Novel nitric oxide producing probiotic wound healing patch: preparation and in vivo analysis in a...

ORIGINAL ARTICLE

Novel nitric oxide producingprobiotic wound healingpatch: preparation andin vivo analysis in a NewZealand white rabbit modelof ischaemic and infectedwoundsMitchell Jones, Jorge G Ganopolsky, Alain Labbe, Mirko Gilardino,Christopher Wahl, Christopher Martoni, Satya Prakash

Jones M, Ganopolsky JG, Labbe A, Gilardino M, Wahl C, Martoni C, Prakash S. Novel nitric oxide producingprobiotic wound healing patch. Int Wound J 2012; 9:330–343

ABSTRACTThe treatment of chronic wounds poses a significant challenge for clinicians and patients alike. Here we reportdesign and preclinical efficacy of a novel nitric oxide gas (gNO)-producing probiotic patch for wound healing.Specifically, a wound healing patch using lactic acid bacteria in an adhesive gas permeable membrane has beendesigned and investigated for treating ischaemic and infected full-thickness dermal wounds in a New Zealand whiterabbit model for ischaemic wound healing. Kaplan–Meier survival curves showed increased wound closure withgNO-producing patch-treated wounds over 21 days of therapy (log-rank P = 0·0225 and Wilcoxon P = 0·0113).Cox proportional hazard regression showed that gNO-producing patch-treated wounds were 2·52 times more likelyto close compared with control patches (hazard P = 0·0375, score P = 0·032 and likelihood ratio P = 0·0355),and histological analysis showed improved wound healing in gNO-producing patch-treated animals. This studymay provide an effective, safe and less costly alternative for treating chronic wounds.

Key words: Dressing • Nitric oxide • Patch • Probiotic • Wound

Authors: M Jones, MD, Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering andPhysiology, Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B4, Canada;JG Ganopolsky, PhD, Micropharma Limited, UQAM, Biological Sciences Building, Montreal, Quebec H2X 3Y7, Canada; A Labbe, PhD,Micropharma Limited, UQAM, Biological Sciences Building, Montreal, Quebec H2X 3Y7, Canada; M Gilardino, MD, Division of Plasticand Reconstructive Surgery, McGill University Health Centre Montreal Children’s Hospital, Montreal, Quebec H3H 1P3, Canada; C Wahl,MD, Micropharma Limited, UQAM, Biological Sciences Building, Montreal, Quebec H2X 3Y7, Canada; C Martoni, PhD, MicropharmaLimited, UQAM, Biological Sciences Building, Montreal, Quebec H2X 3Y7, Canada; S Prakash, PhD, Biomedical Technology and CellTherapy Research Laboratory, Department of Biomedical Engineering and Physiology, Artificial Cells and Organs Research Centre, Facultyof Medicine, McGill University, Montreal, Quebec H3A 2B4, CanadaAddress for correspondence: Professor S Prakash, PhD, Biomedical Eng., Faculty of Medicine, McGill University, 3775 UniversityStreet, Montreal, Quebec H3A 2B4, CanadaE-mail: [email protected]

© 2012 The Authors330 © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc

Novel nitric oxide producing probiotic wound healing patch

INTRODUCTIONThe treatment of chronic wounds poses a sig-nificant challenge for clinicians and patientsalike. Chronic wound healing is often slow orstagnant, causing prolonged patient morbidity.The prevalence of unfavourable wound envi-ronments, such as ischaemia, venous stasis orinfection further hinders successful treatmentof these difficult wounds. In addition, severalpathological healing conditions, including dia-betes and venous stasis, are associated withdysregulation of wound healing mediatorswhich disrupts wound healing and facilitatesthe formation of chronic wounds (1–3).

Key Points

• this study shows for the firsttime, the efficacious and safeuse of a gNO-producing pro-biotic patch for ischaemic andinfected wound healing

• specifically, this study describesthe design of a probiotic patch,its fabrication, in vitro testingand investigates the potentialof a gNO-producing probioticpatch on healing of ischaemicand/or infected full-thicknesswounds in New Zealand whiterabbits

Currently used methods such as the use oftopical antibiotics, as well as other antimicro-bial agents such as colloidal silver polymyxinsand dye compounds have become increasinglyineffective against common pathogens. In fact,recent reviews support the well-accepted factthat there is a worldwide increase in drug-resistant strains of bacteria since the introduc-tion of antimicrobial agents (4–7). Among thesebacteria are methicillin-resistant Staphylococ-cus aureus, vancomycin-intermediate resistantS. aureus, glycopeptide-intermediate S. aureusand community-acquired Enterococcus faeciumand Pseudomonas aeruginosa. Thus, as thecommon antimicrobial agents begin to failalternative treatments which do not rely onconventional antibiotics will be required.

Reduced local and regional circulationis another problem when treating infectedchronic wounds with systemic antibiotics (8).Patients with venous stasis ulcers have venousthrombosis, reduced circulation and poorregional blood flow and patients with diabeticfoot ulcers suffer from poor microcirculationdue to endothelial dysfunction and impairedblood flow and pressure in the microvascu-lature (9–11). Topical applications are oftenmore effective at concentrating the antimicro-bial agent at the wound site. However, theseantimicrobials are often less effective at elimi-nating infection because of reduced circulation,or the ability of bacteria to form biofilmsthat restrict the delivery and efficacy of theantibiotics (8,12). Thus, traditional therapiesfrequently leave infected wounds untreatedand a patient’s limb and life in danger.

Nitric oxide (NO) is a biological media-tor of wound healing that acts as a sig-nalling molecule in endothelial, neuronal andimmunological cells and is involved in many

phases of wound healing including inflam-mation, angiogenesis, collagen deposition,re-epithelialisation and collagen rearrange-ment (1,13). During the inflammatory phase,NO acts as a chemoattractant for monocytesand neutrophils, and stimulates the produc-tion of multiple proinflammatory cytokinessuch as IL-1 and TGF-β (14). Throughout re-epithelialisation NO regulates other chemoat-tractants, such as RANTES, and is importantfor the proliferation of keratinocytes (14). Dur-ing the later stages of wound healing, NO actsin collagen deposition and in the activity ofmatrix metalloproteinase (MMP) involved inthe rearrangement and maturation of the newlydeposited collagen at the wound site (14–17).

It has recently been shown that topical expo-sure of NO gas (gNO) to wounds such aschronic non healing ulcers can be beneficial inpromoting healing and preparing the woundbed for treatment and recovery (18). Applica-tion of exogenous gas has been shown to reducemicrobial infection downregulating inflamma-tion, manage exudates and secretion, upregu-late expression of endogenous collagenase todebride the wound and regulate the formationof collagen (18). Furthermore, treatment regi-mens have been proposed for chronic woundswhich specify high and low gNO treatmentperiods to first reduce the microbial burden andconsequent inflammation, increase collagenaseexpression in a process of debridement andthen restore the balance of NO which inducescollagen expression and aids in wound clo-sure (18). The gNO delivery device, however,used cumbersome and expensive componentsand required that the patient be tethered andremain non ambulatory during the durationof therapy (18). Other methods for deliver-ing gNO have been explored and includeadsorbing NO to polymers (19), zeolite (20) orsilica particles (21,22) requiring vast amountsof purified gNO under pressure, or deliveringexogenous nitrosothiols (GSNO) (23) whichcan be expensive and is limited by shelf life(GSNO is thermally and photolytically liable).These methods are each able to deliver someNO; however, the level is often not physio-logically relevant or the length of gNO releaseis inadequate. For this reason we are propos-ing a probiotic bacteria-based system for theproduction of NO so that the control of gNOproduction relies on the metabolism of bacteriathat can be sustained for the longer durations

© 2012 The Authors© 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc 331


required for treatment of chronic wounds bothin a hospital and community setting.

Here we report design of a novel probioticpatch for treating ischaemic and infected full-thickness dermal wounds and investigation ofthe preclinical efficacy in an experimental NewZealand white rabbit animal model of chronicwound healing.

MATERIALS AND METHODSNO producing probiotic patch designThe gNO-producing dressings were designedusing two Tegaderm™ adhesive patches (3MCanada, London, Ontario, Canada), one in con-tact with the skin and the other acting as atop layer of the device (Figure 1), as describedin our previous study (19). The active ingre-dients of the dressing, namely lyophilisedalginate microbeads containing Lactobacillusfermentum 7230 sourced from NCIMB (Bucks-burn, Aberdeen, Scotland) and a solutioncontaining glucose (10% w/v), NaCl (0·85%w/v) and sodium nitrite (30 mM), were placedin a heat sealed pocket made between theTegaderm™ in contact with the skin and alayer of polyethylene (Figure 1) (19). Controlpatches were constructed similarly, but wereprepared with glucose (10% w/v), NaCl (0·85%w/v) and no sodium nitrite. All chemicalsused were of analytical grade and were pro-cured from Sigma-Aldrich (Oakville, Ontario,Canada). The active area of the patch was asquare of 5 cm × 5 cm (25 cm2).

AnimalsMale New Zealand white rabbits (1·5–2·5 kg)were obtained from Charles River Laboratories

(Wilmington, MA). The animals were housedat the UQAM animal centre in individual cageswith food and water ad libitum. The animalswere kept on a 12-h light–dark cycle and wereleft to acclimatise for 4 weeks prior to surgery.

Surgical proceduresThe animals were sedated with glycopyrrolate(0·1 mg/kg SC) and anaesthesia was startedwith ketamine (35 mg/kg IM) and xylazine(5 mg/kg IM). Once asleep, the anaesthesiawas maintained with 4–5% isofluorane and theanimals were monitored for heart and breatherates. All anaesthetics used were of analyticalgrade and were procured from Sigma-Aldrich.The surgical procedures for the induction ofischaemia and the creation of full-thicknesswounds were based on a modified methodpublished by Chien et al. (24). Briefly, theventral side of each ear was shaved as wellas the areas at the base of the left ear, wherethe incisions were made for the induction ofischaemia. The surgical areas were wiped withethanol and the rabbits were placed on a heatedpad during the duration of the procedure.

Three vertical incisions were made about1 cm from the base of the ear, closely locatedto the central, cranial and caudal bundles(composed of the artery, vein and nerves). Thecentral artery was surgically divided from thenerve and veins. The central artery was ligatedwith 5-0 silk while the venous circulationwas preserved. The entire cranial bundle wasligated with 5-0 silk. The caudal bundle wasidentified and separated from surroundingtissues, but left untouched. After disruptionof the arteries, a subcutaneous tunnel was

L. fermentum

Glucose → Lactic Acid

keq

3 NO2- + 3 H+ ↔ 2 NO + H2O + NO3

-

A

B

Figure 1. (A) Top: chemistry for novel nitric oxide gas (gNO)-producing probiotic patch in which gNO is generated from nitrite andprotons produced by Lactobacillus fermentum NCIMB 7230 bacteria from glucose. (B) Bottom: cross section of novel gNO-producingprobiotic patch design. From the outermost to innermost layers, the patch is composed of (a) adhesive non occlusive Tegaderm™,(b) polyethylene/nylon laminated film, (c) immobilised bacteria L. fermentum NCIMB 7230 and a solution containing 10% glucose,30 mM NaNO2 and 0·85% NaCl and (d) a layer of adhesive non occlusive Tegaderm™.



created between the three incisions. Using asharp pair of scissors, the tissues under theskin were cut and removed to expose cartilageall around the base of the ear, working throughthe three incisions. Once the disruption wascomplete, any bleeding was controlled byapplying pressure with a sterile gauze padbefore closing the incisions using 4-0 nylonsutures.

Four full-thickness wounds were created onthe ventral side of the ears using a sterile sharp6 mm biopsy punch. Very light pressure wasapplied to avoid breaking through the thincartilage of the ear. The wounds were createdon the upper portion of the rabbit’s ears, toallow easy access and to maintain the woundson a flat surface. Once the skin was cut, itwas removed from the cartilage and specialcare was taken to remove the perichondrialmembrane and expose the cartilage. Followingthe creation of the wounds, the ears werecovered with sterile gauze. Cotton was usedto pack the ventral side of the ear. Gauzebandages were used to wrap the ears andwere immobilised with surgical tape. Thewrapping was kept relatively tight to preventthe loss of the dressing but not so tight asto limit blood flow in the non ischaemiccontrol ear. Elizabethan collars were also fittedto the rabbits to prevent damage causedby the rabbits scratching their wounds. Aninjection of antibiotics (enrofloxacin, Baytril�,5 mg/kg IM) procured from Bayer Corporation(Toronto, Ontario, Canada), was administeredintraoperatively, to reduce the risk of systemicinfections. A Fentanyl patch (25 μg/hours) wasapplied for 3 days following surgery on ashaved area located 10 cm distally from thebase of the skull, on the rabbits back.

InfectionExperimental rabbits were infected withS. aureus to assess the effect of gNO-producingpatches on infected wounds. An overnightculture of S. aureus was diluted to approxi-mately 1 × 106 CFU/ml in saline (0·85% NaCl)and applied with sterile cotton tipped appli-cators to the four wounds on each rabbit earfor 10 minutes before the control or treatmentpatches were applied. Wound bacterial countswere determined by plating serial dilutionsof the wound swab samples in Tryptic SoyAgar after an overnight incubation at 37◦C aspreviously described (25).

Wound treatmentStarting a day after surgery, the woundswere treated with control or gNO-producingpatches designed to produce gNO levelsof approximately 200 ppmV for 24 hours(Figure 2). Only one patch per ear was sufficientto cover all four wounds. The patches werereplaced daily after gently wiping the woundsusing sterile gauze. The wounds were dressedas described at the end of the surgicalprocedure. The treatment was maintained for20 or 21 days, irrespective of wound closure.

Wounds of rabbits 1 and 2 were not infected.Wounds on rabbits 3 and 4 were infected withS. aureus. Wounds of rabbits 1 and 3 weretreated with vehicle control patches; woundsof rabbits 2 and 4 were treated with gNO-producing patches.

Data collectionWound healing was monitored daily and pho-tographic records were kept for morphometricanalysis. Starting on the day of surgery, pho-tographs were taken of the wounds on each

Figure 2. Nitric oxide gas (gNO) production by gNO-producing probiotic patch containing 2·6 ml of glucose (10% w/v), sodiumnitrite (30 mM) and 0·5 g lyophilised alginate microbeads containing 20% w/v immobilised Lactobacillus fermentum NCIMB 7230by weight. gNO was detected using a Chemilumiescent Nitric Oxide Analyser (Sievers�, GE) in a custom designed high-densitypolyethylene chamber.



ear. Morphometric analysis was performedby measuring the wound areas from dailyphotographs of wounds treated with gNO-producing or vehicle control patches for eachexperimental condition.

Euthanasia and sample collectionRabbits were euthanised under general anaes-thesia using ketamine (35 mg/kg IM) andxylazine (5 mg/kg IM) followed by inhalationof isoflurane (5% in 1 l/minutes O2). Cardiacpuncture was performed until complete exsan-guination. Blood was collected in EDTA collec-tion tubes for haematological analysis and inglass tubes for serum collection after clotting.Urine samples were also collected directly fromthe bladder after death of the animals. The earswere removed and the four wounds on each earwere cut out and placed in buffered formalinfor histopathological analysis.

HistopathologyHistopathology was performed on all thewounds. Tissue sections were left to fix forat least 24 hours in formalin. The sampleswere bisected, placed in cassettes and pro-cessed to paraffin. Sections were sectioned atapproximately 5 μm, mounted on glass slidesand stained with haematoxylin and eosin andMasson’s trichrome stains. Fixation, mounting,staining and analysis of the stained sampleswere performed by blinded pathologists usinga semi-quantitative grading system. Inflamma-tion was graded from 0 (absent or very fewinflammatory cells) to 3 (abundant inflamma-tory cells). Maturity was graded based on theorganisation and quantity of granulation tis-sue. A grade of 0 indicates the absence ofgranulation tissue, while a score of 4 indi-cates a dense granulation tissue with bundlesof mature dermal-type collagen.

HaematologyHaematological analysis was performed withan ADVIA 120 analyser. The following param-eters were evaluated: red blood cell counts,haemoglobin, haematocrit, mean corpuscu-lar volume, mean corpuscular haemoglobin,mean corpuscular haemoglobin concentration,platelet count, white blood cells (WBC), WBCdifferential counts, cell morphology and reticu-locyte count. Blood smears were also preparedto evaluate morphology.

Blood chemistrySerum samples were analysed for sodium(Na+), potassium (K+), chloride (Cl−), bicar-bonate (HCO3

−), creatinine, urea, glucose,calcium (Ca2+), phosphate (PO4

3−), aspartateaminotransferase, alanine aminotransferase,lipase and C-reactive protein using a Hitachi911 blood clinical chemistry analyser (RocheDiagnostics, Laval, Quebec, Canada). Plasmanitrate and nitrite were determined by chemi-luminescence using a Sievers Nitric OxideAnalyser (GE Analytical Instruments, Boulder,CO) as described earlier (26).

Methaemoglobin measurementsBlood samples were haemolysed with distilledwater and phosphate buffer pH 7·0. Percent-age of blood methaemoglobin in the sampleswere determined according to a modified Eve-lyn–Mallow method (16). The assay is based onthe fact that haemoglobin can be converted tomethaemoglobin after reacting with potassiumferricyanide and that the resulting absorbancemaximum at 632 nm disappears after addi-tion of sodium cyanide. Briefly, OD632 ofhaemolysed blood was measured to determinethe concentration of methaemoglobin. Totalhaemoglobin was determined by OD632 afteraddition of potassium ferricyanide to haemol-ysed blood. The baseline correction values weredetermined by subtracting OD632 values ofboth solutions after the addition of sodiumcyanide. The percentage of methaemoglobinwas calculated as the quotient of the correctedOD632 values.

Statistical analysisStatistical analysis of wound morphomet-ric data was performed by repeated mea-sures ANOVA with mixed models (SAS ver-sion 9·2, Cary, NC). Statistical analysis ofcomplete wound closure was performed byKaplan–Meier curves and Cox proportionalhazard regression analysis. Kaplan–Meiercurves (survival curves) express the likeli-hood of survival over time. Here the statisticalmethod was used as a representation of thelikelihood of wound closure over time. Thesedata were plotted using time-to-closure ofeach wound separately, on a Kaplan–Meiergraph and statistical analysis was performedusing each of the variables present in thestudy: time-to-closure, treatment, ischaemia



N

I

I

NI

N

N

I

I

NI

N

Figure 3. Photographs of infected full-thickness dermal wounds on ears that are either ischaemic ‘I’ or non ischaemic ‘N’ andtreated with nitric oxide gas-producing probiotic patches or treated with vehicle control patches at days 1, 13 and 20 post-surgery.Wound healing was monitored daily and photographic records were kept for computer-aided morphometric analysis.

and infection. The Cox proportional hazardmodel is a statistical method used to deter-mine the multiplicative effect on a subject’shazard rate. The hazard ratio represents theincrease (or decrease) in the likelihood of anevent occurring (wound closure). Data anal-ysis was performed using Epi info™ from theCenter for Disease Control (CDC, Atlanta, GA).

RESULTS AND DISCUSSIONNO producing probiotic patch designFigure 1 shows the chemistry of the gNO-producing probiotic device (top) and depictsthe patch design (bottom). The release of gNOwas evaluated in three representative patchesover a 24-hour period and was measured bychemiluminescence (Sievers�, GE) (Figure 2).There was no gNO detected for control patches(not shown). The patches released a gNO doseof 88·5 nmoles/cm2/hours.

Photographic evaluationand morphometric analysis of woundstreated with gNO-producing probioticpatchesIn general, non infected wounds appeareddry for the duration of the trial, whereasinfected wounds presented purulent exudates.Photographs of infected, ischaemic and nonischaemic wounds, treated with either gNO-producing probiotic patches or vehicle control

patches taken at days 1, 13 and 20 are presented(Figure 3). Non ischaemic wounds closedbetween 13 and 15 days post-surgery, indepen-dent of infection, as previously reported (24).

The efficacy of gNO-producing probioticpatches versus vehicle control patches wasdetermined by morphometric analysis ofwound area under four experimental con-ditions: (i) ischaemic non infected wounds(Figure 4, upper left); (ii) ischaemic infectedwounds (Figure 4, upper right); (iii) nonischaemic non infected wounds (Figure 4,lower left) and (iv) non ischaemic infectedwounds (Figure 4, lower right). Repeated mea-sures analysis of the entire data set, across allexperimental conditions (n = 32), showed thattreatment resulted in a significant decrease inwound area over the duration of this study(P = 0·0001), that the presence of ischaemia sig-nificantly slowed wound closure (P = 0·0009)and that the presence of infection did notsignificantly affect the healing process (P =0·2578). The statistics for ischaemic woundsonly (n = 16) showed that treatment withgNO-producing patches resulted in a signif-icant decrease in wound area over the durationof this study (P < 0·0001). There was also asignificant decrease in wound area for treatedischaemic non infected wounds (P = 0·0006)(Figure 4, upper left) and treated ischaemicinfected wounds (P = 0·015) (Figure 4, upper



A B

C D

Figure 4. Effect of treatment with a nitric oxide gas (gNO)-producing probiotic patch compared with a vehicle control patchunder four experimental conditions as seen by daily morphometric analysis: (A) ischaemic, non infected wounds treated with activegNO-producing probiotic patches versus vehicle control patches; (B) ischaemic, infected wounds treated with active gNO-producingprobiotic patches versus vehicle control patches; (C) non ischaemic, non infected wounds treated with active gNO-producing probioticpatches versus vehicle control patches and (D) non ischaemic, infected wounds treated with active gNO-producing probiotic patchesversus vehicle control is shown. Error bars represent one standard deviation from the mean.

right) compared with the respective vehiclecontrol-treated wounds. Finally, treatment ofnon ischaemic non infected wounds with gNO-producing patches did not significantly accel-erate the rate of healing (P = 0·3539) (Figure 4,lower left) and treatment of non ischaemicinfected wounds did not significantly accel-erate the rate of wound healing (P = 0·1988,through day 11) (Figure 4, lower right) whencompared with treatment with vehicle controlpatches.

Histopathological analysis of woundstreated with NO producing probioticpatchThe histological results show a trend towardsimproved wound healing in tissues of thegNO-producing probiotic patch-treated ani-mals when compared with those treatedwith vehicle control patches. Table 1 showshistological analysis of ischaemic wounds on

ears of rabbits treated with gNO-producingprobiotic patches or those treated with vehi-cle control patches. The results show a trendtowards improved wound healing as evi-denced by less surface depression, less crustingand exudates, less inflammation/infiltration,greater epithelial coverage, reduced hyperpla-sia, greater dermal thickness and improvedoverall epidermal and dermal maturity. Theresults are averages of gNO-producing patch-treated (n = 16) and vehicle control patch-treated wounds (n = 16). In almost every case,there was improvement in the average score(defined in materials and methods) givento histological observations consistent withimproved wound healing (Table 1). At thewound surface less depression was observed(mean grade of 0·14 ± 1·68 versus −1·00 ± 1·77)and less crusting and exudates was observed(mean grade of 0·5 ± 1·07 versus 1·63 ± 1·41).The epidermal layer showed improved mean



Table 1 Histological analysis

Control (n = 16) Treatment (n = 16) Improvement

Wound surface Wound width (microscopic fields at 4× magnification) 1·00 ± 0·27 1·08 ± 0·19 N/CRaised (+)/depressed (−) (−3 to 3) −1·00 ± 1·77 0·14 ± 1·68 ImprovedCentral protrusion (0–3) 0·13 ± 0·35 0·57 ± 0·98Crusting/exudates (0–3) 1·63 ± 1·41 0·5 ± 1·07 Improved

Epidermis Coverage (estimated %) 79·4 ± 29·8 87·5 ± 31·5 ImprovedHyperplasia (multiple of normal epidermal thickness) 2·63 ± 0·74 2·29 ± 0·76 ImprovedMaturity (1–4) 2·38 ± 0·91 3·13 ± 0·64 Improved

Granulationtissue/dermis

Thickness (multiple of normal dermal thickness) 0·84 ± 0·76 1·13 ± 0·64 Improved

Inflammation/infiltration (0–3) 2·38 ± 0·74 2·13 ± 0·83 ImprovedMaturity (0–4) 1·13 ± 0·83 1·88 ± 0·99 Improved

coverage (87·5 ± 31·5% versus 79·4 ± 29·8%),less hyperplasia (mean of 2·29 ± 0·76 versus2·63 ± 0·74 in multiples of epidermal thickness)and improved epidermal maturity (mean scoreof 3·13 ± 0·64 versus 2·38 ± 0·91) for gNO-treated wounds compared with vehicle con-trols. The granulation tissue and dermal layershowed increased thickness (1·13 ± 0·64 ver-sus 0·84 ± 0·76 multiples of dermal thickness),less inflammation and infiltration of inflamma-tory cells (2·13 ± 0·83 versus 2·38 ± 0·74) andimproved maturity (mean grade of 1·88 ± 0·99versus 1·13 ± 0·83) for the case of gNO-treated wounds compared with vehicle con-trols (Table 1). Figure 5 shows representativeMasson’s trichrome stained sections for theevaluation of wound healing, inflammationand maturity.

While a larger sample size would berequired to show significance, these resultsshow improved scores for surface, epider-mal and dermal observations and show atendency towards improved wound healingin the gNO-producing probiotic patch-treatedwounds. In addition, these histological resultsprovide growing support for the hypothesisthat improved wound healing and maturitybecause of the topical application of gNOmay be due to increased collagen deposition,increased stimulation of endothelial progen-itor cells, stimulation of neovascularisation,endothelial cell relaxation and improved ker-atinocyte migration (1,27).

It has been showed that while elevated MMPactivities in wound fluids impair endogenouslyproduced, or exogenously applied, growthfactors from acting as mitogenic agents, NOmay circumvent this difficulty by acting as

a mitogenic agent much the same way asa protein growth factor (KGF, VEGF, FGF,PDGF) (28). In fact, it is believed that NO maybe indistinguishable from protein-like growthfactors, with respect to modulating mitogenicresponse of cells to external stimuli, and mayact to improve wound healing by promotingproliferation of cells in a wound (28). Fur-thermore, it has been shown that epithelialcell migration (29), wound angiogenesis (30)and granulation tissue formation (31), criticalprocesses in wound repair and new tissue for-mation, are mediated by NO. First attemptshave been made using polymeric NONOates,NO-donating agents and free NO gas tosupplement NO during wound healing andthese have shown promising results in woundrepair (2,19,23,32,33). Thus, it is believed thatNO deficiency in chronic non healing ulcerscan be restored improving: (i) antimicrobialactivity; (ii) fibroblast-regulated collagen depo-sition; (iii) angiogenesis by endothelial cellprogenitor proliferation and (iv) keratinocyteproliferation and migration, circumventing ele-vated MMP levels and/or low tissue inhibitorsof MMPs levels and aiding in wound closure.

Safety of NO producing probiotic patchDetermination of the safety and toxicity pro-file of the gNO-producing probiotic patcheswas performed by blood analysis. Table 2shows safety and toxicological data of thegNO-producing probiotic patch-treated andvehicle control patch-treated animals. Non sig-nificant differences were observed betweengNO-producing probiotic patch-treated andvehicle control patch-treated animals in bodyweight, blood morphology, haematology,



A B

C D

Figure 5. Representative Masson’s trichrome stained sections for the evaluation of wound healing, inflammation and maturity byprobiotic patch. (A) Section showing with no granulation tissue and almost absent staining for collagen (grade 1). (B) Shows loosegranulation tissue with low collagen (grade 2). (C) Dense granulation tissue with a high concentration of disorganised collagen (grade3). (D) Dense granulation tissue with bundles of dermal-type collagen (grade 4).

blood chemistry or methaemoglobin levels.Analysis of biochemical markers of safetyshowed that serum samples of gNO-treatedand vehicle control-treated animals werewithin normal ranges except for elevatedpotassium in serum samples for rabbit 3(9·79 mmol/l) and rabbit 4 (11·44 mmol/l)(treated and control, infected rabbits). Theseanimals were found to be mildly hyperkalemicwhich was thought to be the result of either acompensated metabolic acidosis, due to infec-tion and/or poor perfusion, or haemolysisof blood samples prior to centrifugation andserum collection.

Results of a complete haematologic pro-file showed treated and non treated animalsto be comparable (Table 2). One non gNO-treated animal was found to be leucopenicwhich may have resulted from the stress fromthe surgical procedure and daily manipula-tion, or may simply be a statistical outlier;however, no abnormal values were believed to

be associated with gNO-producing probioticpatch treatment.

One possible consequence of gNO absorp-tion is an increase in the blood level ofmethaemoglobin which is a haemoglobinmolecule in which the iron is oxidised fromFe2+ to Fe3+ (16). Analysis of levels ofmethaemoglobin, however, showed normallevels in both the gNO-treated (0·1 ± 0·1%) andvehicle control-treated rabbits (0·3 ± 0·2%),indicating that the gNO-induced formationof methaemoglobin was undetectable or thatclearance was adequate.

Plasma nitrate and nitrite levels did notpresent differences between treated and nontreated rabbits as previously reported (33).These observations are in agreement withmethaemoglobin results and confirm that thetopical delivery of gNO did not alter systemicconcentrations of nitrates and nitrites.

Thus, the results of blood analysis for safetyand toxicity markers corroborate reports that



Table 2 Safety and toxicological data∗

Rabbit 1 Rabbit 3 Rabbit 2 Rabbit 4 Finding

Body weight (kg) −0·1 −0·3 −0·1 −0 No differenceBlood morphology Normal Normal Normal Normal No differenceHaematology White blood cell (×109/l) 6·16 1·54 7·81 4·66

Red blood cell (×1012/l) 5·91 6·10 6·07 5·98Hemoglobin (g/l) 122 121 119 122Hematocrit (l/l) 0·35 0·34 0·35 0·36Mean corpuscular volume (fl) 59·9 55·2 57·7 60·3Mean corpuscular haemoglobin (pg) 20·6 19·8 19·7 20·5Mean corpuscular haemoglobin concentration (g/l) 344 359 341 339Platelets (×109/l) 315 444 247 583Neut (×109/l) 1·85 0·38 1·52 1·32Lymp (×109/l) 3·69 1·07 5·75 2·98Mono (×109/l) 0·06 0·01 0·07 0·05Eos (×109/l) 0·11 0·03 0·15 0·09Luc (×109/l) 0·01 0·00 0·01 0·00Baso (×109/l) 0·43 0·05 0·31 0·22Retic (×1012/l) 0·211 0·086 0·163 0·137

Blood chemistry Na+ (mmol/l) 146·8 146·3 148 148Electrolytes K+ (mmol/l) 5·79 9·79 5·55 11·44

Cl− (mmol/l) 101·5 102·9 107·5 109·2HCO−

3 (mmol/l) 34·13 30·86 27·52 22·76Kidney Creat (μmol/l) 126·84 162·61 144·78 127·26

Urea (mmol/l) 6·99 6·17 7·15 6·57Endocrine Glucose (mmol/l) 13·73 11·22 12·56 14·74

Ca2+ (mmol/l) 3·29 3 3·26 3·16PO4− (mmol/l) 2·11 2·06 2·74 1·97

Liver Alanine aminotransferase (U/l) 50·83 31·01 28·71 32·12Aspartate aminotransferase (U/l) 32·89 33·3 54·95 19·32

Pancreas Lipase (U/l) 190·59 158·6 148·37 194·28Inflammatory C-reactive protein (nmmol/l) 0·14 1·88 −0·38 1·34Methaemoglobin 0·3 ± 0·2% 0·1 ± 0·1% Normal levels

∗Bolded values are those outside of the normal range.

the topical application and inhalation of gNOis non toxic and safe (34).

Wound microbial loadViabilities from wound swab samples weredetermined for infected wounds that weretreated the gNO-releasing probiotic patchesor controls. Whereas the total viability inthe wound swab samples from the controlswas 3 × 106 CFU, in the gNO-treated woundswab samples the viability was less than1 × 106 CFU. These results are in agreementwith the observations previously reported (33).

Kaplan–Meier curve and Coxproportional hazard regressionWhile improved wound healing (decreasedwound area) over time was easily quantifiable,and successfully showed by morphometricanalysis, the definitive endpoint for efficacy in

chronic wound healing is complete wound clo-sure (35). Thus, log-rank and Wilcoxon statis-tics for Kaplan–Meier survival probability plotsand Cox proportional hazard regression analy-ses were performed to determine the increasedor decreased likelihood of complete closure.

A Cox proportional hazard survival regres-sion analysis was performed for all wounds(n = 32) for the variables: treatment, ischaemiaand infection, and for ischaemic wounds(n = 16) for the variables: treatment and infec-tion. Table 3 shows Cox proportional hazardsurvival regression analysis for all wounds(n = 32) and for ischaemic wounds (n = 16)under the conditions: treatment, ischaemiaand infection. Hazard ratios represent anincrease (or decrease) in the likelihood ofan event occurring (wound closure). Thesedata were analysed using EpiInfo software(CDC). The hazard ratio represents the increaseor decrease in the likelihood of an event



Table 3 Cox proportional hazard survival regression analysisfor all wounds∗

Variable Hazard ratio P value

All wounds Treatment (n = 32) 2·45 0·053Ischaemia (n = 16) 0·21 0·0039Infection (n = 16) 0·68 0·42

Ischaemic Treatment (n = 8) 17·95 0·030Infection (n = 8) 0·067 0·037

∗Bolded values indicates a significant difference.

occurring (wound closure). Analysis of allwounds showed a 2·45-fold increase in thelikelihood of closure for treated wounds versuscontrol (P = 0·053) and that ischaemic woundswere 0·21 times less likely to close (P =0·0039). Analysis of ischaemic wounds only(n = 16, 8 treated and 8 non treated) showeda 17·95-fold increase in the likelihood ofclosure for treated wounds versus control (P =0·030), and that infected ischaemic woundswere 0·068 times less likely to close (P =0·037) compared with non infected ischaemicwounds.

The likelihood of wound closure over timewas evaluated in wounds treated with gNO-producing probiotic patches versus woundstreated with vehicle control patches by

a Cox proportional hazard regression plotindependent of covariables (Figure 6). Theanalysis shows that gNO-treated wounds were2·52 times more likely to close than vehiclecontrol (hazard P = 0·038, score P = 0·032 andlikelihood ratio P = 0·036).

The likelihood of wound closure over timewas evaluated in wounds treated with gNO-producing probiotic patches versus woundstreated with vehicle control patches by aKaplan–Meier plot and calculating the log-rank and Wilcoxon statistics (Figure 7). Theanalysis shows that gNO-treated woundsclosed before vehicle control (log-rank P =0·023 and Wilcoxon P = 0·011, 16 wounds pergroup) and that several of the control-treatedwounds remained open.

The likelihood of wound closure overtime was evaluated for ischaemic and nonischaemic wounds (Figure 8). Results showthat ischaemia significantly delays wound clo-sure in this animal model (log-rank P = 0·005and Wilcoxon P = 0·0056, 16 wounds pergroup). Analysing only the infected ischaemicwounds (n = 8, 4 wounds per group) the sameanalysis shows a significant improvement inwound closing for the gNO-treated groupsas compared with the vehicle controls despite

Figure 6. Cox proportional hazard regression plot investigating likelihood (survival probability) of wound closure over time in nitricoxide gas (gNO)-producing patch-treated wounds versus wounds treated with vehicle control patches independent of covariables.These data show that gNO-producing probiotic patch-treated wounds are 2·52 times more likely to close compared with woundstreated with vehicle control patches (hazard P = 0·038, score P = 0·032 and likelihood ratio P = 0·036) and that several nontreated wounds remained open. The light line represents gNO-producing probiotic patch-treated wounds (16 wounds) and the darkgrey line represents wounds treated with vehicle control patches (16 wounds). These data were graphed using EpiInfo software(Center for Disease Control).



Figure 7. Kaplan–Meier plot showing the likelihood (survival probability) of wound closure in wounds treated with nitric oxidegas (gNO)-producing probiotic patches versus wounds treated with vehicle control patches. These data show that gNO-producingprobiotic patch-treated wounds closed before wounds treated with vehicle control patches (log-rank P = 0·0223 and WilcoxonP = 0·011) and that several non treated wounds remained open. The light grey line is gNO-producing probiotic patch-treatedwounds (16 wounds) and the dark grey line represents vehicle control-treated wounds (16 wounds). These data were graphed usingEpiInfo software (Center for Disease Control).

Figure 8. Kaplan–Meier plot showing the likelihood (survival probability) of wound closure over time for ischaemic wounds versusnon ischaemic wounds. These data show that ischaemia delays wound closure (log-rank P = 0·005 and Wilcoxon P = 0·0056) andthat some ischaemic wounds remain open at 21 days. The light grey line represents ischaemic wounds (16 wounds), whereas thedark grey line represents non ischaemic (16 wounds). These data were graphed using EpiInfo software (Center for Disease Control).

the number of wounds (log-rank P = 0·04 andWilcoxon P = 0·046).

Although, morphometric analysis did notshow a significant effect of gNO on woundhealing in infected and non infected nonischaemic wounds, a non significant improve-ment was seen in treated ischaemic and non

ischaemic infected wounds, whereas no suchtrend was evident in non infected wounds.Thus, while morphometric analysis shows thatischaemia plays a role in the pathophysiologyof delayed wound healing, and that gNO-producing probiotic patches can be used toaccelerate wound closure in infected or non



infected ischaemic wounds, only a promis-ing trend exists for treating non ischaemicinfected wounds with gNO. However, basedon the wide spectrum antimicrobial activity ofNO(36), the proven antimicrobial and antifun-gal activity of this gNO-producing probioticpatch (37), and the known pathologic action ofbacterial biofilms in slowing wound closure,a real difference may be apparent in a largerpopulation of wounds.

A total of 32 wounds have been evaluatedin this study, including four rabbits. Eventhough the sample size may be limited,the data analysis is robust and allowed todetect significant differences between gNO-treated and control groups in some cases, asdescribed. This statistical analysis corroboratesresults seen in analysis of morphometricdata and strengthens the hypothesis that thepathophysiology of non healing ischaemicwounds is closely tied to a dysregulationof endogenous NO production and that theexogenous application of gNO may have atherapeutic effect (13,17,19,23).

CONCLUSIONSThis study shows for the first time, the effica-cious and safe use of a gNO-producing probi-otic patch for ischaemic and infected woundhealing. Specifically, this study describes thedesign of a probiotic patch, its fabrication,in vitro testing and investigates the poten-tial of a gNO-producing probiotic patch onhealing of ischaemic and/or infected full-thickness wounds in New Zealand whiterabbits.

The results show that wound healing, asdetermined by morphometric analysis, wassignificantly accelerated in gNO-treated, noninfected and infected ischaemic wounds. Theseresults were corroborated by analysis of accel-erated complete wound closure (Kaplan–Meierand Cox plots). Analysis of all wounds showedthat gNO treatment significantly improved thelikelihood of wound closure and that gNO-treated ischaemic wounds were significantlymore likely to close than vehicle control-treated ischaemic wounds. Finally, histologicalanalysis showed improved wound healing intissues of gNO-treated wounds and safety anal-ysis showed no abnormal haematologic andbiochemical markers of safety associated withgNO treatment.

While several groups are working on gNO-releasing wound healing devices, the barriersto commercial use (sustained gNO release,shelf life, ease of use and cost) have notyet been overcome (18–23). The gNO-releasingprobiotic patch was also efficacious at reducingthe wound bacterial load without alteringthe systemic levels of nitrates, which is inagreement with a previous study in whichrabbits with infected dorsal wounds weretreated with 200 ppmV gNO delivered by atank and regulator (33).

This study shows that gNO-producing pro-biotic patches can be designed and used for top-ical applications, including treating ischaemicand infected full-thickness dermal wounds thatapproximate chronic non healing ulcers, andthat gNO-producing probiotic patches mayprovide an elegant solution to the obstacles tocommercial development. This study also pro-vides further evidence that the dysregulationof gNO may be intimately associated with thepathophysiology of chronic ischaemic woundsand that exogenous application can acceleratewound healing. Human clinical efficacy is yetto be evaluated.

ACKNOWLEDGEMENTSWe gratefully acknowledge financial supportfrom the Industrial Research Assistance Pro-gramme (IRAP) of the National Research Coun-cil (NRC) of Canada and research grant fromMicropharma Limited. Please note that SatyaPrakash and Mitchell Jones have conflict; theyare inventor of the technology mentionedin this research that has been patented byMicropharma. None of the other authors haveany personal or financial conflict of interest.

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