1. Tissue Repair: Regeneration, Healing and Fibrosis
Prof.dr. Manal Fawzy Gadalla
Professor of pathology
Faculty of medicine
Ain Shams University

2. Intended Learning Outcomes (ILOs):
You should be able to:
Define healing and describe its process
Specify the patterns of wound healing.
Differentiate between different types of cells
List the factors that influence wound healing.
Complications of wound healing.
Explain the process of wound healing.

3. 1- Define healing and describe its process
TISSUE REPAIR
Restoration of tissue architecture & function after an injury.
It occurs by two reactions:
Regeneration
Healing by fibrosis.

4. Replacement of the diseased tissue
1- Regeneration:
Replacement of the diseased tissue
By proliferation of parenchymal cells.
Results in complete restoration.

5. II- Repair by laying down of connective (fibrous) tissue:
It occurs if the injured tissues are:
Incapable of proliferation
The supporting structures of the tissue are severely damaged.

6. organization
Fibrosis in a tissue space occupied by inflammatory exudate
E.g. organizing pneumonia & serofibrinous inflammation in pleura.

7. 1- Define healing and describe its process
After injury=====architecture & function after an injury.
occurs:
Regeneration
Healing by fibrosis.
Organizattion

8. 3- Differentiate between different types of cell

9. Resting Interphase DNA synthesis /chromosome duplication
mRNA synthesis, increase protein
DNA synthesis /chromosome duplication
Correction of synthesized DNA
Prophase
Metaphase
Anaphase
Telophase
Interphase

10. The Cell Cycle Rate of cell division: different
Some cells do not divide.
Other cells cycle every hours.
Mitosis is controlled by genes
Genes encode the release of specific proteins
This protiens promote or inhibit mitosis at different steps.

11. Mitosis-promoting proteins:
Cyclins.
Cyclins activate cyclin-dependent kinases (CDKs)
CDK acts in conjunction with cyclins
regulate the phosphorylation of proteins involved in cell cycle progression
leading to DNA replication and mitosis.
After mitosis is completed, cyclins & CDKs are degraded

12. Cell cycle is tightly regulated by stimulators and inhibitors,
Contains intrinsic checkpoint controls to prevent replication of abnormal cells.
Interphase : Period between two mitoses

13. According to the proliferative capacity of the cells in tissue
Tissues are divided into:
Labile
Stable
Permanent

14. Quiescent cells (in G0 of the cell cycle)
Labile cells
Multiply: continous
Injury of tissue composed of labile cells…..Regeneration (Under appropriate conditions & enough stem cells
Stable cells
Quiescent cells (in G0 of the cell cycle)
Multiplication activity: Minimal
Injury… can multply
Permanent cells
Are terminally differentiated cells
Mulitplication activity: lost around birth.

16. Haematopoietic cells of BM, LN & spleen.
Labile cells
Surface epithelia of epidermis, cornea, alimentary tract, respiratory tract, urinary tract, & reproductive tract
Epithelium of the ducts of exocrine organs (e.g., salivary glands, pancreas & biliary tract
Haematopoietic cells of BM, LN & spleen.
Stable cells
parenchymal cells of organs, such as liver, kidney (proximal tubules), pancreas and endocrine glands
Mesenchymal cells like smooth muscle cells, fibroblasts, vascular endothelium, bone and cartilage cells.
Permanent cells (never multiply)
Nerve cells
cardiac muscle cells
skeletal muscle cells
cells of the lens of the eye.
Injury to brain or heart is irreversible (scar.)

17. Continuously dividing tissues (labile tissues) contain:
Stem cells that differentiate to
Replenish lost cells
Maintain tissue homeostasis.

18. Stem Cells Unspecialized cells Can divide through mitosis
Differentiate into specialized cell types.
Self-renew to produce more stem cells.

19. Types of stem cells: Embryonic stem (ES) cells:
Isolated from the inner cell mass of blastocyst
Differentiate into ALL cells of the adult
Preserve small populations of more restricted stem cells
Adult stem cells;
Found in various tissues
Differentiation into ALL CELL TYPES OF THEIR TISSUE i.e: Possess a more restricted range of cell differentiation than ES cells).

20. ES cells differentiation potential diminishes with advancing stages of embryo development:

21. Totipoptent cells
Pleuripotent cells
Multipotent cells
Oligopotent cells
Unipotent (precursor) cells

22. Totipoptent cells
Produce all the differentiated cells,
Including extraembryonic tissues (e.g. placenta),
i.e. have the potential to create a complete organism
Pleuripotent cells
ES cells from the inner cell mass of the blastocyst
Produce nearly all cell types.
No extraembryonic tissue
Cannot develop into a fetus or adult organism
Multipotent cells
Produce multiple, but a limited number of lineages e.g. hematopoietic cells.

23. Oligopotent cells
Produce few cell types e.g. the lymphoid or myeloid stem cells.
Unipotent (precursor) cells
Produce one type e.g., hepatocytes

24. Totipoptent cells
complete organism
Pleuripotent cells
nearly all cell types. But not organism
Multipotent cells
multiple, but a limited number of lineages
Oligopotent cells
few cell types
Unipotent (precursor) cells
one type

25. Factors affecting Cell proliferation tissue repair
List the factors that influence wound healing
Factors affecting Cell proliferation tissue repair
A- Many chemical mediators, such as growth factors, hormones, and cytokines B- Interactions between cells and ECM components

26. Growth factors (Table 4.1)
Is a protein that has the following effects;
Stimulate cellular PROLIFERATION.
Stimulate Migration, Differentiation & Contractility.
Enhance the synthesis of Specialized Proteins (such as collagen in fibroblasts).
Stimulate the function of growth control Genes (Proto-oncogenes).

27. Mechanisms of action.
Induce cell proliferation by binding to specific receptors.
Affect the expression of genes ===products promote replication.
Enhance the synthesis of cellular proteins involved in mitosis.
Prevent apoptosis

28. Epidermal growth factor (EGF)
Cytokine
Source
Functions
Epidermal growth factor (EGF)
Activated macrophages, keratinocytes & other cells
Mitogenic for keratinocytes and fibroblasts.
Transforming growth factor β
(TGF-β)
Platelets,
T lymphocytes,
macrophages, endothelial cells,
keratinocytes,
smooth muscle cells, fibroblasts
Chemotactic for PMNs, macrophages, lymphocytes, fibroblasts, and smooth muscle cells;
Stimulates TIMP synthesis, angiogenesis, and fibroplasia.
Inhibits production of MMPs and keratinocyte proliferation;
Regulates integrin expression and other cytokines
Vascular endothelial cell growth factor
(VEGF)
Mesenchymal cells
Increases vascular permeability; mitogenic for endothelial cells
Platelet-derived growth factor
(PDGF)
Platelets, macrophages, endothelial cells, keratinocytes, smooth muscle cells
Chemotactic for & activating PMNs, macrophages, fibroblasts.
Mitogenic for fibroblasts, endothelial cells, and smooth muscles cells;
Stimulates production of MMPs, fibronectin, and HA
Stimulates angiogenesis and wound remodeling;
Regulates integrin expression

29. Extracellular matrix (ECM)
Dynamic component
Synthesized locally
Assembled into network surrounds cells.
Synthesis and degradation of ECM accompanies :
Morphogenesis.
Wound healing.
- Chronic fibrotic processes.
- Tumor invasion & metastasis.

30. ECM serves several important fuctions:
Mechanical support to tissues
Substrate for cell growth and the formation of tissue
Microenvironments.
Regulates cell proliferation &differentiation.
Intact ECM is required for tissue regeneration,
if ECM is damaged, repair can only be accomplished by scar formation.

31. Forms of ECM Interstitial Matrix:
This is present in the spaces between cells in connective tissue, and between epithelium and supportive vascular and smooth muscle structures.
It is synthesized by mesenchymal cells (e.g., fibroblasts).
Its major constituents are fibrillar and nonfibrillar collagens, as well as fibronectin, elastin, proteoglycans, hyaluronate, and other elements.
Basement Membrane
The basement membrane lies beneath the epithelium and is synthesized by overlying epithelium and underlying mesenchymal cells.
Its major constituents are amorphous nonfibrillar type IV collagen and laminin.

32. Components of the ECM There are three basic components of ECM:
1- Fibrous structural proteins:
such as collagens and elastins,
give tensile strength and recoil.
2- Water-hydrated gels
such as proteoglycans & hyaluronan,
permit flexibility and lubrication.
3- Adhesive glycoproteins:
Connect matrix elements to one another & to cells.

33. Role of the ECM
Provides mechanical support to tissues (role of collagen &elastin).
Control of cell growth & proliferation by signaling through cellular receptors of the integrin family.
Maintenance of cell differentiation. The type of ECM proteins can affect the degree of differentiation of the cells via cell surface integrins.
Scaffolding for tissue renewal; the integrity of the basement membrane or the stroma is critical for regeneration of tissues, whose disruption leads to scar formation.
Establishment of tissue microenvironments; basement membrane acts as a boundary between epithelium and underlying connective tissue and also forms part of the filtration apparatus in the kidney.
Storage & presentation of regulatory molecules e.g. growth factors fibroblast GF & hepatocyte GF are excreted and stored in the ECM. This allows for the rapid use of growth factors after local injury, or during regeneration.

34. Cell and tissue regneration
In tissues formed of labile or stable cells
Damage of the epithelia === proliferation & differentiation of stem cells.
Increased consumption or loss of blood cells.=== Growth factors called colony-stimulative factors (csfs) ===== renewal of hematopoietic cells is
Surgical removal of 40-60% of the liver in LT =====tissue resection triggers a proliferative response of the remaining hepatocytes (stable cells)==== replication & regeneration

35. Extensive regeneration can occur only if
Residual tissue is structurally & functionally intact
If the tissue is damaged by inflammation, regeneration is incomplete and is accompanied by scarring.

36. Repair by connective tissue:
This occurs if:
Non-dividing (permanent) cells are injured.
Tissue injury is severe or chronic, =====Damage to parenchymal cells and epithelium as well as the stromal framework.

37. Cells involved Mesenchymal cells (C.T. Stem cells), Endothelial cells
Macrophages, platelets
Parenchymal cells of the injured organ.

38. Repair by connective tissue deposition consists of four sequential processes:
Angiogenesis
Migration and proliferation of fibroblasts.
Deposition of ECM (scar formation).
Maturation & reorganization of the fibrous tissue (remodeling).

39. Cutaneous wound healing
Involves:
Epithelial regeneration
Connective tissue scar.
Based on the nature of the wound, the healing can occur by :
First
Second intention.

40. Healing by first intention (Primary union)
Healing of a wound which:
Clean and uninfected.
Surgically incised.
Without much loss of cells & tissue.
Edges of wound are approximated by surgical sutures

41. The sequence of events in primary union
Initial haemorrhage:
Immediately after injury
Space is filled with blood
Blood clots seals the wound against dehydration &infection.

42. Within 24 hours;
Neutrophils at the incision margin & toward the fibrin clot.
Basal cells at the cut edge of the epidermis begins mitosis.

43. Within 24 to 48 hours;
Epithelial cells migrate & proliferate along the dermis
Deposition of basement membrane components as they progress.
The cells meet in the midline as a thin but continuous epithelial layer separating the underlying viable dermis from the overlying scab.

44. By day 3:
Granulation tissue invades the incision space.
Collagen fibers formation ,
Vertically oriented
Do not bridge the incision.
Epithelial cell proliferation continues

45. Morphology of granulation tissue
Grossly: granulation tissue appears
Granular
Soft & Moist
Pink and bleeds on touch.
Insensitive
Resistant to bacterial infection

46. Microscopy; it is characterized by:
Proliferation of fibroblasts
New thin-walled delicate capillaries (angiogenesis)
In a loose ECM.

47. By day 5:
Granulation tissue fills the incisional space
Neovascularization reaches its peak
Collagen fibrils : abundant & bridge the incision.
Epidermis recovers its normal thickness:
Mature epidermal architecture with surface keratinization (skin).

48. During the second week:
Fibroblast proliferation & collagen accumulation.
Leucocytic infiltrate, edema & vascularity diminished.
By the end of the first month: Scar

49. Initial haemorrhage: Blood clots seals wound
Within 24 hours; Neutrophils, Basal cells
24 to 48 hours; Epithelial cells proliferate , deposition of basement membrane , thin but continuous epithelial layer separating dermis from the scab..
By day 3: Granulation tissue, Collagen fibers, Epithelial cell proliferation
By day 5: Granulation tissue fills space, Collagen fibrils , Epidermis normal thickness.
During the second week: collagen, inflammation
By the end of the first month: Scar

50. Comprises acellular connective tissue Devoid of inflammatory cells
By the end of the first month: Scar
Comprises acellular connective tissue
Devoid of inflammatory cells
Covered by a normal epidermis.
Dermal appendages destroyed in the line of the incision are permanently lost.
Tensile strength of the wound increases with time

51. In second-intention healing, the basic events are similar to primary union but differ in:
Defect : larger . Hence healing takes place from the base upwards & from the margins inwards.
Inflammatory reaction more intense
Granulation tissue abundant
Followed by accumulation of ECM
Formation of a large scar
Wound contracts within 6 weeks
May be reduced to 5 -10% of its original size
(due to the presence of myofibroblasts (modified fibroblasts).

52. Table 4.2: Differences between primary and secondary union of wounds.
Feature
Primary union
Secondary union
Cleanliness of wound
Clean
Unclean
Infection
Generally uninfected
May be infected
Inflammation
Mild
More intense
Granulation tissue
Scanty
Much larger amount
Wound contraction
Not present
Present
Outcome
Neat linear scar
Contracted irregular wound
Complications
Infrequent.
Epidermal inclusion cyst formation
More frequent
The most important is suppuration

53. Wound Strength
Carefully sutured wounds have approx. 70% of the strength of normal skin.
When sutures are removed, usually at one week, wound strength is approximately 10% of that of normal skin, but this increases rapidly over the next 4 weeks.
Recovery of tensile strength results from collagen synthesis exceeding degradation during the first 2 months, and from structural modifications of collagen (e.g., cross- linking and increased fiber size).
Wound strength reaches approximately 70% -80% of normal by 3 months but usually does not improve beyond that point.

54. Factors affecting wound healing:
Infection: most important cause of delayed wound healing.
Foreign bodies such as fragments of steel, glass, or even bone impair healing.
Nutrition : protein & vitamin C deficiency inhibit collagen synthesis & delay healing.

55. Glucocorticoids (steroids)
Poor perfusion, due to arteriosclerosis, diabetes or obstructed venous drainage results in impaired healing.
Glucocorticoids (steroids)
Have anti-inflammatory effects, and their administration may result in poor wound strength due to diminished fibrosis.
In some cases , e.g. corneal infections, glucocorticoids are sometimes prescribed (along with antibiotics) to reduce the likelihood of opacity that may result from collagen deposition.

56. The type of tissue injured:
Complete restoration can occur only in tissues composed of stable and labile cells.
Injury to tissues composed of permanent cells result in scarring e.g. healing of a myocardial infarct.

57. The site of the injury:
Inflammation in tissue spaces (e.g., pleural, peritoneal and synovial cavities) develops extensive exudate.
Repair may occur by digestion of the exudate, initiated by proteolytic enzymes of leucocytes and resorption of liquefied exudate.
This is called resolution in which normal tissue architecture is restored.
However, in the setting of larger accumulations, the exudates may undergo organization.

58. Factors affecting wound healing:
Infection
Foreign bodies
Nutrition
Poor perfusion,
Glucocorticoids (steroids)
The type of tissue injured
The site of the injury

59. Complications of wound healing
Infection of wound.
Implantation (epidermal) cyst;
Persistence of epithelial cells in the wound after healing.
3- Deficient scar formation;
Inadequate formation of granulation tissue
May lead to wound splitting, incisional hernia and ulceration.

60. 4- Excessive scar formation (keloid):
Mass formed of excessive collagen
Progress beyond the site of initial injury, Covered by stretched epidermis
Recurs after excision
More common in blacks
shows hereditary tendency

61. Exuberant granulation tissue:
Excessive granulation tissue
Protrudes above the level of the surrounding skin
Hinders re-epithelialization

63. Termed contracture or cicatrisation
6- Excessive contraction
Termed contracture or cicatrisation
Results in severe deformity & limit joint mobility
healing of severe burns
7- Pigmentation:
in healed wounds,
may have rust-like color
due of haemosiderin.
8- Malignant change: later, e.g. squamous cell carcinoma in Marjolin`s ulcer.

64. SUMMARY Cell Proliferation, the Cell Cycle, and Stem Cells;
Cell proliferation is regulated by cyclins complexed with CDK
regulate phosphorylation of proteins of cell cycle
leading to DNA replication and mitosis.
Cell cycle is tightly regulated by stimulators and inhibitors
Intrinsic checkpoint prevent replication of abnormal cells.

65. Tissues are divided into labile, stable & permanent, according to the proliferative capacity of their cells.
Continuously dividing tissues (labile tissues) contain stem cells
Stem cells differentiate to replenish lost cells and maintain tissue homeostasis.

66. Cutaneous Wound Healing &Pathologic Aspects of Repair
Main phases are inflammation, formation of granulation tissue, and ECM remodeling.
Cutaneous wounds can heal by primary union (first intention) or secondary union (secondary intention)
Secondary healing involves more extensive scarring and wound contraction.

67. Regeneration and Repair by Connective Tissue
Tissues can be repaired by
Regeneration with complete restoration of form and function, or
Replacement with connective tissue & scar
Repair by connective tissue
Starts with formation of granulation tissue
Laying down of fibrous tissue.

68. Multiple growth factors:
Stimulate the proliferation of the cell types involved in repair.
TGF-β is a potent fibrogenic agent.
ECM deposition depends on the balance between
Fibrogenic agents,
Metalloproteinases (mmps) that digest ECM,
Tissue inhibitors of mmps (timps

69. Wound healing can be altered by many conditions, particularly infection & diabetes.
Excessive production of ECM can cause keloids in the skin.
Persistent stimulation of collagen synthesis in chronic inflammatory diseases leads to fibrosis of the tissue.