Leg ulceration is the most prevalent chronic wound in Western countries, affecting about 1 to 2% of the adult population (Valencia I C, Falabella A, Kirsner R S, Eaglstein W H. Chronic venous insufficiency and venous leg ulceration.  J Am Acad Dermatol 2001; 44: 401-21).  The main causes of leg ulceration are venous hypertension, arterial insufficiency, and diabetes.  Venous ulcers account for approximately 80% of all leg ulcers and are the result of venous hypertension.  The current treatment for venous ulcers includes graduated compression support stockings or compression bandaging of the limb.  Despite the standard of care of compression therapy, 50 to 70% of venous leg ulcers remain unhealed after 12 weeks of treatment and 54 to 78% of ulcers will reoccur (Abbade L P, Lastoria S. Venous ulcer: epidemiology, physiopathology, diagnosis and treatment.  Int Wound J 2006; 3:113-20).  These chronic wounds have a treatment cost in the United States of approximately one billion dollars per year and have a significant impact on the patient's quality of life (Herber O R, Schnepp W, Rieger M A. A systemic review on the impact of leg ulceration on patient's quality of life.  Health Qual Life Outcomes 2007; 5: 44). 
The biology of the chronic venous and diabetic wounds is quite different from acute wounds.  In an acute wound the initial fibrin clot provides hemostasis and the platelets release cytokines, growth factors, and recruit inflammatory cells.  The recruitment of inflammatory cells includes neutrophils and macrophages to eradicate bacteria.  At the leading edge of the wound the protease cut through the fibrin clot.  Matrix Metalloproteinases (MMP) are up regulated by the keratinocyte to cut a path through the matrix proteins to allow the keratinocyte to advance and close the wound.  MMP-9 (gelatinase B) cuts through the basal lamina collagen (type IV) and anchoring collagen (type VII) to allow the keratinocytes to advance and close the wound.  Once the keratinocytes cover the wound, the wound is re-epithelized, the basal lamina is reestablished and the MMP-9 is shut off. 
However, in a chronic wound, the MMP-9 is not shut off.  The elevated levels of this protease continue to destroy the wound matrix that is produced to heal the wound.  The level of MMP-9 in a chronic wound can be five times of its level in an acute wound (Yager et al, 1996, Trengove et al, 1999).  MMP-9 is the major protease that is present in the chronic venous stasis and decubitus ulcers. 
The tissue inhibitor of metalloproteinase (TIMP-1) is absent from chronic wounds and is also decreased with age. 
These chronic wounds also become colonized with bacteria.  The bacterial colonies produce a biofilm which enables the bacteria to act as a multicellular organism.  The biofilm protects the bacteria from the host immune system and all antibiotics.  The bacterial biofilm gains nutrients from its own protease, which are similar to the host MMP-9.  The biofilm then protects the bacteria from the host immune system and all antibiotics.  Thus, in chronic wounds, the bacterial biofilm and the host both produce proteases which are responsible for the degradation of the factors responsible for wound healing. 
Research has shown that silver nanoparticles (AgNPs) can be manufactured into specific shapes that show improved antibacterial properties (Pal, S., Y. K. Tak and J. M. Song, 2007, Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle?  A study of the gram-negative bacterium Escherichia coli.  Appl.  Environ.  Microbiol.  2007, 73(6):1712).  The specific AgNP shapes also demonstrate improved anti-inflammatory and angiogenic properties.  The specific AgNP shapes which have shown improved antibacterial, anti-inflammatory, and angiogenic properties include silver nano prisms (AgNPr), silver nano truncated triangle plates (AgNTTP), and silver nano discs (AgND).  The specific AgNP shapes allow for the use of a lower concentration of silver and produce a more effective wound care treatment than other silver compositions. 
The AgNTTP (silver nano truncated triangular plates) have shown the strongest antibacterial activity due to the surface {111} facet characteristics (Wijnhoven, S. W. P., Peijnenburg, W. J. G. M., Herberts, C. A., Hagens, W. I., Oomen, A. G., Heugens, E. H. W., Roszek, B., Bisschops, J., Gosens, I., Van De Meent, D., Dekkers, S., De Jong, W. H., van Zijverden, M., Sips, A. J. A. M. and Geertsma, R. E., 2009.  Nano-silver—a review of available data and knowledge gaps in human and environmental risk assessment.  Nanotoxicology, 3:2, 109-138).  The reactivity of the nano silver is improved by the high atom density surface {111} facet (Hatchett, D. W. and Henry, S. 1996, Electrochemistry of sulfur adlayers on low-index faces of silver.  Jl.  Phys.  Chem.  100:9854-9859). 
Silver nanoparticles also demonstrate strong anti-fungal properties (Wright, J. B., Lam K, Hansen D., Burrell R. E., 1999, Efficacy of topical silver against fungal burn wound pathogens.  Am.  Jl.  Infect.  Control 27:344-350), anti-viral properties that inhibit HIV-1 replication (Sun, R. W., Chen R., Chung N. P., Ho C. M., Lin C. L., Che C. M. 2005.  Silver nanoparticles fabricated in Hepes buffer exhibit cyto-protective activities toward HIV-1 infected cells.  Chem.  Commun (Camb.):5059-5061), and anti-inflammatory properties (Kirsner R. S., Orsted H., Wrught J. B. 2002.  Matrix metalloproteinases in normal and impaired wound healing: a potential role of nanocrystalline silver.  Wounds 13:4-12). 
As noted above, elevated inflammatory mediators are responsible for chronic wounds not healing.  The chronic inflammatory mediators that are most responsible for non-healing diabetic wounds include matrix metalloproteinases (specifically MMP-9), tumor necrosis factor (TNF-alpha), and interluken (IL-1 and 12). ( Rayment, E. A., Upton Z., Shooter G. K., 2008, Increased matrix metalloproteinase-9 (MMP-9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer.  British Jl.  of Derm.  158, pp 951-961).  The AgNP shapes with {111} facets suppress these proteolytic enzymes that are responsible for the non-healing chronic wounds (Wright J. B., Lam K., Buret A. G., Olson M. E., Burrell R. E., 2002, Early healing events in a porcine model of contaminated wounds: Effects of nanocrystalline silver on matrix metalloproteinases, cell apoptosis, and healing.  Wound Repair Regen.  10:141-151). 
As discussed above, chronic venous, lymphatic and diabetic wounds include a high bacterial load, glycoprotein biofilm, and chronic inflammatory mediators.  The biofilm produced by the bacteria prevents the effectiveness of antibiotics and the host immune system against the bacteria.  However, the incorporation of AgNP into a treatment area will inhibit the bacteria as well as the formation of the biofilm (Percival, S. L., Bowler P. G., Dolman J., 2007, Antimicrobial activity of silver-containing dressings on wound microorganisms using an in vitro biofilm model.  Int.  Wound Jl.  4:186-191). 
One successful device to treat chronic wounds is described in U.S. Pat.  No.  9,220,636, entitled “Sock For Treatment of Foot and Leg Wounds, Methods of Use and Manufacture,” by the present inventor (“the '636 patent”), the disclosure of which is incorporated herein in its entirety.  The '636 patent discloses embodiments of compressions stockings knit from a combination of natural wicking fibers and elastic fibers, and with AgNP including specific AgNP shapes adhered to the natural fibers.  The compression stocking can be applied directly onto the wound with the natural wicking fibers with adhered AgNP coming into contact with the wound.  This compression stocking design has been shown to down regulate the MMP-9 and bacterial proteases, kill the bacteria that are protected by the biofilm, and allow the epithelialization of the wound without harming the keratinocytes. 
Preliminary clinical trial results of a compression stocking incorporating the features disclosed in the '636 patent, referred to as the Vive stocking, are encouraging.  Patients that had failed current standard of care treatment for lower extremity wounds were referred to a wound clinic for evaluation and treatment.  After informed consent was obtained, five patients were started in a preliminary clinical trial using the Improved Sock with wool and alpaca fibers, elastic fibers for compression, and AgNP (See Table 1).  The age of these five preliminary clinical trial patients with chronic wounds (five patients) and acute wounds (one patient) ranged from 58 to 87. 
TABLE 1SockWoundPreviousPreviousWoundTime tochangedPatientDurationWoundAntibioticCompressionSizehealworn 24 hr/AgeComorbidities(months)CultureTreatmentTherapy(cm2)(weeks)day70HL, TR,>12Mag.5012WeeklyCS79EM, SM,3SC, AC403WeeklyHTN244MRSAClindamycin,11WeeklyCiprofloxacin,TMP-SMZ58DM, HTN,>12Doxycycline,SC, XC, SIEntire10DailyCO, LY,Vancomycin,legSAZosyn87SS, VSI,AcuteMRSADoxycyclineEntire14DailyARFlegAbbreviations used in table:Comorbidities: ARF—acute renal failure; CO—COPD; DM—diabetes; EM—emphysema; HL—hyperlipidemia; HTN—hypertension; LY—lymphedema; SA—sleep apnea; SM—smoker; SS—septic shock; TR—trauma; VSI—venous stasis insufficiencyPrevious Compression Therapy: AC—ACell MatriStem ®; CS—Kendal T.E.D. ® hose; DY—Johnson & Johnson DYNA-FLEX ®; Mag-Molnlycke Mepilex ® Ag; SC—Johnson & Johnson Silvercell ®; SI—Silver sulfadiazine; UN—Unna boot; VAC—KCI wound V.A.C. ®
In all cases, the pain decreased.  The only complication occurred in the 58 year old patient, whose wound healed uneventfully, despite a dorsal ankle ulcer from sock fold. 
The chronic wound patients' previous treatment had included compression therapy using Kendal T.E.D™ compression stockings, Johnson & Johnson DynaFlex® compression wraps, and Unna compression boot.  One patient had a wound treated with a KCl wound V.A.C. Wound dressings included silver alginate such as Johnson & Johnson Silvercel®, and Molnlycke Mepilex® Ag.  Treatment with skin graft substitutes included ACell MatriStem®, and Organogenesis Apligraf®.  Prior therapies for the chronic wound patients enrolled in the preliminary clinical trial included several months of a broad spectrum of oral and intravenous antibiotics. 
Despite aggressive standard of care treatment, all of the patients' wounds that were enrolled in the preliminary clinical trial had failed to heal.  Some of the chronic wounds treated with the Vive stocking had been present for several years.  As expected, the larger chronic wounds took longer to heal.  Nonetheless, all of the wounds that had been present for over one year and then treated with the Vive stocking healed in 10 to 12 weeks, and have not recurred. 
The one acute preliminary clinical trial patient with acute fungal and MRSA wounds healed in 2 weeks. 
The most remarkable result from the clinical trial occurred in an 87-year-old woman who presented to the hospital in septic shock with circumferential ulceration of her left leg from the knee to ankle.  She had chronic venous stasis ulcers that failed treatment using compressive wrap with an Unna boot.  Her initial wound cultures were positive for MRSA.  Initial consultation from a general surgeon was to consider an amputation of the leg above the knee.  After informed consent with the patient and her family (including a dermatologist), they elected to try the Vive stocking.  The Vive stocking was changed daily at bedside and washed in regular soap and water.  The patient underwent no anesthesia or surgical debridement.  After a week of rapid improvement the patient was discharged to a skilled nursing facility.  While at the skilled nursing facility the wound care nurse resumed traditional wound dressing including graduated compression.  After two weeks the wound became significantly worse.  The Vive stocking was then reapplied and changed every 2 to 3 days depending on the amount of drainage.  After 14 weeks the treated leg wounds completely healed.  The patient subsequently wore the Vive stocking daily without any swelling or sign of skin breakdown.  No ulcers had reoccurred two months after healing. 
There are currently many medical and non-medical wound care products on the market that contain silver.  These silver wound care dressings have become a standard therapy for patient wound care.  The antibacterial properties of silver are well known but the side effects of systemic absorption of the silver have been the concern of recent studies.  A recent animal study using two well-known commercially available silver dressings shows that silver was detected by plasma mass-spectroscopy in all analyzed organs and tissue samples of the animals using the wound dressings.  Organ concentration levels of silver were detected at the highest levels in the nano crystalline silver dressings and were also detected from the silver sulphate foam.  The highest silver concentrations were detected in the spleen, kidney and liver. ( Pfurtscheller, K., Petnehazy, T., Goessler, W., Bubalo, V., Kamolz, L., Trop, M., Transdermal uptake and organ distribution of silver from two different wound dressings in rats after a burn trauma.  Wound Rep. Reg.  22:654-659.)  As noted in the Pfurtscheller et al.  study, silver leaching can be detrimental to both the patient and the environment. 
The Vive stocking was independently tested to determine if any silver would leach out of the fibers.  Independent testing at Bucknell University, including an atomic absorption (AA) spectrometer flame test and a zone of inhibition (ZOI) antibacterial test, demonstrated that the Vive stocking did not leach silver even after being washed in soap and water. 
Atomic Absorption (AA) Spectrometer Flame Test
This test was advised by Monica Hoover, Director of Environmental Engineering and Science Laboratory, Bucknell University. 
Methods
A standard curve was made using a multi-elemental standard solution prepared for the Aurora Al 1200 Flame Test.  The standard curve quantifies the amount of silver within a solution from a peak height provided by the spectrometer.  Three samples were made with 10 mL of water including the following:
Product A—1 cm by 1 cm piece of a standard silver alginate dressing (SilverCel®)
Product B—AgNP in solution (1 mL of the AgNP not bonded to the stocking)
Product C—1 cm by 1 cm piece of Vive stocking. 
Product A, Product B, and Product C were each combined with 10 mL of water and placed in a separate test tube.  Each test tube was shaken for 10 minutes and a sample was sent through the AA spectrometer.  In other words, Product A was placed in a test tube with 10 mL of water and shaken for 10 minutes.  Then the piece of standard silver alginate dressing was removed from the test tube and the remaining solution was sent through the AA spectrometer.  Similarly, Product C was placed in a test tube with 10 mL of water and shaken for 10 minutes.  Then the piece of Vive stocking was removed from the sample and the remaining solution was sent through the AA spectrometer.  As to Product B, 1 mL of AgNP were added to 10 mL of water and the solution was shaken.  Nothing was removed from the solution of Sample C before sending the solution to the AA spectrometer. 
After the preliminary testing above, it was determined that the original 10 mL solutions of Product A (silver alginate dressing) and Product B (silver solution) required a ten times dilution to be within the range of concentrations produced by the standard curve.  Product C (Vive stocking) did not leach silver upon preliminary testing, so the solution was not diluted.  Table 2 shows the results from the AA spectrometer flame test when the test was performed on Product A with ten times dilution, Product B with ten times dilution, and Product C.
Results:
TABLE 2SamplesPk HeightResult ppmA: SilverCel ® (10x)0.16619.723B: AgNP in solution (10x)0.22027.584A: SilverCel ® (10x)0.19928.16B: AgNP in solution (10x)0.1621.86C: Vive (1x)0.0040C: Vive 1 hr wash0.0110
Each of the tests for Product A and Product B were run twice, as indicated by “A” and “B” in the Table 2.  As shown in Table 2, Product B (silver solution) produced a detectable amount of silver, making Product B (silver solution) important as a positive control.  Product B (silver solution) is the same solution used to coat the wool within the Vive stocking.  Also as shown in Table 2, Product C (the Vive stocking) did not leach any silver after 10 seconds of shaking.  It was then placed on a shaker for an hour with soap to simulate a wash cycle.  Product C (the Vive stocking) did not leach after this period. 
Conclusions
The Vive stocking did not leach silver to the detectable limits of the AA spectrometer Flame Test.  Product A (silver alginate dressing) leached 28 ppm of silver into the water after 10 seconds of shaking. 
Zone of Inhibition (ZOI) Antibacterial Test
This test was advised by Dr. Marie Pizzorno, Professor of Biology, Bucknell University. 
The Vive stocking demonstrated safety to the patient and the environment by not leaching silver.  The next step was to demonstrate that the Vive stocking had equivalent antibacterial activity as Product A (silver alginate dressing) that leached silver.  The test that best replicates the actual usage of the Vive stocking is a zone of inhibition test.  In this test the stocking is applied directly to an agar plate.  The agar best replicates the true environment of the stocking in direct contact with a moist wound. 
Methods
Thirteen nutrient agar plates were prepared with 50 μL of Staphylococcus epidermidis (staph).  Staph was used for testing because it is a gram-positive bacterium that is commonly present in hospitals.  It is a common cause for infections in patients.  Two samples (one silver alginate dressing and one Vive stocking) were cut approximately 1″ in diameter.  The samples were glued to cardboard to prevent rolling.  The samples were placed carefully on top of the bed of Staph.  The plates were incubated for 48 hours at 37° C. To calculate the ZOI of each sample, the dressing widths were subtracted from the zone widths (Landry et al.,  2009).  This was repeated in duplicate perpendicular directions (Landry et al.,  2009). 
Results
FIG.  1 is a representative photo of a sample, reproduced in line drawing format.  A sample plate system 100 includes a plate 101.  A silver alginate dressing sample 102 and a Vive stocking sample 103 are disposed on the plate 101.  Staph 104 is identified by the grey region.  The silver alginate dressing sample 102 has a zone of inhibition 105 and the Vive stocking sample 103 has a zone of inhibition 106. 
As can be seen in FIG.  1, a bed of staph covers the entirety of the plate except for the silver alginate zone of inhibition 105 and the Vive stocking zone of inhibition 106.  The silver alginate zone of inhibition 105 and the Vive stocking zone of inhibition 106 represent areas of limited antibacterial growth of staph.  Thus, a sample with a larger and clearer zone of inhibition is more antibacterial. 
Conclusions
The Vive stocking and the silver alginate wound dressing both limit the growth of Staphylococcus epidermidis (“staph”), as indicated by a clear ring of negated bacteria in each sample.  For example, as shown in FIG.  1, the silver alginate zone of inhibition 105 and the Vive stocking zone of inhibition 106 represent areas of limited antibacterial growth of staph.  The zone of inhibition of the Vive stocking was consistently equal to or greater than the zone of inhibition of the silver alginate dressing. 
Research studies have also shown that wound healing can be enhanced by application of electrical currents to the wounds.  The use of electrical field energy applied to chronic wounds demonstrates improved blood flow, increased angiogenesis and reduced wound necrosis in patients with chronic venous leg ulcers.  A meta-analysis of clinical trials has shown electrical stimulation effectively promotes wound closure. ( Gardner, S. E., Frantz, R. A., Schmidt, F. L., 1999, Effect of electrical stimulation on chronic wound healing: a meta-analysis.  Wound Rep. Reg.  7:495-503; Kawasaki, L, Mushahwar, V. K., Ho, C., Dukelow, S. P., Chan, L. L. H., Chan, K. M., 2013, The mechanisms and evidence of efficacy of electrical stimulation for healing of pressure ulcer: a systematic review.  Wound Rep. Reg.  22:161-173.  Kloth, L. C., Electrical Stimulation Technologies for Wound Healing.  Adv Wound Care (New Rochelle).  2014 Feb. 1; 3(2): 81-90.) 
The Wound Healing Society (WHS) published an updated position paper for wound healing guidelines in 2014.  Guideline #7.2.3 states there is Level 1 evidence that “Electrical stimulation accelerates wound closure and the proportions of wounds that heal in RCTs and prospective and retrospective cohort studies”. ( Braun, L., Kim, P., Margolis, D., Peters, E., Lavery, L., What's new in the literature: An update of new research since the original WHS diabetic foot ulcer guidelines in 2006.  Wound Rep. Reg.  22:594-604.) 
Multilayer compression socks on the market today simply use a two layer sock kit (traditional kit).  This traditional kit includes two layers of sock over the foot, ankle and calf.  The bulk of the two layers of sock over the foot makes wearing regular shoes with the traditional kit difficult.  Also, the two layers of sock over the ankle add pressure to the ankle which has caused ulceration.  Due to the bulk and compression, the traditional kit is challenging for patients put on their calf, ankle, and foot.  For these reasons, patients tend to not be compliant with wearing both sock layers of the traditional kit.  When patients are not compliant, reoccurrence of ulcers is common.  Therefore, using the traditional kit places patients at risk of developing iatrogenic ankle ulcers. 
Thus, there is a need for a wound treatment device that provides the benefits of compression, incorporates AgNP, and produces an electrical current while minimizing the drawbacks of silver leaching, bulkiness, and iatrogenic ankle ulcers to achieve increased compliance and wound healing.