Effectiveness of Copper Nanoparticles in Wound Healing Process Using In Vivo and In Vitro Studies: A Systematic Review

Chronic wounds are defined as wounds that do not heal in an orderly and timely manner through the various stages of the healing process. Copper nanoparticles are essential in dressings for wound healing because they promote angiogenesis and skin regeneration, which hasten the healing process. This systematic investigation sought to explain how copper nanoparticles affect chronic wound healing in vivo and in vitro. We realized a systematic review of original articles studying the effectiveness of copper nanoparticles in the healing process of chronic wounds. The protocol was registered in the PROSPERO database. Several databases were searched between 2012 and January 2022 for English-language papers using MeSH terms and text related to chronic wounds, copper nanoparticles, and wound healing. Quality was evaluated using National Institute for Health and Care Excellence methodology and PRISMA guidelines. We looked at a total of 12 primary studies. Quantitative data were gathered and presented in all studies. Our results suggest that copper nanoparticles could have an excellent healing property, facilitating the liberation of growth factors that help the anti-inflammatory process of the wound and significantly improving antibacterial and antioxidant activities. In addition, copper presents a higher biocompatibility than other metallic ions, promoting regeneration and increasing skin quality.


Introduction
Chronic wounds are characterized as either failing to move through a well-ordered and suitable reparative process to produce anatomic and functional integrity within three months or continuing through the repair process without creating a sustained anatomical and functional outcome [1,2]. Chronic wounds are divided into four groups based on the etiologies that cause them: venous, arterial insufficiency, pressure, and diabetic ulcers [3]. Due to their rising prevalence and high management cost, chronic wounds are significant to the healthcare system. According to a study evaluating the cost, effect, and Medicare policy implications of chronic non-healing wounds, 8.2 million Medicare members in the United States are afflicted [4]. In recent years, impregnated dressings have enhanced wound care wounds by the research question, several databases (MEDLINE, EMBASE, Scopus, and Web of Science) were examined from January 2012 to January 2022 for original articles in English. The reviews covered a variety of biological activities, including antifungal efficacy and antimicrobial, antioxidant, anti-inflammation, and wound healing investigations. Additionally, the reference lists of included studies and relevant reviews were searched.

Identification of Relevant Studies
Two reviewers evaluated titles, abstracts, and papers for inclusion or exclusion. Discussion with a different reviewer helped to settle any discrepancies between the results of the reviewers.

Types of Study and Design
The studies must have reported the role of Cu NPs in wound healing for chronic wounds or the effects of Cu NPs in biological activities and must have been English studies. The inclusion criteria were: (1). primary studies with a quantitative component; (2). studies using descriptive or inferential statistics approaches, with parametric or non-parametric methods; and (3). clinical trials, experimental studies, cross-sectional studies, or randomized controlled trials. Studies were excluded if they met the following criteria: (1). did not include or specify numerical data; (2). were not original investigations published in full; (3). were not published in a peer-reviewed journal; (4). conference abstracts; (5). systematic reviews; (6). editor letters; and (7). studies that did not focus on the role of Cu NPs in wound healing for chronic wounds, or those that did not describe antifungal efficacy and were not antimicrobial, antioxidant, anti-inflammation, and wound healing studies.

Population
For in vivo studies, animal models included only healthy participants, evaluating Cu NPs and their protective capacity on wound healing for chronic wounds or the effects of Cu NPs on biological activities. Pregnant animals, those with burns in other places than the skin, and/or those with comorbidity were excluded.
For studies on the human population, studies that focused just on postmenopausal women or unhealthy participants were excluded.

Quality Assessment/Risk of Bias
One reviewer (GR) assessed quality using the National Institute for Health and Care Excellence (NICE) methodology [39] and another reviewer (NS) analyzed it for accuracy. Disparities between authors were solved by discussion. No studies were excluded based on assessment.

Data Extraction and Synthesis
Data relating to the population and study characteristics of the included studies were extracted by one reviewer and checked by another reviewer (Table 1).
Two researchers went line by line through the results and discussion sections of each text to look for data involving the role of Cu NPs in the healing process or the effects of Cu NPs in biological activities to find information relevant to variables involved in the role of Cu NPs in the healing process using in vivo and in vitro studies. The text was reviewed in greater detail and rearranged into topics (Table 2). These were included if the study's authors built their interpretation and concepts from the initial data. XRD analysis, FTIR spectra, thermal stability, Cu ion release, long-term antibacterial activity, and in vitro cytotoxicity of BC/Cu membranes.
To fabricate BC/Cu composite membrane by in situ chemical reduction method.
Purified BC membranes were first immersed overnight in CuCl 2 aqueous solutions, resulting in Cu 2+ adhesion to BC nanofibers. Following that, NaBH 4 was introduced to the membranes, and the Cu 2 anchored in the membranes was instantly reduced to Cu. After 30 min, the reaction was halted, yielding a stable dark-brown BC/Cu membrane.
In BC/Cu membranes: XRD analysis: No differences were found between XRD curves of BC/Cu and cellulose I crystal. FTIR spectra: No differences were found between BC/Cu and BC membranes. Thermal stability, Cu ion release: In BC/Cu 100 , a considerable weight reduction stage was seen. Antibacterial activity: Significant inhibitions against S. aureus and E. coli after 1, 45, and 90 days were found in all BC/Cu membranes. In vitro cytotoxicity: After being exposed to the membrane extracts from BC/Cu 60 and BC/Cu 100 , cell number was considerably reduced.
[41] CN HEK293 cells and female BALB/c mice were used. To create ultrasmall Cu-based systems, which could serve as a model for future nanosystems employed in the treatment and prevention of disorders connected to ROS.
The Cu 5.4 O USNPs were created as follows: 10 mM CuCl 2 powders in deionized water were dissolved. Next, they were swirled for 10 min at 80 • C. Then, 100 mM L-ascorbic acid solution was added to the CuCl 2 solution above, and the pH of the solution was adjusted to 8.0-9.0 using NaOH solution. After the process, the bigger aggregates were removed by centrifugation, and the supernatant was dialyzed against water for two days to remove tiny molecules.  In vitro: Antibacterial activity, cell migration, and cell angiogenesis.
In vivo: Antibacterial activity and wound healing.
To develop CuS NDs stabilized in albumin to improve healing and antibacterial effects.
CuS NDs were created using a simple one-step hydrothermal method. During the production, BSA was utilized to manage particle size and stability.  In vitro: Antibacterial activity and determination of reactive oxygen species: Escherichia coli and Staphylococcus aureus were used for the antibacterial experiments. Cell viability and proliferation: NIH-3T3 cells were used to evaluate the activity of the hydrogel with or without Cu NPs cuts. qPCR analysis: The mRNA expressions of IL-1β, IL-6, TNF-α, and IL-10 was evaluated.
In vivo: Sprague-Dawley rats were used to evaluate the effects of hydrogel, hydrogel treatment plus laser, Cu NP-embedded hydrogel, and Cu NP-embedded hydrogel treatment plus laser.
In vitro: Antibacterial activity, determination of reactive oxygen species, cell viability and proliferation, and quantitative real-time PCR analysis. In vivo: Wound healing and immunofluorescence staining.
To design GelMA hydrogels combined with BACA/Cu NPs to promote wound healing and antibacterial activities against Gram-positive/ negative bacteria.
Cu NPs were created in a modified version by reacting Cu ions with Na 3 C 6 H 5 O 7 . CuSO 4 ·5H 2 O was then vigorously agitated in ultrapure water for 2 h. Following complete dissolution, a glass container was filled with ethylene glycol and distilled water, and the pH was adjusted to 10 using a strong NH 3 solution. The reducing agent, Na 3 C 6 H 5 O 7 solution, was then added to the previously described combination. After that, the vial was submerged in an oil bath at 90 • C until the blue transparent solution changed to the distinctive brownish-red hue when heated. Cu NPs were cleaned three times with ultrapure water before being stored in an ethanol solution at 20 • C.
In vitro: Antibacterial activity: Cu NP-embedded hydrogels increased Cu release after NIR laser irradiation. Determination of reactive oxygen species: No differences in lipid peroxidation were found. Cell viability and proliferation: All the hydrogel + CuNPs and hydrogel + CuNPs+NIR groups showed a slight decrease in the ability to proliferate NIH-3T3 cells. qPCR analysis: No differences in IL-1β, IL-6, TNF-α, and IL-10 expression were found. In vivo: Wound healing: The wound healing was faster in the Gel-MA/BACA-Cu NPs composite hydrogels treatment plus laser irradiation. In vivo: Wound healing: Wistar rats were used to evaluate the efficacy of the wound dressing. Evaluation of TNF-α and MMP-2: Quantification of TNF-α and MMP-2 was realized on 7th, 14th, and 28th days after damage.
In vitro: Cytocompatibility studies and DNA quantification.
To develop a hydrogel platform composed of biopolymer gelatin and glycosaminoglycans combined with asiatic acid, ZnO, and CuO NPs. To evaluate the efficacy of the wound dressings in burn wounds.
Dissolving gelatin in distilled water yielded a gelatin solution.
After complete dissolution, appropriate amounts of (C 14 H 21 NO 11 )n, C 13 H 21 NO 15 S, C 30 H 48 O 5 , ZnO, and CuO NPs were added sequentially and mixed for 6 h. The solution was collected on a Petri plate after homogenization and lyophilized overnight. The hydrogels were then cross-linked using EDC coupling [47], washed with distilled water, and kept in a refrigerator at 20 • C until use.
In vitro: Cytocompatibility studies: From day 1 to day 3, an increase in cell numbers was observed in GAGs and the asiatic acid hydrogel group. However, a slow proliferation in cell number was observed in ZnO and CuO NP scaffolds. DNA quantification: From day 1 to day 3, an increased L929 cell number was found in the gelatin + GAGs + asiatic group.
In vivo: Wound healing: On day 28, in the hydrogel composite group, a complete healing was observed. Evaluation of TNF-α and MMP-2: On day 7, a lower level of TNF-α was found in the hydrogel composite group in comparison with control. On day 7, MMP-2 levels were higher in the hydrogel than control group.
In vitro: SEM images: Bacterial suspensions in the presence of HvCuO@GOx or PBS with different concentrations of glucose were performed separately at 37 • C under 180 rpm. Scratch assay and endothelial tubule formation: HEK cells were incubated in the presence of HvCuO@GOx, HvCuO, or PBS. All groups were treated with glucose for 2 h. In vivo: Diabetes was induced by an intraperitoneal injection of streptozotocin in mice. Staphylococcus aureus-infected wounds were divided into the following treatments: hydrogel, HSHvCuO, and HSHvCuO@GOx.
In vitro: SEM images, cytotoxicity assay, scratch assay, and endothelial tubule formation. In vivo: Wound healing and treatment, and imaging in vivo.
To design a thermal-responsive spray for the synergistic restoration of DFU using its angiogenesis and antibacterial properties.
Virus-like silica nanoparticles and Cu(NO 3 ) 3 ·6H 2 O were dissolved in deionized water and agitated for 30 min. Then, (CH 2 ) 6 N 4 was added, and the aforementioned mixture was constantly agitated for 2 h. Final samples were centrifuged multiple times with deionized water to eliminate unreacted residues, then dried in an oven. Finally, the virus-like mesoporous silica template was etched with 0.1 M Na 2 CO 3 , agitated, and washed three times with deionized water. HvCuO were produced after drying in an oven overnight.
In vitro: SEM images: In the NPs group, the rough surface was nicely maintained. No changes were found in the morphology of HvCuO@GOx nanoshells even without adding glucose for 12 h. After 2 h, the glucose concentration was dramatically reduced in the HvCuO@GOx presence in comparison with HvCuO. The most effective bactericidal action was found in HSHvCuO@GOx dressing. Cytotoxicity assay: A negligible cytotoxicity was found in HEK and HUVEC cells treated with different HvCuO@GOx concentrations. Scratch assay: A significant cell migration was found in HEK cells treated with HvCuO@GOx. Endothelial tubule formation: A higher number of tubule junctions was found in HUVEC cells treated with HvCuO@GOx at 150 µg/mL concentration. In vivo: Wound healing and treatment: At day 15, the wound healing was almost completely healed in the HSHvCuO@GOx group. Imaging in vivo: The highest quantity of CD34-positive cells was found in the HSHvCuO@GOx group. In vitro: Mechanical properties: A universal machine tester was used to evaluate the mechanical properties of the compounds. Antibacterial activity: The antibacterial ability of compounds was assessed using Escherichia coli and Staphylococcus aureus.
Cell cytotoxicity and proliferation assessment and migration and tubule formation activities: Different ionic extractions were used to culture NIH/3T3 and HUVEC cells.
In vivo: Rats were used to prepare the wound model. Each one was treated with GelMA, 5% Cu-NA@GelMA, or 5% Cu-NA-bFGF@GelMA. The skin was extracted for further histological and immunohistochemical studies on days 3, 7, and 14.
In vitro: Mechanical properties, antibacterial activity, cell cytotoxicity and proliferation assessment, and migration and tubule formation activities.
In vivo: Wound healing and histopathologic evaluation.
To prepare a new Cu-nicotinic acid based on using biomolecules of nicotinic acid. In vitro: Mechanical properties: the best elasticity was found in the CuNA@GelMAs group. Antibacterial activity: CuNA@GelMAs showed good antibacterial ability toward E.
coli and S. aureus. Moreover, an enhancement in antibacterial properties was observed after increasing the CuNA content in the hydrogels. Cell cytotoxicity and proliferation assessment: CuNA-bFGF@GelMA, CuNA@GelMA, and GelMA have shown no significant difference. Migration and tubule formation activities: In HUVEC and NIH/3T3 cells, increased migration and total segment length in the GelMA group were found. In vivo: Wound healing: In CuNA@GelMA and Cu-NA-bFGF@ GelMA treatments, a decreased percentage of wound closures was found in comparison to other groups.
Histopathologic evaluation: More new blood vessels, regular epithelium, and mild inflammatory responses in CuNA@GelMA and CuNA-bFGF@GelMA treatments were found. Histopathological analysis: A more stable and densely perfused vascular network in H-HKUST-1 and HKUST-1 NP treatment in comparison to PBS and PPCN groups was found. In the HKUST-1 and H-HKUST-1 groups, an increased blood vessel number and area, as well as neovascularization, were found. Higher granulation tissue and blood vessel numbers in HKUST-1-and H-HKUST-1-treated wounds were found, respectively. Smaller granulation tissue in the H-HKUST-1-treated wound was found. In vitro: Cytotoxicity and scratch assay: Folic acid, HKUST-1, and F-HKUST-1 were used as treatments in HEKa and HDF cells. Endothelial tubule formation assay: PBS, folic acid, HKUST-1, and F-HKUST-1 were used as treatments in HUVEC cells.
In vivo: Three treatments (HKUST-1, F-HKUST-1, and folic acid) were used to evaluate wound healing at different time points.
In vitro: Cytotoxicity, scratch, and endothelial tubule formation assays.
In vivo: Wound healing and histopathology analysis.
To evaluate the modification of HKUST-1, to release Cu 2+ , reducing cytotoxicity and improving wound healing rates.
HKUST-1 was synthesized in the manner previously reported (17,46). In vitro: Cytotoxicity assay: A lower toxicity in F-HKUST-1 treatment was found (0.5 mM). Scratch Assay: The highest migration rate in F-HKUST-1-treated cells was found.
Endothelial tubule formation assay: The largest number of tubule junctions in F-HKUST-1-treated HUVEC cells was found.
In vivo: Wound healing: At days 19, 21, and 30 post-wounding, an improved wound healing in the F-HKUST-1 group was found (p < 0.05). Histopathology analysis: At day 30, a 107.8 ± 18.1 µm granulation tissue thickness in the F-HKUST-1 group was found.
[53] CN HUVEC cells and female BALB/c mice were used.
In vitro: Antibacterial performance: For the optimal concentration, nanoliquid dressings at different concentrations were used (CuS nanoplates and HNO 3 ). Anti-biofilm assay: Crystal violet assay was used.
In vitro: Antibacterial performance and anti-biofilm assay.
In vivo: Wound healing.
To design a novel nanoliquid dressing based on a mild photothermal heating strategy to provide safe healing of biofilm-infected wounds.
A container was filled with sulfur powder and 1-ODE. After the oxygen was removed, the mixture was heated. Then, the sulfur powder was dissolved and insulated for future use. In another container, there was CuCl 2 powder, OM, and 1-ODE. When the CuCl 2 -containing mixture was heated in a vacuum, a brilliant yellow solution was produced. The container was then quickly injected into a sulfur-containing 1-ODE solution. In between injection cycles, CuS nanocrystals were cultured. After six injection sessions, CuS nanoplates were created. The generated CuS nanoplates were precipitated and centrifuged using an excess of ethanol after the reaction solution was cooled to room temperature. The precipitate was washed and kept at room temperature in chloroform for future use.
In vitro: Antibacterial performance: An~80% cell viability in CuS-CTAB nanoplate-treated or HNO 3 -treated HUVEC cells was found. Anti-biofilm assay: A thickness reduction of biofilms in the CuS−HNO 3 + NIR group was found in comparison to untreated biofilm. In vivo: Wound healing: At day 15, 72.9% and 98.6% of wound closure in normal saline and CuS−HNO 3 + NIR-treated mice were found.

Measurement Wound Changes Evaluation Method References
Cytotoxicity Cytotoxicity of Cu-MBG was well-tolerated (at 0.1 and 1 mg/mL). Metabolic assay PrestoBlue TM [42] The generation of hydroxyl radicals in the biofilm environment may be of insignificant toxicity in diabetic wounds. Endothelial tubule formation [48] Good in vivo safety and biocompatibility have been suggested after CuS NDs + NIR treatment, where no histological changes or toxicity within the treatment period were found. H&E staining [43] After treatment with HKUST-1 and F-HKUST-1, HEKas and HDF cells exhibited enhanced migration. In addition, the highest cell migration in F-HKUST-1 has been found. Its enhanced migration is due to the Cu 2+ presence in HKUST-1 and F-HKUST-1 groups. Scratch assay [52] About NIH-3T3 cells, no significant in vitro cytotoxicity has been described. CCK-8 assay [45] On day 28, no differences in the inflammatory cells were found.
Haematological analysis [46] The use of hydrogel + NPs treatment in second-degree burns is safe because no changes in markers of liver and kidney have been found. Biochemistry analysis [46] No significant cytotoxicity after treatment with different concentration of USNPs was found. Good biocompatibility and normal cytoskeleton morphology in HEK293 cells treated with USNPs (200 ng mL −1 ) have been described. CCK-8 assay [41] After 30 days, no cardiovascular damage after USNP injection has been found. Hemolysis assay [41] After 24 h, no tissue damage or inflammatory lesions in the genitourinary system after USNPs injection have been described. Hemolysis assay [41] A significant inhibition in the MAPK pathway after USNPs treatment was found, showing it might decrease renal injury by decreasing the ROS level. Principal component analysis [41] As the incubation period wore on, the number of cells grew for all groups. The viability of cells decreased for the BC/Cu 60 -and BC/Cu 100 -treated groups (46 ± 6% and 30 ± 8%, respectively). After BC/Cu 20 treatments, NHDF cells did not show decreased cell viability in comparison to the control group; after BC/Cu 60 and BC/Cu 100 treatments, NHDF cells showed decreased cell viability.
CCK-8 assay and calcein staining [40] Antibacterial response Antibacterial effects of Cu-MBG (100 µg/mL) against Pseudomonas aeruginosa and Staphylococcus aureus with 1.2-3.5 log reductions were found. In addition, a reduced existing biofilm after Cu-MBG treatment was described. Brain heart infusion [42] Eradication of bacteria after CuS NDs (45 µg/mL) treatment was found. Likewise, a higher antibacterial effect in the CuS NDs+NIR group than in the CuS NP + NIR group was found. Growth-inhibition assay [43] In bacteria after CuS NPs+NIR and CuS NDs treatments, outer membranes were damaged. However, in bacterial cell walls, a loss of integrity after CuS NDs+NIR treatment was found. In addition, after CuS NDs+NIR treatment, the cytoplasm displayed aggregates in Escherichia coli and Staphylococcus aureus, confirming the cell damage.

TEM [43]
After incubation with AuAgCu 2 O NSs, several dead cells were detected in the Escherichia coli incubation; however, almost complete death of bacteria after AuAgCu 2 O NSs treatment with a laser was found.
SYTO9/PI live/dead fluorescent staining assay [44] After 6 and 24 h of incubation, effective antibacterial effects in GelMA/BACA-CuNPs hydrogels+NIR against Escherichia coli and Staphylococcus aureus have been described.

Measurement Wound Changes Evaluation Method References
The inhibition zone for Escherichia coli and Staphylococcus aureus was 3.1 ± 0.8 mm and 2.6 ± 0.3 mm for the hydrogel; it was 5.3 ± 0.2 mm and 4.9 ± 0.6 mm for the gelatin + ZnO group, whereas the inhibition zone was 4.8 ± 0.7 mm and 3.8 ± 0.3 mm for the gelatin + CuO treatment.
Disc diffusion method [46] BC/Cu membranes showed a higher inhibition zone against Staphylococcus aureus for up to 90 days. Disk diffusion method [40] A slow release of Cu in membranes was found even at 90 days, and remaining membranes with Cu were found. However, a faster release of Cu was found when a 37 • C incubator was used; after 24 h, the release of Cu of membranes was almost complete. Disk diffusion method [40] Wound healing At 24 h, the outgrowth of endothelial cells started for most aortic rings, and the outgrowth area increased from 0.3 to 1.5 mm 2 when VEGF was added. Aortic ring assay [42] Increases in the number of junctions and total vessel length in MBG and Cu-MBG groups were found. CAM assay [42] The vascular network formation was promoted by H-HKUST-1 and HKUST-1 NP treatments. In addition, blood vessel area, blood vessel number, and neovascularization in HKUST-1-and H-HKUST-1 were increased. OCTA [50] The highest functional levels of blood vessel oxygen of the HSHvCuO@GOx group were found, confirming the properties of HSHvCuO@GOx in hypoxia alleviation. Photoacoustic imaging in vivo [48] A total of 14 days was necessary for the F-HKUST-1 group to close 50% of the wound area, whereas a total of 19 days was necessary for the other groups. Dermal excision wound model [52] Tight connections, parallel cell lines, and mesh circles in AuAgCu 2 O NSs treatment groups with or without laser irradiation indicated a late phase of angiogenesis. Matrigel assay [44] Effective antibacterial capacity in hydrogel + CuNPs + NIR after CD86+ and CD206 intensity analysis was found. IHC [45] A larger degradation rate in Cu NP hydrogels than in simple hydrogels was found. Masson's trichrome and H&E staining [45] On day 7, decreased inflammation and high tissue remodeling as a consequence of low levels of TNF-α and high levels of MMP-2 in the hydrogel composite were found. ELISA kit [46] A faster healing rate in the USNPs group in comparison to the control group was found during days 4, 7, 9, and 15 post-surgery (p < 0.01). H&E staining [41] BC

Description of Included Studies
Eight publications from the original studies were carried out in China, two in the US, one in Canada, and one in India.
A total of 12 original articles were analyzed. The papers collected and reported quantitative data through clinical trials or experimental studies (Table 1). Ten studies have used in vivo and in vitro models [41,[43][44][45][46][48][49][50]52,53] while two have used in vitro studies [40,42]. Details of chronic wounds of each study, where available, are shown in Table 1.

Description of Included Studies
Eight publications from the original studies were carried out in China, two in the US, one in Canada, and one in India.
A total of 12 original articles were analyzed. The papers collected and reported quantitative data through clinical trials or experimental studies (Table 1). Ten studies have used in vivo and in vitro models [41,[43][44][45][46][48][49][50]52,53] while two have used in vitro studies [40,42]. Details of chronic wounds of each study, where available, are shown in Table 1.
The above-mentioned in vitro studies used human umbilical vein endotelial cells [43,44,49,52,53], immortalized human epithelial keratinocytes [48,50,52], human dermal fibroblasts [50,52], diabetic (db/db) mice and non-diabetic mice (C57BL/6) [50,52], human embryonic kidney [41], human keratinocytes cells [44], NIH-3T3 cell line [45,49], bacterial cellulose [40], human dermal fibroblasts extracted from split-thickness skin biopsies and obtained during breast reduction and abdominoplasty [42], human foreskin fibroblast cells [43], human corneal epithelial cells [44], fibroblast cell line [46], and recombinant human basic fibroblast growth factor [49]. For the in vivo models, female BALB/c mice [41,43,44,53], type 1 diabetic mice [48], Sprague-Dawley rats [45,49], and Wistar rats [46] were used. Table 3 displays the quality assessment outcomes and evaluation standards for the studies. The studies' overall quality for internal and external validity was often high or moderate. No studies were disqualified due to poor quality. : it is reserved for those aspects in which the study under review fails to report how they have (or might have) been considered; -: it is reserved for those aspects of the study design in which significant sources of bias may persist; +: it indicates that either the answer to the checklist question is not clear from the way the study is reported, or that the study may not have addressed all potential sources of bias for that particular aspect of study design; ++: it indicates that for that particular aspect of study design, the study has been designed or conducted in such a way as to minimize the risk of bias.

Cytotoxicity Assays
Monitoring cell growth inhibition was assessed through cytotoxicity evaluation. However, cell cytotoxicity could be evaluated using other parameters, as shown in Table 1. In this sense, cells exposed to hollow mesoporous CuO nanospheres that resemble viruses (HvCuO@GOx) showed negligible cytotoxicity and biocompatibility when different glucose concentrations (2 mM, 4 mM, 6 mM, 8 mM, 10 mM) were used [48]. Likewise, the Cu nanodots (CuS NDs) dose did not effect the viability of the cells with or without laser irradiation. Even at a dosage of 45 g/mL, both cell lines were still alive, demonstrating the very low cytotoxicity of CuS NDs and photocytotoxicity [43].
In vitro assays have shown that the increase in human dermal fibroblasts' (HDFs) migration due to folic acid-modified Cu-based metal-organic framework (F-HKUST-1) exposure could result from the persistent release of Cu 2+ and folic acid along with modest cytotoxicity [52]. At the same time, according to CCK-8 analysis, a small quantity of ultrasmall Cu 5.4 O NPs (USNPs) were able to shield the cells completely from 250 µM H 2 O 2 ; meanwhile, HEK293 cells had normal polygonal cytoskeleton morphology after being treated to 200 ng mL −1 Cu 5.4 O USNPs for 48 h, showing high biocompatibility [41]. Finally, while BC/Cu 20 membranes did not exhibit cytotoxicity to normal human dermal fibroblasts (NHDFs), BC/Cu 60 and BC/Cu 100 membranes drastically reduced cell viability [40].

Antibacterial Response
The human body uses Cu for the innate immune response by boosting the bactericidal and phagocytic functions of neutrophils and the antimicrobial activity of macrophages [54]. In this sense, the quantity of biofilm present following treatment with Cu-containing mesoporous bioactive glasses (Cu-MBG) is reduced for both species. However, in the infected skin model, the effect of Cu-MBG on Pseudomonas aeruginosa was noticeably less pronounced than its effect on Staphylococcus aureus [42]. In the case of methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase (ESBL) Escherichia coli treated with CuS NDs and NIR irradiation, where ultrasmall NDs stuck to the bacterial surface, their original form was distorted and revealed wrinkled bacterial cellular wall/membranes with visible lesions and holes, which may be due to the strong contact between CuS NDs and the bacterial cell wall [43].
Samples with higher Cu displayed bigger inhibition zones when the antibacterial activity was measured using the disk diffusion method. BC/Cu 20 , BC/Cu 60 , and BC/Cu 100 had diameters of 14.7 mm, 18.0 mm, and 21.3 mm, respectively [52]. In comparison to hydrogel composite, the gelatin + CuO hydrogel (3.8 ± 0.3 in Staphylococcus aureus and 4.8 ± 0.7 in Escherichia coli) and gelatin + ZnO hydrogel (4.9 ± 0.6 in Staphylococcus aureus and 5.3 ± 0.2 in Escherichia coli) showed greater zones of inhibition [46]. Likewise, the viabilities of Staphylococcus aureus and Escherichia coli were reduced when the concentrations of Cu nanoparticles were added to hydrogel samples (93.7% to 9.2% and 92.4% to 8.8%, respectively) [45]. Finally, F-HKUST-1 slowly released Cu 2+ during wound healing processes, which is recognized for having an impact on the expression and synthesis of growth factors, matrix metalloproteinases, collagen, elastin, and integrins [52].

Wound Healing
Wound evaluation has historically relied on a visual inspection by the trained clinician. However, new elements provide accurate assessment modalities. In this sense, the average small vessel length increased significantly for both Cu-MBG and mesoporous bioactive glasses (MBG) [42]. As evidence of HvCuO@GOx (HSHvCuO@GOx) superiority in new vessel development, CD34-positive cells in the HvCuO@GOx group's adhesive hydrogel were higher than those in the pure hydrogel and HSHvCuO groups [48].
On days 7, 14, and 28, it was shown that the composite scaffold's rate of wound contraction (13.2 ± 1.4 mm, 8.5 ± 2.9 mm, and 0.0 ± 0.01 mm, respectively) was significantly higher than cotton gauze's (17.7 ± 3.2, 12.3 ± 2.6, and 3 ± 4.7 mm, respectively) [46]. Similarly, the wound treated with laser and AuAgCu 2 O NSs shrank significantly, indicating a 93.5% healing rate [44]. In fact, an almost completely healed wound in a diabetic mouse model using HSHvCuO@GOx gauze has been found. However, wounds treated with control and HvCuO-based hydrogel did not recover, showing that the HvCuO@GOx-based hydrogel can successfully promote the healing of Staphylococcus aureus-infected wounds [48]. Finally, the incorporation of folic acid into HKUST-1 also reduces Cu 2+ toxicity in vivo, as has been described [52].

Discussion
This systematic review compiles and synthesizes the evidence of twelve quantitative studies that relate the activity of Cu NPs with their different types of antibacterial, cytotoxic, and proangiogenic properties in in vivo and in vitro studies for chronic wound healing.

Summary of Key Findings and Interpretation
The four overlapping healing processes during wound healing are hemostasis, inflammation, proliferation, and remodeling [55]. Both internal and environmental causes may hamper the healing process. Significant factors that prevent wound healing include microbial infections and reactive oxygen species (ROS). They might prolong each recovery step, resulting in less than ideal structural and functional outcomes [56].
In this regard, it is crucial to note that chronic, non-healing wounds have been associated with impaired healing when higher and continued ROS have been detected in vivo [57]. On a molecular level, high ROS and reactive nitrogen species (RNS) can affect the activity of dermal fibroblasts and keratinocytes as well as change and/or degrade extracellular matrix (ECM) proteins both directly and indirectly (through activation of proteolysis). This is in addition to ROS-mediated transcription, which can cause matrix metalloproteases to be induced and prolong pro-inflammatory cytokine secretion [58]. In fact, the adequate equilibrium between low and high levels of ROS is essential in defining functional outcomes: although low levels of ROS are required for promoting efficient healing [59], excessive ROS release damages cells and hinders wound repair [60]. Instead of directly affecting ROS, one option for wound healing might involve influencing the antioxidant system. The in vitro studies analyzed did not exhibit noticeable cytotoxicity during low levels of Cu NPs treatment because it was well-tolerated [40][41][42][43]45,46,[48][49][50]; it does not deform the structure of the cell membrane, and it can be used to relieve oxidative stress at the wound site. However, this is only achieved if a Cu ion is administered in controlled release [50]. In fact, if similar amounts are administered in a short period of time and abruptly, the Cu NPs can become toxic to cells and cause apoptosis [50].
Copper-based combined pharmacological complexes are more effective as antibacterial, antifungal, and antiviral medicines [61,62]. Copper concentration has a direct relationship with the mechanism by which it has a bacteriostatic or bactericidal effect [63]. The highest recorded impact was for copper metal (99.9%), and these results were seen in alloys having at least 70% copper [61,62]. However, due to the structure of the root canal system, which contains microecological niches such as dentinal tubules where antimicrobial drugs cannot reach, nanotechnology presents as an alternative to improve treatment success and endodontic retreatment rates [64]. Although systemic antibiotic administration helps the body fight microbial infections, a locally applied antimicrobial treatment is preferred for a wound [65]. Pomades, gels, and ointments can eliminate the germs that increased in large numbers where the damage was, potentially cutting the length of the healing process [66]. These materials are most helpful to patients with compromised immune systems, such as those with diabetes, hepatitis, and acquired immune deficiency syndrome [67,68]. In fact, according to our systematic analysis, the nanocomposite hydrogels under investigation have solid antioxidant potential against MRSA and ESBL E. coli as well as broad-spectrum antibacterial activity [40,[43][44][45][46]49,53], including P. aeruginosa [42]. When a wound is in the inflammatory phase, large amounts of ROS such as superoxide (O2), peroxynitrite (ONOO−), and hydroxyl radicals (OH−) are generated that damage the body's proteins and DNAs [69]. However, free radicals can be removed from the wound site thanks to the decisive antioxidant action. In this regard, additional research has demonstrated the ability of CuO hydrogels to eradicate human-pathogenic species of Gram-positive and Gramnegative bacteria [6,70,71]. These studies show that using these hydrogels as dressings at the location of a wound speeds up the healing process. These attributes synergistically support wound healing.
In wound therapy, primarily two types of NPs are used: (1) NPs with intrinsic properties that promote wound closure; and (2) NPs used as delivery vectors for therapeutic drugs. The former can be separated into nonmetallic nanomaterials and metallic or metal oxide nanoparticles. To repair the damaged cells and restore epidermal integrity, wound healing requires the migration and proliferation of different cells, angiogenesis, and collagen deposition processes [72]. In fact, collagen formation is a very vital step [73]. Recent investigations have unmistakably demonstrated that NPs represent a crucial therapy platform for skin wounds [41,[44][45][46]50,74,75]. Cu is a well-known NP with a lengthy history of direct angiogenesis involvement and antibacterial action. Additionally, Cu has been found to have a possible involvement in the healing of wounds by controlling the expression of 84 genes linked to angiogenesis and wound repair [5]. In addition, to the best knowledge, studies have reported the effects of CuNPs on keratinocyte and fibroblast cell proliferation and migration during wound healing [41,42,48,50,52].
Increasing the characteristics of polymer nanocomposites, however, is a constant challenge for their broad usage in research [76]. Wound healing, gene therapy, tissue engineering, and controlled drug administration are only a few of the uses for such biomaterials [77]. The biodegradability, nontoxicity, biocompatibility, and environmental susceptibility of polymer nanocomposites have sparked enormous interest and advancement [78].
Because of their bioavailability and repeatability, polysaccharides (e.g., sodium alginate) are often utilized in drug delivery activities. They are biocompatible and biodegradable, with low immunogenicity, and they are attractive candidates for medicine administration [79]. In this sense, sodium alginate/polyvinyl acetate nanocomposites [80], drugloaded nanofibers [81], and polyvinyl acetate/gelatin biopolymeric films [73] could be promising therapeutic options for preventing both resistant infections and life-threatening complications in exudative wounds.

Scope and Limitations
It is essential to highlight that our objective was to evaluate the use of Cu NPs in the healing of chronic wounds. However, it is necessary to consider that multiple physiological processes are involved in the healing process. Within these mechanisms are VEGF-induction, angiogenesis, and the expression and normalization of skin proteins such as collagen and keratin. As a result, the sustained release of non-cytotoxic amounts of Cu ions promotes in vivo wound healing by inducing angiogenesis, collagen deposition, and wound re-epithelialization.
Our findings confirm that Cu NP treatment promotes the formation of new vessels and significantly increases their total length, resulting in a denser and more stable vascular network at the wound site. Furthermore, with new collagen production and epithelial cell regeneration, the time spent in the inflammatory phase was reduced, allowing for a faster transition to the late stage of angiogenesis. However, the activity of Cu NPs is restricted by their biocompatibility in certain biological activities, low toxicity, and antibacterial capabilities, which are always dependent on Cu being present in low-tomoderate concentrations so that it is not damaging to target cells.
In addition, our review had other limitations, i.e., a low quantity of articles linking Cu NPs to chronic wound and wound healing were found. Also, the intervention times were highly variable between studies, with significant differences in the number of weeks and days. Finally, some studies did not provide sufficient data to compare the results obtained before and after the intervention, and some did not even incorporate the baseline measurements for the parameters studied, which limits the extraction of information.

Conclusions
Cu NPs have a high antibacterial response capacity since they are prone to interacting with the bacteria membrane. In addition, they can penetrate the bacteria biofilm and release ions inside it, compromising its integrity, where the rigidity of the membrane is an important determinant of antibacterial efficiency.
Our results have shown that Cu NPs are structurally designed to have a rough surface to facilitate adhesion to the bacterial membrane, helping to reduce or prevent the formation of bacteria. This bactericidal task occurs within a few minutes of encountering the bacteria; therefore, it is fast-acting, efficient, and long-term since it lasts over time without losing its bactericidal activity. However, it has been shown that Cu NDs demonstrate a much higher antibacterial effect than Cu NPs using laser radiation and can almost completely lyse bacterial membranes.