A Randomised Controlled Clinical Study Comparing the Efficacy and Safety of an Autologous Standardised Leukocyte‐Poor Platelet Gel With Standard Care for the Treatment of Chronic Neuropathic Diabetic Foot Ulcers

ABSTRACT

This randomised controlled clinical trial compared the efficacy of a standardised autologous platelet gel (RegenWound gel) (n = 48) with a standard care treatment (hydrocellular or hydrocolloid dressing) (n = 48) for the treatment of hard‐to‐heal foot ulcers in type 1 or 2 diabetes mellitus patients > 18 years old. The primary outcomes were the percentage of ulcers healed 6 weeks after treatment commenced. The secondary outcomes were the average healing time, the time course of the healing process, the local tolerance, and the acceptability of the treatment by the patient compared to the standard treatment. At the 6 weeks end‐of‐treatment visit (ETV), 56.5% of the patients in the RegenWound gel group and 20.0% of the patients in the control group had completely healed. Healing continued to evolve after the ETV and reached 77.3% at end‐of‐study visit 2 (12 weeks) in the RegenWound gel group, compared to 35.1% for the control group. The treatment was well tolerated and safe. RegenWound gel could be an effective treatment for diabetic foot ulcers, with most patients being healed within 6 weeks of treatment, and on average 1 to 2 treatments being needed.

Trial Registration: ISRCTN10032417

Abbreviations

ECM

extracellular matrix

ESV

end‐of‐study visit

ETV

end‐of‐treatment visit

ICER

incremental cost‐effectiveness ratio

ITT

intention‐to‐treat

IV

inclusion visit

PDGF

platelet‐derived growth factor

PP

per protocol

PRP

platelet‐rich plasma

QALY

quality adjusted life year

RWG

RegenWound gel

SC

standard care

STV

study treatment visit

SV

screening visit

TABCT

topical autologous blood clot therapy

TGF‐β

transforming growth factor β

VEGF

vascular endothelial growth factor

Summary.

• There are a growing number of publications on the use of PRP gels as a treatment for chronic diabetic foot ulcers. However, meta‐analyses of these clinical studies are difficult due to the lack of standardisation of the PRP preparation, among others, which are known to influence outcomes. Moreover, the quality of available studies on the use of PRP for the treatment is low, further complicating the analyses. Nonetheless, the consensus is that PRP might be a useful treatment approach for chronic diabetic foot ulcers when standard therapy fails.

• This controlled randomised study using a standardised PRP gel preparation is therefore an important contribution to the field. It demonstrates that PRP is a more effective treatment than standard care. A retrospective analysis of our patients also showed that concomitant treatment with anticoagulants or anti‐platelet medication did not influence clinical outcomes.

• In our manuscript we have referred to the PRP gel used in the study as RegenPRP. We believe it is important to distinguish between standardised and non‐standardised PRP preparations, and even between standardised PRP preparations from different providers. This is because the method for PRP preparation is one of several factors that can influence clinical outcomes. For physicians to make informed choices, it is important to emphasise that these are the results that can be achieved with RegenPRP; different results may be achieved with other methods of preparation, whether commercial or not. However, we will respect the editorial decision on this matter and modify if necessary.

1. Introduction

Foot ulcers are a common occurrence in patients with diabetes, with an estimated 15% to 25% of patients expected to experience them in their lifetime [1, 2]. The high prevalence of diabetes in many countries, which has continued to increase in recent years [3], makes foot ulcers a major and growing public health problem. These ulcers are the cause of severe morbidity, a significant deterioration in the quality of life of patients, result in high therapeutic costs [4] and represent the largest risk factor for lower limb amputation [5]. In France, the risk of amputation of the lower limbs is estimated to be 7 times higher among the population treated pharmacologically for diabetes compared to the non‐diabetic population, with a standardised incidence rate of foot ulcers of 610/100 000 in people with diabetes and 123/100 000 in non‐diabetics in 2013 [6].

The main goal of treatment is therefore to heal the wound and prevent the need for amputation. There are many approaches currently in use, such as debridement of the wound, which aims to convert a chronic wound to an acute one by removing necrotic tissue [7]; pressure off‐loading, which is particularly important to allow plantar foot ulcers to heal as they tend to occur in high pressure areas; treatment of any wound infections to prevent further complications; as well as more advanced wound care approaches such as negative pressure wound therapy; hyperbaric oxygen; and extracorporeal shockwave therapy [8].

The occurrence of foot ulcers in diabetic patients is explained by the presence of neuropathy and/or ischaemia of the lower limbs, with local trauma or repetitive or sustained mechanical stress as a precipitating factor [2]. The neuropathic component is almost always present, either in isolation (pure neuropathic ulcers) or associated with peripheral ischaemia (mixed neuro‐ischemic ulcers), while pure ischaemic ulcers are rare.

Wound repair is a finely orchestrated phenomenon, involving complex biological and cellular events of cell migration and proliferation, extracellular material deposition, and remodelling [9, 10, 11]. Wound repair is usually divided into four phases that are largely intertwined with one another: coagulation, inflammation, migration/proliferation, and remodelling. Initial coagulation is essential for haemostasis and wound protection, but also for cell recruitment and later, the formation of the extracellular matrix (ECM) stimulated by growth factors released by the platelets present in the clot.

The perturbations in the wound healing process observed in diabetes mellitus patients are numerous and complex: abnormalities in polynuclear functions and cell migration, qualitative and quantitative abnormalities in ECM deposition and/or a prolonged inflammatory phase have been described and probably explain the difficulty in obtaining an active local therapeutic product.

In this context, the use of autologous platelet concentrates may be a promising approach for the treatment of chronic wounds, and some systematic reviews highlight the efficacy and safety of these products [12, 13]. Platelet concentrates (e.g., platelet‐rich plasma (PRP)) are blood‐derived products that are enriched in platelets and depleted of red and white blood cells to varying extents depending on the protocol used for their preparation. In addition to growth factors, platelets secrete specific chemotactic signals [14] that attract leukocytes and allow the recruitment of these cells at the wound site, where they have antimicrobial activity and contribute to the debridement of the wound. Platelets also participate in the fight against infection, as they have a direct bacteriostatic effect on certain bacterial strains [15]. Coagulation can be induced in PRP to form a ‘white’ clot (i.e., depleted red blood cells) that can be placed in the wound bed. This clot forms a primary scaffold where cells are attracted to it (migration), multiply (proliferation), and release numerous growth factors.

The aims of this randomised controlled study were to evaluate the efficacy of a standardised autologous leukocyte poor platelet gel prepared with a dedicated medical device (RegenWound gel) used for the treatment of foot ulcers in diabetic patients. The primary outcome was the percentage of ulcers healed 6 weeks after treatment. The secondary outcomes were the average healing time, the time course of the healing process, the local tolerance, and acceptability of the treatment by the patient.

2. Methods

2.1. Study Design

This was a single‐center, open‐label, randomised controlled trial that took place at the Department of Endocrinology and Metabolism, Fondation Hôtel‐Dieu, Le Creusot, France. Patients were randomised to receive either the intervention, RegenWound gel (RWG), or the standard care treatment (SC) with hydrocellular or hydrocolloid dressing according to the recommendations of the HAS (French Health Authority) [16] at the time the study protocol was drawn up, or in the case of deep wounds, sterile non‐woven gauze wicking. Patients for the study were recruited between April 2013 and March 2018. An overview of the study design is given in Figure 1. After the screening visit (SV), the patients had a two‐week wait period during which their lesion was treated with standard care, before proceeding to the inclusion visit (IV) where they were randomly allocated to receive either the RegenWound gel or to continue with standard care. The intervention period lasted a maximum of 6 weeks and included study treatment visits every 2–3 weeks and a new RegenWound gel application when deemed necessary. The patients were observed for a further 3 to 6 weeks (end‐of‐study visits, ESV1 and ESV2). Therefore, for each patient, the study period lasted a maximum of 14 weeks, comprised of 6 weeks of treatment or less if there was complete healing of the treated ulcer, and 6 weeks of post‐treatment follow‐up. All patients were evaluated 6 weeks after the inclusion visit (end of treatment visit, ETV).

FIGURE 1.

Study design.

This study was done in compliance with the ethical principles of the Declaration of Helsinki and recommendations for Good Clinical Practice. The study was approved by the Comité de Protection des Personnes ‘Est I’, Dijon, France.

2.2. Participants

Study participants were diabetes mellitus patients (either type 1 or 2), aged 18 years or older, attending the Department of Endocrinology and Metabolism. The participants had one or more neuropathic ulcers located on the plantar surface of the foot, on the plantar and/or dorsal surface of the toes, grade 3A according to the University of Texas Classification, with a surface area of less than 5 cm² and a depth of more than 5 mm. Arterial disease was assessed at inclusion (arterial Doppler, Doppler ultrasonography or systolic pressure). Only one ulcer per patient was included.

The patients suffered from ulcers that were hard to heal, that is, the ulcer had not decreased by more than 20% in the 2 weeks (recommendation based on investigator's experience) between the screening visit and inclusion visit (measurement of wound surface using the grid tracing method). Any infections or bone complications needed to be treated successfully before the patient could be included in the study. Patients taking anticoagulant or anti‐platelet therapy (Vitamin K agonists, low molecular weight heparin, aspirin, clopidogrel, etc.) were allowed to participate in the study.

The main exclusion criteria were patients with an ulcer whose area had decreased by 20% or more at the inclusion/randomisation visit compared to the area measured at the screening visit; patients with clear clinical signs of acute uncontrolled local or general infection; patients who were not compliant with wound off‐loading. A complete list of inclusion and exclusion criteria is described in Table 1.

TABLE 1.

Main inclusion and non‐inclusion criteria.

Main inclusion criteria

Diabetic patients, type 1 or 2, aged 18 years or older Neuropathic ulcers located on the plantar surface of the foot, on the plantar and/or dorsal surface of the toes, grade 3A according to the University of Texas classification, with a surface area of less than 5 cm2 and a depth of more than 5 mm

Main non‐inclusion criteria

Allergy to any component of the formulation tested Haematological or coagulation disorders Anaemia (HGB < 10 g/dL) Uncontrolled acute local or general infection Autoimmune disease Ulcer whose surface has decreased by 20% at the inclusion visit compared with the surface measured at the pre‐inclusion visit Malignant disease, particularly with haematological or bone involvement, or metastatic disease

After inclusion in the study, any infections present had to be successfully treated. If an infection developed during treatment, the treatment was stopped, and the infection was treated with the appropriate antibiotics.

2.3. Treatments

2.3.1. Preparation of the RegenWound Gel

The RegenWound gel (RWG) was prepared using the RegenKit‐BCT Plus (Regen Lab, Le Mont‐sur‐Lausanne, Switzerland) from a small sample of the patient's blood (20 to 30 mL). This certified medical device contains two RegenBCT tubes for the preparation of a standardised leukocyte poor PRP (Regen PRP) and one RegenATS tube for the preparation of autologous serum containing activated thrombin (ATS) according to the manufacturer's instructions.

The PRP from two tubes (around 10 mL) was combined with 1 mL of ATS and 1 mL of a 10% injectable solution of calcium gluconate (Monico, Venice Mestre, Italy) in a small sterile recipient. After 10 min, the resulting RegenWound gel was ready to be placed on the site to be treated.

After application of RegenWound gel, the wound was covered with a paraffin dressing (Jelonet, Smith & Nephew SAS, Neuilly‐sur‐Seine, France), which was fixed in place using permeable dressing tape, for example, Steristrips (3 M, Cergy‐Pontoise, France) to keep the wound moist, but also to allow the passage of exudates. The primary dressing was covered with a secondary dressing of sterile gauze to absorb exudates, and a bandage, or another means of appropriate fixation.

Every week, a public health nurse visited the patient at home and removed the secondary dressing to check the primary dressing. If the primary dressing was clean, only the secondary dressing was replaced. In case of any doubt, the nurse contacted the investigators to determine the procedure to follow (opening primary dressing, organising an appointment at the treatment center, etc.) A new RWG application was performed when deemed necessary during study treatment visits at the hospital. Criteria for reapplication of platelet gel were as follows: a persistent clean wound with no discharge or inflammatory signs, with or without improvement (surface or depth measurement). The decision to reapply was always made by the main investigator. In some cases, depending on favourable evolution, the gel application was not repeated.

2.3.2. Comparative Treatment (Standard of Care, SC)

The comparative treatment was the standard local treatment that is, primary hydrocellular or hydrocolloid dressing (Molnlycke, Wasquehal, France; Hartmann, Selestat, France) and secondary absorbent dressing. In the case of a deep ulcer, the wound was packed with sterile non‐woven gauze wicking. The dressing was initially redone every day for 7 days by a public health nurse at the patient's home. The frequency of subsequent visits at the patient's home was dependent on the course of the wound healing and continued until the wound was fully healed.

Prior to inclusion in the study, any infection must be successfully treated according to the recommendations described below. If an infection should occur during treatment, the treatment should be interrupted and the infection treated. Depending on the wound, betadine, antibiotics, or Collatamp (collagen + antibiotic) (Syntocoll, Germany) was applied.

Both procedures, RWG and SC treatments, included appropriate offloading (see Table 2: Off‐loading methods).

TABLE 2.

Off‐loading methods.

Off‐loading shoe expressed N (%)	RWG (N = 47)	SC (N = 44)
Bivalve splint	6 (13%)	7 (16%)
Bivalve splint + walking shoes		2 (5%)
Bivalve splint + wheelchair	1 (2%)	2 (5%)
Walking boots	14 (30%)	8 (14%)
Walking shoes	20 (45%)	14 (30%)
Walking shoes + wheelchair		2 (5%)
Walking shoes + Mepiflex film roll		4 (9%)
Wheelchair	1 (2%)	1 (2%)
Resin	2 (4%)
Mepiflex film roll	2 (4%)	2 (5%)
No off‐loading shoes	1 (2%)	2 (5%)

2.4. Clinical Outcomes

The primary outcome was the percentage of ulcers healed at ETV (6 weeks after the first treatment). During this visit, a qualitative assessment of the wound/healing and photography was performed. The ulcer was considered healed when the wound was closed, without signs of inflammation or discharge (the dressing had to be dry). If the wound had not healed, the area and depth of the wound were measured. The results were compared to the standard care treatment. The decision that a wound was closed was always made by the main investigator, head of the department of diabetology and podiatry, or a physician from the department involved in the study.

Secondary outcomes were the mean closure time (expressed in days), the extent of re‐epithelialization, the overall tolerance, and the degree of treatment acceptability and the overall cost of both procedures. These were assessed at each study treatment visit, as well as at the end‐of‐treatment and end‐of‐study visits. Re‐epithelialization was evaluated on a 5° scale (0: Absent; 1: Mild; 2: Moderate; 3: Significant; 4: Very significant). Tolerance was assessed globally using a 10‐point visual analogue scale and the degree of acceptability of the treatment by the patient according to the four available criteria (‘very satisfactory’, ‘somewhat satisfactory’, ‘unsatisfactory’, ‘not at all satisfactory’).

2.5. Safety

For the duration of the study, all adverse events related or unrelated to the products tested were recorded. The serious or non‐serious nature of the event, its severity, date of occurrence, duration, the actions taken, and the causal link with any of the study products were recorded.

2.6. Sample Size

Based on the experience of the Department of Endocrinology and Metabolism of Le Creusot Hospital (France), it was estimated that a 62% success rate could be targeted when ulcers are treated with RegenWound gel vs. 50% with standard care. The test power was set at 80% and the significance threshold at 5%. Based on these assumptions, the sample size required for analysis is 68 patients per group, or 136 subjects in total (nQuery Advisor 6.0 estimate). To consider the risk of discontinuation or patients withdrawing, the total sample size was increased by 10%. It was therefore planned to enrol 150 patients, divided into two groups of 75 patients. However, due to recruitment issues, the study had to be stopped before this number could be achieved. Nonetheless, we have a similar number of patients with the same characteristics in both treatment groups, and ITT and PP analysis of the data gave similar results.

2.7. Randomization

If off‐loading compliance was found to be satisfactory by the investigator, the wound surface was not less than 20% of the surface area measured at the screening visit, and the subject still met the study inclusion and non‐inclusion criteria, the patient was randomly assigned to receive either the first application of RegenWound gel or to continue with the standard treatment. Patients were randomised using a randomization process known as ‘per block’ via Microsoft Access [17]. This is a method for obtaining the same number of patients in each treatment arm sequentially. Enrolment numbers were allocated in chronological order of patient enrolment. Blinding was not possible in this study as the treatments were too different.

2.8. Statistical Analysis

Statistical analysis was performed using SPSS software version 18. Each analysis was done on the intention‐to‐treat (ITT) population and the per protocol (PP) population. The ITT population comprises all subjects included in the study who undertook at least one trial treatment and had at least one primary endpoint value under treatment (post baseline). The PP population includes only subjects who had fully complied with the protocol, that is, subjects with no major deviation. All randomised participants were considered for safety and tolerance assessments. The characteristics of the patients included in the trial were described using descriptive statistics. The characteristics of the two groups of patients at inclusion visit were compared using the Student t test or the Mann–Whitney test for quantitative variables, and the Chi‐squared test or Fisher's exact test for qualitative variables. A bivariate analysis (Student test or Mann–Whitney test) was performed for the primary endpoint, which is the percentage of ulcers healed after 6 weeks of treatment. Secondary criteria (course of the wound healing process, average healing time, local tolerance, patient acceptability and caregiver satisfaction) were analysed using appropriate statistical tests, depending on the nature of the variables.

3. Results

3.1. Patient Characteristics

Ninety‐six patients were screened and randomised between April 2013 and March 2018, and 91 were monitored (intention‐to‐treat (ITT) population) (see Figure 2: Consort diagram). The mean age of this population was 68.13 years in the RegenWound gel (RWG) group and 70.14 years in the comparator (SC) group. Most patients were male in both groups (85.11% in the RWG group, 81.82% in SC group). The mean and standard deviations for age, male/female distribution, depth, and area of the wound were not significantly different between the RWG and SC groups. Among the study participants, 6.6% had type 1 diabetes (5 patients in the RWG group, 1 in the SC group) and 93.4% had type 2 diabetes (42 patients in the RWG group and 43 patients in the SC group). The mean duration of diabetes was about 19 years in both groups. According to the Case Report Forms, 28 of the 91 patients (9/47 in the RWG group, 19/44 in the SC group) had a plantar perforating ulcer and 61 of the 91 included patients presented with a deep ulcer. Specifically, 30 patients treated with RWG presented with a deep ulcer versus 35 patients treated with standard care. More than half the patients had arterial disease (52 out of 91 analysed, including 28/47 in the RWG group and 24/44 in the SC group).

FIGURE 2.

Consort diagram.

The metatarsal area and toes were the most affected areas in both treatment groups. In the RWG group, 17/47 patients had an ulcer in the metatarsal area and 24/47 patients had one in the toe area. In the SC group, 19/44 patients had an ulcer in the metatarsal area and 21/44 patients had an ulcer in the toe area. The mean depth of the ulcer at the screening visit (SV) was 12.89 mm in the RWG group and 14.8 mm in the SC group. The mean depth at the inclusion visit (IV) was 15.13 mm in the RWG group and 16.72 mm in the SC group. The wound surface area of the ulcer was not measured in patients with deep ulcer. In the other patients, the mean wound surface area was 0.64 ± 0.39 cm² (N = 17) at the SV and 0.62 ± 0.49 cm² (N = 16) at the IV for the RWG group, and 0.73 ± 0.45 cm² (N = 12) at the SV and 0.77 ± 0.62 cm² (N = 9) at the IV for the comparator group. The depth and surface area of the ulcer were not significantly different between the two treatment groups (p = 0.492 and p = 0.505, respectively). These analyses confirmed the absence of healing between the SV and IV, and that these were hard‐to‐heal ulcers.

3.2. Efficacy Outcomes

3.2.1. Primary Outcome: Complete Healing at ETV

At the end‐of‐treatment visit (ETV, 6 weeks) (PP population, N = 86), the ulcers of 26/46 patients (56.5%) in the RWG group and 8/40 patients (20.0%) in the SC group had completely healed (p = 0.001) (see Table 3: Wound closure results). Healing continued to progress after the ETV. The percentage of patients in the RWG group who had completely healed was 71.1% at ESV1 (9 weeks) and 77.3% at ESV2 (12 weeks), compared to 30.0% and 35.1%, respectively, for the SC group. These differences between the groups were statistically significant at each visit (p < 0.001).

TABLE 3.

Wound closure results.

Complete healing (%)	Number of patients (PP) (N = 86)
	RWG (N = 46)	SC (N = 40)
ETV (6 weeks)	56.5% (N = 26)	20.0% (N = 8)	p = 0.001
ESV1 (9 weeks)	71.1% (N = 32)	30.0% (N = 12)	p < 0.001
ESV2 (12 weeks)	77.3% (N = 34)	31.5% (N = 13)	p < 0.001

In ITT (N = 91), the ulcers of 26/47 patients (55.3%) in the RWG group and 11/43 patients (25.6%) in the SC group had completely healed at the end of the treatment visit (p = 0.004). The percentage of patients in the RWG group who had completely healed was 69.6% at ESV1 (9 weeks) and 77.3% at ESV2 (12 weeks), compared to 30% and 35.1%, respectively, for the SC group. These differences between the groups were statistically significant at each visit (p < 0.001).

3.2.2. Secondary Outcomes

3.2.2.1. Healing Time

The healing time did not differ significantly (p = 0.07) between the two treatments, although it was shorter in the RWG group (39.00 days, N = 26) than in the usual SC treatment group (46.46 days, N = 11).

3.2.2.2. Re‐Epithelialization

Overall, re‐epithelialisation was more significant in patients in the RWG group (Figure 3a) than the SC group (Figure 3b) during study treatment and follow‐up visits. In the group treated with RWG, very good re‐epithelialisation rates were obtained in 59% and 80% of the patients at ETV and ESV2, respectively, in comparison to 21% and 37% for the SC group.

FIGURE 3.

Extent of re‐epithelialization (%) in the RegenWound gel group (a) and the standard care group (b). The number of patients (N) whose re‐epithelialization results were available at each visit are given in the tables below each graph.

3.2.2.3. Local Tolerance and Acceptability

Overall, local tolerance of the treatment, whether in the RWG group or in the SC group, was assessed as very good (almost 10 (very well tolerated) on the 10‐point visual analogic scale) throughout the study.

Regarding treatment acceptability, patients that received the RWG treatment were mostly ‘very satisfied’ with their treatment, while patients in the SC group mainly reported that they were ‘quite satisfied’.

3.2.2.4. Off‐Loading Compliance

Regarding off‐loading compliance in the RWG group, it was assessed as good to very good in 93% of cases at the IV, and during the follow‐up visit and at the ETV, mostly assessed as good to very good (91% to 100%). In the SC group, the off‐loading compliance was assessed as good to very good in 90% of cases at IV and considered good to very good from 80% to 100% during follow‐up visits.

3.3. Adverse Events

There was a total of 37 adverse events (AEs) reported in 9 subjects in the clinical trial. Of these 37 AEs, 4 AEs were serious adverse events (SAEs). These 4 SAEs were linked with cardiovascular issues and not considered to be related to treatment (Table 4).

TABLE 4.

Summary of adverse events: SAEs (a), AEs (b).

a
Serious adverse events (N = 4)	RWG (N = 3)	SC (N = 1)
Death	1	1
Acute cardiac failure	1
Stroke	1

b
Adverse events (N = 33)	RWG (N = 11)	SC (N = 22)
Infection	2	2
Wound worsening	1	6
Wound recurrence	1	2
New wound		3
Footwear issue		4
Maceration	5
Discharge		1
Inflammation	1	3
Allergy	1
Pressure ulcers		1

Among the 33 other AEs (Table 4b), 11 (33.3%) occurred in the RGW group, while 22 (66.6%) occurred in the control group. None of the AEs were related to the RWG.

3.4. Concomitant Treatments

A retrospective analysis was performed to evaluate the impact of concomitant treatments with anti‐platelet drugs or anticoagulants on healing at ETV. These drugs are usually exclusion criteria for treatment with PRP, as their presence in the blood might affect coagulation, hence interfere with the formation of the platelet gel or the activity of the platelet gel, possibly leading to suboptimal clinical results. In this study, among 90 monitored patients (data are missing for one patient) 61.1% (28/47 in the RWG group and 27/43 in the SC group) were treated with anti‐platelet drugs and 14.4% with anticoagulants (9/47 in the RWG group and 4/43 in the SC group). At ETV, no significant difference was observed regarding the healing among the patients under anti‐platelet drugs (p = 0.278) or under anticoagulants (p = 0.765). More specifically, there were no significant differences among the 37 patients healed at ETV; 20 were taking anti‐platelet drugs (15/26 in the RWG group and 5/11 in the SC group, p = 0.719) and 6 were under anticoagulants (5/26 in the RWG group and 1/11 in the SC group, p = 0.646). Similar results were observed for the 53 patients who had not healed at ETV; 35 were taking anti‐platelet drugs (13/21 in the RWG group and 22/32 in the SC group, p = 0.768) and 6 were under anticoagulants (4/21 in the RWG group and 3/32 in the SC group, p = 0.415). Although these are retrospective results on a small number of patients, they are encouraging, as there is a high percentage of diabetic patients taking anti‐platelet therapy for the prevention of cardiovascular diseases [18]. This also means that we should plan to include patients on these drugs in future clinical trials for a prospective analysis of the treatment efficacy in these subgroups of patients as a secondary criterion.

4. Discussion

This controlled clinical study aimed to compare the efficacy of RegenWound gel for the treatment of chronic diabetic foot ulcers compared to standard care. Our results demonstrate both the clinical efficacy of RegenWound gel for the treatment of diabetic foot ulcers.

The main weakness of this work was that it is not blinded, particularly regarding the evaluation of results. The use of standardised images with blind reading would have improved the value of this clinical trial. The main strength was the design of the comparative study, including hard‐to‐heal ulcers, and conducted in a department well known for its care of diabetic patients.

There is an urgent need to identify alternative approaches for the treatment of diabetic foot ulcers, particularly those that are hard‐to‐heal, due to the substantial impact on the quality of life of the patient and the substantial financial impact on both the patient and the healthcare system. Diabetic patients have altered production of growth factors involved in the wound healing process, such as transforming growth factor β (TGF‐β), vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF). Consequently, hard‐to‐heal foot ulcers in diabetic patients may be associated with perturbations in the natural wound healing process due to altered or insufficient production of the growth factors involved [19, 20]. Indeed, chronic wounds are often associated with a prolonged inflammatory phase, and diabetic foot ulcers have also been associated with the failure of keratinocytes to epithelialize the wound and insufficient production of ECM proteins [19, 21]. Keratinocytes play an important role in the wound healing process, where they respond to growth factors and produce growth factors involved in the wound healing process, such as PDGF [22, 23]. Migration and proliferation of keratinocytes are required for wound closure, and keratinocytes (or products synthesised by them) have a role in the differentiation of fibroblasts to myofibroblasts, which is an essential process for efficient wound closure [22].

While one approach is to treat the wound with specific growth factors, given the importance of the need to modulate both the levels and the production of different growth factors over time to ensure the wound healing process progresses appropriately, this proves challenging. Hence, there is growing interest in the use of PRP gels for the treatment of diabetic foot ulcers. Platelets have been shown to be able to modulate the release of different growth factors over time, and thus platelet concentrates have great potential for the treatment of wounds [24]. Indeed, a previous study demonstrated that treatment with keratinocytes resuspended in PRP prepared with the same kind of medical device as used in this study placed on the wound bed reduced the average healing time from about 14 to 6 days [25].

Since the composition of autologous PRP is known to vary depending on the preparation method, a standardised protocol for the preparation of PRP is essential when conducting clinical trials in order to ensure that comparisons within and between different studies can be made [26]. The technology of the medical device used in this study offers a standardised preparation of autologous PRP gel. These kits consistently produce a PRP with a 1.5 to 1.6× increase in platelets compared to whole blood (Regen PRP), which has been demonstrated to be sufficient to induce wound healing [27].

Regen PRP has already been the subject of two published clinical studies in the field of wound healing. The first one was a retrospective study of diabetic foot ulcer repair in patients with concomitant peripheral arterial disease or critical limb ischaemia (n = 72) [28]. An autologous Regen PRP gel was applied twice a week for a maximum of 16 weeks. A reduction in the ulcer surface area of 50% was observed in the majority of patients in both groups (group A 86% vs group B 73%, p = 0.23). A reduction in ulcer surface area > 90% was achieved for 52/72 patients. This study showed that Regen PRP gel can be an effective means of managing diabetic foot ulcers in a population of patients at high risk of amputation and in whom vascular reconstruction was not indicated.

The second study was a prospective, controlled randomised clinical trial to evaluate the efficacy of autologous Regen PRP gel treatment compared with the usual treatment for chronic leg ulcers, which showed a significant reduction in the ulcer area compared to the control group and was significantly superior to the usual treatment for granulation tissue development [29].

In our study, after a maximum of 6 weeks of treatment, we saw a significant difference in wound healing in favour of patients treated with the RegenWound gel. The percentage of patients in the RegenWound gel treatment group who had completely healed was 55.3% at ETV (6 weeks), 69.6% at ESV1 (9 weeks), and 77.3% at ESV2 (12 weeks), compared with 25.6%, 30%, and 35.1% respectively for the control group. This difference was statistically significant at each time point (p ≤ 0.001).

Over the past decade, many articles have been published illustrating the full potential of autologous PRP treatment for the healing of chronic wounds, mainly in diabetic foot ulcers and venous ulcers [30]. Autologous platelet‐rich plasma has been evaluated for the treatment of diabetic foot ulcers. In a randomised, controlled, double blind, multicentre trial, Driver et al. compared standard care with PRP gel (Autologel, Cytomedix, US) to a control (saline) group for the treatment of nonhealing diabetic foot ulcers for 12 weeks or until healing [31]. The mean area of ulcers at baseline was 3.4 cm² in the Autologel group and 3.6 cm² in the standard care group. 13/19 (68.4%) patients in PRP gel and 9/21 (42.9%) patients in the control group healed at 12 weeks (p = 0,1215). After adjusting for wound size outliers (N = 5), significantly more platelet‐rich plasma gel (13/16, 81.3%) than control gel (8/19, 42.1%) treated wounds healed (p = 0.036). Games et al. compared in a multicentre, observer‐masked, randomised controlled trial the efficacy of a multi‐layered patches comprising autologous leukocytes, platelets, and fibrin (LeucoPatch, Reapplix, Denmark) to standard care in diabetes patients with hard‐to‐heal ulcers [32]. Weekly application was used and the primary outcome was the blinded assessment of healing within 20 weeks. The mean area of ulcers at baseline was 2.3 cm² in the LeucoPatch group and 2.5 cm² in the standard care group. In the LeucoPatch group, 45/132 (34%) ulcers healed within 20 weeks versus 29/134 (22%) in the standard care group (p = 0.0235). The percentage of healing at 12 weeks was 20% versus 13% respectively. Willians et al. evaluated the autologous blood clot therapy (TABCT) in 29 diabetic patients suffering from hard‐to‐heal DFUs in a real‐world setting. Reapplication of TABCT (ActiGraft, RedDress Medical, US) was performed weekly [33]. The autologous blood clot was applied to DFUs of a mean area of 9.36 cm² at baseline (range, 0.5–38.5 cm²) and was found to achieve complete wound closure at 4 weeks in 31% of patients. At Week 12, a total of 28/29 (95%) wounds achieved complete wound closure. Another recent study was conducted with a platelet gel prepared with another device. This was a prospective, controlled, and randomised clinical trial (N = 129) for the treatment of diabetic foot ulcers. At 12 weeks, the platelet gel group had a percentage of 48.5% healed wounds compared to 30.2% in the control group [34]. In general, tolerance was good in the analysed studies, confirming the good acceptability of locally administered autologous blood treatments. Despite differences in terms of methodology, technology used, ulcer characteristics, and treatment application frequency, the results of our study confirm the superiority of PRP‐based treatments over standard treatment in the management of DFUs.

Recent literature reviews or meta‐analyses have clearly shown that, despite some methodological weaknesses in some clinical studies, PRP therapy may be justified in the treatment of chronic diabetic foot ulcers, with significant benefit demonstrated in 87.5% of controlled studies in one analysis [35]. Peng et al. conducted a systematic review of RCTs comparing autologous PRP with conventional treatments for DFUs in accordance with the PRISMA guidelines [36]. A total of 10 RCTs involving 550 patients (279 who received PRP and 271 who received conventional treatment) were included. In this study, PRP was observed to significantly improve the healing rate (risk ratio [RR] = 1.38, 95% confidence interval [CI] 1.05–1.82, p = 0.02) and shorten the healing time (mean difference [MD] = −23.23, 95% CI –45.97 to −0.49, p = 0.05) of patients with DFU when compared to the conventional treatment. The analysed data suggested that the incidence of adverse events was lower in the PRP group than in the conventional treatment group. The authors concluded that compared to conventional treatment, PRP effectively promoted the healing of patients with DFU by evidently improving the healing rate and healing time. Platini et al. conducted a systematic review of randomised controlled trials to assess the safety and efficacy of autologous PRP gel compared with standard treatment in older adult patients with DFUs [37]. Eight RCTs with 598 patients were eligible for this analysis. They concluded that, compared with standard care/conventional treatment, APG could significantly improve the healing rate, shorten the healing time, shorten the length of hospital stay, and lower the amputation rate. Ruiz‐Munoz et al. conducted a systematic review of randomised controlled trials investigating the effect of PRP versus conventional treatments for ulcer healing rate in DFU patients [38]. The results were reported following the guidelines of PRISMA. Eleven articles were included in this review. The studies included a total of 418 individuals in the experimental group and 410 individuals in the control group, resulting in a total of 828 participants. The quality of the studies was assessed using the CASPe tool for critical reading of scientific evidence in the 11 clinical trials included in this review. All studies passed the evaluation, with scores ranging from 10 to 11 out of a total of 11 points, indicating high quality. They found that PRP treatment significantly increases the ulcer healing rate compared to existing conventional treatments and concluded that PRP can be considered as the first choice for addressing ulcer closure and healing in patients with DFU. Deng et al. conducted a systematic review and meta‐analysis of randomised controlled trials to investigate, assess, and synthesise scientific data pertaining to the safety and therapeutic effectiveness of autologous PRP in the management of DFU in comparison with conventional treatment or any other substitute therapy [39]. A total of 22 articles were included. The selected trials involved a total of 1559 individuals who presented diabetic foot ulcers. Of these participants, 785 were treated with platelet‐rich plasma, while the remaining 774 were assigned to a control group. The results of the meta‐analysis indicate that autologous PRP has a significant positive effect on the healing rate (RR = 1.42, 95% CI 1.30–1.56, p < 0.001), reduces the healing time (MD = −3.13, 95% CI 5.86 to −0.39, p < 0.001), accelerates the reduction of ulcer area (MD = 1.02, 95% CI 0.51–1.53, p < 0.001), decreases the rate of amputation (RR = 0.35, 95% CI 0.15–0.83, p < 0.001), and does not increase the incidence of adverse events (RR = 0.96, 95% CI 0.57–1.61, p > 0.05) when compared to conventional therapy. The findings of this systematic review and meta‐analysis indicate that the use of autologous PRP therapy is a viable and secure therapeutic approach for DFU, as it effectively enhances wound healing.

Numerous clinical studies, systematic literature reviews and meta‐analyses have demonstrated the value of PRP in the treatment of chronic wounds, including diabetic foot ulcers, venous leg ulcers and pressure sores. Thus, these publications concluded that PRP improved the healing rate of chronic wounds and raised no safety concerns.

Our results also show that the wound healing process continues after the last treatment. Indeed, by the end of this study, almost 80% of patients treated with RegenWound gel had very good re‐epithelialisation compared to 55% of patients at the ETV, while the corresponding values in the control group were 37% and 21%, respectively. In addition, our retrospective analysis showed that concomitant treatments with anti‐platelet drugs or anticoagulants did not affect the outcomes in both treatment groups and thus are not contraindications for the treatment of diabetic foot ulcers with platelet gel.

Overall, the RegenWound gel treatment appeared to be safe and well tolerated by patients. These findings are in line with recent clinical trials and meta‐analyses demonstrating the efficacy and safety of PRP treatment compared to standard care for the treatment of diabetic foot ulcers [13, 26].

A cost‐effectiveness analysis based on data from our study, and with a focus on France, found that in the base case scenario treatment with PRP represented a cost‐saving compared to the standard treatment, with an incremental cost‐effectiveness ratio (ICER) of PRP introduction of −617 €/QALY (quality adjusted life year), the negative value indicating the dominance of PRP [40]. Similar results were obtained when the healing probability data from a meta‐analysis of 5 clinical trials were used for analysis [26].

As explained above, the main limitation of our study was the inability to mask the participants or the clinical researchers to the study treatment. However, although qualitative assessments were used for the evaluation of the outcomes in this study, these were supported with digital imaging. We had a greater proportion of male compared to female patients, which has been recognised as a feature of clinical trials in this field [32]. The timeframe for this study was also shorter compared to other studies. While most patients had healed within this short timeframe, a longer follow‐up, or the possibility of additional treatments might have revealed additional benefits among those patients that had not achieved complete healing of their lesion by the end of our study.

5. Conclusion

Our study demonstrates that RegenWound gel could be an effective treatment for diabetic foot ulcers, with most patients being healed within 6 weeks of treatment, and on average 1 to 2 treatments being needed. RegenWound gel was well tolerated, and there were no significant adverse events compared to the standard care treatment.

Conflicts of Interest

Antoine Turzi is founder and owner of RegenLab SA, Bruno Boëzennec is an employee of Regen Lab France SAS.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.