Diabetic Foot Wounds Treated With Human Amniotic Membrane and Low-level Laser Therapy: A Pilot Clinical Study

Lower extremity wounds secondary to vascular disease and neuropathy are common complications in patients with diabetes mellitus (DM).¹ These wounds are responsible for diabetes-related lower limb amputations when DM is poorly managed^2,3 and lead to increased health care costs.⁴ Treatment of diabetic ulcers must be adequate to avoid complications, and further studies are needed to discover pathways to faster tissue repair and prevention of lower limb amputations.⁵

Low-level laser therapy (LLLT). The application of LLLT has shown positive results in chronic wound healing,^6-8 including a clinical evaluation of 3 protocols of LLLT in the healing of open wounds in rats.⁶ An experimental randomized clinical study found LLLT to be effective in relieving pain and accelerating the tissue repair process of diabetic foot ulcers.⁷ Low-level laser therapy accelerates tissue repair by emitting photons that reach the mitochondria of fibroblasts, keratinocytes, and endothelial cells. This process promotes the release of chemical mediators of inflammation and cytokines to reduce pain and local edema, and accelerate tissue repair.^8,9 The biological mechanism of LLLT is related to a decrease in inflammatory cells, stimulation of angiogenesis, formation of granulation tissue, and an increase in fibroblast proliferation.^6,7 All these processes promote collagen synthesis.^6,7 A Brazilian study conducted among 60 adult male Wistar rats found that LLLT significantly induced an increase in collagen scarring in the healing of open wounds.⁶ Randomized experimental studies^10,11 have shown that the therapeutic application of LLLT significantly accelerates wound healing. A clinical trial⁷ found that patients with diabetic foot ulcers treated with LLLT showed significant progression (P < .013) in tissue repair compared with the control group (2 mg hydrogel, saline, and gauze).

To apply LLLT, a laser pen that comes into direct contact with the skin with a wavelength of 660 nm, dose 6 J/cm², can be used.

Human amniotic membrane (HAM). Human amniotic membrane also has been shown to be a viable option in chronic wound healing in a randomized clinical trial that compared wound healing among 11 patients treated with conventional wound dressings and 14 patients treated with HAM.¹² Human amniotic membrane acts as a support for the proliferation and differentiation of new epithelial cells due to factors such as fibronectin; elastin; collagen types I, III, IV, V, and VI; and hyaluronic acid.¹³ HAM has been used for more than 100 years, and its application has expanded in the medical field to include the treatment of burns¹⁴; gingival retraction^15,16; focular surface reconstruction^17-19; and the prevention of adhesions after brain, larynx, abdominal, pelvic, and vaginal surgery.^14,20 Due to its anti-inflammatory action, achieved by inhibiting the production of inflammatory cytokines,¹⁵ HAM also has been used in tendon injuries, as observed in an experimental study of 60 rats.²¹ A case study by Shah et al¹⁵ described the benefits of using HAM for coverage of gingival recession.

Both LLLT and HAM may be valid treatment choices in wound healing, but more evidence is needed to compare the efficacy of these therapies in the treatment of foot ulcers in patients with DM. Therefore, this study aimed to compare LLLT and HAM in foot ulcers in patients with DM with regard to the reduction in the wound area as well as the effects on PUSH and pain scores.

Setting and study design. This pilot clinical study was conducted at the Integrated Health Center Lineu Araújo (Teresina-PI, Brazil), a health center that specializes in the treatment of diabetic foot wounds, from November 2016 to August 2017.

Ethical considerations. This project received ethics approval from the Federal University of Piaui (UFPI), CAFS CAAE: 67985917.8.0000.5660. The participation of patients was voluntary, and all patients provided written informed consent prior to study enrollment. The dignity of all participants recruited for the study was protected according to the ethical protocols of the National Health Council, with ethical approval issued by the Federal University of Piaui.

Participants. Patients were invited to participate in the study as they visited the aforementioned center for routine treatment.

Inclusion/exclusion criteria. Age between 30 and 59 years was an inclusion criteria given that the healing process in adults is different from other age groups. Additionally, the authors included those diagnosed with type 2 DM (postprandial capillary glucose levels between 140 and 350 mg/dL), with uninfected stage 2 or 3 diabetic foot ulcers according to the Revised National Pressure Ulcer Advisory Panel Pressure Injury Staging System.²² To be included in the study, the wounds had to have a maximum length and width of 7 cm and 3 cm, respectively. Wounds also had to exhibit scant exudate and be in the granulation phase.

Patients with acute wounds (less than 30 days’ duration); wounds with purulent drainage, hemorrhage, odor, or exudate; and wounds with photosensitive areas, devitalized tissue with necrosis or slough, and without depth were excluded. Immunosuppressed and malnourished patients or those with neoplasms or in critical condition also were excluded.

Procedures. Prior to study enrollment, wounds were inspected, and the length and width were measured with adhesive rulers to establish study eligibility. Additionally, diabetic foot wounds were evaluated for eligibility using tissue debridement, infection or inflammation, moisture balance, and edge effect (the TIME clinical intervention tool). These 4 components of wound healing guide professionals in therapeutic decisions.²³ Wounds that presented charactericsts identified by TIME were not included in the study.

Data were collected by the main investigator (first author), who used a questionnaire that contained the TIME clinical intervention tool information along with Pressure Ulcer Scale for Healing (PUSH), which assesses the evolution of the injury repair process,^24,25 and the Visual Analog Scale (VAS) assessment of pain intensity. The questionnaire also contained anthropometric, demographic, and clinical variables, including age (years), body mass index (BMI), glucose level measured 2 hours after a meal (mg/dL), time since injury (months), and wound size (cm²). No data were collected on blood pressure, offloading, or smoking status, and these were not baseline assessments. Reports from participants regarding neuropathic pain also were recorded. The authors also kept a record of any concerns that patients had about the treatment.

Randomization. There was no randomization; however, patients eligible for inclusion in the research, according to the study criteria, were selected until completing each group at random.

Assessment. All wounds were examined weekly by the first author using the PUSH scale and the VAS. The PUSH scale considers 3 parameters for evaluating the results of the clinical protocol used: length/width, amount of exudate, and appearance of the wound. The sum of the subscores of these parameters generate a total score, ranging from 0 to 17.^24,25 Higher ratings indicate more severe injury conditions, and lower scores show a less-severe wound. Therefore, the PUSH scale generates scores that, in their magnitude and direction, can describe the condition and evolution of a wound.^24,25

The VAS is a 1-dimensional scale widely used to assess pain intensity; the patient views the instrument and chooses the numeral that corresponds to pain intensity (0 means no pain and 10 means unbearable pain).²⁶

An adhesive paper ruler was made to measure the size of the wounds. This ruler was placed on the side of the wound to measure length and width. Images of the wounds were captured weekly with a digital camera (Sony 4.1) in flash mode at a distance of 20 cm (lesion–camera), with zoom and angle standardized. The captured images of the lesions were evaluated macroscopically using the ImageJ program (National Institutes of Health and Laboratory for Optical and Computational Instrumentation), having as a reference the circumference of the wound edges in cm², thus calculating the total area of the wound. Photos were securely stored for future comparisons.

The percent decrease in wound size was calculated in 3 steps: by 1) determining the difference between the starting and final values, 2) dividing the result from step 1 by the absolute value of the starting value, and 3) multiplying the result from step 2 by 100 to calculate percent decrease. Pictures were taken weekly to compare the images of the healing progress. Study participants were followed-up for 4 weeks and evaluated on days 7, 14, 21, and 28. The clinical protocol of this study is described in Table 1.

Treatments

Standard care. The standard care protocol included cleaning the wound with saline (0.9%), applying a secondary dressing (hydrogel) with gauze, and covering the area with a compression bandage. Hydrogel keeps the environment moist and promotes autolytic debridement and is routinely used for wound management.²⁷ All wounds received the same treatment. No offloading treatment was indicated.

Low-level laser therapy. The LLLT equipment used in this study (Brazilian Medical Equipment Industry [IBRAMED]; Amparo, Brazil) utilized a wavelength of 660 nm, 30 mW of power, and a dose of 6 J/cm². The tip of the laser was protected with PVC film and placed perpendicular to the edge of the lesion at a distance of 1 cm. LLLT was used for 13 seconds at each point of the wound. The equipment sets the time according to the dose of 6 J/cm². The wave was kept continuous, with a visible red spectrum beam. Therapists and patients used personal protective equipment to offset possible effects of the laser. The characteristics of the irradiation parameters of the LLLT are described in Table 2.

Human amniotic membrane. Placenta collection was performed on women who underwent cesarean delivery after meeting the mandatory basic requirements for ensuring quality and safety. The researchers also considered the results of previous rigorous clinical, physical, and laboratory screening. The donors signed confirmations of free and informed consent before biomaterial was collected.²⁸ The cryopreservation and preparation of the biomaterial were carried out in the Ophthalmology Department of the Escola Paulista de Medicina-the Federal University of São Paulo (HSP-UNIFESP), in the headquarters of a bank of amniotic membranes for use in ophthalmological treatments according to national guidelines.²⁸

Statistical analysis. Data were tabulated in spreadsheets and analyzed with GraphPad Prism, version 7 (San Diego, CA). Descriptive statistics were used to analyze the participant anthropometric and clinical profiles, with mean and standard deviation (± SD). The Kolmogorov-Smirnov test was used to analyze the sample distribution. The Kruskal-Wallis test with Dunn’s post-test was used to evaluate differences in PUSH and VAS scores and wound size for intergroup analysis, whereas the Mann-Whitney U test was used for the same outcomes in intragroup analysis. The level of significance was 5% (P < .05).

A total of 36 patients were evaluated; 27 (mean age, 51.4 years) were eligible to participate in this study. Mean BMI of the 3 study groups was 26.5 kg/m²; individuals with a BMI from 25 to 29.9 kg/m² are overweight according to World Health Organization classification.²⁹ All patients in the study were hyperglycemic with postprandial blood glucose levels >140 mg/dL, measured 2 hours after a meal, following Brazilian Society of Diabetes criteria.³⁰ The mean intergroup capillary blood glucose was 199.9 mg/dL. All patients had chronic wounds in the foot region at the beginning of treatment, most in the metatarsal region. The wounds had characteristics of neuropathic disease because they were mostly painless, pulsatile, located in a pressure zone, and with hyperkeratosis. Mean time since injury ranged from 2 to 48 months.

The initial wound areas of the groups varied in size. The mean area of wounds was 2.6 cm² for the control group, 1.9 cm² for the LLLT group, and 5.5 cm²  for the HAM group. The samples were homogeneous regarding age, BMI, postprandial capillary glucose, and wound duration. No statistically significant differences were found in anthropometric and clinical variables among the 3 groups (Table 3). Regarding sensitivity, in the control group, some reported no more tactile sensitivity and/or pain, only numbness in the feet.

All groups demonstrated a significant reduction in percent of wound area when comparing day 7 with day 28 (control: 23.5% vs 65.3%, P < .05; HAM: 25.3% vs 66.9%, P < .05; LLLT: 28.1% vs 87.8%, P < .05). In the LLLT group, the wound area’s decrease became significant on day 21 (64.7%,) compared with day 7 (28.1%) (P < .05). However, the intergroup analysis on day 28 showed no significant difference between the percentage of wound area reduction (87.8%, 66.9%, and 65.36%) in the LLLT, HAM, and control groups, respectively (Table 4).

Macroscopic evaluation using the PUSH scale. In the intragroup comparison using the PUSH scale, the control group showed healing progress during the first 2 weeks (9.4 vs 8.8). At day 21 and at day 28, the control group had constant PUSH scale scores with no statistically significant differences (7.6 vs 7.0).

LLLT intragroup comparison showed a reduction in PUSH scores, with a significant difference noted between days 0 and 21 (8.2 vs 4.9, P < .01) and days 21 to 28 (4.9 vs 3.2, P < .001). A reduction in PUSH scores was noted from days 7 to 28 (7.3 vs 3.2; P < .01) and from days 14 to 28 (6.1 vs 3.2; P < .01).

Likewise, the HAM group showed statistically significant healing progress according to PUSH scale
indexes (Figure 1).

VAS scores. Weekly VAS results showed no significant difference in pain in the intragroup analysis in the clinical protocols studied. However, in the intergroup comparison, persons in the control group had higher pain scores than persons in the HAM group (P < .01). Those in the HAM group reported feeling tingling within 7 days; however, after 7 days, they reported an improvement in pain. Patients in both the LLLT and HAM groups reported pain relief (Figure 2). After day 7 of the application of LLLT, macroscopic aspects of the wounds improved (ie, significant reduction of the lesion and keratosis area, contraction of the edges, and growth in the granulation tissue).

No complications, such as allergic reactions, bleeding, or rejection of the HAM patch, were observed in this study.

The results of this pilot clinical study showed that the LLLT group had a higher percentage of wound area reduction compared with the control and HAM groups but the difference was not statistically significant. The use of phototherapy (ie, LLLT) has been shown to stimulate enzymatic action, thus promoting the lysis of keratosis at the edge of the wound, and thus facilitating mechanical debridement.³¹

The ideal primary dressing for wounds must be nontoxic and nonallergenic. This dressing should have antimicrobial properties, not adhere to the skin, and have the ability to absorb excess exudate while maintaining moisture.³² All of these characteristics are found in the HAM patches for wound treatment.^33,34

Adverse effects. No complications, such as allergic reactions, bleeding, or rejection of the HAM patch, were observed in this study, which corroborates the results from a prospective randomized clinical trial¹⁶ conducted among 24 patients with stage 2 and stage 3 pressure ulcers. Similar to the current study, the authors¹⁶ did not report any significant complications related to pain, infection, necrosis, allergic reaction, or bleeding after using HAM.

PUSH for monitoring healing. In the present study, the PUSH scale was effective in monitoring the weekly evolution of chronic wound healing. This is similar to a case study, presented by nurses specializing in rehabilitation, which demonstrated that the PUSH scale had the potential to identify changes in chronic wounds and thus help to guide clinical decision-making.³⁵ An experimental clinical study also showed that PUSH is an easy tool to use for monitoring and documenting the phases of the chronic wound healing process.⁷

Pain. The HAM group reported less pain compared with the control group. Similarly, in a randomized clinical trial, it was found that patients reported decreased pain when pressure ulcers were covered with cryopreserved HAM.¹⁶

An important property of HAM is pain relief; this may be an effect of its ability to decrease inflammation, provide wound bed hydration, and protect nerve endings.¹² The pain reduction results in this study corroborate randomized experimental clinical studies with HAM. A clinical trial¹⁶ among patients with stage 2 and stage 3 pressure ulcers found that wounds treated with cryopreserved HAM exhibited less pain as compared to conventional wound dressings. In a randomized clinical trial,¹² pain improved in the majority (78.6%) of patients.

Studies also corroborate that LLLT can induce significant biological changes, with increased production of mitochondrial adenosine triphosphate, the release of prostaglandin E₂, as well as stimulation of the beta-endorphin production and several other mediators that contribute to the reduction of pain sensitivity.^36,37 In addition, an experimental, randomized, controlled, prospective, interventional clinical case study using a quantitative approach (n = 32 patients with DM) also showed LLLT to be effective in pain relief in diabetic foot ulcers.¹¹

This study concluded that clinical protocols that used LLLT and HAM were more effective in treating chronic wounds than standard treatment.

This study has several limitations. First, the small sample size may affect the reliability of the results. Second, it was difficult to find patients with chronic foot wounds in the established age range (30 to 59 years). These patients needed to have lesions with similar characteristics and sizes, according to the inclusion criteria. Third, the study lacks a longer follow-up period given that the study concluded before wounds were completely healed. Fourth, the lack of control for offloading, smoking, and arterial blood pressure levels could potentially confound the study results.

This study aimed to compare LLLT, HAM, and control treatments in foot ulcers in patients with DM with regard to the reduction in the wound area as well as the effects on PUSH and pain scores. The wound area reduction as well as PUSH and VAS scores showed significantly more improvement over time for patients with DM receiving LLLT or HAM than for the control group, but differences between groups were not significant. The PUSH and VAS scales were effective in documenting healing progress and pain. Further controlled clinical studies are needed to confirm these findings in the HAM and LLLT groups.

Dr. Santos is a professor of nursing, Department of Nursing, Federal University of Piaui, Teresina, Piaui, Brazil.  Dr. Nicolau is a professor of biomedical engineering, Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, São Paulo, Brazil. Dr. Sant’Anna is a professor of histology, Universidade Estadual de Campinas, São José dos Campos, São Paulo, Brazil. Dr. Paterno is a professor of nephrology, Department of Medicine; Dr. Crostovam is a professor of ophthalmology and visual sciences, Escola Paulista de Medicina; and Dr. Gomes is a professor of ophthalmology, Universidade Federal de São Paulo, São Paulo, Brazil. Mr. Santos is a research assistant, College of Medicine, University of Saskatchewan, Regina, Canada. The work he performed related to this article was not related to his employment. Dr. Arisawa is a professor of oral biopathology, São José dos Campos, São Paulo, Brazil. Address all correspondence to: José Diego Marques Santos,  BScN, MSc, Rua Gonçalves Dias, 4841, Lourival Parente, Teresina, Piauí, Brasil. Cep: 64022230; email: jd_ms@live.com.