Management of Diabetic Foot Ulcers Using Topical Probiotics in a Soybean Based Solution
Chao-Chih Yang, Meng Si Wu, Honda Hsu
Chao-Chih Yang MD, Attending Plastic Surgeon and Chief of Division of Plastic Surgery, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taiwan Meng-Si Wu MD, Attending Plastic Surgeon, Lecturer, Division of Plastic Surgery, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan and School of Medicine, Tzu Chi University, Hualien, 97004, Taiwan Honda Hsu MBChB, Attending Plastic Surgeon, Associate Professor, Division of Plastic Surgery, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Dalin, Taiwan and School of Medicine, Tzu Chi University, Hualien, 97004, Taiwan
Diabetic foot ulcer is a common illness seen worldwide. It has the potential to affect everyone regardless of gender, race, religion, and socioeconomic status. Approximately 34% of all patients with diabetes mellitus will develop some form of lower extremity ulcer, and of these, an estimated 12% will ultimately require lower limb amputation1.
Standard management of these ulcers include strict glycemic management, infection control both locally and systemically, revascularization of the ischemic limb, debridement, mechanical off-loading and foot care education2. Currently, there is a wide range of advanced wound care therapies available for the management of these ulcers, and all are effective to a certain degree3,4.
The majority of us are unaware of the beneficial role of probiotics when administered topically in the management of wounds. Numerous reports show that oral administration of probiotics plays a beneficial role in managing diabetes mellitus, respiratory tract infections, gastrointestinal disorders, urogenital infections, certain skin inflammatory diseases, and chronic wounds5- 12. There are now emerging reports of the beneficial effects of using probiotics topically in the management of wounds. Soy protein has been shown to be beneficial to wound healing by increasing dermal extracellular matrix (ECM) synthesis and stimulating re-epithelialization. It has bioactive peptides similar to extracellular matrix (ECM) proteins present in human tissues. These ECM- mimetic peptides can promote cell adhesion, proliferation, and migration, critical for supporting wound healing13,14. But the application of topical probiotics in a soybean concentrate for advanced wound care has not been well described.
This study aims to look at the efficacy of using topical probiotics in a soyabean based concentrate in the management of diabetic foot ulcers when administered by a wider variety of clinicians at multiple locations with a more heterogeneous patient population.
Materials and Methods
A retrospective, multicentre clinical trial was conducted to evaluate healing outcomes in diabetic patients with chronic lower extremity ulcers treated with twice-daily topical application of probiotics in a soybean-based concentrate (Lactera / Sanarever) as an adjunct to standard wound care. The study population consisted of patients with diabetes under management by clinicians specialising in wound care at three different hospitals in different regions in Taiwan.
Diabetic patients with non-infected foot ulcers between October 2020 and October 2021 were included in the study. The inclusion criteria were: age 20 or 22 older, Type 1 or Type 2 diabetes, ulcer size 1cm and < 25cm , ulcer duration of 4 weeks or more that has been unresponsive to standard wound care, HbA1c <12%, and those that are able to comply with standard wound care. The exclusion criteria were: ulcer with exposure of tendon or bone, severely infected wound, those currently under radio or chemotherapy, those with known or suspected malignancy of the ulcer, use of biomedical/topical growth factor within previous 30 days, pregnant or breastfeeding, taking medications considered to be immune system modulators, allergy to known sensitivity to probiotics or soybean, inability to comply with wound care or current participation in another clinical trial.
The patients were seen weekly or fortnightly for up to 16 weeks or until wound healing whichever came earlier. At each visit wound size were measured and documented with photographs. During the progress of the study, normal clinical care was given. If surgical debridement was undertaken, these were recorded. Complete re-epithelialisation of the wound without the need for dressing was used as the definition for complete healing. The primary study outcome was the incidence of complete wound closure. Secondary outcomes included time to healing and incidence of ulcer recurrence at the site of the study ulcer during the follow-up phase.
A total of 22 patients fulfilled the inclusion criteria and were enrolled in this study. There were 16 men and 6 women with a mean age of 61 years old (range 31-89 years old). The defect size ranged from 1cm2 to 10cm2 (Range 4.6cm2).
These patients often had multiple comorbid illnesses. Among them, 9 (41%) patients had hypertension, 6 (27%) patients had end-stage renal diseases and were on hemodialysis, 10 (45%) patients had a history of peripheral vascular disease and were under treatment, and 2 (9%) patients with a history of congestive heart failure. The mean length of days required till complete healing was 51 days (range 21-112 days) (Table 1A total of 83.3% of the patients showed complete healing of their ulcers at the end of the 16 weeks, 72% showed complete healing in 12 weeks, 56% in 6 weeks and 22% in 4 weeks. The wounds showed an average decrease in size of 0.59cm2 (9%) per week calculated using a generalized estimating equation. It took approximately 19 days to achieve 25% of the surface area, 39 days to achieve 50%, 58 days to achieve 75%, and 78 days to achieve 100% healing (Table 2). One patient was lost to follow up, and one patient required debridement followed by local flap reconstruction. One patient with Charcot's foot showed complete healing at 6 weeks but developed recurrence of the ulcer 3 weeks later as he did not wear his off-loading shoes. The probiotics in a soybean-based concentrate were re-started and off-loading shoes were worn. The ulcer healed in 2 weeks and no further ulceration was seen once proper compliance to wearing his off-loading shoes was followed.
Two illustrative cases are shown in Figures 1a-d and 2a-e.
Diabetic foot ulcer is a serious complication of diabetes that can impact significantly on the patients' quality of life if not properly managed. Successful management of diabetic foot ulcers requires a multi-disciplinary stepwise approach. The management is bound to fail if this is not adhered to. The underlying management principle is a need for strict glycemic control, adequate debridement, infection control, revascularization in the presence of peripheral arterial occlusive disease, and the need for proper off-loading of the plantar ulcers to allow for wound healing and to prevent recurrent ulcers. For those wounds with exposed bone or tendon, surgical reconstruction with local or a free flap is required. Where bone or tendon is not exposed, then skin grafting or coverage with an advanced wound care dressing can lead to successful wound healing15.
Comparing our findings to other liquid dressings such as B-glucans by King et al. 16, they found that in a total of 26 wounds, complete healing was seen in 7 patients at 12 weeks, and 8 patients showed a greater than 50% decrease in wound size at the end of the 12 weeks. Our findings were comparable, even slightly superior to their findings in that we found a total of 83.3 percent of the patients achieved total wound healing at the end of the 16 weeks and that by 6 weeks, 56% of the patients had achieved complete wound healing. We had a healing rate of 0.59cm2 (9%) healing rate over the 16-week period.
Our results were also comparable to a new macrophage-regulating drug (ON101) on the healing efficacy in the management of diabetic foot ulcers17; they achieved a 61.9% complete healing at the end of 16 weeks, with 82.8% achieving a greater than 50% reduction in ulcer size. However, their study had a much larger sample size. Animal studies show that topical application of probiotics on wounds demonstrated successful reduction of the two most common skin pathogens, S. aureus and P. aeruginosa18,19. Two clinical studies have used topical application of L. plantarum on infected wounds. In one study, it was applied to burn wounds and in the other to chronic foot ulcers20,21. These studies showed that the topical application of L. plantarum on burns was as effective against pathogens as the topical application of silver ions20. There was a statistically significant decrease in pathogen load after day 10 compared to day 1 when treated with topical probiotics21.
In the investigated clinical studies, the most commonly used probiotics strains were L. plantarum, L. casei, and L. acidophilus. These strains of lactobacilli aid in epithelialization during wound repair and can inhibit wound colonization by other pathogens20,21.
Several mechanisms of direct probiotic action against pathogens have been proposed. These include the production of antimicrobials, stimulation, and modulation of host immune responses, displacement of pathogens from host epithelial cells by competition with pathogenic bacteria for nutrients and binding sites on the host cell, and the elimination of pathogens by co- aggregation and quorum sensing22.
Antimicrobials produced by probiotic strains include organic acids, hydrogen peroxide, diacetyl, reuterin, and bacteriocins. Hydrogen peroxide, produced by lactobacilli, were accountable for decreased counts of anaerobic Gram-positive bacteria. Reuterin (3- hydroxypropionaldehyde), a well-known antimicrobial metabolite produced by Lb. reuteri, is thought to exert its effect by oxidizing thiol groups in the target microorganism. Diacetyl, another metabolic product of lactobacilli, also exhibits antimicrobial potential against both Gram-negative and Gram-positive pathogens. Bacteriocins, small peptides produced by probiotics, also show a wide range of antimicrobial activity both in vitro and in vivo23-28.
A further possible mechanism is the ability of probiotics to co-aggregate with other microorganisms, and this co-aggregation confers further antimicrobial properties. In addition to the production of antimicrobial substances and co- aggregation, probiotic strains can displace pathogens29-31. Displacement is attributed to the competition by specific surface molecules produced by lactobacilli for enterochromaffin adhering 31. A further important antimicrobial mechanism is the inhibition of the pathogen's quorum sensing system. Most pathogens, including species commonly found in chronic wounds (e.g. Pseudomonas aeruginosa and S. aureus), utilize quorum sensing for virulence, biofilm formation, and resistance to host defenses31-33. Probiotics interfere with the pathogen's quorum sensing by inhibiting the production of quorum sensing signaling molecules (acyl-homoserine-lactone). This inhibition subsequently reduces the formation of biofilms32. In addition to their antimicrobial effects, probiotics also enhances epithelial barrier function, thereby limiting pathogen invasion. They strengthen the epithelial barrier by increasing expression and regulating the localization of tight junction (TJ) proteins34.
Soy protein, the major component of the soybean, is a very promising natural biomaterial. Soybean paste was commonly used in traditional folk medicine in the Far East. It was traditionally applied to lacerated or abrased skin wounds for wound care. This protein extracted from soybeans has been thoroughly studied in the last couple of decades due to the potential health benefits as well as being a "green" and renewable substitute for petroleum- or animal-derived polymers in biomedical applications. It comprises approximately 38% proteins, 30% carbohydrates, 18% oil, and 14% minerals35. Soy protein has been found to have bioactive peptides similar to ECM proteins present in human tissues. These ECM-mimetic peptides can promote cell adhesion, proliferation, and migration critical for supporting tissue regeneration36,37.
In cutaneous wound healing, soy protein has attracted increased attention as a safe and cost-effective alternative to animal protein and endogenous estrogen. Previous studies have shown that peptides in soy protein improved wound healing by increasing dermal ECM synthesis and stimulating re-epithelialization. Soy phytoestrogens have been demonstrated to accelerate the healing process via estrogen receptor signaling pathways38. They also possess anti-bacterial, anti-inflammatory, and anti-oxidant properties that support and enhance wound healing37. Genistein, which is one of the two primary estrogen isoflavones in soybeans, has a potential beneficial effect on wound healing, especially in epithelialization as well as preventing hypertrophic scar formation38. Due to the above-mentioned advantages, Soy protein shows great promise as a natural biomaterial to be used in wound healing.
In any form of wound management, the main goal is to achieve rapid healing with retained functional and aesthetic results. An ideal dressing is to restore the best milieu achievable for the best possibility of healing while simultaneously protecting the wound against bacteria and hostile environmental threats. The dressing should also be easy to apply and remove. In this study, the formulation of probiotics in soybean-based concentrate was prepared in a liquid state. This approach gives it the additional benefit that it can be sprayed in an even distribution over the entire surface of the ulcer, even in wounds with irregular surfaces where some dressings might find it difficult to conform to the surface of the wound. It was extremely easy to apply, even for patients at home, with little concern that spraying the solution on surrounding normal skin would lead to maceration of the skin. The disadvantage was that extra coverage of the wound was required, which leads to added expenditure.
Our results showed that using a topical application of probiotics in a soybean- based concentrate was effective in the management of Wagner 1 and 2 diabetic foot ulcers. Over 80% of the patients could achieve complete healing within the 16-week follow-up. It was extremely easy to apply without discomfort.
This study provides the first description of using probiotics in a soybean-based concentrate in the management of wound healing, specifically in the management of small diabetic foot ulcers. Our multi-center study showed that topical application of probiotics in a soybean-based concentrate solution was easy to apply and can be used as an add-on therapy to the standard management of wound care using wet to dry methods. It successfully healed up to 83% of Wagner 1 to 2 diabetic foot ulcers within a 16-week period. The limitations of this study were that it was retrospective in nature, a control group was not available, and that a further dressing was required for coverage of the wound. In this study, we used the standard wet to dry wound care.
This study provides a new perspective and a therapeutic potential of probiotics in a soybean-based concentrate as a safe form of management in patients with small chronic diabetic foot ulcers. It showed that it was effective in managing small diabetic foot ulcers without bone or tendon exposure. Probiotics in a soybean-based concentrate is an exciting new prospect as a topical treatment for all those involved in the management of patients with diabetic foot ulcers.
69-year-old man with type II diabetes mellitus who developed an infected wound of the right lateral foot. Debridement was done, followed by a normal saline wet dressing. Once the wound was clean to the naked eye (3cm x 2cm), a liquid dressing using probiotics in soybean concentrate was applied twice daily.
Follow-up at two weeks shows the wound decreasing in size (2cm x 0.7cm) with epithelialization seen.
After 4 weeks the wound is responding well and had decreased to 0.5cm x 0.5cm
Complete healing was seen after 6 weeks.
85-year-old male patient with a chronic infected wound of the left lateral foot. After debridement, liquid dressing using probiotics in soybean concentrate was started. The initial wound size measured 6cm x 2cm
The wound at 2 weeks follow-up showed a decrease in size to 5cm x 1.5cm. There was slight tendon exposure at the distal portion, and office debridement was done.
After 8 weeks, the wound had decreased to 5cm x 0.3cm.
The wound had almost healed at 12 weeks, now measuring 0.3cm x 0.3cm
Follow-up at 16 weeks shows complete epithelialization. But the patient’s family described that the wound had healed a week earlier.
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