Scaffolds in the microbial resistant era: Fabrication, materials, properties and tissue engineering applications

Due to microbial infections dramatically affect cell survival and increase the risk of implant failure, scaffolds produced with antimicrobial materials are now much more likely to be successful. Multidrug-resistant infections without suitable prevention strategies are increasing at an alarming rate. The ability of cells to organize, develop, differentiate, produce a functioning extracellular matrix (ECM) and create new functional tissue can all be controlled by careful control of the extracellular microenvironment. This review covers the present state of advanced strategies to develop scaffolds with antimicrobial properties for bone, oral tissue, skin, muscle, nerve, trachea, cardiac and other tissue engineering applications. The review focuses on the development of antimicrobial scaffolds against bacteria and fungi using a wide range of materials, including polymers, biopolymers, glass, ceramics and antimicrobials agents such as antibiotics, antiseptics, antimicrobial polymers, peptides, metals, carbon nanomaterials, combinatorial strategies, and includes discussions on the antimicrobial mechanisms involved in these antimicrobial approaches. The toxicological aspects of these advanced scaffolds are also analyzed to ensure future technological transfer to clinics. The main antimicrobial methods of characterizing scaffolds’ antimicrobial and antibiofilm properties are described. The production methods of these porous supports, such as electrospinning, phase separation, gas foaming, the porogen method, polymerization in solution, fiber mesh coating, self-assembly, membrane lamination, freeze drying, 3D printing and bioprinting, among others, are also included in this article. These important advances in antimicrobial materials-based scaffolds for regenerative medicine offer many new promising avenues to the material design and tissue-engineering communities.


Introduction to tissue engineering and microbial resistance
Tissue engineering is currently attempting to provide breakthrough technologies capable of achieving successful results in regenerative medicine [1]. The tissue engineering regeneration strategy relies on the creation of biomimetic 3D cellular microenvironments (artificial ECM or scaffolds) that control and guide local tissue regeneration, usually made from a combination of natural and/or synthetic biodegradable biomaterials, cells and biomolecules (bioactive factors) [2]. The natural and synthetic polymers commonly used in tissue engineering include chitosan, alginate, gelatin, agarose, collagen, hyaluronic acid, carrageenan (CG), polycaprolactone (PCL), polyvinyl alcohol (PVA), polylactic acid (PLA), polyglycolide acid (PGA), poly lactic-co-glycolic acid (PLGA), poly (hydroxybutyrate-co-valerate) (PHBV) and many others [3][4][5][6][7][8][9]. Depending on the type of application, the scaffold will require specific physical-chemical (biodegradability, mechanical properties, etc.) and morphological properties (surface topology, pore size, pore distribution and interconnection, etc.) to mimic the cellular environment in vivo [10]. Most of the degradable polymers used to produce scaffolds can also release biomolecules that promote tissue regeneration, including growth factors, or antimicrobials to fight infections. The ability to manipulate physical-chemical variables (cross-linking, blends, copolymerization, etc.) enables the release dynamics to be tailored to the requirements of the application [11,12]. However, antibiotic resistance in pathogenic microorganisms has reached alarming levels and has become a serious global public health problem [13]. The use of alternative antimicrobial agents capable of dealing with antibiotic-resistant bacteria such as metal ions [14][15][16], quaternary ammonium compounds [17,18], antimicrobial peptides [19], peptoids [20], α-peptides [21], β-peptides [22], carbon-based nanomaterials [23][24][25] or combined strategies [26,27] are being given a lot of attention by researchers for their important contributions to future healthcare systems. Growth factors are often studied in cell-free tissue-engineering approaches to facilitate tissue regeneration [28]. However, their use can generate problems associated with immunogenicity, cancer risk and associated problems in cellular homeostasis [29,30]. In this context, the use of inorganic biomolecules is being studied for regeneration applications since they induce tissue regeneration without the drawbacks of growth factors [30,31]. Biometals have shown potential results in regenerative medicine, mostly because of their affordability, stability, and capacity to trigger cellular responses via signaling pathways. Biometals like zinc integrated into scaffolds are also being researched as regenerative agents [32][33][34][35][36] Their antibacterial qualities give them additional advantages for the prevention of infections following scaffold implantations. Biomolecules with both bioactivity and antibacterial characteristics have thus been the basis of newly discovered methods for regenerative medicine applications [37][38][39]. Some of the biomaterials used as scaffolds for tissue engineering (with no additional components) also possess intrinsic antimicrobial properties, providing a cellular microenvironment capable of stimulating cellular response and simultaneously inhibiting microbial growth [40][41][42] (Fig. 1).
Since surgical infections in tissue engineering are associated with significant postoperative morbidity, increased healthcare costs and high risk of death in case of multidrug-resistant pathogens, the scientific community have been working hard on the development of antimicrobial scaffolds for the last ten years. This article reviews the current state of antimicrobial scaffolds produced for bone, oral tissue, skin, muscle, nerve, trachea, cardiac and other tissue engineering applications. The latest scaffolds developed to prevent infections produced by bacteria and fungi are also discussed in depth with detailed descriptions.
Electrospinning uses polymers for scaffold design, generating polymeric fibers controlled by an electric field between two electrodes [3,8] to produce porous substrates made of ultra-fine fibers with a large surface area, which makes them ideal environments for cell growth and subsequent tissue organization [68], e.g. antimicrobial scaffolds made of polymers with intrinsic antimicrobial activity such as chitosan (CS) (Fig. 3) [69,70].
The phase separation scaffold production technique is based on separating the polymeric solution into two phases by temperature changes [43]. The polymer is dissolved in a solvent to produce porous scaffolds with bioactive molecules integrated into that structure after evaporation and sublimation [44]. Phase separation can be combined with other techniques to design 3D structures with a controlled pore morphology [71]. This technique is widely used to make polymer-based scaffolds such as PLA for regenerative medicine applications [72]. The polymeric matrix can be combined with other materials with intrinsic antimicrobial properties.
The great advantage of gas foam scaffolding manufacturing techniques is that they do not require chemicals or high temperatures, which can damage cells, tissue and the microenvironment [43]. Nucleation of pores is created due to gas phase separation from the polymer, expanding the scaffold volume while reducing the polymer density [45]. The gas foaming method is often used to produce new nanocomposite scaffolds charged with a material with antimicrobial properties [46].
The porogen leaching method is commonly used to produce scaffolds with the required geometry, pore size and pore interconnection using a porogen such as salt, wax, sugar, polymers, glass, fibers, polymer microspheres, meshes, etc. [47,48,73,74]. A porogen of the desired size and shape is leached away from the polymer mixture by a suitable solvent to make the scaffold with the required characteristics [75]. This technique is often used in combination with melt molding [76][77][78] to produce degradable polymer scaffolds, which are the basis of many new developments in antimicrobial scaffolds.
Scaffolds can also be produced via polymerization in the presence of a solvent, e.g. poly (methyl methacrylate) (PMMA) sponges can be made via polymerization in solution with ethanol [51,52,79]. Poly (2-hydroxyethyl acrylate) (PHEA) porous hydrophilic sponges have also been created by polymerization in solution in the presence of water, ethanol or methanol [50,53,80]. Hybrid PHEA/PMMA sponges can be obtained by combining polymerization in solution with plasma polymerization [81][82][83]. This means antimicrobial scaffolds could be synthetized by polymerization in solution combined with the incorporation of Fig. 3. Scanning electron microscope images of chitosan and PVA blended electrospun fibers at a magnification of x10000. Chitosan and PVA were dissolved in formic acid at 7% w/w and in distilled water at 9% w/w, respectively. The two solutions were mixed and electrospun in the indicated chitosan: PVA specified volume ratios of 50:50 (A), 30:70 (B) and 0:100 (C). Electrospun fibers made of a mixture of chitosan dissolved in formic acid (or 0.2 M acetic acid) at 2% w/w and mixed with a solution of 9% w/w PVA in a volume ratio of 50:50 (D). Adapted with permission from Ref. [69]. Copyright 2004 Elsevier. antimicrobial nanomaterials such as graphene oxide (GO) [84]. The self-assembly technique is based on the spontaneous organization of several molecules in a given medium, forming an ordered structure with a specific function [57]. It commonly used, for example, in amphiphilic peptides in aqueous solution that link their hydrophobic residues through non-covalent bonds [85], forming 3D nanofibers for tissue engineering [58].
The membrane lamination method is used to construct layer by layer anatomically accurate three-dimensional scaffold assemblies during the manufacturing process [59,60]. The fiber mesh coating method consists of depositing a polymer solution on a porous polymer fiber mesh and subsequently allowing the solvent to evaporate [55,56].
The freeze dying technique is based on the sublimation principle and is used to manufacture porous scaffolds for tissue engineering [1,61,62]. Scaffolds with high porosity can be produced by dissolving a polymer in a solvent. After freezing the mixture, the solvent is removed by lyophilization [86]. This technique is simple and can manufacture highly porous scaffolds of a certain pore size, which are determining factors in tissue engineering [87].
These scaffolds not only showed good antibacterial and cytocompatibility results in vitro, but also performed well in an in vivo rabbit model, demonstrating their potential for bone regeneration due to their compatibility, antimicrobial capacity and mechanical properties [63]. The production of porous metal alloys with powerful antimicrobial properties by AM for potential biomedical applications has recently been reported [89,90].
The reproducible, automatic 3D bioprinting technique uses biomaterials, cells and growth factors to produce artificial living tissues or even an entire organ [10,91,92]. Multicellular building blocks (bioinks) are distributed layer by layer and scaled to manufacture the final construct [10].
Bioprinting includes a number of different methods: laser-induced forward transfer, inkjet printing, or robotic dispensing [66] (Fig. 5), with specific requisites for bioinks. Bioprinting aims to engineer solid organs by computer-controlled systems capable of depositing biomaterials with or without cells to create solid and viable organs.
However, the diversity of solid organs in terms of specific cellular and structural microenvironments, together with the demands of nutrients, is still a challenge [93,94]. Different types of tissue approaches have recently been investigated, such as blood vessels [95,96], skin [97,98], cardiac tissue [99,100], bladder and urethral tissue [10,101], cartilage [96,102] or bone [102,103], among others.
We firmly believe that bioprinting is a rapidly emerging technology that will provide a real clinical solution to the shortage of organ donors while avoiding the associated risks of transmitting diseases and immunological rejection. This method will certainly revolutionize the biomedical sector in the near future. Also, if the bioprinting design is performed with the additional aim of incorporating antimicrobial agents, the chances of success of the engineered constructs increase exponentially. A variety of antimicrobial materials can thus be used to enhance the current bioink formulations to improve biocompatibility and combat the spread of multidrug-resistant infections [104,105].

Antimicrobial scaffolds for tissue engineering
Tissue engineering has undoubtedly become a promising strategy for repairing damaged or diseased tissue [3] by means of highly porous materials or scaffolds capable of providing structural support for the engineered cellular environment with rapid diffusion of nutrients and metabolites [106]. If these scaffolds also incorporate new antimicrobial materials that can prevent microbial infections they will be even more promising. Controlled drug delivery materials, medical prostheses and medical devices are examples of other biomedical applications [3]. Enormous progress has been made in material engineering and the design of biomaterials that can mimic ECM [107]. Many preclinical and clinical trial studies have looked into the effect of stem cell-based therapies for tissue regeneration [108,109]. For example, human induced pluripotent stem cells are a powerful tool for the generation of specialized cells to treat diseases such as nonalcoholic steatohepatitis (NASH) [110]. Mesenchymal stem cells (MSCs) are gaining a lot of interest as perfect candidates for cell therapy and tissue engineering, due to their ability to differentiate into different cell types [111,112]. However, their potential in bioengineering is reduced when the reactive oxygen and nitrogen species levels overcome the physiological levels, which can worsen differentiation and proliferation while it favors senescence and cell death [113]. In this regard, nano-antioxidants in the form of chemical compounds, biometabolites, or protein precursors/proteins are effective in the treatment of MSCs to optimize their clinical use.
Biomaterials used as scaffolds for tissue engineering are preferably endowed with antimicrobial intrinsic or extrinsic agents to provide a 3D environment with bioactive and biocidal properties. This section describes a broad range of current antibacterial, antifungal and antibiofilm scaffolds according to their specific tissue engineering application, such as bone, oral tissue, muscle, nerve, trachea, cardiac, and skin, among others (Fig. 6).

Antimicrobial fillers
Scaffolds containing antimicrobial fillers such as antibiotics, antiseptics, polymers, peptides, carbon nanomaterials, metals, ceramics or combined and alternative strategies have been developed to prevent and/or treat infections in tissue engineering. For example, polyhydroxyalkanoate/chitosan (PHA/CS) and 2D molybdenum disulfidedoped (2D MoS 2 ) scaffolds have been proposed for biomedical and antimicrobial applications such as wound healing and antibacterial treatment of skin infections caused by methicillin-resistant Staphylococcus aureus (MRSA) [114]. These materials are biocompatible and also show promise for drug delivery. Other approaches consisted of creating PCL nanofibers containing Ag nanoparticles by electrospinning to produce antibacterial scaffolds [115]. Cell viability studies on this material have revealed that cytotoxicity is highly dependent on the concentration of silver nanoparticles. Brennan et at. evaluated the degradation products resulting from the acid digestion of scaffolds composed of ECM for antibacterial effects against Staphylococcus aureus and Escherichia coli. The results suggest that several low-molecular-weight peptides with antibacterial activity exist within the ECM, which may help explain the resistance to bacterial infection provided by these biobased scaffolds [116].
Biocompatible boron nitride doped polyhydroxyalkanoate/chitosan (PHA/Ch-hBN) nanocomposite scaffolds have been successfully designed and manufactured with superior antibacterial activity by means of the solvent casting technique [117]. In another study, CS-blended PLA nanofibers were successfully produced by electrospinning [118]. CS-blended PLA nanofibers exhibited antibacterial activity against E. coli and no cytotoxicity in mouse fibroblasts (L929 cell line), making them potential candidates for biomedical applications. Many types of antibacterial scaffolds that are safe and efficient for biological applications have thus been produced containing different types of antimicrobial fillers to provide the required characteristics for specific tissue engineering applications.

Antibacterial scaffolds for bone regeneration
The most widely used practice to treat large bone defects has been autologous bone grafting [119]. Unfortunately, this strategy is associated with the morbidity of the donor site, the need for one or more surgical interventions and the small amount of bone that can be removed from the patient [120]. A lot of research has been done to make bone substitutes that are structurally and functionally similar to real bone, such as highly porous 3D scaffolds that help to achieve the diffusion of nutrients and metabolites and antibacterial activity following a broad range of strategies (see Table 1).
However, the antibacterial properties of recent promising scaffolds proposed for bone tissue engineering have not been studied to date. Some examples of these scaffolds include an arabinoxylan-co-acrylic acid/ HAp/TiO 2 nanocomposite scaffold [230] and a carrageenan/acrylic-acid/graphene/hydroxyapatite hybrid nanocomposite scaffold [231], both produced by freeze-drying. Arabinoxylan (ARX) and carrageenan are natural biological macromolecule with promising applications in biomedicine [230,231]. The antimicrobial properties of freeze-dried silver coated biocompatible scaffolds containing acrylic acid/guar gum, nano-hydroxyapatite, titanium nanoparticles and graphene oxide has so far not been tested [232]. These scaffolds showed promising results against mouse pre-osteoblast (MC3T3-E1) cell lines and increasing the amount of TiO 2 in combination with GO improved physicochemical and microstructural properties, mechanical properties (compressive strength and Young's modulus), and porous properties (pore size and porosity). Another scaffold with a nacre-mimetic architecture and consisting of SrFe 12 O 19 -doped nano-layered double hydroxide/chitosan has recently been developed for bone tissue engineering [233]. The slow release of Mg 2þ and Sr 2þ of these scaffolds can maintain bone homeostasis and promote the formation of new blood vessels. However, their antibacterial performance has not yet been evaluated, so that it should be noted that a complete antimicrobial evaluation of developed scaffolds is essential for tissue engineering applications.

Antibacterial scaffolds with antibiotics
The use of scaffolds for controlled localized drug release is one of the most promising techniques in tissue engineering. The aim of this method is to act on the focus of the problem and so avoid using large concentrations of possibly toxic antimicrobials to the organism or even produce microbial resistance [234]. Vancomycin (VAN) [124,125] is one of the most commonly used antibiotic with this release technique as an antibacterial agent [127,128]. VAN-laden mesoporous bioglass/PLGA composite scaffolds have been developed for this purpose [125]. These scaffolds showed a sustained release of the antibacterial drug for more than eight weeks in vitro producing inhibition of S. aureus growth and biofilm formation. These results, along with the ability to promote osteoinduction, make these scaffolds a very promising biomaterial for bone tissue engineering. VAN-PCL scaffolds maintained their antibacterial effect for more than 4 weeks [126] and showed complete inhibition of S. aureus [215]. A new scaffold composed of hydroxyapatite (HAp), SA and CS loaded with vascular endothelial growth factor (VEGF) and vancomycin was recently reported [130]. Gentamicin [121] and tetracycline hydrochloride (TCH) [235] are two other antibiotics used as antibacterial agents in PCL scaffolds, which revealed a significant antibacterial effect, although their toxicity in cells or animal models has not been assessed.
Some studies have developed an antibacterial bone graft by immobilizing levofloxacin hydrochloride-loaded mesoporous silica microspheres on the surface of a nano-HAp/polyurethane (PU) bioactive composite scaffold. The results show considerable antibacterial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria with a drug release for up to 42 days [127]. This approach could be a very promising strategy against chronic osteomyelitis, whose mainstay treatment is the aggressive excision of necrotic bone and infected soft    [195] Calcium phosphate 3D printing (direct extrusion) and crosslinking S. aureus L929 fibroblast cells 2020 [196] Phosphate-free glass-ceramic scaffolds Freeze-drying E. coli Adipose MSCs 2020 [197] PHBV Scaffolds Incorporated with Zinc Oxide Selective laser sintering E. coli MG-63 osteoblast-like cells 2020 [198] Forsterite scaffolds 3D printing and polymer-derived ceramics (PDCs) strategy E. coli and S. aureus Not studied 2020 [199] Silver-coated grafted beta-glucan/hydroxyapatite nanocomposite scaffolds tissue and prolonged local antibiotic delivery [236]. 3D scaffolds for bone regeneration based on agarose, nanocrystalline apatite, VEGF, and the antibiotic cephalexin were also capable of inhibiting the growth of S. aureus bacteria [128]. Krishnan et al. developed porous gelatin-hydroxyapatite (G-HAp) scaffolds loaded with various amounts of ciprofloxacin (CPFX). They observed a reduction in the growth of S. aureus and concluded that it has the potential to be used as a local drug delivery system. This scaffold can release effective antibiotics for reducing S. aureus for 60 days, with no detrimental effects on human adipose-derived mesenchymal stem cell (ADMSCs) viability or osteogenic potential [122]. 3D printed PLA/collagen/nano HAp loaded with minocycline showed increased osteogenic activity and reduced S. aureus biofilm formation [37]. Alkaloids from Semen Strychine, which possess antibacterial, anti-inflammation and analgesic effects, were incorporated into polyetheretherketone/polyglycolide acid (PEEK/PG) scaffolds to provide a sustained release of the antimicrobial compound against E. coli and S. aureus, as well as biocompatibility [129].
A multidrug sequential release of antibiotic agents from a hierarchical 3D scaffold was reported by García-Alvarez et al. [123] and scaffolds based on nanocomposite bioceramic and PVA with three antibiotics were produced by rapid prototyping. These three antibiotics (rifampin, levofloxacin and vancomycin) were located in different compartments of the scaffold to obtain different release kinetics. The scaffolds showed good bioactivity in preosteoblasts and were able to inhibit bacteria growth and destroy Gram-positive and Gram-negative bacteria biofilms. Laponite nanoplates/amoxicillin-functionalized PLA nanofibrous scaffolds with osteoinductive and antibacterial activity have recently been developed by electrospinning [131].

Scaffolds with antibacterial polymers/peptides
The intrinsic antibacterial properties of chitin, CS, cellulose and several polysaccharides of microbial origin are well known [237][238][239]. Hu et al. reported a vanillin-bioglass crosslinked 3D CS scaffold with good biocompatibility, strong antibacterial activity and capable of promoting osteoblastic differentiation prepared using a novel crosslinking technique with vanillin [139]. In other bone regeneration studies, biocomposite scaffolds containing CS were synthesized to obtain bioactive and antibacterial scaffolds [133,136]. Scaffolds based on nano-HA, starch, CS, alginate and S-nitroso-N-acetyl-penicillamine were manufactured by freeze-drying, obtaining porous scaffolds and an interconnected structure favorable to cell attachment and the growth of new tissue. Zhou et al. prepared a scaffold from a CS derivative (with an acrylamidomethyl group) with good prolonged antibacterial ability against S. aureus and E. coli [133]. Tissue regeneration, osseointegration, and bacterial accumulation in biomedical implants can be improved by surface modification [240]. For example, the surface modification of 3D printed PCL/HAp scaffolds has been performed with an antimicrobic polypeptide [138], providing favorable biocompatibility, osteoconductivity and antibacterial activity.
Electrostatic deposition of cationic oligopeptides in a PCL/CS nanofiber scaffold inhibited S. aureus while promoting osteoblast adhesion, spread, and proliferation [132]. Another strategy consists of either incorporating antibacterial peptides into the scaffolds or coating the scaffolds with them [137,140,141]. A mineralized collagen scaffold containing PLGA microspheres loaded with two antibacterial synthetic peptides was found to promote osteogenic capacity and antibacterial properties [137]. 3D printed scaffolds based on PLA/gelatin/nano HAp and the peptide ponericin showed that E. coli and S. aureus were inhibited for up to 24 h, and the inhibition could remain for up to 72 h [135]. Karamat-Ullah et al. developed a 3D hybrid aerogel-based scaffold combining an antibacterial peptide-modified silk fibroin (SF) with silica using micro-extrusion-based printing and directional freeze-casting/drying. This hybrid scaffold was found to be bactericidal against both Gram-positive and Gram-negative bacteria, and to be biocompatible with mouse embryonic pre-osteoblast (MC3T3-E1) cells [140]. 3D printed technology has also produced hydroxypropyl trimethylammonium chloride chitosan (HACC) grafted PLGA/HAp scaffolds that showed antibacterial activity against S. aureus and bone regeneration in infected bone defect models [134].
A novel flax/silk protein-based nanofibrous scaffold has recently been developed for bone regeneration [142]. This scaffold showed biocompatibility in MG-63 osteoblast cells and long-term antibacterial activity against E. coli and S. aureus. Flax holds bioactive peptides, which could promote antioxidant activity, antibacterial performance and anti-inflammation capacity [142].
Smart electroactive polymers have been developed to produce changes in electric charge distribution. These biomaterials, particularly conductive polymers, can deliver electrical signals by controlling the electric field applied to promote cell proliferation and differentiation, stimulating the regeneration of muscles, organs, and bones [241][242][243]. Electrostimulation applied to material surfaces appears to have an effective antibacterial activity against biofilm formation [244]. Electroactive polymers are promising materials for exploration in microbiology to develop novel strategies for fighting antibacterial resistance [241]. These materials can be useful as scaffolds for tissue regeneration to prevent infections associated with biofilm formation in implants, such as osteomyelitis in bone regeneration.
Composites containing carbon nanomaterials with antibacterial and osteogenic activity have recently been reported [256]. Some researchers have developed 3D printed scaffolds with electroactive properties that are composed of percolated PCL composites with thermally reduced graphene oxide (TrGO), whose antibacterial activity has been tested for use in tissue engineering applications [145] (Fig. 7).
The results showed that applying an electrical stimulus of 30 V for 3 h to the surface of the 3D-printed electroactive scaffolds containing GO completely eradicated bacterial growth (S. aureus) on the scaffold surface. However pure scaffolds without GO possessed bacterial attachment after electrostimulation [145]. In addition to the greater bactericidal effect, the presence of highly conductive rGO linked to electrostimulation seems to increase cell viability. Recently, Lu et al. reported 3D printed scaffolds based on rGO, gelatin, CS and tricalcium phosphate (TCP) with good antibacterial activity (against S. aureus and E. coli) and without adverse effects on osteoblast viability and proliferation [147].
GO, which has low electrical conductivity, has also demonstrated antimicrobial properties. Melo et al. prepared a layer-by-layer PCL-3D printed fibrous scaffold with GO at different concentrations. The results showed good antibacterial behavior against Gram-positive and Gramnegative bacteria, and the ability to promote cell adhesion [146]. In another study, antibacterial capacity and in vitro osteogenesis were demonstrated by a GO-decorated microporous scaffold prepared with polyetheretherketone (PEEK), a semi-crystalline polymer used for orthopedic and spinal implants [144]. Khan et al. fabricated a composite hydrogel based on the polysaccharide ARX, GO nanosheets, HAp and PVA with bonding interactions between the components. The seeded preosteoblasts showed significant proliferation with no significant toxicity, while antibacterial activity against selected Gram-positive and Gram-negative bacteria was confirmed [148]. In a second study, the authors used GO and HAp to prepare nanocomposite scaffolds with bacterial cellulose and β-glucan. It was found that increasing the amount of GO has a positive impact on antibacterial activity and cell behavior [149].
A combination of different carbon nanomaterials has also been reported. For example, polymeric matrices of PLA and high concentrations of GO/carbon nanotubes (50/50% w/w of filler) were prepared by solvent casting and treated with oxygen plasma to enhance wettability [143]. The scaffolds had significant cell adhesion, showed no cytotoxicity and reduced bacterial proliferation. Several approaches using carbon nanomaterials in combination with silver nanoparticles have also given good results both as antibacterial and osteogenic biomaterials [176,217]. It should be noted that particle size is the most important characteristic that affects the antimicrobial behavior of carbon nanomaterials. The high surface-to-volume ratio of the nanoparticles (NPs) can adhere to microbial cells and affects cell membrane integrity, structural components and metabolic processes [257], which makes these new materials very promising in the field of tissue engineering, both as bioactive and biocide agents.

Scaffolds with metals/ceramics/glass
Several studies in the field of antimicrobial scaffolds are based on silver (Ag), zinc (Zn), magnesium (Mg) or strontium (Sr) ions, due to their known antibacterial properties. Nanocomposite antibacterial scaffolds were prepared by loading AgNPs with an adsorption process. Overall, the results show that AgNPs confer good antibacterial properties on composite scaffolds to impede early infections [158]. Scaffolds composed of TiO 2 nanotubes manufactured by 3D printing and silver ions were subsequently incorporated into their surface. The antimicrobial effect against S. aureus was found to continue for two weeks [172].
Some researchers have developed AgNPs containing antibacterial scaffolds composed of pentaerythritol triacrylate-co-trimethylolpropane tris (3-mercaptopropionate) (PETA) and HAp. These scaffolds showed osteoinductive and degradable properties capable of stimulating the proliferation of bone progenitor cells, did not affect cell viability and inhibited the proliferation of S. aureus and E. coli [159]. Gelatin/bioactive glass/AgNP scaffolds showed good cytocompatibility to human mesenchymal stem cells (hMSCs) and antibacterial capacity against E. coli and S. aureus [160]. Researchers investigated the antimicrobial activity of Ag octahedral nanoparticles containing PCL scaffolds, which showed antibacterial activity, osteogenic differentiation and no adverse effects on hFOB and hMSCs cells [174,202]. Polymeric PLA scaffolds with metals such as silver, which give a final result of antibacterial activity, showed osteogenic differentiation and no cytotoxicity to human cells [179]. A continuous Ag þ release can last more than 3 weeks, which can be useful in long-term bone implants. Zhang et al. synthesized a brushite/Ag 3 PO 4 -coated Mg-Nd-Zn-Zr scaffold to substitute bone [192]. This new scaffold demonstrated high antibacterial activity against Gram-positive and Gram-negative bacteria, with appropriate degradation characteristics and cytocompatibility. Arjunan et al. manufactured a pure Ag scaffold and demonstrated its antibacterial efficacy against S. aureus [193]. SF films with AgNPs have also been developed, since silk fibroin is a suitable biomaterial for bone tissue engineering. SF/AgNPs scaffolds showed cytocompatibility and an effective antibacterial effect against Gram-negative and antibiotic-resistant bacteria [189]. Silver-coated bioactive nanocomposite scaffolds have been developed using a polymeric matrix of beta-glucan biopolymer, acrylic acid, and nano-hydroxyapatite through free radical polymerization and freeze drying [200]. These scaffolds showed an antibacterial effect against DH5 alpha E. coli with no cytotoxicity in MC3T3-E1 cells.
Some researchers have combined silver with other materials to prepare antibacterial scaffolds. For example, silver-doped HAp scaffolds (HAp/Ag) reduced E. coli, S. aureus and S. epidermis bacterial populations while maintaining cytocompatibility with mammalian cells [175,191,258]. PVA-starch/HAp/Ag scaffolds prepared by freezing-thawing also demonstrated antibacterial activity against Gram-positive Bacillus and Gram-negative E. coli [184]. In another study, Deng et al. developed Ag-decorated 3D printed PEEK scaffolds via catecholamine chemistry. The antibacterial tests performed indicated that these Ag-PEEK scaffolds showed significant antibacterial effects against Gram-negative and Gram-positive bacteria and could support the proliferation of MG-63 osteoblast cells [173]. In another study, Wang et at [187]. proposed Ag-incorporated zincosilicate zeolite scaffolds with compressive strength and a Young's modulus similar to human cancellous bone. The scaffolds showed good antibacterial and bioactivity, indicating their potential as antimicrobial materials for bone substitutes. Antibacterial degummed silk fibers (ADSF) in combination with nano-HAp and PLA have been prepared, including Ag-nanoparticles as a reinforcing material [190]. The biological and antibacterial assessments showed that the ADSF/nano HAp/PLA composites had good bioactivity and antibacterial properties.
Bioactive glass-ceramic scaffolds [152,156] or coral hydroxyapatites [155] combined with Ag ions have been reported as a good approach for preparing scaffolds with antibacterial properties. In a similar approach, gelatin composite scaffolds were made by gelatin, alginate, PVA, nano-silver and HAp. These reinforced scaffolds showed antibacterial activity against Gram-positive and Gram-negative bacteria, as well as good biocompatibility in MC3T3-E1 preosteoblast cells [188].
Scaffolds with TiO 2 have shown strong antibacterial activity [180,181]. PCL/TiO 2 nanocomposite coatings were developed with a good bioactive performance against osteoblast cell lines and excellent antimicrobial behavior against S. aureus [178]. TCP/silver/PLGA scaffolds (TCP/Ag/PLGA) with a proven prolonged antibacterial effect against E. coli [151] have also been reported. Some researchers developed HAp scaffolds with Ag/TiO 2 /PA66 [154], Sr, Zn [164,177], or Ti6Al4V (pTi), CS and selenium (Se) (pTi/CS/HAp-Se) [186]. They observed osteoblast proliferation, tumor cell growth inhibition and bacterial viability with pTi/CS/HAp-Se scaffolds. Jiang et al. developed nano HAp/PU scaffolds with varying concentrations of Ag 3 PO 4 particles for the repair of infectious bone defects [166]. The incorporation of Ag 3 PO 4 in nano HAp/PU scaffolds increased their antibacterial potential against both Gram-positive and Gram-negative bacteria. The antibacterial tests and cytocompatibility evaluation revealed that nano HAp/PU scaffolds with 3% w/w Ag 3 PO 4 had stronger antimicrobial effects and satisfactory cytocompatibility.
A novel porous nano HAp/polyamide 66 (nHP66)-based nanoscaffold material containing varying concentrations of silver ions (Ag þ ) (TA-nHP66) and oxidized titanium (TiO 2 ) was developed successfully in an experimental osteomyelitis study in rabbits [168]. Porous osteoinductive TA2-nHP66 scaffolds with a composition of 0.64% w/w of Ag þ and 2.35% w/w of TiO 2 , were shown to have strong antibacterial activity against S. aureus and E. coli in vitro and S. aureus in vivo. A new biomaterial composed of PLA, halloysite nanotubes loaded with ZnO nanoparticles was prepared by 3D printing [195]. The scaffolds so prepared showed osteoinductive potential. The external coating with gentamicin preserved the osteogenic properties and reduced bacterial growth. Zhu et al. prepared a forsterite scaffold by combining 3D printing and polymer-derived ceramics that contain biometal Mg [199]. The scaffolds showed efficient photothermal-induced antibacterial activity.
Bioactive glass scaffolds have many advantages such as osteoconductivity and osteoinductivity, making them an ideal scaffold for bone tissue engineering applications [259]. The development of multifunctional bioactive scaffolds that combine angiogenesis activity, a capacity, and antibacterial performance for regenerating lost bone tissues is of great importance in this field [260]. The antimicrobial activity of selenium nanoparticles (SeNPs) has also been reported [261,262]. Adding Ag þ to bioactive glasses has been investigated to produce antibacterial glasses [263]. Bioactive glass scaffolds have also been developed with delivery systems. Poly (octanediol citrate) bioactive glass scaffold containing zinc and gallium ions demonstrated antibacterial activity against Gram-positive and Gram-negative bacteria as well as cytocompatibility with human cells [169]. Silver-doped bioactive glass scaffolds showed antibacterial activity against S. aureus and E. coli. The scaffolds mimicked cancellous bone in terms of architecture and mechanical properties [170,183]. Some researchers obtained bioactive glass scaffolds from a soda-lime glass powder consisting of microspheres belonging to the [171] and compared this new scaffold with the 45S5 Bioglass® scaffold and found its antibacterial activity to be higher against C. krusei. Other researchers modified the 45S5 Bioglass® scaffold to improve its characteristics. For example, Gorriti et al. added free boron to 45S5 Bioglass® scaffold and the bactericidal effect increased by 55% [153]. A new 45S5Bio-glass®/PLGA/SeNPs scaffold was fabricated to combine the antimicrobial properties of SeNPs with the osteoinductive capacity of bioactive glass to achieve bone regeneration [163].
Scaffolds made of borosilicate bioactive glass doped with varying amounts of Ag 2 O showed a sustained release of Ag þ over more than 8 weeks and resistance against colonization by the bacterial strains E. coli and S. aureus [165]. Phosphate-free glass-ceramic porous scaffold is another example of antibacterial bioglass scaffolds. This can be synthesized by a three-step method involving slurry preparation, induction of porosity by surfactant-assisted foaming, followed by freeze-drying and sintering [197]. Hayashi et at. fabricated antibacterial honeycomb scaffolds by a procedure consisting of the replacement of their principal component (carbonate apatite) for silver phosphate on their surface [203]. Scaffolds containing 9.9⋅10 À4 % w/w silver phosphate showed antibacterial activity against S. aureus and allowed MC3T3-E1 preosteoblast proliferation and differentiation. They also prevented bacteria from growing in a rabbit with a femoral defect, which had S. aureus in it and new bone started to grow two weeks after surgery.
Hypoxia is one of the key factors that can affect scaffold implantation and lead to cell necrosis and microbial infection [264]. To solve this problem, oxygen-releasing bioceramic scaffolds were fabricated from biphasic calcium phosphate (BCP) powder [185] (Fig. 8).
Calcium phosphate scaffolds with specific designs in terms of pore size, shape, and porosity can be precisely produced by AM technology [196]. In situ, the porous 3D printed bioceramic material was crosslinked with SA and freeze-dried. Scanning electron microscope (SEM) images revealed that the crosslinked scaffold has a multi-level porous structure compared to the uncross-linked one (Fig. 9).
At the same time the scaffolds were loaded with berberine, a quaternary ammonium compound with antibacterial activity and showed both antibacterial and bone-promoting functions. In vitro studies indicated that the 3DP scaffolds had low cytotoxicity with a beneficial effect on MC3T3 cell adhesion and proliferation [196].
Copper-containing mesoporous bioactive glass (Cu-MBG) scaffolds stimulate the hypoxia-inducible factor (HIF)-1a and VEGF expression in human bone marrow-derived mesenchymal stromal cells (BMSCs). Antibacterial Cu-MBG scaffolds promoted the osteogenic differentiation of human BMSCs and maintained a sustained release of ibuprofen [157]. Magnesium (Mg) has also been reported to possess antibacterial activity [265]. For example, a PLGA/Mg scaffold fabricated by a low-temperature rapid-prototyping technique showed an ability to inhibit bacterial growth and biofilm formation [162].
Zinc is an essential element with intrinsic antibacterial and osteoinductive capacity [266]. Zinc cross-linked scaffolds significantly reduced the growth of Bacillus subtilis and E. coli by 70 and 81%, respectively [167]. PCL-ZnO nanofibrous scaffolds have been developed with antibacterial activity against S. aureus and are also capable of inducing early mineralization with ZnO concentration-dependent degradation [182]. ZnO-enriched meso-macroporous glass scaffolds were prepared by S anchez-Salcedo et al. The results showed that the porous structure was suitable for osteoblast growth and that the Zn ions released exhibited antibacterial properties against S. aureus [161]. ZnO nanoparticles have also been incorporated into PHBV to produce antibacterial porous scaffolds [198].
PLGA is one of the most commonly used polymer biomaterials for producing bone tissue engineering scaffolds, since this biodegradable copolymer does not have any side effects when used as a medical material [267,268]. A novel PLGA/Cu(I)@ZIF-8 scaffold for infected bone repair was created by combining antibacterial copper-loaded-zeolitic-imidazolate-frameworks (ZIF-8) and PLGA [194], as shown in Fig. 10.
3D-printed biocompatible scaffolds based on calcium-deficient hydroxyapatite (CDHA) with gold nanoparticles showed effective antibacterial activity against Micrococcus luteus for bone tissue engineering  Reprinted with permission from Ref. [196]. Copyright 2020 Elsevier. Reprinted with permission under a Creative Commons CC BY 4.0 License from Ref. [194]. Copyright 2020 Springer Nature. A. Serrano-Aroca et al.
Materials Today Bio 16 (2022) 100412 [204]. Bio-ceramic clinoenstatite (MgSiO 3 ) scaffolds of different micropore sizes were fabricated by the space holder method and subsequent sintering [201]. They showed good mechanical strength as well as biocompatibility in MG63 cells and controlled drug-release potential of metronidazole (MET) towards the Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans bacteria.

Antibacterial scaffolds produced by combined and alternative strategies
Biological and antibacterial properties of titanium implants are required to prevent implant-associated infections and promote cell attachment of orthopedic devices. Several antimicrobial scaffold delivery systems have been developed in this research line as an emerging technology for the reconstruction of bone and cartilage tissue defects [269]. A reinforced hybrid scaffold consisting of continuous and porous layers of titanium and ultrahigh-molecular-weight polyethylene (UHMWPE), a polymer with good compatibility, and a subsequent amoxycillin impregnation were prepared to prevent the appearance of opportunistic infections [219]. As a result, the contact of Gram-positive and Gram-negative bacterial cultures (S. aureus, S. epidermidis and E. coli) with the hybrid polymeric scaffolds suppressed microorganism growth and colony formation. A new bioactive monticellite-ciprofloxacin (Mon-CPFX) scaffold was created by the same researchers using the space holder method [220]. This scaffold showed good cell attachment and growth, suitable compression performance and drug release and an excellent antibacterial capacity [220]. In another study, Thanyaphoo reported on Si-nano HAp scaffolds loaded with vancomycin or recombinant human bone morphogenetic proteins (rhBMP-2) which showed potential to be used as a drug delivery system to kill S. aureus [208].
Ceramic porous scaffolds loaded with antibiotics have also been proposed as an alternative approach. Bakhsheshi-Rad et al. prepared biocompatible bredigite-amoxicillin scaffolds with good antibacterial activity against both S. aureus and E. coli bacteria [214]. Doxycycline in a low concentration in a Mg-Ca-TiO 2 composite scaffold showed no cytotoxic behavior against MG63 cells but did have efficient antibacterial activity against Gram-positive and Gram-negative pathogens [218].
Layer-by-layer electrospinning was used to construct chlorhexidinedoped PLGA/PCL (PPC), PLGA/PCL (PP), and β-tricalcium phosphatedoped-PLGA/PCL (PPβ) [216]. The three-layer electrospun membranes showed high strength, good cell adhesion, promoted osteoconductive properties and enhanced antimicrobial properties. Other researchers have engineered scaffolds that combine several strategies. For example, Xie et al. studied the antibacterial activity of AgNPs combined with Hap, CS, and bone morphogenic protein-2 (BMP-2) [209]. Ti bars with BMP/CS/Ag/HAp coatings were implanted into rabbit femurs [209]. In another study, an Ag-loaded strontium hydroxyapatite (SrHAp)/CS scaffold (Ag-SrHAp/CS) was prepared to analyze its biocompatibility, osteoinductivity, and antibacterial activity [211]. The Ag ions released from the scaffold inhibited the growth and attachment of S. aureus. In another study, Mg and Zn ions were combined with the antibiotic tetracycline to prepare a scaffold by the space-holder technique [221]. The results indicated that the engineered Mg-Zn scaffolds containing 1-5% of tetracycline had good potential for bone tissue healing due to their good biocompatibility and antibacterial activity.
The biodegradable polyester PLA, approved by the Food and Drug Administration (FDA) for direct contact with biological fluids, is a promising biodegradable polymer for the fabrication of biocompatible scaffolds [270]. However, it is not antibacterial and needs to be combined with antibacterial agents to provide protection against infections. These combinations can be very simple; for example, mixing, polymeric PLA scaffolds with metals such as silver or carbon nanomaterials, which provide antibacterial activity, osteogenic differentiation and no cytotoxicity to human cells [179,217]. PLA is a relatively hydrophobic polymer that can be combined with hydrophilic polymers such as collagen (COL), minocycline hydrochloride (MH) and citrate-hydroxyapatite nanoparticles (cHApNPs) to reduce bacterial adhesion and biofilm formation [37]. The presence of minocycline hydrochloride also enhances the biological properties of the composite material. These scaffolds can inhibit S. aureus, which is a major pathogen in bone-associated infections because of its ability to adhere and form biofilms on bone and/or implants [271]. The gelatin-siloxane hybrid scaffold with gentamicin sulfate is another example of a scaffold with excellent bioactivity and antibacterial capacity [205]. Li et al. coated the 45S5 Bioglass® scaffold with genipin cross-linked gelatin (GCG) and further incorporated it with poly (p-xylyleneguanidine) hydrochloride (PPXG) to produce a biocompatible scaffold with antibacterial activity against Gram-positive and Gram-negative bacteria [210].
The combination of antimicrobial polymers (particularly CS) and antibiotics is another strategy to treat bone biofilm infection or provide antibacterial activity. Nano HAp/CS/Konjac glucomannan scaffolds [206] and PCL/CS [215] loaded with vancomycin have demonstrated antimicrobial activity. Another strategy is the combination of multiple antibacterial agents to prevent the use of toxic levels. The synergistic effects of silver ions and the antibiotic vancomycin, together with the antimicrobial CS, were assessed in a scaffold prepared from CS/gelatin/Ag and loaded with vancomycin [213]. A CS/carboxymethyl cellulose with Zn and Fe integrated hydroxyapatite (ZFHAp) scaffold has also been proposed [225]. The combination of the antibacterial properties of CS and the release of Zn ions (5% of ZFHAp) resulted in a robust antibacterial activity and good biocompatibility with bone cells.
Some researchers have developed different mesoporous bioactive glass (MBG) scaffolds [207], which are very brittle and lack antibacterial activity. To avoid these disadvantages, a novel modified MBG scaffold was developed with prolonged antibacterial activity and demonstrated biocompatibility with hMSCs [207]. Scaffolds produced by other alternative strategies, such as baghdadite-vancomycin scaffolds reloaded with a drug for 6 h presented antibacterial activity against S. aureus [124]. Hu et al. demonstrated that berberine/Ag nanoparticle embedded biomimetic calcium phosphate scaffolds showed enhanced antibacterial performance [223].
Xyloglucan-co-Methacrylic Acid/Hydroxyapatite/SiO 2 nanocomposite scaffolds showed important properties for bone tissue engineering such as potent antimicrobial activity against several Grampositive and Gram-negative strains, porosity with substantial mechanical strength, biodegradability, biocompatibility and cytocompatible behavior [222].
Recently, Yu et al. developed a novel antibacterial PGA-based scaffold produced by cation exchange of montmorillonite (MMT) with Cu þ2 and the intercalation of cetyltrimethylammonium bromide (CTAB) into the interlayer of MMT [226] that showed superior antibacterial activity.
An antibacterial metal in combination with carbon nanomaterials has been proposed as a new approach. Ag-GO nanohybrids, prepared by AgNPs in situ grown on GO, were introduced into PLA to produce biocompatible and antibacterial scaffolds using the SLS technique [227].
In another study, Zn-doped HAp/rGO nanocomposites were prepared using a mechanochemical process [224]. Zn doping in combination with rGO promoted alkaline phosphatase (ALP) activity and proliferation of MSCs as well as antibacterial performance.
Polymeric nanocomposite scaffolds composed of cellulose and codispersed nanosystem (Fe 3 O 4 /GO) were very recently produced by free radical polymerization and freeze drying [228]. These electroactive scaffolds showed good biocompatibility in a pre-osteoblast (MC3T3-E1) cell line and potent antibacterial activity against Gram-positive S. aureus and Gram-negative E. coli and Pseudomonas aeruginosa. Using another approach combining different strategies, a 3D-printed PCLA scaffold with nano-hydroxyapatite coating doped green tea epigallocatechin-3-gallate promoted bone growth and inhibited multidrug-resistant bacteria colonization [229].

Antibacterial scaffolds for skin regeneration
Soft tissue infections in open fractures, burns or diabetic complications are some of the main causes of high morbidity [272]. Functional antibacterial skin tissue scaffolds are being developed to treat large and deep skin defects (see Table 2). Several strategies are being investigated to treat and promote wound healing, such as the release of antibiotic drugs or antibacterial biometals, the use of polymers, biopolymers or peptides with intrinsic antibacterial properties, the incorporation of nanomaterials with antibacterial properties, such as carbon nanomaterials, or the combination of different strategies. This section describes the different approaches developed in recent years.

Antibacterial scaffolds with antibiotics
Sustained, long-term and localized release of antibiotics loaded into scaffolds during fabrication is another strategy used to provide antibacterial activity for the early eradication of skin infections. PLGA electrospun scaffold containing CPFX delayed drug delivery by 24 h and showed an antibacterial effect toward P. aeruginosa, S. aureus and S. epidermidis [274]. The results indicated that physically adsorbed CPFX provided more antibacterial properties than CPFX blended with PLGA in the first 6 h, indicating that physisorption is a simple approach for a strong short-term antibacterial effect. In another study, Iga et al. developed a fast degradable hybrid porous scaffold modified with CPFX with different PU/PLA rates (Fig. 11). The resulting antibacterial scaffold showed suitable mechanical characteristics, morphology and degradation rate [275], while the antibacterial properties against S. aureus depended on the amount of ciprofloxacin added to the hybrid scaffolds but was not dependent on the PLA content.
Biodegradable scaffolds in the form of non-woven nanofibrillar matrices made of mixtures containing PCL and PLA and loaded with CPFX were obtained by jet-spraying [273]. The antibiotic release was efficient, inhibiting E. coli and B. subtilis growth, while showing good biocompatibility with dermal fibroblasts.
Many approaches to antibiotic delivery from scaffolds produce a burst release, but maintaining long-term inhibitory concentration is still a problem. Akkineni et al. prepared scaffolds based on alginate and methyl cellulose or alginate methylcellulose and Laponite by 3D printing to modulate the antibiotic release kinetics [272].
A biocompatible porcine acellular dermal matrix hydrogel blended with vancomycin has been developed for hemorrhage control, antibacterial action, and tissue repair in infected trauma wounds [276]. A patterned microstructural nanofibrous mats/gentamicin-loaded hydrogel composite scaffold has recently been proposed for skin tissue engineering [277]. The biocompatibility of the scaffold was proven by cytotoxicity and haemolysis studies.

Scaffolds with metals/glass
Scaffolds made of mesoporous bioactive glass nanoparticles modified with Ag (Ag-MBGN) were tested to check their antibacterial activity in vitro and in a 3D skin model for potential use in wound dressing [283] (Fig. 12).
Despite the good antibacterial activity obtained in in vitro tests, Ag-MBGN could not effectively inhibit P. aeruginosa in the 3D model that invaded deeper into the dermis, so that further research is necessary. Nonetheless, this is a promising scaffold for wound dressing due to its cytocompatibility and partial antibacterial capacity [283]. In another study, bioactive self-healing antibacterial injectable hydrogels based on bioactive glasses containing Cu þ2 (Cu-BGs) as antibacterial agent were reported [289]. A double network of poly (ethylene glycol diacrylate) and SA containing Cu-BGs exhibited strong antibacterial activity over a wide range of bacteria. In vitro experiments showed that the self-healing hydrogels stimulated the viability, proliferation, and angiogenic capability of endothelial progenitor cells. In vivo studies demonstrated their efficiency in restoring blood vessels.
Aktürk et al. reported starch-coated silver nanoparticles (S-AgNPs) incorporated into a PVA matrix to fabricate nanofibers crosslinked with glutaraldehyde. These materials demonstrated an antibacterial effect against E. coli and S. aureus due to the release of silver nanoparticles, which were not toxic to HaCat keratinocytes and human epidermal keratinocytes and so were promising for wound dressing applications [284]. Another study with PVA/Starch scaffolds containing AgNPs confirmed the results and indicated good properties such as biodegradability, biocompatibility, and antibacterial activity against P. aeruginosa [285]. Alternative Ag-based scaffolds for tissue engineering with good antibacterial activity against E. coli, S. aureus, P. aeruginosa, and C. albicans have also been developed [279,288]. PCL/gelatin (Ge) nanofibrous scaffolds coated with silver (PCL/Ge/Ag) were non-toxic to cells and demonstrated antibacterial capacity against B. cereus and E. coli [280]. PCL scaffolds containing different ratios of calcium peroxide with or without ascorbic acid exhibited antimicrobial capacity and were not toxic [278]. In another study, a PCL/poly (glycerol sebacate) (PGS) scaffold with calcium peroxide provided sustainable oxygen release for from several days to a week and showed good antibacterial activity [286]. In vivo experiments have demonstrated that PCL nanofibrous mat containing silver sulfadiazine as an antibacterial compound can be considered a powerful wound dressing because of its effects on skin tissue repair and remodeling, plus its antibacterial capacity against both Gram-positive and Gram-negative bacteria [287]. Fibrous PCL scaffolds containing Ag-doped magnetite nanoparticles were reported by Anhmed et al. The roughness and hydrophilicity of the polymeric nanofibers were modified by the Ag-doped nanoparticles, which showed positive results on cell adhesion and growth. Both the viability of human melanocytes and the antibacterial performance (against E. Coli and S. aureus) increased with the concentration of Ag in the magnetite nanoparticles. In vivo results demonstrated that skin wound healing in rats also increases monotonically with the concentration of Ag in the magnetite phase [290].
In a recent study, biocompatible nanobiocomposite scaffolds were engineered based on crosslinked lignin-agarose hydrogel, silk fibroin, and zinc chromite (ZnCr 2 O 4 ) nanoparticles as antibacterial agents [291]. Toxicity was less than 13% with a good antibacterial activity, preventing the formation of P. aeruginosa biofilm. In vivo experiments showed that wounds in mice treated with these nanobiocomposite hydrogels were completely healed in five days.
Effective hemostasis and antibacterial activity are the urgent challenges for deep, narrow, irregular or non-compressible wounds. Ai et al. prepared a 3D printed injectable wound-cooling hemostatic system based on SA/SiO 2 with the addition of Ag nanoparticles [281]. The hydrogel showed good biocompatibility and a robust antibacterial capacity against E. coli, P. aeruginosa, S. aureus and MRSA. The in vivo test on a femoral artery injury model showed a rapid hemostatic response.
In a new approach, P erez-Diaz et al. developed a biomatrix based on radiosterilized pig skin (RPS) as a carrier to deliver MSCs into wound beds. In addition, AgNPs were incorporated into the biomatrix at different concentrations [282]. The nanocomposites showed antibiofilm properties with samples containing 250 and 1000 ppm of AgNPs, although MSCs survived and proliferated on the nanocomposites impregnated with up to 250 ppm of AgNPs.
Even though zinc-based material scaffolds have been studied much less than silver-based ones, these alternative approaches with zinc are very promising for skin tissue engineering applications. For example, gelatin-based and Zn 2þ -incorporated composite hydrogels have been developed for accelerated infected wound healing [292]. This hydrogel exhibited sustainable release behavior of Zn 2þ with good biocompatibility toward NIH-3T3 cells and strong antibacterial abilities against E. coli and S. aureus.

Scaffolds with antibacterial polymers/peptides
Chitosan is an ideal biopolymer for tissue engineering because of its antibacterial properties, biocompatibility, control of inflammatory mediators, ability to aid in faster healing and ability to regulate coagulation A.   [295,297,333,334]. PCL scaffolds with CS coverage demonstrated cell adhesion and viability as well as a slower bacterial growth rate toward S. aureus and S. epidermis [294]. When treating wounds in rat liver, 3D porous sponges with quaternary chitin/partially deacetylated chitin nanofibers as their skeleton (QCNS) outperformed traditional hemostatic agents (gauze, gelatin sponge, and Celox™). QCNS were shown to be an excellent hemostatic dressing for noncompressible wounds because of its excellent cytocompatibility, hemocompatibility, and antibacterial activity [293,296]. Scaffolds that include peptides as antibacterial agents have also been reported for skin tissue engineering and wound healing. Kandhasamy et al. developed silk fibroin electrospun fiber mats containing antibacterial Vitamin K3 carnosine peptides for diabetic wound healing applications [299]. The fiber mats presented good biodegradability, adhesiveness and sustained drug release. Human HFF1 and NIH 3T3 fibroblasts were tested for cell viability and antibacterial performance against S. aureus, E. coli and P. aeruginosa. The mats' diabetic wound curative capacity in vivo was tested in male diabetic Sprague Dawley rats. The results showed that the fiber mats with peptides promoted wound healing in a shorter time than those without peptides. In a similar approach, a cationic antimicrobial peptide (CM11 peptide) was loaded into silk fibroin/gelatin bilayer sponges as wound dressing [300]. The sponges loaded with the peptide showed a controlled release without cytotoxicity on human foreskin fibroblasts (Hu02 cell line) and a significant antibacterial performance against Gram-positive and Gram-negative bacteria.

Scaffolds with carbon nanomaterial
Graphene nanosheets can be used as reinforcement and cellinstructive materials in soft tissue scaffolds [335]. Scaffolds with carbon nanomaterials embedded within the polymeric matrix have also been proposed for skin tissue engineering and wound healing. Nanofibrous PHBV/collagen with GO (0.3% w/w) as an antibacterial agent was prepared for wound coverage [301]. The incorporation of collagen and GO reduced the diameter of the nanofibers and increased porosity. The nanofibers showed enhanced cell proliferation (3 T3-L cell line) and antibacterial capacity against E. coli and S. aureus. In another study, Thangavel et al. prepared a nanocomposite dressing based on isabgol, a natural carbohydrate polymer, and rGO nanoparticles for enhanced vascularization and wound healing using normal and diabetic rats as models [302]. Isabgol/rGO scaffold dressing showed good biocompatibility and antibacterial activity. rGO made the wounds in the Wistar rats shrink and cut down on the time it took for the wounds to heal, which means that this method could speed up the healing of both normal and diabetic wounds.
In another approach, an antibacterial PU-modified GO composite was developed with a skin-like bilayer structure for wound healing applications [303]. The results of tests on a mouse model showed there was a big difference in how quickly the wounds healed. This could be because of the free-bacteria environment and re-epithelization during healing, both brought about by the engineered membrane.
Zmejkoski et al. recently reported a novel hydrogel composite based on bacterial cellulose impregnated with graphene quantum dots (GQDs) for wound healing treatment [304]. The cellulose polymeric matrix was loaded with ca. 12% of GQDs. The hydrogel composites were biocompatible and showed robust antibacterial performance against Gram-positive and Gram-negative bacteria, also good wound fluid absorption and water retention, which indicate their potential in wound healing applications.
Hurtado et al. recently reported a new biodegradable semi- interpenetrated polymer network of PHBV and calcium alginate to provide an alternative strategy to enhance the poor adhesion properties of calcium alginate [305]. These hydrogels were also synthesized with the addition of 10% w/w of graphene nanoplatelets (GNPs), which had no cytotoxic effect on human keratinocytes and provided superior antiviral activity against a surrogate viral model of SARS-CoV-2. However, the antibacterial activity of these hydrophilic materials has so far not been studied.

Scaffolds produce by combined strategies and alternative methods
Several studies explored the combination of antibacterial polymers and antibiotics. Collagen/CS scaffolds loaded with CPFX were prepared by freeze-drying. The scaffolds were highly biocompatible (fibroblast cells) and efficient against E. coli and S. aureus [320]. In a new approach, collagen/CS oxygenating scaffolds loaded with the same antibiotic were prepared with calcium peroxide as a chemical oxygen source [321]. Oxygen-producing biomaterials have been shown to promote wound healing. In this study, both oxygen and drugs showed a sustained release. In vitro cultures indicated that the scaffold had suitable cell adhesion and fibroblast migration and robust antibacterial activity. In vivo studies Fig. 11. Scanning electron microscope (SEM) images of unmodified and Cipro-modified (2 or 5% w/w) HPPS, obtained by using 5 or 10% w/w of PLA and a SEM image of ciprofloxacin used for the scaffolds modification Reprinted with permission under a Creative Commons CC BY 4.0 License from Ref. [275]. Copyright 2020 MDPI. performed on a rat skin flip model showed better wound healing and less necrosis, indicating the promising potential of this strategy. Some antibacterial scaffolds are produced by combining antibacterial polymers with metals. A chitosan scaffold covered with different metals such as selenium or silver demonstrated antibacterial activity against S. aureus, E. coli and MRSA, while Ag-CS scaffolds showed also cytocompatibility towards fibroblasts [308,319].
Other strategies consist of developing scaffolds with natural antimicrobial extracts. For example, Radhika et al. extracted collagen and gelatin from Priacanthus humrur skin [41]. They created an antibacterial collagen/gelatin/CS porous novel scaffold by freeze drying and subsequent crosslinking of polymers. In another study, CS matrices were loaded with grape seed extract or royal jelly to produce scaffolds with antimicrobial, anti-inflammatory and wound healing capabilities [309] cytocompatible with human lung fibroblast. It should be noted that honey in hydrogels and cryogels also reduces bacterial adhesion [307,315]. Biocompatible PU and PLA polymer scaffolds are commonly proposed for skin tissue engineering [336]. Other polymer scaffolds such as PU, PLA, PLGA, PEG-DA or PHA have been investigated with natural extracts for tissue engineering, particularly for skin, oral tissue, and cardiac regeneration [289,306,337]. The incorporation of cellulose acetate (CA) within the PLA matrix enhanced the physicochemical properties of the scaffolds. The scaffold exhibited promising results in in vivo wound healing assays and antibacterial activity against both S. aureus and E. coli [316]. Çakir et al. demonstrated that silk fibroin sponges with heparin and silver sulphadiazine can mimic the physical structure of natural skin tissue. These scaffolds showed an antibacterial effect against Gram-positive bacteria [311]. Silk fibroin/CS scaffolds exhibited excellent biocompatibility and antibacterial capacity against S. aureus and E. coli [318].
Antibacterial scaffolds made of quaternary ammonium organosilane (QOS) collagen nanofibers increased the flexibility of rigid collagen nanofibers and had good properties like thermal stability, antibacterial activity and stimulated cellular growth and proliferation, and are therefore very promising for effective use as an interactive wound dressing material [312]. PLA modified with l-ascorbic acid or fumaric acid via a plasma treatment method changed the surface morphology and topography, so that the scaffold showed antibacterial capacity against Gram-positive and Gram-negative bacteria [317]. In another combined strategy, PHA with graphene-decorated silver nanoparticles have shown antibacterial properties against Gram-positive and Gram-negative bacteria [310]. The antibacterial activity lasts for up to 24 h of incubation, which is a factor to consider for effective wound dressings. Graphene-silver nanocomposites (rGO-Ag) with an antimicrobial peptide (polylysine) have been reported. The peptide functionalization of the rGO-Ag nanocomposites increased the antibacterial efficacy against S. aureus biofilm and reduced the dissolution of Ag ions and in vitro toxicity. The ex vivo rat disinfection model was shown to be capable of eliminating biofilm formation and disruption [322].
Graphene foams have also been proposed as carriers of metal ions against antibiotic-resistant bacteria, with a special potential for wound dressing applications [323]. Another strategy consists of CS/PVA/GO based pH-responsive composite hydrogels crosslinked with TEOS and produced by solution casting for wound healing [326], showing antibacterial activity against Gram-positive and Gram-negative pathogens and good biocompatibility. Biopolymer-based composite hydrogels with antibacterial and antitumor properties were made from sodium alginate (SA) and GO covalently linked and crosslinked with tetraethyl orthosilicate (TEOS) by the solvothermal method [331].
Antibacterial, degradable and pH-responsive CS/guar gum/PVA blended hydrogels with different crosslinking amounts of TEOS for wound dressing [325]. The antimicrobial study revealed that these composites are highly antibacterial against Gram-positive (S. aureus and Bacillus cereus) and Gram-negative (P. aeruginosa and E. coli) bacterial strains. ARX-based nanocomposite hydrogels functionalized into carboxymethylarabinoxylan (CMARX) with different amounts of TEOS loaded with the chemotherapeutic agent Fluorouracil (5FU) onto rGO showed antibacterial activity against S. aureus and P. aeruginosa and anticancer activity against Uppsala 87 Malignant Glioma (U-87) cells [324]. ARX, CG, and rGO composites cross-linked with the optimal amount of TEOS were shown to be hemocompatible, pH-responsive and broad spectrum antibacterial, thus very promising for sustained drug release for skin wound care and treatment [328]. ARX, CS and rGO sheets were combined and crosslinked using TEOS as a crosslinker to produce smart pH-sensitive biocompatible composite hydrogels with broad-spectrum antibacterial activity for wound healing [327]. Multifunctional hydrogels based on ARX-functionalized-GO and bacterial cellulose-functionalized-GO using the hydrothermal method through cross-linking GO-arabinoxylan and PVA with TEOS recently prevented infections (E. coli, S. aureus, and P. aeruginosa) and were thus shown to be promising for wound healing [329,330]. Advanced hydrogels based on arabinoxylan-functionalized-GO promoted wound healing in vitro and in vivo [329]. The advanced hydrogels based on bacterial cellulose-functionalized-GO showed potential anticancer activity against U87 cells and could be used for the controlled release of curcumin [330].
A simple and safe PDA-based photothermal platform has recently been developed for photothermal antibacterial therapy in wound healing [332]. This biocompatible platform composed of polyethyleneimine, pectin and polydopamine@Cu nanoparticles showed a highly efficient bacteria-killing ability.

Antibacterial scaffolds for oral regeneration
Maxillofacial defect restoration is a great challenge due to the complicated pre-existing anatomy of the skull [338], for which new strategies for oral tissue regeneration using antimicrobial scaffolds have been developed (See Table 3).
Scaffolds with antibiotics have been proposed for oral tissue regeneration using a combination of metronidazole and CPFX [340]. Their antimicrobial activity has been confirmed against Enterococcus faecalis, Porphyromonas gingivalis, and Fusobacterium nucleatum. MET/CPFX scaffolds enhanced the viability and proliferation of dental pulp stem cells [339,341]. Albuquerque et al. reported an electrospun antibacterial scaffold prepared with polydioxanone (PDS) nanofibers loaded with TAP (CPFX, metronidazole, and minocycline) against P. gingivalis-infected dentin biofilm. The results indicated the potential of these nanofibrous scaffolds for intracanal disinfection before regenerative endodontics [342]. A novel antibacterial HAp scaffold against S. mutans has been developed by immobilizing chlorhexidine (CHX)-loaded human serum albumin (HSA) nanoparticles on its surface via surface charge interaction [343].
A biopolymer made of a polyelectrolyte complex (PEC) composed of CS, γ-polyglutamic acid (γ-PGA) and carboxy-methyl-cellulose (CMC) was developed to fabricate dental scaffolds [344]. These PEC scaffolds showed biocompatibility and antibacterial activity against E. coli and S. aureus. In another study, Li et al. reported the antibacterial activity of a non-cross-linked CS scaffold against typical oral pathogens such as Porphyromonas gingivalis and Streptococcus mutans [40]. These scaffolds had good physical and biological properties such as biodegradability, physical stability and biocompatibility. Polyvinyl-siloxane (PVS) is a suitable material to prepare dental scaffolds because it is biocompatible, it can be modeled and can be produced with higher resistance to colonization to prevent bacterial infections by adding BaTiO 3 to it [346]. The engineered scaffolds based on this approach increased the beneficial antibacterial capacity against S. epidermis by 25%.
Other researchers have developed scaffolds using metal ions, particularly silver, for example, Ag 3 PO 4 -lidocaine-loaded-PCL scaffolds using pneumatic extrusion-based 3D printing were developed by Shao et al. [345]. The scaffolds demonstrated both antibacterial and analgesic activity in addition to cytocompatibility, which depended on the lidocaine and Ag concentrations. Following this strategy, 3D hybrid scaffolds consisting of extracellular matrix components, collagen, chondroitin 4-sulfate, and fibronectin, functionalized with AgNPs were prepared to improve periodontitis treatments [347].
Dental scaffolds with drug delivery capability, such as hematitedoped bioglass/CS scaffolds with CHX were investigated for the repair of infected root canals. They were also found to have osteoinduction capacity [348] and bacterial growth of E. faecalis was eliminated after 14 days. In a different strategy, epigallocatechin gallate (EGCG) was used as an antibacterial cross-linking agent in hydrogel collagen scaffolds to promote proliferation and differentiation of human dental pulp cells (hDPCs) while impeding bacterial infections [350].
Novel approaches based on carbon nanomaterials have also been reported. Wu et al. proposed a new antibacterial scaffold with GO and calcium phosphate incorporated in a CS hydrogel [351]. The antimicrobial scaffold proved to be effective in preventing E. faecalis biofilm and also had good biocompatibility to support human dental stem cell attachment.
Novel carrageenan-based injectable hydrogel scaffolds containing Cissus quadrangularis extract have shown biocompatibility and antioxidant activity for facilitating dentin-pulp complex regeneration [352].

Antibacterial scaffolds for muscle, nerve, trachea, cardiac and other tissue engineering applications
A variety of scaffolds (sometimes combined with stem cells) have been developed and optimized for muscle, nerve, trachea, cardiac and other tissue engineering applications [353][354][355][356]. However, introducing foreign bodies into the human body increases the risk of bacterial infection. Despite disinfection procedures, there is a risk of contamination by pathogens that can cause infections during surgical interventions. Bacterial infections can appear long after surgery and can be responsible for implant failure and distress to patients, reducing their quality of life [357].
Research on antimicrobial scaffolds is focused mainly on bone, skin, and oral tissue applications. However, different approaches to developing antimicrobial scaffolds for other tissues such as muscle, nerve, cardiac or trachea have been proposed. This section reports on general strategies with antimicrobial scaffolds or other strategies for other types of tissue not included in the previous sections (Table 4).
Different strategies have been reported based on scaffolds loaded with antibiotics for general tissue engineering applications. Chen et al. developed polylactic acid/poly (ethylene glycol) (PLA/PEG) scaffolds to deliver multiple biomolecules (including growth factors) and drugs for wound dressing, periodontal membranes, or more complicated tissues in which growth factors and anti-infection precautions are critical. The scaffolds were assessed by loading the model drug TCH. These scaffolds reduced the activity of S. aureus [358]. Visscher et al. reported dual macro/micro porous scaffolds prepared by combining 3D printing with the traditional salt-leaching technique [359]. This antibacterial platform was evaluated for the local release of the antibiotic Cefazolin, loaded via a solution drop-loading technique, had no cytotoxic effects on 3 T3 fibroblasts and did not cause in vitro blood clots. In another recent approach, composites consisting of recombinant spider silk proteins and mesoporous silica nanoparticles loaded with specific antibiotics and antimycotics showed antimicrobial activity over 15 days. 2D films and scaffolds, prepared by 3D printing, exhibited good biocompatibility, promoting cell adhesion and proliferation [360].
Scaffolds with antibacterial metals have also been proposed as a general approach for tissue engineering [115,[361][362][363][364] or for specific applications, such as muscle [353,365]. A CuO nanoparticle decorated biobased hyperbranched epoxy/CuO-nanofibrillar cellulose nanocomposite scaffold was prepared to acquire efficient antimicrobial activity for smooth muscle cell regeneration [353]. In situ injectable hydrogel has the advantage of being able to match the shape of the damaged tissue and reduce patients' distress with a minimally invasive method [379]. Ge et al. engineered a conductive, antioxidative, and antibacterial hydrogel with oriented channels to enhance skeletal-muscle regeneration [365]. Biometal gold@dopanime nanoparticles were incorporated as an antibacterial agent. In vitro experiments in C2C12 murine myoblasts showed that these advanced materials could promote myotube formation. In vivo assessment, performed on a rat tibialis anterior muscle defect model, showed that these scaffolds facilitated skeletal muscle regeneration. Scaffolds with antibacterial polymers have been proposed as a general strategy for tissue engineering [118,366,367,[369][370][371][372] or for specific applications [354,368]. Zhao et al. proposed an in situ forming antibacterial conductive degradable hydrogel employing quaternized chitosan (QCS) and grafted polyaniline (PANI) with oxidized dextran as a crosslinker for electrical signal-sensitive tissues, such as muscle, cardiovascular, and nerve [354] (Fig. 13).
The introduction of PANI into the QCS copolymer significantly reduced its cytotoxicity, greatly stimulated the proliferation of C2C12 cells and improved its antibacterial activity, especially the QCS40P3- Odex hydrogel with a killing percentage of up to 95% [354]. Injectable hydrogels have also been studied for cell delivery therapy in myocardial [368] and bladder regeneration [5]. A self-healable conductive injectable hydrogel made of chitosan-graft-aniline tetramer (CS-AT) and dibenzaldehyde terminated poly (ethylene glycol) (PEG-DA) as cell delivery platform showed very promising results for myocardial infarction [368]. The resulting hydrogel not only does not cause cytotoxicity but also shows antibacterial activity against E. coli and S. aureus and is also capable of producing good targeted cell release in vivo [368]. A conductive peptide-PANI composite hydrogel with antimicrobial activity that can bind to deoxyribonucleic acid (DNA) was recently reported [373]. The hydrogel supported the organization of cardiomyocytes into a spontaneously contracting system and demonstrated antibacterial activity against E. coli and S. epidermidis.
In another approach, Ghannadian et al. compared different fabrication techniques to prepare PCL scaffolds by electrospinning, rotary jet spinning, and airbrushing (AB) for the treatment of musculoskeletal defects without infections. The products of AB significantly reduced bacterial surface colonization of Gram-positive and Gram-negative bacteria [370].
Graphene-based nanomaterials are promising compounds for cardiac tissue engineering due to their excellent electrical and mechanical properties [380]. Collagen patches charged with different concentrations of rGO have been developed to achieve good long-term cardiac regeneration [374] (Fig. 14).
The results showed that the rGO coating significantly improved the mechanical properties, electroactivity of the collagen scaffolds and the viability of human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. The antibacterial properties of the Collagen-rGO scaffolds against Escherichia coli, S. aureus, and Streptococcus pyogenes were confirmed by field emission scanning electron microscopy [374].
These results indicate that the rGO coating has promising properties for collagen scaffolds that provide a desirable microenvironment for the regeneration of vascular tissue. In another approach, a biocompatible tissue-engineered trachea was developed with electrospun patterned PLA/GO and IL fibrous membranes with synergistic antibacterial properties [355].
Scaffolds produced by combining strategies and alternative methods have also been reported for several tissue engineering applications [114,116,117,376,377]. Zuo et al. prepared a stable 3-dimensional printed polylactic/cellulose acetate scaffold with the antimicrobial agent 1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC) for biomedical applications and food packaging [376]. Essential oils of aromatic foliage have also been proposed both as bioactive and biocide agents. Amma et al. incorporated Nigella sativa essential oil into a biogenic scaffold [377]. The quinine constituent of N. sativa has been reported to stop microbial growth. The scaffold, prepared by grafting HAp and the essential oil, enhanced myoblast differentiation and antibacterial activity against S. aureus.
The polydopamine (pDA) coating method was used to construct a multilayer ε-polylysine and fibrin (FIB) modification on the surface of the 3D HDPE scaffold via the LBL self-assembly approach. The LBL strategy enhanced bioactive and antibacterial properties against Gram-positive S. aureus and Gram-negative E. coli.

Antifungal scaffolds
As the incidence of infections caused by invasive fungal pathogens Fig. 13. Hydrogel morphologies after swelling in phosphate buffered saline.
Scale bar was 500 μm. Reprinted with permission from Ref. [354]. Copyright 2015 Elsevier. has increased dramatically in the last twenty years, the fabrication of new antifungal scaffolds with antimicrobial properties is becoming more important [381,382]. Table 5 shows the antifungal scaffolds developed so far for tissue engineering applications. Incorporating bioactive materials into biodegradable polymers can provide drug-releasing bioactive scaffolds for potential use in novel controlled drug delivery, wound dressing, tissue engineering, stem cell regeneration and differentiation [400]. In vitro efficacy and toxicity of three classes of US Food and Drug Administration-approved antifungal-loaded fiber mats produced by electrospinning have been compared [401]. New chitosan-based mucoadhesive nanofiber mats were simultaneously loaded with VAN and Amphotericin B (AMB) as antibacterial and antifungal agents for the treatment of oral aphthous ulceration [384]. Films and scaffolds based on recombinant spider silk proteins with silica NPs (see antibacterial scaffolds in Section 3.4) were also loaded with the antibiotic and antimycotic AMB (in addition to specific antibiotics) to assess their antimycotic potential [360]. The derived composite materials showed good antimicrobial properties against the E. coli bacteria and P. pastoris yeast. AMB was also incorporated into PHA matrices combined with the antifungal agent nystatin at different concentrations [385]. The formulations, tested against different pathogenic fungi (C. albicans species and C. parapsilosis) as well as filamentous fungi, demonstrated a robust antifungal effect. The antifungal PHA composite inhibited the formation of C. albicant biofilm, although it was not efficient in the eradication of mature biofilms. Very recently, silk sericin/PVA hydrogel loaded with azithromycin was synthesized using a freeze/thaw process [386]. The hydrogel exhibited antimicrobial activity against S. aureus, P. aeruginosa, E. coli, and C. albicans and excellent cytocompatibility. accelerating the healing of infected burns while reducing systemic burn effects.
Metals (particularly Ag) and antimicrobial oxides have also been studied as antifungal agents in recent years. Hipler et al. prepared several textile commercial fibers (Sea-Cell fibers) from brown, red, green and blue algae loaded with Ag ions for potential application as antifungal and antibacterial textiles for skin conditions [387]. The fibers demonstrated antifungal activity against three Candida species and antibacterial activity against S. aureus and E. coli in a dose-dependent manner. A potential treatment for superficial candidiasis proposed the use of nanocomposites based on AgNPs in combination with CS. Inhibitory effects against several Candida species were found for concentrations Fig. 15. Preparation scheme of the multifunctional auricle scaffold by 3D printing and subsequent activation by polydopamine (pDA) and coated layer-by-layer with EPL and FIB. The pDA-EFE auricle scaffold obtained showed bioactive, antibacterial, angiogenesi enhancing, and tissue ingrowth-promoting properties. Reprinted with permission from Ref. [378]. Copyright 2022 Elsevier. between 0.06 and 1 μg/mL. When the antifungal fluconazole and the antibiotic and antifungal AMB were combined with the AgNPs, the composite showed an additive antifungal effect. The AgNPs/CS composites exhibited low cytotoxicity in mammalian cells [387].
Oxide metals are widely used compounds in scaffolds due to their broad-spectrum antimicrobial capacity [402]. PU membranes modified by nano-ZnO have exhibited important antifungal activity and can be successfully employed in biomedicine [388]. In another study, Trcin et al. prepared antimicrobial PCL/titanium dioxide (TiO 2 ) and PCL/cefuroxime scaffolds by electrospinning. The scaffolds supported cell growth and differentiation of limbal stem cells and provided antimicrobial activity, particularly against the bacteria P. aeruginosa, S. aureus and the fungi P. albicans. These fiber mats would be suitable for the management of superficial fungal infections of the cornea and corneal tissue regeneration [392]. Borophosphate glasses doped individually with a few antimicrobial oxides such as CeO 2 , ZnO and CuO were prepared by the melt quenching technique [391]. The results revealed antimicrobial activity against some fungi and bacteria, that the addition of the antimicrobial oxides had a positive effect on the glass bioactivity and could play a part in biomedical applications [391]. In another study, the deposition of Ag/HAp thin films on Ti modified with TiO 2 nanotubes substrates, followed by a heat treatment at 500 C in water vapor for 6 h, produced efficient antifungal shield barriers for treating bone defects [389]. Yazdimamaghani et al. developed hybrid scaffolds consisting of gelatin and AgNPs produced by a green method and investigated their antimicrobial properties against Gram-positive S. aureus, Gram-negative E. coli and C. albicans [390]. The scaffolds interfered with the virulence factor of the C. albicans for invasion into the tissue and prevented hyphae-formation. The in vitro hMSC cell culture study on the samples revealed appropriate cytocompatibility [390].
Polymers with antimicrobial behavior in combination with antimicrobial agents have also been reported. Dermirci et at. developed hydrogels from Halomonas levan polysaccharide that possess antimicrobial activity and are loaded with AMB as antifungal agent with different crosslinking densities. The hydrogels showed good cytocompatibility with mouse fibroblasts in addition to exhibiting high antifungal activity against C. albicans due to the release of AMB [383].
Another strategy consists of producing scaffolds with antimicrobial peptides as an alternative approach for fungal control [403]. Antifungal Cm-p1 (Cencritchis muricatus peptide 1) was electrospun into a nanofiber scaffold for drug delivery to reduce the growth of C. albicans [394].
Other strategies have been developed based on antifungal agents not included in the previous categories or employing combined strategies. Thus, new PCL-based fibers useful for wound dressing were produced by the melt co-extrusion process with the clotrimazole antifungal and showed high antifungal capacity for 3 weeks, which was more than the same scaffolds manufactured by electrospinning [395]. Silk fibroin/-PU/polyvinylpyrrolidone (SF/PU/PVP) nanofibers were prepared by electrospinning with the incorporation of the antifungal drug sertaconazole nitrate. The resulting nanofiber material exhibited fungicide activity against C. albicans from both silk fibroin (SF) incorporated into the PU/PVP nanofibers and PU/PVP nanofibers coated with SF as well as good biocompatibility. SF as an efficient polymer to sustain or control the release of antifungal agents can be considered a potential topical drug delivery system for the treatment of fungal infections as a topically applied scaffold [397]. Essential oils have also been proposed as antimicrobial agents. Mishra et. Al proposed a gelan/PVA nanofiber mat loaded with eucalyptol/β-cyclodextrin with antifungal capacity against C. glabrata and C. albicans that inhibited biofilm formation by 70% [398]. Finally, it is worth noting the development of combined strategies based on antifungal drugs and carbon nanomaterials. Misra et al. proposed a new approach combining the antifungal drug Tolnaftate (Tf) and graphene nanoplatelets, with polymethacrylate as a polymeric matrix, to prepare nanofibrous scaffolds for the treatment of topical infections [396]. They found superior antifungal activity of the Tf-graphene-loaded nanofibers as compared to Tf-nanofibers without graphene, demonstrating the efficacy of this strategy. 10-undecenoic acid based polyurethane/PCL fibers have recently been proposed as wound dressing materials to treat fungal diseases [399]. These scaffolds showed antifungal activity against C. albicans and C. tropicalis.

Antibiofilm scaffolds
Biofilms are the principal source of persistent infection and can become a serious problem in medical devices [404] because they protect microorganisms against drugs [405]. New antibiofilm scaffolds based on different strategies have been developed to solve this problem.
A novel three-dimensional scaffold containing multiple antibiotics (rifampin, levofloxacin, and vancomycin) has been designed to treat bone infections by rapid prototyping of a mixture of nanocomposite bioceramic and PVA with a coating of gelatin glutaraldehyde [123]. These antibacterial 3D scaffolds rapidly release rifampin, followed by the sustained and prolonged release of vancomycin and levofloxacin to destroy bacterial biofilms and inhibit bacteria growth in very short periods.
Antimicrobial metal Ag has also been reported as a tool to prevent or destroy microbial biofilms. PVA-Ag and CS-Ag nanocomposites obtained from AgNPs mixed with PVA or CS showed higher thermal stability than pure PVA and CS and enhanced AgNP antimicrobial and antibiofilm activities, which resulted in the efficient eradication of bacterial and biofilm growth of multi-drug resistant clinical isolates [364]. The presence of antimicrobial Ag showed significantly low cytotoxicity against liver cells. In another study, impregnated silver nanoparticles on radiosterilized pig skin nanocomposites inhibited bacterial growth and prevented biofilm while allowing non-cytotoxicity in mesenchymal stem cell culture at low AgNPs concentrations [282]. MRSA is considered a common colonizer of burn wounds and accounts for high morbidity and mortality rates all over the world [406]. Two formulations containing moxifloxacin for topical delivery were prepared and confirmed their efficacy in an MRSA-infected burn wound in BALB/c mice [407]. In vivo studies showed that the two gels have the same efficacy in eradicating bacteria from the wound site when treatment was started during the early stages of infection. On the other hand, in a delayed treatment, a new gel was more efficient than a traditional gel in burn wounds infected with S. aureus, both planktonic and biofilm [407]. Colonization of the lungs by biofilm-forming pathogenic microorganisms is a major cause of mortality in cystic fibrosis (CF) due to the difficulty of dealing with the biofilm exopolysaccharide matrix produced by the pathogens and the viscous mucus [408]. The use of alginate in combination with NO has shown improved activity compared to common antibiotics for chronic CF infections [372].
Another strategy to fight bacteria and achieve osteo proliferation is to use palladium nanoparticles (PdNPs) that are anchored to polypyrrolefunctionalized rGO nanocomposite (Pd/βy/rGO NC). These PdNPs were able to prevent the bacterial biofilm formation caused by common human pathogens such as P. aeruginosa, K. pneumoniae, B. subtilis and E. coli [375]. Proper wound healing is often affected by bacterial infection. Murugesan et al. prepared a nanocomposite that combined another carbon nanomaterial, multi-walled carbon nanotubes (MWCNT) modified with a polypyrrole (PPy) matrix with the incorporation of PdNPs [371]. This MWCNT/PPy/Pd hybrid composite prevented the formation of biofilms in B. subtilis, P. aeruginosa, E. coli and K. pneumoniae bacteria and showed a significant dose-dependent toxicity in Vero and HeLa cells.
Adeli-Sardou et al. reported that lawsone incorporated into PCL/ gelatin nanofibers via electrospinning exhibited potential antibacterial and antibiofilm activity [313]. All lawsone-containing scaffolds showed antibacterial activity against S. aureus and MRSA and PCL/Ge/lawsone 10% prevented the growth of P. mirabilis. In conclusion, significant anti-biofilm activity was observed in all biofilm strains [313].
Bioactive glasses (BGs) have been proposed as promising materials for the reconstruction of periodontal and peri-implant bone defects due to their favorable structural and antimicrobial properties [409]. Porous novel complex drug carrier porous nano-HAp/CS/konjac glucomannan scaffolds were combined with liposomes containing vancomycin to provide sustained release and impede biofilm formation [206].

Antimicrobial characterization of scaffold materials
The agar disk diffusion test is one of the most useful antimicrobial tests in the field of biomaterials, which is based on incubating the bacteria on a plate in the presence of a disk of the scaffold material [410]. This test is recommended for a broad range of microorganisms such as Gram-positive bacteria (e.g., S. aureus), Gram-negative bacteria (e.g., E. coli) and yeast (e.g. Candida albicans). If the material shows antimicrobial activity, an inhibition halo forms around it where the bacteria will not be able to grow. The antimicrobial activity of the scaffolds can be analyze by measuring the diameter of the halo and comparing it with the positive and the negative control [37,410] (Fig. 16).
The normalized halo must be determined by applying Equation (1) to compare the antimicrobial activity of several scaffold materials [410].
The antimicrobial action of the materials tested for the growth of microorganisms is expressed by the normalized width of the antimicrobial "halo" (nw halo ), determined by the inhibition zone diameter (d iz ) and scaffold disk diameter (d). The diameters can be measured by image analysis software (e.g. the recently developed Image J or Antibiogramj free open source software [411]) from a photograph of the microbial culture. To ensure reproducibility, each antimicrobial test is carried out at least three times in quadruplicate on different days. This test is similar to the antibiogram test [412], in which disks impregnated with different antibiotics or the same antibiotic in different concentrations are used to test their antibacterial capacity. Another method commonly used to characterize scaffold antimicrobial properties is based on the ISO standard 22,196:2007 to measure the antimicrobial activity on material surfaces (contact method). In this method, the microorganisms are placed directly on the scaffold and their growth inhibition can be determined by the colony counting procedure after a certain amount of contact time [410].
However, as bacteria and fungi can resist antimicrobials by forming biofilms [413], the study of biofilm formation on scaffolds is an important issue in tissue engineering. The capacity of a scaffold to impede biofilm formation can be studied by putting it in contact with a bacterial culture on well culture plates [414] or in bioreactors [415] (Fig. 17).
The presence of bacterial biofilm can also be analyzed by SEM [37,414], confocal microscopy [417,418] and/or atomic force microscopy [419,420].
The antimicrobial capacity of a material can also be measured by putting it in contact with a bacterial suspension in its culture medium and measuring optical density at different times [209]. Rising absorbance indicates increased bacterial activity.

Antimicrobial mechanisms
Microbial infections on an implanted scaffold surface can eventually lead to biofilm formation and thus impede the use of the scaffolds in humans. Despite an effective host immune system, the scaffold surface can be rapidly occupied by microorganisms, resulting in persistent infection, implanted scaffold failure and can even cause the patient's death [421]. These problems are difficult to solve because microorganisms such as bacteria and fungi possess complex mechanisms to adhere to scaffolds that vary according to the microbial strain. Several antimicrobial scaffolds have been developed by incorporating antibiotics in the scaffold material matrix. However, as bacterial resistance is increasing at an alarming rate [13], this strategy will probably not provide long-lasting solutions to tissue engineering. In this regard, other antimicrobial strategies consisted of scaffolds capable of releasing other types of antimicrobial agents such as antiseptics, antimicrobial polymers, peptides, metals, carbon nanomaterials and combinatorial strategies. Another strategy consists of developing scaffolds made of smart materials, i.e. stimuli-responsive biomaterials, such as toxin-triggered, pH-responsive or dual stimulus-responsive adaptive antimicrobial materials [422][423][424]. Biofilm can also be combated by modifying the scaffold surface by diverse strategies to produce an antifouling (superhydrophobic, non-charged or highly hydrated) surface that prevents the bacteria adhering to the implant or a bactericidal surface that kills the bacteria in contact with the implants [425,426]. The mechanisms of bacterial adhesion, biofilm formation and the released substances are discussed in detail in Ref. [421].
The exact antimicrobial mechanism of nanoparticles and nanomaterials is not yet clearly understood, but it may be attributed to the production of reactive oxygen species (ROS) that damage the cell membrane [425]. It is well-known that when the production of excessive ROS exceeds the bacteria's scavenging ability it will cause fatal damage to the microorganism [229]. 2D MoS 2 exhibits broad antibacterial activity associated with the production of ROS in polyhydroxyalkanoate/chitosan (PHA/CS) and 2D molybdenum disulfide-doped (2D MoS 2 ) scaffolds [114]. Scaffolds with TiO 2 also possess effective antibacterial activity because TiO 2 damages the bacteria by generating ROS and destroying their structure and functions [180]. The bactericidal effect of metals such as silver or gold is also attributed to their ROS-scavenging properties [427]. 3D-printed biocompatible scaffolds based on calcium-deficient hydroxyapatite (CDHA) with gold nanoparticles were able to produceROS effective against Micrococcus luteus ( Fig. 18 (a)) [204].
The potential antibacterial mechanism of Zn-embedded biomaterials is also mainly related to the production of ROS [428]. For example, the antibacterial mechanism of a gelatin-based and Zn 2þ -incorporated composite hydrogel (Gel@Zn) for rapid infected wound healing consisted of reducing the ATP level, generating ROS and leakage of protein [292] (Fig. 18(b)). PLGA/Cu(I)@ZIF-8 scaffolds produced by combining antibacterial copper-loaded-zeolitic-imidazolate-frameworks (ZIF-8) and  PLGA [194] generate ROS in the presence of H 2 O 2 , which contributes to their superior antibacterial activity in vitro and in vivo. PGA-based scaffolds produced by cation exchange of MMT with Cu þ2 and the introduction of CTAB into the MMT interlayer showed strong antibacterial activity due to the high level of ROS release [226].
The study of the antibacterial mechanism of a PCLA scaffold with nano-hydroxyapatite coating doped green tea epigallocatechin-3-gallate against MRSA showed that the wall and membrane structure of the bacteria were seriously damaged [229] so that the intracellular components such as nucleic acid and proteins flowed out of the cell. It was also determined that the scaffold promoted the production of ROS in MRSA cells, which attack important macromolecules in bacterial cells (e.g., nucleic acid, proteins and lipids) and eventually cause cell death.
The antibacterial activity of xyloglucan-co-methacrylic acid/hydroxyapatite/SiO 2 nanocomposite scaffolds was shown to be related to the penetration of silica and hydroxyapatite nano-particles into bacteria to interact with the cellular protein [222].
The intrinsic antimicrobial activity of dopamine is due to its ability to effectively eradicate bacteria, microbes, plankton, or biofilm and it has been used to produce antimicrobial scaffolds [429,430]. Small molecules of catechol and proteins of amine in the presence of alkaline condition (aqueous) and oxygen allow dopamine to undergo polymerization to form a thin adherent PDA film, which prevents the attachment and growth of bacteria by taking over the bacteria's nutrient supply [431]. In most cases, this antibacterial activity has been attributed to the benzene ring of the dopamine molecule and the formation of local toxic effects by active groups formed on the outer membrane of the bacteria cell, which affect the permeability of the cell membrane by obstructing the components required for the bacteria to survive.
The antimicrobial mode of action of quaternary ammonium compounds against pathogens is attributed to positively charged nitrogen atoms [18], which eradicate microorganisms by damaging their membranes. The antimicrobial mechanism of antimicrobial polymers such as chitosan is also attributed to their positively-charged linear structure [432].
Carbon nanomaterials prevent the formation of microbial biofilms mainly through preventing microbial adhesion by reducing surface free energy and increasing hydrophobicity and killing them mostly by oxidative stress and photothermal/photodynamic effects [433]. The release of AgNPs from the Ag-GO nanohybrids introduced into PLA promoted the generation of bacteria-inhibiting ROS [227].
Nonetheless, there are still many questions to answer regarding the antimicrobial mechanism of action of many of the scaffolds included in this review and further research is required to obtain a complete understanding of all these antimicrobial processes.

Toxicological aspects
Scaffolds with antibacterial and/or antifungal properties must not be toxic to human beings and should be subjected to toxicological assays to ensure their safe use in tissue engineering. The different approaches to developing antimicrobial scaffolds use antibacterial and antifungal agents that can be toxic at certain concentrations, so that it is crucial to find a balance between the scaffolds' biocidal properties and cell biocompatibility. For example, the cell viability of PCL-AgNPs prepared by electrospinning revealed that cytotoxicity was highly dependent on the concentration of AgNPs [115].
Biocompatibility was considered and tested by in vitro or in vivo tests in most of the studies in this review. The cytotoxicity of antibacterial bone regeneration scaffolds was assessed using different cell lines such as osteoblasts, preosteoblasts, MSCs, osteosarcoma cells and fibroblasts (Table 1). In scaffolds that contain antibiotics, the biocompatibility of those loaded with levofloxacin hydrochloride was studied in a fibroblast cell line [127]. Toxicity assays performed with other cell lines like MSCs have been carried out on scaffolds loaded with CPFX [122], vancomycin [125,126,130], and minocycline [37]. MC3T3-E1 preosteoblasts were used to analyze bioceramic-PVA scaffolds loaded with several antibiotics (rifampin, levofloxacin and vancomycin) [123] and scaffolds containing VEGF and cephalexin [128]. MG-63 osteoblast cells were also used as cell lines in scaffolds containing vancomycin. Cytotoxicity assays with scaffolds loaded with strychnine were performed on the human osteoblast cell line hFOB1.19 [129]. However, the toxicological aspects of scaffolds developed with gentamicin [121] and TCH [235] have not yet been studied.
The biocompatibility of scaffolds prepared with antibacterial polymers or those that include peptides as antibacterial agent has been evaluated in MC3T3-E1murine preosteoblast [135,[137][138][139][140] and ET3 mouse fibroblasts [136]. Human osteoblasts [132] and MSCs [133,141] from rabbits have also been used in some studies. In the study performed by Li et al. in which HACC-grafted PLGA/HAp scaffolds were prepared by FDM 3D printing, cytotoxicity and in vivo performance were assessed by rabbit MSCs and New Zealand white rabbits, respectively [63]. Cytotoxicity assays on scaffolds that incorporate carbon nanomaterials have been performed using MC3T3-E1 preosteoblast cells [148,149], human osteoblasts [147], human MSCs [145], osteosarcoma cells [144] and fibroblasts from human [146] and murine [143] origin. In addition, antibacterial scaffolds with antibacterial metals have been assessed in several cell lines to prove their biocompatibility. In several studies, murine preosteoblasts MC3T3-E1 [162,165,179,181,187,188,190,192,195,203], MSCs [157,159,160,172,176,177,189,191,194], osteoblastic cell line [155,178,182,202], osteoblast-like cells [156,158,161,166,167,173,180,183,198], fibroblasts [184,196], and breast cancer cells [186] were used as models. In vitro bioactivity in an acellular simulated body fluid (SBF) was examined in some studies [154,169,170] and also metal ion penetration with bovine bone specimens [169]. Other studies also reported in vivo assessment of biocompatibility in rat [194] or rabbit [168,203] models. Finally, in scaffolds developed with combined or alternative strategies, biocompatibility was analyzed in MC3T3-E1 preosteoblasts [216,223], osteoblasts [208,212,221] and osteoblast-like cells [210,213,214,217,220,223,225], MSCs [176,207,209,211,215,224], although it was not assessed in other studies [206,219,226]. In vivo assays performed on rat [208] and rabbit models [209] were also reported.
The toxicity of antibacterial scaffolds developed for skin tissue engineering has been assessed in a wide variety of cells, such as fibroblasts, melanocytes, and keratinocytes (Table 2). Nanofibrilar scaffolds loaded with the antibiotic CPFX (concentration up to 0.025 g/mL) prepared by jet-spraying were assessed in dermal fibroblasts [273]. Cell colonization was complete after 12 days, suggesting that cells were proliferating within the nanofibers with no evident cytotoxicity. In other studies on antibiotic-loaded scaffolds, cytotoxicity was not analyzed [272,274,275]. Biocompatibility in scaffolds loaded with antibacterial metals was assessed in different cell lines. AgNPs or Ag ions incorporated into the polymer matrix were evaluated in fibroblast cells [280,283,287], MSCs [282] and human melanocytes [290]. Ai et al. prepared swellable hemostatic scaffolds by 3D printing, which incorporated AgNPs [281]. Besides a broad-spectrum antibacterial effect, the system also demonstrated good biocompatibility with several cell lines (A549 adenocarcinomic human alveolar basal epithelial cells, U251 fibroblast-like cells, and epithelial-like cells (HepG2 and HBE cell lines). The in vivo assay in a rabbit femoral vascular injury model also indicated a rapid hemostatic effect. The cytotoxicity of oxygen-generating nanofiber with calcium peroxide as an antibacterial agent was assessed in human osteoblasts [278], and BMCSs [286] were used as models to evaluate biocompatibility. Both studies found the scaffolds to have good biocompatibility. In vitro, the cytocompatibility of bioactive glass nanocomposite hydrogels containing Cu ions was analyzed with endothelial progenitor cells, and a rat model was used in the in vivo assay [289]. The hydrogels significantly promoted cell viability, proliferation and angiogenic ability, while accelerating wound healing and skin tissue regeneration in a diabetic wound. Finally, the biocompatibility of hydrogels containing silk fibroin and zinc chromide NPs as antibacterial agent was analyzed in Hu02 fibroblast and a mouse model to assess their efficacy in wound healing [291]. After five days, the wounds of mice treated with the nanocomposite scaffold were almost completely healed. Some studies, however, did not include biocompatibility assays [279,284,285,288]. Fibroblasts [293,294,299,300,302,304], fibroblast-like cells [301], epidermal [296], endothermal [297] and keratinocytes [298,303] were used as cell models to evaluate the cytotoxicity of antibacterial scaffolds prepared with antibacterial polymers, peptides and scaffolds incorporating carbon nanomaterials. The approaches reported by the in vivo assays also showed good wound healing efficacy [293,299,302,303]. Finally, biocompatibility of antibacterial scaffolds prepared by combining strategies and alternative methods has been evaluated using fibroblasts (both human and murine) [307][308][309]312,314,320], fibroblast-like cells [316,321,322], epithelial cells [306], osteoblasts [312], and also Schwann cells [318] and red blood cells [322]. In vivo experiments showed the effectiveness of the technique in wound healing in the mouse model [307,316], absorbable sutures in a rabbit model [318] and a bacterial infection model performed on rats [322]. Nevertheless, some studies did not report on biocompatibility assessment [41,310,311,313,315,317,319,323]. The toxicity of antibacterial scaffolds developed for oral tissue regeneration (Table 3) has been studied mostly in hDPCs as a model. This cell line has been used in different strategies that involve the incorporation of antibiotics [340,341] and other antibacterial agents [350,351]. Gingival cells [40,347] and murine preosteoblasts MC3T3-E1 [344,345] have also been used as cell models to assess biocompatibility in antibacterial scaffolds for oral tissue regeneration. In vivo assessment was performed in bioglass/CS/chlorhexidine gluconate scaffolds developed for dental application, in which osteoinductive ability was proven using a Wistar-Furth rat model [348]. Conversely, toxicology assays were not included in several other studies [339,342,343,346].
Toxicological assays were performed on antibacterial scaffolds for muscle, nerve, trachea and other tissue engineering applications for oral regeneration (Table 4). Myoblast (C2C12 and L6 cell lines) were used as cell models to assess the biocompatibility of scaffolds for muscle regeneration using different strategies such as antibacterial metals incorporated in polymeric matrices [353,365] or essential oils as antibacterial agents [377]. Scaffolds based on chitosan-aniline tetramer showed good biocompatibility in C2C12 myoblasts and ADMSCs [368]. The biocompatibility of cardiac regeneration scaffolds that incorporated the PANI antibacterial polymer was shown using 3T3 fibroblast-like cell lines [373], while antibacterial scaffolds that incorporated rGO as antibacterial nanomaterial were proven with HUVEC [374]. The toxicity of antibacterial fibrous membranes based on PLA/GO/IL for trachea regeneration was assessed using L929 fibroblast cells, while they confirmed the favorable biocompatibility and promotion of tissue regeneration in a rabbit model. Antibacterial scaffolds for non-specific tissue-engineered applications were assessed using different cell lines such as MSCs [115,354,363], fibroblasts [118,366,367], fibroblast-like cells [359,360], osteoblasts [370], osteoblast-like cells [375], human osteosarcoma cell line [371], liver cells [364] or keratinocytes [114,117], although biocompatibility was not assessed in several studies [116,349,358,361,362,369,372,376].
The cytotoxicity studies carried out on antifungal scaffolds for tissue engineering are included in Table 5. Scaffolds for skin regeneration that include antifungal properties based on the incorporation of antibiotics, metals, peptides and also the use of antimicrobial polymers have been assessed using mainly human [383] or murine fibroblasts [383,393,397]. Biocompatibility studies of scaffolds based on PLC/TiO 2 /cefuroxime for cornea regeneration were performed with human limbal stem cells [392], which showed good behavior regarding cell adhesion, proliferation, and differentiation. Mofidfar et al. prepared polymeric nanofibers of PCL containing the antifungal compound clotrimazole by co-extrusion with poly (ethylene oxide), which was subsequently removed. The scaffold showed effective antifungal behavior in an in vivo study (mouse model) as well as good biocompatibility [395], indicating a good potential for wound healing. Cytotoxicity studies were carried out on general-purpose antifungal scaffolds for tissue engineering using several cell lines such as MSCs [390], fibroblasts [360,393], human endothelial cells, human carcinoma cells [389] and human endothelial cells [394], although biocompatibility assays were not reported in several studies [384,385,387,388,391,396,398].

Conclusions and future perspectives
A lot of progress has been made in the development of scaffolds with antimicrobial activity against bacteria and fungi for a broad range of tissue engineering applications, including bone, oral tissue, skin, muscle, nerve, trachea, cardiac and other applications. Scaffolds produced by different methods to provide antimicrobial activity are essential to avoid microbial infections, which can dramatically affect an implant's success. Antimicrobial activity against bacterial and fungal growth and biofilm formation can be achieved by combining scaffold materials with a broad range of antimicrobial agents such as antibiotics, antiseptics, antimicrobial polymers, peptides, metals, carbon nanomaterials and combined strategies. As multidrug-resistant infections are increasing at an alarming rate, alternative regenerative medical platforms are essential to ensure safe clinical treatments. This review has described the state of the art of antimicrobial scaffolds capable of impeding bacterial and fungal infections in tissue engineering. However, the antimicrobial mechanisms involved in these tissue engineering approaches capable of impeding infections and biofilm formation still need further investigation. The toxicological aspects of these antimicrobial scaffolds have been ensured in most of these studies for safe clinical transfer. There is now a broad range of antimicrobial characterization techniques available to study the antimicrobial behavior of a scaffold against bacterial and fungal growth and biofilm such as the agar disk diffusion test, contact method or biofilm formation in well culture plates or in bioreactor. A broad range of fabrication methods of antimicrobial scaffolds have been included in this review. The best method and materials for tissue engineering depend on the specific applications involved. The antimicrobial approaches now able to prevent infections, including those produced by multidrugresistant strains, show great promise for future clinical tissue engineering applications.

Author contributions
A.S.A. conceived the idea for this work, wrote the draft manuscript, prepared the figures, performed major editing, reviewed and proofread the manuscript. A.C-V. and R. SiS, M.E-T, A.A.A.A, M.M.T. and Y⋅K.M. reviewed, edited and proofread the manuscript.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.