THE JOURNAL OF CLINICAL AND APPLIED RESEARCH AND EDUCATION

Diabetes Care

In This Issue of

Diabetes Care          By Max Bingham, PhD

 

Global Mechanisms Proposed for Cardioprotective Effects of SGLT2 Inhibitors

The beneficial effects of sodium–glucose cotransporter 2 inhibitors (SGLT2i) in type 2 diabetes are mostly attributed to their ability to enhance glucose excretion and lower hyperglycemia. But they can also promote positive cardiovascular outcomes. Less clear is quite how they manage to achieve the effects, and although many hypothetical mechanisms exist, they only partly explain what might be going on. Avogaro et al. (p. 501) attempt to bring the different strands of evidence to- gether and propose a hypothesis that suggests SGLT2i might modify the trajectory of cell responses to high glucose levels from one of defense to dormancy. They suggest this might be the mechanism that explains the cardiac and renal protective effects of SGLT2i treatments. On that basis they call for dedicated studies to test the hypothesis to ultimately gather the support needed for human studies. They explain that high blood glucose is effectively a toxic environment that likely shifts cell responses to a state of defense characterized by immune responses, anabolic metabolism, inflam- mation, adiposity, and also cardiovascular events. In contrast, they suggest that switching to a dormancy program would curtail many of these issues and that evidence suggests that SGLT2i may actually be able to force this switch—effectively explaining the positive cardiorenal outcomes of

the trials. They acknowledge that most of the cited evidence comes from animal studies but suggest that, together with the more limited human data, the evidence points towards SGLT2i having a dormancy effect at a cellular level. Commenting further, author Angelo Avogaro told us: “There is still a lot to be understood about what SGLT2i do to humans beyond their glycosuric effects. Many hypotheses have been proposed, but we found it fascinating that they may switch the milieu of the cells to a state similar to that observed in mammalian animals during hibernation. If this is the case, this evolutionary hypothesis should be rigorously tested in future studies.”

Oxygen Therapy Improves Diabetic Ulcer Wound Healing: RCT Data

Treating diabetic foot ulcers for 12 weeks with a topical wound oxygen therapy in addition to standard care increases the likelihood that they heal, according to Frykberg et al. (p. 616). Specifically, they found that the therapy resulted in a >4.5-fold increased likelihood of healing compared with placebo and notably could be administered at home by patients. The results come from a double-blind randomized controlled trial (RCT) that compared an oxygen treatment ap- proach (Topical Wound Oxygen [TWO2]) or placebo (circulating air) delivered via a device called a HyperBox (AOTI Ltd., Galway, Ireland). Both approaches were applied on top of standard care for wounds, which were long-standing and had not healed prior to the trial. The company-sponsored trial was stopped early (as planned) after the active treatment showed clear success in healing wounds compared with placebo. Seventy-three individuals had been enrolled up to that point.

The primary outcome was the percentage of ulcers achieving 100% healing at 12 weeks. The authors found that the active treatment had a closure rate of nearly 42%, while the placebo had a closure rate of 13.5%. This resulted in an odds ratio of ~4.5, which was statistically significant, and it increased to 6.0 once ulcer grade was accounted for. Additionally, more than half of ulcers were closed at 12 months after the active treatment but only about one-quarter following placebo. Quality of life measures also improved more following the active treatment. There were high compliance rates in both groups, and no device-related adverse events were experienced in either group. Commenting further, author Robert Frykberg told us: “We believe that in this rather robust double-blinded RCT we have clearly demonstrated the positive effects of cyclical, pressurized topical oxygen therapy in the healing of chronic diabetic foot ulcers. Accordingly, we now have the evidence required to recommend the use of this therapy as an adjunct to good standard care for the management of difficult-to-heal diabetic foot ulcers.”

Avogaro et al. Reinterpreting cardiorenal protection of renal sodium–glucose cotransporter 2 inhibitors via cellular life history reprogramming. Diabetes Care 2020;43:501–507

Kaplan-Meier curve showing the separation between placebo (SC

+ Sham) and active therapy (SC + TWO2) study groups throughout the 12-week trial. SC, standard care.

Frykberg et al. A multinational, multicenter, randomized, double- blinded, placebo-controlled

trial to evaluate the efficacy of cyclical Topical Wound Oxygen (TWO2) therapy in the treatment of chronic diabetic foot ulcers: the TWO2 study. Diabetes Care 2020;43:616–624

A Multinational, Multicenter, Randomized, Double-Blinded, Placebo-Controlled Trial to Evaluate the Efficacy of Cyclical Topical Wound Oxygen (TWO2) Therapy in the Treatment of Chronic Diabetic Foot Ulcers: The TWO2 Study

Robert G. Frykberg,¹ Peter J. Franks,² Michael Edmonds,³ Jonathan N. Brantley,⁴ Luc Te´ot,⁵ Thomas Wild,⁶

Matthew G. Garoufalis,⁷ Aliza M. Lee,⁸ Janette A. Thompson,⁹ Ge´rard Reach,¹⁰ Cyaandi R. Dove,¹¹ Karim Lachgar,¹² Dirk Grotemeyer,¹³ and Sophie C. Renton,¹⁴ on behalf of the TWO2 Study Group*

OBJECTIVE

Topical oxygen has been used for the treatment of chronic wounds for more than 50 years. Its effectiveness remains disputed due to the limited number of robust high-quality investigations. The aim of this study was to assess the efficacy of multimodality cyclical pressure Topical Wound Oxygen (TWO2) home care therapy in healing refractory diabetic foot ulcers (DFUs) that had failed to heal with standard of care (SOC) alone.

RESEARCH DESIGN AND METHODS

Patients with diabetes and chronic DFUs were randomized (double-blind) to either active 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 three predetermined analyses and hard stopping rules once 73, 146, and ultimately 220 patients completed the 12-week treatment phase.

RESULTS

At the first analysis point, the active TWO2 arm was found to be superior to the sham arm, with a closure rate of 41.7% compared with 13.5%. This difference in outcome produced an odds ratio (OR) of 4.57 (97.8% CI 1.19, 17.57), P 5 0.010. After adjustment for University of Texas Classification (UTC) ulcer grade, the OR increased to 6.00 (97.8% CI 1.44, 24.93), P 5 0.004. Cox proportional hazards modeling, also after adjustment for UTC grade, demonstrated >4.5 times the likelihood to heal DFUs over 12 weeks compared with the sham arm with a hazard ratio of 4.66 (97.8% CI 1.36, 15.98), P 5 0.004. At 12 months postenrollment, 56% of active arm ulcers were 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 therapy was superior in healing chronic DFUs compared with optimal SOC alone.

¹Diabetic Foot Consultants, Midwestern Univer- sity, Glendale, AZ

²Centre for Research and Implementation of Clinical Practice, London, U.K.

³King’s College Hospital, London, U.K.

⁴McGuire Veterans Affairs Medical Center, Richmond, VA

⁵Montpellier University Hospital, Montpellier, France

⁶Medical Center Dessau, Brandenburg Medical School Theodor Fontane, Dessau, Germany ⁷Edward Hines Jr. VA Hospital, Chicago, IL ⁸Salem Veterans Affairs Medical Center, Salem, VA

⁹Washington DC Veterans Affairs Medical Center, Washington, DC

¹⁰Hoˆpital Avicenne and Paris 13 University, Bobigny, France

¹¹Advanced Foot & Ankle Center, Las Vegas, NV ¹²Hoˆpital Simone Veil, Eaubonne, Paris, France ¹³Hoˆpitaux Robert Schuman – Hoˆpital Kirchberg,

Luxembourg City, Luxembourg

¹⁴Northwick Park Hospital, London, U.K.

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

Received 8 March 2019 andaccepted 3 September 2019

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

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

*A complete list of the TWO2 Study Group collaborators, Steering Committee, and Data Monitoring Committee can be found in the Supplementary Data online.

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

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

See accompanying article, p. 515.

With the growing worldwide prevalence of diabetes there has been a resultant increase in the incidence of diabetic foot ulcerations (DFUs) with attendant mor- bidity, mortality, and health care costs (1–3). Common diabetes comorbidities including 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 when combined with numerous underlying cellular or metabolic and ulcer-related factors (hypoxia, inflammation, biobur- den, etc.) will quite frequently lead to impaired wound healing and to possible amputation (5,6).

Over the last decade it has become clear that basic standards of care for DFUs mandate rigorous attention to proper de- bridement and off-loading (7–9). While a number of new adjunctive therapies have become available, including growth fac- tors, cellular and acellular tissues, topical negative pressure, oxygen therapies, etc., most therapies suffer from inadequately designed or nongeneralizable studies that cannot attest to their efficacy, safety, and cost-benefit (1,10,11).

Oxygen is an essential component in the wound-healing cascade. Energy me- tabolism (ATP synthesis), reactive oxygen species generation, redox signaling, H₂O₂ production, antioxidant generation, col- lagen synthesis, deposition of extracel- lular matrix, VEGF gene expression, and angiogenesis are among processes de- pendent on a sufficient supply of oxygen for their activities (12–15).

Hyperbaric oxygen therapy (HBOT) has been studied extensively for its efficacy in healing DFUs and amputation preven- tion, but despite several recent random- ized clinical trials, the results remain inconsistent regarding its effectiveness in healing DFUs (10,16–19). Topical ox- ygen therapies (TOTs), used in clinical practice for .50 years, supply oxygen directly to the hypoxic wound surface without the potential complications posed by HBOT (13,15,20,21). Despite long-standing clinical evidence support- ing the effectiveness of topically applied oxygen for chronic wounds, hyperbaric oxygen proponents have raised concerns about such benefits without systemic hyperoxygenation (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 oxygen increased the wound tissue partial pres- sure of oxygen (PO₂) levels 10-fold after 4 min and that repeated treatments ac- celerated wound closure compared with control (air-exposed) wounds. Histological examination showed a stronger presence of VEGF, signs of improved angiogenesis, and more advanced remodeling with bet- ter quality collagen. Their findings sug- gest several biological mechanisms for the enhanced healing found in other topical oxygen studies. While numerous reports have similarly suggested the po- tential benefits of topical oxygen in heal- ing chronic wounds, its effectiveness in healing DFUs remains disputed due to a combination of poorly designed studies, inconsistent results, and the paucity of ro- bust investigations through randomized controlled clinical trials (RCTs) (15,24–26). In recognition of the need for more rigorous studies of this therapy, a ran- domized, double-blinded, sham-controlled clinical trial was designed to explore the efficacy of cyclical pressurized Topical Wound Oxygen (TWO2) therapy in heal- ing refractory DFUs that had failed to heal with optimal standard of care (SOC) alone. We herein present the results of the TWO2 diabetic foot ulcer study.

RESEARCH DESIGN AND METHODS

Study Design

The TWO2 study was designed as a pro- spective, multinational, multicenter, double- blinded, placebo-controlled, randomized clinical trial with 17 diabetic foot centers participating across the U.S., U.K., France, Germany, and Luxembourg. The protocol was approved by the governing institu- tional review or local ethics board of each of the participating centers throughout the U.S. and Europe. The study was per- formed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines of the International Conference on Harmonization. Written informed con- sent was provided by all participants prior to performance of study procedures. An independent data monitoring committee and a study steering committee were established to monitor the conduct and analysis of the study.

Sample Size and Design Rationale Limited information was available on RCTs looking at the efficacy of cyclical pressurized topical oxygen for healing DFUs. Aburto and Frye (27), in a ran- domized study of topical oxygen, dem- onstrated better healing in DFU patients after 90 days (90% vs. 40%) compared with the control group. Blackman et al.

(20) enrolled 28 patients with DFUs and obtained a similar result (82.4% vs. 45.5%). In combining the results of these two studies, the control group achieved a healing rate of 9 of 21 (42.8%), and in the active group healing occurred in 23 of 27 (85.2%). Using these figures, we would anticipate a tentative expected control rate of 43%, and it was proposed that a conservative estimate of difference be- tween groups would be half that experi- enced in these trials at 21%. In order to address the unknown outcomes, we used a group sequential design with three predetermined analysis points. With three analyses, the level of signif- icance needed to be adjusted to maintain the integrity of the analysis. The Pocock stopping boundary method requires a more stringent P value threshold (P , 0.022) at each of the three analysis points to achieve an overall probability of P ,0.05 at the final evaluation. For achieve- ment of a minimal level of significance between study arms, it was calculated that 110 patients would be required in each study arm (n 5 220). The resultant analyses would therefore be performed after one-third (73), two-thirds (146), and finally all (220) enrolled patients completed the active phase of the study. Since analysis would be exclusively of the intention-to-treat (ITT) cohort, all pa- tients would be analyzed as per the 12-week primary end point (healed vs. unhealed). Furthermore, no up-rating of this sample size was made to take into consideration patients lost to follow-up.

Patients

Inclusion criteria for participation in the trial were as follows: patients with type 1 or 2 diabetes with nonhealing, full-thickness, University of Texas Clas- sification (UTC) grade 1 or 2 DFU measuring $1 cm² and ,20 cm² post- debridement. All ulcers included were to be between 4 weeks and 1 year in duration and to have been receiving stan- dard care for at least 4 weeks. Patients with modest limb ischemia were per- mitted with an ankle brachial index (ABI) .0.7. To account for falsely elevated ABI measurements (7), we performed a secondary confirmatory measurement of distal perfusion adjacent to or distal to the index ulcer in all patients, in- cluding a transcutaneous oxygen pres- sure (TcPO₂) .30 mmHg, skin perfusion pressure .30 mmHg, toe pressure .30 mmHg, or a Duplex ultrasound showing biphasic waveforms below the knee. Detailed study enrollment cri- teria can be found in Table 1.

Randomization

Patients were randomly assigned in a 1:1 ratio double blinded to either the SOC plus sham therapy (SC1Sham) arm or to an SOC plus active TWO2 therapy (SC1TWO2) arm. The randomization list of 220 codes in A or B format was generated by the blinded statistician using a random permuted block design, with blocks of 2, 4, 6, and 8. Study arm allocation was randomly assigned by a centralized study coordinator for each patient at the randomization visit.

Interventions

All patients were recruited as outpatients in participating wound care centers. At the screening visit and after obtaining informed consent, the pa- tient’s wound was sharply debrided and digitally photographed. All patients were then provided with the same study foam dressings and hydrogel (Kendall; Covidien), instructions, and the study off- loading device (Optima Diab; Salvatelli srl, Civitanova Marche, Italy). After a run-in period of 2 weeks, patients re- turned for their randomization visit. Only if the wound area reduction was ,30% were patients subsequently random- ized double-blind into either the active (SC1TWO2) or sham (SC1Sham) study arm.

The U.S. Food and Drug Administration– cleared, CE-marked TWO2 therapy device (HyperBox; AOTI Ltd., Galway, Ireland) operates by inflation of a single- use extremity chamber over the pa- tient’s limb; then, humidified oxygen is cycled between 10 mb and 50 mb within the chamber. A 10 liters per minute oxygen concentrator was used to provide the oxygen supply rather than oxygen cylinders.

Both the active and sham devices looked and operated identically. How- ever, the sham device did not deliver pressurized oxygen into the extremity chamber, even though values displayed on the device controls looked as if this was being performed. The sham treatment therefore consisted only of unrestricted nonpressurized ambient room air in the nonocclusive extremity chamber.

Delivery, installation, and training on the use of the blinded study device was performed by blinded home equipment providers. No study-related procedures or treatments were provided by these representatives. Patients treated them- selves at home for 90 min daily five times per week with either the allo- cated TWO2 or sham therapy. Dressing changes were performed at home by either the patient or their personal care- giver. No study therapy was done at the study centers.

Patients visited a local study center weekly for the duration of the study for wound assessment, debridement, and digital wound photographs. Patients re- corded therapy and off-loading compli- ance daily on diary cards that were verified at each study visit. Additionally, therapy hours were verified by the TWO2 device itself. The active treatment phase was continued until the ulcer healed or for a maximum of 12 weeks.

 

Data Collection and Outcome

Measures

The treatment phase of the study was 12 weeks. The randomization visit mea- surement after debridement served as the index (baseline) measurement. If mul- tiple ulcers were present, the largest area ulcer at the baseline visit was designated the index ulcer. Weekly digital wound im- ages were transmitted electronically and were assessed for area changes and clo- sure confirmation by a single blinded cen- tral assessor using automated CE-marked wound measurement software (MOWA; Healthpath srl, Rome, Italy).

Once a wound was initially determined to be closed by the blinded study site investigator, that visit served as the first of two confirmatory visits. Wound clo- sure (complete epithelialization) was confirmed at the second closure visit 2 weeks later (28). Upon completion of the 12-week treatment phase, patients entered the posttreatment follow-up period 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. During the follow-up phase, patients without healed ulcers received standard care according to their clinician’s recommen- dation and were asked not to participate in another wound care trial.

The primary study end point was the percentage of ulcers in each group achieving 100% healing at 12 weeks. Secondary end points included wound 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).

 

Table 1—Inclusion/exclusion criteria Inclusion criteria Males and females aged between 18 and 89 years	Exclusion criteria 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 • If the index ulcer is postamputation, date of surgery must be .30 days • If .1 ulcer is present, largest is considered as the study index ulcer • Index ulcer must be $1 cm from any other ulcers present on the foot	Index ulcer has exposed bone Active Charcot foot on the study limb Uncontrolled diabetes: HbA1c .12% (108 mmol/mol) Renal 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, toe pressure .30 mmHg, or Duplex ultrasound with biphasic waveforms below the knee	Chronic steroid use or immunosuppressive agents within the last 3 months or anticipated to require them during the duration of the study
No planned revascularization procedure or vascular surgery within the last or next 30 days	Subject participated in another investigational device, drug, or biological trial within last 30 days
Subject and caregiver willing and able to comply with all specified care and visit requirements	Index ulcer exhibits signs of severe clinical infection that requires hospitalization 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 size reduction	Subject has had a deep vein thrombosis within the last 30 days Subject has received growth factor therapy, autologous platelet-rich plasma gel, bilayered cell therapy, dermal substitute, extracellular matrix, etc., within the screening period

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

RESULTS

Between November 2014 and December 2017, 136 patients were screened for the study. Of these, 63 patients (46%) were excluded from randomization for not meeting the inclusion criteria. Thirty- four patients (25%) returned from the 2-week run-in with wound size reduc- tions $30%, 10 (7%) had ABI values or second vascular assessments out of range, and 19 (14%) either were not willing to comply fully with the protocol or had other laboratory values out of range. Therefore, 73 patients were ran- domized into the active phase of the study (see Fig. 1).

At baseline, 65 patients (89%) had type 2 diabetes and 8 patients (11%) had type 1 diabetes. Fourteen index ulcers (39%) in the active arm, compared with six index ulcers (16%) in the sham arm, were assessed to be UTC grade 2 (penetrating to tendon or capsule). Conversely, 22 ulcers (61%) in the active arm, compared with 31 ulcers (84%) in the sham arm, were assessed to be UTC grade 1 wounds (P 5 0.038). Additionally, 10 patients (28%) in the active arm, compared with 4 patients (11%) in the sham arm, had a previous diagnosis of PAD (P 5 0.066). Seventeen patients (47%) in the active arm had a history of prior amputations on the index limb in contrast to eight (22%) in the sham arm (P 5 0.018) (see Table 2).

Primary Outcome

At the first ITT analysis point of 73 pa- tients, the independent data monitoring committee recommended that enroll- ment should conclude per the predeter- mined stopping rules, as the active arm was shown to be superior to the sham arm for the primary outcome. In the ac- tive arm 15 wounds (41.7%) completely healed versus 5 wounds (13.5%) in the sham arm at 12 weeks [Pearson x² 5 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. Examination of the potential confounding by other baseline variables revealed that UTC ul- cer grade substantially changed the OR in favor of the TWO2 group (OR 5 6.00 [97.8% CI 1.44, 24.93], P 5 0.004). The active TWO2 arm showed .3.5 times the likelihood to completely heal over 12 weeks compared with the sham arm with an HR of 3.64 (97.8% CI 1.11, 11.94), P 5 0.013. With inclusion of the UTC ulcer grade into the model, the HR increased to 4.66 (97.8% CI 1.36, 15.98), P 5 0.004. The Kaplan-Meier curve shown in Fig. 2 clearly shows the separation between groups throughout the active phase of the study. The patients then entered into the follow-up phase of the study where they were assessed for index ulcer re- currence, healing, and QOL changes for 12 months post enrollment (see Table 3).

Table 2—Baseline characteristics Sham TWO2 (n 5 37) Age, years, mean (SD)                             61.9 (9.5)		Active TWO2 (n 5 36) 64.6 (10.3)	Total (n 5 73) 63.3 (9.9)	P 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 (%)				0.32
Dorsal 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-extremity amputation, n (%)	8 (21.6)	17 (47.2)	25 (34.3)	0.02
Comorbidities, n (%)
Hypertension	30 (81)	28 (78)	58 (79)	0.73
Cardiovascular disease	9 (24.3)	13 (36.1)	22 (30.1)	0.27
PAD	4 (10.8)	10 (27.8)	14 (19.2)	0.07
Venous disease	1 (2.7)	2 (5.6)	3 (4.1)	0.54
Renal disease	6 (16.2)	10 (27.8)	16 (21.9)	0.23
Neurologic disease	31 (83.8)	28 (77.8)	59 (80.8)	0.52
Peripheral edema	1 (2.7)	3 (8.3)	4 (5.4)	0.29
Hyperlipidemia	25 (67.6)	23 (63.9)	48 (65.8)	0.74
Smoker, n (%)	10 (27)	13 (36)	23 (31.5)	0.41
Peripheral arterial circulation parameters Mean ABI (SD) Mean toe systolic blood pressure (SD), mmHg	1.00 (0.23)	1.07 (0.23)	1.03 (0.23)	0.20
	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)       0.61 CRP, nmol/L                                         140 (173)          65.7 (96.2)          99.6 (139)       0.05 Creatinine, mmol/L                           105.2 (30.1)       113.2 (81.3)         108.7 (61)       0.57 HbA1c, %                                             8.14 (1.49)         8.43 (1.75)         8.25 (1.64)       0.46 HbA1c, 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 in each cell, white race was compared with all other races combined. **Due to low frequency in UTC categories, UTC I was compared with UTC II.

Secondary Outcome Measures

Ulcer Recurrence

At 12 months postenrollment, only 1 of 15 healed ulcers (6.7%) in the active arm recurred, compared with 2 of 5 healed ulcers (40%) in the sham arm, falling just short of statistical sig- nificance (P 5 0.070). In total, 20 (56%) active arm (SC1TWO2) ulcers were closed at 12 months postenrollment compared with 10 (27%) of the sham arm (SC1SHAM) ulcers [x² (1 df) 5 6.13, P 5 0.013].

Wound Area Reduction

Of the patients with open ulcers at the end of the 12-week active phase, the mean (SD) absolute reduction in ulcer area from baseline was 1.97 (2.75) cm² for the active arm compared with 0.40 (1.75) cm² for the sham arm [t (df) 52.12 (35), P 5 0.041].

For the patients with larger open ulcers .4 cm² at the end of the active phase, the mean (SD) absolute reduction in ulcer area from baseline was 4.12 (1.51) cm² for the active arm compared with a 1.34 (1.18) cm² increase for the sham 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 all functional domains. This positive in- crease was observed in both full and partial responders. The greatest im- provement was seen for the well-being component, with mean (SD) score dif- ference between baseline and the end of 12-week treatment in the active arm of 9.1 (13.9) compared with 20.1 (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 active and sham arms was high, with 94% and 96% completing treatments, respectively. Off-loading device compliance in both the active and sham arms was also high, with 97% and 99% using the off-loading .75% of the time.

Adverse

Events During the study, there were equal num- bers of serious adverse events (10) and adverse events (8) experienced in both study arms. There were no TWO2 device– related adverse events reported. Two index limb amputations (5%) occurred in the active arm compared with three index limb amputations (8%) in the sham arm.

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 from methodological weaknesses, such as a lack of blinding, uncontrolled SOC, or in- appropriate analyses of the ITT populations.

The present TWO2 study has demon- strated, in a randomized, sham-controlled trial, that cyclical pressurized TOT ad- junctive to optimal SOC is significantly superior to standard care alone in heal- ing recalcitrant DFUs within a 12-week home-based treatment period. To this end, trial enrollment was terminated at the first predetermined analysis point, since the primary end point had been achieved after the initial 73 randomized patients had completed their 12-week treatment phase.

Despite the loss of 25% of patients in the 2-week run-in period prior to random- ization, a four-and-a-half–fold increased likelihood of healing was achieved at 12 weeks in patients allocated to the active TWO2 therapy. With adjustment for UTC ulcer grade, this effect increased even further. A very high degree of com- pliance with treatment and off-loading was demonstrated in both groups. Clin- ically, the durability of healing as mea- sured by index ulcer recurrence at 12 months was sixfold better than that in the sham group and that seen in other studies (2). Of interest, and distinct from other topical oxygen studies, this RCT allowed for patients with up to UTC grade 2 ulcers with modest degrees of ischemia. Although not statistically significant, nearly 28% of patients ran- domized to the active therapy had a prior history of PAD compared with just 10% in the control group. How- ever, despite double-blinded random- ization, a significant 47% of active therapy patients had a history of lower- extremity amputations compared with just 22% in the sham arm.

This study is consistent with results reported in several previous studies us- ing topical oxygen in DFU (20,30–32) and venous leg ulcers (33,34), as well as animal studies (23). Several other re- views of this approach have also sug- gested mechanisms of action and putative benefits of topically applied oxygen in the management of chronic wounds (13,15,24,26). Blackman et al. (20), in a prospective open-label study, examined the clinical efficacy of TWO2 therapy in healing DFU patients in a community wound care clinic. Patients were allocated to topical oxygen or oth- erwise treated with advanced moist wound therapy. At 12 weeks, 82.4% of the ulcers in the TWO2 therapy arm and 45.5% in the control arm healed completely (P 5 0.04). Median time to complete healing was 56 days in the active and 93 days in the control arm (P 5 0.013). Another unblinded comparative study investigated the benefits of con- tinuous diffusion of oxygen compared with variable standard care for DFUs (31). Notwithstanding methodological weak- nesses, they found significantly faster rates of healing in the topical oxygen group compared with the standard care group and most notably in deeper ulcers. A more recent randomized placebo- controlled trial using a continuous dif- fusion of oxygen device for only UTC grade 1A ulcers reported a higher pro- portion of healed DFUs (32.4% vs. 16.7%, P 5 0.033) and a faster time to closure (P 50.015) in the active group at 12 weeks (30). This study was also planned with a group sequential design; however, their interim analysis end point was not met, and their ITT analysis did not include 35% of randomized patients who were subsequently removed from the trial.

Strengths and Limitations

This TWO2 study followed the guidance for wound-healing therapies put forth by the U.S. Food and Drug Administration (28) as well as subsequent publications from leading authorities calling for more robustly designed sham-controlled RCTs (1,29,35). Nonetheless, and despite ran- domization of known and unknown po- tential confounders between groups, it does have limitations. One is the rela- tively small number of patients included in the primary end point analysis of our ITT population, although the group was similar in size to those of other wound care RCTs (2,36). In a group sequential design study, predetermined hard stop- ping rules are put in place that in our case were met at the first analysis point of 73 patients. At that point, the primary outcome was achieved by finding signif- icantly more patients in the active group had healed compared with the sham- treated group (41.7% vs. 13.5%, P 5 0.007). This approach is used when the magnitude of the treatment effect is uncertain, as it allows for stopping a trial once a wide treatment effect is proven. This also ethically ensures that patients are not further randomized to an inferior arm. In our study, a large margin of effect (68%) and relative performance ratio (309%) were achieved.

Table 3—Summary of the results: ITT analysis
Sham TWO2       Active TWO2	Pearson x2 or OR or
(n 5 37)             (n 5 36)	HR (97.8% CI), P value
Primary outcome
Ulcers completely healed at 12 weeks, n (%)                                                   5 (13.5)               15 (41.7)	x2 7.27 (1 df), P 5 0.007
By randomized treatment group, univariate	OR 4.57 (1.19, 17.57), P 5 0.010
	HR 3.64 (1.11, 11.94), P 5 0.013
After adjustment for UT grade	OR 6.00 (1.44, 24.93), P 5 0.004
	HR 4.66 (1.36, 15.98), P 5 0.004
Margin of effect/relative performance                                                                         68%/309%
Secondary outcomes
Healing durability
Ulcer recurrence at 12 months, n (%)                                                          2 (40.0)                 1 (6.7)	P 5 0.070
Ulcers closed at 12 months, n (%)                                                                 10 (27)                 20 (56)	P 5 0.013
Margin of effect/relative performance                                                                     52%/207%
Healing trajectories
Absolute change in ulcer area over 12 weeks, cm2                                   0.40 (1.75)           1.97 (2.75)	P 5 0.041
Absolute change in ulcer area in ulcers .4 cm2 over 12 weeks, cm2        21.34 (1.18)         4.12 (1.51)	P 5 0.021
Time to complete wound closure, weeks                                                     6.3 (1.9)               8.2 (4.2)	P 5 0.350
QOL
CWIS well-being improvement between baseline and week 12               20.1 (16.9)           9.1 (13.9)	P 5 0.033
CWIS social life improvement between baseline and week 12                  4.1 (12.4)            7.9 (16.9)	P 5 0.340
CWIS 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 0.668
TWO2 therapy and off-loading compliance
Used TWO2 therapy device 5 days/week, 90 min/day, n (%)                      35 (96)                 34 (94)	P 5 0.978
Used off-loading device .75% of the time, n (%)                                        36 (99)                35 (97)	P 5 0.984
Safety analysis Incidence of serious adverse events, n                                                                 10                        10 Wound infection                                                                                                  2                          3 Osteomyelitis                                                                                                       5                          2 Hypoglycemic event                                                                                            1                          0 Urinary tract infection                                                                                         0                          2 Significant necrotic tissue                                                                                   1                          0 Cardiovascular event                                                                                           0                          1 UTC grade 2 ulceration                                                                                       0                          1 Severe maceration/dermatitis                                                                             1                          0 Pneumonia                                                                                                           0                          1 Incidence of adverse events, n                                                                               8                          8 UTC grade 1 ulceration                                                                                       0                          3 Ulcer decline                                                                                                        0                          2 Minor infection                                                                                                    1                          1 Minor osteomyelitis                                                                                             0                          1 Minor necrotic tissue                                                                                          1                          0 Cellulitis                                                                                                                1                          0 Swelling/edema                                                                                                   1                          1 Maceration                                                                                                          2                          0 Dermatitis                                                                                                            1                          0 Contusion                                                                                                             1                          0 Incidence of adverse device events                                                                        0                          0 Data are means (SD) unless otherwise indicated. Boldface type indicates significant differences.	P 5 0.943                   P 5 0.950

The quality of DFU studies is often measured by the results obtained in the control groups. In our sham-treated control group, 13.5% of patients achieved complete ulcer healing within the 12-week outcome period. This rate is similar to that of some studies and lower than others (17,30,37,38). Interestingly, a recent topical oxygen RCT reported an active group healing rate lower than ours at 32.4% and a similar control healing rate (30). For the more chronic ulcers, their placebo arm healing rate dropped to 13.2%. Despite the large margin of effect between our active and sham groups, we attribute our osten- sibly low sham healing rate to the chronicity of the ulcers, complexity of the patients, and the control of, rather than a failure of, SOC treatment. In this regard, the average duration of ulcers enrolled in the trial was .5 months, with a nonsignificant 14-day longer duration in the control group. After the 2-week run-in period, 25% of enrolled patients were excluded from randomi- zation due to a reduction in wound area $30%. The study off-loading device, itself proven to be as efficacious as gold standard total contact casting (39), may have enabled progress toward healing that excluded patients likely to heal with such standard care alone. This allowed only patients with wounds more difficult to heal (true SOC failures) to be random- ized into this trial. Since there was a very high degree of compliance with both blinded treatments and off-loading throughout the study, we have no reason to believe that the control group healing result was due to any shortcoming in the SOC protocol.

Our sham therapy itself provided nothing more than nonpressurized room air that was free to circulate within the extremity chamber. Room air cannot conceivably be detrimental to the control patients or have a negative impact on ability to heal. Even at the 12-month follow-up evaluation point, long after the active therapy had ended, there was still a clear separation between study groups, with the sham control patients achieving a healing rate of only 27%. Analysis for predictors of healing at 12 weeks resulted only in the treatment effect and UTC ulcer grade being signif- icant. Furthermore, we found no difference in compliance with the therapy or off- loading between study groups. In the ab- sence of otherwise explanatory data to account for the control healing rate, we are left with our presumption that those randomized into the study had ulcers that were truly hard to heal and that the dif- ference in healing rates between active and sham groups was indeed a treatment effect.

The mean age of our study population was ;63 years old, which mirrors that seen in other DFU studies. Eighty-six percent of our study patients were men, likely resulting somewhat from the fact that one-half of the U.S. study sites were Veterans Affairs wound care clinics. Multiple studies have shown DFUs to be more prevalent in men than women to a degree similar to that seen in this RCT (4,10,38). With no significant differences in covariates seen between the two study groups, our findings support the premise that these results are generalizable to simi- larly afflicted patient populations.

Conclusion

The results of the TWO2 study demon- strate that cyclical pressurized TOT in conjunction with both optimal off-loading and good standard wound care can heal significantly more DFUs at 12 weeks compared with optimal SOC alone. In fact, we found a .4.5-fold increased likelihood of healing within this time period for our actively treated patients. This therapy was safe, without compli- cations, and provided more durable heal- ing for those who had wound closure during active treatment. Uniquely, the therapy has additional benefit in that it can be administered by the patient at home without the expense and difficul- ties of daily travel to a specialized center. In contrast to recently reported systemic HBOT studies (16,18,40), this robust double-blinded, sham-controlled trial provides evidence to support use of this adjunctive cyclical pressurized TOT for chronic DFUs.

Acknowledgments. The authors thank all study patients, clinic personnel, study coordinators, and colleagues who assisted with patient referrals.

Duality of Interest. This study was sponsored by AOTI Ltd. (Galway, Ireland). The sponsor incurred all costs for the study including all institutional fees, monitoring, data warehousing, statistical services, and the provision of study devices and supplies. R.G.F. received research support from thesponsorduringtheconductofthestudy while employed at the Phoenix VA Healthcare System and 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 received research support from the sponsor during the study. No other potential conflicts of interest relevant 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 or study treatments.

Author Contributions. R.G.F. assisted with the conception, design, and analysis of the study and wrote the manuscript. P.J.F. provided the sta- tistical design, performed the analyses, and assisted 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 the discussion and critically reviewed and provided edits to the manuscript. R.G.F. and P.J.F. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented as a late-breaking abstract at the 78th Scientific Sessions of the American Di- abetes Association, Orlando, FL, 22–26 June 2018.

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