3- Wound Fealing BLMarch2021 2156 INTRODUCTION: HEALING: Process of replacement of dead tissue by living tissue. Divided into regeneration and repair. 1. Regeneration: + Process in which lost/ damaged tissue is replaced by tissue of similar type. + Results in the complete restoration of lost or damaged tissue by proliferation of residual uninjured cells and replacernent from stem cells 2. Repair + Process in which lost/ damaged tissue is replaced by fibrous tissue or scar. + Replacement of lost tissue may occur by regeneration with complete restoration or by replacement by CT to form scar + Healing—combination of regeneration and repair + Healing process + May itself cause tissue dysfunction (ex: in pathogenesis of atherosclerosis} FACTORS DECIDING THE PATTERN OF HEALING: Proportion of regeneration and repair process in healing depends on PROLIFERATIVE CAPACITY OF THE TISSUE: A/c to proliferative capacity of the cells, the tissues of the body—3 groups: 1. Labile (continuously dividing | tissues: Cells proliferate throughout life, replacing the lost cells from the stem cells Examples: + Hematopoietic cells of the bone marrow + Surface epithelia of the skin, oral cavity, vagina and cervix, + Columnar epithelium of the GIT and uterus Labile tissues with regenerative capacity: -Hematopoietic cells -Epithelium of skin and GIT. 2. Stable [quiescent] tissues. Cells of stable tissue normally don't proliferate ; can proliferate in response to injury or loss of tissue. Examples + Parenchymal cells of liver, kidneys and pancreas * Mesenchymal cells: fibroblasts, vascular endothelial cells, smooth muscle cells, chondrocytes and osteocytes. 3. Permanent (nondividing| tissues: Cells cannot proliferate after birth Repair—by scar formation. Example: + Neurons: Damaged neurons are replaced by the proliferation of the glial cells + Skeletal muscle cells * Cardiac muscle cells.Limited stem cell replication and differentiation—in some areas of the adult brain, and heart muscle cells can proliferate after myocardial necrosis Permanent tissues—cells cannot proliferate after birth. Ex: neurons, cardiac muscle. EXTENT OF TISSUE INJURY: Mild and short duration: damaged tissue is healed by regeneration w/o significant scarring. Severe and chronic: healing occurs by fibrous tissue forming scar -Severe tissue injury damages both parenchymal cells and the extracellular matrix framework (£CM) -Chronic inflammation STEM CELLS: ability of self- renewal and capacity to generate differentiated cell lineages PROPERTIES 1. Self-renewal capacity: and capacity to generate differentiated cell lineages, 2. Asymmetric replication: division of stem cell into two cells * One daughter cell which gives rise to mature cells + Other cell—undifferentiated stem cell—retains the self- renewal capacity. Stem cell: + Self-renewal capacity + A dormant phase of cell cycle + Asymmetric replication * Capacity to generate differentiated cell lineages: ‘TYPES 1. Embryonic stem cells: During development of embryo, the blastocysts contain undifferentiated pluripotent stem cells—embryonic stem cells or ES cells—can form cells of all three germ cell layers. Normal function—to give rise to all cells of the body. 2. Adult (somatic) stem cells: less undifferentiated than ES cells found in adults Found among undifferentiated cells within a tissue. More limited capacity to generate different cell types than ES cells Usually differentiate into particular tissue Normal function—tissue homeostasis 3. Induced pluripotent stem cells: (iPS cells]: achieved by transferring the nucleus of adult cells to an enucleated oocyte. Use—for therapeutic cloning in the treatment of human diseases, ‘Types of stem cells: embryonal, adult, induced Embryonal cells are pluripotent cells capable of forming cells of all three germ layers SITES OF STEM CELLS: Reside in special microenvironments called niches. 1, BONE MARROW: contains two types of stem cells. jemat op oietic stem cells [HSCs| Can generate ail of the blood cell lineages—used for treatment of hematologic diseases Can be collected directly from the bone marrow, from umbilical cord blood andfrom the peripheral blood + Marrow stromal cells [MSCs] Can generate chondrocytes, osteoblasts, adipocytes, myoblasts, and endothelial cell precursors depending on the tissue to which they migrate. Stem cells—used in bone marrow transplantation in the treatment of various types of leukaemia and lymphoma. 2. INTESTINAL EPITHELIUM: immediately above paneth cells in the smaill intestine or at the base of the crypt in the colon. 3. LIVER: contains stem cell in the canals of Hering—capable of differentiating into hepatocytes and biliary cells 4. CORNEA: in the limbus region b/w the conjunctiva and the cornea, 5. SKIN: in the bulge area of the hair follicle, in the sebaceous glands, and in the lower layer of the epidermis. CELL CYCLE AND CELL PROLIFERATION: + Inflammation—primary response of living tissue to injury + Damage or loss of tissue—by transient increase in cellularity due to proliferation of cells by wither regeneration and / or by repair + Characterized by DNA replication and mitosis. Sequence of events that control DNA replication and mitosis—cell cycle. * Cell proliferation is a regulated process, which involves activators and inhibitors, as well as checkpoints PHASES OF CELL CYCLE: * Gi [pre-synthetic) + S (DNA synthesis} + G2 [pre-mitotic] + M [mitotic] phase Checkpoints: Checks whether there is any damage to DNA and chromosomes in the replicating cells. Make sure that only normal cells complete replication. Two! 1. G1/S checkpoint monitors the integrity of DNA before replication 2. G2/M checkpoint checks DNA after replication and monitors whether the cell can safely enter mitosis Proliferation of cells occur when quiescent cells enter the cell cycle.Newco “wo checkpoints in call ee: ‘enters resting pase Arcs a 2 G/Mcheckpoint. New col Clleces tightly regulated process andhas checkpoint controls om emer which prevent the proliferation of abnormal cells [Major action of growth factors i to stimulate genes that control ‘growth. Many of them are called as proto-oncogenes. ‘Quiescent cls, which have not entered the cll cle, are inthe Gstate. 6, phase: Mos variable phase in celleyle. checkpoint {6,10 phase: Most crtical phase inthe cll cycle. Fig. 3.1:Cel cycle showing diferent phases (GG, Gy S, and M) Cells frm lable tissues (eg epidermis) may remain in cycle continuous state cls (eg hve cel) ae quiescent but can eter the cell eye; permanent els (eg neurons) havelest the capac to peoferate ancl ‘yeast inthe G, phate or ext the cycle and are in Gyphaie GROWTH FACTORS: + Stimulate the survival and proliferation of particular cells and mosst of them are proteins Mechanism of action: induce cell proliferation by binding to specific receptors, and deliver positive growth signals to the target cells—stimulate the expression of genes whose products have several functions: * Activation of cell cycle + Relieve blocks which prevent cell cycle progression + Prevention of apoptosis + Increases the synthesis of cellular proteins.‘TABLE 3.1: List of rowth factors and cytokines involved in wound healing and regeneration GF tally EGFR 1. Epidermal growth (EGF) 1. GERI (R881) 2. Transforming growth fctora(TGF-0) 2. ERBB2 (HER 2 or HERDY Platelet-derived growth factor POGF) POGFRa and B ‘= boforms A,B,C Vascular endothelial cll growth factor (VEGF) VEGFR-,VEGFR-2 and, 1+ soforms A,B, C.D veceR3 ‘Fibroblast growth factor (FGA farily FoFRS 1-4 Keratinocyt growth factor (FGF-7) TGF receptors types and i) Growth inhibitor for most epithelia cells Potent fibrogenic agent Strong ant ieflammatory effect ‘TNF receptor (TNFR), or death TNF activates macrophages regulates other receptor for TN Interieukin-1_ cytokines and has mulple funcbons receptor (LR) fort and Interoukin 6 receptor 1-68) aso known as CD126 (Cluster of differentiation 126) fork6 SIGNALING MECHANISMS OF GROWTH FACTOR RECEPTORS: Receptor- mediated signal transduction process—activated by the binding of ligands (ex: growth factors and cytokines) to specific receptors. Receptor activation leads to expression of specific genes Modes of signalling: ‘Signaing molecule ‘Adjacent target cot Slonaing molecule secreting col ‘wih rocepior © Target coll at a distant ste NF: Considered as master switch tothe nudeus. Growth factors: Produced wansientl in response to stimull and act by binding to speci receptors. ‘Growth factors: Some of them may also act at stant site (eg HGF). ‘Some tumors may produce excessive growth factors andior their receptors resting in their uncontrolled proliferation. ‘Growth factors act in autocrine paracrine oF endocrine signaling. ‘tokines Several cytokines in inflammation may ‘ko act through endocrine signaling. Figs 3.2A 10. Modes of signaling: (A) Autocrine; 8) Paractine and (C) Endocrine sgnaing Depending on the source of the ligand and the location of its corresponding receptors (Le. inthe same, adjacent, or distant cells), the modes of signalling—3 types:1. Autocrine signalling: + Signalling molecules act on the cells which secretes them. + Ex: Liver regeneration, proliferation of antigen-stimulated lymphocytes, tumors. 2. Paracrine signalling: + Signalling molecule—produced by one cell type that acts on adjacent target cells (usually of a different type] which expresses the appropriate receptor. + Ex: healing by repair: factor produced by macrophage (one cell type] has growth effect on fibroblast (adjacent target cells of different type] 3. Endocrine signalling: + Hormones: produced by cells of endocrine organs — usually carried by the blood and act on target cells that are at a distant from the site of its synthesis HEALING BY REPAIR, SCAR FORMATION AND FIBROSIS: + Healing: by regeneration or repair or combination of both. + Mild and transient injury or damage persists—inflammation becomes chronic— excessive deposition of CT—fibrosis (repair] STEPS IN HEALING BY REPAIR (SCAR FORMATION): INFLAMMATION Whenever there is tissue injury, inflammatory reaction begins — tries to limit the damage and remove the injured tissue. Also promotes the deposition of ECM components at the site of injury and stimulates angiogenesis ANGIOGENESIS: Process of formation of new blood vessels from existing vessels. Steps in angiogenesis * Vasodilatation in response to nitric oxide and increased permeability of the pre- existing vessel due to VEGF. + Separation of pericytes from the abluminal surface of blood vessel. Breakdown of the basement membrane to facilitate formation of a vessel sprout + Migration and proliferation of endothelial cells toward the site of injury fibroblast growth factors (FGFs], mainly FGF-2. + Maturation of endothelial cells and remodelling into capillary sprouts/tubes + Formation of mature vessel: It involves recruitment of pericytes and smooth muscle cells to form the peri-endothelial layer + Suppression of endothelial proliferation and migration, and deposition of basement membrane.Quescent ood Vasodlataton = wo reo / Toca een Basement- oe ‘Angiogenic factors memocone HM 2, = (VEGF. Noten sais) Senn cit me = 8 Pericyte detachment, oe mate (ecm) Endothelial cot ccmeetsn sect Pam Congaton of Tome Formation of > Figs3.3A to: Mechanism of angiogenesis (A) Normal blood vee: B) Vasodiataton Fist, pericytes separate folowed by moblzation and proliferation of endothelial cells from the existing vessel to {erm cally sprouts towards the ste of jay langagenic simul (© Endothelial cel peoferate, loosely folowing eachother. and are ent pores an e (0) Final, bod vessel sprous wife with new crculatory systems the perendotheta FORMATION OF GRANULATION TISSUE: + First 24-72 hours of the repair process begins with proliferation of fibroblasts and vascular endothelial cells + Forms a specialized type of tissue—granulation tissue—halimark/ characteristic of tissue repair + Term: derived from its pink, soft, granular appearance on the surface of healing wounds. + Mier + Presence of new small blood vessels (angiogenesis): new blood vessels are leaky— allow passage of plasma proteins and fluid into the extravascular space— responsible for edema often seen in granulation tissue. + Proliferation of fibroblasts. 0}‘Stati’ squamous epinetum — Ferblast Granulation tissue Prolersing capes . Forbin oSe Indenmatony cts — LY) ece ' note4e = + Amount of granulation tissue formed depends on the -size of the tissue deficit created by wound -intensity of inflammation SCAR FORMATION: Leukocytes, edema, and angiogenesis disappear, accomplished by the increased accumulation of collagen. + Granulation tissue scaffolding is converted into a pale, avascular scar. * Components of scar: spindle-shaped fibroblasts, dense collagen, fragments of elastic tissue, and other ECM components, + By the end of the first month, the scar consists of acellular connective tissue without inflammatory infiltrate CONNECTIVE TISSUE REMODELLING: + Long-lasting phase of tissue repair. * Indicates that the equilibrium/ balance b/w ECM synthesis (collagen deposition] and degradation has been restored. ROLE OF MACROPHAGES IN REPAIR: + Clear the offending agents and dead tissue + Provide GPs for the proliferation of various cells + Secrete cytokines that stimulate fibroblast proliferation and CT synthesis and deposition. CUTANEOUS WOUND HEALING: HEALING BY PRIMARY UNION OR BY FIRST INTENTION: + Healing of a clean, uninfected surgical incision in the skin joined with surgical sutures + Surgical incision - death of a minimum number of epithelial and CT cells. * Disruption of epithelial basement membrane continuity is also minimal, + Re-epithelialization—by regeneration and there is a relatively thin scar + Simplest type of cutaneous wound healing. STAGES IN THE HEALING BY FIRST INTENTIONEptnotum prokferates and rows across the Bod cot ‘ie te Noutophis Blo. Death of minimal umber of ‘pital and connective issue cos ‘A First 24 hours ransom Epinetat_| prolteraton | ‘complet ‘Manna scar wit good ers union, . Weeks to months wen Figs 3.5Ato: Heating by primary intention (A) A wound th osely apposed edges and minenal ssueloss The Blod clots and fils the gap between the edges ofthe wound) Epithelium atthe edges prolate Minimal amount of rasan tissues formed) he epthaa rolteation complete ard the wounds weak (D) bos with sols First 24 hours: + Formation of blood clot: in the space b/w sutured margins. Blood clot contains not only trapped red cells but also fibrin, fibronectin and complement components. Clot stops bleeding and acts as a scaffold for migrating and proliferating cells. Dehydration at the external surface of the clot leads to formation of a scab over the wound. + Neutrophil infiltration: within 24 hrs of wound, neutrophils appear at the margins of the incision. Neutrophils use the scaffold produced by the fibrin clot for its migration. Release proteolytic enzymes which clean out debris + Epithelial changes: At the cut edges of the wound, the basal cells of the epidermis begin to show mitotic activity. Epithelial cells from both the edges of wound proliferate and migration across the wound along the dermis, ‘Two days + Neutrophils—replaced by macrophages. + Epithelial cells fuse in the midline below the surface scab and epithelial continuity is re-established in the form of a thin continuous surface layer: 3-7 days: * Granulation tissue begins to invade incision space. It progressively grows into the incision space/wound and fills the wound area by 5-7 days. Collagen is progressively laid down, + Surface epidermis achieves its normal thickness and differentiation. It matures with surface keratinization. * Acute inflammatory response begins to subside, 10-14 days: * Leukocytic infiltration, edema, and angiogenesis disappear during the second week. + Increased accumulation of collagen and regression of vascular channels, Thegranulation tissue scaffolding is converted into a pale, avascular scar. Wound normally gains about 10% strength of normal skin, Further fibroblast proliferation occurs with collagen deposition. Weeks to months: * Scar appears as acellular CT covered by intact epidermis and w/o inflammatory infiltrate. * Collagen deposition along the line of stress and wound gradually achieves maximal 80% of tensile strength of normal skin. HEALING BY SECONDARY UNION OR BY SECOND INTENTION: * When injury produces large defects on the skin surface with extensive loss of cells and tissue, the healing process is more complicated. + Healing in such cutaneous wound is referred to as healing by secondary union or by second intention, + Basic mechanisms of healing by primary (first intention] and secondary (second intention) union are similar. FEATURES OF HEALING BY SECONDARY INTENTION: tissue and the edges ae far ap edges and lage amount of granuation + Larger wounds—more exudate and necrotic tissue, Clot or scab—at the surface of wound—large + Full epithelialization of wound surface—slow because larger gap. + Sever inflammatory reaction because of larger defect and greater necrotic tissue. + Larger defect requires more amount of abundant) granulation tissue, + Extensive deposition of collagen with substantial scar formation. + Wound contraction—in large surface wounds—impt. Feature in healing by secondary union. + Myofibroblasts of granulation tissue have ultratructural features of smoothmuscle cells—contract in the wound tissue and the responsible for wound contraction. + Advantages of wound contraction—decreases the gap b/w its dermal edges of the wound, reducing the wound surface area WOUND STRENGTH. Major portion of the CT in repair is fibrillar collagens (mostly type I collagen|-- responsible for the development of strength in healing wounds. Time of a skin wound to achieve its maximal strength: + At the end of the first week: sutures—removed from an incisional surgical wound, wound strength is about 10% that of normal unwounded skin. + 4 weeks: wound strength quickly increases over the next 4 weeks and then slows down. + 3 months: wound strength reaches a 70-80% of the tensile strength of unwounded skin. Wound strength: 10% after 1 week Rapidly increases during next 4 weeks 70% at the end of 3% month Differences b/w healing 4 ik 2s a and secondary intention: Nanreotwoond Ceansuial Uncen Marine = 7s rrargin sures Used or apposition Cannot be wed of margins Infection Aosent May be infected Amount of Scant atthe Abundant and fl Graulatontssue incised gapand —thegap. ‘ong stare ack outcome Neatinesrscar regular Complications Rave Infection and FACTORS THAT INFLUENCE WOUND HEALING: Local factors: 1. Infection: single most impt cause for delay in healing. Causes persistent tissue injury and inflammation. 2. Mechanical factors: movement of wounded area may compress the blood vessels, and separate the edges of the wound and can result in delayed healing. 3. Foreign bodies: unnecessary sutures or foreign bodies (fragments of steel , glass), or even bone can delay healing. Infection: most cornmon cause of delay in wound healin, 4. Sze and type of wound: small surgical incisional or other injuries heal quickly with less scar formation than large excisional wounds or wounds caused by blunt trauma, 5. Location of injury: wound over the skin covering bone with little intervening tissue prevents wound contraction (ex: skin over the anterior tibia). The edges of skin lesions (ex: burns) in such locations cannot be apposed. 6. Blood supply: wounds in areas with good blood supply, such as the face, heal faster than those with poor blood supply, such as the foot. Varicose veins of the legs decrease the venous drainage—can cause nonhealing ulceration. Bed sores [decubitis ulcers}-- due to prolonged, localized pressure, which diminishes botharterial and venous blood flow. 7. Ionizing radiation: decreases repair process 8. Complications: may delay wound healing, Systemic facto 1. Nutritional deficiencies: delays wound healing—include: + Protein deficiency (ex: malnutrition} + Vitamin C deficiency: inhibits collagen synthesis and retard healing. + Trace elements: copper and zinc deficiency. Vitamin C: i, Essential for synth of collagen ii, Antioxidant ili, Reducing agent Vitamin C deficiency: i, Decreases cross linking of trophocollagen ii, Decreases tensile strength of wound 2. Age: wound healing rapid in young than aged. 3. Metabolic status: diabetes mellitus—delayed healing due ti microangiopathy 4. Circulatory status: inadequate blood supply (due to arteriosclerosis) or venous abnormalities (ex: varicose veins) that retard venous drainage , delay healing. Zinc—cofactor in collagenase 5. Hormones: glucocorticoids - anti-inflammatory effects—inhibit collagen synth, thereby impair wound healing 6. Hematological abnormalities: quantitative or qualitative defects in neutrophils and bleeding disorders may siow the healing process. COMPLICATIONS OF WOUND HEALING: Inadequate granulation tissue formation: or a deficient scar formation - wound dehisence and ulceration. Wound dehiscence: most common life- threatening complication after abdorninal surgery, 1. Dehiscence: wound splitting open—rupture of a wound—most common life- threatening complication after abdominal surgery. Due to increased abdominal pressure/ mechanical stress on the abdorninal wound from vomiting, coughing or ileus 2. Ulceration: Due to inadequate angiogenesis during healing, Wounds in leg of patients with atherosclerotic peripheral vascular disease or varicose veins usually ulcerate Nonhealing wounds also develop in regions devoid of sensation—neuropathic ot trophic ulcers-- in diabetic peripheral neuropathy, nerve damage from leprosy. 3. Incisional hernia: resulting from weak scars of the abdominal wall due to a defect caused by prior surgery—due to insufficient deposition of extracellular matrix or inadequate cross- linking in the collagen matrix. Excessive scar formation: Can result in 1. Hypertrophic scar: accumulation of excessive amounts of extracellular matrix, mostly collagen may give rise to a raised scar at the site of wound known as a hypertrophic scar — usually develop after thermal or traumatic injury, which involves the deep layers of the dermis 2. Keloid: if the scar tissue grows/ progress beyond the boundaries of the original wound and does not regress—keloid—exuberant scar that recurs with still largerkeloid after surgical excision. Keloids—more frequent among dark skinned people. Excessive deposition of type III collagen One of the complication of wound healing characterized by excessive production of ECM 3. Exuberant granulation: =Pyogenic granuloma or granuloma pyogenicum: + Consists of the localized formation of excessive amounts of granulation tissue, + Projects above level of the surrounding skin and prevents re-epithelialization. This mass—proud flesh. -Desmoids of aggressive fibromatoses + Incisional scars or traumatic injuries may be followed by excessive proliferation of fibroblasts and other CT elements + May recur after excision + Aggressive fibromatosis usually develops in the anterior abdominal wall. Excessive contraction: + Decrease in size of a wound due to myofibroblasts — contraction. + Exaggeration of this contraction - contracture—deformities of the wound and the surrounding tissues. * Consequences: * Compromise movements: for ex: contractures that follow severe burns can compromise the movement of the involved region and joint movements * Obstruction: for ex: in GIT contracture [stricture] can cause intestinal obstruction. Contracture: exaggeration of wound contraction Common sites for contractures—palms, soles and the anterior aspect of the thorax. Othe: 1. Infection of wound by microbes 2. Epidermal cysts can develop due to persistence of epithelial cells at the site of wound healing 3. Pigmentation - due to either colored particle left in the wound or due to hemosiderin pigment 4. Neoplasia: squamous cell carcinoma may develop in Marjolin's ulcer—scar that follows burns in skin. Fibrosi ‘TGF-B: Important fibrogenic agent + Normal wound healing—associated with deposition of collagen + Excessive deposition of collagen and other ECM components in a tissue— fibrosis—in chronic inflammation + TGF-8 is an important fibrogenic agent + Examples of disorders with fibrosis: Cirrhosis of liver, pneumoconioses, chronic pancreatitis and glomerulo nephritis Complications of wound healing: A. Deficient scar formation B, Excessive formation of the repair components C. Formation of contractures D. Others.