INTRODUCTION

Exposure to ultraviolet (UV) radiation causes cumulative damage that accelerates normal chronologic aging and exacerbates injury to skin tissue, resulting in photodamage. Consumer interest in correcting signs of photodamage, such as wrinkles, dyspigmentation, sagging, and surface roughness, is increasing as the population ages, particularly as the ‘Baby Boomer’ segment reaches middle age. Treatments include topical retinoids and antioxidants, chemical peels, dermabrasion, laser, and various lifting surgeries, depending on the severity of skin damage.

Within the last decade researchers have focused on the pathophysiology of photodamage and have found correlations with certain aspects of acute and chronic wound healing. Of specific interest to cosmeceutical manufacturers are the effects of growth factors in the process of wound healing. Growth factors are regulatory proteins that mediate signaling pathways between and within cells. After a wound has been inflicted, a variety of growth factors flood the wound site and interact synergistically to initiate and coordinate each phase of wound healing. This process is complex and not completely understood. Most studies have evaluated the role of single growth factors in controlled wound-healing environments. These studies demonstrate the importance of growth factors in the repair of damaged tissue, but research into the phases of wound healing has demonstrated that it is the interaction of multiple growth factors that is vital to tissue regeneration. Cosmeceutical manufacturers have taken notice of the positive results of clinical studies showing accelerated wound healing and have begun to include growth factors in products designed to mitigate damage from chronologic aging and sun exposure.

PHOTODAMAGE EFFECTS ON SKIN TISSUE

Photodamage occurs predominantly within the epidermis and the upper papillary dermis. Histologic studies demonstrate that UV exposure disrupts the normal architecture of connective tissue within the dermis. The dermal extracellular matrix is composed primarily of type I collagen, although type III collagen, elastin, proteoglycans, and fibronectin are also included in smaller amounts. UV exposure decreases collagen and elastin and alters the cross-linked structure of collagen and elastin fibers within the dermal extracellular matrix. Abnormal elastic material containing elastin and fibrillin accumulates and appears to occupy areas of lost collagen. This deposition of abnormal elastic material is called solar elastosis. Glycosaminoglycans (GAGs), a type of proteoglycan that is a component of the extracellular matrix, are polysaccharide molecules that bind to water, forming a hydrated, space-filling polymer between collagen and elastin fibers that helps support skin tissue. In photodamaged skin GAGs are abnormally deposited in the elastotic tissue rather than between collagen and elastin fibers. The clinical result of decreased collagen and elastin and disruption of the normal support architecture is the appearance of wrinkles, sagging skin, uneven pigmentation, hyperpigmentation, and thickened or leathery skin texture. Although chronologic aging skin also develops wrinkles, photodamaged skin is differentiated histologically by the presence of solar elastosis.

BIOCHEMICAL PATHWAYS OF SKIN AGING

Research in the area of photoaging over the past decade has resulted in an improved understanding of the molecular mechanism of the aging process. Figure 19.1 shows a summary of major pathways involved in the aging process. Absorption of UV radiation by chromophores in the skin as well as cellular oxidative metabolism results in formation of reactive oxygen species (ROS). ROS increase oxidative phosphorylation of cell-surface receptors causing activation of the transcription factor activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB).

Fig. 19.1 Biochemical pathways involved in intrinsic and extrinsic aging processes

AP-1 stimulates transcription of matrix metalloproteinase (MMP) growth factor genes in fibroblasts and keratinocytes, and inhibits type 1 procollagen gene expression in fibroblasts. MMPs produce degradation of fibrillar type I and type III collagen. Activity of MMP is decreased by binding with tissue inhibitors of metalloproteinase (TIMPs). ROS inactivate TIMPs, thereby increasing MMP activity. AP-1 mediated reduction in synthesis of procollagen appears to result from two mechanisms—interference of AP-1 with type 1 and type 3 procollagen gene transcription and blocking the profibrotic effects of transforming growth factor-beta (TGF-β) by impairment of the TGF-β type 2 receptor/Smad pathway.

Activation of NF-κB stimulates transcription of proinflammatory cytokine genes including interleukin-1 (IL-1), TNF-α, IL-6, and IL-8. Inflammation resulting from these cytokines increases secretion of ROS and more cytokines, further enhancing the effect of UV exposure. Inflammation causes protease-mediated degradation of elastin and UV exposure causes formation of abnormal elastin by fibroblasts. UV light is also an inhibitor of leukocyte elastase, thereby increasing accumulation of elastotic materials. The accumulation of elastotic materials is accompanied by degeneration of the surrounding collagenous network.

The overall effects of these interlinked biochemical activities is reduction of procollagen synthesis, increase of collagen degradation in the dermal extracellular matrix, and increase in irregular elastin deposition.

GROWTH FACTORS IN WOUND HEALING

Hundreds of growth factors have been identified. Those that are important in wound healing include cytokines involved in immune response and phagocytosis, and growth factors that induce the synthesis of new collagen, elastin, and GAGs (components of the dermal extracellular matrix) that are abnormally affected by UV radiation. Table 19.1 lists the functions of the most important growth factors in wound healing.

Table 19.1 Growth factors in wound healing

Sources: Fitzpatrick RE, Rostan EF 2003 Reversal of photodamage with topical growth factors: a pilot study. Journal of Cosmetic and Laser Therapy 5:25–34; Moulin V 1995 Growth factors in skin wound healing. European Journal of Cell Biology 68:1–7.

Wound healing is dependent on the synergistic interaction of many growth factors. After injury, cytokines and other growth factors flood the wound site to mediate the inflammatory response, promote new cell growth, and decrease wound contraction and scarring. The process of wound healing is commonly divided into four overlapping phases that describe physiologic responses to injury. These phases include hemostasis, inflammation, proliferation, and remodeling. Figure 19.2 summarizes each phase of wound healing along with dominant growth factors and cytokines. During hemostasis, platelets release various cytokines and other growth factors at the wound site to promote chemotaxis and mitogenesis. In the inflammatory stage, neutrophils and monocytes migrate to the wound site in response to specific cytokines and growth factors to initiate phagocytosis and to release additional growth factors that will attract fibroblasts. The proliferation phase is marked by epithelialization, angiogenesis, granular tissue formation, and collagen deposition. During proliferation, keratinocytes restore barrier function to the skin and secrete additional growth factors that stimulate the expression of new keratin proteins. Fibroblasts produce collagen that is deposited in the wound bed. This cycle of collagen production and growth factor secretion continues in a type of autocrine feedback loop of continuous wound repair.

Fig. 19.2 Phases of wound healing and role of growth factors

The remodeling phase is the final step in the wound repair process and typically lasts several months. It is during remodeling that the extracellular matrix is reorganized, scar tissue is formed, and the wound is strengthened. Type III collagen deposited during the proliferation phase is gradually replaced by type I collagen, which is more tightly cross-linked and provides more tensile strength to the matrix than type III collagen. Cells at the wound site secrete several growth factors with specific functions related to remodeling and matrix formation. For example, collagen and fibronectin synthesis are initiated by TGF-β, while PDGF and TGF-β stimulate fibroblasts to produce GAGs and modulate the proliferation of smooth muscle cells. Other growth factors modify the vasculature. Over time, cell density increases and skin tissue is strengthened.

Specific growth factors directly initiate activity that promotes wound healing as well as modifying the activities of matrix cells and other growth factors. Growth factors are capable of both stimulating and inhibiting specific actions. The activity of growth factors is modulated through other growth factors and through various intrinsic factors that interact to achieve homeostasis and balance during wound healing. Research continues to uncover more information about the functions of individual growth factors during wound healing and, importantly, the synergistic interaction of growth factors with each other and with other components of wound healing. Whether the presence or absence of a single growth factor is significant in wound healing is not yet known. Current understanding suggests that it is the interaction of many growth factors that is significant, with no single growth factor being solely determinant in the outcome of wound healing.

TREATING PHOTODAMAGED SKIN

The most aggressive approach in the treatment of photodamage has been to remove damaged skin and foster the growth of healthy new epidermal and papillary dermal layers. Acid peels and dermabrasion are effective in destroying damaged skin, but it is difficult to precisely control the amount of surface tissue removed. Adverse effects from these procedures may include erythema, scarring, hyperpigmentation, or hypopigmentation. Ablative skin resurfacing with the CO₂ laser has also been widely used to remove photodamaged skin by vaporizing outer skin layers. Lasers can be precisely controlled so the amount of tissue removed is predictable. However, the removal of the epidermis results in a partial thickness open wound that may take weeks to heal and presents the same risks for erythema, scarring, and dyspigmentation as acid peels and dermabrasion.

Nonablative laser resurfacing appears to stimulate dermal healing and new collagen formation without removing the epidermis. Studies of nonablative laser treatment of photodamaged skin have demonstrated statistically significant improvement in clinical grading of skin damage with only transient erythema and minimal side effects, and biopsies have quantified post-treatment formation of new dermal collagen. Histologic evaluation of traditional ablative laser wounds reveals the expression of EGF, TGF-β, PDGF, and fibroblast growth factors, as well as wound healing activity that is identical to the phases of wound healing initiated from surgically inflicted wounds. Similarly, nonablative laser techniques appear to result in subclinical thermal wounds that initiate the wound-healing process and presumably the release of growth factors.

TOPICAL APPLICATION OF GROWTH FACTORS

Individual growth factors (e.g. TGF-β, EGF, PDGF, etc.) have been shown to accelerate wound healing in both acute and chronic wounds. Photodamaged skin is similar to a chronic wound that may not progress to complete tissue remodeling. Total healing of photodamaged skin is unlikely because the surface area of the injury is too large to be completely repaired, and ongoing cumulative damage continues to occur daily. It is estimated that only 15 minutes of sun exposure can induce enough damage to collagen and elastic fibers to require remodeling activity. Table 19.1 lists some important growth factors and cytokines that affect the proliferation of dermal fibroblasts and extracellular matrix production. Providing some of these agents to cells responsible for extracellular matrix production and remodeling may result in rejuvenation of aging skin. Several cosmeceutical products containing a combination of multiple human growth factors and cytokines are currently marketed for skin rejuvenation. Clinical results are now available for some of these products and the results show that human growth factors when applied topically provide beneficial effects in reducing the signs of facial skin aging.

In one study, a mixture of multiple growth factors derived from a three-dimensional tissue culture of human fibroblasts was used to evaluate the effects of growth factors applied topically to treat photodamaged skin. The objective in using multiple growth factors was to stimulate the remodeling phase of wound healing in which many growth factors work synergistically. The selection of growth factors used in the pilot study was based on preliminary data that demonstrated the efficacy of this combination in stimulating the proliferation of fibroblasts and keratinocytes and increasing collagen secretion. The study included 14 patients with photodamaged skin (Fitzpatrick class II). Each patient applied the mixture of growth factors twice daily for 60 days. Baseline and 60-day results were evaluated using 3 mm punch biopsies and optical profilometry. A total of 11 of 14 patients (78.6%) showed clinical improvement at 60 days. New collagen formation in the grenz zone increased by 37%, and epidermal thickening increased by 27%. The results also showed a statistically significant reduction in fine lines and wrinkles and reduction in periorbital photodamage. Clinical grading showed a 12.2% improvement (P = 0.0003) in periorbital area after 60 days compared to baseline. Optical profilometry shows a 14.1% decrease in roughness average (RA) measurement (P = 0.008) and a 36.2% decrease in shadow measurements (P = 0.02). Additional evaluation of this combination of growth factors in a longer duration, double-blind, placebo-controlled study showed statistically significant improvement in several parameters of photoaging with growth factor-containing product when compared against a vehicle control.

COMBINATION APPROACHES: LASER PLUS TOPICAL GROWTH FACTORS

Laser resurfacing and topical application of growth factors have each been shown to improve clinical signs of photodamage and to stimulate the formation of dermal collagen. Depending on whether CO₂ or nonablative resurfacing is used, some degree of erythema will occur, and the process of wound healing will take time. A human fibroblast-derived temporary skin substitute was approved in 1997 for use as a temporary wound covering for mid-dermal to indeterminate depth wounds. This skin substitute has been used extensively in patients with partial thickness burns. In addition to providing a protective barrier, this temporary skin substitute contains growth factors secreted by the tissue culture and allows fibroblasts to proliferate and secrete dermal collagen, matrix proteins, and growth factors. A study of the use of fibroblast-derived temporary skin after CO₂ laser resurfacing demonstrated faster healing and less pain and inflammation than traditional postoperative measures.

The degree of clinical improvement and histologic improvement seen with nonablative resurfacing is very similar to that seen with the use of topical growth factors. This is logical, since they both appear to involve the same mechanisms for improvement. The simultaneous use of both would be expected to result in an enhanced response and this approach is taken by many physicians.

For noninvasive, nonablative laser resurfacing, the post-treatment application of growth factors in a topical formulation may provide benefit in accelerated or improved wound healing.

PLANT-DERIVED GROWTH FACTORS

Kinetin, a plant-derived growth hormone discovered almost 50 year ago, has been recently used in several cosmeceutical products as an antiaging ingredient. Kinetin is found in the DNA of almost all organisms tested so far, including human cells. In plants, kinetin regulates cellular differentiation by an endocrine pathway with unknown mechanism; however, its function in human cells is not known. Kinetin and other cytokinins are products of oxidative metabolism of the cell. Kinetin is formed in the nucleus by reaction of hydroxyl free radicals with DNA whereas reaction of hydroxyl radicals with RNA results in formation of zeatin, another cytokinin used in cosmeceutical products.

A large number of in vitro and in vivo studies in many species show a number of pharmacologic actions leading to potential antiaging effects of kinetin and other cytokines. The effects, however, are not related to the growth factor/hormone-related properties but are primarily due to their strong intracellular antioxidant properties. A pilot study on 0.1% kinetin showed moisture retention and reduction in appearance of fine wrinkles and pigmentation; however, more controlled studies are required to substantiate the antiaging benefits.

MESOTHERAPY

Mesotherapy is the microinjection of growth factors or other active molecules into the mesoderm with the premise that the active molecules are directly delivered to the target tissue using fine gauge short needles to a 2–5 mm depth of penetration. Multiple close-spaced injections are made to ensure adequate coverage of the treatment area. There are no clinical studies evaluating the efficacy of the procedure and safety remains a concern due to lack of availability of sterile growth factor solutions.

RISKS ASSOCIATED WITH GROWTH FACTORS

There are no proven risks associated with the topical application of growth factors, other than the potential for allergic reactions in patients with hypersensitivities to these substances. The protein molecules are too large to be absorbed in large quantities, although there is growing evidence that small amounts of macromolecules and particles can penetrate into the upper epidermis where they can induce cytokine cascade for potential effects in dermis. However, theoretical concerns have been raised about the potential for growth factors to stimulate the development of melanomas. This theory is based on the presence of receptors for some growth factors, such as VEGF, in various types of melanoma. In addition, certain growth factors are expressed by cancerous cells, while others are thought to alter the environment around cancerous cells to promote tumor growth. For example, VEGF, which is a key factor in tumor neoangiogenesis, is expressed by some types of skin tumor. Whether increased VEGF expression contributes to tumor growth is uncertain. Exogenous VEGF added to melanoma cells was shown in one study to increase cell proliferation, but increased expression of VEGF in another study did not result in melanoma cell proliferation. By contrast, VEGF expression in squamous cell carcinomas of the head and neck was shown to produce a significant inhibitory effect on cell proliferation and tumor cell migration. Whether VEGF contributes to tumor cell proliferation or is produced in response to the growth of a tumor is unknown. To date, studies of growth factors and their association with tumors have focused on their expression by the tumor. It is unlikely that topically applied VEGF would affect tumor proliferation. Similarly, TGF-β has been alternately reported to decrease or promote cancer progression. Generally, this growth factor has been found to have inhibitory effects on tumor growth, but the activity of TGF-β in cancerous tissue is complex and has not been fully explained. As with VEGF, studies have focused on TGF-β expressed by tumor and other types of cell: topical application is unlikely to either inhibit or promote cancer growth.

Another potential concern about the application of growth factors is whether they could contribute to hypertrophic scarring. Specifically, it has been postulated that TGF-β may increase the risk for scarring during wound healing due to its function in activating fibroblasts, which synthesize collagen, and because elevated levels of TGF-β have been noted at the site of dermal injury. But these findings cannot be extrapolated to suggest a causative role for TGF-β in the development of hypertrophic scarring. It has been suggested that an abnormal response of proliferative scar fibroblasts to TGF-β stimulation may contribute to the development of keloid and burn scars, but evaluation of patients with a genetic proclivity for the development of keloid scars failed to demonstrate a relationship between TGF-β plasma levels and keloid formation. Increased levels of TGF-β have been noted at the site of dermal wounds, but whether this growth factor contributes to scarring or is produced in response to scar development is unclear. With regard to the topical application of growth factors, there is no clinical evidence that they induce abnormal scarring, and anecdotal observations have failed to reveal any activity of growth factors that would produce an abnormal wound-healing response. In fact, the current understanding of growth factor activity in the wound-healing environment reveals a balance of both stimulatory and inhibitory actions that are carefully modulated to achieve homeostasis.

PRODUCT QUALITY CONSIDERATIONS

Growth factors and other biologically active peptides are inherently unstable in nonphysiologic environments. Cosmeceutical products containing surface active additives, alcohols, and other protein-denaturing excipients must be tested for the presence of active proteins during the claimed shelf-life of the product, something most cosmeceutical manufacturers have not done so far.

CONCLUSIONS

Studying the role of growth factors in cutaneous wound healing has led to research demonstrating positive cosmetic and clinical outcomes in photodamaged skin (Fig. 19.3). Although the topical use of growth factors is an emerging treatment approach, initial studies suggest that dermal collagen production and clinical improvement in photodamage appearance are substantial. Further, the increase in dermal collagen produced by topical growth factors can be measured quantitatively by biopsy. Although the functions of growth factors in the natural wound-healing process are complex and incompletely understood, it appears that wound healing is dependent on the synergistic interaction of many growth factors.

Fig. 19.3 An example of the clinical effects observed while using a growth factor containing moisturizer. (A) Baseline. (B) Month 3. (C) Month 6

Currently, most studies of single growth factors provide limited understanding within a narrow scope. The most promising research suggests that multiple growth factors used in combination may stimulate the growth of collagen, elastin, and GAGs. The use of a multiple growth factor topical formulation appears to provide a promising first-line treatment for mild to moderate photodamaged skin. Combination with laser therapy for more severe damage has not been adequately studied but may provide additional benefit. The plant-derived cytokines kinetin and zeatin appear to have strong intracellular antioxidant properties which could help in neutralizing cellular damage from environmental challenges, but do not play a role in modulating wound healing in humans. Their role as antiaging agents is controversial. Mesotherapy is unproven at this time.