GRANULATION
TISSUE”, “ORGANIZATION”,
“ORGANIZING INFLAMMATION”
Minarcik robbins 2013_ch3-regen
Minarcik robbins 2013_ch3-regen
Minarcik robbins 2013_ch3-regen
WOUND HEALING
• 1st INTENTION • 2nd INTENTION
• Edges lined up

•
•
•
•

Edges NOT lined up
Ergo….
More granulation
More
epithelialization
• MORE FIBROSIS
Minarcik robbins 2013_ch3-regen
“HEALTHY” Granulation Tissue
FIBROSIS/SCARRING
• DEPOSITION OF COLLAGEN by
FIBROBLASTS
• With time (weeks, months,
years?) the collagen becomes
more dense, ergo, the tissue
becomes “STRONGER”
Wound RETARDING factors
(LOCAL)
• DECREASED Blood supply
• Denervation
• Local Infection
• FB
• Hematoma
• Mechanical stress
• Necrotic tissue
Wound RETARDING factors
(SYSTEMIC)
• DECREASED Blood supply
• Age
• Anemia
• Malignancy
• Malnutrition
• Obesity
• Infection
• Organ failure

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Minarcik robbins 2013_ch3-regen

  • 2. LEARNING OBJECTIVES • Review the normal physiology and concepts of cell proliferation, cell growth, cell “cycle”, and cell differentiation • Understand the basic factors of tissue regeneration • Understand the relationships between cells and their ExtraCellular Matrix (ECM) • Understand the roles of the major players of healing---angiogenesis, growth factors (GFs), and fibrosis • Differentiate 1st & 2nd intention healing
  • 3. DEFINITIONS: • REGENERATION: Growth of cells to replace lost tissues • HEALING: A reparative tissue response to a wound, inflammation or necrosis, often leads to fibrosis • GRANULATION TISSUE • “ORGANIZING” INFLAMATION
  • 4. REGENERATION • Replacement of lost structures • Is dependent on the type of normal turnover the original tissue has • Can be differentiated from “compensatory” growth
  • 5. HEALING (repair) • Needs a wound, inflammatory process, or necrosis • Many disease appearances anatomically are the result of “healing” such as atherosclerosis • Often ends with a scar • Fibrosis, as one of the 3 possible outcomes of inflammation, follows “healing” • Requires a connective tissue “scaffold” • Fibrosis occurs in proportion to the damage of the ECM
  • 7. Cell Population Fates • PROLIFERATION – Hormonal, especially steroid hormones – eg., EPO, CSF • DIFFERENTIATION * – UNIDIRECTIONAL, GAIN (specialization) and LOSS (versatility) • APOPTOSIS *One of the most KEY concepts in neoplasia
  • 10. CELL CYCLE • G0 – Quiescent (not a very long or dominent phase) • G1 – PRE-synthetic, but cell GROWTH taking place • S – Cells which have continuous “turnover” have longer, or larger S-phases, i.e., DNA synthesis – S-phase of TUMOR CELLS can be prognostic • G2 – PRE-mitotic • M (Mitotic:, P,M,A,T, Cytokinesis)
  • 11. CELL TYPES • Labile: eg., marrow, GI • Quiescent: liver, kidney • NON-mitotic: neuron, striated muscle
  • 13. EMBRYONIC STEM CELLS • DIFFERENTIATION • KNOCKOUT MICE (mice raised with specific gene defects) • REPOPULATION OF DAMAGED TISSUES, in research
  • 14. ADULT STEM CELLS • MARROW (HEMOCYTOBLAST) (hematopoetic stem cells) • NON-MARROW (RESERVE)
  • 16. ADULT TISSUE DIFFERENTIATION and REGENERATION PARALLELS EMBRYONIC DEVELOPMENT
  • 17. Growth Factors (GFs) • Polypeptides • Cytokines • LOCOMOTION • CONTRACTILITY • DIFFERENTIATION • ANGIOGENESIS
  • 18. Growth Factors (GFs) • • • • • • • • Epidermal Transforming (alpha, beta) Hepatocyte Vascular Endothelial Platelet Derived Fibroblast Keratinocyte Cytokines (TNF, IL-1, Interferons)
  • 20. CELL PLAYERS (source AND targets) • • • • • • • • Lymphocytes, especially T-cells Macrophages Platelets Endothelial cells Fibroblasts Keratinocytes “Mesenchymal” cells Smooth muscle cells
  • 21. E (Epidermal) GF • Made in platelets, macrophages • Present in saliva, milk, urine, plasma • Acts on keratinocytes to migrate, divide • Acts on fibroblasts to produce “granulation” tissue
  • 22. T (Transforming) GF-alpha • Made in macrophages, T-cells, keratinocytes • Similar to EGF, also effect on hepatocytes
  • 23. H (Hepatocyte) GF • Made in “mesenchymal” cells • Proliferation of epithelium, endothelium, hepatocytes • Effect on cell “motility”
  • 24. VE (Vascular Endothelial) GF • • • • • Made in mesenchymal cells Triggered by HYPOXIA Increases vascular permeability Mitogenic for endothelial cells KEY substance in promoting “granulation” tissue
  • 25. PD (Platelet Derived) GF • Made in platelets, but also MANY other cell types • Chemotactic for MANY cells • Mitogen for fibroblasts • Angiogenesis • Another KEY player in granulation tissue
  • 26. F (Fibroblast) GF • Made in MANY cells • Chemotactic and mitogenic, for fibroblasts and keratinocytes • Re-epithelialization • Angiogenesis, wound contraction • Hematopoesis • Cardiac/Skeletal (striated) muscle
  • 27. T (Transforming) GF-beta • Made in MANY CELLS • Chemotactic for PMNs and MANY other types of cells • Inhibits epithelial cells • Fibrogenic • Anti-Inflammatory
  • 28. K (Keratinocyte) GF • Made in fibroblasts • Stimulates keratinocytes: – Migration – Proliferation – Differentiation
  • 29. I (Insulin-like) GF-1 • Made in macrophages, fibroblasts • Stimulates: – Sulfated proteoglycans – Collagen – Keratinocyte migration – Fibroblast proliferation • Action similar to GH (Pituitary Growth Hormone)
  • 30. TNF (Tumor Necrosis Factor) • Made in macrophages, mast cells, T-cells • Activates macrophages (cachexin) • KEY influence on other cytokines • The MAJOR TNF is TNF-alpha
  • 31. Interleukins • Made in macrophages, mast cells, T-cells, but also MANY other cells • MANY functions: – Chemotaxis – Angiogenesis – REGULATION of other cytokines
  • 32. INTERFERONS • Made by lymphocytes, fibroblasts • Activates MACROPHAGES • Inhibits FIBROBLASTS • REGULATES other cytokines
  • 33. SIGNALING • Autocrine (same cell) • Paracrine (next door neighbor) (many GFs) • Endocrine (far away, delivered by blood, steroid hormones)
  • 36. ExtraCellular Matrix (ECM) • • • • Collagen(s) I-XXVII Elastin Fibrillin CAMs (Cell Adhesion Molecules) – Immunoglobulins, cadherins, integrins, selectins • Proteoglycans • Hyaluronic Acid
  • 38. Collagen One - b ONE (main component of bone) Collagen Two - car TWOlage (main component of cartilage) THREEculate (main component of reticular fibers) Collagen Four - FLOOR - forms the basement membrane Collagen Three - re
  • 39. GENETIC COLLAGEN DISORDERS • • • • • • • • • • • I OSTEOGENESIS IMPERFECTA, E-D II ACHONDROGENESIS TYPE II III VASCULAR EHLERS-DANLOS V CLASSICAL E-D IX STICKLER SYNDROME IV ALPORT SYNDROME VI BETHLEM MYOPATHY VII DYSTROPHIC EPIDERMOLYSIS BULLOS. IX EPIPHYSEAL DYSPLASIAS XVII GEN. EPIDERMOLYSYS BULLOSA XV, XVIII KNOBLOCH SYNDROME
  • 40. DEFINITIONS: • REGENERATION: Growth of cells to replace lost tissues • HEALING: A reparative tissue response to a wound, inflammation or necrosis
  • 41. HEALING • FOLLOWS INFLAMMATION • PROLIFERATION and MIGRATION of connective tissue cells • ANGIOGENESIS (Neovascularization) • Collagen, other ECM protein synthesis • Tissue Remodeling • Wound contraction • Increase in wound strength (scar = fibrosis)
  • 42. ANGIOGENESIS (NEOVASCULARIZATION) • From endothelial precursor cells • From PRE-existing vessels • Stimulated/Regulated by GF’s, especially VEGF • Also regulated by ECM proteins • aka, “GRANULATION”, “GRANULATION TISSUE”, “ORGANIZATION”, “ORGANIZING INFLAMMATION”
  • 46. WOUND HEALING • 1st INTENTION • 2nd INTENTION • Edges lined up • • • • Edges NOT lined up Ergo…. More granulation More epithelialization • MORE FIBROSIS
  • 49. FIBROSIS/SCARRING • DEPOSITION OF COLLAGEN by FIBROBLASTS • With time (weeks, months, years?) the collagen becomes more dense, ergo, the tissue becomes “STRONGER”
  • 50. Wound RETARDING factors (LOCAL) • DECREASED Blood supply • Denervation • Local Infection • FB • Hematoma • Mechanical stress • Necrotic tissue
  • 51. Wound RETARDING factors (SYSTEMIC) • DECREASED Blood supply • Age • Anemia • Malignancy • Malnutrition • Obesity • Infection • Organ failure

Notes de l'éditeur

  1. Of the three possible outcomes of inflammation, one was “return to normal”. This is healing.
  2. Lots of BASIC CELL BIOLOGY in this chapter!
  3. Regeneration is a normal process, “healing” follows damage
  4. An example of compensatory growth is when one kidney becomes larger after a nephrectomy, or the left portion on the right lobe of the liver “enlarges” after a left lobectomy.
  5. Healing (repair), like inflammation, can be thought of as a predictable sequence of events, just like in the Cecil B. DeMille “Inflammation” epic!
  6. Cells derived from stem cells can do only three things: 1) multiply 2) differentiate, or 3) die (apoptosis). I can’t think of anything else they can do.
  7. There isn’t a single day in the life of a pathologist when he does not think of the concept of “differentiation” a lot, particularly in reference of neoplasms! Although most neoplasms do not look as “differentiated” as their mature tissues of origin, there is NO such thing as reverse differentiation.
  8. Cells from these three areas behave similarly and look similarly, basically, epithelium vs. connective tissue. If you wanted to narrow this list down to only 2 histologic concepts, then epithelial (ectoderm and entoderm) and stromal (connective tissue or mesodermal)
  9. Four Phases of the Cell Cycle: Growth (G1), DNA synthesis (S-phase), Premitotic (G2), and Mitotic (P, M, A, T)
  10. Differentiate TOTI- from OMNI- from PLEURI- potent cells. These terms are often used interchangeably, but perhaps not totally correctly
  11. A knockout mouse is a genetically engineered mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA. The loss of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable physical and biochemical characteristics.
  12. Adult stem cells, or “near” stem cells, are often given the term “totipotential”, and are ubiquitous, even in blood.
  13. Diagram for the ever mysterious concept of “cell differentiation”, elucidated by “growth factors”. MSCs can be thought of as being the mother of all mesodermal cells. They look totally boring because of no cytoplasmic differentiation, i.e., mesenchyme. Why is the word “pluri”-potent used here instead of totipotent or omnipotent?
  14. Epithelium is generally thought of as arising from ectoderm or entoderm, while connective tissue, i.e., “stroma”, is thought of as arising from mesoderm.
  15. Please remember that these are the “general” features of GF’s
  16. EGF, TGF, HGF, VEGF, PDGF, FGF, FGF, KGF, Cytokines
  17. Typical protein (polypeptide) configurations of GF’s
  18. The fact that the GF’s are made by the cells involved in inflammation and healing shows the PARACRINE nature of their behavior. For EACH growth factor, you should have an idea WHERE it’s made, WHAT it does, and WHERE it goes. Often, the name helps you.
  19. You can this that this GF works on both ectodermally as well as mesodermally (mesenchymal) derived cells. Because it is called EGF, do you suspect that it’s was first discovered as having an effect on epithelium rather than connective tissue cells?
  20. TGF is similar to EGF
  21. VEGF is probably the most widely studied of all GFs in relationship to diseases.
  22. PDGF is a lot like VEGF
  23. Note that just as EGF was also for connective tissue, FGF is also for epithelial cells (keratinocytes)!!!
  24. This particular GF looks like it has many inhibitory functions, rather than stimulatory ones, i.e., control! Might you think of TGF-beta as having many opposite effects of TGF-alpha? Yes!
  25. KEY interplay between mesoderm and ectoderm, like embryonic “induction”
  26. LIKE INSULIN!
  27. Often called, with IL-1, the mother of all cytokines!
  28. We are up to 36 interleukins by now, probably by the end of this lecture, 37? http://en.wikipedia.org/wiki/Interleukin
  29. About a dozen varieties. http://en.wikipedia.org/wiki/Interferon
  30. Autocrine, paracrine, endocrine concepts
  31. As a major general ACTION of GF’s, transcription factors take orders from GFs, in humans. In molecular biology and genetics, a transcription factor (sometimes called a sequence-specific DNA-binding factor) is a protein that binds to specific DNA sequences, thereby controlling the movement (or transcription) of genetic information from DNA to mRNA via RNA polymerase.
  32. The ever increasing cast of ECMs: CAMs=ImmunoGlobulin Super Family (IGSF), Cadherins, Selectins, Integrins 27 types of collagen of which Type-I is the most common
  33. This is one of the rare mnemonics that I don’t hate.
  34. Ehlers-Danlos: Hyperelastic skin Achondrogenesis, Ib, 2: Small body, short limbs Stickler syndrome: distinctive facial appearance, eye abnormalities, hearing loss, and joint problems, myopia, absent nasal bridge, hyperelasticity Alport syndrome: glomerulonephritis, end stage kidney disease, and hearing loss Bethlem myopathy: progressive myopathy, ankles, fingers, joints Dystrophic epidermolysis bullosa: a blistering disease Knobloch syndrome: ophthalmic, retinal abnormalities I would recommend that you become familiar with the general features of each “collagen disorder” and know how to relate it to the specifically numbered collagen defect.
  35. Worth repeating!
  36. Healing starts BEFORE the end of inflammation. If you remember the 3 possible final outcomes of acute inflammation, 1) complete regeneration, 2) chronic inflammation, and 3) fibrosis, “healing” is the usual process BEFORE you get to one of those 3 final outcomes. Note the yellow background on this slide. Not only is familiarity needed in all these steps but the PRECISE order is also need. You might call this Hollywood Epic, Part II. The healing saga or epic, is now seen as the continuation of the inflammation saga or epic! The ORDER is LOGICAL!
  37. More likely than not, any GF will probably have a positive direct or indirect effect on angiogenesis, also called neovascularization, also called “organization”, or “organizing” inflammation, or “granulation”, or “granulation tissue”.
  38. Granulation on the left (many blood vessels), fibrosis on the right (trichrome stain stains collagen blue-green) Inflamation Granulation Fibrosis Inflamation Granulation Fibrosis Inflamation Granulation Fibrosis Inflamation Granulation Fibrosis Inflamation Granulation Fibrosis Inflamation Granulation Fibrosis Inflamation Granulation Fibrosis Inflamation Granulation Fibrosis
  39. It is tempting to estimate the actual times of events in tissue injury and repair. Another way to describe, in three words, the three phases of “repair”. In which phase would you see “fibrin”? Ans: Inflam. In which phase would you see a dense “scar”? Ans: Maturation Which phase is characterized by prominence of “budding” blood vessels? Ans: Prolif.
  40. Healing by SECOND intention involves a much greater destruction of the ECM, so therefore it is more likely to produce a greater amount of FIBROSIS. The degree of FIBROSIS is directly proportional to the amount of DESTRUCTION or DISRUPTUION of the ECM.
  41. The main difference between 1st and 2nd intention is: Are the edges of the wound lined up (1st), or not (2nd).
  42. These processes also parallel the appearance and regression of cells, namely, in order, neutrophils, macrophages, endothelial cells, fibroblasts. This is my favorite graph, because it summarizes the whole chapter!
  43. OFTEN, totally HEALTHY granulation tissue can be described as “INFECTED”. Don’t get caught making this mistake. Why are there “lines” on the upper left image?
  44. Wound “contraction”. What is a keloid?