Biologic Therapeutics and Molecular Profiling to Optimize Wound Healing

Abstract

Non-healing wounds represent a significant cause of morbidity and mortality for a large portion of the adult population. Wounds that fail to heal are entrapped in a self-sustaining cycle of chronic inflammation leading to the destruction of the extracellular matrix. Among cancer patients, malnutrition, radiation, physical dehabilitation, chemotherapy, and the malignancy itself increase the likelihood of chronic wound formation, and these co-morbidity factors inhibit the normal wound healing process. Current wound treatments are aimed at some, but not all, of the underlying causes responsible for delayed healing of wounds. These impediments block the normal processes that allow normal progression through the specific stages of wound healing. This review summarizes the current information regarding the management and treatment of complex wounds that fail to heal normally and offers some insights into novel future therapies [1,2].

Introduction

Non-healing wounds (chronic wounds or chronic ulcers) are trapped in a constant inflammatory state due to a failure to progress through the normal stages of wound healing. This inflammation results in an abnormal wound microenvironment and results in significant, chronic disability with frequent relapse [3].

Wounds heal through four distinct yet overlapping phases: 1) hemostasis, 2) inflammation, 3) proliferation, and 4) remodeling (Figure 1; Table 1). In a chronic wound, changes associated with senescence, ischemia, and bacterial colonization transform the normal progression of healing into a self-sustaining cycle of inflammation and injury [4]. Continuous recruitment of neutrophils results in abnormally elevated levels of matrix metallo-proteinases (MMP) [5–9], especially MMP-8 and neutrophil-derived elastase. A normal wound inhibits the excess matrix metallo-proteinases MMPs through the nonspecific proteinase inhibitor, α2-macroglobulin, and specifically by a small group of proteins called tissue inhibitors of matrix metallo-proteinases (TIMP)[5, 10]; however, the abnormal MMP (degradative):TIMP (protective) ratios observed in chronic wounds lead to extracellular matrix (ECM) degradation [11–13], changes in the cytokine profile [14], and reduced concentration of proliferative factors [15, 16]. The release of reactive oxygen molecules by neutrophils both damages the wound and serves to recruit additional neutrophils. The continuous feedback loop prevents ECM deposition despite continued recruitment of fibroblasts [17–19].

Figure 1.

Comparison of the phases of normal wound healing with the appearance of specialized cells.

Table 1.

Phases of Wound Healing

Phase	Primary Cell Type Recruited	Function
Hemostatic	Platelets	Provisional fibrin matrix and recruits inflammatory cells
Inflammatory	Macrophages Neutrophils	Preparation of the wound bed by killing bacteria and removing devitalized tissue as well as recruiting fibroblasts
Proliferative	Fibroblasts	Stable extracellular matrix formation
Remodeling	Fibroblasts	Mature scar formation

Malignancy and its treatment may interfere with all four stages of wound healing through decreased tissue perfusion and hypoxia, impaired inflammatory response rates, and changes in fibroblastic proliferation. As many of the components of wound healing are involved in tumor progression, targets of chemotherapy and radiation often involve proteins related to tissue repair as well as cellular proliferation, and certainly, preoperative radiation therapy results in an increased risk of wound complications [20]. This effect may be seen on the genomic and proteonomic level [21]. Malnutrition and/or obesity further complicate the process. Hypoalbuminemia and anemia have been correlated with diminished wound healing [22].

Evaluation

Chronic wounds are not subtle; however, they must be carefully evaluated to ensure maximation of treatment options and to prevent further degradation. The extent of the wound, its underlying etiology, duration, location, size, odor, appearance, and appearance of the surrounding tissue should all be documented [23,24]. Chronic ulcers are commonly divided into four categories (Table 2) [25,26].Wounds located on an extremity are classified as non-limb threatening, limb threatening, or life threatening. Superinfection may be local (cellulitis or abcess) or systemic with signs ranging from erythema and lymphadenopathy to vital sign instability and altered mental status.

Table 2.

Stages of Tissue Breakdown

I	Non-blanchable erythema of intact skin.
II	Partial-thickness skin loss involving the epidermis, dermis, or both. Superficial and may present as an abrasion, blister, or shallow crater.
III	Full-thickness skin loss involving damage to subcutaneous tissue that may extend down to, but not through, fascia. Presents clinically as a deep crater with or without undermining of adjacent tissue.
IV	Full-thickness skin loss with extensive damage to muscle, bone, or supporting structure. Extend all the way to bone and are likely to have deep seated soft tissue infections or osteomyelitis.

Chronic ulcers with a high bacterial load will not heal; a biopsy from the base is indicated to determine the underlying organism [27]. Radiographic films will assist in identifying subcutaneous air or a foreign body; however, if osteomyelitis is suspected, MRI or bone scans are required for evaluation. Additional evaluation of the vascular system on extremities may require venous and arterial dopplers or angiography [28].

Management

Management always aims to treat the underlying etiology; in the cancer patient population, however, this often results in competing therapeutic interests of chemotherapy and/or radiation. Advanced-staged cancer with its subsequent complications may result in a chronic state of malnutrition that further prevents adequate response to tissue injury. Nevertheless, optimization of patient care can be achieved through measures that reduce the effects of co-morbidities (e.g., glycemic control and vitamin supplementation), increase perfusion, and control the inflammatory response [26,29–32].

Reduction of Bacterial Load

Regardless of the underlying cause, a clean wound bed is the minimum effort required to prevent further sequelae and reduce inflammation. Irrigation followed by debridement will reduce the bacterial load without damaging healing tissue. Utilization of appropriate wound dressings provides barrier protection against further colonization and irritation.

Irrigation may be performed initially with saline or lactated Ringers using an 18 gauge needle and 60 mL syringe. Excessive pressure from devices such as power sprays may damage granulation tissue and force bacteria and debris further into the wound bed. While effective at removing superficial bacteria, skin cleansers such as hydrogen peroxide and Betadine will also kill host tissue. Debridement is commonly performed surgically; however, enzymes such as proteolytics and collagenases have been used. Both serve to reduce bacterial concentration and remove necrotic tissue [25]. Less widespread, sterile maggots are another method of debridement [33,34].

Prevention of Further Injury

The wound requires a protective barrier to allow healing to progress. Commercially available preparations include hydrocolloids, films, foams, alginates, and hydrogels. The choice of dressing will depend on the location, state of the wound, and type of wound (Table 3) [35]. Occlusive dressings such as hydrocolloids should be avoided in infective wounds that require draining. Absorbant dressings such as foams and alginates may cause damage if dry and are best suited for wounds that produce copious amounts of exudate. Wounds with good granulation tissue would benefit from moisture-providing hydrogels and hydropolymers. The Wound VAC (vacuum-assisted closure) offers an attractive alternative as it keeps the wound bed moist while removing exudates; similar to occlusive dressings, however, these negative pressure devices are contraindicated for infected wounds [36, 37].

Table 3.

Wound Dressings

Dressing	Type	Function and Use
Hydrocolloids	Occlusive	Autolytic debridement through endogenous proteolytic enzymes; not for use with infected wounds
Films, Foams, Alginates	Occlusive	Wounds kept moist through fluid entrapment; may result in skin tears and tissue maceration
Foams	Absorbant	Not as adherent as films; may need secondary dressing for reinforcement
Alginates	Absorbant	Made from seaweed; best for highly exudative wounds as may cause damage if dry in wound bed
Hydrogels	Hydrophilic polymers	Best for wounds that have formed granulation tissue

Ischemia-reperfusion injury may result from pressure or trauma. When possible, pressure relief should be initiated either through re-positioning, specialty mattresses, or scheduled turning as this will minimize additional inflammation and tissue damage [38,39]. In addition to the specific factors and situations discussed above, there are many other factors that should be considered that can impair normal wound repair (Table 4).

Table 4.

Factors That May Impair Normal Wound Healing

Age
Co-Morbidities
Cardiac
Congestive Heart Failure
Connective Tissue Disease
Diabetes Mellitus
Hepatic
Hypertension
Renal
Lifestyle
Illicit Drug Use
Nutrition
Obesity
Smoking
Therapeutic Modalities
Chemotherapy
NSAIDs
Radiation
Steroids

Other Techniques

Exogenous cytokines and growth factors can, in theory, shift the degradative: protective ratio to one more favorable to healing. Of those evaluated, only recombinant PDGF (Regranex) has show efficacy in multi-center randomized controlled trials; however, response to this expensive therapy was limited due to the ability of proteolytic enzymes to degrade both PDGF and TGF-β[40]. In patients whose inflammation level can be kept low, healing may progress.

Bioengineered dermal replacements such as Apligraf and Dermagraft have also shown evidence of augmentation of healing [41,42]. These living cell materials contain highly active cells that produce and supply a spectrum of growth factors that can promote the repair process. However, these high-tech materials will only supplement the healing process; usage is best reserved as an adjuvant when the chronic inflammation is controlled or reduced.

The treatment of bacterial wound colonization with topical antibiotics is controversial. The line between colonization and infection has not been well defined, nor has the level at which colonization leads to an inflammatory response. Lastly, bacterial biofilms, which are resistant to topical antibiotics, theoretically serve as a source for an uncontrolled inflammatory response.

Although ubiquitous, bacterial colonization does not always lead to an inflammatory response [43]. Topical antibiotics should be reserved for wounds with a large exudative component, with oral or systemic medicationsonly for patients with a known infectious process (e.g., cellulitis or osteomyelitis). As the organisms are generally polymicrobial, outpatient management options require broad spectrum coverage such as oral amoxicillin plus clavulanate (Augmentin) 875 mg P.O. twice a day with trimethoprim-sulfamethoxazole double strength (Bactrim DS) twice a day [44]. Intravenous antibiotics such as vancomycin 1 gm every 12 hours with piperacillin plus tazobactam (Zosyn) 3.375 gram every 6 hours may be required for severe systemic infections [44].

Although only available at select centers, hyperbaric oxygen has been shown to be an effective treatment of radiation-induced wounds [45].The partial pressure of oxygen is increased to greater than one atmosphere to allow elevated concentrations in the blood and tissue [46]. Complications include reversible myopia, central nervous system toxicity, middle ear, dental, sinus or pulmonary barotraumas, and pneumothorax. Tension pneumothorax is the only absolute contraindication; however, relative contraindications include inability to equalize pressures and coronary artery disease.

Recently, our laboratory found that Androstenediol (AED), an immune-regulating hormone, has the ability to reverse glucocorticoid inhibition of wound healing by immunomodulation [47, 48]. In addition, AED has the ability to improve outcomes from overwhelming bacterial and viral infections. Furthermore, AED stimulates host cellular and humoral immunity and can protect against lethal whole body irradiation. It is also important that there is no known toxicity associated with the administration of AED. These combined biological attributes afforded by AED may prove useful to overcome wound healing inhibition following irradiation. These studies are currently underway in our Laboratory of Tissue Repair.

The Future

Studies by Brem et al. have shown that chronic ulcers contain subpopulations of cells with differing capacities to heal [49]. Cells from the margin of ulcers were compared to cells distant form the ulcer and found to have very unique patterns of gene expression. These new molecular strategies to investigate and understand the many complexities of chronic ulcers will provide new therapeutic approaches to treat and promote the healing of these costly problems.

Summary

In chronic wounds, the balance between tissue degradation and synthesis is shifted toward degradation, resulting in impaired wound healing. Care of the cancer patient requires treatment modalities aimed at restoring the balance with recognition that this process may be further impaired through chemotherapy, radiation, malnutrition, or the malignancy itself. Irrigation and debridement are the primary methods of initial management to break the cycle of chronic inflammation. Dressings provide a protective barrier and should match the wound characteristics to prevent further damage; negative pressure devices may help prepare the wound bed to facilitate wound healing. Other adjunctive methods include antibiotics, growth factors, hyperbaric oxygen, and skin and dermal substitutes. Great care should be taken to tailor these methods to the appropriate patient. No treatment strategy will promote healing until the wound is modulated by changing the protease-laden, inflammatory microenvironment into one that is moving forward to produce healthy granulation tissue. Then, and only then, can the wound be stimulated to heal by a variety of therapies.