A Multinational, Multicenter,Randomized, Double-Blinded,Placebo-Controlled Trial toEvaluate the Efficacy of CyclicalTopical Wound Oxygen (TWO2)Therapy in the Treatment ofChronic Diabetic Foot Ulcers: TheTWO2 StudyDiabetes Care 2020;43:616–624 | https://doi.org/10.2337/dc19-0476

OBJECTIVE

Topical oxygen has been used for the treatment of chronic wounds for more than50 years. Its effectiveness remains disputed due to the limited number of robusthigh-quality investigations. The aim of this study was to assess the efficacy ofmultimodality cyclical pressure Topical Wound Oxygen (TWO2) home care therapyin healing refractory diabetic foot ulcers (DFUs) that had failed tohealwith standardof care (SOC) alone.

RESEARCH DESIGN AND METHODS

Patients with diabetes and chronic DFUs were randomized (double-blind) to eitheractive TWO2 therapy or sham control therapydboth in addition to optimal SOC.The primary outcome was the percentage of ulcers in each group achieving 100%healing at 12 weeks. A group sequential design was used for the study with threepredetermined analyses and hard stopping rules once 73, 146, and ultimately220 patients completed the 12-week treatment phase.

RESULTS

At thefirst analysispoint, theactiveTWO2armwas foundtobesuperior to the shamarm, with a closure rate of 41.7% comparedwith 13.5%. This difference in outcomeproduced an odds ratio (OR) of 4.57 (97.8% CI 1.19, 17.57), P 5 0.010. Afteradjustment for University of Texas Classification (UTC) ulcer grade, the OR increasedto 6.00 (97.8% CI 1.44, 24.93), P5 0.004. Cox proportional hazards modeling, alsoafter adjustment for UTC grade, demonstrated >4.5 times the likelihood to healDFUs over 12weeks comparedwith the shamarmwith a hazard ratio of 4.66 (97.8%CI 1.36, 15.98), P5 0.004. At 12 months postenrollment, 56% of active arm ulcerswere closed compared with 27% of the sham arm ulcers (P 5 0.013).

CONCLUSIONS

This sham-controlled, double-blind randomized controlled trial demonstrates that,at both 12 weeks and 12 months, adjunctive cyclical pressurized TWO2 therapywas superior in healing chronic DFUs compared with optimal SOC alone.

1Diabetic Foot Consultants, Midwestern Univer-sity, Glendale, AZ2Centre for Research and Implementation ofClinical Practice, London, U.K.3King’s College Hospital, London, U.K.4McGuire Veterans Affairs Medical Center, Rich-mond, VA5Montpellier University Hospital, Montpellier,France6Medical Center Dessau, Brandenburg MedicalSchool Theodor Fontane, Dessau, Germany7Edward Hines Jr. VA Hospital, Chicago, IL8Salem Veterans Affairs Medical Center, Salem,VA9Washington DC Veterans Affairs MedicalCenter, Washington, DC10Hopital Avicenne and Paris 13 University,Bobigny, France11Advanced Foot & Ankle Center, Las Vegas, NV12Hopital Simone Veil, Eaubonne, Paris, France13Hopitaux Robert Schuman - Hopital Kirchberg,Luxembourg City, Luxembourg14Northwick Park Hospital, London, U.K.

Corresponding author: Robert G. Frykberg, rgfdpm@diabeticfoot.net

Received 8March 2019 andaccepted3 September2019

Clinical trial reg. no. NCT02326337, clinicaltrials.gov.

This article contains Supplementary Data online athttps://care.diabetesjournals.org/lookup/suppl/doi:10.2337/dc19-0476/-/DC1.

*A complete list of the TWO2 Study Groupcollaborators, Steering Committee, and DataMonitoring Committee can be found in theSupplementary Data online.

This article is featured in a podcast available athttps://www.diabetesjournals.org/content/diabetes-core-update-podcasts.

© 2019 by the American Diabetes Association.Readers may use this article as long as the workis properly cited, the use is educational and notfor profit, and the work is not altered. More infor-mation is availableathttps://www.diabetesjournals.org/content/license.

See accompanying article, p. 515.

Robert G. Frykberg,1 Peter J. Franks,2

Michael Edmonds,3 Jonathan N. Brantley,4

Luc Teot,5 Thomas Wild,6

Matthew G. Garoufalis,7 Aliza M. Lee,8

Janette A. Thompson,9 Gerard Reach,10

Cyaandi R. Dove,11 Karim Lachgar,12

Dirk Grotemeyer,13 and Sophie C. Renton,14

on behalf of the TWO2 Study Group*

616 Diabetes Care Volume 43, March 2020

PATH

OPHYSIOLO

GY/COMPLICATIONS

With the growing worldwide prevalenceof diabetes there has been a resultantincrease in the incidence of diabetic footulcerations (DFUs) with attendant mor-bidity, mortality, and health care costs(1–3). Common diabetes comorbiditiesincluding peripheral neuropathy, defor-mity, and peripheral arterial disease(PAD) are among a number of well-established risk factors for DFUs (2,4).These person-level conditions whencombined with numerous underlyingcellular or metabolic and ulcer-relatedfactors (hypoxia, inflammation, biobur-den, etc.) will quite frequently lead toimpaired wound healing and to possibleamputation (5,6).Over the last decade it has become

clear that basic standards of care for DFUsmandate rigorous attention to proper de-bridement and off-loading (7–9). While anumber of new adjunctive therapies havebecome available, including growth fac-tors, cellular and acellular tissues, topicalnegative pressure, oxygen therapies, etc.,most therapies suffer from inadequatelydesigned or nongeneralizable studies thatcannot attest to their efficacy, safety, andcost-benefit (1,10,11).Oxygen is an essential component in

the wound-healing cascade. Energy me-tabolism (ATPsynthesis), reactiveoxygenspecies generation, redox signaling, H2O2

production, antioxidant generation, col-lagen synthesis, deposition of extracel-lular matrix, VEGF gene expression, andangiogenesis are among processes de-pendent on a sufficient supply of oxygenfor their activities (12–15).Hyperbaric oxygen therapy (HBOT)has

been studied extensively for its efficacyin healing DFUs and amputation preven-tion, but despite several recent random-ized clinical trials, the results remaininconsistent regarding its effectivenessin healing DFUs (10,16–19). Topical ox-ygen therapies (TOTs), used in clinicalpractice for .50 years, supply oxygendirectly to the hypoxic wound surfacewithout the potential complicationsposed by HBOT (13,15,20,21). Despitelong-standing clinical evidence support-ing the effectiveness of topically appliedoxygen for chronic wounds, hyperbaricoxygen proponents have raised concernsabout such benefits without systemichyperoxygenation (22).To study the effect of topically admin-

istered oxygen on cutaneous wounds,Fries et al. (23) conducted a controlled

porcine dermal wound-healing experi-ment. They found that topical oxygenincreased the wound tissue partial pres-sure of oxygen (PO2) levels 10-fold after4 min and that repeated treatments ac-celerated wound closure compared withcontrol (air-exposed) wounds. Histologicalexamination showed a stronger presenceof VEGF, signs of improved angiogenesis,andmore advanced remodeling with bet-ter quality collagen. Their findings sug-gest several biological mechanisms forthe enhanced healing found in othertopical oxygen studies.While numerousreports have similarly suggested the po-tential benefits of topical oxygen in heal-ing chronic wounds, its effectiveness inhealing DFUs remains disputed due to acombination of poorly designed studies,inconsistent results, and the paucity of ro-bust investigations through randomizedcontrolled clinical trials (RCTs) (15,24–26).

In recognition of the need for morerigorous studies of this therapy, a ran-domized, double-blinded, sham-controlledclinical trial was designed to explore theefficacy of cyclical pressurized TopicalWound Oxygen (TWO2) therapy in heal-ing refractory DFUs that had failedto heal with optimal standard of care(SOC) alone. We herein present theresults of the TWO2 diabetic foot ulcerstudy.

RESEARCH DESIGN AND METHODS

Study DesignThe TWO2 study was designed as a pro-spective, multinational, multicenter, double-blinded, placebo-controlled, randomizedclinical trial with 17 diabetic foot centersparticipating across the U.S., U.K., France,Germany, and Luxembourg. The protocolwas approved by the governing institu-tional review or local ethics board of eachof the participating centers throughoutthe U.S. and Europe. The study was per-formed in accordance with the Declarationof Helsinki and Good Clinical Practiceguidelines of the International ConferenceonHarmonization.Written informed con-sent was provided by all participants priorto performance of study procedures. Anindependent data monitoring committeeand a study steering committee wereestablished to monitor the conduct andanalysis of the study.

Sample Size and Design RationaleLimited information was available onRCTs looking at the efficacy of cyclical

pressurized topical oxygen for healingDFUs. Aburto and Frye (27), in a ran-domized study of topical oxygen, dem-onstrated better healing in DFU patientsafter 90 days (90% vs. 40%) comparedwith the control group. Blackman et al.(20) enrolled 28 patients with DFUs andobtained a similar result (82.4% vs.45.5%). In combining the results of thesetwo studies, the control group achieved ahealing rate of 9 of 21 (42.8%), and in theactive group healing occurred in 23 of27 (85.2%). Using these figures, we wouldanticipate a tentative expected controlrate of 43%, and it was proposed that aconservative estimate of difference be-tween groups would be half that experi-enced in these trials at 21%. In orderto address the unknown outcomes, weused a group sequential design withthree predetermined analysis points.With three analyses, the level of signif-icance needed to be adjusted tomaintainthe integrity of the analysis. The Pocockstopping boundary method requires amore stringent P value threshold (P ,0.022) at eachof the three analysis pointsto achieve an overall probability of P ,0.05 at the final evaluation. For achieve-ment of a minimal level of significancebetween study arms, it was calculatedthat 110 patients would be required ineach study arm (n5 220). The resultantanalyses would therefore be performedafter one-third (73), two-thirds (146),and finally all (220) enrolled patientscompleted the active phase of the study.Since analysis would be exclusively of theintention-to-treat (ITT) cohort, all pa-tients would be analyzed as per the12-week primary end point (healed vs.unhealed). Furthermore, no up-rating ofthis sample size was made to take intoconsideration patients lost to follow-up.

PatientsInclusion criteria for participation in thetrial were as follows: patients withtype 1 or 2 diabetes with nonhealing,full-thickness, University of Texas Clas-sification (UTC) grade 1 or 2 DFUmeasuring $1 cm2 and ,20 cm2 post-debridement. All ulcers included wereto be between 4 weeks and 1 year induration and to have been receiving stan-dard care for at least 4 weeks. Patientswith modest limb ischemia were per-mitted with an ankle brachial index(ABI).0.7. To account for falsely elevatedABI measurements (7), we performed

care.diabetesjournals.org Frykberg and Associates 617

a secondary confirmatory measurementof distal perfusion adjacent to or distalto the index ulcer in all patients, in-cluding a transcutaneous oxygen pres-sure (TcPO2) .30 mmHg, skin perfusionpressure .30 mmHg, toe pressure.30 mmHg, or a Duplex ultrasoundshowing biphasic waveforms belowthe knee. Detailed study enrollment cri-teria can be found in Table 1.

RandomizationPatients were randomly assigned in a1:1 ratio double blinded to either theSOC plus sham therapy (SC1Sham) armor to an SOC plus active TWO2 therapy(SC1TWO2) arm. The randomization listof 220 codes in A or B format wasgenerated by the blinded statisticianusing a random permuted block design,with blocks of 2, 4, 6, and 8. Study armallocation was randomly assigned by acentralized study coordinator for eachpatient at the randomization visit.

InterventionsAll patients were recruited as outpatientsin participating wound care centers.At the screening visit and afterobtaining informed consent, the pa-tient’s wound was sharply debridedand digitally photographed. All patientswere then provided with the same studyfoam dressings and hydrogel (Kendall;

Covidien), instructions, and the study off-loading device (Optima Diab; Salvatellisrl, Civitanova Marche, Italy). After arun-in period of 2 weeks, patients re-turned for their randomization visit. Onlyif the wound area reduction was ,30%were patients subsequently random-ized double-blind into either the active(SC1TWO2) or sham (SC1Sham) studyarm.

The U.S. Food and Drug Administration–cleared, CE-marked TWO2 therapydevice (HyperBox; AOTI Ltd., Galway,Ireland) operates by inflation of a single-use extremity chamber over the pa-tient’s limb; then, humidified oxygenis cycled between 10 mb and 50 mbwithin the chamber. A 10 liters perminute oxygen concentrator was usedto provide the oxygen supply ratherthan oxygen cylinders.

Both the active and sham deviceslooked and operated identically. How-ever, the sham device did not deliverpressurized oxygen into the extremitychamber, even though values displayedon the device controls looked as ifthis was being performed. The shamtreatment therefore consisted only ofunrestricted nonpressurized ambientroom air in the nonocclusive extremitychamber.

Delivery, installation, and training onthe use of the blinded study device wasperformed by blinded home equipment

providers. No study-related proceduresor treatments were provided by theserepresentatives. Patients treated them-selves at home for 90 min daily fivetimes per week with either the allo-cated TWO2 or sham therapy. Dressingchanges were performed at home byeither the patient or their personal care-giver. No study therapy was done at thestudy centers.

Patients visited a local study centerweekly for the duration of the study forwound assessment, debridement, anddigital wound photographs. Patients re-corded therapy and off-loading compli-ance daily on diary cards that wereverified at each study visit. Additionally,therapy hours were verified by the TWO2device itself. The active treatment phasewas continued until the ulcer healed orfor a maximum of 12 weeks.

Data Collection and OutcomeMeasuresThe treatment phase of the study was12 weeks. The randomization visit mea-surement after debridement served asthe index (baseline) measurement. If mul-tiple ulcers were present, the largest areaulcer at the baseline visit was designatedthe index ulcer. Weekly digital wound im-ages were transmitted electronically andwere assessed for area changes and clo-sure confirmation by a single blinded cen-tral assessor using automated CE-marked

Table 1—Inclusion/exclusion criteria

Inclusion criteria Exclusion criteria

Males and females aged between 18 and 89 years Evidence of gangrene on any part of affected limb

Documented diagnosis of type 1 or 2 diabetes Documented evidence of osteomyelitis on any part of affected limb

Foot ulcer at or below ankle with duration .4 weeks to ,1 year Index ulcer has exposed bonec If the index ulcer is postamputation, date of surgery mustbe .30 days

Active Charcot foot on the study limb

c If.1 ulcer is present, largest is considered as the study index ulcerUncontrolled diabetes: HbA1c .12% (108 mmol/mol)

c Index ulcermust be$1 cm fromany other ulcers present on the footRenal dialysis or creatinine .2.5 mg/dL (221 mmol/L)

Ulcer size$1 and#20 cm2 after debridement at start of run-in period Known immune insufficiency

Ulcer of UTC grade 1A, 1B, 1C, 1D, 2A, 2B, 2C, or 2D Active treatment for malignancy (not specific to study limb)

ABI .0.7 with a TcPO2 .30 mmHg, skin perfusion .30 mmHg, toepressure.30 mmHg, or Duplex ultrasound with biphasic waveformsbelow the knee

Chronic steroid use or immunosuppressive agents within the last3 months or anticipated to require them during the duration of thestudy

No planned revascularization procedure or vascular surgery within thelast or next 30 days

Subject participated in another investigational device, drug, or biologicaltrial within last 30 days

Subject and caregiver willing and able to comply with all specified careand visit requirements

Index ulcer exhibits signs of severe clinical infection that requireshospitalization or immediate surgical intervention

Subject has a reasonable expectation of completing the study Subject is pregnant at the time of screening

Subject completed 2-week run-in period with ,30% wound sizereduction

Subject has had a deep vein thrombosis within the last 30 daysSubject has received growth factor therapy, autologous platelet-rich

plasma gel, bilayered cell therapy, dermal substitute, extracellularmatrix, etc., within the screening period

618 TWO2 Study Diabetes Care Volume 43, March 2020

wound measurement software (MOWA;Healthpath srl, Rome, Italy).Onceawoundwas initially determined

to be closed by the blinded study siteinvestigator, that visit served as the firstof two confirmatory visits. Wound clo-sure (complete epithelialization) wasconfirmed at the second closure visit 2weeks later (28). Upon completion of the12-week treatment phase, patientsentered the posttreatment follow-upperiod for an additional 38 weeks,whereby they returned for wound clo-sure assessment and quality of life(QOL) questionnaires.The maximum duration for participa-

tion in the study was 54 weeks. Duringthe follow-up phase, patients withouthealed ulcers received standard careaccording to their clinician’s recommen-dation and were asked not to participatein another wound care trial.The primary study end point was

the percentage of ulcers in eachgroup achieving 100% healing at 12weeks. Secondary end points includedwound area reduction, 12-month in-cidence of both recurrence and com-plete healing, incidence of amputation,Cardiff Wound Impact Schedule (CWIS)QOL assessment, and adverse events(1,28,29).

Statistical AnalysisAll analyses were performed solely on theITT study population using Stata 12 (Stata-Corp, College Station, TX). Results arereported to one decimal place; P valuesand SDs have been reported to twosignificant figures. For the primary endpoint of ulcers achieving 100% healingat 12 weeks, statistical significance wasassessed at the Pocock 2.2% level (P ,0.022). Logistic regression analysis wasused to determine the influence ofpossible confounding variables. Model di-agnostics were used to check regressionmodel assumptions and transformations ifthey did not hold. For this analysis, a back-ward elimination process was used incor-porating the following variables: age, sex,ulcer area, ulcer duration, presence ofneuropathy, UTC grade, and HbA1c (%).The same potential confounders were ex-amined within the Cox proportional haz-ards model. Confounders were includedin both models if they changed the oddsratio (OR) or hazard ratio (HR) by.10%.The final logistic regression model andlongitudinal hazard models included

97.8% CIs. For all other analyses, statis-tical significance was assessed at thetwo-sided 5% level (P, 0.05) with 95%CIs provided as appropriate. The stat-istician conducting all analyses wasblinded to treatment allocation (withgroups identified as A and B) untilresults had been finalized.

RESULTS

Between November 2014 and December2017, 136 patients were screened for thestudy. Of these, 63 patients (46%) wereexcluded from randomization for notmeeting the inclusion criteria. Thirty-four patients (25%) returned from the2-week run-in with wound size reduc-tions $30%, 10 (7%) had ABI values orsecond vascular assessments out ofrange, and 19 (14%) either were notwilling to comply fully with the protocolor had other laboratory values out ofrange. Therefore, 73 patients were ran-domized into the active phase of thestudy (see Fig. 1).

At baseline, 65 patients (89%) hadtype 2 diabetes and 8 patients (11%)had type 1 diabetes. Fourteen index ulcers(39%) in the active arm, comparedwith sixindex ulcers (16%) in the sham arm, wereassessed to beUTC grade 2 (penetrating totendon or capsule). Conversely, 22 ulcers(61%) in the active arm, compared with31 ulcers (84%) in the sham arm, wereassessed to be UTC grade 1 wounds (P50.038). Additionally, 10 patients (28%) inthe active arm, comparedwith 4 patients(11%) in the sham arm, had a previousdiagnosis of PAD (P5 0.066). Seventeenpatients (47%) in the active arm had ahistory of prior amputations on the indexlimb in contrast to eight (22%) in the shamarm (P 5 0.018) (see Table 2).

Primary OutcomeAt the first ITT analysis point of 73 pa-tients, the independent data monitoringcommittee recommended that enroll-ment should conclude per the predeter-mined stopping rules, as the active arm

Figure 1—CONSORT diagram of study flow. lab, laboratory; LTFU, lost to follow-up.

care.diabetesjournals.org Frykberg and Associates 619

was shown to be superior to the shamarm for the primary outcome. In the ac-tive arm 15 wounds (41.7%) completely

healed versus 5 wounds (13.5%) in thesham arm at 12 weeks [Pearson x25 7.27(1 df), P 5 0.007]. The difference in

outcome produced an OR of 4.57 (97.8%CI 1.19, 17.57), P 5 0.010. Examinationof the potential confounding by otherbaseline variables revealed that UTC ul-cer grade substantially changed the ORin favor of the TWO2 group (OR 5 6.00[97.8% CI 1.44, 24.93], P 5 0.004). Theactive TWO2 arm showed .3.5 timesthe likelihood to completely heal over12 weeks compared with the sham armwith anHR of 3.64 (97.8%CI 1.11, 11.94),P5 0.013.With inclusion of theUTC ulcergrade into the model, the HR increasedto 4.66 (97.8% CI 1.36, 15.98), P5 0.004.The Kaplan-Meier curve shown in Fig. 2clearly shows the separation betweengroups throughout the active phase ofthe study. The patients then entered intothe follow-up phase of the study wherethey were assessed for index ulcer re-currence, healing, and QOL changes for12 months postenrollment (see Table 3).

Secondary Outcome Measures

Ulcer Recurrence

At 12 months postenrollment, only 1 of15 healed ulcers (6.7%) in the activearm recurred, compared with 2 of5 healed ulcers (40%) in the shamarm, falling just short of statistical sig-nificance (P 5 0.070). In total, 20 (56%)active arm (SC1TWO2) ulcers wereclosed at 12 months postenrollmentcompared with 10 (27%) of the shamarm (SC1SHAM) ulcers [x2 (1 df)5 6.13,P 5 0.013].

Wound Area Reduction

Of the patients with open ulcers at theend of the 12-week active phase, themean (SD) absolute reduction in ulcerarea from baseline was 1.97 (2.75) cm2

for the active arm compared with 0.40(1.75) cm2 for the shamarm [t (df)52.12(35), P 5 0.041].

For the patients with larger openulcers .4 cm2 at the end of the activephase, the mean (SD) absolute reductionin ulcer area from baseline was 4.12(1.51) cm2 for the active arm comparedwith a 1.34 (1.18) cm2 increase for thesham arm [t (df) 5 2.85 (8), P 5 0.021].

QOL

The wound care–focused CWIS QOL in-dex improved during the study for pa-tients whose ulcers healed across allfunctional domains. This positive in-crease was observed in both full andpartial responders. The greatest im-provement was seen for the well-being

Table 2—Baseline characteristics

Sham TWO2(n 5 37)

Active TWO2(n 5 36)

Total(n 5 73) P

Age, years, mean (SD) 61.9 (9.5) 64.6 (10.3) 63.3 (9.9) 0.21

Sex, male, n (%) 31 (84) 32 (89) 63 (86) 0.53

Race, n (%)White/Hispanic 24 (65) 26 (72) 50 (68.5) 0.90*Black 5 (14) 5 (14) 10 (14)Asian 1 (2.7) 2 (5.6) 3 (4.1)American Indian 1 (2.7) 0 (0) 1 (1.4)Not reported 6 (16.2) 3 (8.3) 9 (12.3)

Type 2 diabetes, n (%) 33 (89) 32 (89) 65 (89) 0.97

BMI (kg/m2), mean (SD) 31.2 (7.6) 30.8 (5.9) 31 (6.8) 0.85

Wound area (cm2), mean (SD) 3.22 (2.54) 3.02 (2.66) 3.13 (2.57) 0.74

Wound perimeter (cm),mean (SD) 6.85 (4.18) 6.22 (2.85) 6.54 (3.55) 0.45

Ulcer duration (days),mean (SD) 174.6 (94) 160.3 (96) 166.4 (95) 0.53

Wound classification, n (%)UTC grade 1A 27 (73) 20 (56) 47 (64)UTC grade 1B 2 (5.4) 1 (2.8) 3 (4.1)UTC grade 1C 2 (5.4) 1 (2.8) 3 (4.1)UTC grade 2A 4 (10.8) 9 (25) 13 (17.8) 0.04**UTC grade 2B 0 (0) 1 (2.8) 1 (1.4)UTC grade 2C 2 (5.4) 4 (11.1) 6 (8.2)

Neuropathic foot, n (%) 29 (78) 28 (78) 57 (78) 0.95

Charcot deformity, n (%) 3 (8.1) 1 (2.8) 4 (5.4) 0.32

Ulcer location, n (%) 0.32Dorsal foot 5 (13.5) 8 (22.2) 13 (17.8)Leg below malleoli 4 (10.8) 1 (2.8) 5 (6.8)Pedal foot 22 (59.5) 18 (50) 40 (54.8)Toe 6 (16.2) 9 (25) 15 (20.5)

Previous history of lower-extremityamputation, n (%) 8 (21.6) 17 (47.2) 25 (34.3) 0.02

Comorbidities, n (%)Hypertension 30 (81) 28 (78) 58 (79) 0.73Cardiovascular disease 9 (24.3) 13 (36.1) 22 (30.1) 0.27PAD 4 (10.8) 10 (27.8) 14 (19.2) 0.07Venous disease 1 (2.7) 2 (5.6) 3 (4.1) 0.54Renal disease 6 (16.2) 10 (27.8) 16 (21.9) 0.23Neurologic disease 31 (83.8) 28 (77.8) 59 (80.8) 0.52Peripheral edema 1 (2.7) 3 (8.3) 4 (5.4) 0.29Hyperlipidemia 25 (67.6) 23 (63.9) 48 (65.8) 0.74

Smoker, n (%) 10 (27) 13 (36) 23 (31.5) 0.41

Peripheral arterial circulationparameters

Mean ABI (SD) 1.00 (0.23) 1.07 (0.23) 1.03 (0.23) 0.20Mean toe systolic blood

pressure (SD), mmHg 83.00 (32.75) 84.50 (30.55) 83.77 (30.63) 0.84

Blood work values, mean (SD)Prealbumin, mmol/L 4.29 (1.45) 4.44 (0.93) 4.36 (1.18) 0.61CRP, nmol/L 140 (173) 65.7 (96.2) 99.6 (139) 0.05Creatinine, mmol/L 105.2 (30.1) 113.2 (81.3) 108.7 (61) 0.57HbA1c, % 8.14 (1.49) 8.43 (1.75) 8.25 (1.64) 0.46HbA1c, mmol/mol 65 (16.3) 69 (19.1) 67 (17.9) 0.46

All comparisons are nonsignificant except for values in boldface type. *Due to low frequency ineach cell, white race was comparedwith all other races combined. **Due to low frequency in UTCcategories, UTC I was compared with UTC II.

620 TWO2 Study Diabetes Care Volume 43, March 2020

component, with mean (SD) score dif-ference between baseline and the endof 12-week treatment in the active armof 9.1 (13.9) compared with20.1 (16.9)in the sham arm [t (df) 5 2.18 (53),P 5 0.033].

TWO2 Therapy and Off-loading Compliance

Therapy compliance in both the activeand sham arms was high, with 94% and96% completing treatments, respectively.Off-loading device compliance in both theactive and sham arms was also high, with97% and 99% using the off-loading.75%of the time.

Adverse EventsDuring the study, there were equal num-bers of serious adverse events (10) andadverse events (8) experienced in bothstudy arms. Therewere no TWO2device–related adverse events reported. Twoindex limb amputations (5%) occurredin the active arm compared with threeindex limb amputations (8%) in the shamarm.

CONCLUSIONS

TOT has been reported to improve heal-ing of DFUs in several earlier prospec-tive randomized studies (20,27,30,31).However, these studies suffered frommethodological weaknesses, such as alack of blinding, uncontrolled SOC, or in-appropriate analyses of the ITTpopulations.

The present TWO2 study has demon-strated, in a randomized, sham-controlledtrial, that cyclical pressurized TOT ad-junctive to optimal SOC is significantlysuperior to standard care alone in heal-ing recalcitrant DFUs within a 12-weekhome-based treatment period. To thisend, trial enrollment was terminated atthe first predetermined analysis point,since the primary end point had beenachieved after the initial 73 randomizedpatients had completed their 12-weektreatment phase.

Despite the loss of 25% of patients inthe 2-week run-in period prior to random-ization, a four-and-a-half–fold increasedlikelihood of healing was achieved at12 weeks in patients allocated to theactive TWO2 therapy. With adjustmentfor UTC ulcer grade, this effect increasedeven further. A very high degree of com-pliance with treatment and off-loadingwas demonstrated in both groups. Clin-ically, the durability of healing as mea-sured by index ulcer recurrence at12 months was sixfold better than thatin the sham group and that seen in otherstudies (2). Of interest, anddistinct fromother topical oxygen studies, this RCTallowed for patients with up to UTCgrade 2 ulcers with modest degreesof ischemia. Although not statisticallysignificant, nearly 28% of patients ran-domized to the active therapy had

a prior history of PAD compared withjust 10% in the control group. How-ever, despite double-blinded random-ization, a significant 47% of activetherapy patients had a history of lower-extremity amputations compared withjust 22% in the sham arm.

This study is consistent with resultsreported in several previous studies us-ing topical oxygen in DFU (20,30–32)and venous leg ulcers (33,34), as well asanimal studies (23). Several other re-views of this approach have also sug-gested mechanisms of action andputative benefits of topically appliedoxygen in the management of chronicwounds (13,15,24,26). Blackman et al.(20), in a prospective open-label study,examined the clinical efficacy of TWO2therapy in healing DFU patients in acommunity wound care clinic. Patientswere allocated to topical oxygen or oth-erwise treated with advanced moistwound therapy. At 12 weeks, 82.4% ofthe ulcers in the TWO2 therapy arm and45.5% in the control arm healed com-pletely (P5 0.04). Median time to com-plete healing was 56 days in the activeand 93 days in the control arm (P 50.013). Another unblinded comparativestudy investigated the benefits of con-tinuous diffusion of oxygen comparedwith variable standard care for DFUs (31).Notwithstanding methodological weak-nesses, they found significantly fasterrates of healing in the topical oxygengroup compared with the standard caregroup andmost notably in deeper ulcers.A more recent randomized placebo-controlled trial using a continuous dif-fusion of oxygen device for only UTCgrade 1A ulcers reported a higher pro-portion of healed DFUs (32.4% vs.16.7%, P 5 0.033) and a faster timeto closure (P50.015) in theactivegroupat 12 weeks (30). This study was alsoplannedwith a group sequential design;however, their interim analysis end pointwas not met, and their ITT analysis didnot include 35% of randomized patientswho were subsequently removed fromthe trial.

Strengths and LimitationsThis TWO2 study followed the guidancefor wound-healing therapies put forth bythe U.S. Food and Drug Administration(28) as well as subsequent publicationsfrom leading authorities calling for morerobustly designed sham-controlled RCTs

Figure 2—Kaplan-Meier curve showing the separation between study groups throughout the12-week trial.

care.diabetesjournals.org Frykberg and Associates 621

(1,29,35). Nonetheless, and despite ran-domization of known and unknown po-tential confounders between groups, itdoes have limitations. One is the rela-tively small number of patients includedin the primary end point analysis of ourITT population, although the group wassimilar in size to those of other woundcare RCTs (2,36). In a group sequentialdesign study, predetermined hard stop-ping rules are put in place that in our case

were met at the first analysis point of73 patients. At that point, the primaryoutcome was achieved by finding signif-icantly more patients in the active grouphad healed compared with the sham-treated group (41.7% vs. 13.5%, P 50.007). This approach is used whenthe magnitude of the treatment effectis uncertain, as it allows for stopping atrial once a wide treatment effect isproven. This also ethically ensures that

patients are not further randomized to aninferior arm. In our study, a large marginof effect (68%) and relative performanceratio (309%) were achieved.

The quality of DFU studies is oftenmeasured by the results obtained inthe control groups. In our sham-treatedcontrol group, 13.5% of patients achievedcomplete ulcer healingwithin the 12-weekoutcome period. This rate is similar tothat of some studies and lower than

Table 3—Summary of the results: ITT analysis

Sham TWO2(n 5 37)

Active TWO2(n 5 36)

Pearson x2 or OR orHR (97.8% CI), P value

Primary outcomeUlcers completely healed at 12 weeks, n (%) 5 (13.5) 15 (41.7) x2 7.27 (1 df), P 5 0.007By randomized treatment group, univariate OR 4.57 (1.19, 17.57), P 5 0.010

HR 3.64 (1.11, 11.94), P 5 0.013After adjustment for UT grade OR 6.00 (1.44, 24.93), P 5 0.004

HR 4.66 (1.36, 15.98), P 5 0.004Margin of effect/relative performance 68%/309%

Secondary outcomesHealing durabilityUlcer recurrence at 12 months, n (%) 2 (40.0) 1 (6.7) P 5 0.070Ulcers closed at 12 months, n (%) 10 (27) 20 (56) P 5 0.013Margin of effect/relative performance 52%/207%

Healing trajectoriesAbsolute change in ulcer area over 12 weeks, cm2 0.40 (1.75) 1.97 (2.75) P 5 0.041Absolute change in ulcer area in ulcers .4 cm2 over 12 weeks, cm2 21.34 (1.18) 4.12 (1.51) P 5 0.021Time to complete wound closure, weeks 6.3 (1.9) 8.2 (4.2) P 5 0.350

QOLCWIS well-being improvement between baseline and week 12 20.1 (16.9) 9.1 (13.9) P 5 0.033CWIS social life improvement between baseline and week 12 4.1 (12.4) 7.9 (16.9) P 5 0.340CWIS physical symptom improvement between baseline and week 12 4.6 (11.8) 12.1 (23.2) P 5 0.130

Index limb amputations, n (%) 3 (8) 2 (5) P 5 0.668TWO2 therapy and off-loading complianceUsed TWO2 therapy device 5 days/week, 90 min/day, n (%) 35 (96) 34 (94) P 5 0.978Used off-loading device .75% of the time, n (%) 36 (99) 35 (97) P 5 0.984

Safety analysisIncidence of serious adverse events, n 10 10 P 5 0.943Wound infection 2 3Osteomyelitis 5 2Hypoglycemic event 1 0Urinary tract infection 0 2Significant necrotic tissue 1 0Cardiovascular event 0 1UTC grade 2 ulceration 0 1Severe maceration/dermatitis 1 0Pneumonia 0 1

Incidence of adverse events, n 8 8 P 5 0.950UTC grade 1 ulceration 0 3Ulcer decline 0 2Minor infection 1 1Minor osteomyelitis 0 1Minor necrotic tissue 1 0Cellulitis 1 0Swelling/edema 1 1Maceration 2 0Dermatitis 1 0Contusion 1 0

Incidence of adverse device events 0 0

Data are means (SD) unless otherwise indicated. Boldface type indicates significant differences.

622 TWO2 Study Diabetes Care Volume 43, March 2020

others (17,30,37,38). Interestingly, arecent topical oxygen RCT reported anactive group healing rate lower thanours at 32.4% and a similar controlhealing rate (30). For the more chroniculcers, their placebo arm healing ratedropped to 13.2%. Despite the largemargin of effect between our active andsham groups, we attribute our osten-sibly low sham healing rate to thechronicity of the ulcers, complexity ofthe patients, and the control of, ratherthan a failure of, SOC treatment. In thisregard, the average duration of ulcersenrolled in the trial was .5 months,with a nonsignificant 14-day longerduration in the control group. After the2-week run-in period, 25% of enrolledpatients were excluded from randomi-zation due to a reduction in woundarea $30%. The study off-loading device,itself proven to be as efficacious as goldstandard total contact casting (39), mayhave enabled progress toward healingthat excluded patients likely to heal withsuch standard care alone. This allowedonly patients with wounds more difficultto heal (true SOC failures) to be random-ized into this trial. Since there was a veryhigh degree of compliance with bothblinded treatments and off-loadingthroughout the study, we have no reasonto believe that the control group healingresult was due to any shortcoming in theSOC protocol.Our sham therapy itself provided noth-

ing more than nonpressurized room airthat was free to circulate within theextremity chamber. Room air cannotconceivably be detrimental to the controlpatients or have a negative impact onability to heal. Even at the 12-monthfollow-up evaluation point, long after theactive therapy had ended, there wasstill a clear separation between studygroups, with the sham control patientsachieving a healing rate of only 27%.Analysis for predictors of healing at12 weeks resulted only in the treatmenteffect and UTC ulcer grade being signif-icant. Furthermore, we found no differencein compliance with the therapy or off-loading between study groups. In the ab-sence of otherwise explanatory data toaccount for the control healing rate, weare left with our presumption that thoserandomized into the study had ulcers thatwere truly hard to heal and that the dif-ference in healing rates between active andshamgroupswas indeed a treatment effect.

The mean age of our study populationwas ;63 years old, which mirrors thatseen in other DFU studies. Eighty-sixpercent of our study patients weremen, likely resulting somewhat fromthe fact that one-half of the U.S. studysites were Veterans Affairs wound careclinics. Multiple studies have shownDFUs to be more prevalent in menthan women to a degree similar tothat seen in this RCT (4,10,38). Withno significant differences in covariatesseen between the two study groups,our findings support the premise thatthese results are generalizable to simi-larly afflicted patient populations.

ConclusionThe results of the TWO2 study demon-strate that cyclical pressurized TOT inconjunctionwith bothoptimal off-loadingand good standard wound care canheal significantly more DFUs at 12 weekscompared with optimal SOC alone. Infact, we found a .4.5-fold increasedlikelihood of healing within this timeperiod for our actively treated patients.This therapy was safe, without compli-cations, and provided more durable heal-ing for those who had wound closureduring active treatment. Uniquely, thetherapy has additional benefit in that itcan be administered by the patient athome without the expense and difficul-ties of daily travel to a specialized center.In contrast to recently reported systemicHBOT studies (16,18,40), this robustdouble-blinded, sham-controlled trialprovides evidence to support use of thisadjunctive cyclical pressurized TOT forchronic DFUs.

Acknowledgments. Theauthors thank all studypatients, clinic personnel, study coordinators,and colleagues who assisted with patientreferrals.Duality of Interest. This study was sponsored byAOTI Ltd. (Galway, Ireland). The sponsor incurredall costs for the study including all institutionalfees, monitoring, data warehousing, statisticalservices, and the provision of study devices andsupplies. R.G.F. received research support fromthesponsorduring theconductof thestudywhileemployed at the Phoenix VA Healthcare Systemand has received subsequent speaking honoraria.P.J.F.,M.E., J.N.B., L.T., T.W.,M.G.G., A.M.L., J.A.T.,G.R., C.R.D., K.L., D.G., and S.C.R. all receivedresearch support from the sponsor during thestudy. No other potential conflicts of interestrelevant to this article were reported.Aside from delivery and home setup of

study devices, no sponsor employee or agent

participated in any aspect of patient care orstudy treatments.Author Contributions. R.G.F. assisted with theconception, design, and analysis of the study andwrote the manuscript. P.J.F. provided the sta-tistical design, performed the analyses, andassisted with writing the manuscript. M.E.,J.N.B., L.T., T.W., M.G.G., A.M.L., J.A.T., G.R.,C.R.D., K.L., D.G., and S.C.R. contributed to thediscussion and critically reviewed and providededits to the manuscript. R.G.F. and P.J.F. are theguarantors of this work and, as such, had fullaccess to all the data in the study and takeresponsibility for the integrity of the data andthe accuracy of the data analysis.Prior Presentation. Parts of this study werepresented as a late-breaking abstract at the78th Scientific Sessions of the American Di-abetes Association, Orlando, FL, 22–26 June2018.

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