Mechanism of Wound Healing - Detailed Answer (20 Marks)

INTRODUCTION

1. Haemostasis (immediate, minutes to hours)
2. Inflammation (days 1-7)
3. Proliferation / Fibroplasia (day 3 to week 4)
4. Remodelling / Maturation (week 1 onward, up to 2 years)

Figure: Phases of wound healing showing three main phases on a log-scale timeline. Adapted from Greaves et al. (Scott-Brown's Otorhinolaryngology, Vol 1, Fig 10.2)

TYPES OF WOUND HEALING

Figure: Healing by primary, secondary and tertiary intention (Scott-Brown's, Fig 10.1)

1. Primary Intention (Healing by First Intention)

2. Secondary Intention (Healing by Second Intention)

3. Tertiary Intention (Delayed Primary Closure)

PHASE 1: HAEMOSTASIS

• Vasoconstriction lasting 10-15 minutes (mediated by catecholamines and prostaglandins)
• Platelet activation: Exposure of sub-endothelial collagen triggers platelet adhesion, aggregation, and degranulation
• Coagulation cascade (extrinsic pathway) is activated, resulting in fibrin deposition
• A clot forms composed of collagen, platelets, thrombin, and fibronectin

• Covers and protects the wound from contamination
• Provides an initial scaffold for cell attachment and migration
• Acts as a reservoir of growth factors

PHASE 2: INFLAMMATION

Early Inflammation - Neutrophil Phase (Days 1-2)

• TGF-β, complement constituents (C3a, C5a)
• Bacterial peptide products
• Fragments of ECM proteins, IL-1, TNF-α, platelet factor 4

1. Margination - neutrophils adhere to endothelial cells of surrounding blood vessels
2. Diapedesis - movement through the vessel wall into tissue
3. Chemotaxis - directed migration toward the wound

• Phagocytosis and intracellular destruction of bacteria
• Release of proteolytic enzymes (collagenases) to digest devitalized tissue and bacteria
• Formation of oxygen-free radicals via the myeloperoxidase pathway to sterilize the wound
• Release of IL-1α, IL-1β, and TNF-α - early activators of growth factor expression in macrophages, keratinocytes, and fibroblasts

Note: Neutrophils are not essential for healing in a sterile wound - their primary role is infection control. (Schwartz's)

Late Inflammation - Macrophage Phase (Days 2-3)

T-lymphocytes (Days 5-7)

• CD8+ suppressor T cells: depletion enhances wound healing
• CD4+ helper T cells: depletion has no effect
• Lymphocytes exert a downregulating effect on fibroblast collagen synthesis via IFN-γ, TNF-α, and IL-1 through direct cell-cell contact - suggesting extracellular matrix synthesis is regulated not only by soluble factors but also by direct cell-cell contact (Schwartz's)

PHASE 3: PROLIFERATION (FIBROPLASIA)

3A. Re-epithelialization

1. Increased mitotic activity within basal cells of wound edges begins within 12 hours
2. Keratinocytes migrate - as a sheet, extending lamellipodia across the defect, migrating between the fibrin clot and the collagenous dermis
3. "Epiboly" - creation of a delicate epithelial covering over the raw area
4. Migration is guided by integrin receptor-ECM interactions
5. Keratinocytes produce plasminogen activators which activate plasmin and collagenase-1 (MMP-1) to dissolve fibrin and type I collagen for migration
6. Contact inhibition stops migration once epithelial sheets meet
7. Keratinocytes return to normal differentiation; epithelial stratification is restored

3B. Granulation Tissue Formation

• New blood vessels (angiogenesis)
• Fibroblasts producing ECM
• Loose collagenous matrix (pink, granular appearance on examination)

Fibroplasia

1. Fibroblasts proliferate and migrate into the wound
2. They produce: fibronectin, hyaluronan, collagen (types I and III), elastin, and proteoglycans
3. Initial matrix: fibrin and fibronectin → gradually replaced by collagen and glycoproteins
4. Lactate (accumulates ~10 mmol in wound) is a potent regulator of collagen synthesis via adenosine-related mechanisms

• Fibroblasts synthesize procollagen chains (protocollagen, ~1000 amino acids), each with glycine in every third position + proline or lysine
• In the endoplasmic reticulum: hydroxylation (prolyl hydroxylase requiring O₂, iron, α-ketoglutarate, vitamin C as cofactors) + glycosylation → forces α-helical configuration
• Three α-helical chains entwine to form procollagen (right-handed superhelix with registration peptides)
• Extracellularly: registration peptides are cleaved by procollagen peptidase → collagen monomer
• Further polymerization and cross-linking via intra/intermolecular covalent bonds → mature collagen fibrils (Schwartz's)

• Type III collagen predominates early (scar collagen) - more flexible, less strong
• Type I collagen predominates in mature scar - thicker fibers, greater tensile strength
• Aberrant collagen deposition → keloid (excess) or poor wound healing (deficit)

Angiogenesis

1. Degradation of basement membrane of parent vessel
2. Endothelial cell migration toward angiogenic stimuli
3. Endothelial cell proliferation
4. Formation and maturation into capillary tubes
5. Invasion creates a microvascular network

• VEGF (vascular endothelial growth factor) - produced by macrophages and keratinocytes in response to hypoxia
• FGF-1 and FGF-2 (acidic and basic fibroblast growth factor) - released from disrupted cells; most potent angiogenic factors
• PDGF, TGF-β
• Hypoxia is the strongest physiologic inducer of angiogenesis

3C. Wound Contraction

• Fibroblasts differentiate into myofibroblasts (contain smooth muscle actin - α-SMA)
• Myofibroblasts attach to the ECM and to each other
• Through contractile activity, they pull wound edges together
• Driven by TGF-β

• Reduces the amount of scar tissue needed
• In humans, skin is relatively immobile - contraction alone rarely closes a wound completely
• Excessive contraction → contracture (impairs function, especially at mobile sites like the neck, joints)
• Insufficient contraction → delayed wound closure, bleeding, infection

PHASE 4: REMODELLING AND SCAR MATURATION

1. Equilibrium is reached between collagen formation and degradation around day 21
2. Matrix metalloproteinases (MMPs) - collagenases, gelatinases, stromelysins - synthesized by fibroblasts, neutrophils, and macrophages degrade excess collagen
3. TIMPs (tissue inhibitors of metalloproteinases) regulate MMP activity
4. Type III collagen is progressively replaced by type I collagen
5. Collagen fibers reorganize from a disorganized array of fine fibers → thicker fibers aligned parallel to skin stress lines (Langer's lines)
6. Myofibroblasts and vascular cells undergo apoptosis → granulation tissue transforms into scar
7. Reduced: cellularity, vascularity, macrophage activity, capillary outgrowth

• At 1 week: 3% of normal dermis
• At 3 weeks: 20% of normal dermis
• At 3 months: 80% of normal dermis
• The scar never fully regains normal dermal strength (maximum ~80%)

GROWTH FACTORS AND CYTOKINES IN WOUND HEALING (SUMMARY TABLE)

FACTORS AFFECTING WOUND HEALING

Local Factors

• Infection (most common cause of delayed healing)
• Foreign bodies, devitalized tissue
• Ischemia / poor blood supply
• Mechanical trauma, excessive tension
• Radiation damage
• Wound type and size

Systemic Factors

• Age: Elderly patients show delayed epithelialization, altered inflammatory response, reduced collagen content, impaired angiogenesis
• Nutritional status: Protein-calorie malnutrition delays healing; key nutrients include arginine, vitamins A and C, and zinc. Serum albumin <30 g/L predicts delayed healing
• Diabetes: Immune dysfunction, slower collagen synthesis, decreased angiogenesis, poor tensile strength - multifactorial
• Malnutrition: Decreased fibroblast proliferation, decreased proteoglycan and collagen synthesis
• Medications: Corticosteroids, immunosuppressants, and chemotherapy agents alter the healing environment in the first 3 days - critical molecular modulators are released during this window
• Smoking: Reduced oxygenation, vasoconstriction, impairs all phases
• Immunosuppression: Failure of inflammatory phase, absent molecular modulators
• Weight loss >10% (common in cancer patients) correlates with decreased wound healing

EXCESSIVE / ABNORMAL WOUND HEALING

Hypertrophic Scars

• Excess collagen deposition within the original wound boundaries
• Elevated, red, firm scar
• Common at mobile sites (neck, joints)
• Management: topical silicone, intralesional corticosteroid, compression therapy, surgical excision (Bailey & Love)

Keloid Scars

• Extend beyond original wound borders - locally destructive
• Occur more commonly in darker skin types (African, Asian)
• Most commonly affect: face, earlobes, deltoid area, presternal region
• Appear months after injury
• More resistant to treatment; may require repeated excision + adjuvant radiotherapy (Bailey & Love)

Chronic Wounds

• Healing delayed >3 weeks
• Deficient in growth factors; decreased keratinocyte and fibroblast migration
• Increased reactive oxygen species and tissue proteases
• Microbial contamination; fibroblasts become senescent

RECENT EVIDENCE (PubMed, 2022-2024)

• Peña & Martin, 2024 (Nat Rev Mol Cell Biol) - "Cellular and molecular mechanisms of skin wound healing" - updated review confirming the four-phase model with emphasis on immune cell crosstalk and stem cell roles (PMID: 38528155)
• Sorg & Sorg, 2023 (Eur Surg Res) - "Skin Wound Healing: Of Players, Patterns, and Processes" - comprehensive review of cellular players (PMID: 36417847)

SUMMARY