Combination of Regeneration(Epithelium) and Repair (Dermis)

Lip Teh

Skin Healing

Introduction

• combination of regeneration(epithelium) and repair (dermis)
• processes of
• wound contracture
• connective tissue and matrix deposition
• epithelialisation
• stimulatory and inhibitory cell processes

Anatomy

• Skin consists of 2 essential layers: the epidermis, which provides a barrier against the environment, and the dermis, which provides strength, durability, and elasticity.
• important components of the dermis that contribute to its function include the basement membrane complex at the dermal-epidermal junction, collagen, elastin, proteoglycans, and a distinctive vascular plexus. The basement membrane complex at the dermal-epidermal junction contains type IV collagen, laminin, and highly specialized type VII collagen. Type VII collagen forms anchoring fibrils and filaments, which ensure strong physical bonding of the epidermis to the dermis.
• The bundle orientation of collagen and elastin differs between the papillary dermis and the reticular dermis. Collagen bundle orientation is random in the papillary dermis, but it is perpendicular to the lines of tension in the deeper reticular dermis. Similarly, elastin fibers are sparse and finely reticular in the papillary dermis, whereas they are thicker and form a complex 3-dimensional array in the reticular dermis.
• dermal vasculature forms a distinct plexus in the papillary dermis. This plexus configuration plays an important role in the remodeling process because collagen deposition tends to occur along the pathways of neovascularization. If the plexus is absent, collagen remodeling occurs along the pathways of an altered vascular pattern, as evident in granulation tissue in scar formation. Proteoglycans of the dermis provide a reservoir for growth factors (eg, basic fibroblast growth factor [FGF] binds to heparin sulfate). Dermal proteoglycans also direct the assembly of collagen (eg, decorin, tenascin) or are involved directly in angiogenesis and the regulation of cellular functions (eg, hyaluronic acid, chondroitin sulfate).
• Following full-thickness skin injury, the epidermis heals rapidly by regeneration. However, the dermis is not capable of regeneration and, therefore, heals by repair. This repair process begins with the formation of granulation tissue, which subsequently matures into the scar tissue. This process often leads to disfigurement and functional impairment of the integument.
• Scar tissue is different from the dermis because it is a relatively simple undifferentiated structure consisting of parallel arrays of collagen bundles that orient parallel to the lines of stress. Scar tissue also lacks elastin, normal distribution of proteoglycans, and the organs of the normal dermis.

Types of wound healing

primary, delayed primary, secondary and partial thickness wounds

1. Primary
2. sealed within hours, matrix metalloproteinases regulate collagen and ECM degradation during wound remodelling to produce a fine scar

1. Delayed primary
2. contaminated or poorly delineated wound is left open to prevent wound infection.
3. collagen metab is undisturbed and the wound achieves strength as if it had been primarily closed

1. Secondary healing
2. wound contracture 2 to myofibroblasts. These appear in the wound by the 3rd day, are maximal between 10 and 21 days and then begin to disappear as wound contracture is complete

1. Partial thickness wounds
2. mainly by epithelialisation over the exposed dermis
3. epithelium grow from transected sweat and sebaceous ducts and hair follicles. There is no wound contracture and minimal collagen deposition.

Connective Tissue Structure

Matrix metallo proteinases

• produced by fibroblasts, PMN's, macrophages
• inhibited by TGFb TGFb promotes matrix accumulation

Fibronectins

• matrix molecules(gylcoproteins) produced by epithelial cells, fibroblasts and macrophages
• incorporated into the blood clot and fibrin network found in the tissue stroma and the basal lamina
• Among the first proteins laid down in a fresh wound, thereby forming part of the provisional wound matrix
• Important in cell-cell communication, cell migration, wound contraction, collagen matrix deposition, epithelialisation
• Have the ability to bind a wide variety of molecules involved in wound healing, including collagen types I to IV, actin, fibrin, hyaluronic acid, dermatan and heparan sulfates, fibronectin itself, and fibroblast surface receptors
• Acts as a scaffold for collagen deposition
• During wound healing, fibronectins become cross-linked to fibrin clot and facilitate fibroblast attachment.
• Their quantity decreases as the wound matures and type I collagen replaces type III.

Ground substance

• Made up of proteoglycans which are covalently linked to glycosaminoglycans
• The majority of GAGs in the body are linked to core proteins, forming proteoglycans (also called mucopolysaccharides).
• GAG types:
• chondroitin sulfate, heparin sulfate and keratan sulfate all have a protein core and are sulfated
• hyaluronic acid does not have a protein core, is not sulfated and consists of repeating units of disaccharides, and it appears earlier in the wound the other three
• They create a negatively charged, hydrated environment that facilitates cell mobility. Eventually, they provide the viscoelastic properties of normal connective tissues

• Cells interact with collagen, laminin, fibronectin and proteoglycans found in the extracellular matrix using integrins
• Extracellular glycoproteins (Fibronectin and Laminin) are large molecules with many adhesion sites for collagen, GAGs and cell surface receptors
• Integrin diversity and specificity is generated by combining different alpha and beta subunits
• Mechanical aspects of integrin interactions:
• Fibroblasts and muscle cells realign the extracellular matrix – this in turn orients the movement and shape of fibroblasts and other cells
• Cells must attach to a substrate in order to migrate.
• Cell migration is an important aspect of developmental movements
• Cells such as bone forming (osteoblasts), fibroblasts, endothelial cells sense physical stresses via integrin interactions.
• Disrupting integrin function prevents cell responses to mechanical stress
• Mechanical attachment of cells to the extracellular matrix is required for normal tissue function
• Mutation of integrins recognizing laminin 5 results in epidermolysis bullosa
• Regulated loss of integrin function is also required for normal development
• Mammary epithelial cells stop expression of laminin and integrins during invasion of the underlying mesenchymal tissues (known as involution).

Proteoglycan aggregates are formed by linking core

proteins to a GAG core with linker proteins.

Collagen / Anchor / Proteoglycan / Cell-Surface Receptor / Cells
I / fibronectin / chondroitin and dermatan sulfates / integrin / fibroblasts
II / fibronectin / chondroitin sulfate / integrin / chondrocytes
III / fibronectin / heparan sulfate and heparin / integrin / quiescent hepatocytes, epithelial; assoc. fibroblasts
IV / laminin / heparan sulfate and heparin / laminin receptors / all epithelial cells, endothelial cells, regenerating hepatocytes
V / fibronectin / heparan sulfate and heparin / integrin / quiescent fibroblasts
VI / fibronectin / heparan sulfate / Iitegrin / quiescent fibroblasts

Tissue injury

Cellular responses

Overview:

1. coagulation
2. inflammation
3. In the inflammatory phase, bacteria and debris are phagocytized and removed and factors are released that cause the migration and division of cells involved in the proliferative phase.
4. proleferative phase
5. characterized by
6. fibroplasia
7. angiogenesis
8. collagen and matrix deposition
9. granulation tissue formation
10. epithelialization,
11. wound contraction

1. matrix synthesis.

Coagulation

• Transient vasoconstriction and coagulation followed by dilatation and capillary leak of plasma to the wound area.
• intrinsic pathway is activated when blood comes into contact with sub-endothelial connective tissues or with negatively charged surface that are exposed as a result of tissue damage. Quantitatively it is the most important of the two pathways, but is slower to cleave fibrin than the extrinsic pathway.The Hageman factor (factor XII), factor XI, prekallikrein, and high molecular weight kininogen (HMWK) are involved in this pathway of activation
• Theextrinsic pathway is an alternative route for the activation of the clothing cascade. It provides a very rapid response to tissue injury, generating activated factor X almost instantaneously, compared to the seconds or even minutes required for the intrinsic pathway to activate factor X. The main function of the extrinsic pathway is to augment the activity of the intrinsic pathway.Two main components - tissue factor or factor III, and factor VII. Tissue factor is present in most human cells bound to the cell membrane. The activation process for tissue factor is not entirely clear. Once activated, tissue factor binds rapidly to factor VII which is then activated to form a complex of tissue factor, activated factor VII, calcium, and a phospholipid, and this complex then rapidly activates factor X.
• The intrinsic and extrinsic systems converge at factor X to a single common pathway which is ultimately responsible for the production of thrombin (factor IIa).
• Fibrin and fibronectin cross-link together and form a plug that traps proteins and particles and prevents further blood loss
• This fibrin-fibronectin plug is also the main structural support for the wound until collagen is deposited - migratory cells use this plug as a matrix to crawl across and platelets adhere to it and secrete factors
• When blood comes in contact with collagen, triggering blood platelets to begin secreting inflammatory factors. Platelets also express glycoproteins on their cell membranes that allow them to stick to one another and to aggregate, forming a mass.
• Platelets provide the initial signals to begin the repair process.

• Release of alpha granules (contains PDGF, TGF-beta) and proinflammatory factors like serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, and histamine
• Vasodilation and activation of inflammatory cascade

Inflammation

• Complement activation
• release of C' products, TGFB lead to chemotaxis for PMN's 1st 48hours and macrophages by margination, diapedesis

Neutrophilsare responsible for

1. debris scavenging,
2. bacteriocidal
3. complement-mediated opsonization of bacteria
4. oxidative burst mechanisms (ie, superoxide and hydrogen peroxide formation).

• Absence of PMN's does not alter wound healing.

Macrophage is central to wound healing. They replace PMNs as the predominant cells in the wound by two days after injury

1. phagocytise bacteria and damaged tissue
2. debrides damaged tissue by releasing proteases
3. release of growth factors for fibroblasts and also for angiogenesis

• They express:
• collagenases, which debride the wound
• interleukins and tumor necrosis factor (TNF), which stimulate fibroblasts (produce collagen)
• stimulated by the low oxygen content of their surroundings to produce factors that induce and speed angiogenesis
• transforming growth factor (TGF), which stimulates keratinocytes
• Absence leads to severe  healing with poor debridement, delayed fibroblast proliferation, inadequate angiogenesis, and poor fibrosis
• Macrophages are attracted by:complement, FDP's, bacterial degradation products, TGFb, PDGF, clotting components, immunoglobulin G (IgG) fragments, collagen and elastin breakdown products, and cytokines, such as leukotriene B4, platelet factor IV

Lymphocyte is the last to appear- esp under the influence of IL1, C' system and IgG, may have a role in collagen and ECM remodeling

Proliferative Phase (to 3weeks)

• Fibroblast appear by day 4, major cell type by 5-7 day, last 2-4weeks
• Provisional matrix made up of fibrin, fibronectin and hyaluronic acid provide a scaffold for cell migration through integrin mediated matrix:fibroblast interactions.
• Fibroblast are responsible for synthesis, deposition and remodeling of the ECM
•  production of collagen(Types I and III) in a linear fashion up to 14 to 21 days
• Type I:Type III ratio changes from 4:1 to 2:1
• Cytokines: TGF-B, FGF

• Epithelialisation
• begins within hours
• dedifferentiation - cell line loses its specialised function and undergoes mitosis
• mobilization - cells enlarge, flatten, detach from neighbouring cells and basement membrane. Retraction of intracellular tenofilaments; dissolution of desmosomes and hemidesmosomes; formation of peripheral cytoplasmic actin filaments allowing movement.
• migration - motile epithelial cells move in a direction where contact will not occur. requires production of collagenase by epidermal cells.
• mitosis - maximal increase 48 to 72 hours after injury. Fixed basal cells away from the injury divide to replace the migrating cells. The cells, which have migrated, divide thereby thickening the new epithelial layer.
• contactinhibition - migrating epithelial cells meet cells of a similar type and adhesion occurs
• resting stage - entire process slows down upon adhesion of cells
• cell maturation - Once re-epithelialisation is completed, differentiation re-establishes progression from basal mitotic cells to stratum corneum
• integrins - maintain integral cell contact through a bridge between extracellular structural protein matrix and the cell’s internal cytoskeleton
• During migration, integrins on the pseudopod attach to the ECM, and the actin filaments in the projection pull the cell along
• epithelial cells require viable tissue to migrate across, so if the wound is deep it must first be filled with granulation tissue
• cell movement occurs more rapidly against 2 surfaces than 1 (wound cover - artificial, scab) and if basal lamina is intact, or by absence of debris, infection, dry eschar.
• Because they must dissolve any scab that forms, keratinocyte migration is best enhanced by a moist environment, since a dry one leads to formation of a bigger, tougher scab. To make their way along the tissue, keratinocytes must dissolve the clot, debris, and parts of the ECM in order to get through. They secrete plasminogen activator, which activates plasmin to dissolve the scab
• If the basement membrane remains intact, the epithelial cells migrate upwards in the normal pattern. This is equivalent to a first-degree skin burn. The epithelial progenitor cells remain intact below the wound, and the normal layers of epidermis are restored in 2-3 days.
• If the basement membrane has been destroyed, similar to a second- or third-degree burn, then the wound is reepithelialized from the normal cells in the periphery and from the skin appendages, if intact (eg, hair follicles, sweat glands).
• If the wound is very deep, skin appendages will be ruined and migration can only occur from wound edges
• The leading edge cells are phagocytic to clear away debris
• Marginal basal cells begin to migrate across the wound along fibrin strands stopping when they contact each other (contact inhibition).
• Within the first 48 hours the entire wound is epithelialized.
• Growth factors- EGF, basic FGF and keratinocyte growth factor (KGF)
• Angiogenesis
• occurs concurrently with fibroblast proliferation
• marked by endothelial cell migration and capillary formation.
• The migration of capillaries into the wound bed is critical for proper wound healing.
• attracted to the wound area by fibronectin found on the fibrin scab and by growth factors released by other cells
• also stimulated by hypoxia and presence of lactic acid in the wound
• The granulation phase and tissue deposition require nutrients supplied by the capillaries, and failure for this to occur results in a chronically unhealed wound.
• ECM and endothelial receptors for provisional matrix proteins are necessary for angiogenesis as is protease expression and activity
• Angiogenic factors- TNF-alpha and basic FGF, VEGF; also low oxygen tension, lactic acid
• Granulation tissue formation
• Fibroblasts differentiate and produce ground substance and then collagen.
• The ground substance is deposited into the wound bed; collagen is then deposited
• Collagen levels rise for ~ three weeks with a concomitant rise in tensile strength and a fall in the number of synthesizing fibroblasts until the rates of degradation and synthesis are equivalent – “collagen homeostasis”.
• Important cytokines: PDGF, insulinlike growth factor (IGF), and EGF.

Remodelling (>3 weeks)

• Accelerated synthesis and degradation of collagen (no net increase) with regression of new capillaries and organisation of collagen fibres determined by mechanical stresses.
• Scar maturation and increase in tensile strength.
• Collagen type III replaced by collagen type I until normal skin ratio of 4:1 (type I: typeIII) is obtained.
• Degradation of GAG’s in ground substance and reduction of water content to levels found in normal dermis ensues. Stable crosslinking of collagen is established.
• Length of phase is multifactorial. (wound factors/patient factors)
• The tensile strength of a wound is a measurement of its load capacity per unit area. A wound’s breaking strength is defined as the force required to break it regardless of its dimensions.
• Collagen fibers are largely responsible for the tensile strength of wounds
• The entire process is a dynamic continuum with an overlap of each phase and continued remodeling. The wound reaches maximal strength at 60days, with a tensile strength that is 70%-80% of normal skin. Collagen deposition continues for a prolonged period, but the net increase in collagen deposition plateaus after 21 days
• Contraction
• Contraction:Active, normal part of healing whereby the defect in soft tissue is closed.
• Contracture:Abnormal compaction of scar tissue beyond the point of reepithelialisation causing distortion of the anatomical appearance and functional capacity of adjacent tissues and organs
• Occurs in full thickness wounds
• 2 theories – mediated by

1. fibroblasts
2. fibroblasts organize the disorganized haphazardly deposited collagen matrix, effectively compacting the ECM. ECM reorganization leads to wound contraction and, in some cases, scar contracture.
3. myofibroblasts
4. specialized mesenchymal cells that normally appears during the wound-healing process and generates contractile force to the extracellular matrix to pull both edges of the wound until it disappears by apoptosis.
5. Show features of fibroblast and smooth muscle cell
6. In scar contracture, the myofibroblast remains in the dermis and continuously contracts the scar, eventually causing scar contracture.
7. fibronexus represents an intercellular junction between myofibroblasts, but in particular is a device for providing contact between myofibroblasts and matrix that mediates continuity between intracellular contractile filaments and extracellular matrix proteins.

• Forces of contraction act to close the wound until balanced by tension in the surrounding tissues
• Full-thickness skin grafts inhibit wound contraction by speeding up the rate at which myofibroblasts disappear from the wound. Theamount of dermis grafted, not the absolute thickness of the graft, determines the degree to which wound contraction is prevented.
• A study measured Fibronectin and actin content in excised wounds in 18 rats. In six rats, wounds were allowed to granulate and contract. Wounds were grafted with full-thickness skin in six rats and with split-thickness skin grafts in the remaining six. Multiple biopsies were taken for immunochemistry and electron microscopy. Actin and fibronectin content was greatest in ungrafted wounds. This content was reduced in wounds by skin grafting, with full-thickness grafts having a generally more inhibitory effect than split. In grafted wounds, actin was observed primarily in the wound bed, whereas fibronectin was present in both the wound bed and graft. Fibronectin appeared coincidently with actin in split-thickness skin grafts and their wounds. In contrast, full-thickness skin grafts had fibronectin staining before actin. The degree of wound contraction inhibition by different types of skin grafts appears to correlate with the fibronectin and actin staining pattern
• Transforming growth factor beta 1 (TGF-β1) stimulates fibrosis and scar contraction in the following manner: by increasing the production of collagen, fibronectin, and glycosaminoglycan;by augmenting the tissue inhibitor of metalloproteinase and α2-macroglobulin(inhibits collagenase) while at the same time inhibiting the production of protease and by increasing the expression of alpha-smooth muscle actin from the fibroblasts.
• Tranilast [N-(3,4-dimethoxycinnamoyl) anthranilic acid], which has been used as an anti-allergic drug, suppresses collagen synthesis in fibroblasts by inhibiting the action of TGF-β1
• Epithelialization by itself does not inhibit wound contraction.
• Contraction and possible resulting deformity can be minimized by replacing missing skin with grafts, particularly thick transfer or free grafts which can also fill the depth defect left by tissue loss. The thicker the graft the better is the inhibition of contraction.
• Areas of fixed tissues (immobile skin) and little excess skin frequently cannot close with contraction. These areas remain as an ulcer or heal by little more than epithelialization and are less resistant to additional and often minor trauma..

Collagen