WOUND HEALING AND REPAIR

Presenter: Nyangara Rajabu, MMed(OBGY) Facilitator: Dr Mgaya

CONTENTS
History Introduction Types of wounds Regeneration and Repair Phases of wound healing Nature of wound healing Factors influencing healing Complications of wound healing References

HISTORY

The earliest accounts of wound healing date back to about 2000 B.C Galen of Pergamum emphasized the importance of maintaining a moist environment to ensure adequate healing. Ambriose Par found that simply dressed gunshot wounds heal faster and are less painful than when treated with boiling oil, the previously accepted method. Ignaz Philipp Semmelweis advocated need for washing hands Joseph Lister began soaking his instruments in phenol and spraying the operating rooms, reducing the mortality rates from 50 to 15%.

INTRODUCTION
The repair of tissue damage broadly separated into two processes, regeneration and healing Regeneration refers to growth of cells and tissues to replace lost structures. Wound healing is the effort of tissues to restore normal function and structure after injury -To reform barriers to fluid loss and infection, -limit further entry of foreign organisms and material, -re-establish normal blood and lymphatic flow patterns, -restore the mechanical integrity of the injured system

TYPES OF WOUND
Abrasions. Also called scrapes, they occur when the skin is rubbed away by friction against another rough surface (e.g. rope burns and skinned knees). Avulsions. Occur when an entire structure or part of it is forcibly pulled away, such as the loss of a permanent tooth or an ear lobe. Animal bites may cause avulsions. Contusions. Are also called bruises and are the result of a forceful trauma that injures an internal structure without breaking the skin. Blows to the chest, abdomen, or head with a blunt instrument (e.g. a football or a fist) can

TYPES OF WOUND cont...

Crush wounds. Occur when a heavy object falls onto a person, splitting the skin and shattering or tearing underlying structures. Cuts. Slicing wounds made with a sharp instrument, leaving even edges. They may be as minimal as a paper cut or as significant as a surgical incision. Incised wound: Any sharp cut in which the tissues are not severed; a clean cut caused by a keen cutting instrument. The wound may either be aseptic or infected, depending on the circumstances.

TYPES OF WOUND cont...

Lacerations. Also called tears, these are separating wounds that produce ragged edges. They are produced by a tremendous force against the body, either from an internal source as in childbirth, or from an external source like a punch. Open wound. A contusion, in which the skin is also broken, such as gunshot, incised or lacerated wound. Penetrating wound. A wound in which the skin is broken and the agent causing wound enters subcutaneous tissue or a deep lying structure

REGENERATION AND REPAIR

Healing involves two processes: 1. Regeneration 2. Repair

Regeneration
Definition: means proliferation of the parenchymal cells resulting in complete restoration of the original tissues. It requires cell proliferation which is largely regulated by micro environment that can either stimulate or inhibit cell growth.

Regeneration cont...
Cell proliferation depends on the cell growth cycle which consists of 4 unequal phases: M (mitotic) phase G1 (pre synthetic) phase S (DNA synthesis) phase G2 (pre mitotic) phase Quiescent or resting cells will be in a physiologic state called G0.

The Cell Cycle and Different Cell Populations

Cells of the body are usually classified into three groups depending on their capacity for regeneration: 1.Labile cells 2.Stable cells 3.Permanent cells

1. Labile cells
Also called continuously dividing cells Follow the cell cycle from one mitosis to other Continue to proliferate throughout life, replacing tissues that are continuously destroyed These include: surface epithelial cells of epidermis, alimentary tract , respiratory tract, urinay tract, vagina, cervix, uterine endometrium, haematopoietic cells of bone marrow and cells of lymph node and

2. Stable cells
Also called quiescent cells These cells lose or decrease their capacity to proliferate after adoloscene but retain the capacity to multiply in response to stimuli throughout adult life These include: parenchymal cells of organs like liver, pancreas, kidney, adrenal and thyroid ; mesenchymal cells of smooth muscle cells , fibroblasts, vascular endothelium, bone and cartilage cells.

3. Permanent cells
Also called non dividing cells. Have left the cell cycle and cannot undergo mitotic division in post natal life. These include: neurons of nervous system, skeletal muscle and cardiac muscle cells.

Repair
Is the replacement of injured tissue by fibrous tissue. Processes involved in repair: 1. Granulation tissue formation 2. Contraction of wounds

PHASES OF WOUND HEALING

Normal wound healing follows a predictable pattern that can be divided into overlapping phases defined by characteristic cellular populations and biochemical activities: (a) Hemostasis & inflammation (b) Proliferation (c) Maturation and Remodeling

HEMOSTASIS AND INFLAMMATION

In the inflammatory phase clotting takes place in order to obtain hemostasis, or stop blood loss, and various factors are released to attract cells that phagocytise debris, bacteria, and damaged tissue and release factors that initiate the proliferative phase of wound healing
The events can be divided into: 1.Vascular events 2.Cellular events

1. Vascular events

Earliest manifestation is vasodilatation, it follows a transient constriction of arterioles lasting a few seconds. Wounding disrupts tissue integrity and direct exposure of extracellular matrix to platelets Initial contact between platelets and collagen requires the von Willebrand factor (vWF) Binding results in changes in platelet conformation, triggering intracellular signal transduction pathways that result in platelet activation and the release of biologically active proteins. Platelet granules are storage organelles that contain -Platelet-derived growth factor (PDGF), -Transforming growth factor(TGF)-, -Insulin-like growth factor (IGF)-1, -Fibronectin, -Fibrinogen, -Thrombospondin, -vWF.

Vascular events cont...

The dense bodies contain the vasoactive amines, such as serotonin, which cause vasodilation and increased vascular permeability. The clotting cascade is initiated through both the intrinsic and the extrinsic pathways. Vasodilation is followed by increased permeability of the microvasculature, followed by stasis, which leads to accumulation of leucocytes along the vascular endothelium which then migrate through the vascular wall into the interstitial tissue. Increased permeability is due to - formation of endothelial gaps in venules, - direct endothelial injury, - delayed prolonged leakage, - leucocyte mediated endothelial injury, -increased transcytosis and leakage from new vessels The combination of intense vasodilation and increased vascular permeability leads to clinical findings of inflammation, rubor (redness), tumor (swelling),calor (heat), and dolor (pain).

2. Cellular events
1. Polymorphonuclear cells or Neutrophils First infiltrating cells to enter the wound site, peaking at 24 to 48 hours Neutrophil migration is stimulated by - Increased vascular permeability, - local prostaglandin release, and - the presence of chemotactic substances, such as complement factors, interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-), TGF , platelet factor 4, or bacterial products. PMNs are also a major source of cytokines early during inflammation, especially TNF-, also release proteases such as collagenases. Following functional activation neutrophils scavenge necrotic debris, foreign material, and

PMN cont.
Stimulated neutrophils generate free oxygen radicals with electrons donated by the reduced form of nicotinamide adenine dinucleotide phosphate, (NADPH). The electrons are transported across the membrane into lysosomes where superoxide anion (O2-) is formed. This very potent free radical is bactericidal, but it is also toxic to neutrophils and surrounding viable tissues. Migration of PMNs stops when wound contamination has been controlled, usually within the first few days after injury. PMNs do not survive longer than 24 hours

PMN cont.
If wound contamination persists or secondary infection occurs, continuous activation of the complement system and other pathways provides a steady supply of chemotactic factors, resulting in a sustained influx of PMNs into the wound. PMNs are not essential to wound healing because their role in phagocytosis and antimicrobial defense may be taken over by macrophages. Sterile incisions will heal normally without the presence of PMNs

2. Macrophages

Second population of inflammatory cells that invades the wound. Macrophage is the one cell that is truly central to wound healing, serving to orchestrate the release of cytokines and stimulate many of the subsequent processes of wound healing Derived from circulating monocytes, achieve significant numbers in the wound by 48 to 96 hours post injury and remain present until wound healing is complete. Chemotactic factors specific for monocytes include -bacterial products, -fibronectin, -complement degradation products (C5a), -collagen, -thrombin,

Functions of macrophages
1. Macrophages induce PMN apoptosis 2. Macrophages have specific receptors for IgG (Fc-receptor), C3b (CR1 and CR3), and fibronectin (integrin receptors), which permit surface recognition of opsonized pathogens and facilitate phagocytosis. 3. Activated wound macrophages also produce nitric oxide which has antimicrobial properties. 4. Phospholipase is induced, causing enzymatic degradation of the cell membrane phospholipids, releasing thromboxane A2 and prostaglandin F2 5. Releases leukotriene B4 ,a potent neutrophil chemoattractant, and C4 and 15- and 5hydroxyeicosatetraenoic acid. 6. Release proteinases, including matrix metalloproteinases (MMP-1, MMP-2, MMP-3, and MMP-9) which degrade the ECM and are crucial for removing foreign material, promoting cell movement through tissue spaces, and regulating ECM turnover. 7. Release growth factors that stimulate fibroblast, endothelial

3. T lymphocytes Another population of inflammatory/immune cells that routinely invades the wound. Less numerous than macrophages, numbers peak at about 1 week post injury Bridge the transition from the inflammatory to the proliferative phase of healing Depletion of most wound T lymphocytes decreases wound strength and collagen content Also exert a down regulating effect on fibroblast collagen synthesis by cellassociated interferon-, TNF-, and IL-1.

PROLIFERATION PHASE
Second phase of wound healing and roughly spans days 4 through 12 It is during this phase that tissue continuity is reestablished Fibroblasts and endothelial cells are the last cell populations to infiltrate the healing wound, and the strongest chemotactic factor for fibroblasts is PDGF. Recruited fibroblasts first need to proliferate, and then become activated, to carry out their primary function of matrix synthesis remodeling. The proliferative phase is also called the reconstruction phase. The events in this stage can be subdivided into:

Angiogenesis Fibroplasia and granulation tissue formation Epithelization Contraction

1. Angiogenesis
Also called neovascularization, the process of angiogenesis occurs concurrently with fibroblast proliferation when endothelial cells migrate to the area of the wound. Because the activity of fibroblasts and epithelial cells requires oxygen and nutrients, angiogenesis is imperative for other stages in wound healing, like epidermal and fibroblast migration. The tissue in which angiogenesis has occurred typically looks red (is erythematous) due to the presence of capillaries. Stem cells of endothelial cells, originating from parts of uninjured blood vessels, develop pseudopodia and push through the ECM into the wound site to establish new blood vessels. Endothelial cells are attracted to the wound area by fibronectin found on the fibrin scab and chemotactically by angiogenic factors released by other cells, e.g. from macrophages and platelets when in a low-oxygen environment. Endothelial growth and proliferation is also directly stimulated by hypoxia, and presence of lactic acid in

Angiogenesis cont..
To migrate, endothelial cells need collagenases and plasminogen activator to degrade the clot and part of the ECM. Zincdependent metalloproteinases digest basement membrane and ECM to allow cell migration, proliferation and angiogenesis When macrophages and other growth factorproducing cells are no longer in a hypoxic, lactic acid-filled environment, they stop producing angiogenic factors. Thus, when tissue is adequately perfused, migration and proliferation of endothelial cells is reduced. Eventually blood vessels that are no longer needed die by apoptosis.

2. Fibroplasia and granulation tissue formation Simultaneously with angiogenesis, fibroblasts begin accumulating in the wound site. Fibroblasts begin entering the wound site two to five days after wounding as the inflammatory phase is ending, and their numbers peak at one to two weeks post-wounding. By the end of the first week, fibroblasts are the main cells in the wound. Fibroplasia ends two to four weeks after wounding. In the first two or three days after injury, fibroblasts mainly proliferate and migrate, while later, they are the main cells that lay down the collagen matrix in the wound site. Fibroblasts from normal tissue migrate into the wound area

Fibroplasia and granulation tissue formation cont....

Initially fibroblasts use the fibrin scab formed in the inflammatory phase to migrate across, adhering to fibronectin. Fibroblasts then deposit ground substance into the wound bed, and later collagen, which they can adhere to for migration Granulation tissue functions as rudimentary tissue, and begins to appear in the wound already during the inflammatory phase, two to five days post wounding, and continues growing until the wound bed is covered. Granulation tissue consists of new blood vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and the components of a new, provisional extracellular matrix (ECM)

Extracellular Matrix
The wound is strengthened by proliferation of fibroblasts and myofibroblasts which get structural support from the extracellular matrix (ECM). ECM has five main components: i. collagen ii. adhesive glycoprotein iii. basement membrane iv. elastic fibres v. proteoglycans.

i.

Collagen deposition

One of fibroblasts' most important duties is the production of collagen. Fibroblasts begin secreting appreciable collagen by the second or third postwounding day, and its deposition peaks at one to three weeks. Collagen production continues rapidly for two to four weeks, after which its destruction matches its production and so its growth levels off. Collagen deposition is important because it increases the strength of the wound; before it is laid down, the only thing holding the wound closed is the fibrin-fibronectin clot, which does not provide much resistance to traumatic injury. Also, cells involved in inflammation, angiogenesis, and connective tissue construction attach to, grow and differentiate on the collagen matrix laid down by

Collagen deposition cont....

Collagen synthesis, as well as post translational modifications, is highly dependent on systemic factors such as an adequate oxygen supply the presence of sufficient nutrients (amino acids and carbohydrates) cofactors (vitamins and trace metals) the local wound environment (vascular supply and lack of infection).

ii. Adhesive glycoprotein

Fibronectin is the best characterised glycoprotein in ECM and has binding binding properties to other cells and ECM. It is of 2 types
Plasma fibronectin, synthesized by the liver cells and is trapped and is trapped in basement membrane such as in filtration through the renal glomerulus. Tissue fibronectin, is formed by fibroblasts , endothelial cells and other mesenchymal cells. It is responsible for the primitive matrix in wound healing.

Tenascin or cytotactin is the glycoprotein associated with fibroblasts and appears in wound after 48 hours of injury. It disappears from mature scar tissue. Thrombospondin is mainly synthesised by granules of platelets. Function as adhesive protein for keratinocytes and platelets but is inhibitory to

iii. Basement membrane Flexible, thin (40- to 120-nm thick) mats of specialized ECM that separate cells and epithelia from the underlying or surrounding connective tissue In the skin, the basal lamina is tethered to the underlying connective tissue by specialized anchoring fibrils This composite of basal lamina and collagen is the basement membrane Most mature basal laminae contain type IV collagen and the glycoproteins laminin. The basal lamina serves numerous functions 1. As a molecular filter, preventing passage of macromolecules (i.e., in kidney glomerulus) 2. As a selective barrier to certain cells (i.e., the lamina beneath the epithelium prevents fibroblasts from contacting epithelial cells, but does not stop macrophages or lymphocytes) 3. As a scaffold for regenerating cells to migrate 4. Is important in tissue regeneration where the basal lamina

iv. Elastic fibres

Provide the resilience to allow for recoil after transient stretch. Elastin is composed of hydrophobic and alanine and lysine-rich -helical segments that alternate along the polypeptide chain Elastic fibers consist of an elastin core covered with a sheath of microfibrils, which are composed of several distinct glycoproteins, such as fibrillin. Microfibrils appear before elastin in developing tissues and seem to form a scaffold on which the secreted elastin

v. Proteoglycans

These are agroup of molecules having 2 components- an essential carbohydrate polymer (called glycosaminoglycan), and a protein bound to it, and hence the name proteoglycan. Various proteoglycans are distributed in different tissues as under:
Chondrointin sulphate- abundant in cartilage , dermis Heperan sulphate- in basement membranes Dermatan sulphate- in dermis Keratan sulphate- in cartilage Hyaluronic acid- in cartilage , dermis

In wound healing the deposition of proteoglycans precedes collagen laying

3. Epithelialization

The formation of granulation tissue in an open wound allows the reepithelialization phase to take place, as epithelial cells migrate across the new tissue to form a barrier between the wound and the environment. Basal keratinocytes from the wound edges and dermal appendages such as hair follicles, sweat glands and sebaceous (oil) glands are the main cells responsible for the epithelialization phase of wound healing. Keratinocytes migrate without first proliferating. Migration can begin as early as a few hours after wounding.

If the basement membrane is not breached, epithelial cells are replaced within three days by division and upward migration of cells in the stratum basale in the same fashion that occurs in uninjured skin. However, if the basement membrane is ruined at the wound site, reepithelization must occur from the wound margins and from skin appendages such as hair follicles and sweat and oil glands that enter the dermis that are lined with viable keratinocytes. If the wound is very deep, skin appendages may also be ruined and migration can only occur from wound edges. Migration of keratinocytes over the wound site is stimulated by lack of contact inhibition and by chemicals such as nitric oxide. Before they begin to migrate, cells must dissolve their desmosomes and hemi desmosomes, which normally anchor the cells by intermediate filaments in their cytoskeleton to other cells and to the ECM. Transmembrane receptor proteins called integrins, which are made of glycoproteins and normally anchor the cell to the basement membrane by its cytoskeleton, are released from the cell's intermediate filaments and relocate to actin filaments to serve as attachments to the ECM for pseudopodia during migration.

4. Contraction Contraction is a key phase of wound healing. Contraction commences approximately a week after wounding, when fibroblasts have differentiated into myofibroblasts. In full thickness wounds, contraction peaks at 5 to 15 days post wounding. Contraction can last for several weeks and continues even after the wound is completely reepithelialized. A large wound can become 40 to 80% smaller after contraction. Wounds can contract at a speed of up to 0.75 mm per day, depending on how loose the tissue in the wounded area is.

At first, contraction occurs without myofibroblast involvement. Later, fibroblasts, stimulated by growth factors, differentiate into myofibroblasts. Myofibroblasts, which are similar to smooth muscle cells, are responsible for contraction. Myofibroblasts contain the same kind of actin as that found in smooth muscle cells. Myofibroblasts are attracted by fibronectin and growth factors and they move along fibronectin linked to fibrin in the provisional ECM in order to reach the wound edges. They form connections to the ECM at the wound edges, and they attach to each other and to the wound edges by desmosomes. As the actin in myofibroblasts contracts, the wound edges are pulled together. Fibroblasts lay down collagen to reinforce the wound as myofibroblasts contract The contraction stage in proliferation ends as myofibroblasts stop contracting and commit apoptosis. . These events signal the onset of the maturation stage of wound healing.

MATURATION AND REMODELLING PHASE

When the levels of collagen production and degradation equalize, the maturation phase of tissue repair is said to have begun. The maturation phase can last for a year or longer, depending on the size of the wound and whether it was initially closed or left open. During maturation, type III collagen, which is prevalent during proliferation, is gradually degraded and the stronger type I collagen is laid down in its place. Originally disorganized collagen fibers are rearranged, cross-linked, and aligned along tension lines. As the phase progresses, the tensile strength of the wound increases, with the strength approaching 50% that of normal tissue by three months after injury and ultimately becoming as much as 80% as strong as normal tissue. Since activity at the wound site is reduced, the scar loses its red appearance as blood vessels that are no longer needed are removed by apoptosis. The phases of wound healing normally progress in a predictable, timely manner; if they do not, healing may progress inappropriately to either a chronic wound such as a venous ulcer or pathological scarring such as a keloid scar.

Keloid scar

NATURE OF WOUND HEALING

Accomplished by on of the following two ways: i. Healing by primary intention ii. Healing by secondary intention

i. Healing by primary intention

Healing of wounds with following characteristics: -clean and uninfected -surgically incised -without much loss of cells and tissue -edges of wound are approximated by surgical sutures

Sequence of events involved are: Initial haemorrhage

Space between approximated surfaces of the incised wound is filled with blood which than clots and seals the wound against dehydration and infection.
Within 24hours appearance of polymorphs from the margins of incision. By the 3rd day polymorphs are replaced by macrophages Basal cells of epidermis from both the cut margins start proliferating and migrating towards incisional space in the form of epithelial spurs. The migrated epithelial cells separate the underlying viable dermis from the overlying necrotic material and clot, forming scab which is cast off. The basal cells from the margins continue to proliferate and by the 5th day a multilayered new epidermis is formed By 3rd fibroblasts also invade the wound area. By 5th day new collagen fibrils start forming which dominate till healing is completed. In 4 weeks, the scar tissue with scanty cellular and vascular elements, a few inflammatory cells and epithelialised surface is formed.

Acute inflammatory response

Epithelial changes

Organisation

Suture tracks

Each suture track is a separate wound and incites the same phenomenon as in

Healing by secondary intention

Healing of a wound with the following characteristics: -open with a tissue defect, at times infected -having extensive loss of cells and tissues -the wound is not approximated by surgical sutures but is left open

Sequence of events involved are: Initial haemorrhage As a result of injury, the wound is filled with blood and fibrin clot which dries. Inflammatory phase There is an initial acute inflammatory response followed by the appearance of macrophages which clear off the debris as in primary union Epithelial changes The epidermal cells from both the margins of wound proliferate and migrate into the wound in the form of epithelial spurs till they meet in the middle and re-epithelialise the gap completely . However the proliferating epithelial cells do not cover the surface fully until granulation tissue from the base has started filling the wound space. In this way, pre existing viable connective tissue is seperateed from the necrotic material and clot on the surface, forming scab which is cast off. In time the regenerated epidermis becomes stratified and keratinised.

Granulation tissue Main bulk of secondary healing is by granulations. Granulation tissue is formed by proliferation of fibroblasts and neovascularisation from the adjoining viable elements. The newly formed granulation tissue us deep red, granular and very fragile . With time the scar on maturation becomes pale and white due to increase in collagen and decrease in vascularity. Specialised structures of skin like hair follicles and sweat glands are not replaced unless their viable residues remain which may regenerate Wound contraction Due to the action of myofibroblasts present in granulation tissue, the wound contracts to one-third of its original size. Wound contraction occurs at a time when active granulation tissue is being formed. Presence of infection Bacterial contamination of open wounds delays th process of healing due to release of bacterial toxins that provoke necrosis, suppuration and thrombosis. Surgical removal of dead and necrosed tissue, debridement , helps in preventing the bacterial infection of open wounds.

FACTORS INFLUENCING HEALING

LOCAL FACTORS 1. Infection single most important reason for delayed wound healing 2. Foreign bodies
-suture material, bone and wood splinters . -interfere with healing and cause inflammatory reaction and infection.
3.

Mechanical factors
-Early movement -Pressure -Delays healing process

4.

Ionising radiation : delays granulation tissue

SYSTEMIC FACTORS 1. Malnutrition

-Protein deficiency delays wound healing -Vitamin C deficiency (inhibition of collagen synthesis)
2.

Metabolic status
-e.g. Diabetes mellitus -Cortisone treatment
.inhibits inflammation and collagen
synthesis

3.

Circulatory status
-Inadequate blood supply due to arteriosclerosis -Varicose veins

Age : Older patient at higher risk of poor wound healing 5. Medication: Anti-inflammatory (aspirin),
4.

COMPLICATIONS OF WOUND HEALING

1.Infection 2. Pigmentation 3. Implantation 4. Deficient scar formation 5. Hypertrophied scar & Keloid formation 6. Excessive contraction 7. Neoplasia 8. Incisional hernia

REFERENCES
Robins textbook of Basic Pathology ; 8thEdition Essentials of Pathology for Dental students; 3rd edition Harsh Mohan www.google.com Wikipedia http://emedicine.medscape.com