III.             

V.  BIOLOGIC PROPERTIES OF SILVER PRODUCTS RELATED TO BURNS

There appear to be three properties of silver that have positive effects on the burn wound. These include antimicrobial, pro-healing, and anti-inflammatory properties. Clearly these properties overlap.

A. Antimicrobial Properties

 The antimicrobial activity of silver ions is well defined. Silver ions rapidly kill microbes by blocking the cell respiration pathway. The speed of action is almost instantaneous once the silver reaches the microbe. The efficacy of microbe killing is based not only on the amount of silver ion present, but likely also the presence of other silver radicals.

Silver has been delivered to wounds in the form of a solution, a cream, or newer silver delivery systems.

Some strains of gram-negative bacteria and pseudomonas aeruginosa appear to be resistant to silver sulfadiazine and some fungi, such as candida tropicalis, are resistant to silver nitrate. It is possible that the rate of bacterial and fungal kill of silver-containing products is important, as a more rapid kill kinetics ensures less likelihood of bacteria or fungi achieving physiological adaptation to silver.^(29,30) Thus developing silver resistance is less likely. In order to achieve an optimum bactericidal effect, a large number of silver ions must be available in solution, as efficacy depends on the aqueous concentration of these ions in contact with microbes.

Although silver ions are rapidly inactivated in the wound, the rapid bactericidal effects, sustained release of silver ions and radicals released from the silver nanocrystal delivery system should minimize the likelihood of bacteria developing resistance to silver. Although the concentrations of aqueous silver ions released from nanocrystals are about 30 times less than those found with silver nitrate and silver sulfadiazine, a more rapid kill curve is produced.^(31,33)

The results of several studies that evaluated the antimicrobial activity of pure silver delivery (Acticoat), compared with conventional topical silver-containing agents illustrates both the antimicrobial spectrum and the rapid kill rate. Acticoat is active against both gram positive and gram-negative organisms, including both aerobic and facultatively anaerobic organisms as well as fungi. Pure silver ions and radicals are active against all pathogens found in burn wounds, particularly Pseudomonas aeruginosa, common bacterial causes of skin infections, such as Staphylococcus aureus, Candida species, and all methicillin resistant staphylococcus and vancomycin resistant enterococcus.^(26,31)

The study presented compares the antimicrobial efficacy of the silver released from the three silver products currently used on burns.

Silver was extracted from the pure silver delivery system by incubating the dressing in water at 37°C in a shaking incubator, and silver concentrations were measured using atomic absorption spectrophotometry. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using five bacterial isolated of clinical interest, and results were compared for Acticoat, silver nitrate and silver sulfadiazine, as shown in Table 6. Acticoat had the lowest MIC and MBC values of the three silver containing agents. Kill kinetics were also studied, using 2.0 cm x 2.0 cm pieces of Acticoat dressing, and the same sized pieced of dressing impregnated with either silver nitrate (100 µl of 1% solution -producing a final concentration of 0.5% silver nitrate) or silver sulfadiazine (370mg of a 1% cream). Bacterial survival was measured using plate counting. Nanocrystal silver demonstrated the fastest kill times for the five bacteria used. In most instances, bacterial survival was undetectable 30 minutes after inoculation with the pure silver, whereas at least 2-4 hours elapsed before no viable bacteria were detected with silver nitrate or silver sulfadiazine.^(14,31)

Reactive Species of Silver Released from Silver Products

In summary, the rate and degree of killing of all microbes tested is the highest with the silver delivery from nanocrystals compared to silver nitrate and SSD. It is likely that the reasons for the more effective silver in nanocrystals is the more rapid rate of delivery and the presence of other silver radical species, found with silver release from nanocrystals.

B. Prohealing Effects.

Past observations and recent studies using a pure silver delivery system have demonstrated an increased re-epithelialization rate of non-infected partial-thickness wounds and burns compared to other silver compounds or other dressings.^(34-38,39)

The direct healing effect of pure silver has yet to be defined (Table 8).  Silver has a number of effects which would indirectly increase healing, namely controlling infection, decreasing excess inflammation, maintaining moist healing and decreasing wound surface mechanical trauma caused by frequent dressing changes.

1) Direct Effect of Silver on Healing

Wound Effects of Silver

MMP - metalloproteinase

However, several studies comparing silver released from a silver nanocrystal delivery system with other topical antibiotics have demonstrated increased re-epithelialization of partial thickness wounds and meshed skin grafts (Figure 5).^(34-38)  A comparison was made of the rate of re-epithelialization of 2:1 meshed skin grafts on excised burn wounds.

Closure with silver significantly faster than standard antibiotic solution

On each individual excised, one half of the meshed grafts were maintained moist by using a dilute Neosporin solution under fine mesh gauze while the other half was covered with the nanocrystal silver delivery moistened twice a day. Bacterial growth was negligible in both wound groups. However, re-epithelialization to mesh closure was significantly faster with silver released from the nanocrystals. This data strongly suggests a direct wound healing effect of pure silver (Figure 6 ).⁽³⁷⁾

2) Eliminating Silver Complexes

The use of silver salts, and other complexes like sulfadiazine have been necessary in the past to provide silver to the wound because of the lack of stable silver delivery systems. Although these silver complexes have had potent antimicrobial properties, the effect on wound healing has been largely negative. Using pure silver will at least remove any negative effects.^(43-45)

3) Controlling Surface Microbes

Increasing evidence has demonstrated that wound colonization can result in a bacterial burden to healing. The concept that one needs 105 organisms per gram of tissue to be deleterious is no longer considered accurate. Certainly 105 organisms or greater is destructive to healing but fewer bacteria can also impede healing as the immune defenses of a wound and of a patient vary considerably, e.g. the elderly diabetic versus the healthy young adult.⁽⁴⁶⁾

Improved antimicrobial activity should therefore assist healing independent of any direct healing property of silver.

4) Moist Wound Healing

Prior to the late 20th century, burns were felt to heal better if left exposed producing a "scab". This exposure process also produced surface desiccation and eschar formation now known to deepen the wound. The thinking may have been correct given the fact that no antibacterial agents were available to treat an infection if it occurred and desiccation does decrease bacterial proliferation.

The most detailed descriptions of the benefits of scab formation were found in medical books from Ancient Greece. Although the initial ingredients first applied to the burn and other wounds varied considerably, wound dryness was always sought. The description of the healing process, leading to scar as the normal endpoint, would indicate that outcomes were less than ideal.

The concept of "dryness is good" remained popular for wound care until the mid-20th century (and even longer for the burn wound).

A landmark study in 1962, by Winter et.al.⁽⁴⁷⁾, demonstrated that partial thickness wounds re-epithelialized more rapidly under occlusive dressings, with the reason being that occlusive dressings maintained a moist wound surface. This environment accelerated the re-epithelialization process. Numerous studies followed which demonstrated that wound occlusion and moisture improved all phases of healing.^(48-53) The data, demonstrating that a moist wound surface increases re-epithelialization and all other components of wound healing, is now well established. Any surface desiccation leads the risk of further tissue damage.(Table 9)

Problems of Surface Desiccation

In general, research has shown that a moist wound environment is associated with less severe and prolonged inflammation; more rapid keratinocyte proliferation and migration; earlier differentiation of keratinocytes to restore surface barrier function; more rapid fibroblast proliferation, increase in collagen synthesis; earlier, angiogenesis; and earlier full-thickness wound contraction.  A more rapid decrease in wound volume and surface area has also been well documented with moisture.  It has been suggested that moist environments improve these measures of healing repair and regeneration by limiting necrosis due to desiccation.

This use of a moist healing environment has not routinely been used in burns because of the concern for infection.   Also, the antibiotic creams do not produce a moist healing environment. In fact, the creams silver sulfadiazine and sulfamylon, will extract water from the wound surface as a result of the hyperosmolar environment,  which results with drying of the cream.

Advantages of a Moist Wound Surface

Silver nitrate solution maintains a moist surface but this approach is not very popular because of increased nursing care demands as the dressings must be constantly moistened.

The nanocrystal silver delivery system maintains a moisture layer between the wound surface and the inner silver membrane. This moisture layer is also a potent antimicrobial silver solution on the burn wound surface.

Moisture is maintained on a deep burn by wetting the surface of the Acticoat. Moisture on a partial thickness wound is provided by the wound itself.

The ability to maintain both a moist healing environment and an antimicrobial environment is a unique property of this silver system, especially beneficial for partial thickness burns, excised or grafted wounds.

5) Decreasing Mechanical Trauma

Frequent dressing change and wound manipulation create not only systemic toxicity but also local wound trauma. The high rates of bacteremias with wound manipulation have been well described as has the typical pyrogen release and post-dressing change hyperthermia. Also wound surface manipulation especially on the re-epithelializing wound will injure new tissue formation Mechanical removal of creams, ointments and dressings will all cause local trauma. The nanocrystalline silver dressing can remain in place for days and if wetted prior to removal, mechanical trauma is minimal. In addition, the exudate beneath is usually minimal and there is no film or byproduct, which requires mechanical removal.

C. Anti-Inflammatory Properties

1) Nanocrystal Silver and MMP's

Silver has been reported to decrease burn wound surface exudates thereby avoiding excess surface inflammation and rapid autolysis. Excess inflammation is well recognized to impair healing of any wound. Increased metalloproteinase activity (MMP) has been reported to be present on the surface of the burn wound. The upregulation of MMP activity can increase collagenolysis activity on the wound surface, a necessary factor for undermining an eschar during re-epithelialization,^(54-57) but excess MMP's can be deleterious. Before proceeding it is important to better understand wound inflammation, MMP's, their positive and negative effects. However, increased MMP's also degrade growth factors and can thereby impede re-epithelialization. Silver decreases but does not prevent MMP activity on the wound, as some MMP's are necessary for surface collagenolysis.

Silver decreases surface wound zinc, zinc being the necessary metal cofactor for MMP activity. Silver has been shown to decrease MMP activity on an in vitro wound model as well as on the surface of non-healing wounds.

The current data on silver's ability to decrease MMP activity in the non-burn wound is of interest but its effect on burn wound MMP's has yet to be determined. However, a number of studies are underway. A decrease in wound exudates alone is a beneficial effect of the nanocrystal silver delivery system.

In recent animal studies, nanocrystalline silver dressings promoted a more rapid healing in full thickness wounds while decreasing the concentration of metalloproteinase. All phases of wound healing were promoted with nanocrystalline silver compared to other non-silver containing antibiotics, and the rate of healing corresponded with the decrease in the very elevated levels of MMP's. It is important to point out that MMP activity was not eliminated but rather attenuated to a level more compatible with that seen with a normal healing wound. A total elimination of MMP's would be deleterious as some surface collagenase and protease activity is required to remove the surface debris needed prior to healing.

2) What Are Metalloproteinases (MMP's) And How Do They Alter Healing?

The metalloproteinases⁽⁵⁶⁾ are a family of proteases (enzymes which break down tissue) characterized by:

a. dependence of the metal zinc for activation
b. present in wounds where their role is to break down damaged tissue, denatured protein and matrix, in order to make way for new tissue initiated by growth factors.
c. High sulfur content including sulfhydryl and disulfide bonds
d. Present in highest concentration on the wound surface and in matrix
e. Can inactivate growth factors

The burn wound healing process is recognized to be a dynamic balance between growth factors or the synthetic aspects and proteinases which produce tissue breakdown and remodeling.

MMP’s involved in wound repair:

These proteinases therefore break down the elements of a wound namely collagen, elastin, and matrix. In addition, these proteases will inactivate growth factors if present in excess as in a chronic wound or an acute burn wound.

It is now well recognized that the acute inflammatory response is needed to activate the healing process, but persistent inflammation is deleterious to the healing of any wound.

A key component of excess inflammation is excess surface protease activity destroying the required balance between growth factors and protease activity.

Excess healing leads to excess scar and healing itself requires some MMP activity to break down damaged tissue, making room for migration of new tissue formation. Excess MMP activity is normally controlled by endogenous tissue inhibition of metallo proteinases. TIMP's.

3) Mechanism of MMP Production

The MMP production is activated by cytokines and a variety of inflammatory mediators. The factor induced a variety of cells.

The Burn Wound Factory for MMP’s and Growth Factors

In addition, factors such as available zinc for activation and sulfur for sulfide bonds is essential in addition to gene expression for MMP production modulated by both cytokines and growth factors.

4) How are Excess MMP's Controlled?

Since the MMP's if produced in excess are deleterious to healing, there is present a counteraction system to maintain optimum balance in the wound. This process works well in the normal uninfected acute wound when balance can be maintained. Balance is lost in the chronic wound and to a lesser degree in the acute burn wound.

a) Endogenous tissue inhibitors of MMP's (TIMP's)

 

There are a number of TIMP's released by macrophages and fibroblasts, in response to MMP production which protect tissue from protease breakdown by a competitive binding to tissue protease sites.

This system is limited to a finite production, which can be overwhelmed by the greater potential for protease production with inflammation. At present, considerable research is underway to product TIMP's. This genetic engineering process is expensive as will be the products, if they work!

 

b) Exogenous blockers of MMP action

 

There are a number of inhibitors which have been found to block MMP's in vitro or in vivo, most of which cannot be effectively used on a wound surface.⁽⁵⁸⁾

• Zinc binding or removal is highly effective in blocking MMP action. Currently the only safe agent that works is silver ion. It has now been established that nanocrystalline silver decreases but does not eliminate wound MMP activity. The mechanism remains unclear but decreasing zinc may be one mechanism.^(59,60)

 

What are the Characteristics of the Acute Burn Wound?

The acute burn wound, especially the partial thickness injury, is similar to any acute non-infected traumatic wound, relative to the stages of healing with one major exception: There is now recognized to be an over-expression of surface MMP's, even in the absence of non-viable tissue.⁽⁶¹⁾

The increase in surface MMP's is 2-3 fold higher than a non-burn acute cutaneous wound and matrix MMP activity is 20 to 30 fold higher. This excess up-regulation of MMP's after burn exceeds the level of local inhibition, resulting in a net proteolysis in burn wounds which could certainly impede initial healing and accentuate scar. The mechanism is likely due to the severe inflammatory response in initiated by a burn as opposed to an incision or traumatic wound. Autolysis is beneficial in a deep burn but not in a partial thickness burn.

Because of this MMP imbalance, nanocrystalline silver release is more likely to be effective in improving healing in an acute burn wound. However, data on the burn wound verifying this concept is not yet available.