May J. Reed, Teruhiko Koike, and Pauli Puolakkainen
The purpose of this chapter is to review the changes in wound healing that occur in the aged. Unlike pathological conditions such as infection or diabetes as a cause for impaired wound repair, aging may simply reduce the speed at which an individual heals. Thus, a goal of this review is to assess whether aging represents truly impaired or merely slowed wound healing.
This chapter focuses on cutaneous wound healing in response to an acute incisional wound, as after surgery, or an acute excisional wound, as after a punch biopsy. Although the general principles that follow will be relevant for most tissues, there are age-associated alterations in certain organs of the body (e.g., sclerosis of the kidney and large blood vessels) that create unique challenges during wound repair. These changes are generally confounded by diseases (such as diabetes) that are more prevalent with age and are, therefore, beyond the scope of this review. Moreover, this review does not address chronic wounds or pressure wounds, pathological conditions that are accentuated by, but not unique to, aged tissue.
Human skin or the epidermis (primarily squamous epithelial cells or keratinocytes), dermis (largely fibroblasts and an extracellular matrix [ECM] of collagens I and III, elastin, and glycosaminoglycans), subcutaneous fat, and associated appendages (hair follicles, sensory receptors, sweat and sebaceous glands). As the largest organ of the body, the skin has a critical role as the protective barrier from environmental insults. As such, it is not surprising that in the absence of injury, changes in the skin with age have primarily cosmetic consequences. However, age does have a detrimental effect on the skin's reserves and subsequent response to injury. Although changes caused by intrinsic aging have been recognized in the skin of elderly individuals for many decades, only recently have these changes been analyzed independently of chronic environmental insults such as sun exposure (1).
To provide a framework for a review of aging and wound healing, this chapter begins with a summary of how age affects the morphology and function of the skin (2-4). As the outermost layer, the epidermis is the first line of defense after tissue injury. The total thickness and the four layers of stratified squamous epithelial cells (keratinocytes) are maintained in aged skin, but the cells become more variable in size, shape, and orientation. In addition, the turnover time, the number of days for keratinocytes in the basal layers of the epidermis to migrate and be shed from the skin surface, increases by 50% in the aged. Other cells in the epidermis such as melanocytes (which provide pigment) and Langerhans cells (which are responsible for the immune response in the epidermis) are also reduced in number. Whereas barrier function against water is maintained in aged skin, there is a significant decrease in the ability of the epidermis to regenerate in response to noxious substances such as ammonia (5).
The dermis of humans and other taut-skin animals is divided into the superficial papillary dermis and the deeper reticular dermis. In aged skin the normal ridges at the epidermal-dermal junction flatten, thereby reducing the contact surface between the epidermis and dermis (6). This loss of surface area results in fewer proliferative epidermal cells in the basal layers and a decrease in the strength of epidermal-dermal adhesion. The generalized atrophy and thinning of the dermis with age is a result of decreases in cell number and changes in the surrounding matrix. The former includes fewer fibroblasts, less vascularization, and a decrease in the population of mast cells and macrophages that function as antigen-presenting cells (APCs) in the dermis (for reviews see refs. 3 and 7). Changes in dermal protein content with age include a decrease in collagen, the primary structural protein, as a result of both decreased production and increased degradation (8-11). The physical properties are also altered; whereas collagen in young skin is oriented in ropelike bundles, in aged skin the protein becomes disorganized with randomly oriented bundles that appear to unravel and show variable bundle width. Moreover, in the deeper dermis the fiber density is increased with age owing to a decrease in space between the bundles (12,13). It is controversial as to whether the composition of glycosaminoglycan (e.g., hyaluronic acid and dermatan sulfate) is significantly decreased with age (for review see ref. 7). These hydrophilic dermal proteins are referred to as the ground substance of the dermis and provide an optimal microenvironment for cell adhesion and migration. Glycosaminoglycans also maintain skin turgor, and alterations in this class of dermal proteins in the aged may contribute to a flaccid appearance of the dermis. Elastin, the protein most responsible for maintaining skin elasticity (also termed recoil), is a large, highly stable protein with minimal turnover with age. Although elastin content does not decrease significantly (and may actually increase) in the aged, there is a greater prevalence of fibers with a disordered morphology resulting in a loss of cutaneous elasticity. The morphological changes are myriad: there are defects termed lacunae and cysts in the elastin fibers; the candelabra-like organization of elastin at the epidermal-dermal junction disappears; and the fibers appear granular, fragmented, and about to disintegrate into fibrils (12).
In summary, the decreases in cell number and the discussed changes in the matrix result in the thinning and wrinkles that are so characteristic of aged skin. Accompanying functional changes include compromised vascular responses to stress (e.g., excessive cold or heat) as a result of a diminished dermal microcirculation (14). In addition, alterations in skin appendages in the aged result in slowed growth of the hair and nails; decreased secretions from the sweat and sebaceous glands; and diminished sensation to light touch, pressure, and pain.
It has recently been appreciated that most of the undesirable changes in aged skin are the result of chronic environmental damage, in particular long-term ultraviolet (UV) (sun) exposure, superimposed on intrinsic aging. This photoaging process creates a low-grade inflammatory response characterized by amorphosis of elastic fibers, activated dermal fibroblasts, and stimulation of the microvasculature in the form of telangiectasias. Fisher et al. (1) have shown that exposure to UV irradiation leads to sustained elevations of matrix metal-loproteinases (MMPs), such as collagenase-1, gelatinase B, and stromelysin-1, the primary enzymes that control matrix turnover. As a consequence, repeated exposure to UV irradiation degrades collagen and other skin proteins, resulting in a significantly negative impact on the composition of the ECM. Thus, whereas excess MMP activity was previously attributed to an intrinsic dys-regulation of metalloproteinases with aging, it is now generally accepted that UV activation of MMPs contributes significantly to the detrimental changes that occur in aged skin (4).
The study of wound repair in aged humans, in the absence of comorbidities, remains a significant challenge. In addition to a high prevalence of diseases that are more common in the aged, such as diabetes and vascular disease, elderly individuals present with variations in nutritional status and exposure to drugs (both prescription and over the counter) that can affect wound repair
(for reviews, see refs. 15-17). Relevant nutritional deficiencies that are more common in the aged include vitamin C and zinc. Many medications prescribed to the elderly, such as corticosteroids and anticoagulants/antiplatelet agents, can interfere with healing. Over-the-counter medications and high doses of vitamins, in particular vitamin E, can adversely affect the clotting cascade, thereby altering tissue repair. As already noted, the aged demonstrate large variations in exposure to solar damage. Moreover, it is well known that differences among individuals in a given cohort increase with age. Even in a population that is matched for comorbidities and environmental insults, individuals will have inherent differences in the rate at which their skin will age (18). This is especially the case for studies of the "old-old" (those over 85 yr of age), who are much more difficult to study than "young-old" (65-74 yr) and "middle-old" (75-84 yr) humans.
In contrast to humans, laboratory animals have identical genetic backgrounds and controlled environmental exposures. Thus, they age at similar rates and provide useful models for the study of wound repair in aging. Most of the animals have loose skin (with the exception of pigs) and, therefore, do not closely mimic the taut skin of humans. Moreover, whereas mice develop atrophy of the skin with age, rats do not and some strains of rats exhibit thickened skin. In spite of these limitations, studies of mice, rats, and rabbits have yielded important, and largely reproducible, information on general changes in wound healing that occur with age (for review see ref. 7). Published models have included 22- to 40-mo-old mice and rats and 48- to 60-mo-old rabbits (19-23). The age at which an animal is defined as aged is dependent on the strain (hybrids often live longer) and environmental factors such as diet (caloric-restricted mice and rats live longer). One accepted guideline is that an animal is definitively "aged" at the point in its life-span that 50% of its age-matched cohort has expired.
In addition to studies in vivo, there are numerous in vitro models for the examination of changes in wound repair that are relevant to aging. These include wound explants from the aforementioned aged animals as well as dermal fibroblasts and keratinocytes from aged human donors (24,25). Cells aged in vitro, via serial passage in culture, provide additional information on cellular changes relevant to aging (26,27). Explants and cells in culture, in contrast to live animals, provide a more feasible method of examining specific cellular functions that are relevant to wound repair such as proliferation, migration, and biosynthesis. These methods are discussed in other chapters of this volume.
4. Alterations in Wound Healing in the Aged 4.1. General
The first published observation of altered wound healing with age can be traced to the reports of DuNouy (28), who assessed healing in soldiers of different age groups (20-40 yr) during World War I. However, the type of wound, infectious complications, and the overall young age of the patients diminished the validity of his conclusions. Halasz (29) reported an increase in the incidence of wound dehiscence with age in 3000 patients having undergone duodenal surgery. Again, concurrent morbidity and infectious complications were not recorded. More recently, the majority of studies have found a delay in healing, but less scarring, in the wounds of aged animals and humans relative to their young counterparts (19,20,22,23,30,31).
Wound healing is generally divided into an early inflammatory phase, a middle phase of proliferation/granulation tissue formation, and a final phase of remodeling (for review see ref. 32). Whereas Subheading 4.2. reviews the effect of age on wound repair in light of those phases, this section reviews general principles of cell and tissue behavior.
Examination of the mechanisms of altered wound repair in aging have found decreases in cell proliferation and migration, deficits in matrix secretion, and a paucity of mitogenic growth factors. Each of the cellular components (keratinocytes, fibroblasts, and endothelial cells) show reduced proliferation in the wounds of aged animals. Early studies demonstrated decreased weight of granulation tissue or delayed appearance of cellular invasion in wounds from older animals (33,34). More recent studies have measured proliferation (using specific markers such as incorporation of the DNA analog bromodeoxyuridine) to specifically document less proliferation of fibroblasts and endothelial cells in the wounds of aged animals relative to young controls (20,30).
Generalized changes in matrix deposition in the wounds of aged animals have also been noted. Viljanto's (35) group reported that with increasing age there was a concomitant decrease in the amount of hydroxyproline (a biochemical measure of collagen content/production) incorporated into cellulose sponges within human wound sites. Sussman (36) noted thicker wounds associated with greater breaking strength in young rats as compared with old animals. These studies suggested that connective-tissue deposition, especially collagen I, was reduced in the wounds of the aged; more recent data support these findings (19,20).
Fibronectin is a large extracellular protein that has also been examined in aging and wound repair. It functions to regulate inflammation and cell adhesion by providing a provisional matrix, along with hyaluronan and collagen III, during the early phases of wound healing (37). Although aged cells have been associated with an increase in fibronectin synthesis in culture (38), studies of aged tissues and wounds from aged animals have shown a decrease in fibronectin expression in association with reduced levels of collagen (39,40).
Wound healing requires the invasion and migration of cells into the area of injury. As such, the proteolysis of the ECM is requisite for proper repair.
This process is regulated by MMPs that are secreted largely by keratinocytes and fibroblasts. Synthesis and activation of MMPs are controlled by several mechanisms including growth factor stimulation, exposure to the ECM, and changes in pH (41). Among MMPs, the expression of collagenases (MMP-1, MMP-13) stromelysins (MMP-3), and gelatinases (MMP-2, MMP-9) have been reported during wound healing (42-44). Whereas the collagenases degrade intact collagen, the gelatinases break down collagen that has been previously denatured (gelatin). The membrane-type MMPs (MT-MMPs) are not only responsible for the activation of MMP-2 at cell surfaces; recent data have demonstrated collagenase activity from cell-surface MT-MMP during migration into collagen (45,46). In early wound repair, MMP-1 enables keratinocytes to migrate between dermal collagen and the fibrin clot (42,43,47-49). During granulation tissue formation, all the MMPs assist the movement of fibroblasts into crosslinked fibrin and the newly deposited ECM. Angiogenesis, the formation of new vessels from preexisting vasculature, occurs during this time and vascularizes the newly formed granulation tissue. Angiogenesis is also dependent on MMP activity to permit the invasion of microvascular endothelial cells into the structural matrix (50,51).
The activity of MMPs, although necessary, must be tightly controlled. Indeed, an excess of proteolytic activity can destroy the support matrix necessary for cell migration and tissue repair. Long-standing observations have associated nonhealing wounds with excessive proteolytic activity (52). In this context, it is not surprising that slowed healing in the aged has been attributed, in part, to increases in the expression of MMPs and decreases in their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) (53-57). Recently, it has become clear that the changes in proteolytic activity with age are not invariant but are cell and tissue specific. For example, whereas dermal keratinocytes and microvascular endothelial cells show a deficit in MMP-1 synthesis with aging, dermal fibroblasts from aged donors show an excess of MMP-1 secretion (24,25). Moreover, studies of transgenic mice demonstrate that both deficits and excesses in MMP activity can inhibit the healing of acute wounds (58,59). Taken together these data highlight the importance of an optimal level of controlled proteolysis for efficient repair of aged tissues.
Growth factors such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-P (TGF-P), and vascular endothelial growth factor (VEGF) are expressed in tissues during wound healing (60-64). Although their precise spatial and temporal distribution varies with the model examined, it is generally accepted that these factors perform critical regulatory functions during wound healing. Functional roles assigned to these factors include, but are not limited to, cell proliferation, migration, biosynthesis, and control of the secretion of MMPs and TIMPs. The multifunctional roles assigned to these factors is most notable with TGF-P 1; this factor is a chemoattractant to fibroblasts, enhances their deposition of collagen and fibronectin, and decreases the synthesis of MMP-1 at the same time it increases TIMP-1 by these same cells (61,65). With increased age, decreased circulating and tissue levels of most of these factors have been reported and are thought to contribute to altered healing in aging (21,66). Whether the decrease in availability is accompanied by a lack of response to exogenous replacement of these growth factors is controversial. There are numerous reports of deficient growth factor responses, attributed largely to defective receptors, in aged cells and tissues (67-71). However, there are many studies demonstrating that aged cells and tissues are able to respond to stimulation with these same factors (19-21,31,72). For example, dermal fibroblasts from aged donors show a deficit in biosynthesis of collagen and collagen gel contraction, but a similar biosynthetic and contractile response to young donors on stimulation with TGF-P1 (72). TGF-P1 applied to dermal wounds accelerated the healing of wounds in aged rats (19,20). Exogenous replacement of estrogen and VEGF has also been shown to reverse impairments in tissue repair and vascularization (21,31). Indeed, a recent study has demonstrated that topical replacement of estrogen in the skin of elderly females can indirectly enhance expression of TGF-P 1 in the wound, thereby accelerating healing (31). Observations such as these underscore the complexity of attempting to analyze the effect of any single growth factor on tissue repair in the aged.
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