6. Modifications after wound regeneration
After the remodeling stage, wound closure istraditionally complete.However, there are still some problems, such as scar hyperplasia, loss of hair follicles and sensory disturbances. In the past, post-healing repair was often overlooked. With the improvement of medical technology, more attention has been given to aestheticsand functional recovery. As a new therapeutic material, nanomaterials have also been developed and applied forthe post-healing repair of wounds.
6.1Scar prevention
Scarsmanifest as excessive fibroblastproliferation, disordered cell growth and abnormal collagen deposition, which not only affect the aesthetic appearance but also cause poor local traction and elasticity, resulting in limb dysfunction;there is also a risk of cancer. Currently,scars are mostly treated by surgical resection, local injection and radiation. Nanomaterials havebeen gradually applied in the treatment of scars. For example, some scholars developed glucocorticoid-loadedhydrogel particles to realize the long-term release of glucocorticoids, and the effectson the inhibition of scars has been confirmed in rabbits(Guo et al., 2018). AuNPs have been used as carriers of photosensitizersbecause ofthe photothermal effects of AuNPs and local surface plasmon resonance (LSPR) to enhance oxidative stress, leading to fibroblast apoptosis or necrosis(Zhang et al., 2017).However, these methods are used after the scar has formed, although ideally, scar formation would be prevented before the wound is healed. Nanomaterials have been explored in this area. Studies have found that some nanomaterials, such as carbon nanotubes,can inhibit the excessive proliferation of fibroblasts by inhibiting the TGF-β-SMAD pathway. Moreover,these nanomaterials can regulate the expression of various enzymes in the cellular microenvironment, such as promoting the expression of MMP-1 and inhibiting collagen and fibronectin levels in the ECM. In addition, nanomaterials can induce the oriented movement of fibroblasts(Poormasjedi-Meibod et al., 2016; Weng et al., 2018).All of these observations indicate that nanomaterials have the potential to prevent scar formation before epithelialization. However, there is still a lack of in-depth mechanistic research, such as whether these nanomaterials can affect other scar pathways, such as Wnt/β-catenin, and regulate macrophage polarization to inhibit scarring hyperplasia (Hesketh et al., 2017; Yu et al., 2016)(Figure 2).In addition, most of the current studies have beenin vitro experiments, and there is a lack of in vivo confirmation studies, which are difficult to carry out. Reviewing the process of wound healing, the hyperplasia process of granulation tissue before epithelialization requires the activation of the TGF-β-SMAD pathway to promote the proliferation of fibroblasts. This processalso promotes the deposition of collagen during the remodeling phase, which is exactly the opposite of the antiscarring mechanism. Therefore, the application of nanomaterials to prevent scars before epithelialization may inhibit the active proliferation of granulation tissue. If we want to prevent scar formation before epithelialization, we need to consider other aspects. For example, we can implant nanotubes and other nanomaterials duringthe granulation tissue proliferation stage or use 3D printing technology to fill wound defects with nanoscaffolds to guide the directed and moderate growth of fibroblasts. We can also develop new nanomaterials to reduce the high levels of cytokines such as IL-6, IL-8, and IL-17 in the scar and adjust the expression of miR-146a, miR-21, neurotransmitters such as bradykinin, and substance P. Further experiments are needed as to whether these ideas are sufficient(Lebonvallet et al., 2018; Lee et al., 2018; Zhang et al., 2018).
6.2Hair follicle reconstruction
When the defect of the wound reaches the dermis, it will lead to loss of the hair follicle, so regeneration of the hair follicle is another part of wound healing. However, in wounds with skin substitute treatment and scar formation, regeneration of the hair follicles is the technical bottleneck for the complete restoration of skin structure and function. The application of nanomaterials in medicine has attracted the attention of scholars, who have begun to note the influence of nanomaterials on hair follicles. Synthetic composite electrospun membranes can promote the recruitment and proliferation of hair follicle stem cells by releasing zinc ions(Zhang et al., 2019). The nanofiber scaffold seeded with hair follicle stem cells can promote the attachment, proliferation and differentiation of hair follicle stem cells on the scaffold(Hejazian et al., 2012). These studies indicate that nanomaterials have the potential to promote hair follicle regeneration, but there are few studies in this field. Most of the studiesare in vitro experiments with limited types of nanomaterials. Although an in vivo study inSprague-Dawley (SD)rats showed that the number of hair follicles and hair follicle stem cells in the nanodressinggroupwas higher than that in the control group, further experiments are needed to explain the mechanism. The reconstruction and regeneration of hair follicles are difficulties that are faced in wound recovery(Zhang et al., 2019).Currently, surgical hair transplantation is widely used for hair follicle reconstitution, but the survival rate is limited. This may be related to factors such as damage to the transplanted hair follicle during the operation and local inflammation after transplantation. Inspired by the fact that nanomaterials can promote the proliferation of hair follicle stem cells and inhibit inflammation, we couldtry to use anti-inflammatory nanomaterials or composite nanomaterials loaded with stem cells, PRP and other drugs that promote the survival of hair follicles as auxiliary treatments to improve the viability and success rate of hair transplants (Figure 2).
After hair follicle reconstruction, whether nanomaterials can promote hair growth can also be studied in the future. It was reported that poly(glutamic acid) (PGA)NPscan increase the expression levels of the proteinscyclin D1 and CDK4 and induce the development of the hair follicle cycle by activating the Wnt/β-catenin pathway. These proteinsalso increase the expression of type II keratin and melanin and promote the proliferation of dermal papilla cells toencourage hair growth(Lee et al., 2019).However, most current studies have focusedon nanomaterials as drug carriers for hair loss, and there is still a lack of research on the effects of the nanomaterials themselves on hair growth. Because of itsspecial structure, drugs can be stored within the hair follicle and slowly released for a long time. Nanomaterials are small in size and more easily enter the hair follicle for storage. Compared with other drugs for hair loss treatment, nanomaterials have the advantages of higher efficiency and longer lasting effects. Further exciting progress is expected in the exploration ofnanomaterials to promote hair growth.
6.3Skin sensory regulation
There are many cutaneous nerves and peripheral nerves in the skin, and skin wounds are bound to damage these nerves. Local infection, inflammation or improper nerve regeneration will cause paresthesia such as pain, itching, and hypoesthesia after healing, which affect the quality of life of patients.
Nanomaterials are widely used in the development of topical drug delivery systems due to their superior drug-carrying properties. To increase percutaneous drug penetration, prolong the release time and reduce side effects, some analgesics, such as nonsteroidal anti-inflammatory drugs, local anesthetics, capsaicin, and antipruritic drugs, such as glucocorticoids, have been developed as nanoemulsions, transfersomes, solid lipid nanoparticles (SLNs) and as other systems for the treatment of pain and itching after healing.In addition, topical ZnO NPs have been observed to have anti-itch effects(Aman et al., 2019; Andreu et al., 2018; Bikkad et al., 2014; Ghiasi et al., 2019; Nafisi et al., 2018).These formulations are feasible for most kinds of wounds, such as postoperative wounds, diabetic wounds, and burn wounds. However, postherpetic neuralgia woundsare a special circumstance. Neuralgia is severe and continuous and requires more powerful analgesics. We couldtry to apply an anestheticnanosystem for local pain relief or to develop delivery nanosystems for neuralgia drugs such as gabapentin and pregabalin. The actual clinical effects still need to be confirmed by further studies. In addition, there are few studies on whether nanomaterials can relieve pain and itching symptoms. The mechanisms of pain and itching overlap, and both are related to the sensory transmission of nerve cells mediated by neuropeptides, proteases and other mediators. Whether certain nanomaterials can achieve pain relief and antipruritus by inhibiting the secretion of these nerve mediators or inhibiting nerve signal transduction is a direction that can be explored in the future.
Hypoesthesia after wound healing is difficult. Generally, physical therapy is used to improve symptoms, but the therapeutic effect is poor. In recent years, electronic skin represented by carbon-based nanomaterials has emerged in the field of body surface monitoring. Electronic skincan collect information of temperature, pain sensation, and even taste sensation from the skin and convert it into electronic signals for transmission(Qiao et al., 2018; Zhao et al., 2018b).Then,the application of this kind of equipment to patients with hypoesthesia after a large area of wound healing could be imagined. When the patientsare exposed to a harsh environment of heat, chemical damage and electrical stimulation, they cannot respond in time due to hypoesthesia. The application of electronic skin can transmit danger signals on a more appropriate timescale and protect the body from danger. Unfortunately, the current technology just convertsthese signals into digital signals, which cannot be directly transmitted to human nerves and fed back to the brain. If this difficulty is overcome, holistic functional wound healing wouldbe further realized(Ma et al., 2019).Moreover, before electronic skin is applied to the body, it is necessary to consider the harmonious symbiosis between the electronic skin and the surrounding normal nerves, muscles, lymphatics and glands, as well as ensure the precise transmission of nerve instructions. These may be the next directions for researchers (Figure 2).
7. The toxicity of nanomaterials in wound healing
Nanomaterials have an excellent ability to promote wound healing, and there is still great potential for their application and development in the future. However, it should be noted that the wound surface is not protected by intact skin. The nanomaterials directly contact the tissue inside the wound, and the biological safety of the products must be considered before application.
The most reported transdermal toxicities of nanomaterials are skin irritation and allergies(Ema et al., 2013; Palmer et al., 2019).Theseside effectshave individual differences and may be unavoidable. There havealso been frequent reports ofnanomaterials causingoxidative stress, autophagy and apoptosis in keratinocytes and fibroblasts(Wang et al., 2018). These toxicities depend on the particle size, shape, surface charge and concentration of the nanomaterial. Therefore, these factors should be adjusted to reduce toxicity to skin cells when developing new nanomaterials used in wounds(Hashempour et al., 2019).In addition, it has been reported that nanomaterials cause DNA damage and decrease gene methylation, suggesting the potential of cell canceration(Ali et al., 2016; Sooklert et al., 2019).However, there is still no direct evidence to prove that nanomaterials can cause malignant transformations and inherited gene mutations in skin cells. Thisdoes not mean that nanomaterials are safe. It is not known whether the long-term percutaneous exposure and deposition of nanomaterials in the skin will cause severe consequences, which will need to be confirmed by long-term exposure experiments in the future.
Nanomaterials will come in contact with blood cells in ruptured blood vessels in wounds and enter the blood circulation. This phenomenonbrings two consequences, one of which is hemolysis. Some metal nanomaterials, such as AgNPs and ZnO NPs, have been found to cause hemolysis. To solve this problem, we can adjust the physical and chemical properties of the materials or wrap biologically active substances such as phospholipids and polysaccharides onto the surface of the nanomaterials(Bakshi, 2017).Another consequence is that the nanomaterials will spread throughout the body into various organs after entering the blood, causing multisystem effects. Compared with the original concentration of the nanomaterials, the concentration of nanomaterials after entering the blood circulation is greatly reduced, and they can be partly excreted through urine and feces. Weight loss and even death have been observed in animal experiments, but in practical use, there is no conclusive evidence regarding whether nanomaterials will cause organ failure and/or tumors, whether exposure during pregnancy will affect offspring and what the safe concentrationis(Hadrup et al., 2018).
In general, nanomaterial toxicity studies in wound healing havemainly concentrated on local acute adverse reactions. There are relatively few studies on systemic and chronic toxicity. On the other hand, most of the studied nanomaterials are metal nanomaterials, carbon-based nanomaterials and nanotubes, whereas nanofibers, films and other novel nanomaterials are still rarely studied(Teixeira et al., 2020).Future toxicity studies urgently need to solve these problems.
8.Outlook and future challenges
8.1 Limitations of the existing research
In summary, it has been demonstrated that nanomaterials have a positive effect duringeach process of wound healing. But most current studies have beenconducted in vitro, and the in vivo experiments need to be improved. The animals used for studies are rats, mice, rabbits and pigs. Due to the differences in animal cost, size and availability, rats and mice are most commonly used. However, the skin morphologiesand wound healing processes of these rodents are different from those of humans. In comparison, the skin of pigs is the most similar to that of humans. Because of the high cost and cumbersome operation of experiments with large animals, pigs are not widely used in wound healing research(Abazari et al., 2020).In addition, many wounds lack a standardized and controllable modeling method. For example, wound biofilms, diabetic wounds and wounds of immune skin diseases are usually imitated woundphenomena, and it is difficult to reflect the mechanism of human skin wounds.
Drugs and methods for wound treatment are changing rapidly, and nanomaterials are being applied to wounds in various formulations. However, the mechanism by which nanomaterials promote wound healing hasstill only been superficially studied. For example,macrophage polarization and the TGF-β1/SMAD signaling pathway are the most commonly reported pathways in the inflammatoryand proliferation phases, respectively. The related mechanism by which nanomaterials promote wound repair after reconstruction is rarely reported. There are many types of nanomaterials with different characteristics, and the mechanism of wound healing promotion should also be multifaceted. Deeper and more innovative mechanisms need to be explored in the future, which would behelpful to improve treatment methods and avoid unnecessary side effects(Dukhinova et al., 2019; Janjic et al., 2017).
8.2 Future challenges
Because nanomaterials have superior drug-carrying properties, an increasing number of new drugs have beenloaded onto nanomaterials, such as stem cells and PRP. For the treatment of immune skin diseases, the emergence of a variety of biological agents has gradually replaced traditional drugs. If these monoclonal antibodies can be loaded onto nanomaterials, it may lead to an improvement in the absorption rate and efficacy of the drug.
Although the existing research has confirmedthat nanomaterials can play a positive role in all phases of the promotion of wound healing, the same materials cannot be beneficial throughoutthe whole process, and different types of materials may be required at different stages. Since it is impossible to visually judge the dividing point of each stage, it is important to develop a real-time indicator of the wound condition. In recent years,self-powered implantable electronic skins based on ZnO nanowires modified by enzymes (urease and uricase) have been developed for transcutaneous detection of human health, including blood pressure, temperature, humidity, electrolyte metabolites, etc(Asif et al., 2015; Ma et al., 2019).Through this technology, electronic skin that monitors the pH value, humidity, inflammatory factors and signaling pathway proteins can be developed in the future. Then, the therapist can accurately control the treatment of wounds and select appropriate nanomaterials according to the real-time situation.
With the development of medicine, the treatment of wounds not only pursues the filling of defects but also requires comprehensive functional and aesthetic recovery. Nanotechnology is rapidly developing and is considered to be able to solve a number ofproblems in various situations. Currently, scholars have successfully applied nanomaterials to promote wound healing and prevent scar formation. Nanotechnology still has great potential in the regeneration of hair follicles, the regulation of paresthesia and the improvement of abnormal pigmentation. Especially with the emergence of electronic skin in recent years, the combination of nanotechnology and electronic technology has provided new ideas for the recovery of paresthesia after wound repair and brought an intelligent concept for wound healing.
9.Conclusion
In recent years, an increasing number of nanomaterials have been used to treat wounds. This work reviewed the possible ways that nanomaterials can facilitate wound healing and their related mechanisms, which improves our understanding of the role of nanomaterials in wound healing. We pointed out that most of the current studies have focused on promoting hemostasis, antiinfection, immunoregulation and proliferation, but there is a lack of research on the in-depth mechanisms and post-wound modifications. Additionally, we proposed some methods and new thoughts for subsequent studies, especially for functional and aesthetic problems after wound healing. We hope our work will provide inspiration for more exciting progress in the future.