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TGF-ß2 [23, 24]. A single lineage of broblasts that express
the gene Engrailed-1 (En1) is the main culprit of brosis and
scar formation during cutaneous wound healing. This difference in capacity to form a scar is cell intrinsic and permanent
[25]. Elegant studies using genetic fate-mapping techniques
at the single cell level found that a lineage of broblasts that
do not express EN1 are responsible for forming the structural
ECM during embryonic development. This lineage subsequently declines during development with a concurrent
increase in EN1 expressing broblasts, providing a rationale
for why adult skin in higher-order vertebrates is prone to
scarring and why embryonic skin can regenerate with no scar
[26]. Furthermore, this transition can be reversed by transplanting EN1 naïve cells into the local wound microenvironment. A subpopulation of EN1 naïve cells in postnatal mice
give rise to postnatally EN1 expressing cells by activating
EN1 expression during wound healing. EN1 expression is
induced by mechanical tension and canonical mechanotransduction signaling pathways. Wound regeneration (versus
scarring) can be induced in adult mice by blocking this pathway with small molecule inhibitors and genetic knockout
techniques [27]. A recent study constructed a broblast atlas
by integrating single-cell transcriptomic data from about
230,000 broblasts across 17 tissues, 50 datasets, and 11 disease states, and both mice and humans found the existence of
two universal broblast subtypes across all tissues dened
by the expression of Pi16 (peptidase inhibitor 16) and
Col15a1 (collagen-type XV alpha-1 chain). The roles of
these universal broblasts include ECM secretion, collagen
production, and basement membrane maintenance.
Furthermore, these subtypes serve as a reservoir that yields
two broad classes of specialized broblasts: steady-state
broblasts in tissues and activated broblasts in disease and
perturbed states such as wound healing. Lrrc15+ (leucine
rich repeat containing 15) cluster was implicated in wound
healing as well as arthritis, brosis, and pancreatic ductal
adenocarcinoma. This cluster also showed high expression
of Cthrc1 (collagen triple-helix repeat containing 1), Acta2
(actin alpha 2), Postn (periostin), Adam12 (ADAM metallopeptidase domain 12), and collagens, suggesting they were
composed of predominantly myobroblasts [28].
Furthermore, IGF1 (insulin-like growth factor) was found
implicated in myobroblast formation, and WNT4 (WNT
family member 4) was primarily involved in the ability of
broblasts to support epidermal stratication and maintenance [29]. These studies are just beginning to shed light on
the key organizing principals of the broblast lineage in
development and disease. They revealed that nonoverlapping
disease-associated broblasts reside in specic locations/tissues which are different from normal and/or embryonic
broblasts involved in tissue repair and regeneration. This
revelation has profound implications in drug discovery and
therapeutic development, whereby one can specically target broblasts involved in wound healing pathology [30].
In response to skin injury, local dermal broblasts are
activated and begin to proliferate. Twist1 (Twist family
BHLH transcription factor 1)-Prrx1-(Twist1-paired-related
homeobox 1)-TNC (tenascin-C)-positive feedback loop is
implicated in broblast activation in wound healing [31]. A
few days later during the process, broblast migrate into the
provisional matrix of the wound clot where they begin to
produce the major constituents of the ECM, which is rich in
collagen [32–34]. A critical discriminator between broblasts in dermal development versus wound healing is that
dermal maturation occurs without active broblast migration
[35]. The ECM acts as both a scaffold and a reservoir,
whereby it provides structure to the healing wound and
attachment sites for the various cells and sequesters signaling molecules that are involved in crucial wound healing
process such as angiogenesis, cell proliferation, cell migration, and inammation [36, 37]. Additionally, broblasts are
indispensable for producing a stromal address which directs
leukocyte behavior within tissues including trafcking, differentiation, and survival [38]. Dysregulation of this stromal
address leads to chronic inammatory disease [38].
Dermal broblasts at the site of injury begin to proliferate
as an early response to wounding. A few days after wounding, broblasts migrate into the provisional matrix of the
wound clot to lay down their own collagen-rich matrix [32–
34]. This ECM acts as a “scaffold” during tissue repair, pro-
viding structural support and attachment sites for cell surface
receptors while simultaneously acting as a regulated “reservoir” for signaling molecules that modulate diverse processes such as angiogenesis, cell proliferation, cell migration,
and inammation [36, 37]. In order to migrate into the clot,
dermal broblasts must downregulate their collagen receptors and upregulate integrins that bind ECM proteins such as
brin, bronectin, and vitronectin [39, 40]. During migration, broblasts sense and respond to signals coming from
both their local matrix environment and from the surrounding growth factor milieu.
About 1 week after wounding, the wound clot is fully
invaded by activated broblasts. Transforming growth factor
beta-1 (TGFß-1) is a potent pro-brotic signaling molecule
that with other growth factors stimulates broblasts to synthesize and remodel the new collagen-rich matrix [33, 34,
39]. Simultaneously, a proportion of the wound broblasts
transform into myobroblasts, which express α-smooth muscle actin and resemble smooth muscle cells in their capacity
for generating strong contractile forces [41, 42].
Conversion from broblasts to myobroblasts is mediated not only by growth factors, especially TGF-ß1 [41, 43,
44], but also by mechanical tension [45–48]. Myobroblasts
align parallel to mechanical tension building up in the granu-
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lation tissue, and their appearance in the wound coincides
with a strong induction of contractile properties. Collagengel models have been useful for the study of various tensile
forces acting on and exerted by wound broblasts before,
during, and after their contraction. Several growth factors
present in the wound site and ECM reservoir are stimulators
of broblast-driven gel contraction, which can presumably
induce granulation tissue contraction in vivo [49, 50].
Platelet-derived growth factor (PDGF)-AA and PDGF-BB
isoforms and TGF-ß1 lead to efcient collagen-gel contraction and can be considered stimulatory [49–52], while IL-1α
and IL-1ß were shown to be inhibitory, which is due to
increased matrix metalloproteinase activity [53, 54].
Contraction stop signals have been studied in a similar
manner by releasing mechanically stressed anchored gels
from their substrate attachments, which simulates the loss of
resistance after a wound has closed. Within minutes of
release from resisting forces, PDGF and EGF receptors on
the cell surface become deactivated [55], and the relaxed
cells return to a quiescent state similar to that existing before
the injury. Fibroblasts present in the granulation tissue
undergo apoptosis, triggered by TGF-ß1 and FGF at the
injury site, after wound contraction has ceased [56, 57].
Together, these mechanisms promote a return to the normal
physiologic state and location of broblasts after the process
of healing has completed.
Given the importance of broblasts and keratinocytes in
proper wound healing, human skin substitutes have been
developed as a wound treatment modality. Several cell therapies are approved for use in diabetic foot ulcers (DFUs) and
two in particular utilize broblasts. Graftskin (Apligraf) is a
human skin equivalent composed of a dermal layer containing human broblasts and connective tissue and an epidermal layer consisting of keratinocytes [58]. Similarly,
Dermagraft is a human dermal substitute consisting of cryopreserved human broblasts, ECM, and a bioabsorbable
scaffold [59]. Thus, broblasts and keratinocytes are vitally
important for the maintenance of the epidermal barrier as
well as the process of wound healing after injury while also
showing great promise in their practical translation to the
bedside. Please see treatments section for additional
information.
Endothelial Cells (ECs)
Local ECs are additional responders to the wound healing
signals released by keratinocytes and broblasts. Normally,
ECs are located in the wall of the vascular lumen forming the
tubular structure of blood vessels and the barrier between
blood and extravascular tissue. They express integrins and
other cell adhesion molecules to allow selective permeability
between these two compartments and are highly upregulated
during angiogenesis [60, 61]. The process of angiogenesis is
facilitated by growth factors, cytokines, cell-cell and cellmatrix interactions, and even exosomes from certain stem
cells that activate these ECs [62]. These activated ECs along
with platelets, macrophages, and broblasts release proangiogenic cytokines that lead to the invasion and migration
of ECs into the ECM, EC proliferation, and new immature
vascular formation [63, 64]. Before angiogenesis can begin,
ECs must detach from neighboring ECs primarily by digesting the basement membrane and components of the ECM
[64, 65]. This is achieved by proteolytic enzymes, including
serine proteases, urokinase plasminogen activator, and
matrix metalloproteinases (MMPs) that are released by activated ECs [66]. The addition of an MMP synthetic inhibitor
to EC cultures signicantly decreases angiogenic activity
[66], highlighting the importance of this group of enzymes to
EC function. Once they are liberated, ECs migrate to the site
of new vessel formation via vascular endothelial growth factor (VEGF)-stimulated chemotaxis where they will proliferate [64, 66, 67]. Additionally, specic adhesion molecules,
especially integrins, mediate endothelial cell-matrix interactions to ensure migration to the site of new vessel formation
[64, 67]. Functional capillary formation in wound healing
relies on induction of capillary growth, removal of most
endothelial cells during resolution, and successful maturation [68]. In the maturation phase, blood vessel pericytes
increase and spread, and endothelial cells produce a functional basement membrane [68]. Finally, wound resolution
and remodeling phases are induced by the inhibition of endothelial cell movement governed by anti-angiogenic factors
capable of inducing vascular regression and capillary pruning [68].
Neutrophils
Inammation is a key process of normal wound healing and
is tightly regulated both temporally and spatially by multiple
cell types. Immediately following injury, activation of the
clotting cascade ensues causing platelet aggregation and
leading to the formation of a brin clot which initiates hemostasis. These processes are important to stop blood and uid
loss as well as to provide a provisional matrix that facilitates
the inltration and recruitment of inammatory cells and
other cells to the injury site [1, 69]. Platelets degranulate and
release a variety of growth factors and cytokines that act as
chemoattractants for cells such as neutrophils, macrophages,
endothelial cells, broblasts, and keratinocytes resulting in
the initiation of the inammatory phase [1, 70]. The rst
inammatory cells to migrate to the wound site are the neutrophils which are the predominant cell type during the rst
2 days. Their role is to remove dead cells and infectious
microorganisms by phagocytosis and generation of reactive
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oxygen species or by releasing neutrophil extracellular traps
(NETs) [71–74]. Neutrophil activity has to be rapid to minimize host tissue damage and excessive inammation, and the
cells undergo apoptosis or die by NETosis once infection is
under control [75]. After a few days, neutrophil inltration
ceases, and expended neutrophils are phagocytosed by
macrophages.
Macrophages
Macrophages appear at the wound site within 2days after
injury and play an important role in clearing matrix, cell
debris, and microorganisms. Both inammatory monocytes,
recruited from the bone marrow that later become macrophages, and resident macrophages are recruited to the
wound site [76, 77]. These macrophage populations change
their expression proles according to cytokine and growth
factor stimuli [78, 79]. During normal wound healing, macrophages transition from a pro-inammatory or “M1” phenotype to a wound healing-associated or “M2” phenotype
[80, 81]. M1 macrophages dominate earlier during injury
and express pro- inammatory mediators and cytokines
including TNFα, IL-1, IL-6, IL-12, and inducible nitric
oxide synthase (iNOS), whereas M2 macrophages dominate
later during injury and express anti-inammatory genes as
well as promote ECM synthesis and cell proliferation [81–
83]. The M2 macrophage population include multiple sub-
types such as the pro- healing subtype (M2a), pro-remodeling
(M2b), and anti-inammatory (M2f) [84]. Several factors
mediate the phenotypic switch that include anti-inammatory cytokines (e.g., IL-4 and IL-13) and phagocytosis of
apoptotic neutrophils that triggers the release of pro-healing
growth factors and suppress pro-inammatory factors to
augment the tissue repair process [84–86]. Diabetic mouse
models demonstrate a dysregulated and persistent M1 macrophage polarization compared to normal wounds [87].
Macrophages also release a battery of growth factors, chemokines, and MMPs [77], which all aid in driving cell proliferation and ECM synthesis. Sustained inammasome
activation in macrophages from diabetic wounds has been
demonstrated to contribute to the inhibition of wound healing and pharmacological inhibition of inammasomes
resulted in phenotypic switch to pro- healing macrophages
with increased growth factor production and improved healing [88].
Mast Cells
Mast cells are one of the rst responders to injury and are
involved in neutrophil recruitment, clot stability, angiogenesis, and brogenesis [89–91]. Mast cells quickly become
activated and increase in number over time during wound
healing. At the site of injury, mast cells undergo degranulation and release their pre-stored factors that inuence the
repair process [89–91]. Diabetic wounds are characterized
by increased degranulated mast cells, and the inhibition of
mast cell degranulation promotes wound healing and shifts
macrophages to the pro-healing M2 macrophage phenotype
[91–93]. However, mice decient for mast cells display
impaired healing, indicating that certain mast cell mediators
are required for proper healing [92].
Inammatory cells also exert their inuence on the surrounding tissue by generating nitric oxide (NO) and large
amounts of ROS [94]. NO and ROS are known to drive certain aspects of repair [95, 96], but, at the same time, affected
wound cells must protect themselves by detoxifying programs [94, 97]. NO is a very transitory molecule, whose levels together with inducible NO synthase (iNOS) activity
shows a distinct time course during normal healing [98, 99].
Recent studies have demonstrated the essential need for
inammatory cells that include macrophages and neutrophils
that coordinate the early phase of the tissue repair process by
exerting profound inuences on other cell types present in
the wound environment [100–103]. One of the important
roles of inammatory cytokines is to regulate angiogenesis,
which they accomplish in concert with signals from other
wound cells and from serum (see section on angiogenesis).
However, nonhealing wounds fail to progress through the
normal phases of wound repair, but instead remain in a suppressed and prolonged inammatory state [101–103].
Imbalances in wound proteases and their inhibitors in chronic
wounds, because of sustained production of inammatory
mediators and inux of inammatory cells, prevent matrix
synthesis and remodeling, essential for progression to a
healed wound [104–109].
Pathophysiology ofWound Healing
inDiabetes Mellitus
Over 415million people worldwide have diabetes with an
estimated 29.1million affected people in the United States
alone. These numbers incur annual costs of more than
$245billion, rendering it a major public health and socioeconomic concern [110]. The prevalence of diabetes is on an
upward trend, possibly affecting one-third of the US adult
population by 2030 [111]. Diabetes has many devastating
complications, one of which is DFUs, which occurs in 15%
of diabetic patients often leading to lower-limb amputations
[1]. Following amputation, DFU patients have a 5-year mortality rate of nearly 50% [112]. DFUs often lead to lengthier
hospitalization with associated high treatment costs, pain,
and reduced quality of life [113, 114] and are a signicant
cause of morbidity and mortality [115].
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Wound healing is a dynamic process comprising of
overlapping phases of hemostasis, inammation, proliferation, and remodeling that involve multiple cell types. This
highly organized and coordinated series of processes results
in the restoration of tissue integrity and functions.
Deregulation in any of these processes leads to a delayed or
nonhealing phenotype as seen in DFUs [1, 112, 116].
Factors that contribute to the delayed wound healing seen
in diabetes are multifactorial and include macro- and
microvascular, neuropathic, immune functions, and biochemical abnormalities. Indeed a recent study provided
evidence that additional diabetic- associated problems may
play an important role in the development of DFUs since
only subtle changes were found between non-ulcerated
non-neuropathic diabetic foot skin and healthy nondiabetic
foot skin using comparative genomic analyses as well as
detailed histomorphological evaluations [117, 118]. Poor
glucose control and hyperglycemia are additional factors
that may contribute to the metabolic pathophysiology of
diabetes-related complications [119, 120]. DFU development involves both extrinsic and intrinsic factors. Extrinsic
factors include callous formation, excessive pressure,
repeated trauma, and wound infection [121], while intrinsic
factors that contribute to an impairment in diabetic wound
healing include prolonged inammation, persistent infection, imbalanced proteolytic activity, improper formation
and remodeling of the ECM, reduced growth factors, poor
angiogenesis and various cell type and stem cell dysfunction, cellular senescence, and reduced reepithelialization
[1, 122–125]. Prolonged hypoxia [126] and reduced levels
of neuropeptides [127] have all been shown to contribute to
the impaired wound healing in DFUs.
Keratinocytes’ andFibroblasts’ Role
inImpaired Wound Healing
Upon cutaneous injury, epidermal keratinocytes become
activated. However, this activation is not properly executed
in chronic wounds, and migration and proliferation of these
cells are affected as a result. The EGF family, particularly
family members involved in wound healing such as EGF,
heparin-binding EGF (HB-EGF), and TGF-α can bind and
activate the EGF receptor (EGFR), thus leading to the stimulation of keratinocyte migration and proliferation [15]. Lee
etal. (2005) showed that EGF-mediated migration is blocked
by glucocorticoids, and this occurs through repressing K6/
K16 transcription [128]. In the nonhealing edge of chronic
wounds, EGF receptor (EGFR) is expressed in the cytoplasm
of keratinocytes instead of the membrane as with normal epidermis [129], suggesting that these cells are unable to
respond to EGF ligands. This may be a likely reason why
topical application of EGF, in an attempt to heal human
chronic wounds, is met with limited success [130].
Nonhealing ulcer keratinocytes are hyperproliferative in
both basal and suprabasal layers of the epidermis, giving rise
to parakeratosis and hyperkeratosis, indicating impaired differentiation (Fig.7.1) [131, 132]. The differentiation mark-
ab
Fig. 7.1 Histology of DFU (a) compared to adjacent non-ulcerated
diabetic foot skin (b). DFUs display a hyperproliferative nonmigratory
epidermis with parakeratosis. Signicantly thicker cornied layer
(hyperkeratosis) and the presence nuclei in the cornied layer (para-
keratosis) are characteristic for DFUs (a) and not present in adjacent
non-ulcerated diabetic skin (b). E epidermis, D dermis, CL cornied
layer, I inammatory cellular inltrate; arrows indicate nuclei in the
cornied layer. Scale bar=200μm
ab
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ers K1/K10, laggrin, and a subgroup of small proline-rich
proteins were found to be suppressed, while the late differentiation markers transglutaminase 1 and involucrin were
induced in nonhealing venous ulcers [132]. In a prospective
clinical study, it was shown that the nonmigratory and hyperproliferative phenotype of keratinocytes seen at the nonhealing wound edge of chronic ulcers was attributed to nuclear
presence and overexpression of the oncogene c-myc and
β-catenin (Fig. 7.2), contributing to epidermal stem cell
depletion [122, 133]. In addition, stabilization of nuclear
β-catenin inhibited wound healing and keratinocyte migration by blocking EGF response, inducing c-myc and repress-
Fig. 7.2 Wound edge of the chronic DFU shows overexpression of
c-myc. Immunohistochemistry with c-myc-specic antibody of healing
(a) and nonhealing DFU (b). Nuclear presence and overexpression of
c-myc (brown) contributes to hyperproliferative phenotype in the nonhealing DFU.Healing DFU has less c-myc positive nuclei; dashed lined
demarcates epidermal-dermal boundary
ing K6/K16 in vitro [133]. Mechanisms of β-catenin
activation in DFU keratinocytes also involve membranous
glucocorticoid receptor (mbGR) signaling [134, 135]. While
glucocorticoids are established inhibitors of reepithelialization acting through the phosphorylation and nuclearization
of GRs, mbGR inhibits wound reepithelialization by activating a Wnt-like phospholipase/protein kinase C signaling
resulting in atypical activation of β-catenin and c-myc [134,
136]. Importantly, the inhibitory effects of glucocorticoids
can be reversed by topical application of statins, widely used
inhibitors of cholesterol synthesis, resulting in enhanced
wound closure through inhibition of GR activation and suppression of local, cutaneous cortisol synthesis [137, 138]. In
addition, topical mevastatin-induced expression of noncoding RNA Gas5 resulted in suppression of c-myc and accelerated wound closure [138]. Despite the hyperproliferative
phenotype characterized by overexpression of oncogenes,
DFU epidermis remains protected from tumor development
due to newly discovered mechanism governed by miRNA193b- 3p targeting proto-oncogene KRAS (Kirsten rat sarcoma viral proto-oncogene) and KIT (KIT proto-oncogene)
[139]. Furthermore, miRNA-193b-3p has a dominantnegative effect on cellular migration by targeting components of keratinocyte actin cytoskeleton, and inhibition of
stress ber formation mediated by RhoA, required for
directed migration and successful wound closure [139].
Moreover, the epidermis of chronic nonhealing DFU has
also been shown to exhibit decreased expression of the precursor of the alpha 3 chain of laminin [140], and increased
levels of Cav1 (Caveolin 1) involved in sequestration of the
growth factor signaling and diminished Rho GTPases activity contributing to impaired reepithelialization and inhibition
of wound closure [141, 142]. The lack of keratinocyte migration in DFU and insufcient inammation may also be a
consequence of the deregulated expression of immune inhibitory receptor ligand-programmed death ligand 1 (PDL1)
[143]. Transmembrane protein PDL1 was found downregulated in DFU epidermis, contributing to delayed wound closure, while PDL1 treatment improved wound closure and
modulated prolonged inammation in a mouse diabetic
wounds [143].
Additional characteristic of the DFU epidermal keratinocytes is their inability to properly respond to DNA damage.
DFU keratinocytes are characterized by accumulated DNA
breaks as a result of suppression of the plethora of DNA
repair genes (double-strand break repair protein, RAD50;
MutS homolog 2, MSH2; WEE1 G2 checkpoint kinase;
tumor protein P53; and receptor tyrosine kinase, KIT) [144].
This marked downregulation of the DNA repair genes is governed by miRNA15b-5p, overexpressed due to the accumulation of Staphylococcus aureus, the most common DFU
pathogen [144].
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Altered Fibroblast Function inDFU
Fibroblast dysfunction is a dening feature of the DFU [42,
145–151]. Wound broblasts from diabetic patients display
impaired migration, proliferation, ECM production and
deposition, resistance to growth factors, altered morphology,
and increased cell death [42, 125, 151–156]. Isolated DFU
broblasts also display induced cell differentiation and
senescence pathways [125]. Cellular aging and senescence
and the profound altered metabolic changes of diabetic broblasts stimulated by chronic hypoxia and hyperglycemia are
broadly implicated in DFUF dysfunction [126, 157].
Recent studies using single-cell RNA sequencing technology have also implicated broblast heterogeneity in
DFUs [103, 158]. Compared to acute wounds, diabetic ulcer
foot broblasts (DFUF) displayed increased expression of
pro-brotic and inammatory markers and decreased expression of anti-apoptotic markers. Healing DFUFs showing an
enrichment of a newly found population of broblasts overexpressing matrix metalloprotease 1 (MMP1), matrix metalloprotease 3 (MMP3), matrix metalloprotease 11 (MMP11),
hypoxia-inducible factor-1 alpha (HIF1α), chitinase-3-like
protein 1 (CHI3L1), and tumor necrosis factor-inducible
gene 6 (TNFAIP6) compared to nonhealing DFU [103].
Age is a signicant risk factor for diabetic foot complications and development of diabetes itself [159–161].
Inextricably tied to aging is cellular senescence which is the
irreversible state of cell cycle arrest triggered by an array of
cellular stressors [162–164]. Although senescent cells are
dormant, they still are metabolically active and secrete factors that inuence cell behavior and may alter response to
wounding. Activation of senescence in broblasts is protective in the early phases of acute wound healing and contributes to prevention of brosis during the later stages of wound
resolution [165]. On the contrary, aberrant broblast senescence in DFU is associated with wound chronicity and the
failure to heal [148, 166, 167]. Elegant studies revealed that
aged diabetic wounds exposed to senescent macrophages
directly induces broblast senescence and transformation to
a pro-brotic phenotype. Specically, these macrophages
produce factors called senescence-associated secretory phenotype (SASP) that contain CXCR2 which act via the activation of nuclear P21 [166]. Reversal of this SASP production
led to enhanced healing in diabetic mouse model and human
exvivo models [166].
Chronic exposure to hypoxia is an important factor in
wound chronicity [168]. DFUs are subjected to chronic
hypoxia due to the poor vasculature exhibited in diabetic
patients and increased pressure points due to neuropathy.
Low-oxygen tension in an acute setting is benecial for
wound healing as it activates the myobroblast expression of
genes necessary for physiologic healing such as TGF-β and
collagen A1 (COLA1) as well as the homing of peripheral
stem cells to the wound [3, 169, 170]. Chronic oxygen depri-
vation impedes the hydroxylation of proline and lysine residues, which are oxygen dependent, during collagen synthesis
leading to aberrant ECM synthesis and organization [171–
176]. In effect broblast growth, function, and activity are
negatively affected leading to wound chronicity [153, 168,
177, 178]. Hypoxia adaptation is largely controlled through
the activation of the master gene regulator hypoxia-inducible
factor (HIF1α) [179]. There is a complex interplay between
hyperglycemia and HIF1α, which ultimately leads to HIF
destabilization, dysfunctional broblast responses, and
impaired wound healing [126, 180–183]. Hyperbaric oxygen
therapy, a widely used treatment modality for DFUs, works
to restore oxygen levels to the wound microenvironment to
activate broblasts to restore proper ECM production necessary for wound healing [180, 184, 185]. Interestingly there is
little high-quality evidence to support HBOT use, and almost
all RCTs involving HBOT have shown little to no benet
[186, 187]. New attention has been directed toward topical
oxygen therapy (TOT) for DFUs, with recent trials showing
increased statistically signicant healing outcomes compared to standard of care [187–194].
Hyperglycemia, the hallmark of diabetes, promotes broblast dysfunction and wound chronicity largely through the
formation of advanced glycation end products (AGE) and
imposing major epigenetic and metabolic changes [195].
The formation of AGEs (discussed in more detail in ECM)
has been shown to induce apoptosis and cell cycle arrest and
decreased ECM production in dermal broblasts [196–201].
Interesting studies using cultured broblasts from nonulcerated diabetic skin showed similar expression and function as broblasts isolated form nondiabetic healthy subjects,
implying that hyperglycemia does not directly impair broblast activity prior to wounding [117]. Indeed, much evidence has shown that broblasts isolated from DFUs show
decreased wound healing responses and a stronger resistance
to growth factors such as EGF, IGF, and PDGF than nonulcerated diabetic controls [145, 149, 202]. However, more
recent studies claried broblasts isolated from diabetic
non-wounded skin do undergo important biological changes
that renders them less responsive to important wound healing factors such as TNF-α, reduced their ability to stimulate
wound closure in mice, and decreased their production and
assembly of ECM similarly to DFUF [42, 145, 203, 204].
Important epigenetic differences between broblasts isolated
from diabetic patients is maintained even when re-exposed
to physiologic glucose levels, suggesting that hyperglycemia
imparts a robust metabolic epigenetic memory program in
broblasts that may prime diabetic skin to chronic wounds
when injured [205]. Studies using broblasts isolated from
monozygotic twins discordant for type 1 diabetes have found
important changes in broblast behavior to stressors that is
hardwired by diabetes exposure [206, 207]. These insights
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are supported by newer studies showing unique broblast
subpopulations shared in non-ulcerated and ulcerated diabetic skin compared to healthy skin [103, 208]. In DFU
broblasts plasticity is signicantly impaired through aberrant NOTCH1 signaling, long-noncoding RNAs, miRNAs,
and chromatin remodeling pathways [125, 209–217].
Fibroblasts isolated from DFUs and implanted in a 3D model
recapitulated the chronic DFU through impaired stimulation
of angiogenesis, decreased reepithelialization, and compromised ECM assembly and deposition [145]. Taken together,
epigenetic changes lock DFU broblasts in a proinammatory, anti-angiogenic, and anti-brotic state that
opposes wound healing [208, 218, 219]. This epigenetic
memory is likely a factor in the less-than-optimal healing
results seen with autologous skin grafts and allogenic
broblast- based therapies in the treatment of DFUs [59, 220,
221].
One potential remedy to bypass DFU broblasts memory
is to reprogram these broblasts into “normal,” pro-healing,
broblasts. In a series of recent studies, broblasts were collected from chronic DUFs and reprogrammed into induced
pluripotent stem cells (iPSC) to regain normal function.
These iPSCs were then transformed back into normal broblasts [222–224]. In an array of functional assays, these
reprogrammed DFUFs displayed enhanced healing capabilities that was, in part, attributed to miRNA-induced epigenetic reprogramming [224, 225].
Imbalance ofExtracellular Matrix (ECM)
Components inChronic Wounds
DFUs display an irregular, thin ECM that has increased
inammatory inltrates and decreased granulation tissue and
broblasts [226–228]. In normal wound healing, the acellular ECM is an important scaffold and hub that acts as an
everchanging highway and signaling reservoir for the plethora of migrating cells to complete the healing process. The
ECM must recruit and house inammatory cells as well as
repair local damage during the inammatory phase, encourage angiogenesis and cell migration during the proliferative
phase, and contract and reorganize during remodeling [229].
MMPs and their negative regulators tissue inhibitor of matrix
metalloproteinase (TIMP) are key enzymes involved in ECM
dynamics [229]. Major structural components of the ECM
known to be affected by these altered proteases include collagen, dermatopontin, and bronectin as well as important
growth factors, receptors, and cell adhesion molecules necessary for proper wound healing and cell behavior [230–
235]. MMPs are divided into seven broad subtypes according
to their substrate, including collagenases, gelatinases,
stromelysins, metalloelastases, matrilysins, membrane type,
and other [236]. In acute wounds, the ratio of MMPs and
TIMPs are tightly controlled in a spatiotemporal manner to
direct proper ECM reorganization for healing. In DFUs, this
control is ameliorated leading to chronically elevated levels
of certain MMPs and reduced levels of TIMPs favoring ECM
degradation [229, 234, 237]. The ratio of different MMP/
TIMP has been proposed in many studies to predict healing
out comes of DFUs [238–240]. Serine proteases are the other
major ECM remodeler implicated in DFU chronicity and
include mast cell tryptase and chymase, tissue-type plasminogen activators, neutrophil elastase, cathepsin G, and urokinase plasminogen activators [236, 241]. Neutrophil elastase
and cathepsin G, important components of neutrophil extracellular traps, are enriched in chronic DFU dermis and may
be an important biomarker to differentiate DFUs that are
prone to heal versus fail to heal [242]. Inhibiting NETosis in
diabetic wound models can accelerate wound healing [242,
243]. Topical sucrose octasulfate-impregnated dressings
have shown promising results in healing DFUs by inhibiting
the action of MMPs [187, 244].
The formation of advanced glycation end products (AGEs)
is a well-known metabolic consequence of hyperglycemia
and aging and specically targets many components of the
ECM [201, 245–249]. This intricate process involves a nonenzymatic reaction wherein a condensation reaction occurs
between the carbonyl group of a reducing sugar molecule and
a free amino group of a protein, forming an intermediate
Schiff base. Subsequently, this Schiff base transforms into an
Amadori product, which undergoes irreversible oxidation
into an AGE [250]. Collagen, vitronectin, vimentin, laminin,
and bronectin are recognized targets of this glycation [251–
256]. AGEs cause increased intermolecular cross-linking
between collagen bers resulting in the stiff, inelastic matrix
observed in diabetic tissues including the skin, heart, and
renal vasculature [245, 257–259]. AGEs also target the amino
acid side chains of collagen affecting their charge and interaction with other ECM components. Glycated collagen impairs
the migration and adhesion of inammatory cells, endothelial
cells, and broblasts [260–262]. Growth factors and their
canonical receptors are subject to this glycation with alteration of their activity [255, 263]. Specic receptors for AGEs,
termed RAGE, are located on most skin-residing cells and
facilitate complex cellular signaling cascades that impede
proper wound healing [264–267].
Certain growth factors and their receptors are indispensable for ECM formation and reorganization including
VEGF, IGF-1, TGF-B, PDGF, FGFs, and TNF-a. All these
factors are erroneously regulated in the DFU milieu and are
implicated in chronic DFU pathology [268–272].
Interestingly the only three FDA-approved therapies that
have shown efcacy for treating DFUs are Regranex
(PDGF-BB-based biologic therapy), a major EMC growth
factor, and Apligraf and Dermagraft which contain broblasts and ECM components [273].
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Dermal broblasts from diabetic skin closely resemble
those in healthy foot skin, suggesting that diabetes itself does
not impact function of broblasts prior to wounding [117].
Namely, a study of the mRNA and micro-RNA (miRNA)
expression proles of diabetic and healthy, nondiabetic foot
skin broblasts has revealed no signicant differences in
gene expression levels [117].
However, broblasts from diabetic foot ulcers exhibit
major changes including altered morphology, ECM deposition, increased apoptosis, diminished response to growth factors, reduced proliferation, and reduced migration [42, 125,
145, 146, 274]. Patient-derived broblasts from DFUs
seeded into three-dimensional models led to decreased stimulation of angiogenesis, impaired ECM synthesis, and recapitulated the nonmigratory and hyperproliferative epidermal
phenotype in organotypic cultures in the presence of keratinocytes [145]. In a study that used genomic approaches to
analyze the pathophysiology of DFU broblasts, miR-21-5p,
miR-34a-5p, and miR-145-5p, were found to be induced in
DFU-derived broblasts contributing to inhibition of cell
migration and proliferation, as well as induced differentiation and cell senescence [125].
I. Pastar et al.
Matrix Metalloproteinases andTissue
Inhibitors ofMetalloproteinases
An imbalance between ECM protein synthesis and remodeling by MMPs and the tissue inhibitors of metalloproteinases (TIMPs) is seen in DFUs. Increased MMP production
causes ECM degradation [275]. MMPs 1, 2, 8, 9, 14, and 26
are highly expressed in DFUs [231, 276–278] with concomitant reduction in the expression of their inhibitors
[104]. High MMP9 and a high MMP9/TIMP1 ratio has
been shown to be a predictor of poor wound healing [278],
whereas a higher MMP1/TIMP1 ratio is correlated with
healing (Fig.7.3) [239]. Lobmann etal. (2002) showed an
increase in MMPs with reduced concentrations of
TIMP-2 in patients with DFUs, compared to traumatic
wounds of nondiabetic patients, suggesting that the
increased proteolytic environment reduces ECM formation
and contributes to the failure of diabetic wounds to heal
[279]. The rise in MMP activity not only causes matrix
degradation, which delays cell migration and inhibits collagen deposition, but also breaks down growth factors and
their target cell receptors [15, 279].
Other common causative factors for chronic wounds
include deregulation of certain cytokines, growth factors,
and their receptors and corresponding signaling molecules.
Examples of these include TGF-β, FGF, insulin-like
growth factor 1 (IGF-1), interleukins, VEGF, TNF-α,
PDGF, EGF, EGFR, granulocyte-macrophage colony stimulating factor (GM-CSF), and receptors such as TGF-β
Fig. 7.3 Deregulation of MMPs and TIMPs in DFUs. Schematic overview of upregulated (arrows up) and downregulated (arrows down)
MMPs, TIMPs, and their ratios in DFUs
receptors, EGFR, and bone morphogenetic protein receptor [10, 15, 122, 280].
Angiogenesis inDiabetes
During wound healing, new capillaries form and replace
damaged capillaries in a process known as neovascularization. Neovascularization is important to reestablish oxygen
and nutrient supply to the wound and remove waste products
[64]. Angiogenesis, one form of vascularization, is the formation of new blood vessels from preexisting ones and is
usually caused by tissue injury or neoplastic transformation
[281, 282]. During the proliferative phase of wound healing,
endothelial cells, stimulated by VEGF, FGF2, or low-oxygen
tension/hypoxia, migrate to the wound and induce angiogenesis and sprout capillaries to vascularize the tissue that is
being formed [283, 284]. Angiogenesis arises through a
nely balanced process involving pro-angiogenic and antiangiogenic mediators, cells ECM, cytokines, and growth
factors, and a shift in this balance leads to impaired angio-
7 Physiology andPathophysiology ofWound Healing inDiabetes
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genesis (Table7.1) [282, 285, 286]. Vasculogenesis is the de
novo formation of blood vessels from bone marrow-derived
endothelial progenitor cells (EPC) [287]. These newly
formed vascular structures mature into capillaries, arterioles,
arteries, venules, and veins.
Impaired angiogenesis and vasculogenesis, as a result of
deregulation and cleavage of growth factors, and their receptors lead to insufcient oxygenation and sub-optimal delivery of nutrients to the wound contributing to poor diabetic
wound healing (Tables 7.1 and 7.2) [68, 285, 288].
Dysfunctional angiogenesis is also a key player in many
diabetes- related microvascular complications including diabetic nephropathy, diabetic peripheral neuropathy, and diabetic retinopathy [282, 285]. The capillary architecture in
DFU is compromised including structural components, maturation, and capillary permeability and pericyte coverage
[289]. The density of capillaries and levels of pro-angiogenic
stimuli are also decreased in DFU [68, 290].
VEGF, a pro-angiogenic growth factor, is important for
endothelial cell proliferation and migration, ECM degradation, vessel permeability, and vasodilation [285]. Deregulated
Table 7.1 Overview of angiogenesis in acute and chronic wound healing
Normal angiogenesis Angiogenesis in DFU
Pro-angiogenic cytokines (including VEGF) are released from
platelets, monocytes, and broblasts
Endothelial cells (ECs) disrupt their interactions with neighboring ECs Resident ECs on the chronic wound may lose their ability to support
ECs digest the basement membrane and extracellular matrix (ECM)
components (via matrix metalloproteinases)
ECs, broblasts, platelets, smooth muscle cells, and monocytes release
more pro-angiogenic cytokines
ECs invade ECM and migrate/proliferate to form robust new vessels.
These vessels are disorganized, tortuous, dense, leaky, and poorly
perfused [68]
Antiangiogenic and maturation factors induce remodeling of the
capillary bed where excess vessels are pruned and vessel integrity is
restored [68]
VEGF and associated signaling pathway contributes to
diabetes- related pathologies [282, 285]. In some organ systems, high levels of VEGF acts as a pathologic angiogenic
stimulus (i.e., ocular neovascularization), while in others,
reduced levels of VEGF activity leads to pathology (i.e.,
nephropathy, peripheral neuropathy, and wound healing)
[282, 285, 291, 292]. Macrophages have been implicated in
dysfunctional angiogenesis in DFU largely through their role
in VEGF/VEGFR signaling [289, 290, 293–296].
Another contributing factor to the impaired angiogenesis
seen in diabetics is the bone marrow-derived EPC (Tables
7.1 and 7.2). Dysfunctional EPC, diminished EPC numbers,
and defective recruitment, as well as transition of EPC phenotype to a pro-inammatory one, have all been observed in
diabetic patients [296–298]. Increased number of CD34+
CD45dim measured at the beginning of care in DFU patients
are positively associated with healing status and affected by
wound area and duration, with increasing numbers showing
benecial effect on wound closure [297, 298]. Furthermore,
lncRNA antisense noncoding RNA in the INK4 locus
(ANRIL) is found downregulated in peripheral blood sam-
Fibroblasts become senescent in chronic wounds and lose their ability
to provide angiogenic functions [64, 410]
new vessel formation [285]
Impaired balance between the accumulation of ECM components and
their remodeling by MMPs [279, 411]
The chronic wound environment impairs cellular proliferation and
angiogenesis [412]
EC adhesion, migration through the ECM, and proliferation are
impaired in diabetic wounds [282, 285, 413]. The density and number
of newly formed capillaries is signicantly attenuated in DFU [68]
Antiangiogenic and maturation factors are altered in DFU, resulting in
leaky and dysfunctional capillaries [68]
Table 7.2 Overview of normal vasculogenesis process and its impairment in DFUs
Normal vasculogenesis Vasculogenesis in DFU
Multipotent adult progenitor cells (MAPCs) differentiate into
hematopoietic precursor cells or early endothelial progenitor cells (EPCs)
in the bone marrow
Increased vascular endothelial growth factor-A (VEGF-A) induces
vascular endothelial growth factor receptor-1 (VEGF-R1) activation and
subsequently increased matrix metalloproteinase-9 (MMP-9) secretion
Increased MMP-9 mediates the conversion of membrane-bound Kit
ligand (mKitL) to soluble Kit ligand (sKitL), which mobilizes EPCs from
the bone marrow to circulation
Early EPCs in the circulation further differentiate to late EPCs and gain
specic endothelial cell (EC) surface markers
Late EPCs arrive to the site of new vessel formation and further
differentiate into mature ECs or act as a source of pro-angiogenic
cytokines
Impaired VEGF-induced proliferation response in EPCs [413]
Hyperglycemia-mediated inhibition of VEGF [413, 414]
Decreased number and function of circulating EPCs impairs
healing [414, 415]
Decreased EPCs in the bloodstream is correlated with nonhealing
DFUs and can be used to predict healing potential [297, 298]
Diminished blood supply to peripheral wound [414]
EPCs demonstrate abnormal mobilization and homing mechanisms
in diabetics [414–416]
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ples, DFU mouse models, and EPCs exposed to hyperglycemia, while increased ANRIL expression promoted wound
healing in DFU mouse models by modulating the expression
of HIF1α [299]. Additional lncRNA, growth arrest-specic 5
(GAS5), was shown to promote healing by activating the
HIF1A/VEGF pathway by binding the TATA box-binding
protein-associated factor 15 (TAF15) [300]. E2F2 transcription factor accelerated wound healing in mouse models
likely through activating expression of cell division cycleassociated 7-like (CDCA7L) expression in EPCs and activating angiogenesis [301]. Causes of EPC dysfunction and
reduced recruitment from the bone marrow in diabetic individuals include hyperglycemia, increased oxidative stress,
chronic inammation, and NADPH oxidase activation [68,
297, 298, 302]. In rodent diabetic wound models, researchers
were able to modulate EPC dysfunction in response to hyperglycemia and increase wound healing by exposing them to
exosomes rich in circular RNA, mmu_circ_0000250, which
stimulated SIRT1/miR-128-3p-mediated autophagy [303].
A better understanding of the mechanisms that cause dysfunctional angiogenesis is of vital importance particularly in
light of the upward trend in diabetes mellitus microvascular
complications. Further understanding of the role of
angiogenesis in these pathologies is necessary to pave the
way forward for the development of novel therapies.
Inammation andInfection in
Diabetic Wound Healing
Unresolved inammation is a key aspect of chronic wounds
that prevent them from progressing through the normal
phases of wound healing [116]. The current paradigm characterizes chronic wounds as being “stuck in a chronic inammatory phase” in which excessive inammation contributes
to the inhibition of wound healing and is the rationale to
therapeutically target the inammatory response to limit
inammation. However, this has proven to be clinically
unsuccessful [304]. More recent studies have demonstrated
that chronic wounds display an ineffective inammatory
response characterized by impaired immune cell recruitment
and function that results in a deregulated yet prolonged
inammatory response, which fail to reach the optimal levels
of inammation that support progression of the healing in an
acute wound [101, 102]. The imbalance between proinammatory and anti-inammatory signals results in inhibition of wound healing through increased cortisol levels
[136–138] that in turn induce expression of caveolins resulting in impaired actin cytoskeletal signaling [101–103, 142,
305]. These previously unrecognized ndings describe a
paradigm shift demonstrating a diminished inammatory
state present in chronic wounds and provides the insights
regarding lack of clinical effectiveness of therapeutic targeting of inammation.
A persistent inammatory state contributes to the poor
wound healing phenotype seen in chronic wounds and may
be caused by multiple factors. Uncontrolled inammation is
known to induce MMP expression, cause tissue damage,
decrease collagen synthesis, and inhibit epithelialization
[121, 275, 306]. Elevated levels of advanced glycation endproducts (AGEs) in serum of diabetic individuals result in a
subclinical chronic inammatory state and affects synthesis
of collagen [121, 306]. Hyperglycemia has been shown to
elevate oxidative and inammatory stress via ROS and tumor
necrosis factor alpha (TNF-α), sustaining inammation [307,
308]. Neuropathy and uncontrolled diabetes can affect inl-
trating cell numbers in the skin. Studies have shown an
increased number of inammatory cells present in the forearm skin of neuropathic individuals [272, 309], but not nonneuropathic ones [117].
During normal wound healing, macrophages shift from a
pro-inammatory or “M1” phenotype to a healingassociated or “M2” phenotype [80, 81] and express proinammatory and anti-inammatory genes, respectively
[82, 83]. Indeed, relative expressions of pro-inammatory
and anti- inammatory genes have been shown to be different in healing versus nonhealing human chronic DFUs
[310]. Macrophages exhibited a prolonged pro-inammatory response with high-expression levels of pro-inammatory molecules such as TNF-α, IL-1β, and MMP-9 during
diabetic- impaired healing [80, 88]. Macrophage dysfunction contributed to the defective healing seen in wounds of
diabetic humans and mice [88]. In other studies, Nod-like
receptor protein (NLRP)-3 inammasome contributed to the
sustained inammatory state displayed by macrophages and
is in part mediated by IL-1β in diabetic human and mouse
wounds [88, 311]. Prolonged production of IL-1β has also
been reported to reduce peroxisome proliferator-activated
receptor γ (PPARγ) expression in diabetic wounds contributing to impaired healing [80]. Recent studies have demonstrated impaired recruitment of macrophages at the wound
edge of DFUs [101]. In addition, single-cell transcriptomics
revealed increased numbers of M1 macrophage population
in healing DFUs compared to nonhealing DFUs, supporting
the notion that an acute wound-like inammatory response
needs to be reactivated in DFUs to allow progression of
their healing [101–103]. These ndings are in contrast to
animal studies that demonstrated increased M1 populations
inhibit healing, suggesting that increasing the M2 population would stimulate healing in diabetic wounds. However,
these ndings reect the differences in human vs. mouse
models of wound healing and further supports that nonhealing chronic wounds have a suppressed inammatory
response [312, 313].
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