medicina

Review
Diabetic Wound-Healing Science
Jamie L. Burgess, W. Austin Wyant , Beatriz Abdo Abujamra, Robert S. Kirsner * and Ivan Jozic *

Wound Healing and Regenerative Medicine Research Program, Department of Dermatology and Cutaneous
Surgery, University of Miami Miller School of Medicine, Miami, FL 33136, USA; jlb452@med.miami.edu (J.L.B.);
waw8@med.miami.edu (W.A.W.); Bxa520@med.miami.edu (B.A.A.)
* Correspondence: RKirsner@med.miami.edu (R.S.K.); i.jozic@med.miami.edu (I.J.)

Abstract: Diabetes mellitus is an increasingly prevalent chronic metabolic disease characterized by

prolonged hyperglycemia that leads to long-term health consequences. It is estimated that impaired
healing of diabetic wounds affects approximately 25% of all patients with diabetes mellitus, often
resulting in lower limb amputation, with subsequent high economic and psychosocial costs. The
hyperglycemic environment promotes the formation of biofilms and makes diabetic wounds difficult
to treat. In this review, we present updates regarding recent advances in our understanding of the
pathophysiology of diabetic wounds focusing on impaired angiogenesis, neuropathy, sub-optimal
chronic inflammatory response, barrier disruption, and subsequent polymicrobial infection, followed
by current and future treatment strategies designed to tackle the various pathologies associated
with diabetic wounds. Given the alarming increase in the prevalence of diabetes, and subsequently
diabetic wounds, it is imperative that future treatment strategies target multiple causes of impaired
healing in diabetic wounds.

Keywords: diabetes; wound healing; diabetic foot ulcer (DFU)

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Citation: Burgess, J.L.; Wyant, W.A.;

Abdo Abujamra, B.; Kirsner, R.S.;
Jozic, I. Diabetic Wound-Healing
1. Introduction
Science. Medicina 2021, 57, 1072. Currently, close to 500 million people are estimated to be suffering from diabetes
https://doi.org/10.3390/ mellitus (DM), with a predicted startling increase in the upcoming years. In the US alone,
medicina57101072 over $300 billion is spent annually on both medical costs and as a result of lost workdays
due to DM [1,2]. Moreover, one estimate suggests that between one in three to one in every
Academic Editor: Manfredi Rizzo five patients with DM will develop a chronic non-healing wound in their lifetime, such
as a diabetic foot ulcer (DFU), with an alarming recurrence rate (40% within one year and
Received: 27 August 2021
65% within five years) and no reliable methods available to predict its occurrence [3,4].
Accepted: 4 October 2021
Considering additional factors identified by Armstrong et al., the overall lifetime incidence
Published: 8 October 2021
of foot ulcers in diabetic patients could be as high as 19–34% [5]. Thus, it is not surprising
that a large proportion requires lower limb amputations, affecting patients’ quality of life
Publisher’s Note: MDPI stays neutral
and requiring costly treatments; it is estimated that the DFU market alone is set to increase
with regard to jurisdictional claims in
from 7.03 billion USD in 2019 to 11.05 billion USD by 2027, making it imperative that
published maps and institutional affil-
more effective diagnostic and treatment strategies are developed to combat this debili-
iations.
tating disease [2,4]. The diabetic foot ulcer has an exceptionally complex pathology due
to persistent hyperglycemia and associated diabetic complications, including (1) barrier
disruption and infection, (2) high oxidative stress, (3) neuropathy, (4) microvascular com-
plications, and (5) suboptimal chronic inflammatory response, in addition to psychological
Copyright: © 2021 by the authors.
problems, including a patient’s mental health, self-esteem, and family cohesion (among
Licensee MDPI, Basel, Switzerland.
others) (Figure 1). Below, we will outline recent advances in our understanding of the
This article is an open access article
pathophysiology of diabetic wounds and then review current/upcoming diagnostic and
distributed under the terms and
treatment strategies for this devastating disease.
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Medicina 2021, 57, 1072. https://doi.org/10.3390/medicina57101072 https://www.mdpi.com/journal/medicina

Medicina 2021, 57, 1072 2 of 24

Figure 1. Pathophysiology of diabetic wounds. Diabetic wounds exhibit deregulated angiogenesis, chronically sustained
sub-optimal inflammatory response, increased levels of reactive oxygen species, and persistent bacterial colonization that
often develops into a hard-to-treat biofilm. Created with BioRender.com, 29 July 2021.

2. Pathophysiology Associated with Diabetic Wound Healing

2.1. Hyperglycemia
In patients with DM, hyperglycemia can contribute to impaired wound closure and
development of DFUs through atherosclerosis, impaired functioning of various skin cells,
and peripheral neuropathy. Although hypoglycemia has also been associated with the
vascular complications of diabetes [6], most of the literature, and thus this section, focuses
on the deleterious effects of hyperglycemia as it relates to the development and progression
of DFUs. Hyperglycemia contributes to the development of atherosclerosis, thereby pre-
venting circulating nutrients from reaching wounds, impairing healing [7]. Moreover, in
patients with DM, hyperglycemia has been found to be a potential cause of dysfunction of
endothelial cells [8], which are critical for the healing of DFUs [9,10] via pressure-induced
vasodilation, a response that is normally protective for the skin [8].
In addition to endothelial cells, hyperglycemia also disrupts processes that are critical
for re-epithelialization, namely, the protein synthesis, migration, and proliferation of
keratinocytes and fibroblasts [11–14]. In patients with DFUs, the expression of several
keratinocyte proteins related to re-epithelization are disrupted, including cytoskeletal
keratin proteins (K2, K6, and K10), which are important for keratinocyte differentiation,
and a laminin-5 ↵3 chain precursor protein (LM-3A32), which regulates the binding of
epithelial cells to the basement membrane [15]. Subsequently, reduction of LM-3A32
perturbs keratinocyte survival and differentiation and thus re-epithelization [16].
Another mechanism by which hyperglycemia impairs wound healing is via free
radical damage as a result of reduced activity of the antioxidant enzymes glutathione
peroxidase and superoxide dismutase [17]. This may partly explain why other studies
have found that long-standing uncontrolled hyperglycemia is correlated with higher
levels of markers associated with the skin aging process, namely, advanced glycation end
products (AGEs) and their receptors [11]. Hyperglycemia can also lead to the production
of reactive oxygen species (ROS) via the polyol, hexosamine, protein kinase C, and AGE
pathways [18]. Although it is understood that ROS are required for the early stages of
wound healing [19,20], the imbalance of ROS production has been shown to be detrimental
to later stages of wound healing. Specifically, elevated ROS levels can damage the blood
supply, metabolism, and structure of peripheral nerves. In affected nerves, this can lead to
dysfunction of sensory, motor, and/or autonomic functioning, with each deficit uniquely
increasing the risk of developing a DFU [21]. Together, these changes brought on by
uncontrolled high blood glucose levels make the skin more susceptible to injury and
infection, which impairs wound healing (see the section below on infection).
Medicina 2021, 57, 1072 3 of 24

2.2. Neuropathy
In addition to increasing the risk of DFU formation, each type of neuropathy (sensory,
motor, and/or autonomic) can uniquely contribute to impaired DFU healing. For example,
autonomic neuropathy decreases sweat gland activity, leaving skin dry and cracked, thereby
increasing the risk for pruritus and infection, which inhibits wound healing [18]. In addition
to dry skin and poor circulation, diabetic neuropathy is, for unclear reasons, associated
with pruritus [22,23]. Meanwhile, motor neuropathy increases pressure on the plantar
surface of the foot, leading to tissue ischemia and death [18]. Overall, neuropathic skin has
a reduced density of neurons and exhibits reduced skin healing [24].
Besides optimizing blood sugar control, patients may prevent dry skin, and thus pruri-
tus, by avoiding exposure to hot water and utilizing moisturizers, particularly ones without
perfumes or dyes. Other treatments for improving wound closure in patients with diabetes
and neuropathy include antibiotics if an infection is present, as well as debridement and
wound cleansing [25]. Recent evidence also suggests that hyperlipidemia, specifically
hypertriglyceridemia, may play a role in the development of diabetic neuropathy and
therefore, lipid-lowering drugs may prevent or even reverse the damage to nerve fibers in
patients with diabetic neuropathy [26]. This approach to prevention is not often employed
as therapies targeting neuropathy currently focus on reducing the pressure placed on the
foot and relieving the need to itch [25]. Since neuropathy predominantly affects nerves
that are dependent on nerve growth factor (NGF) in diabetic patients, exogenous NGF
supplementation has demonstrated improved wound contraction, leukocytic chemotaxis,
and keratinocyte turnover [27] and, in one study, clinically improved healing [28].

2.3. Microvascular Complications

2.3.1. Peripheral Arterial Disease
Peripheral arterial disease (PAD) is prevalent in patients with DFU and contributes to
worse outcomes and increased risk of limb amputation [29,30]. One cross-sectional study
found PAD in 43% of cases of DFUs [29]. Similarly, a retrospective comparison of patients
with Charcot foot found a high prevalence of PAD, which was predicted by the presence of
DFUs [31]. There are many revascularization techniques that have shown that reperfusion
of the ulcer area in eligible patients decreases amputation risk and death [32–34]. One
method to evaluate the benefit of revascularization for the treatment of PAD in diabetic
patients is with the wound, ischemia, and foot infection (WIFI) classification system devel-
oped by the Society for Vascular Surgery. While WIFI Q1–3 cases had approximately 83–87%
healing rates, in WIFI Q4 cases, where revascularization had an uncertain benefit, patients
had increased limb amputation rates even when revascularization was performed [35]. As
another predictor of healing after revascularization, a prospective cohort study found that
an increase in >2 C of surface skin temperature after endovascular therapy is associated
with increased wound healing [36].

2.3.2. Hypoxia
As a natural consequence of poor circulation in patients with DM, DFUs result in hy-
poxic environments. In the setting of hypoxia, the various cell populations of the skin have
differential gene expression. Using a cell culture model, Alessandro et al. found endothe-
lial cells and differentiated macrophages encoded genes for angiogenesis, cytokines, and
growth factors, while keratinocytes and dermal fibroblasts had gene expression changes for
cell metabolism proteins [37]. A recent study monitoring skin hypoxia using flow-mediated
skin fluorescence found that lower levels in DFUs corresponded to a worse healing progno-
sis and other complications [38]. In an attempt to compensate for these hypoxic conditions,
hyperbaric oxygen therapy has been extensively used in the treatment of DFUs, though the
exact mechanisms of hyperbaric oxygen treatment on DFU gene expression are still under
investigation (see below for hyperbaric treatment strategy).
Medicina 2021, 57, 1072 4 of 24

2.3.3. Anemia
In recent studies, anemia has been demonstrated to be prevalent in patients with DM,
especially in the setting of DFUs [39–44]. However, there are conflicting reports on the
correlation between anemia and DFU prognosis. A meta-analysis found that increasing
anemia severity was associated with DFU severity and could serve as a predictor of ampu-
tation and mortality [45]. Retrospective cohort studies identified anemia as significantly
associated with larger, deeper ulcers, more severe infections, high amputation risk, and
increased mortality rates [46,47], while observational studies in Nigeria have found anemia
to be associated with poor wound healing, amputation, and increased mortality [48,49].
Conversely, other studies found anemia to be a non-significant predictor of clinical outcome
for patients with DFUs [50–52]; thus, the context under which anemia may be a prognostic
factor for DFU wound healing is still debatable and requires further elucidation.

2.4. Barrier Disruption and Infection

2.4.1. Transepidermal Water Loss (TEWL)
A healthy skin barrier relies on a well-regulated balance of lipids, cell–cell junctions,
antimicrobial peptides, and enzymes to prevent water loss and infection. The uppermost
skin layer (stratum corneum) is composed of the terminally differentiated, denucleated
keratinocytes filled with keratin fibers and cross-linked envelope proteins called corneo-
cytes that are surrounded by a hydrophobic lipid layer, and as such, protects against
transepidermal water loss (TEWL). With age, the skin naturally has decreased lamellar
body secretions, depletion of lipids, slower barrier repair, and increased TEWL [53,54].
Though the global stratum corneum water content decreases with age [55], the surface
stratum corneum water content has been shown to be similar in both young and aged
skin [56].
Diabetic skin has been found to be remarkably similar to aged skin, with decreased
lipid content, decreased stratum corneum hydration, and increased AGEs, though some
studies noted no significant changes to TEWL [11,57,58] while other studies noted an
increase [57,59]. A comparative analysis between humans and rats suggested that in
diabetics, a paradoxically insignificant change in TEWL could be due to a decrease in
sweating to maintain water loss homeostasis [10]. Similarly, a 2017 case–control study
found changes in TEWL in diabetics with a dysfunctional peripheral sympathetic nervous
system, while no significant findings were found in cases with sensorimotor neuropathy
compared to controls [60]. A recent mouse model study by Horikawa et al. comparing
skin dryness in type 1 and type 2 diabetes found that type 1 diabetes increased AGEs
and matrix metalloproteinase-9 (MMP-9), leading to a decrease in collagen IV, while
type 2 diabetes reduced hyaluronic acid levels and increased inflammatory cytokines
levels [61]. Skin hydration appeared to be correlated with microcirculation [62] and was
found to be a significant predictor of wound healing when hydration was measured prior to
interventions such as recanalization [63]. Additionally, hyperglycemia further increases the
risk of infection by changing the distribution of tight junction protein 1, altering epidermis
histology, and modifying the basal cell ultrastructure, all of which disrupt the normal
function of the skin barrier [64].

2.4.2. Antimicrobial Peptides

In conjunction with maintaining water homeostasis, the structural and functional
integrity of the skin is essential for preventing infection. Healthy skin has evolved immune
mechanisms, such as antimicrobial peptide (AMP) production, to help regulate the nat-
ural skin microbiome and keep pathogens in check. In response to Staphylococcus aureus
(S. aureus) infection in healthy skin, dermal fibroblasts can differentiate into adipocytes
to produce cathelicidin (LL-37) [65,66], which has also been demonstrated to promote
wound healing by stimulating keratinocyte migration and angiogenesis [67]. However,
there is little to no production of cathelicidin in DFUs [68], which contributes to the im-
paired wound healing phenotype. Expression of other AMPs, such as human -defensins,
Medicina 2021, 57, 1072 5 of 24

have been shown to be upregulated in DFUs, but subsequent AMP production has been
proposed to be insufficient for microbial regulation [68,69]. To further complicate matters,
AMP production has been shown to be influenced by common drugs used in diabetes
treatment, such as in the case of RNase 7 downregulation by metformin [69]. Low AMP
production combined with evidence of increased AGEs, impaired lamellar body produc-
tion, and decreased stratum corneum lipid content in the setting of diabetes sets the stage
for an impaired wound healing environment [14]. Recent studies have investigated the
potential to utilize AMPs to promote wound healing. Treatment of keratinocytes with 1,25-
dihydroxyvitamin D3 induced cathelicidin and human -defensin 2, increased keratinocyte
migration, and showed effective antimicrobial activity [70]. Direct delivery of antimicrobial
peptides using hydrogels [71], gold nanoparticles [72], and nanopolymers [73] showed
antimicrobial activity and enhanced wound healing using mice and in vitro models.

2.4.3. Bacterial Diversity

With the advent of high-throughput technologies, including 16S rRNA sequencing,
microarray, and whole-genome sequencing, the characterization of the diabetic skin and
microbiome has expanded. There is growing evidence of microbiome dysbiosis of not only
the skin [74] but also the gut [75] of diabetics that may contribute to the development and
complications of diabetes. Diabetic skin has been shown to have higher colonization of
both S. aureus and S. epidermidis [74]. A recent analysis of German patients with DFUs
identified Staphylococcus, Pseudomonas, and Enterobacteriaceae as the most common bacterial
colonizers [76]. When stratifying DFUs by infection severity, a recent study identified
Staphylococcus and Streptococcus as the most abundant species in mildly to moderately
infected DFUs, while more severely infected DFUs had increased bacterial diversity [77].
Bacteria such as Staphylococcus and Streptococcus express proteolytic factors that disrupt
the skin barrier. Specifically, it has recently been shown that SpeB from Streptococcus
cleaves desmoglein 1 and 3, compromising the epidermal barrier and promoting skin
infection [78]. The increased prevalence of S. aureus colonization of intact diabetic skin and
DFUs contributes to the high rate of diabetic foot ulcers infections [74] and subsequent
spread of infection to the bone and bloodstream. In fact, osteomyelitis was identified as a
significant predictor of wound healing [48] and amputation [79] in a Nigerian multi-center
observational study. Unfortunately, systemic antibiotics have limited delivery to chronic
wound sites, especially in the presence of a bacterial biofilm, so new therapies have been
focused on topical delivery of drugs with varied release mechanisms [80,81].

2.4.4. pH and Microbiome

While the pH of intact skin has generally not shown a significant difference in dia-
betics versus controls, one study identified a slightly higher skin pH in the intertriginous
areas of diabetics [59,82]. The wound environment of DFUs is significantly more alkaline
than acute wounds, contributing to the complex host–microbiome interaction. In a test of
different bacterial strains, including Pseudomonas, alkaline pH conditions increased biofilm
formation [83], with pH also exhibiting differential effects on bacterial resistance to antibi-
otics [84]. Considering antibiotic resistance testing generally occurs near physiological pH,
this difference in bacterial sensitivity should be accounted for when prescribing treatment
for an infection found in an abnormally alkaline DFU environment.

2.5. Inflammation and Immune System Deficiency in Chronic Wounds

The healing dynamics in acute wounds consist of four overlapping phases, including
hemostasis, inflammation, proliferation, and remodeling [85,86]. Diabetic individuals
can develop various complications, including chronic wounds such as non-healing DFUs,
which arise from perturbation of each stage of wound healing [87]. First, unlike acute
wounds, DFUs are characterized by the non-resolving inflammation phase, where a large
number of neutrophils and macrophages are found in the wound bed [85,88,89], as well
as the chronic release of proinflammatory cytokines including interleukin (IL)-1, IL-6,
Medicina 2021, 57, 1072 6 of 24

tumor necrosis factor (TNF)-↵, and plasma C reactive protein [85,88,90,91], and bacterial
proliferation [88,92] being the most explored factors that contribute to the impaired healing
process. Another feature of DFUs is the sustained hypoxia state derived from insufficient
angiogenesis, in which its state is strengthened by the continuous inflammatory response,
resulting in an increase of ROS and dysfunctional healing process [86,93,94]. The down-
regulation of connective tissue growth factors in DFUs correlates with decreased levels
of transforming growth factor (TGF)- and collagen levels, delaying wound closure by
affecting fibroblast proliferation and vascular cell populations in both mouse models and
humans [95–98]. Studies have shown that overexpression of TNF and downregulation
of TGF- 1 in macrophages leads to elevated IL-10 levels, reduced collagen production,
and increased tissue damage [87]. Altogether, these factors contribute extensively to the
prolonged inflammatory state in DFUs and thus inhibit successful wound closure.
Other studies have shown that the failure to progress from the inflammatory to pro-
liferative could result from the activation of the p38 mitogen-activated protein kinase
signaling pathway. In this case, p38 induces the release of cytokines and downregulates
miR-21, which is involved in the termination of the inflammatory phase [85,99–101]. Re-
cently, studies have elucidated the correlation between angiogenesis and inflammation in
the progression of chronic wounds. A possible mechanism involves the downregulation
of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) [102] in
DFUs, dysregulating angiogenesis by lowering vascular endothelial growth factor (VEGF)
expression and increasing inflammation in the wound bed [102,103]. Conversely, induc-
tion of MALAT1 via the hypoxia-inducible factor (HIF)-↵ signaling pathway has been
demonstrated to restore regular fibroblast activity and promote wound healing in a diabetic
mouse model [102,104].
Furthermore, impaired immune cell function has been well documented in diabetic pa-
tients [105] who exhibit impaired phagocytic activity and leukocytes dysfunction [106,107].
Macrophages are widely investigated when studying the immune system in chronic
wounds of diabetic humans and mice in part because they produce and release cytokines,
are influenced by the surrounding microbiome, and coordinate the transition from the
inflammatory to proliferative phase [108–111]. In acute wounds, as the inflammatory stage
is resolved, M1 macrophages are replaced by M2, whereas in DFUs, M1 macrophages
continue to predominate the wound microenvironment [108,112,113]. Likewise, in dia-
betic patients, chronic inflammation causes accumulation of T-cells, which may be the
reason for high levels of TNF-↵ and C-C Motif Chemokine Receptor 4 (CCR4) chemokines,
significantly affecting the immune response and facilitating the proliferation of oppor-
tunistic pathogens [114–117]. Studies have also shown that the severity of DFUs may be
in part determined by the deficiency of the immune response in DFUs via deregulation
of IL-6, macrophage migration inhibitory factor (MIF), and interferon-inducible protein
(IP)-10 [118], and a compromised neutrophil response [119–121]. In recent years, it has
been proposed that high platelet-to-lymphocyte (PLR) and neutrophil-to-lymphocyte ra-
tios (NLR) may be a biomarker for DFU severity [122,123], where high PLR levels reflect
the increased platelet activity, inflammation, and the risk for thrombosis and atheroge-
nesis [122,124,125], while high NLR levels lead to the upregulation of cytokines and of
proteolytic enzymes that can cause tissue damage [122,123].

2.6. Psychological Impacts of Diabetes Mellitus

Although DM can have a negative impact on a patient’s mental health, self-esteem,
and family cohesion [126], there are conflicting reports on whether or not a DFU diagnosis
impacts a patient’s standard of living [127,128]. Moreover, patients with a DFU do not
appear to have significantly worse mental health than those without [127,129]. Nonetheless,
there is evidence to suggest that DFUs can lead to emotional distress, reduced quality of life
(QoL), and physical dysfunction [130]. Physical dysfunction is one of the most important
psychosocial aspects for patients living with a DFU since it may restrict their activities
of daily living [131] and prevent them from being able to fulfill their normal family and
Medicina 2021, 57, 1072 7 of 24

social roles [132]. Moreover, patients with DFUs have worse QoL than patients who healed
without amputation or underwent minor amputation [133], which further solidifies the
notion that physical dysfunction is central to the psychosocial experience of patients with
DFUs. Even most patients who underwent major transtibial amputation experienced
improved quality of life [134]. For patients with DFUs that experience significant negative
mental health effects, there are psychological interventions available that may reduce
anxiety, depression, and patient global assessment scores [135]. Another intervention,
characterized by psychotherapy during hospitalization, reduced anxiety, depression, and
scores on the Problem Areas in the Diabetes Scale [136].

3. Treatment Strategies
Due to the above-referenced multifactorial pathophysiology of diabetic wounds,
DFUs remain a clinical challenge. Wound-healing strategies can fall under standard of
care therapies and advanced therapies, with the standard of care treatment involving
wound debridement, offloading, and glycemic and infection control, whereas advanced
therapies include hyperbaric oxygen therapy (HBOT), wound dressings, negative pressure
wound therapy (NPWT), and growth factor therapies including platelet-rich plasma, stem
cells, and cell- and tissue-based products [2,4] (Table 1). Considering the clinical need,
stimuli-responsive and multifunctional treatment strategies that can accelerate diabetic
wound healing are likely to be an important part of future diabetic wound management [1].

Table 1. Summary of current treatment strategies for diabetic wounds.

Strategy Intervention Origin of Evidence Reference

Surgical debridement Meta-analysis—human [137]
Debridement
Autolytic debridement Clinical trial [138]
Propylbetaine-polihexanide Porcine models, clinical trial [139–141]
Wound Cleansing Solutions
Polyhexamethylene biguanide Clinical trial [142,143]
Hyperbaric Oxygen Therapy Mice, rabbits, clinical trials [144–151]
Oxygen Therapy
Topical Oxygen Therapy Clinical trials [152,153]
Negative Pressure Wound Therapy Negative Pressure Wound Therapy Clinical trials, porcine [154–159]
Diabetic footwear Meta-analysis—human [160,161]
Off-Loading
Surgical off-loading Meta-analysis—human [162–164]
Nerve Growth Factor (NGF) Case report—human [27]
Growth Factor Therapies
Belcaplermin—human-platelet-derived
FDA approved, Clinical trial [165–167]
growth factor (PDGF)
Stem cell hydrogels Mice, rats, clinical trial [168–170]
Hydrogels
Desferrioxamine-laden silk nanofibers
In vitro, rats [171]
hydrogels
Decellularized purified reconstituted
Clinical trial [172]
bilayer matrix
Matrices
Acellular dermal matrix Clinical trial [173,174]
Stromal vascular fraction gel Clinical trial, mice, In vitro [175,176]
Dressings Sucrose octasulfate Clinical trial [177–179]
Skin Substitutes Apligraf, Dermagraft, etc. Meta-analysis, clinical trial [180–182]
Platelet gel Clinical trial [183,184]
Platelet Products Platelet-rich plasma Clinical trial [185–196]
Autologous platelet-derived product Clinical trial [197–199]
Unmodified stem cells Clinical trial, mice, rats [200–206]
Modified stem cells In vitro, mice, rats [103,180,181]
Stem Cells
Cell-free stem cell therapies Mice, rats [182,207–209]
Adipose stem cell sheets Clinical trial [168]
Medicina 2021, 57, 1072 8 of 24

3.1. Debridement
As part of standard care, debridement of the wound bed helps to reduce bacterial
burden, including biofilm, and increase the immune system’s effectiveness, among other
mechanisms of action [139]. Whereas the presence of bacterial biofilms in acute wounds act
as both a mechanical barrier and as an innate progression of wound healing, uncontrolled
biofilm formation can become multidrug-resistant and make it difficult for the healing
process to occur in DFUs [139,210,211]. Surgical debridement of wounds is thought to
promote healing by removing non-viable tissue and perhaps interact synergistically with
other co-administered treatments and is included as the standard of care for DFUs [212–214].
In a retrospective study of patients with DFUs treated for biofilm-associated infections,
sharp debridement combined with meshed skin grafts and NPWT resulted in a mean
wound healing time of 3.5 ± 1.8 weeks [215]. Secondary analysis of debridement modalities
found that surgical debridement was associated with shorter healing time [137], though
there is a lack of strong evidence for surgical debridement efficacy in promoting wound
healing [216]. Other studies in porcine models and in humans have demonstrated that
another form of debridement, enzymatic debridement, may decrease wound size, reduce
inflammation, and increase granulation tissue, with the caveat that they may require a
secondary dressing to penetrate the rooted layers of the wound in order to control the
biofilms present [139–141]. Likewise, it has been shown that hydro-active dressing soaked
with polyhexamethylene biguanide (in humans) can promote macrophage activation in the
wound, inhibiting bacterial proliferation and dampening inflammation [138,142,143].

3.2. Hyperbaric Oxygen Therapy

Despite lacking a multitude of high-quality trials in the use of HBOT, recent work
has emphasized that hyperbaric oxygen therapy can be effective for the treatment of
patients with Wagner grade 3 and 4 ulcers [144], showing a concurrent improvement in
HbA1c, leukocyte levels, and serum creatinine [145]. While a recent study in rabbits treated
with HBOT found no significant changes to the expression of genes involved in wound
healing [146], a study using a diabetic mouse model found accelerated wound healing
and a significant reduction in MMP-9 levels with treatment [147]. In a small study with
17 patients, hyperbaric oxygen therapy was noted to induce cytoplasmic translocation of
HIF-1a and nuclear factor (NF)-kB localization as well as increased VEGF, IL-6, insulin-like
growth factor binding protein 3, adiponectin, fibrosis, and angiogenesis while decreasing
interferon (IFN)- levels [148]. In addition, the prolonged use of hyperbaric oxygen therapy
has also been shown to decrease the recruitment and adhesion of neutrophils, increase
oxygen dispersion to damaged tissues, reduce inflammation, and accelerate healing in
patients with diabetic ulcers [149–151]. More recently, studies of topical oxygen therapy
rather than HBOT have been shown to promote healing in DFUs and promote an aerobic
wound microbiome [152,153].

3.3. Negative Pressure Therapy and Off-Loading

Recent randomized controlled trials examining the effectiveness of NPWT in the
treatment of DFUs have provided mixed results. Landmark studies suggested when used
prior to a wound closure therapy, NPWT in more difficult surgically treated DFUs helped
improve overall healing. Recently, however, one study found that there was no significant
difference in wound closure between NPWT and standard moist wound care [154], and
another study found no significant difference in wound closure between NPWT and a tradi-
tional Vacuum-Assisted Closure (VAC(® )) Therapy System [155]. NPWT remains a part of
standard care. It should be noted, however, that NPWT may provide other benefits beyond
wound closure. For example, treatment with NPWT significantly reduced leukocyte count,
pain, and systemic inflammatory response; discharge criteria and granulation tissue were
also present significantly earlier when using this treatment [156]. Whether NPWT increases
or decreases blood flow and oxygenation in treated tissues is controversial, as different
investigative techniques have yielded varying results. For example, while using NPWT,
Medicina 2021, 57, 1072 9 of 24

laser Doppler showed a significant increase in blood flow [157], thermal imaging revealed
no significant change in blood flow [158], and transcutaneous partial oxygen pressure
demonstrated a significant reduction in tissue oxygenation levels in DFUs, the effects of
which may be beneficial since relative ischemia is a stimulus for neovascularization [159].
Off-loading is the best-studied and most reliable element of standard care for patients
with DFUs. The treatment involves reducing foot pressure, specifically high plantar foot
pressure, which may help prevent ulcer formation [217]. This is particularly important
in patients with neuropathy because walking with elevated plantar pressures has been
associated with the presence of ulcers [218]. Casting and non-removable walkers to date
have the best results, but recently, “diabetes footwear”, which includes shoes and insoles
designed to reduce stress on the foot, has emerged as an option for reducing plantar
pressures in patients with DM [160]. A recent systematic review and meta-analysis found
that the best footwear for reducing plantar pressures includes the features of metatarsal
additions, apertures, and arch profiles [161]. In addition to modifying the footwear of
patients with diabetes, there are also surgical versions of off-loading available [162]. Some
examples of surgical off-loading include Achilles’ tendon release and foot reconstruction,
both of which are designed to optimize the foot for long-term offloading [163]. Some
studies have even shown that the healing and amputation rates for patients with DFUs are
significantly better with surgical off-loading compared to non-surgical treatment [164].

3.4. Growth Factor-Based Therapies

Due to their involvement in basically every phase of wound healing, as well as their
general deregulation in chronic wounds, growth factors (including keratinocyte growth fac-
tor (KGF)-2, platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGFb),
epidermal growth factor (EGF), etc.) have long been considered as potential strategies in
diabetic wound healing and have exhibited promise in small animal models of diabetic
wound healing [219–221]. Likewise, NGF supplementation has demonstrated some poten-
tial to promote healing after 5–14 weeks of treatment; however, these results came from a
very small sample size [27]. The US Food and Drug Administration has approved only one
topical-growth-factor (GF)-based therapeutic, Becaplermin (0.01% Regranex® gel), with
efficacy to promote healing of DFUs [165–167]. Although growth factor therapies exhibited
promising results in vitro and in small animal models in vivo, all but one ultimately failed
to achieve efficacy in accelerating diabetic wound closure for a number of reasons. For one,
locally prolonged bioavailability and hourly interaction of the ligand with the receptor are
necessary for successful wound closure, but wounds are known to be a harsh microenviron-
ment full of proteases and peptidases [222], making it hostile for local GF stability, chemical
integrity, and bioavailability [223,224]. This has therefore rendered topical delivery of GF
therapy futile without encapsulation into a protective delivery vehicle [225,226]. Moreover,
DFUs often exhibit deregulation and mislocalization of GF receptors [227–229], as well
compartmentalization within microdomains (i.e., caveolae), which prevent activation of
downstream signaling events [230–232]. Therefore, even if GFs can be delivered to the
wound, the lack of available and functional receptors precludes their ability to bind to
the appropriate GF receptor and elicit a signaling cascade that will ultimately result in
accelerated directional cell migration and subsequent wound closure. Thus, unless these
underlying problems are not corrected by future formulations that both encapsulate GFs
and allow for their sustained slow release, as well as clear GF receptors from sequestration
of specialized membrane microdomains, GF-based therapies will continue to be futile.

3.5. Hydrogels/Matrices/Dressings and Skin Substitutes

A standard regimen for treating DFUs is the use of dressings in conjunction with vari-
ous secondary treatments. In recent years, the use of various types of hydrogels composed
of adipose-derived stem cells [168], bone marrow mesenchymal stem cells (MSCs) [169],
human adipose stem cells containing hyaluronic acid [170], as well as desferrioxamine-
laden silk nanofibers [171] has garnered a lot of attention. Moreover, in a pilot study,
Medicina 2021, 57, 1072 10 of 24

Kaufman et al. showed that using a decellularized purified reconstituted bilayer matrix
has substantially reduced healing time [172]. Similar results were observed when using the
IntegraTM Flowable wound matrix [173,174]. Other studies have demonstrated that the
combination of extracellular matrix and stromal vascular fraction gels, besides promoting
healing, also stimulated collagen synthesis and neoangiogenesis using both mouse models
and human subjects [175,176]. In the treatment of neuroischemic DFUs, a multicenter,
randomized controlled trial found sucrose octasulfate dressings significantly improved
wound closure in 48% of patients compared to the control (30%) [177]. Follow-up studies
found sucrose octasulfate dressings improved transcutaneous oxygen pressure, and early
treatment of DFUs with these dressings could lower treatment costs while improving
wound healing rates [178,179]. A recent review by the Agency for Healthcare Research and
Quality has identified 76 skin substitute products currently sold in the United States, with
a majority being acellular products made up of decellularized dermal, placental, or animal
tissue [233]. The best, highest quality clinical data exist for bioengineered skin substitutes
with living cells [233]. Meta-analyses have found that skin substitute treatment of DFUs
results in a shorter time to wound closure and lower amputation rate when compared to
standard of care [234–236].

3.6. Platelet Gels and PRPs

For over 30 years, the use of autologous platelet-rich plasma (PRP) and platelet gel
products has been reported to accelerate the healing of chronic wounds. This results from
the presence of several growth factors, including PDGF, TGF- 1, and EGF, as well as
antimicrobial effects that stimulate tissue regeneration, cell proliferation and differentia-
tion, ↵-degranulation, and chemotaxis [185–190]. Interestingly, allogeneic-PRP is much
less investigated than autologous-PRP, though it is an effective and safe treatment for
diabetic chronic wounds [191–193]. Currently, PRP is combined with different activators
and used either in injection therapies or gels—for instance, the use of platelet-enriched
fibrin in combination with collagen-based dressings [194], thrombin/fibrinogen formula-
tions [195,237], or calcium gluconate [183,184]. The LeucoPatch® device, a PRP activated
with fibrin embedded in a leukocyte wound dressing produced by the patient’s own blood,
has shown significant improvement in healing outcomes [197–199]. Moreover, because it is
painless, platelet products have been reported to be more acceptable to patients with DFUs
and have stimulated healing more than regular saline dressings that are standard care for
non-healing DFUs [196].

3.7. Stem Cells

Stem cell therapy for the treatment of DFUs has been a recent topic of great interest.
Murine diabetic models have found that the use of adipose- [200], umbilical- [201], bone-
marrow- [202], and smooth-muscle [203]-derived stem cells or combination therapies
with MSCs [204,205] accelerated wound healing. As diabetic-derived stem cells have
an impaired healing phenotype, modification of MSCs by selective gene overexpression
such as stromal-cell-derived factor (SDF)-1↵ [180], overexpression of c-Jun [181], depletion
of miR-205-5p [103], or by photobiomodulation [238] have shown promising results in
promoting wound healing and provides potential pathways for autologous stem cell
treatment. In humans, the injection of autologous micro-fragmented adipose tissue [239]
and combination therapies with umbilical cord MSCs improved healing outcomes [240].
A recent meta-analysis found lower amputation rates and increased wound healing in
autologous stem cell treatment randomized controlled trials [241]. Cell-free therapies
using adipose MSC conditioned media [182] and exosomes [207–209] to treat wounds
are also in development. Some current clinical trials in progress utilize MSCs derived
from the umbilical cord (NCT04104451), human placenta (NCT04464213), and adipose
tissue (NCT03865394, NCT03916211). Therapy using allogenic adipose stem cell sheets has
shown potential for improving DFU wound healing [168] and is being tested in clinical
trials (NCT02619877, NCT03754465, NCT04497805, NCT03370874, NCT04569409). Another
Medicina 2021, 57, 1072 11 of 24

clinical trial (NCT00955669) found autologous bone marrow MSCs promoted limb blood
flow and healing [206]. With such rapid development in the last couple of years, stem cells
could be the next generation of therapies for DFU wound healing.

4. Diagnostic Measures
4.1. Biomarkers
Biomarker identification is essential for the assessment of DFU healing progress and
prognosis. DFU biomarkers can be analyzed on a range of specimens, including tissue
biopsies, serum, and wound exudate fluid. Of note, though, specimens must be of sufficient
quality for proper biomarker analysis, as one study noted the high prevalence of poor-
quality tissue specimens in DFUs, which may affect clinical trial designs [242]. One set
of biomarkers of interest are inflammatory biomarkers in DFU osteomyelitis, such as
erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP), which was the focus
of one recent clinical trial (NCT04025853). However, there is much controversy about
the efficacy of inflammatory biomarkers for osteomyelitis. Some studies have shown
inflammatory biomarkers correlate with developing osteomyelitis [243] or could be used
to monitor response to therapy [244]. Additional studies have found procalcitonin to be
a predictor of DFU severity, osteomyelitis occurrence, and amputation risk [245–247]. In
contrast, other studies noted procalcitonin was not effective at differentiating uninfected
and infected DFUs [248], with CRP serving as a more sensitive osteomyelitis biomarker
instead [249,250], though ESR and CRP were noted for being unreliable in the setting
of sensory neuropathy [251]. Another clinical trial (NCT02927678) utilizing white blood
cells—single-photon emission computed tomography/computed tomography found that
it could be used for prediction of osteomyelitis remission after 1 year [252]. However,
prediction relied on experienced nuclear physicians for analysis, which can significantly
differ based on the training level of the physician, a drawback that could be compensated
for with the use of a composite scoring system [253].
Other ongoing clinical trials utilize TEWL as a marker for DFU recurrence and tissue
biomarkers such as c-myc and phosphorylated glucocorticoid receptor (NCT04591691)
for assessing wound healing. While a full evaluation of DFU biomarkers is outside the
scope of this review, briefly, some recent reports of wound exudate biomarkers for DFU
wound healing include epithelial neutrophil-activating protein (ENA)-78 [254], c-x-c motif
chemokine ligand 6 (CXCL6) [255], and MMP-9 [256]. A number of serum biomarkers
have been identified, including albumin [257], PLR and NLR [122,258], angiopoietin-
like 2 (ANGPTL2) [259], lipoprotein-associated phospholipase A2 and interleukin-18 (IL-
18) [260], pentraxin 3 [261], T-cell differentiation markers [262], stem/progenitor cells [263],
as well as neutrophil extracellular traps (NETs)-specific markers [264]. Recent genomic
analyses of DFU have utilized circRNAs [265–267], lncRNAs [268], miRNAs [99,100,269],
genetic polymorphisms [270–272], cytokine arrays [273], and network maps [274] for the
identification of other potential biomarkers for DFU diagnosis and prognosis. With the
advent of bioinformatic analyses predicting factors for diabetic complications [275], the
integration of computational algorithms with clinical observations and biomarker results
will likely become the new standard for DFU care.

4.2. Biosensors and Imaging

With technological advancements in chip technology, there has been a rise in the
medical application of biosensors to predict clinical outcomes such as DFU prevention and
monitoring of wound-healing progress. One clinical trial (NCT02586519) uses a pressure-
sensing insole coupled to a smartwatch system to provide real-time feedback on plantar
pressure offloading. Reports by the investigators found that patients optimally received one
alert every 2 h, and a reduction of DFU recurrence was associated with the use of this smart
sensor technology [276,277]. Recent guidelines for DFU prevention have incorporated
regular monitoring of foot skin temperature to assess for early signs of inflammation and
enable early intervention to prevent ulceration [278]. A number of other wearable devices
Medicina 2021, 57, 1072 12 of 24

have been created for thermal foot monitoring, including smart insole systems [279,280]
and socks [281,282], though the efficacy of these wearables in DFU prevention or wound-
healing monitoring need to be further tested. Interestingly, a thermal foot-monitoring smart
mat reported by Frykberg et al. has been shown to predict ulcer development an average of
35 days prior to ulcer presentation [283,284]. In addition to prevention, thermal monitoring
has also been shown to be a useful predictor of DFU wound healing. A small observational
study by Gethin et al. noted that lowered pH and surface temperature correlated with DFU
healing status [285]. In an Australian-based pilot study, authors reported a lower ratio
of wound bed area as measured by thermal images is predictive of week 4 DFU healing
status [286]. Other sensors are being developed that could perhaps be integrated into
dressings to provide feedback on wound temperature and pH [287].

5. Conclusions
There has been an incredible increase in knowledge on diabetic wound healing mech-
anisms in recent years, but there are still unmet needs in clinical diabetic wound manage-
ment. A better understanding of the changes in wound status would allow diagnoses to
be made faster, easier, and cheaper. Personalized management and treatment of diabetic
wounds can become possible with smart wound dressings, hydrogels, and other technolo-
gies. Close monitoring and timely treatments based on these technologies may be able
to prevent non-healing wounds from developing further as patients often fail to realize
the severity of their wounds. As the population of diabetic patients increases, there is a
growing need for chronic wound management, and further research is needed to uncover
how to accelerate diabetic wound healing and improve patients’ quality of life.

Author Contributions: B.A.A., I.J., J.L.B., R.S.K. and W.A.W. All participated in the conceptualization,
writing of the manuscript, and the creation of the figures. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was funded in part by the Stanley J. Glaser Research Award, American Skin
Association Research Scholar Award and startup funds from the Dr. Philip Frost Department
of Dermatology, University of Miami Miller School of Medicine (to I.J.), as well as NIH grants
U01DK119085, 1R01AR073614, and U24DK122927 (to R.S.K.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The figures were created with BioRender.com, 29 July 2021.
Conflicts of Interest: The authors declare no conflict of interest.

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