Review Article | Open Access
Shedding Light on a New Treatment for Diabetic Wound Healing: A Review on Phototherapy
Impaired wound healing is a common complication associated with diabetes with complex pathophysiological underlying mechanisms and often necessitates amputation. With the advancement in laser technology, irradiation of these wounds with low-intensity laser irradiation (LILI) or phototherapy, has shown a vast improvement in wound healing. At the correct laser parameters, LILI has shown to increase migration, viability, and proliferation of diabetic cells in vitro; there is a stimulatory effect on the mitochondria with a resulting increase in adenosine triphosphate (ATP). In addition, LILI also has an anti-inflammatory and protective effect on these cells. In light of the ever present threat of diabetic foot ulcers, infection, and amputation, new improved therapies and the fortification of wound healing research deserves better prioritization. In this review we look at the complications associated with diabetic wound healing and the effect of laser irradiation both in vitro and in vivo in diabetic wound healing.
1.1. Diabetes and Wound Healing
Diabetes Mellitus (DM) is a chronic metabolic disorder due to an absence of, insufficiency in, or resistance to insulin. Complications arise as a result of elevated glucose levels and protein glycation and include cardiovascular disease, retinopathy, nephropathy, angiopathy, and neuropathy. Patients are more likely to have foot problems due to blood vessel and nerve damage and often suffer from sensory loss. Small sores can develop on the feet and often go unnoticed. These later develop into deeper ulcers which become slow to heal, and further complications such as infection arise which often necessitate amputation due to the spread of infection to the underlying tissue and bone. It is estimated that 15–25% of patients will develop foot ulcers [1, 2], of which 6% of these will require hospitalization to treat these ulcers . Around 15–20% of these patients will require lower limb amputation  and around 50% of all non traumatic amputations are as a result of DM . To further highlight the seriousness of diabetes associated lower-limb amputations, the 5-year mortality rate following amputation stands at 39–80% .
To control the development of lower-limb ulcers, patients are required to check their feet daily, wear the correct footwear, and regularly visit their health care provider. It is estimated that more than 200 million people worldwide suffer from DM, and in 2004, 3.4 million people died as a result of DM . DM and its associated complications impact heavily on the patient, their family, health care departments, and countries. The treatment of chronic wounds is becoming more of a burden due to the increase in health care costs, an aging population, and an increase in the incidence of diabetes .
Despite the huge amount of research into the underlying pathogenesis of impaired diabetic wound healing, there is still no clear answer and it appears to be a net result of micro- and macrovascular disease  and inadequate angiogenesis (Figure 1). Neuropathy and sensory loss have also been recognized as a major cause of prolonged healing in diabetic patients. In addition, advanced glycation end products also contribute to the pathogenesis , and hyperglycemia adds to the oxidative stress.
There is a decrease in wound strength, reduced angiogenesis, and poor wound contraction [8, 9]. In DM, there is a disruption in clot formation and the inflammatory phase is dysregulated [8, 10], often with a prolonged and excessive inflammatory response. Hypoxia is associated with diabetic wounds and further amplifies the inflammatory response . Formation of the extracellular matrix (ECM) is a crucial step in wound healing and provides structural integrity to tissue. In diabetic wound healing, there is a malformation of the ECM due to the disruption in ECM-growth factor interactions and impaired migration and proliferation of fibroblasts . Collagen is an important component of the ECM and is synthesized and maintained by a balance between matrix synthesis and degradation. In DM, there is an imbalance between matrix degrading enzymes, matrix metalloproteases (MMPs), their inhibitors, and tissue inhibitor metalloproteinases (TIMPs). The loss of collagen which is associated with diabetes can be due to decreased levels of its synthesis, enhanced metabolism, or a combination of both . Nonhealing diabetic foot wounds display elevated MMP activity, with a 30- to 60-fold increase in MMP-2 and MMP-9 [11, 13]. Dysregulated cellular functions also play a part, such as defective T cell immunity, leukocyte chemotaxis, phagocytosis, and bactericidal capacity .
Infection of diabetic ulcers remains a real problem. It can become life threatening and is one of the most common causes of lower-limb amputation and appropriate treatment is essential . Wounds are commonly infected with Pseudomonas aeruginosa and Staphylococcus . Infection can spread from one ulcer to another as the foot has several intercommunicating compartments, and combined with sensory loss patients can continue walking on these infected ulcers further facilitating their spread . Ischemia complicates matters further by reducing defense mechanisms. Administration of antibiotics has its own complications especially with the emergence of antibiotic resistant bacteria, poor arterial supply which affects antibiotic delivery, correct duration of treatment, and toxicity and allergy to patients.
1.2. Treatment of Diabetic Wounds
Management of the diabetic foot is multidisciplinary and can become problematic. Treatment is both local (treating of the diabetic foot) and systemic (glycemic control). Treatment of the diabetic foot is extensive and can encompass mechanical and surgical debridement, management of the wound base, antibiotic therapy to treat infection, revascularization, prophylactic foot surgery, mechanical off-loading, accommodative orthotics, and an alteration of footwear [2, 14]. Hyperbaric oxygen therapy (HBOT), which entails delivering 100% oxygen at pressures above one atmosphere, increases the amount of oxygen dissolved in the blood and has been used to treat a variety of wounds [12, 15]. HBOT has been used as an adjunctive treatment for diabetic foot ulcers; however its evidence of efficiency is limited . Kaya and colleagues  treated 184 patients with diabetic foot ulcers with HBOT. Sixty-three percent of patients responded to treatment, while 17% showed no improvement and 21% underwent amputations. Complications associated with HBOT are not common but can include claustrophobia, ear, sinus, or lung damage, temporary worsening of short sightedness, and oxygen poisoning .
A number of clinical applications have been found for lasers in a variety of medical specialities and have been used in dentistry, dermatology, osteology, physiotherapy, acupuncture, surgery, photodynamic cancer therapy, and chiropractic and veterinary science. Lasers have also been used in the treatment of chronic wounds, including diabetic ulcers.
Phototherapy, also known as photobiomodulation, low-level laser therapy (LLLT), involves the application of light (often laser light of a specific wavelength or a light emitting diode, LED) to stimulate cellular processes.
The effects of phototherapy are chemical and not thermal. Energy which is delivered to cells produces insignificant and minimal temperature changes, typically in the range of 0.1–0.5°C . Cellular responses are the result of changes in photoacceptor molecules, or chromophores. Photoacceptors take part in cellular metabolism and are not connected to a light response, such as chlorophyll which is a photoreceptor . Once the photon energy is absorbed, the photoacceptor assumes an electronically excited state , which in turn stimulates cellular metabolism [19, 20] by activating or deactivating enzymes which alter other macromolecules such as DNA and RNA [21, 22]. The energy which is absorbed by the photoacceptor can be transferred to other molecules causing chemical reactions in the surrounding tissue; this then gives rise to observable effects at a biological level [18, 23]. Photon energy is absorbed by the chromophores and there is an increase in adenosine triphosphate (ATP) [24, 25] and cell membrane permeability, which leads to activation of secondary messengers which in turn activate a cascade of intracellular signals . There is also an increase in mitochondrial membrane potential and proton gradient .
The exact mechanisms of action following laser irradiation are not well understood, and a number of theories exist, the most studied and best understood being that of cytochrome-c oxidase (cyt ), the terminal enzyme in the eukaryotic mitochondrial respiratory chain (complex IV). Cytochrome c oxidase facilitates the transfer of electrons to molecular oxygen. The end product of this complex is the production of ATP. Cytochrome c oxidase has two heme moieties (heme and heme ) and two redox-active copper sites (CuA and CuB), and these are the possible absorbing chromophores for visible red and near infrared (NIR) light [18, 27, 28]. When photon energy is absorbed by cytochrome c oxidase, there is a change in the mitochondrial redox state and/or pumping of protons across the inner mitochondrial membrane  and an increase in ATP synthesis. There is also an increase in intracellular calcium ([Ca2+]i) which stimulates DNA and RNA synthesis . It has been speculated by Karu  that photoirradiation may intensify the transfer of electrons within cytochrome c oxidase by making more electrons available. An increase in the transfer of electrons and protons accelerates oxidative metabolism which ultimately leads to increased ATP . Photoirradiation causes the reduction or oxidation of cytochrome c oxidase and is dependent on the initial redox status of the enzyme at the time of irradiation . Silveira and colleagues [25, 29] showed that LILI produced an increase in mitochondrial complexes I, II, III, and IV, as well as succinate dehydrogenase. Hu and colleagues  also found an increase in cytochrome c oxidase activity and concluded there was a cascade of reactions which altered cellular homeostasis. Houreld et al.,  showed that irradiation of isolated mitochondria resulted in an increase in cytochrome c oxidase (complex IV) activity. There is also an increase in the concentration of active mitochondria in irradiated cells. Both effects lead to an increase in ATP. The effect of laser irradiation on the mitochondria at a transcriptional level was also investigated, and there is evidence that that there is an upregulation of genes involved in complexes I, IV, and V  (Figure 2).
A second possibility is the localized transient heating of the photoacceptor which may cause structural changes and trigger mechanisms such as activation or inhibition of enzymes . Another theory is the release of nitric oxide (NO) from reduced cytochrome c oxidase which reverses the signalling consequences of excessive NO binding [28, 33, 34], as NO in very low concentrations inhibits cytochrome c oxidase by competing with oxygen [33, 34].
2.1. In Vitro Effects of Photoirradiation
A number of studies, on various cell types, have shown positive effects of photoirradiation. Studies have been conducted on stem cells [35–38], keratinocytes [39, 40], mast cells [41, 42], fibroblasts [43–48], smooth muscle cells , osteoblasts [50, 51], and schwann cells  to name but a few.
Impaired diabetic wound healing has been associated with impaired cellular function, and there is a decrease in cellular migration, proliferation, NO synthesis, growth factors, and collagen synthesis. There is also an increase in proteinases that degrade the extracellular matrix and collagen (MMPs) and cells appear to be stuck in the inflammatory phase of wound healing. The increase in oxidative stress also leads to increased cell death. Laser irradiation in vitro has shown that these cells respond in a favourable fashion, even irradiation of diabetic cells (Table 1). There is an increase in cellular migration [45, 46], proliferation [44, 46, 53–56], viability [44, 46], collagen production [45, 49, 57–59], ATP , mitochondria concentration , cytochrome c oxidase activity , NO , growth factors [57, 58, 61, 62], and gene regulation . There is also a decrease in MMPs , apoptosis [46, 55, 63] and proinflammatory cytokines .
Irradiation of hypoxic cells has also shown favourable effects, with an increase in ATP and cyclic adenosine monophosphate (cAMP) , proliferation [46, 51], viability , transforming growth factor-1 (TGF-1) , intracellular Ca2+  and mitochondrial membrane potential , and a decrease in apoptosis and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-) . Irradiation of hypoxic/ischemic cells resulted in reduced ROS, which results in increased angiogenesis . Laser irradiation restores homeostasis of injured and stressed cells, resulting in improved repair and wound healing.
Not all studies have shown positive effects. Pereira et al.  and Marques et al.  showed that laser irradiation of fibroblast cells had no effect on the synthesis of procollagen. In fact there were ultrastructural changes to the endoplasmic reticulum which may have resulted in a disruption in protein synthesis . Damante et al.  demonstrated that irradiation at 660 nm had no effect on basic fibroblast growth factor (bFGF). Interestingly, irradiation of the same cells at 780 nm significantly increased bFGF. Hakki and Bozkurt  irradiated human gingival fibroblasts by different laser parameters and found no increase in proliferation at each of the parameters used. However, they did find an increase in the transcription of various growth factors, namely, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-). Irradiation of hypertrophic scar-derived fibroblasts and normal dermal fibroblasts at a wavelength of 880 nm and a fluence of 2.4 and 4 J/cm2 had an inhibitory effect . Pereira and colleagues  found no benefit when they irradiated human dental pulp stem cells at 660 nm using various fluencies (0.05, 0.30, 7, and 42 J/cm2). Schwartz-Filho and colleagues  showed that irradiation at a wavelength of 685 nm with a density of 25, 77, or 130 J/cm2 had no effect on osteogenic cell growth or viability.
These adverse effects and difference can be explained by differences in laser parameters. The effects of laser irradiation are highly dependent on the laser parameters such as wavelength, power density, and fluence. Cells respond to LILI in a dose- and wavelength-dependent manner, and the number of exposures as well as the time between exposures plays an important role [47, 70–72]. Higher fluencies have a negative effect on cells, while too low fluences have no effect. The influence of wavelength was demonstrated by Gupta et al.,  who demonstrated that irradiation at 635 and 810 nm had a positive effect on wound healing, while a wavelength of 730 and 980 nm had no effect. This can be explained by the absorption spectrum of chromophores which absorb light at different wavelengths.
2.2. In Vivo Effects of Photoirradiation
A limited number of clinical studies have been done on diabetic wound healing (Table 1). A reason for the small number of randomized trials may be due to ethical issues associated with doing human clinical trials . A number of studies using phototherapy in animal models have been done (Table 1).
Al-Watban  irradiated Sprague-Dawley rats () to different wavelengths (532, 633, 810, 980, 10,600 nm, and 510–872 nm LED cluster) and fluencies (5, 10, 20, and 30 J/cm2). He showed that phototherapy at a wavelength of 633 nm accelerated healing and was the best for alleviating diabetic wounds and burn healing. It was suggested that phototherapy with 633 nm should be given three times a week at a fluence of 4.71 J/cm2 per dose for the treatment of diabetic burn wounds or 2.35 J/cm2 for diabetic wounds. Chung et al.  treated full-thickness circular wounds (5 mm) in diabetic mice (type 2 diabetes) with a 660 nm laser to various fluencies (0–10 J/cm2) for seven consecutive days. Wounds were splintered to minimize contraction; the main process of healing would thus be epithelization and granulation. On day 14 mice were euthanized and the wound excised. At a fluence between 3.7 and 10 J/cm2, splinted irradiated wounds responded better and healed quicker than nonirradiated splintered wounds. Santos et al.  irradiated cutaneous flaps with poor circulation in diabetic Wistar rats either with 680 or 790 nm (2.5 J/cm2 per point). It was shown that angiogenesis was increased in irradiated rats compared to control, non-irradiated rats, more so when a wavelength of 790 nm was used.
Irradiation of diabetic male Wistar rats reduced the expression of MMP and accelerated collagen production . Firat and colleagues  irradiated full thickness wounds in diabetic male Wistar rats with a 940 nm diode laser (10 J/cm2). Histopathological analysis revealed that there was a decrease in inflammatory cells and an increase in collagen and vascularization. Blood tests showed that there was a decrease in oxidative stress. LILI has been found to promote healing in both soft and hard tissue. Irradiation at 980 nm has been shown to stimulate wound healing and the formation of new bone . Peplow and colleagues  demonstrated that the effect of irradiation is due to cellular and biochemical changes in the wound environment, rather than a hypoglycemic effect. One of the long-term complications of diabetes includes musculoskeletal abnormalities, and it is a source of disability in these patients. Laser irradiation (780 nm, 5 J/cm2) of cryoinjured diabetic male Wistar rats showed improved muscle repair, with enhanced reorganization of the myofibers and the perimysium and reduced fibrosis .
Caetano et al.  conducted a randomized placebo-controlled double-blind study on 20 patients with a total of 32 chronic venous ulcers. Patients were divided into three groups: in group one (placebo), ulcers were cleaned with saline and treated with 1% silver sulfadiazine (SDZ) cream and patients received a placebo phototherapy; in group two ulcers were treated similarly and patients received phototherapy (combined 660 nm and 890 nm at a fluence of 3 J/cm2); and in group three (controls) ulcers were treated similarly and received no phototherapy. Patients that received phototherapy responded to the laser treatment, and ulcers healed significantly faster than the control and placebo group, particularly larger ulcers. Minatel et al.  showed that combined irradiation with 660 and 890 nm promoted granulation and healing of diabetic ulcers that failed to respond to other forms of treatment. Kaviani et al.  performed a randomized study on diabetic patients with foot ulcers that would not respond to other treatments. Patients were divided into two groups: group one received conventional treatment and placebo irradiation, while group two received conventional treatment and phototherapy (685 nm, 10 J/cm2). The size of the ulcers in the phototherapy treated group was significantly smaller and the average time of healing was 11 weeks as opposed to 14 weeks as observed in the placebo group.
Infection in diabetic wounds is a major problem, and eradication with antibiotics proves difficult due to decreased blood flow. Laser irradiation has also been shown to inhibit bacteria. Enwemeka et al.  showed a dose-dependent decrease in the number of methicillin-resistant Staphylococcus aureus (MRSA) treated in vitro at a wavelength of 470 nm (blue light). Irradiation at a wavelength of 408 nm was suggested by Ankri and colleagues  in treating infected wounds to clear the infection, followed by irradiation at 730 nm to speed up the healing process. This is an important breakthrough as combined irradiation with visible red and blue light can potentially be used to treat infected diabetic ulcers.
DM is the leading cause for lower limb amputations. Current treatments are challenging, lengthy, costly, and associated with failure to heal and relapse. The patient’s quality of life is affected, and a burden is placed on both patients and caregivers. There is a need to develop additional therapies to treat diabetic ulcers. Due to its stimulatory effect and no reported sideeffects, laser therapy has been used to treat chronic wounds, including diabetic ulcers. Phototherapy has been shown to be beneficial in treating diabetic ulcers which are unresponsive to conventional treatments. This has led to an improvement in the quality of patient’s lives. By studying the effects of LILI in vitro, the underlying mechanisms are being identified. The number of clinical studies in DM is limited, and there is methodological heterogeneity which explains the varied results seen. Better designed, well-controlled, randomized, and double-blind studies are needed for this type of therapy to become accepted and used as an adjuvant therapy for the treatment of diabetic ulcers. Phototherapy can be an important tool in speeding up the healing process as well as alleviating pain and inflammation. There is also a need to inform clinicians and other health care providers of the beneficial effects of phototherapy.
Conflict of Interests
The material in this research paper neither has been published nor is being considered elsewhere for publication.
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Copyright © 2014 Nicolette N. Houreld. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.