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).