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1 Tissue Repair Robert F. Diegelmann, Ph.D. OBJECTIVES After this presentation and discussion, the participants should be able to: 1. Define the biochemical responses to tissue injury 2. Describe the mechanisms of tissue repair 3. Define the clinical processes of tissue repair 4. Outline the cellular, biochemical & molecular elements of normal tissue repair 5. Identify the cytokines and growth factors involved in tissue repair 6. Discuss factors which may alter tissue repair 7. Give examples of fibrotic responses to injury 8. Identify the underlying pathophysiology of chronic wounds 9. Understand the current research information about wound healing and how this data can help develop new clinical strategies to treat the many tissue repair problems. Figure 1 I. OVERVIEW The process of wound healing consists of an orderly sequence of events characterized by the specific infiltration of specialized cells into the wound site. The platelets and inflammatory cells are the first cells to arrive and they provide

2 key functions and signals needed for the influx of connective tissue cells and a new blood supply. These chemical signals are known as growth factors or cytokines. The fibroblast is the connective tissue cell responsible for collagen deposition needed to repair the tissue injury. Collagen is the most abundant protein in the animal kingdom, as it accounts for 30% of the total protein in the human body. In normal tissues, collagen provides strength, integrity and structure. When tissues are disrupted following injury, collagen is needed to repair the defect and hopefully restore structure and thus function. If too much collagen is deposited in the wound site, then normal anatomical structure is lost, function is compromised and the problem of fibrosis results. Conversely, if insufficient amounts of collagen are deposited, the wound is weak and may dehisce. Therefore, to fully understand wound healing it is first essential to understand the basic cell biology, immunology and biochemistry involved in the repair process. II. DEFINITIONS A. Wound a disruption of normal anatomic structure and function B. Healing a complex, dynamic process that result in the restoration of anatomic continuity and function C. Acute Wounds normally proceed through an orderly and timely reparative process and results in sustained restoration of anatomical and functional integrity D. Chronic Wounds have failed to proceed through an orderly and timely process to produce anatomical and functional integrity, or proceeded through the repair process without establishing a sustained anatomical and functional result III. RESPONSES OF TISSUES TO INJURY A. Normal 1. Resolution Following minimal tissue injury and inflammation, the small blood vessels in the area regain their usual semi permeability, fluid flux ceases, migration of leukocytes stops and the area is left precisely as it was before the reaction started. 2. Regeneration Following loss of tissue, there is minimal inflammation followed by proliferation of cellular elements and replacement of extracellular matrix identical to that lost with the net results being an exact structural and functional replacement.

3 3. Repair by Scar Following injury and inflammation, there is proliferation of fibroblasts and deposition of connective tissue elements with restoration of function. B. Pathologic 1. Fibrosis Following injury and inflammation, the normal cellular elements and extracellular matrix of the damaged tissues are replaced by a distorted accumulation of scar tissue and function is lost. 2. Dehiscence During the process of repair by scar, either insufficient amounts of cross linked collagen are deposited in the wound site or there is an excessive amount of collagen degradation occurring. Thus, the repair site is weak and will split open if tension is applied. 3. Contracture Excessive scar contraction, which leads to malformation and loss of movement. Frequently seen after extensive burn injury. Some fibroblasts become specialized cells called myofibroblasts. They have characteristics of both fibroblasts and smooth muscle cells and they function to cause wounds to contract. This is a normal and beneficial process. However, if too much contraction occurs, then the pathologic process of contracture can result. IV. MECHANISMS OF WOUND REPAIR A. Connective tissue deposition the process where fibroblasts in the surrounding tissue proliferate and move into the wound site. These newly recruited fibroblasts then produce the collagen and proteoglycans (ground substance) which form the scar tissue. B. Epithelization residual epithelial cells in the hair follicles and other appendages migrate across the surface of the wound and proliferate to provide new surface epithelial cells. C. Contraction this is a normal process whereby the wound margins are pulled to the center of the wound by cells called myofibroblasts. When excessive wound contraction occurs, the pathologic process of contracture can result.

4 V. CLINICAL TECHNIQUES USED FOR WOUND CLOSURE A. Primary Intention: Matrix deposition is the main mechanism B. Delayed Primary Intention: Used if the wound is contaminated. Some Contraction initially, but then mostly Matrix deposition once the wound is closed. C. Secondary Intention: Mainly Contraction, but also involves Matrix deposition & Epithelization. D. Partial Thickness Healing: All Epithelization. VI. SEQUENCE OF EVENTS FOLLOWING INJURY A. Hemostasis 1. Fibrin clot formation provides an initial wound matrix and a guide for the migration of inflammatory cells and fibroblasts into the wound site 2. Platelets provide initial signals to begin the repair process a. Platelet derived growth factor (PDGF) attracts fibroblasts to the wound and stimulates them to proliferate b. Transforming growth factor beta (TGFβ) also attracts fibroblasts to the wound site and stimulates them to produce collagen Figure 2

5 At the initial time of tissue disruption, platelets release coagulation factors and cytokines to initiate the healing process. B. Inflammation 1. Polymorphonuclear neutrophils (PMN) important cells to prevent wound infection a. Vessel adherence (pavementing, margination) b. Diapedesis movement of PMNs through vessel walls c. Chemotaxis directed movement to the site of injury in response to chemical signals d. Phagocytosis (Opsonization) ingestion and destruction of bacteria and foreign materials Figure 3 Within the first day following tissue injury, neutrophils attach to surrounding vessel walls (margination) and then move through the vessel walls (diapedesis) to migrate (chemotaxis) to the wound site. 2. Macrophages the most essential inflammatory cell for wound healing a. Interleukin 1 (IL 1) another signal to stimulate fibroblasts to proliferate

6 b. TGFβ (Transforming Growth Factor beta) c. PDGF (Platelet Derived Growth Factor) d. Tumor necrosis factor (cachectin, TNF) an additional chemical signal to stimulate fibroblasts to proliferate Figure 4 The fibroplasia phase is characterized by movement of wound macrophages into the site of injury, which in turn attract fibroblasts. The fibroblasts then repair the site by producing new connective tissue matrix. 3. Lymphocytes a. TGFβ b. TNF 4. Mast cells products from these cells produce the characteristics redness and itchiness around the wound c. Vasoactive amines histamine, and serotonin d. Heparin e. Proteolytic enzymes f. Prostaglandins, leukotrienes and thromboxanes

7 g. TNF Figure 5 The remodeling phase is characterized by equilibrium between collagen synthesis and collagen degradation in an effort to reestablish the connective tissue matrix that was destroyed by the tissue injury. Characteristics of Wound Inflammation 1. Redness (Rubor) 2. Swelling (Tumor) 3. Heat (Calor) 4. Pain (Dolor) C. Collagen Metabolism 1. Collagen Facts a. most abundant protein b. organic mass for bone, skin, tendon, blood vessels, cornea, etc. c. framework for liver, spleen, kidney d. strength and structure for of all tissues 2. Types; where is collagen found in the body, which cells produce it, and how do the specific types relate to function

8 3. Biosynthesis; how is collagen synthesized and why is this information important for the clinical care of patients Other components of the Extracellular Matrix and Their Functions 4. Elastin protein forming the major constituent of elastic tissue fibers 5. Glycosaminoglycans 6. Proteoglycans 7. Adhesive glycoproteins group of compounds consisting of a protein combined with a carbohydrate (such as galactose or mannose). D. Collagen Degradation and Scar Remodeling 1. Collagenases and other matrix metalloproteinases (MMPs); specific enzymes essential for collagen remodeling 2. How is collagenase activity controlled in tissues? VII. NORMAL TISSUE REPAIR a. Plasminogen, plasmin b. Tissue inhibitor of metalloproteinase (TIMP) c. Alpha 2 macroglobulin Synthesis < > Degradation in order for synthesis and thus function to be restored after injury, an equilibrium between synthesis and degradation must be re established. VIII. LIST OF FACTORS THAT MAY ALTER WOUND HEALING (Do NOT memorize) A. Local factors 1. Blood supply; tissue hypoxia prevents healing a. Pressure b. Venous stasis c. Arterial occlusion d. Volume

9 2. Denervation: soft tissue is not protected without sensation, therefore trauma will cause wounds and these wounds are difficult to heal because of constant re injury. 3. Hematoma generates free oxygen radicals which causes tissue damage and delays healing and also becomes a site for infection 4. Infection retards healing. Infection is the number one cause of delayed wound healing. If the wound contains more than 10 5 organisms per gram of tissue, it should be treated with gentle, sterile irrigation and kept in a moist sterile environment. Chronic use of caustic agents such as peroxide and iodophores should be avoided because they are toxic to new wound tissue. Never put anything into a wound that you would not put into your eye 5. Irradiation causes very complex wound healing problems and reduced re vascularization 6. Mechanical stress too much stress can cause delayed healing 7. Dressing materials there are many new dressings available today and it is important to select the correct material for reach specific wound situation 8. Surgical technique tissues must be handled in a gentle fashion 9. Irrigation can the golden period be prolonged? 10. Electrocoagulation can cause increased tissue damage 11. Suture materials can cause varying degrees of inflammation 12. Antibiotics topical more effective than systemic 13. Type of tissue all tissues do not heal the same. Wounds on the face heal at faster rates compared to wounds on the lower leg; bladder greater than gut 14. Factitious wounds this problems occurs frequently and it must be recognized B. General factors 1. age healing processes decrease with age 2. anemia need adequate oxygen for optimal healing 3. anti inflammatory drugs suppress the healing response 4. cytotoxic and anti metabolic drugs retard healing 5. diabetes mellitus these patients may have wound healing problems due to vascular problems and reduced inflammatory cell function, sensation loss, and alterations in matrix metabolism. 6. hormones steroids depress the immune response to infection and can reduce the healing response 7. systemic infection can delay wound healing 8. jaundice thought to have adverse effects on tissue repair 9. malignant disease some evidence suggests cancer patients have delayed wound healing 10. malnutrition can cause deleterious effects on collagen metabolism 11. obesity more susceptible to infection

10 12. temperature excessive heat or cold can damage tissue 13. trauma, hypovolemia, and hypoxia all delay healing 14. uremia thought to delay wound healing 15. vitamins C and A are essential for proper tissue repair. Vitamin E, in high doses, can retard healing. Vitamin D is essential for bone healing. 16. trace metals IX. SCAR WARS A. Excessive collagen breakdown In some rare situations, excessive collagenase is produced and the wound is very weak and may dehisce. B. Excessive scar excessive collagen deposition 17. Skin a. Keloid b. Hypertrophic Scar 18. Tendon adhesion 19. Nerve transmission loss 20. Joints ankylosis 21. Vessels atherosclerosis 22. Tubular structures a. Crohn s disease b. Biliary stenosis/atresia c. Esophageal stricture 23. Breast contracture 24. Liver cirrhosis 25. Bone fibrotic non union C. Keloid and hypertrophic scar more specifics 1. Keloid extends beyond the boundary of the wound, usually does not regress, and often recurs after excision 2. Hypertrophic scar remains within the boundary of the line of incision or injury and gradually regresses with time 3. Proposed etiologies many speculations, but non proven

11 4. Genetic influences approximately 10% of the Black population; less than 1% of Caucasians 5. Incidence certain anatomical locations are more prone to keloid formation 6. Biochemistry a. Studies on collagen biosynthesis keloid fibroblasts have an increased capacity to produce collagen b. Possibly a selection of high collagen producing fibroblasts 7. Treatment modalities D. Chronic Ulcers a. Surgical considerations b. Steroids c. Cytotoxic agents d. Pressure e. Irradiation f. Laser g. Collagen cross link inhibitors 1. Diabetic ulcers 2. Venous stasis ulcers 3. Pressure ulcers Current data suggest that pressure ulcers are locked into a chronic state of inflammation driven by the neutrophil. Latest research information Current evidence on the role of excessive neutrophils in pressure ulcers Effect of a hydroactive dressing on proinflammatory mediators and collagenase E. Comparison Of Acute Wounds And Chronic Ulcers ACUTE WOUNDS Orderly & timely Closed wound Viable tissue Single injury Controlled inflammation Controlled bacteria CHRONIC ULCERS Complex & delayed Open ulcer Compromised tissue Repeated injury Uncontrolled inflammation Uncontrolled bacteria

12 F. Fetal Wound Healing 1. Research studies of fetal wound healing may provide new insights into the biology of tissue repair and may offer new clinical modalities to that adult wound healing problems. Figure 6: Cytokines involved in Wound Healing (Do NOT memorize) PMNs, polymorphonuclear leukocytes; MMP, matrix metalloproteinase; HA, hyaluronic acid, TIMP, tissue inhibitor of matrix metalloproteinase.

13 X. WOUND HEALING RESPONSE CURVE Taking into consideration all the complexities involved with the inflammatory response and all the steps involved in the regulation of collagen metabolism, it is important to note that the vast majority of wounds heal normally. The challenge before us now is to focus on the patients with wound healing problems associated with either excessive healing or deficient healing. We have the hope in the future of utilizing new research information and new clinical approaches to reduce the incidence of these wound healing problems. Figure 7

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