A Review of Metal Nanoparticles Embedded in Hydrogel Scaffolds for Wound Healing In Vivo

An evolving field, nanotechnology has made its mark in the fields of nanoscience, nanoparticles, nanomaterials, and nanomedicine. Specifically, metal nanoparticles have garnered attention for their diverse use and applicability to dressings for wound healing due to their antimicrobial properties. Given their convenient integration into wound dressings, there has been increasing focus dedicated to investigating the physical, mechanical, and biological characteristics of these nanoparticles as well as their incorporation into biocomposite materials, such as hydrogel scaffolds for use in lieu of antibiotics as well as to accelerate and ameliorate healing. Though rigorously tested and applied in both medical and non-medical applications, further investigations have not been carried out to bring metal nanoparticle–hydrogel composites into clinical practice. In this review, we provide an up-to-date, comprehensive review of advancements in the field, with emphasis on implications on wound healing in in vivo experiments.


Introduction
Wound healing is an intricate physiological process consisting of a series of molecular and cellular events that facilitate the regeneration of the skin, a protective barrier against the external environment. Since its inception, hydrogels have advanced the field of wound healing, insofar as to promote damaged tissue healing within a hydrated milieu [1]. As well, the integration of therapeutic nanoparticles (NP) and biomolecules into hydrogels for local wound application has been shown to enhance and accelerate healing [2,3]. In this era of increasing antibiotic resistance, nanoparticles with antimicrobial properties are mainstay alternatives to the incorporation of antibiotics into hydrogels. The literature also contains evidence for the application of other inclusions such as metals, growth factor-releasing nanoparticles, and enzyme-releasing nanoparticles. Continuing advancements in hydrogel synthesis and nanoparticle integration offer a vast range of possibilities for creating more effective therapeutic gels.
Chronic wounds are increasingly a cause of public health concern. Indeed, under the growing population of patients with obesity and advanced age, the burden of pressure sores, venous insufficiency, and diabetes is expected to increase along with secondary instances of chronic wounds [4]. As such, a corresponding rise in treatment costs for chronic wounds is expected to strain current systems of healthcare delivery [5]. If undertreated, the persistence of chronic wounds can lead to delayed healing, progression of infectious wound infiltration, and reduced patient quality of life [5]. This outcome is especially likely in underserved populations, thereby grounding chronic wounds as a growing global Gels 2023, 9,591 3 of 49

Search Methods for Identifying Studies
On 30 October 2022, Ovid MEDLINE and EMBASE were searched by SS using the study eligibility criteria. The search strategy was developed in consultation with an institutional librarian. Notable search restrictions included the English language. Study record management and eligibility assessment were completed in Covidence (Melbourne, Australia). Figure 1. depicts the PRISMA flow diagram for study inclusion for this review.
On 30 October 2022, Ovid MEDLINE and EMBASE were searched by SS using the study eligibility criteria. The search strategy was developed in consultation with an institutional librarian. Notable search restrictions included the English language. Study record management and eligibility assessment were completed in Covidence (Melbourne, Australia). Figure 1. depicts the PRISMA flow diagram for study inclusion for this review.
Excluded studies fell under the following categories: wrong intervention, where studies did not include metal nanoparticles embedded in hydrogel scaffolds; wrong outcomes, where studies did not include review wound healing outcomes; wrong indication, where studies did not have the primary objective to investigate the effect of the intervention on wound healing; wrong study design, where in vivo investigations were not conducted; abstract only; non-English studies; and wrong setting, where studies discussed hydrogel preparation.

Data Collection and Risk of Bias Assessment
Title and abstract screening were completed by two independent reviewers (FB and JD) using Covidence (Melbourne, Australia). Studies were excluded if they were in vitro studies or if they did not describe wound healing outcomes with hydrogel scaffolding. Full-text articles were subsequently evaluated by two independent review authors (FB and JD), with reasons documented for study exclusion. Data extraction proceeded independently and in duplicate (FB, HS, JD, and LR). Variables recommended for extraction included hydrogel types; notable structural and in vitro properties; antimicrobial agent delivered; particle size; dosage delivered; rate of release; antimicrobial capacity; inhibition zone; cell type; cytotoxic effects; validated animal model; application method for wound healing; and year of publication. Conflicts at any stage in these review steps were adjudicated by a lead study author (SS and BT). For title and abstract screening, full-text evaluation, and data extraction, all review authors (SS, BT, FB, and JD) evaluated the first 100 records to calibrate inter-rater inconsistencies. Risk of bias evaluation was not conducted as the included studies were overwhelmingly conducted in a controlled nonclinical setting. Excluded studies fell under the following categories: wrong intervention, where studies did not include metal nanoparticles embedded in hydrogel scaffolds; wrong outcomes, where studies did not include review wound healing outcomes; wrong indication, where studies did not have the primary objective to investigate the effect of the intervention on wound healing; wrong study design, where in vivo investigations were not conducted; abstract only; non-English studies; and wrong setting, where studies discussed hydrogel preparation.

Data Collection and Risk of Bias Assessment
Title and abstract screening were completed by two independent reviewers (FB and JD) using Covidence (Melbourne, Australia). Studies were excluded if they were in vitro studies or if they did not describe wound healing outcomes with hydrogel scaffolding. Fulltext articles were subsequently evaluated by two independent review authors (FB and JD), with reasons documented for study exclusion. Data extraction proceeded independently and in duplicate (FB, HS, JD, and LR). Variables recommended for extraction included hydrogel types; notable structural and in vitro properties; antimicrobial agent delivered; particle size; dosage delivered; rate of release; antimicrobial capacity; inhibition zone; cell type; cytotoxic effects; validated animal model; application method for wound healing; and year of publication. Conflicts at any stage in these review steps were adjudicated by a lead study author (SS and BT). For title and abstract screening, full-text evaluation, and data extraction, all review authors (SS, BT, FB, and JD) evaluated the first 100 records to calibrate inter-rater inconsistencies. Risk of bias evaluation was not conducted as the included studies were overwhelmingly conducted in a controlled non-clinical setting.

Data Synthesis and Analysis
The primary objective of this review was to produce a summary catalogue of nanoparticleembedded hydrogels that, to date, have been investigated for their antimicrobial properties in wound healing in vivo. The secondary objective was to synthesize the evidence of study findings for each nanoparticle agent. A thematic analysis approach was used to summarize nanoparticles features as they relate to hydrogel types; notable structural and in vitro Gels 2023, 9, 591 5 of 49

Infected Wound Healing
As the majority of wound beds are moist, warm, and nutritious, they are highly susceptible to infection and provide optimized environments for the growth and colonization of bacteria [21]. The most predominant species found in infected wounds include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Klebsiella species, Streptococcus species, and Proteus species [22]. Bacteria are present on virtually all open wounds [4]. When the growth of bacteria does not exceed host defenses, wound beds are considered colonized and may actually hasten wound healing via the promotion of wound bed perfusion [23]. Once host defenses are no longer able to maintain the balance between bacterial growth and death, the wound becomes infected and enters a non-healing state [23]. Infected wounds are often polymicrobial, with Staphylococcus aureus and anaerobes being the most common [22]. Infected wounds pose a burden to both the patient as well as the health care system due to increased costs of care [24].
Most commercial products are not catered towards wounds with active infection. Such wounds progress to requiring systemic antibiotic therapy; however, this also has complications such as systemic toxicity, associated with end organ damage [25]. Additionally, systemic therapy does not penetrate well into ischemic or necrotic tissues. The use of local antibiotics, usually topically, has grown more popular, with Acticoat, high-density polyethylene mesh with nanocrystalline silver coating, as a prime example [21]. However, with the increasing use of antibiotics, antibiotic-resistant microorganisms have considerably increased [26]. This is an issue encountered both systemically and topically. As such, new strategies have been sought out to curb wound infections without the risks of antibiotics as listed above.

Current Wound Dressing Materials and Limitations
There is a longstanding history of wound dressings that dates back to ancient times, all of which demonstrate designs to manipulate the wound environment for the facilitation of wound healing [4]. Today, a myriad of wound care products are available, all majorly created with the understanding that a moist environment is crucial for cell migration and the facilitation of wound healing and contraction [16]. Further, these products aim to remove excess exudate, minimize trauma on removal, and protect against contaminants [4]. Currently, the following dressing materials described below are available [4]: Peri-wound protection: Protects the integrity of the tissue surrounding the wound; includes ointments and barrier creams.
Gauze: Provides absorption but promotes desiccation of the wound base, which hinders healing. Moreover, binding of the gauze to the wound bed often results in trauma upon removal [27]. Moreover, as gauze dressings can become fully saturated with wound exudate, they are not effective barriers against infection. Modern gauze dressings have substances embedded to optimize the wound environment.
Films: Adhesive, non-absorbent, semi-occlusive dressings that can be used as primary or secondary dressings that facilitate the exchange between oxygen and water vapor between the wound bed and the external environment while still providing barrier functions against infection.
Foams: Non-adhesive, absorbent, semi-occlusive dressings that protect against shear with non-traumatic application and removal.
Hydrogels: Water-based, facilitating moist wound-healing environments. Application and removal cause minimal trauma to the wound bed. Further, hydrogels promote autolytic debridement.
Hydrocolloids: Adhesive, absorbent, occlusive dressings that are gel-forming, able to absorb large amounts of exudate while still maintaining moist wound-healing environments.
While wound dressings shield wounds from external contamination, functionalization and delivery of antibiotic properties directly to the wound bed have been investigated in efforts to accelerate and ameliorate the healing of infected wounds [28]. The incorporation Gels 2023, 9, 591 6 of 49 of bioactive agents with such dressings has grown in popularity with increasing potential to be used for wound treatment. Due to their localized effect, this method is postulated to be more effective in the medicinal treatment of non-healing chronic wounds [29]. Despite this, the direct application of bioactive agents is still found to be more effective due to faster absorption [30].

Biopolymers in Wound Healing
As previously described, many different varieties of antibacterial wound dressings have been developed for wound treatment, including gauze, films, foams, hydrocolloids, alginates, collagens, and hydrogels. One issue with common wound dressings is their propensity to adhere to the wound site and edges, disrupting healing processes and causing damage to the wound bed [31]. Hydrogels, three-dimensional scaffold networks, are promising dressings as they provide suitable environments for cellular growth and adhesion [6]. They have high water content and also swell in hydrated environments, thus being able to absorb wound exudate while still maintaining a moist wound environment [4]. As hydrogels can retain large amounts of water, they are flexible and elastic [31]. Another issue with common scaffolds is that they have to be implanted at the wound bed, whereas hydrogels are able to conform to wound topography, filling all defects of the wound from the bottom up [6].
Hydrogel dressings can be made from many materials. Most commonly, polymers of synthetic molecules are used, such as polyacrylamide or polyvinylpyrrolidine [6]. These polymers are non-toxic and can maintain their shape; however, they have limited tissue adhesion properties and, as such, must be combined with other materials for the adjustment of their mechanical properties [31].
Another commonly used biopolymer is cellulose, a component of plant cell walls that is cost-efficient and ameliorates wound healing through the release of growth factors to stimulate granulation tissue formation, re-epithelialization, and angiogenesis as well as proliferation and movement of fibroblasts to the wound bed [32,33]. While more costly, chitin is another frequently used polysaccharide biopolymer for the formation of hydrogels [34]. Chitin is transformed into chitosan, which is soluble in aqueous solutions. In addition to its strong mechanical properties for wound healing, it also offers some antimicrobial properties as it is a cation, thus being able to treat infections through the disruption of cell membranes [35,36].
Alginate is another biopolymer that can be used in the preparation of hydrogels, but also in other dressings such as foams due to its absorbent properties [37][38][39][40]. It has good cytocompatibility and is non-toxic. It can be used in combination with other biopolymers, such as synthetic polymers, to enhance the biological and mechanical properties of the 3D hydrogel scaffold [41,42]. Gelatin, another naturally occurring polymer, is derived from collagen and is also used for its biocompatibility and biodegradable properties [43]. Likewise, fibrin, derived from fibrinogen, offers similar properties when used for wound healing, including reduction in inflammation, promotion of cell adhesion properties, and immunomodulation [43].
All the polymers mentioned above, both biological and synthetic, have been used in conjunction with metal nanoparticles for an enhanced healing effect. Other notable materials observed to be used in such combinations include dextran, elastin, silica, tannic acid, and lignin.

Nanotechnology for Wound Healing
Nanotechnology is defined as the manipulation of materials on an atomic or molecular scale [48]. Ever evolving, nanotechnology has revolutionized many industries, especially Gels 2023, 9, 591 7 of 49 within the fields of nanoscience, nanoparticles, nanomaterials, and nanomedicine. Specifically, the field of nanomedicine has risen in popularity with myriad applications, including vaccine production, wearable devices, implants, drug delivery, and antibacterial applications [49]. In tissue engineering and regenerative medicine, nanomaterials have shown low toxicity and customizability, making them versatile agents to incorporate into medical practice [49]. For instance, metal nanoparticles such as silver (Ag) [50], gold (Au) [51], copper (Cu) [52], and zinc oxide (ZnO) [53] have demonstrated marked antimicrobial properties. While these intrinsic properties are advantageous for wound healing, these metal nanoparticles can also display anti-infective properties within drug-delivery vehicles.

Nanoparticles Used in Wound Healing
Metal nanoparticles have been considered in clinical applications for reasons including small size, high surface-to-volume ratio, shape, stability, low toxicity, and economic reasons, given their affordability [54,55]. Additionally, they can conveniently integrate into wound dressings [54]. One of the primary mechanisms in which antibacterial activity is offered by metal nanoparticles is through their bacteriostatic properties via attachment to DNA or RNA, via electrostatic interactions, halting further replication [56]. MicroRNAs, short, non-coding RNA molecules that have regulatory roles in gene expression, play a large role in wound healing processes, including inflammation, angiogenesis, cell proliferation, and ECM remodeling. In aberrant wound healing, such as infectious states, microRNAs can be targeted by metal nanoparticles through encapsulation, shielding charge groups and allowing for cellular uptake [57]. The modulation of microRNA allows for the enhancement of gene expression factors, promoting the production of factors essential for wound healing. Further, targeted delivery of these therapeutic agents minimizes off-target effects [57].
Another mechanism is through bactericidal properties via the creation of reactive oxygen species [56]. When embedded in hydrogel scaffolds, a substitute is created for damaged ECM which facilitates fibroblast proliferation and matrix formation for enhanced regeneration and repair [58,59]. As such, these nanoparticles can be used in lieu of antibiotics and are thought to accelerate and ameliorate healing while preventing infection [54,55]. A graphical summary of wound healing mechanisms per nanoparticle is depicted in Figure 2. fection [54,55]. A graphical summary of wound healing mechanisms per nanoparticle is depicted in Figure 2.

Silver (Ag) Nanoparticles
The use of silver for the treatment of wounds and infection prevention dates back to at least 4000 B.C.E. with documented medical applications dating back to the 1700s; however, in large quantities, silver can also impair healing due to its toxic effects on keratinocytes and fibroblasts [60,61]. Today, silver continues to serve many applications in wound healing. For example, silver nitrate is used as a commonplace treatment for chronic wounds while silver sulfadiazine is used for burns. Nanotechnology has

Silver (Ag) Nanoparticles
The use of silver for the treatment of wounds and infection prevention dates back to at least 4000 B.C.E. with documented medical applications dating back to the 1700s; Gels 2023, 9, 591 8 of 49 however, in large quantities, silver can also impair healing due to its toxic effects on keratinocytes and fibroblasts [60,61]. Today, silver continues to serve many applications in wound healing. For example, silver nitrate is used as a commonplace treatment for chronic wounds while silver sulfadiazine is used for burns. Nanotechnology has changed the use of silver for wound healing with the creation of silver nanoparticles (AgNPs), which are the most commonly used metal nanoparticles in wound management with many applications, including wound infections, ulcers, and burns. Known for their wide range of antimicrobial activity, effective against bacteria, viruses, fungi, and protozoa, as well as promotion of wound healing, AgNPs have been shown to disturb quorum sensing, effectively reducing biofilm formation [62][63][64].
The antibacterial effect is demonstrated via bactericidal and inhibitory mechanisms. In terms of bactericidal activity, apoptosis is induced in bacteria through AgNP interactions with sulfur and phosphorous-containing proteins, effectively disrupting cell membranes [65]. Moreover, as DNA consists of sulfur and phosphorous, AgNPs act on these bases to destroy DNA, further facilitating the apoptosis of bacterial cells [65]. Moreover, the continuous release of AgNPs, specifically at lower pH whereby acidic environments facilitate the oxidation of AgNPs to Ag + , negatively charged proteins are bound to, allowing for disruption of bacterial cell walls and membrane [65]. Through this mechanism, cell respiration is also disrupted through damage to bacterial mitochondria [66,67]. Despite these cytotoxic effects, which are AgNP-dose-and size-dependent, the proliferation of fibroblasts and keratinocytes is not affected [68]. In terms of inhibitory mechanisms, the presence of AgNPs in the wound environment allows for the formation of reactive oxygen species (ROS), which further disrupt bacterial cell viability through oxidative stress [66,67]. Wound healing is accelerated through these antibacterial properties as microbes can delay all stages of wound healing.
In addition to the antimicrobial properties of AgNPs, they also promote wound healing [69][70][71][72]. Firstly, they assist in the differentiation of fibroblasts into myofibroblasts, which allows for wound contractility [73]. Moreover, they stimulate the proliferation and relocation of keratinocytes to the wound bed [74]. As such, quicker wound epithelialization and scarless wound healing are promoted [73]. Accelerated and complete healing with increased epithelialization was observed in a study wherein an AgNP hydrogel was applied to a partial-thickness cutaneous wound in mice [75,76].
AgNPs also have anti-inflammatory effects through cytokine modulation, reducing levels that allow for decreased lymphocyte infiltration, further enhancing re-epithelialization [52,77]. One study demonstrated a significant reduction in inflammatory cytokines and oxidative stress, effectively promoting healing, while another study in a burn wound model in mice demonstrated reduced interleukin-6 (IL-6) and neutrophils and increased the levels of IL-10, vascular endothelial growth factor, and TGF-ß [50].
The summary of all in vivo studies related to AgNP-loaded hydrogels is shown in Table 1.

Gold (Au) Nanoparticles
AuNPs are commonly used in tissue regeneration, wound healing, and drug delivery of bioactive compounds due to their biocompatibility, high surface reactivity, and antioxidative effects [118,119]. While some antimicrobial effects are seen, unlike AgNPs, AuNPs do not offer much antimicrobial activity alone [120].
Antimicrobial action is demonstrated via two principal mechanisms, similar to AgNPs: bactericidal and inhibitory. Cell death is induced via the disruption of ATP synthase, leading to decreased ATP stores and an eventual collapse in energy metabolism [121]. This is due to the ability of AuNPs to alter membrane potential on entry into the cell [121]. Additionally, the creation of ROS is facilitated by AuNPs, further facilitating cell death. The smaller the size of the AuNPs, the greater the surface area and interface for interaction with microbes, demonstrating a stronger antimicrobial effect [122].
While some antibacterial effects are seen, AuNPs are principally used in tissue repair given their anti-inflammatory properties via cytokine modulation and antioxidant properties [123,124]. Substantial antioxidant properties are seen as AuNPs are able to bind free radicals such as nitric oxide (NO) or hydroxyl (OH-) [125][126][127]. This strong catalytic activity in free radical scavenging is further observed through the ability of AuNPs to increase nuclear factor erythroid 2-related factor (NRF2), which allows for antioxidant gene activation [128,129]. Furthermore, while being able to facilitate the creation of ROS, they are also able to receive electrons and remove or deactivate ROS, with greater effects seen the higher the surface area of the AuNPs is [119].
In addition to tissue repair, wound healing is found to be accelerated and ameliorated with the use of AuNPs through the promotion of collagen expression, growth factors, vascular endothelial growth factor (VEGF), fibroblast proliferation, decreased cellular apoptosis, and angiogenesis [67,130].
Despite these beneficial effects, AuNPs must usually be incorporated with other biomolecules for efficacy in wound healing applications. Examples include the incorporation of AuNPs in chitosan or gelatin for the enhancement of wound healing or in collagen for a similar effect [51,52]. One study of a rat full-thickness excisional wound model demonstrated accelerated healing and wound closure with improved hemostasis and reepithelization compared to the Tegaderm dressing and pure chitosan hydrogel controls in a chitosan-AuNP hydrogel [131]. Recent studies have also incorporated phototherapy in conjunction with AuNPs to achieve antimicrobial activity [53,132].
The summary of all in vivo studies related to AuNP-loaded hydrogels is shown in Table 2.

Copper (Cu) and Copper Oxide (CuO)
Previous studies have demonstrated that CuNPs have antimicrobial activity as well as properties that facilitate tissue repair. CuNPs have shown antibacterial activity against bacterial strains such as Escherichia coli and Staphylococcus aureus but also fungicidal effects [138][139][140][141][142][143]. The principal mechanism of action is through adhesion of the CuNP to bacteria due to their opposing electrical charges, resulting in a reduction reaction that weakens and destroys the bacterial cell wall. CuNPs have also shown antibacterial activity through the enhancement of immunity with the promotion of interleukin-2 (IL2) production, as well as its ability to serve as a cofactor for various enzymes such as cytochrome oxidase [144]. Additionally, CuNPs have an influence on cytokine regulation, thus also having anti-inflammatory properties [145]. In terms of tissue repair, ECM synthesis is promoted through the stimulation of ECM components such as fibrinogen and fibroblasts as well as the production of integrins and collagen [146,147]. One study observing the use of a CuNP-embedded hydrogel in the treatment of full-thickness excisional wounds in rats demonstrated an accelerated wound healing rate [52]. Despite these positive effects, CuNPs are prone to rapid oxidation, promotion of the production of free radicals, and instability, thus limiting its use [148,149].
CuO NPs have been used in multiple biomedical settings, such as in drug delivery, as anti-cancer agents, and wound healing given their biocompatibility, low toxicity, and antimicrobial properties [150,151]. The specific mechanism for the antibacterial effects of CuO remains unknown; however, it is postulated that it is related to the generation of ROS within bacterial cells [152]. However, with CuO NPs, antibacterial activity was partially related to bacterial properties. For example, different effects were noted with increased bactericidal activity in Gram-negative organisms, such as E. coli, compared to Gram-positive organisms, such as S. aureus [153]. Despite these antibacterial properties, one concern is toxicity, the induction of oxidative stress, and subsequent DNA and mitochondrial damage [154,155].
The summary of all in vivo studies related to Cu and CuO NP-loaded hydrogels is shown in Tables 3 and 4, respectively.

Zinc (Zn) and Zinc Oxide (ZnO)
Zn and ZnO NPs are some of the most commonly used NPs in wound healing applications due to their anti-inflammatory and antimicrobial properties [166]. As inorganic agents, they are more stable than their organic agent counterparts. They are also advantageous in their ability to remain within the wound bed for longer periods of time [166,167]. The antimicrobial effects of Zn and ZnO NPs are due to disruption of cell membranes and oxidant injury [166,167]. Zinc also serves as a cofactor for metalloproteinases and other enzymatic complexes, promoting migration of keratinocytes and regeneration of the ECM [166,167]. A previous study examining full-thickness wounds in a rat model showed accelerated and ameliorated healing compared to control with improved re-epithelialization as well as increased collagen deposition and tissue granulation [167]. Moreover, both Zn and ZnO NPs have demonstrated good biocompatibility and low cytotoxicity [166].
Like other NPs, the Zn and ZnO NP effect is dependent on the size, surface-areato-volume ratio, and concentration of the NPs [168]. Smaller NPs have been shown to be more cytotoxic given their larger surface-area-to-volume ratio, whereas larger NPs demonstrate increased cytocompatibility [169]. In fact, a previous study demonstrated that ZnO NPs are highly compatible with fibroblast cells and promote their growth, migration, and adhesion [142].
Summaries of all in vivo studies related to Zn and ZnO NP loaded hydrogels are shown in Tables 5 and 6, respectively.

NIH/3T3s
No toxicity was observed against NIH/3T3s when co-incubation with 40% and 70% of ZnG/rhEGF@Chit/ Polo in the growth medium.

Scald wounds in rats
Supplements wound healing to promote the vascular remodeling and collagen deposition, facilitates fibrogenesis, and reduces the level of interleukin 6 for wound basement repair, and thus is a good wound therapy.

Titanium Oxide (TiO 2 )
A study of the antimicrobial effects of TiO 2 NPs demonstrated little effect but showed accelerated wound healing in a full-thickness excisional wound model [183].

Cerium Oxide (CeO 2 )
CeO 2 NPs have the highest antioxidant activity of all NPs and are most active in the scavenging of free radicals [184]. This is due to the oxygen vacancies of CeO 2 , leading to the reduction of Cerium from Ce +4 to Ce +3 , for example [184].

Manganese Oxide (MnO 2 )
MnO 2 is also a potential candidate to be used in nanoparticle-hydrogel composites. Known to relieve oxidative stress, MnO 2 is able to catalytically decompose H 2 O 2 into O 2 , thus effectively providing a targeted approach to hypoxic relief [185]. One study evaluating MnO 2 nanoparticles in the healing of chronic diabetic wounds in vivo demonstrated the eradication of biofilms, attenuation of hyperglycemia, hemostasis, and the creation of an optimized wound environment which reduced inflammation, accelerated granulation tissue formation and re-epithelialization, and accelerated wound healing [185]. The summary of all in vivo studies related to MnO 2 NP-loaded hydrogels is shown in Table 7. Less commonly used in antibacterial wound dressing applications, iron nanoparticles have been shown to induce bacterial death, membrane damage, DNA degradation, and lipid peroxidation [187][188][189]. One study demonstrated high antibacterial activities against S. aureus and E. coli both in vitro and in an in vivo infected full-thickness excisional wound model in mice where accelerated wound healing and anti-inflammatory properties were observed [187].
The summary of all in vivo studies related to FeNP-loaded hydrogels is shown in Table 8.

Gallium (Ga) Nanoparticles (GaNP)
Gallium is very infrequently used in wound healing applications. Given that gallium and iron have equal ionic radii, one study hypothesized that the substitution of iron with gallium would impair bacterial iron metabolism and exert an antimicrobial effect [194]. This has previously been observed in vitro, whereby gallium resulted in reduced bacterial survival [194][195][196]. Moreover, given the inability of gallium to be reduced in physiological environments, a property not shared with iron, gallium also disrupts enzyme activity [197]. A 2022 study by Qin et al. demonstrated the good antimicrobial effect of gallium embedded in an alginate-base hydrogel, with good biocompatibility against NIH3T3 cells in vitro as well as accelerated wound healing with good biocompatibility, angiogenesis, and collagen deposition compared to the control in an S. aureus infected full-thickness excisional wound model in mice [194].
The summary of all in vivo studies related to GaNP-loaded hydrogels is shown in Table 9.

Combinations of Metal Nanoparticles
Occasionally, metal NPs can be used in conjunction with each other to provide synergistic antibacterial effects. For example, one study investigated the effects of AgNP and CuNP within a chitosan hydrogel, demonstrating good antibacterial activity against S. aureus and E. coli with good biocompatibility and accelerated healing compared to control in an S. aureus infected full-thickness excisional wound model in type 1 diabetic rats [198]. Another study looked at the synergy between ZnO and AgNPs, demonstrating an excellent bactericidal effect against E. coli and S. aureus [199]. Interestingly, AgNPs were observed to exhibit a small amount of cytotoxicity when tested against mouse calvarial (MC3T3-E1) cells alone, but when used in conjunction with ZnO NPs, lower cytotoxicity was seen [199]. In an S. aureus infected partial thickness wound model in rats, the release of Ag + and Zn 2+ was found to stimulate immune function to produce a large number of white blood cells and neutrophils (2-4 times more than the control), thereby producing the synergistic antibacterial effects and accelerated wound healing [200].
The summary of all in vivo studies related to combinations of metal-NP-loaded hydrogels is shown in Table 10.

Challenges and Future Directions
While there are myriad advantages in the addition of metal NPs to hydrogel scaffolds, there are limitations to their use as well. Principally, cytotoxicity is of concern as the interactions of NPs and cells have not yet fully been elicited [65]. While exploration into the cytotoxicity of NPs has been investigated, the field lacks uniformity such that tests of different cell types have demonstrated different cytotoxic responses. Further, there are multiple factors that influence NP activity, including the size, shape, concentration within the hydrogel, and surface charge [93]. Another concern regarding NPs is release and uptake. Poor release or uptake limits the efficacy and quality of treatment provided whereas excess release can result in deposition in surrounding tissues or organs, toxicity, or other undesired effects [31,65,202]. For example, overuse of silver-containing dressings impair healing processes due to cytotoxicity against keratinocytes and fibroblasts as well as systemic adverse effects such as argyria [202][203][204]. Thus, despite an extensive repertoire of current studies, future investigations and development would benefit from further toxicity and cytocompatibility assessments both in vivo and in vitro and iterate on current formulations for hydrogels with metal nanoparticles. We also recommend continued translational research on the application of these dressings in clinical practice.

Conclusions
The main aim of this review is to highlight the antimicrobial activity of metal nanoparticles embedded within hydrogel scaffolds for wound healing. Here, we elaborately discussed how these nanoparticles interact with pathogens but also with the wound environment to eradicate infection and enhance the healing process. When used in conjunction, hydrogels and metal nanoparticles demonstrate a synergistic effect. Excellent activity was demonstrated by select metal nanoparticles embedded within hydrogels. Accelerated and ameliorated wound healing was also observed with the promotion of re-epithelialization, angiogenesis, increased deposition of collagen and granulation tissue, and downregulation of inflammatory processes. Despite the large body of research in the field, there is a gap in preclinical and clinical studies, which, if addressed, could facilitate the use of these dressings in common clinical practice. Funding: This research received no external funding.

Institutional Review Board Statement:
This study extracts data in its entirety from published primary research. Under Article 2.4 of the Tri-Council Policy Statement, this study is exempt from institutional review board approval. This study will not identify any individual or generate new forms of identifiable information.