4. Immunoregulation
Local ischemia, necrosis and
microorganisms in wounds trigger the inflammatory response. At this
stage, macrophages and neutrophils invade the wound to inhibit the
microorganisms and clear necrotic tissue and cells. Nanomaterials can
promote beneficial inflammation and immune regulation, making them a new
therapeutic strategy for wound treatment.
4.1 Acute wounds
The inflammatory period of acute wounds, such as traumatic wounds and
surgical wounds, usually lasts 2-3 days. In this stage, macrophages and
neutrophils are recruited and secrete inflammatory factors such as IL-1,
IL-10 and TNF-α to clear local microorganisms and necrotic tissue, which
is considered to be conducive to wound repair(Vigani et al., 2019).
Nanomaterials have the potential to stimulate innate immunity. Some
metal nanomaterials,including TiO2 NPs, CuONPs and
carbon-based nanomaterials, such as graphene,have beenreported to
recruit and activate macrophages and neutrophils. This may be related to
activation by the nanomaterials that mimic
pathogen-associated molecular
patterns (PAMPs), inflammatory receptors such as the
Toll-like receptors (TLRs), and
nucleotides combined with the
oligomeric structure domain (NOD) receptor NLRP3(Boraschi et al., 2017;
Kinaret et al.,
2020).Therefore,
we can infer that application of these nanomaterials in the early stage
can promote the inflammatory period of clean wounds. However, little
attention has been paid to this aspect because the release time of
proinflammatory nanomaterials is not controllable, which may cause a
continuous inflammatory response and impede wound healing. For this
reason, proinflammatory nanomaterials can be applied within 3 days of a
clean acute wound or nanomaterials loaded with neutrophil and macrophage
activators such as IL-8 and IFN-γ can be developedfor release over the
first 3 days to achieve short-term effects. The actual effects need
further experimental confirmation.
4.2 Chronic wounds
Chronic wounds, such as burn wounds and diabetic wounds,are usually
trapped in a state of persistent inflammation due to burn stimulation
and chronic infection, which is characterized by increased levels of
proinflammatory cytokines and a nonhealing ability. Current studies have
suggested that persistent inflammationis mainly caused by the disordered
transition of macrophages from the M1 to M2 phenotype. Macrophages that
are trapped in the M1 phenotype continuously secreteproinflammatory
factors, which leads to severe tissue damage. Researchers have explored
several ways to reverse thissituation. Some nanomaterials, such
asTiO2 NPs and nanofibrous scaffolds, can promote the
transition from the M1 to the M2 phenotype. IL-10, a polyamine secreted
by M2 macrophages, has anti-inflammatory and tissue repair effects to
promote wound healing(Dukhinova et al., 2019; Kaymakcalan et al., 2018;
Sun et al., 2018). Moreover, further mechanistic studies have shown that
nanomaterials could activate the complement system in wounds by
regulating the TLR/NFκB, MAPK/mTOR, and KGF2/p38 signaling pathways,
reducing the expression of proinflammatory cytokines such as TNF-α, IL-1
and IL-6, and increasing the expression of anti-inflammatory cytokines
such as IL-4 and IL-10 (Sun et al., 2019; Zhang et al., 2020)(Figure
1C).
The application of nanomaterials to regulate the inflammatory state of
chronic wounds is a feasible treatment strategy. However, this approach
has its limitations. For example, the inflammatory factors in burn
wounds are mainly TNF-α, IL-1β and IL-6, while high expression of IL-18
is always observed in diabetic wounds. Nanomaterials such as
silver nanoparticles (AgNPs) and
nanofibers have beenreported to reduce the levels of IL-1β and IL-6, but
the inhibitory effects of IL-18 havenot been verified, suggesting that
these nanomaterials may have poor anti-inflammatory
effectsagainstdiabetic wounds. Therefore, nanomaterials with excellent
anti-inflammatory properties should be developed in the future for the
treatment of various kinds of wounds(Mohammadi et al., 2019; Wasef et
al., 2020). In addition, the inflammatorystage usually overlaps with the
proliferation stage, and it is difficult to identify the end point of
the inflammatoryperiod. Therefore, the negative effects of nanomaterials
on proliferation should be taken into consideration when being applied
for the immunoregulation ofchronic wounds. Further research could
identify a marker that denotes the transition from theinflammatory phase
to the proliferation phase to indicate the usable time of
animmunomodulatory nanomaterial.
4.3 Immunological wounds
Immune factors can also cause skin woundsthat patients with immune skin
diseases are often accompanied by ulcers. For example, allergic
vasculitis is accompanied by activation of the NFκB pathway and
increases in TNF-α and IL-6, and dermatomyositis shows a proinflammatory
phenotype with elevated levels of IL-6 and IL-10 after activation of
TLR7. Behcet’s disease and pyoderma gangrenous also displayelevated
expression levels of IL-1 and IL-6(Chen et al., 2014; Kozono et al.,
2015; Piper et al., 2018; Talaat et al., 2019; Wallach et al.,
2018).Immune disorders are the main cause of wound formation in
theseconditions, so immunotherapy plays a crucial role in wound healing.
For this purpose, we can obtain inspiration from the anti-inflammatory
properties of nanomaterials. For example, AgNPs and nanofibers can
reduce the expression levels of IL-1, IL-6 and TNF-α, and metal
nanomaterials such as ZnO NPs and TiO2 NPs can inhibit
the TLR and NFκBpathways by activating the transcription factorsPPARγ
and arginase 1. Therefore, it can be inferred that nanomaterials may
play a role in controlling immunological skin wound inflammation through
the above pathways, but there is still a lack of relevant research(Chen
et al., 2019; Dukhinova et al., 2019; Zhang et al., 2020). On the other
hand, immune skin wounds are often accompanied by an adaptive immune
response. The activation of CD4+ and
CD8+ T cells and the decline in Treg cells are
considered tobe associated with the pathogenesis of immune skin
diseases(Hoeppli et al., 2019; Leccese et al., 2019; Quaglino et al.,
2016). Treg cells maintain peripheral immune tolerance in the body and
can secrete anti-inflammatory factors or exosome vesicles to reduce T
cell proliferation and promote T cell apoptosis. Therefore, we propose a
new concept of nanomaterial-mediated immune tolerance for the treatment
of immune wounds. Similar to the principle of nanovaccines, specific
antigens carried by peptides can betransmitted through nanomaterials.
The antigens are then recognized and presented by
immature dendritic cells (DCs),
inducing Treg cell proliferation but not a proinflammatory response.
Immature DCs then transmit tolerance signals by default to maintain
peripheral tolerance(In’t Veld et al.,
2017).This
method has been studied in patients with multiple sclerosis. If the
desired effects in patients with immune skin diseases are achieved, it
will be of great help to patients in the stable stage to prevent
recurrence.
5. Reconstruction
phase
Woundsareruptures or defects of the skin. After the local infection is
controlled, the proliferation of skin and subcutaneous tissue is the
most important process duringwound healing and involves the formation of
blood vessels, the proliferation of fibroblasts and keratinocytes,
andthe regeneration of skin appendages. Overthe past few decades, many
studies have focused on the proliferation effects of nanomaterials
themselves or their use as carriers on wounds. Different from
traditional wound dressings, the porous structure of a nanomaterial can
provide a scaffold for new cells and proteins. The nanomaterialcan
interact with the tissues around the wound, activate the repair system
in the body, and stimulate the secretion of various growth factors to
promote wound healing.
5.1 Granulation tissue
Initial wound repair relies on the accumulation of granulation tissue.
Fibroblasts together with new capillaries proliferate rapidly and
synthesize collagen fibers and matrix components.Many studies have shown
that nanomaterials can promote the formation of granulation tissue.
Inorganic nanomaterials such as ZnO and terbium hydroxide NPscould
induce oxidative stress in
vascular endothelial cells(VECs). The accumulated ROS in VECs activate
the p38 MAPK/Akt/eNOS signaling pathway, leading to the formation of NO,
which stimulates angiogenesis(Nethi et al., 2019). Alternatively,these
inorganic nanomaterialscould activate the Notch signaling pathway in
VECs, the Notch1, Notch3, and Notch ligands Dll 1 and Jagged 1 and their
target genes Hes 1 and Hey 1, which is another mechanism to promote
angiogenesis by nanomaterials(Zhao et al., 2018a).
Moreover,nanomaterials could also promote the proliferation of
fibroblasts. Human skin fibroblasts were treated with composite
nanofibers containing chitosan and AgNPs in vitro. After exposure to the
nanofibers, the cell cycle progressed from stationary G0/G1 phase to S
and G2 phases with active DNA synthesis and division. During this
process, the TGF-β1/SMAD signaling pathway was activated, and the
results could be reversed by TGF-β1 receptor inhibitors(Zi-Wei et al.,
2017).Moreover, the nanomaterials prevented the apoptosis of
fibroblasts. A hyaluronic acid-based nanosystem was found to shield cell
death receptorsandprevent cell apoptosis by activating the CD44 receptor
in the cell membrane. In addition, the activation of CD44 stimulates the
signaling cascade of the RHAMM receptor and tyrosine kinase 2 pathways,
which leads to increased cell motility and growth (Vigani et al.,
2019)(Figure 1D). The proliferation of blood vessels and fibroblasts is
synchronous. For chronic old wounds with reducedblood supply,
nanomaterials can be used as wound dressings to promote the repair of
wound defects. However, the timing and duration of administration should
be taken into consideration. A clean wound is a prerequisite for
granulation growth. Insufficient proliferation of granulation will lead
to the collapse of the wound surface, while excessive proliferation of
granulation will hinder the process of epithelialization or lead to scar
hyperplasia. Future research could explore the indicators that signify
the treatment endpoints to guide the administration duration of use in
practical applications.
5.2 Epithelization
After the granulation tissue fills in the wound defect, keratinocytes
proliferate to form an integrated epidermis. In vivo studies in rats
have confirmed that nanomaterials can promote the viability and
proliferation of keratinocytes and accelerate the
epithelialization
of wounds. They can upregulate the expression of repair-related genes,
such as TGF-β, Smad2, KRT6a, and IVN,in HaCaT cells and activate the
TGF-β-VEGF-MCP 1, TGF-β-Smad2,and HER2-ErbB2 pathways to promote the
epithelialization process. In addition, nanomaterials can stimulate the
secretion of various growth factors,such as FGF2, PDGF and EGF, activate
the ERK and P38 signaling pathways and promote the proliferation and
migration of fibroblasts and HaCaT cells (Bhattacharya et al.,
2019)(Figure 1D). Keratinocytes are involved in the formation of the
skin barrier, electrospun tilapia collagen nanofibers have been observed
to upregulateinvolucrin, filaggrin and TGase1, which are important
components of the skin barrier(Zhou et al., 2016).Therefore, in the
later stage of wound repair, application of these nanomaterials can
facilitate wound closure and repair the skin barrier. However, for some
wounds with large areas, such as burn wounds, the healing speed of this
approach may not be enough. Therefore, several methods should be
combined. For example,
platelet-rich plasma (PRP) is a
natural repository of growth factors. PRP-containing nanoscaffolds have
been developed to release PRP to promote epithelialization. In the
future, PRP can also be loaded into nanomaterials with
pro-epithelialization potential(do Amaral et al., 2019). Alternatively,
new nanomaterial-based wound dressings can be developed by loading
autologous, allogeneic, or tissue-engineered skin pieces to accelerate
the adhesion of skin, the formation of skin paddles and the
proliferation of keratinocytes.
5.3 Adipose tissue
Duringthe
repair of some deep wounds, local collapse usually appears after wound
closure. This collapse is probably caused by the dysplasia of
subcutaneous adipose tissue during the healing process. Whether
nanomaterials can be used to promote the
reconstruction
of subcutaneous adipose tissue defects has not yet been studied. The
present research has explored the influences of nanomaterials on
adipose-derived
stem cells (ADSCs), which have multidirectional differentiation
potential to differentiate into fat, cartilage and bone. Nanomaterials
can promote the hypermethylation of the Dlg3 gene promoter, which leads
to a decrease in Dlg3 expression. Downregulation of Dlg3 promotes the
proliferation
ofADSCs
and reduces cell apoptosis(Lin et al., 2018a). On the other hand,
nanomaterials can promote the adipogenic differentiation of ADSCs. The
adipogenic markers of ADSCs, such as PPARγ and FABP4,aretypically
expressed after culture with scaffolds made by electrospinning(Gugerell
et al., 2014).This suggests that nanomaterials can be used in deep
wounds to promote ADSCs proliferation and adipocyte differentiation, and
that the accumulation of adipocytes can repair defective subcutaneous
tissue. However, most of the current studies have beenin vitro
experiments, and confirmation studies inin vivo models are still
lacking. In addition, different types of nanomaterials have different
influences on ADSCs. For example, nanoscaffolds and some new
nanomaterials, such as tetrahedral
DNA nanostructures (TDNs),have beenconsidered to promote adipogenic
differentiation, while SiO2 NPs and fullerenes were
found to have the opposite effect(Saitoh et al., 2012; Yang et al.,
2017).There is still a wide space
for research on developing new nanomaterials to promote the
reconstruction of subcutaneous adipose tissue and the related
mechanisms.
The application of nanomaterials in
the wound healing process should follow the sequence of subcutaneous
tissue repair promotion, granulation tissue hyperplasia and
epithelialization, and different nanomaterials may be required for each
stage. However, because of the different types and depths of wounds, the
repair time of each stage is also different. Therefore, distinguishing
the dividing point of these stages has currently become a difficulty. In
the clinic,the dividing point usually relies on the subjective judgment
of the physician. If a marker can be obtained, we can extract the local
tissue of the wound to determine the stage of tissue repair and select
an appropriate nanomaterial. In this way, accurate wound repair can be
achieved in the future, which not only reduces healing time but also
maintains aesthetics as much as possible.
5.4 Amelioration of the
microenvironment
As nanomaterial research has developed, scholars have begun to pay
attention to the impacts of these nanomaterials on the cellular
microenvironment. It has been found that nanomaterials such as
nanofibers and hydrogels have hydrophilic surfaces, exhibita high water
retention capacity and provide a moist environment for wound healing(Fu
et al., 2014). AgNPs, gold
nanoparticles (AuNPs), copper nanoparticles (CuNPs) and other
nanomaterials can reduce the expression of matrix
metalloproteinasesMMP-1,MMP-3, andMMP-8 and inhibit their decomposition
of collagen(Frankova et al., 2016; Lee et al., 2015).These nanomaterials
accelerate the remodeling of the ECM by promoting the deposition of type
I and III collagen and fibronectin, providing a beneficial environment
for cell proliferation and wound repair. In addition, studies have been
conducted to use nanomaterials, collagen extracted from marine fish skin
or pig decellularized ECM to prepare composite materials. These
nanodressings have high histocompatibility, which can reduce both
irritation to tissue and the inflammatory response. These
nanodressingsare especially suitable for wounds that require long-term
coverageof the dressing, such as burn wounds and diabetic wounds(Lin et
al., 2018b; Ramanathan et al., 2017).Nanomaterials can also monitor the
physical and chemical status of the ECM. Some scholars developed a novel
electronic dressing that can continuously display the pH changes in the
wound environment and control the pH value through an electric field to
keep it in a suitable range for cell proliferation(Nischwitz et al.,
2019).This technique can be used to control wound infection by
artificially regulating the pH value to inhibit the growth of
microorganisms, and it can also guide the release of drugs by monitoring
the pH value. Furthermore, in the future, we can develop electronic
dressings that monitor the state of extracellular hypoxicconditions and
Ca2+ concentration, which can affect MMP activity and
the synthesis of collagen. According to the real-time status, we can
adjust these parametersto be in the appropriate range. The
implementation of these ideas heralds the advent of anera of wound
repair with precision control(Khadjavi et al., 2015; Navarro-Requena et
al., 2018).