Biology of Wound Healing

Regeneration Versus Repair

In children and adults, the wound healing response characteristically leads to fibrosis, i.e. scar formation. In addition, adnexal structures (e.g. hair follicles, sweat and sebaceous glands) as well as components of the dermal extracellular matrix may fail to regenerate, resulting in a loss of normal skin function and impaired morphology. Because it is less than ideal, this type of wound healing is sometimes referred to as a reparative process . In contrast, during embryogenesis injured fetal skin can heal completely without fibrosis. Thus, the process resembles regeneration .

The underlying mechanisms that determine whether tissue regeneration versus tissue repair will occur still remain a mystery. Insights will hopefully come from experimental studies in other multicellular organisms (e.g. amphibians, fish) that retain throughout their adult life the capability to regenerate tissues after injury, as well as from studies of wound repair in fetal skin. In the future, these insights could lead to methods for transforming repair into regeneration and as a result, provide novel therapies that allow wound healing with minimal scarring.

Effect of Immune Response on Wound Healing

The immune system, including both innate and adaptive arms of the immune response, plays a critical role in wound healing. By influencing multiple repair mechanisms (e.g. angiogenesis, connective tissue deposition, epithelialization), inflammation has an impact on all stages of the repair response and ultimately the extent of scarring ( Fig. 141.1 ). In a number of models, there appears to be an inverse correlation between the intensity of the inflammatory immune response and the ability to undergo regeneration, with inappropriate immune reactions leading to tissue damage and impairment of tissue repair. However, this paradigm has recently been challenged by studies in several model organisms in which inflammatory signals were found to be crucial for promoting timely repair and for inducing fundamental processes involved in regeneration . Of note, these novel and unexpected findings emerged from investigations that employed well-established models within the field of tissue regeneration (not immunology).

Amphibians

Among the vertebrates, amphibians and fish are exceptional in their capacity to regenerate anatomically complete and fully functional tissues and organs during adulthood. In particular, urodele amphibians (newts and salamanders) can regenerate a range of organs and tissues . Cellular and molecular studies have focused primarily on limb regeneration after amputation, and recently such studies in adult salamanders demonstrated that the immediate influx of macrophages after tissue damage (preceding blastema formation) was an essential component of regeneration .

Zebrafish

Zebrafish also represent a traditional and valuable model for the study of regeneration. Adult zebrafish maintain regenerative capacity, not only after caudal fin amputation, but also following skin injury . Of interest, in both fin and skin regeneration there is an infiltration of myeloid inflammatory cells, suggesting that in principle regeneration can occur in the presence of inflammatory signals. Thus, by examining skin wound healing in zebrafish it may be possible to distinguish between beneficial and harmful inflammatory mediators and how they influence scar formation.

Mammals

The effects of inflammation on regeneration and repair have also been studied in mammals, primarily in mice, but also in humans. In addition to transgenic mouse models, refined mouse models that permit inducible and time-restricted depletion of specific immune cells have unraveled specific and critical functions of individual immune cell lineages in skin repair. For example, they provided evidence that, besides their central role in clearing cell detritus and microorganisms, macrophages exert distinct functions during the various phases of repair . Notably, macrophages recruited immediately after injury are essential for the induction of vascular sprouts and angiogenesis . Also, as the wound begins to contract, there is activation of focal adhesion kinase/extracellular signal-regulated kinase (FAK-ERK) within fibroblasts which leads to release of chemokine ligand 2 (CCL2), one of the prime chemokines known to drive monocyte/macrophage recruitment . Thus, an intimate interaction between macrophages and fibroblasts is envisioned.

In the presence of a functional immune system, higher vertebrates, including rodents and humans, can undergo post-amputation fingertip regeneration . Previously it was thought that the blastema, an undifferentiated pluripotent cell population presumably derived from mature cells via dedifferentiation, was responsible for mouse distal digit regeneration. However, recent genetic fate mapping and clonal analysis of individual cells revealed that a wide range of lineage-restricted tissue stem/progenitor cells contributed to restoration of the mouse distal digit, rather than pluripotent blastema cells . In humans, conservatively managed amputation injuries in children (but not adults) can be followed by restoration of the contour, fingerprints, and normal sensation and function of the digit, with minimal scarring . While the molecular mechanisms underlying this phenomenon are not yet understood, investigation of conserved mechanisms in more easily trackable non-human models will hopefully allow extrapolation to humans.

Clinical Implications

The underlying etiology of chronic, non-healing ulcers is multifactorial (see Ch. 105 ). It includes hypoxia due to vascular disease or sustained pressure, infection, and persistent inflammation . How the latter impairs the healing response is still not well understood, but increased protease activity (e.g. metalloproteinases [MMPs], serine proteinases) and generation of reactive oxygen species are thought to play a major role. Unravelling the underlying molecular pathophysiology of chronic wounds in humans will clearly involve studies of both regeneration and repair.

Primary Versus Second Intention Healing

When an acute wound, such as one resulting from a surgical excision, is allowed to heal on its own, this is termed second (secondary) intention healing (see Ch. 146 ). In primary intention healing, closure of the wound is accomplished by approximating the wound edges; the latter includes side-to-side closures, flaps, and grafts. Both primary and second intention forms of wound healing proceed through the three phases of healing.

In second intention healing, the time interval until complete re-epithelialization depends upon several factors. These include wound depth, anatomic location (e.g. facial wounds heal faster than acral wounds), any secondary infection, vascular supply, and geometric shape (for a given area, a more narrow diameter will heal faster). For smaller wounds, especially those that are partial-thickness, primary and second intention healing can produce similar cosmetic results. Once wounds have a diameter >8 mm, primary intention healing leads to better cosmetic results. Tertiary intention healing refers to wounds that are closed with the goal of primary intention healing, but dehiscence occurs and the wound is then allowed to heal by second intention.

The decision to choose primary versus second intention wound healing depends on a number of variables, including a patient's overall medical status and cosmetic concerns, as well as the anatomic location, depth and size of the wound, and whether it is located on a convex or concave surface. In addition to comorbidities such as atherosclerosis, venous hypertension and diabetes mellitus, the risk of infection (which may be increased in open wounds) and the patient's preference also influence the decision.

Three Phases of Wound Healing

Tissue injury causes leakage of blood constituents into the wound site and release of vasoactive factors ( Table 141.1 ), resulting in the activation of the clotting cascade. Clotted blood provides a matrix that allows for cell adhesion and migration. Not only do platelets trapped within the clot play an important role in hemostasis, they also represent a rich source of growth factors (e.g. PDGF) and proinflammatory cytokines that mediate the recruitment of inflammatory cells and fibroblasts into the wound site ( Fig. 141.3 ). The early inflammatory phase of repair is characterized by local activation of innate immune functions and chemoattraction, both of which lead to an early influx of polymorphonuclear leukocytes (PMNs; neutrophils). This is followed by invasion of blood monocytes, which differentiate into tissue macrophages .

While one function of the inflammatory infiltrate is to combat invading microbes, another equally important function is the release of cytokines and growth factors (e.g. IL-1, IL-6, vascular endothelial growth factor [VEGF], tumor necrosis factor [TNF], TGF-β; Table 141.2 ), which are critical for the initiation of the second proliferative phase of skin repair (see Fig. 141.3 ). During this phase of tissue formation, newly formed granulation tissue, consisting primarily of invading macrophages and fibroblasts as well as endothelial cells (as vessel precursors), covers and fills the wound area. Fibrin, fibronectin, vitronectin, type III collagen, and tenascin are components of the provisional extracellular wound matrix which facilitate cell adhesion, migration and proliferation. At the wound edge, epidermal–mesenchymal interactions stimulate keratinocyte proliferation and migration, leading to re-epithelialization .

Upon completion of epithelialization, cell proliferation and neovascularization cease, scar tissue forms and the wound enters the remodeling ( maturation ) phase, which lasts for several months. This last phase is characterized by a balance between the synthesis of new components of the scar matrix and their degradation by proteases. This balance determines whether there is normal versus abnormal scar formation (e.g. atrophic scars, hypertrophic scars, keloids). The mechanisms underlying granulation tissue regression and its transformation into scar tissue during this phase are largely unknown. Typically, there is also regression of vascular structures, transformation of fibroblasts into myofibroblasts, substitution of provisional extracellular matrix (ECM) with a permanent collagenous matrix, and a final resolution of the inflammatory response. However, if the inflammatory response persists, a disturbed healing response may result, leading to chronic skin ulcers (see Fig. 141.3 ). The mechanisms that direct resolution of the inflammatory response are not precisely understood, but most likely involve increased production of anti-inflammatory mediators, active suppression of proinflammatory factors, normalization of microvascular permeability, induction of apoptosis of inflammatory cells, and epithelialization.