Cellular Senescence and Matrix Metalloproteinase Activity in Chronic Wounds. Relevance to Debridement and New Technologies

Abstract

A prolonged inflammatory response may adversely affect wound closure. Delayed wound closure and extended exposure to chronic wound fluid may also affect cellular activity in a wound bed and result in cellular senescence. Prolonged inflammation and cellular senescence may adversely affect the efficacy of topically-applied biological agents, including growth factors. Appropriate wound bed preparation and debridement are necessary to improve clinical outcomes of new technologies.

Chronic wounds have been defined as those that “have failed to proceed through an orderly and timely process to produce anatomic and functional integrity, or proceeded through the repair process without establishing a sustained anatomic and functional result.”[1] Although they vary in etiology and pressure, venous and diabetic ulcers are tissue defects in which regeneration is delayed or absent even though periwound tissue may appear normal and intact. The majority of chronic wounds respond well to standard care, which includes a triad approach that addresses extrinsic, intrinsic, and wound environment factors. Extrinsic factors include repetitive trauma, pressure, activity, ambulatory status, and the individual’s environment. Intrinsic factors encompass medications, concurrent disease states, and the overall medical condition of the patient. The wound environment refers to the status of the wound bed. A failure to address all three elements may result in impaired healing. In approximately 15% of patients, however, wound closure and tissue repair may be impaired despite a complete regimen of good care. In these cases, the clinician must determine if any factor or condition was missed during treatment. This article focuses on specific factors related to the wound environment that may either delay healing or reduce the effectiveness of treatment modalities, including newer genetic recombinant technologies.

Treatment Modalities

The treatment of the wound environment has been relatively simple during the last century, consisting primarily of routine visits to maintain wound cleanliness and the application of gauze dressings. While a plethora of wound dressings and devices have become available during the last two decades,[2]-[5] new technologies involving growth factors and bioengineered tissues are relatively recent. These dressings and devices help optimize the wound environment for tissue repair. They may protect the wound from external contaminants, and prevent trauma and desiccation, but are not known to mediate cellular activity. Growth factors and tissue replacement therapy introduce living cells or chemical mediators that promote wound repair through modulation of cell function. The ultimate success of both dressings and more advanced modalities is highly dependent on the status and presentation of the wound base. Considerations that may delay wound repair include fibrotic and desiccated tissues, impaired vascularization affecting oxygenation, deficient levels of cytokines, and growth factors promoting cell senescence and necrosis. Optimal activity occurs when active, viable populations of cells are present in a clean, moist environment that contributes to vigorous granulation and epithelialization.

The wound environment must be optimized prior to treatment. Surgical debridement must be aggressive and down to the level of viable, bleeding tissue. Sharp surgical debridement of all necrotic, nonviable tissues and wound debris from the wound bed is the most effective and rapid way to create a clean, viable environment. Furthermore, removal of bacteria and other unwanted wound contents might expedite wound closure, and increase the availability of viable cells and an optimal response to cytokines. A correlation between wound response, debridement, and growth factor application was demonstrated in a large, randomized, double-blinded controlled trial by Steed et al.[6] Wounds that were debrided and followed by the application of platelet-derived growth factor (rrPDGF-BB) had a significantly better healing response than those debrided and treated with moist, saline gauze alone. The significant difference between the two groups may be related to greater availability of receptors following debridement. Cells with receptors must be exposed in order to bind with growth factors or other chemical mediators. Growth factor binding cannot occur when these receptors are not available, resulting in ineffective cytokine and gene therapies.

In a study of patients with diabetes by Sihl,[7] it was postulated that even with appropriate care at least 15% to 20% of diabetic patients do not follow expected healing rates. The deviance from expected closure rates was thought to be related to cellular abnormalities in healing and the decreased availability of platelet-derived growth factor in diabetic ulcers. Based on the response to these treatments, some investigators have evaluated new therapies by classifying patients as either responders or nonresponders.[8] Robson et al[9] classified responders in a 35-day clinical trial as patients who achieved at least an 85% decrease in wound closure. Bello and Phillips[10] classified venous ulcers with closure rates of less than 0.032 cm/week as nonresponders and those with a rate of 0.109 cm/week as responders. Variations in response may be attributed to the natural heterogeneity that exists in the patient population.

The heterogeneous response to cytokine treatment may be attributed to variations in cell viability existing within chronic wounds. While the actual basis for these differences among patients is not known, Vande Berg et al[11] discovered that pressure ulcer fibroblasts became prematurely senescent. Mendez et al[12] later noted that fibroblasts cultured from venous ulcers also exhibited senescent characteristics. Senescent cells are characterized by cells that are viable but have lost their proliferative capacity. Fibroblast viability and senescence can vary in pressure ulcers within each wound as well as among patients[11] and the senescent population of ulcer fibroblasts decreases as the wound heals.[13] A recent study showed that cytokine therapy, particularly basic fibroblast growth factor, was effective in stimulating pressure ulcer repair and was correlated with a decrease in the senescent ulcer fibroblast population.[14] While these findings provide a possible explanation for variation or lack of response to some pressure ulcer therapies, cellular senescence cannot be viewed as the only reason for decreased response to wound repair. Patient age, past medical history, bacterial contamination, prolonged inflammation, and excessive metalloproteinase activities are also relevant factors.

Matrix metalloproteinases are enzymes subclassified in the metalloendopepdidase family that have been shown to be critical factors in wound repair.[15], [16] The subset of enzymes involved in the repair process includes stromelysins, gelatinases, collagenases, and membrane-type proteinases. This article does not elaborate on the role of matrix metalloproteinases and their substrate specificity, and the reader is referred to the review by Woessner.[17]

Matrix metalloproteinases degrade extracellular matrix components following tissue trauma, thereby assisting wound debridement. Proteases released during the acute inflammatory phase assist with the removal of damaged and denatured extracellular matrix components in preparation for the subsequent proliferative phase. Once this nonsurgical, natural form of debridement is achieved, granulation, new tissue formation, and cell migration may proceed toward the completion of wound repair.

It is important for the clinician to understand the role that the matrix metalloproteinase family has in acute wound repair and its relevance to the application of new technologies. Matrix metalloproteinase activity during the inflammatory phase of repair should not be confused with the release of matrix metalloproteinases during the remodeling phase of healing. During the inflammatory phase, matrix metalloproteinase activity assists with debridement and preparation for the migratory phase of healing. During the final phase of healing, matrix metalloproteinases assist with remodeling of scar and tissue after wound closure. Macrophages, keratinocytes, and fibroblasts may produce matrix metalloproteinases in the inflammatory phase. Tissue inhibitors of metalloproteinases act to block tissue destruction by matrix metalloproteinases as the inflammatory phases terminate and proceed into a proliferative phase. A balance between matrix metalloproteinases and tissue inhibitors of metalloproteinase is necessary throughout normal wound healing for successful and optimal closure. The cellular interplay between these wound components mediates the repair process in a complex manner. Agren[18] found that following complete epithelialization and scab sloughing in partial-thickness wounds, peak levels of MMP-1 occurred on days 3 to 5, and decreased to normal skin levels by day 7. Large, unsutured full-thickness granulating wounds showed a high content of MMP-1 on day 5 with a sharp decline by day 7, continuing in the same manner to day 21. In surgical incision wounds, peak levels were noted on day 1 and then gradually declined. Collectively, these observations showed that matrix metalloproteinase levels mirrored the progression of the wound healing process in acute wounds.

Disruptions and imbalances between matrix metalloproteinases and their regulatory components may affect wound closure and contribute to wound chronicity. Chronic wounds are known to deviate from the sequence of repair in the acute wound by elevated pro-inflammatory cytokines, high protease activity, and diminished growth factor activity. While the pro-inflammatory response is limited in the acute wound, it may persist in the chronic wound environment. This difference is of particular importance to a clinician applying topical growth factors or skin replacements containing slow-release growth factors to assist closure of chronic wounds. Growth factors and skin equivalents within the hostile environment of chronic wounds may be subject to degradation by prolonged inflammation, resulting in high levels of proteinases, negating the benefit of applying these materials and delaying tissue repair.

The inflammatory response to healing is one of the greatest differences between chronic and acute wounds. In the acute wound, the following events take place: a single tissue injury results in limited and transient stimuli of inflammatory cytokines; growth factors are secreted from macrophages, fibroblasts, vascular endothelial cells, and keratinocytes; extracellular matrix production and angiogenesis take place; and scar formation occurs. In recalcitrant or chronic wounds, recurrent tissue trauma and the presence of contaminants could contribute to pro-inflammatory cytokine response with subsequent elevated protease and impaired growth factor activity. Tarnuzzer and Schultz[19] and Mast and Schultz[20] examined the wound environment and found that the average level of proteases was 116-times higher in chronic wounds as compared to acute wounds. The authors stated that increased protease production was due to repeated and persistent trauma, ischemia, or low-grade bacterial infection. Within the chronic wound, neutrophils and macrophages were shown to impair wound healing by producing elevated levels of matrix metalloproteinase and decreased levels of tissue inhibitor of metalloproteinase. Thus, the clinician must adjust intrinsic and extrinsic factors that prolong pro-inflammatory response and delay closure. This can be accomplished by removing sources that lead to repeated trauma, ongoing bacterial contamination, and ischemia.

Conclusion

The use of advanced treatment modalities, including growth factors and tissue replacement, may offer a way to expedite wound closure in recalcitrant wounds. Wounds that show no response to appropriate therapy, including debridement and control of extrinsic and intrinsic factors after approximately 2 weeks, may be considered nonresponsive. The application of growth factors should follow wound debridement once the wound has been stabilized. The overall medical status of the patient must also be considered. Bacteria, repetitive trauma, and ischemia may contribute to increased growth factor degradation and decreased product efficacy. Cellular senescence creates an environment void of receptors for growth factors. Debridement addresses cellular senescence, necrotic tissue, and removal of superficial contaminants. The application of topical materials is meant to assist with cellular modulation and will only be beneficial in a stable wound environment. Failure to address and correct problems in the wound bed or the external environment will lead to poor treatment outcomes.