Magnetic fields as a potential therapy for diabetic wounds based on animal experiments and clinical trials

Abstract Diabetes mellitus (DM) is a chronic metabolic disorder with various complications that poses a huge worldwide healthcare burden. Wounds in diabetes, especially diabetic foot ulcers (DFUs), are difficult to manage, often leading to prolonged wound repair and even amputation. Wound management in people with diabetes is an extremely clinical and social concern. Nowadays, physical interventions gain much attention and have been widely developed in the fields of tissue regeneration and wound healing. Magnetic fields (MFs)‐based devices are translated into clinical practice for the treatment of bone diseases and neurodegenerative disorder. This review attempts to give insight into the mechanisms and applications of MFs in wound care, especially in improving the healing outcomes of diabetic wounds. First, we discuss the pathological conditions associated with chronic diabetic wounds. Next, the mechanisms involved in MFs’ effects on wounds are explored. At last, studies and reports regarding the effects of MFs on diabetic wounds from both animal experiments and clinical trials are reviewed. MFs exhibit great potential in promoting wound healing and have been practised in the management of diabetic wounds. Further studies on the exact mechanism of MFs on diabetic wounds and the development of suitable MF‐based devices could lead to their increased applications into clinical practice.


| INTRODUC TI ON
Skin, the largest organ in human body, has important immune and protective traits also with amazing ability to self-repair. [1][2][3] After injury, multiple biological pathways become activated resulting in re-establishment of tissue integrity. Based on the time required for healing, wounds can be classified into acute and chronic wounds.
Acute wounds can be repaired by themselves through the normal healing process resulting in the functional restoration; chronic wounds cannot be repaired through the normal and timely way resulting in prolonged or incomplete repair. 4 Normal wound repair follows coordinated sequence of haemostasis phase, inflammation phase, proliferation phase and remodelling phase involving crosstalk between different cells, extracellular matrix (ECM) and cytokines in time and spatial dimensions. 5,6 Chronic wounds develop when there are disruptions in normal healing process and are big challenges to people with diabetes. 7 In diabetes mellitus (DM), chronic wounds are common on the lower extremities, particular happening at foot, it is so-called diabetic foot ulcer (DFU). 8,9 Development of chronic wounds in people with diabetes possibly results in high risk of limb amputation if they are not treated effectively. 10  have been focused on wound care with an emphasis on the physical approaches and the development of related device for enhancing the healing rate of diabetic wounds. 11,12 Over the history, people have explored magnetic fields (MF) from not only nature but also artificial sources for therapeutic uses. Nowadays, MF has been developed as an alternative, noninvasive and safe therapeutic tool for tissue repair due to the beneficial effects on cell migration, proliferation and adhesion. [13][14][15][16][17][18] In spite of MFs' potential for their therapeutic application, the safety of MFs including static magnetic field (SMF), extremely low-frequency electromagnetic field (ELF-EMF) and pulsed electromagnetic field (PEMF) is still widely discussed and considered.
International Commission on Non-Ionizing Radiation Protection (ICNIRP) demonstrates that no evidence supports the adverse effect on human after exposure to up to 8 T SMF. 19 There is no adverse effect to experimental mice that are short-termly or longtermly exposed to high or even ultrahigh SMF. [20][21][22] At present, it reaches no exact conclusion about the adverse impact of dynamic MFs on human body, moreover, dynamic MFs are widely used in clinics for treatment of bone diseases and neurodegenerative and related disorder. [23][24][25][26] MFs affect cellular function and activities by their actions of electric/magnetic properties or magnetic property alone on cellular processes and functional molecules and act as a kind of potential therapy for wound repair as far as wounds in DM. 16,17,[27][28][29] The present review focuses on the pathological conditions associated with chronic wounds in DM. Then, the possible mechanisms involved in MFs' effects on wound healing are explored. Last, we review the therapeutic effects of MFs on diabetic wounds both from animal experiments and clinical studies and attempt to arouse the interest of pushing forward the applications of MFs on wound healing in DM.

| IMPAIRED WOUND HE ALING IN D IAB E TI C MELLITUS
DM is characterized by hyperglycaemia which is a significant cause in the development of inflammation in diabetic complications. 30 Hyperglycaemic condition and oxidative burden cause modifications and dysfunctions to cells that participate in wound repair and promote inflammation resulting in inhibitory effects on wound healing. 31,32 Oxidative stress in DM may generate from glucose metabolism and auto-oxidation or through the formation of reactive oxygen species (ROS) and advanced glycation end products (AGEs). A state of persistent hyper glycaemic condition the delay of wound healing in DM and promotes the development of chronic wounds. 33 Wound healing is a highly coordinated biological process. [34][35][36] After injury, various types of cells, such as platelets, neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells migrate to wounds to initiate and regulate the repair process. Intrinsic abnormalities and pathological factors in DM disturb the normal activities of cells participate in wound healing and affect their secretions and the communication network which further interrupts the coordinated cascade of events in wound repair process 37 (Figure 1). Impaired wound healing is commonly encountered in people with diabetes and leads to severely unfavourable outcomes. 32

| DM affects the function of platelets in wound healing
Platelets initiate the earliest events after injury and form a platelet plug in haemostasis phase. Platelet-derived growth factor (PDGF) stimulates the migration of cells to wounds in inflammation phase, enhances the proliferation of fibroblasts and production of ECM in proliferation phase and regulates matrix metalloproteinases (MMPs) in remodelling phase. 38 The abnormalities of platelets in DM are characterized to be hyperactive with increased autophagy, activation, adhesion and aggregation. [39][40][41][42][43] These abnormalities in platelets lead to wound healing dysfunction. Strategies using functional platelets and the combination of their secretions exhibit significant outcomes in managing wounds in DM. 44,45

| DM affects the function of neutrophils in wound healing
Neutrophils are mobilized to inflammatory site upon injury and involved in the early stage of inflammation phase to form a weblike structure called neutrophil extracellular traps (NETs). [46][47][48][49][50][51] After completing their function, neutrophils must be eliminated or migrate away within a defined time period. Otherwise, excessive infiltration and retention of neutrophils lead to delayed wound healing. 52,53 The expressions of microRNA in neutrophils involved in inflammatory response are changed in DM. 54 DM condition increases the release of NETs through facilitating NETosis, and the exacerbated release of NETs is a key factor for the delayed wound healing. [55][56][57] Inhibition of NETosis by using gonadotropin-releasing hormone (GnRH) antagonist improves the delayed wound healing in diabetic mice. 58

| DM affects the function of macrophage in wound healing
Macrophages affect the whole wound healing process by displaying different polarization phenotypes. 59 The events associated with the end of inflammation phase to the start of proliferation phase are the removal of macrophages by lymphatics and the transition of macrophage polarization from M1 to M2 phenotype. 60 The hyperglycaemic environment imprints epigenetic modulation in macrophages towards a pro-inflammatory phenotype which fails to transit to the anti-inflammatory state. 31 AGEs modulate macrophage polarization to M1 phenotype which impairs wound healing in DM. 61 The imbalance transition between two functional phenotypes of macrophage induces the delayed healing process in diabetic wounds. 62 Treatments with macrophage-secreting cytokine transforming growth factor (TGF-β1) or macrophage-derived exosomes accelerate diabetic wound healing. 63,64

| DM affects the function of keratinocytes in wound healing
Keratinocyte is the major cell type of epidermis, its migration and proliferation are important for re-epithelialization in wound healing process. 76 Under diabetic condition, keratinocytes exhibit reduced proliferation potential, less migration capacity, abnormal gap junction and expression of MMPs. [77][78][79] Diabetic condition reduces keratinocytes migration through indirectly changing the activity of macrophage and creating a micro-environment with high level of tumour necrosis factor α (TNFα). 80 Human keratinocytederived micro-vesicle that expresses miR-21 mimic promotes F I G U R E 1 Diabetic condition disturbs the normal activities of cells participate in wound healing. Abbreviations: NET, neutrophil extracellular trap fibroblasts migration, differentiation, contraction and induce a proinflammatory response. 81

| DM affects the function of endothelial cells in wound healing
Vasculogenesis occurs at the late stage in wound healing process.
Endothelial cells are responsible for the development of new vessels. Oxidative stress induced by hyperglycaemia causes damage to endothelial cells which further leads to endothelial dysfunction which is implicated as an underlying deterrent to diabetic wound healing. [82][83][84] The migration pathway of endothelial progenitor cells from bone marrow to the diabetic wounds is dysfunctional. 84,85 In diabetic condition, phagocytes activated by inflammatory cytokines inhibit the migration and recruitment of endothelial progenitor cells to the wound sites. 86 The ways to increase the migration and proliferation of endothelial progenitor cells by using platelet-rich plasma or anti-diabetic drug promote diabetic wound healing. 87,88

| THE EFFEC TS OF MAG NE TIC FIELDS ON WOUNDS
Based on the intensity and direction, MFs are classified as static magnetic field (SMF) and dynamic MF. The intensity and direction are both constant in SMFs, while they are varied with time in dynamic MFs. Studies have proved that MFs improve cell migration and adhesion which could be extremely beneficial to tissue regeneration and wound healing. 28,29 As a result, MFs, especially dynamic MFs, have been widely developed as an effectively therapeutic tool for repairing tissues.

| The effects of SMFs on wound healing
SMF is generated by permanent magnets or by passing direct current through a coil. There is no electric energy in SMF, therefore there is no heat and electrical harm to the tissues. It has been shown great potential in the field of tissue regeneration. 89 SMF assists in wound healing through promoting cell migration by affecting cellular orientation, morphology and migration. 89 Different intensities (10, 50, 80 and 100 mT) of SMFs cause no effect on cell viability and no damage on cell membrane of mouse embryonic fibroblasts; however, cell morphology becomes elongated with protrusions and short microvilli possibly as a result of re-arrangement of cytoskeleton. 90 The field orientation of SMF affects the bio-effects differently when applied to wound healing. 91,92 Exposure to SMF of perpendicular other than parallel direction to the incision increases the strength of cutaneous wounds that are closed primarily. 93 Wound tissues that exposed to the North pole of SMF show better healing outcome. 94 Also, local exposure to 180 mT SMF with two attracted neodymium magnets encourages fast wound healing. 95 140 120 μT increases proliferation potential and upregulates endothelial nitric oxide synthase (eNOS) expression in human umbilical vein endothelial cells. 96 A double-blind placebo-controlled pilot study also demonstrates that application of SMF device promotes leg ulcer healing. 97 Contrasting studies, however, suggest that there are no differences between gross healing parameters, mechanical strength and hydroxyproline deposition regarding wound healing processes in rat with a magnet in contact with wound or not. 98

| The effects of dynamic MFs on wound healing
Dynamic MFs are also able to affect cell morphology, differentiation and function. 28,99 As to their applications in wound healing, it mainly includes ELF-EMF and PEMF. 100,101 ELF-EMF represents a form of non-ionizing and low energy radiation with frequency induce a variety of biological effects. 101 Several studies demonstrate that ELF-EMF exhibits driving actions on the progression of wound healing. On one hand, ELF-EMF modulates cytokine profile which drives transition from chronic proinflammatory state to anti-inflammatory state in wound healing process. 102 On the other hand, exposure to ELF-EMF also drives a shift in wound healing process from inflammation phase to proliferation phase. 103 ELF-EMF exposure enhances the proliferation of keratinocyte HaCaT cells and improves early NOS activity, while decreases cyclooxygenase 2 (COX-2) which indicates its role in accelerating the transition from inflammation phase to remodelling phase. 104 These results hint that ELF-EMF may play different roles in different phases of wound healing and promote the progression of wound healing.
Moreover, ELF-EMF has been shown to alter the function of other participants in wound healing. Exposure to ELF-EMF with frequency of 50 Hz and intensity of 1 mT increases cytokine release and activates the expression of MMP-9 in human immortalized keratinocytes. 105 The upregulation of MMP-9 represents the effect of ELF-EMF on promoting cell migration and inducing phagocytosis in inflammation phase of wound healing. 106 ELF-EMF increases cellular ROS production in human keratinocyte cell line NCTC 2544. 107 On the contrary, ELF-EMF activates glutathione peroxidase with decrease in malondialdehyde in the live tissue of rats during wound healing process. 108 ELF-EMF promotes the proliferation and differentiation of transplanted epidermal stem cells in the full-thickness defect nude mice with more mature generated skins and viable cell layers and rich hair follicles' structure at the wound sites. 109 ELF-EMF also directly acts on the ion channel to affect cellular function. Exposure to 50 Hz ELF-EMF activates macrophage/monocyte through regulating Ca 2+ ion channel. 110 After being exposed to ELF-EMF, the morphology of macrophages change to elongated shape, because the cluster of cation channel receptor alters Ca 2+ homeostasis and further affects actin polymerization. 111 PEMF is a kind of low-frequency magnetic field with specific wave shape and amplitude. 18

| MAG NE TI C FIELDS PROMOTE D IAB E TI C WOUND HE ALING
After being exposed to the actions of MFs, cellular participants, cytokines and ion channel exhibit alterations in their performance in wound healing process. 100   enhances wound closure and re-epithelialization with production of myofibroblasts which play a key role in wound closure and collagen synthesis in wound healing process. 124 In an animal study, diabetic rats exposed to LF-PEMF show reduced time of wound healing and increased tensile strength of scar. 125

| The effects of MFs on diabetic foot ulcers from clinical trials
DFUs, one of the most common and severe complications of DM, are characterized with severely impaired wound healing. 126,127 The aetiology of DFU is multifactorial and classified into neuropathic, ischaemic and neuro-ischaemic ulcers. 128 Thus, in view of the complexity of origins of DFU, it is of great importance to understand the differences and healing process in these types of DFUs for effective prevention and management.
The physical interventions to improve the healing outcomes of DFUs include negative pressure, electrical fields, lasers, ultrasound, shockwaves and dynamic MFs. [132][133][134] Accumulative studies from cellular level and animal models investigate the effects of MFs on wound healing process and demonstrate their positive effects.
There are also commercially available MFs devices developed for clinical applications on wound healing. Trials that apply MFs on DFUs management are summarized in Table 3.  In this review, factors that may explain the discrepancy in MFs' effectiveness in diabetic wound healing are concluded as following. First, the constructed and used diabetic wound models should be considered. As seen in Table 1 The third factor is the exposure manner of MFs ( Figure 3). It can be classified into local exposure and whole-body exposure. SMFs generated from permanent magnets are easily used to directly place near the wound sites, and it is also possible to achieve whole-body exposure. The diabetic wounded animals are exposed to dynamic MFs whole-body, it is hard to investigate whether the positive effects on wound healing are ascribed to their effects on wound sites or the regulation on the whole body. The effects of MF exposure manners make the mechanism involving in diabetic wound healing even more complex to explain.

| FAC TOR S AFFEC T THE OUTCOME S OF MAG NE TI C FIELDS ON D IAB E TI C WOUNDS
The bio-effects of MFs are largely dependent on the stimulation time, so the forth factor is the duration of MFs exposure. Wound healing is a complex process that matters of time. It is important to choose the suitable duration and frequency of MFs exposure and in which wound healing stage MFs exposure can work best. As to dynamic MFs exposure, the tissues experience a heated process, the short duration of dynamic MFs exposure may protect the biological tissues from development of increased temperature.
As far as we known from Tables 1-3

| CON CLUS IONS
To summarize, MF as a kind of noninvasive and safe physical therapeutic approach has been shown great potential of application prospects in diabetic wound healing with no significant side effects.
Although a majority of studies have indicated the positive effects of MFs on diabetic wounds, there is still no general agreement on the exact mechanisms related to such biological or therapeutic effects, and there remain many unknown aspects to focus on. It is also encouraged to develop more domestically portable equipment generating MFs to manage chronic wounds for people with diabetes at home, and push forward more accessible usage of physical therapy to reduce both the mental and financial burden in people with diabetes.

ACK N OWLED G EM ENTS
This work was carried out with the support of the 2019 Zhejiang Postdoctoral Science Fund, the National Natural Science Foundation of China (NO. 81803032 and NO. 52037007) and the Natural Science Foundation of Shaanxi Province (NO. 2019JQ632).

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
HH Lv wrote the manuscript. JY Liu drew the figures. CX Zhen, YJ Wang, YP Wei and WH Ren revised the manuscript. P Shang supervised the manuscript. All authors read and approved the final version of the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the conclusions of this work are available from the first author and the corresponding author.