Diabetes mellitus (DM) is a clinical syndrome characterized by high blood glucose levels, mainly related to a deficiency or absence of insulin secretion by β-pancreatic cells or a deficiency in peripheral insulin signaling
[20][1]. In 2030, the number of people living with diabetes is expected to increase by 25% based on the reported number of cases in 2019
[21][2]. Patients with diabetes are likely to suffer from many complications related to this condition, such as nephropathy, retinopathy, and neuropathy
[22][3]. Among the different types of diabetes, type 1 and type 2 are the most prevalent
[23[4][5],
24], with type 2 comprising almost 90% of reported diabetic cases
[25][6]. Whereas type 1 diabetes mellitus (T1DM) is related to an autoimmune response, leading to β-cell destruction and insufficient insulin production, type 2 (T2DM) is mainly characterized, at least in the early phase of the disease, by insulin resistance
[25][6]. Patients with T1DM are more likely to develop microvascular complications, while T2DM patients are more prone to macrovascular complications
[24][5]. The development of diabetic foot is considered a major public health problem, since the patients present with impaired wound healing, leading to diabetic foot ulcer (DFU) development
[26][7].
1.1. Diabetic Foot Ulcers
Diabetic wounds (DWs) represent one of the most frequent and devastating complications of DM, directly impacting patients’ morbidity and quality of life
[5][8]. DWs might differentiate according to internal or external origins. The wounds of external origin, such as from cuts and injuries, might remain unnoticed by patients due to the peripherical neuropathy caused by diabetes
[6][9]. On the other hand, the wounds of internal origin, such as ulcers and calluses, cause skin destruction and have an increased rate of bacterial infection
[6][9]. In such cases, the DFU begins as a superficial ulcer, progressing to a deep-tissue infection and then in the final step to osteomyelitis
[27][10]. The three major factors contributing to DFU are neuropathy, vasculopathy, and infection
[28][11].
1.1.1. Diabetic Peripheral Neuropathy and Vasculopathy
Diabetic neuropathy is a neurodegenerative disorder of the peripheral nervous system that targets sensory, autonomic, and to a lesser extension motor neurons
[29][12]. Foot ulceration can develop via motor neuropathy due to the foot’s intrinsic muscle weakness; sensory neuropathy, which promotes unnoticed trauma; and finally autonomic neuropathy, which decreases sweating and leads to xerosis development
[9][13]. Peripheral nerve dysfunction has been strongly correlated with microvascular complications caused by diabetes
[23][4]. In fact, the prolonged inflammation status caused by hyperglycemia in the microcirculation might lead to the thickening of the capillaries’ basement membrane and endothelial hyperplasia, which impair nutrient and white blood cells movement, leading to tissue ischemia
[23][4]. Microvascular disease can also affect the nervous system through nerve fiber deterioration, altering the thermal and vibration sensitivity thresholds, thereby favoring the development of neuropathy
[20][1]. In this sense, small undetected wounds may turn into serious complications, such as DFUs.
Diabetes arterial hypertension is also another suggested risk factor for microvascular dysfunction, neuropathy, and cardiovascular disease development
[23,30][4][14]. The evidence suggests that controlling hypertension levels in patients with diabetes through the use of medicaments decreased the risk of developing microvascular and large-vessel dysfunctions
[31][15]. In diabetes, the endothelial dysfunction and inflammation caused by large-vessel diseases, such as atherosclerosis and vascular calcification, are amplified, increasing the risk of DFU development
[30][14]. Despite both micro- and macrovascular diseases being considered risk factors for peripheral arterial disease (PAD), a five-year follow-up with T2DM patients showed that only microvascular disease was an independent predictor of PAD, being associated with lower-limb ulceration development and amputations
[32][16].
1.1.2. Infection and Wound Healing
In general, wounds can be characterized as acute, which have a normal healing process, and chronic, where the healing process is impaired
[33][17]. A normal wound healing process is composed of four overlapping steps, namely hemostasis, inflammation, proliferation, and remodeling
[34][18]. These phases are orchestrated by different cell types that progressively operate within the wound milieu, namely monocytes, macrophages, neutrophils, keratinocytes, and fibroblasts
[35,36][19][20]. The first phase is characterized by the release of growth factors by the platelet plug that is formed during primary hemostasis, such as transforming growth factor beta (TGF-β) and epidermal growth factors
[5][8]. During the subsequent inflammatory phase, neutrophils recruited to the wound site initiate phagocytosis to remove foreign materials, bacteria, and damaged tissue
[34][18]. Monocytes are stimulated to migrate to the tissue and release inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6)
[37][21]. After neutrophil infiltration ceases, macrophages continue the phagocytosis process to clean the wound site in preparation for the next phase
[33,34][17][18]. Following the proliferative phase, the processes of angiogenesis, collagen deposition, granulation tissue formation, re-epithelialization, and wound contraction occur
[18][22]. The re-epithelialization process results from the keratinocytes’ migration and proliferation to the skin surface. Afterwards, during the remodeling phase, the previously deposited collagen is remodeled and the unnecessary cells present in the wound undergo apoptosis
[33][17].
The chronic wound state is characterized by a persistent injury state during wound healing due to the lack of or the severe slowdown of at least one of the healing phases
[35][19]. Chronic wounds are usually infected and exhibit a persistent aberrant inflammatory profile, with hyperproliferative keratinocytes, elevated matrix metalloproteases, and poor fibroblast infiltration and angiogenesis
[36,38][20][23]. Chronic wounds represent one of the major public health issues worldwide, impairing patients’ quality of life and mobility
[39][24]. Almost 90% of chronic wounds contain microorganisms such as bacteria and fungi, creating a multispecies biofilm that protects them from antimicrobial therapies and the immune system
[6,8][9][25]. Diabetic foot infections caused by
Pseudomonas aeruginosa,
Escherichia coli,
Citrobacter spp.,
Acinetobacter spp., and
Staphylococcus aureus usually develop non-healing chronic wounds
[16][26]. The polymicrobial biofilm infection can impair wound healing with more than a single bacterial infection, becoming even more resistant to antimicrobial therapies
[40][27].
The wound healing process in patients with diabetes is impaired due to the multifactorial disruption of cellular coordination and the molecular process underlying wound repair
[5][8]. Moreover, diabetes causes microvascular complications, favoring the local wound ischemia and delaying the healing process
[6][9]. The increased blood viscosity, dysfunctional activity of polymorphonuclear neutrophils, and declined delivery of nutrients and oxygen to the wound site promoted by diabetes are also important factors that justify the wound healing delay
[33][17]. Moreover, DWs usually exhibit a persistent inflammatory phase, impairing tissue granulation and the connective tissue’s tensile strength
[6][9].
Diabetes impairs the functions of neutrophils and macrophages, including cell adherence, chemotaxis, phagocytosis, and cytokine production and secretion
[41][28]. The hyperglycemia and oxidative stress that diabetes patients are exposed to lead to epigenetic changes, thereby altering the macrophages’ polarization
[42][29]. In fact, lower numbers of macrophages are found in DWs, contributing to impaired tissue repair
[43][30]. Macrophage migration inhibitory factor (MIF) was also found to decrease in DM
[44][31], which could affect the recruitment of endothelial progenitor cells
[45][32].
Beyond macrophage dysfunction
[43][30], animal models of diabetes show impaired fibroblast response to growth factor stimulation
[46][33], as well as impaired keratinocyte and fibroblast migration and proliferation
[47][34]. Diabetes might also impair wound healing by decreasing the growth factors responsible for the extracellular matrix formation, such as keratinocyte growth factor (KGF) and fibroblast growth factor (FGF)
[48,49][35][36]. Beyond this, the expression of receptors can also be reduced, such as transforming growth factor β receptor type 1 (TGF-βR1), which is responsible for fibroblast-to-myofibroblast differentiation during the granulation tissue formation process, leading to deficient re-epithelization and wound contraction
[50][37]. In fact, IGF-1 expression in animals with diabetes was also reported to be delayed and decreased during the healing process when compared with non-diabetic animal models
[51][38].
Delayed vascular regeneration was also observed in diabetes due to many factors, such as receptor dysfunction, a reduction in the number of essential ligands or cells, and impaired epithelial-to-mesenchymal transition (EMT)
[49][36]. In fact, a reduced functional capillary density and decreased angiogenesis-positive area were observed in animal models with diabetes, showing impaired vascular regeneration
[26][7]. Moreover, the levels of the transcription factor zinc finger E-box binding homeobox 1 (ZEB1) were observed to be responsive to the hyperglycemia status, and the dysregulation in ZEB1 expression led to a defective EMT towards wound epithelialization and poor angiogenesis
[52][39].
The severity of DFU is classified by the Wagner system into grade I, indicating a superficial uninfected ulcer; grade II, indicating a deep ulcer with exposed tendons; grade III, indicating a deeper ulcer with exposed bone and infection; grade IV, indicating partial gangrene of the foot; and grade V, indicating complete gangrene
[7][40]. In a study performed in 194 patients with DFU grades between I and III, 16% did not heal in 6 months, 15% underwent amputation, and 4% died
[53][41]. Therefore, diabetes is known to compromise wound healing via multifactorial mechanisms, increasing several concerns about the development and prevalence of DFUs in this population.
1.1.3. Types of Treatment for Wound Healing and their Limitations
The standard treatment of DFUs comprises wound debridement, offloading, and infection control with antibiotics
[3][42]. This involves removing infected and necrotic tissue with a scalpel, providing a specialized cast model to distribute the body’s weight in the foot, and controlling infection through the use of antibiotic treatments, respectively
[3][42]. In this sense, wound management follows the TIME concept (tissue control, infection–inflammation, moisture balance, and edge of wound)
[27][10]. Tissue control corresponds to wound debridement, infection–inflammation comprises the control of the bioburden continuum, moisture balance is related to regulating the wound exudate and restoring the moisture balance, and finally the edge of the wound comprises the promotion of epithelial advancement
[27][10].
The ideal wound dressing material must be non-adherent to the wound, be able to create and maintain a moist environment, reduce the excess of wound exudate, and allow gaseous exchanges
[54][43]. Since a reduction in wound healing time is crucial in lowering the risk of infection and complications in DWs
[16][26], new alternatives such as bioengineered tissue products and natural and synthetic skin grafts have been developed for wound healing
[9,55][13][44]. In this sense, biomaterials, such as alginates, collagen, fibronectin, and chitosan, have been exploited to accelerate wound healing, stimulating cell proliferation and angiogenesis
[55][44] in the form of nanofibrous bandages, films, hydrogels, hydrocolloids, tulle, foams, or gauzes
[20][1]. For instance, a chitosan topical gel and film were effective in promoting tissue granulation and DFU closure
[56][45].
The development of a three-dimensional scaffold improves the wound healing result even more, since it acts as a wound dressing, protecting against external infections and also providing an appropriate surface chemistry with nano- and microstructures that facilitate cellular attachment, proliferation, and differentiation
[14][46]. Furthermore, scaffolds are biodegradable, and upon application at the wound site they start to degrade themselves and release drugs in a time-dependent manner into the wound
[9][13]. Despite all of these alternatives, many of these treatments are still expensive, require extensive care, and do not full recover the skin’s functionalities
[13,14][46][47].
For infection control, the treatment with antibiotics might be performed based on a previous culture of the infected tissue to assess the bacterial colonization profile. The most reported antibiotic therapy options for DFUs in clinical trials are cephalexin and amoxicillin–clavulanate for mild infections and ampicillin–sulbactam, ertapenem, imipenem–cilastatin, vancomycin, and piperacillin–tazobactam for moderate to severe infections
[57][48]. The systemic administration of antibiotics has been considered an increasing limitation, especially against multidrug-resistant bacteria
[16,58][26][49]. Standard antibiotics seem to have a minimal long-term effect on treating chronic wounds, since they are not able to fully penetrate biofilms or attack all species of bacteria embedded in the extracellular polymeric matrix
[59][50]. In the European Union, antibiotic resistance causes 25,000 deaths per year
[60][51], and if not controlled this number might reach 10 million deaths worldwide by 2050
[61][52].
Among the multidrug-resistant pathogens,
S. aureus, methicillin-resistant
S. aureus (
MRSA),
P. aeruginosa,
E. coli, extended-spectrum β-lactamase-producing (ESBL)
E. coli, and vancomycin-resistant
enterococci (
VRE) have been reported as being challenging in infection treatment
[12][53]. Importantly, 18% of the hospitalized patients with DFUs were positive to multidrug-resistant organisms, mostly
MRSA [10][54]. Similarly, another study reported the presence of resistant bacteria in 21.8% of patients, of which 62.7% also presented with
MRSA [11][55]. Despite efforts toward the development of new antibiotics, the extent of bacterial resistance is increasing worldwide and represents a great concern for public health
[62][56]. In this context, alternative treatments have been increasingly stimulated to overcome the growing bacterial resistance to available conventional therapy
[15][57].
In order to promote more accessible and efficient treatments with reduced side effects and risks, natural therapies have been increasingly investigated to treat microbial infections in DFUs. For instance, herbal products, such as curcumin
[13][47], allicin
[63][58], and
Aloe vera [64][59], possess antimicrobial, anti-inflammatory, and antioxidant activities that accelerate wound healing
[16][26]. Similarly, traditional Chinese medicines such as Tangzu Yuyang ointment
[65][60] and
Centella asiatica [66][61] have also been considered options in the management and healing of DFUs due to their anti-inflammatory effects. Although these options hold potential benefits for wound management, the antibacterial effects of traditional Chinese medicines are still not very well documented in vivo for DFU treatment
[27][10].
Honey is among the natural resources with major potential to promote wound healing, since it is accessible and has convincing antibacterial, antioxidant, and anti-inflammatory properties
[17][62]. Furthermore, honey’s biological structure meets several of the ideal wound dressing requirements, since it provides a moist environment for wounds and protects against injury during dressing changes
[18,67][22][63]. All of these advantages make honey a promising and cost-effective natural medicine to treat DFUs
[19][64].
2. Honey in Diabetic Foot Treatment
Several studies carried out with animal models have shown the efficacy of honey in DW treatment
[17][62]. In animal models of diabetes, honey is able to accelerate the wound healing, promoting epithelialization, improving the tissue granulation, and increasing the wound contraction
[136,138,139][65][66][67]. Streptozotocin-induced mice with diabetes treated with honey from the nectar of
Thymus serpillum and
Astragalus microcephalus showed reduced wound areas with greater wound contraction and epithelization than animals treated with isotonic saline solution
[111][68]. The treatment with propolis honey hydrogel dressings derived from Seoul Propolis Co., Ltd. (Daejon, Korea), in
db/db female mice was effective against
S. aureus and
E. coli and improved the amounts of wound area reduction and contraction
[41][28]. The topical application of manuka (
Lepstospermum) and jamun (
Syzygium cumini) honeys in animals with diabetes not only accelerated the wound closure and re-epithelialization but also improved the collagen deposition and modulated the essential angiogenic markers, namely hypoxia-inducible factor and vascular endothelial growth factor
[136][65].
In diabetic mice, heme oxygenase-1 (HO-1), which is a cytoprotective, pro-angiogenesis, and anti-inflammatory enzyme, is usually found to decrease, impairing the regulation of angiogenesis
[140][69]. The application of a chestnut (
Castanea sativa) honey hydrogel in db/db mice was able to rapidly upregulate HO-1 proteins at the wound site, which might mediate the coordination of keratinocytes and enhance the expression of Ki-67 proliferation markers
[138][66]. The topical application of mad honey, a
Rhododendron honey containing grayanotoxins, also decreased the gene expression of inflammatory markers such as TNF-α and metalloproteinase 9 (MMP-9) and increased the IL-10 expression
[139][67]. According to the
resea
uthorchers, the honey’s characteristics, such as its optimum phenolic and flavonoid contents, were responsible for the mad honey’s antioxidant properties. The information from experimental studies involving models of diabetic animals treated with honey is summarized in
Table 1.
In humans, honey has been shown to be effective in DFU treatment, decreasing the bacterial load and inflammation, regenerating the granulation tissue, and reducing the wound size, as well as the rate of amputation
[141,142,143,144][70][71][72][73]. A clinical study over 16 weeks carried out in 63 patients with DFU treated with manuka-honey-impregnated dressings showed accelerated rates of wound healing and disinfection, as well as a nullified need for antibiotics or hospitalization, in comparison with conventional saline-soaked dressings
[110][74]. Another clinical trial compared the DFU treatments from debridement to wound closure in 33 patients and found a higher wound healing rate in those treated with a thin layer of Australian honey dressings covered with gauze (mean: 14.4 days; range: 7–26) than with povidone-soaked gauze (mean: 15.4 days; range: 9–36)
[145][75]. A reduced wound size and higher wound healing rate were observed in patients submitted to beri (
Ziziphus jujuba) honey dressings (
n = 136; median 18 days) than in patients treated with saline dressings (
n = 97; median 29 days)
[146][76]. Currently, five clinical cases have exhibited the effectiveness of medical grade honey in reducing the wound area, exudate volume, and severity of infection, as well as wound-related pain, in diabetic patients
[147][77]. Allergy and irritation symptoms were also decreased in DFU patients treated with Jordanian natural honey
[142][71]. Despite these benefits, in cases of severe vascular compromise, exposed bone, or established osteomyelitis, honey was not as effective
[19][64]. The characteristics of the studies carried out with human DFUs treated with honey are detailed in
Table 2.
Table 1. General characteristics of studies where diabetic animals were treated with honey.
Authors (Country) |
Animal Models |
Reagents Time of Intervention |
Honey Characteristics |
Results |
sodium alginate gel.