The application and challenges of antimicrobial drug-loaded scaffold materials for the treatment of bone infections

Abstract

Bone infections are a complex clinical issue, typically caused by bacterial infections, leading to damage and dysfunction of bone tissue. Traditional treatments primarily rely on antibiotics (ABs); however, they have limitations such as low permeability and the development of resistance. In recent times, the progress in tissue engineering has introduced novel approaches to tackle bone infections. Biocompatible scaffolds not only serve as supportive structures for bone defects but also function as drug delivery systems, enabling targeted release of antimicrobial drugs to enhance anti-inflammatory and antibacterial effects. The drug release rate from drug-loaded scaffolds can be controlled, which helps to manage infections while promoting bone healing and enhancing the synergistic effects of antibacterial action and bone repair. This review discusses the latest advancements in scaffold materials for the release of antimicrobial drugs, analyzes the current challenges in research, and outlines future directions for development, providing a basis for clinical applications.

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1. Introduction

Bone infection refers to a series of musculoskeletal system diseases caused by microorganisms through exogenous or hematogenous routes.¹ These infections primarily include implant-associated osteomyelitis, fracture-related infections, acute hematogenous osteomyelitis, and infectious bone defects.^2,3 Bone infections are common complications following orthopedic surgery, which can lead to joint deformities, limb amputation, or even death, imposing a significant economic burden on society. Despite employing top-tier medical care, osteomyelitis continues to be difficult to treat, with failure rates of treatment ranging between 10% and 40%.^4,5 The typical causative agents of osteomyelitis often encompass Staphylococcus aureus (S. aureus),⁶ Streptococcus pyogenes,⁷ Streptococcus pneumoniae,⁸ Escherichia coli (E. coli),⁹ and Pseudomonas aeruginosa.¹⁰ Among these, S. aureus exhibits strong invasive abilities, colonization, and proliferation of bone cells, accounting for over 70% of the pathogen spectrum in osteomyelitis.¹¹ Necrotic bone is often caused by and infection forms a microenvironment characterized by acidity, immune suppression, and poor antibiotic penetration due to its association with a limited vascular system.^12,13 In the majority of instances, this leads to the formation of persistent biofilms, resulting in chronic infections that require surgical intervention.¹⁴ The long-term presence of intracellular bacteria can exacerbate recurrent chronic bone infections; therefore, selecting an antibiotic that effectively eliminates intracellular bacteria will be a significant milestone in the management of bone infections.

The main mechanisms by which antibiotics (ABs) inhibit bacteria associated with bone infections include four types:¹⁵ blocking bacterial cell wall formation, disrupting the cell membrane, hindering protein synthesis, and inhibiting nucleic acid replication and transcription. The current commonly employed approaches for managing bone infections primarily involve systemic antibiotic administration combined with local drug delivery treatment. For cases with bone necrosis, surgical debridement to remove the dead bone is also necessary.¹⁶ However, due to the persistent presence of bacteria deep within the bone, these traditional treatment methods often struggle to effectively cure bone infections. Systemic use of ABs also has some drawbacks, such as insufficient local drug concentrations to combat the infection, vulnerability to resistance and systemic adverse effects.^17,18 Additionally, when bone infections occur, they are often accompanied by bone loss. Therefore, effective management of bone infections demands not only the efficient elimination of pathogens but also the rapid regeneration of bone in regions with defects.¹⁹ Prompt surgical debridement in conjunction with appropriate ABs can effectively treat acute osteomyelitis. However, traditional treatment strategies often yield poor results in chronic infections associated with bone defects. Repair of bone defects can only commence once the infection is under control. Although techniques such as bone grafting, bone transport, osteoinductive membrane formation, and the implantation of antibiotic-loaded slow-release carriers are widely used in clinical practice for bone infections, challenges remain, including prolonged treatment durations, insufficient donor bone availability, and complications at the donor site.²⁰ Therefore, the development of antibacterial bioactive bone fillers represents a significant clinical advancement in treating these conditions.

In recent years, with the rapid development of bone tissue engineering (BTE), bone repair scaffolds have become an indispensable core component in this field. Other therapeutic functions rely on scaffolds to effectively perform their roles.^21,22 The creation of bone infection repair scaffolds with superior mechanical strength, strong bone-forming capacity, and localized antimicrobial properties has emerged as a key area of research.^23,24 Ongoing studies on anti-infection bone scaffolds mainly concentrate on the choice of materials, scaffold fabrication techniques, drug delivery mechanisms, and non-antibiotic biomaterials. For irregular open bone defects in clinical settings, it is important to further load traditional bone scaffolds with antibacterial agents to promote bone reconstruction.²⁵ The selection of these antibacterial agents significantly impacts both infection healing and the quality of bone reconstruction. The advantages and disadvantages of various antibacterial agents, along with their target bacterial species, are presented in Table 1. Tissue engineering involves the integration and synergistic interaction of cells, bioactive factors, and scaffold biomaterials. This approach aims to enhance the microenvironment at the site of local infected bone defects, thereby promoting bone healing and regeneration.^26,27 After effectively filling bone defect sites, composite biological scaffolds can rapidly release antibacterial components to inhibit pathogen proliferation and prevent excessive early inflammation. Research on anti-infection bone scaffolds requires collaborative innovation across material science, pharmacology, and tissue engineering. By optimizing scaffold materials, structures, drug loading, and integrating with biologic therapies, tissue engineering methods offer potential solutions for tackling the dual challenges of infection management and bone restoration in infected bone defects. This review innovatively integrates the latest advancements in the field of bone infection treatment, covering both traditional and emerging antimicrobial strategies. First, it provides an in-depth analysis of common antimicrobial agents, including the application and challenges of ABs, the mechanisms and potential of antimicrobial peptides (AMPs), and the types of antimicrobial nanomaterials and their precision antimicrobial technologies. Second, the review focuses on BTE scaffolds, systematically summarizing the characteristics and prospects of various drug-loading methods (such as direct loading, coating, and composite preparation), which provide theoretical support for drug release, long-term antimicrobial effects, and bone repair. It also explores the clinical applications of antimicrobial bone scaffolds (Fig. 1). This interdisciplinary integrative perspective offers new insights for the study of bone infection treatment and promotes innovative development in this field.

Table 1 Advantages and disadvantages of different antibacterial agents and species of action

Antimicrobial agents	Advantage	Disadvantage	Species of action
Antibiotic	Broad-spectrum antibacterial, easy to prepare and inexpensive	Resistance is readily developed and systemic use has low toxicity	Gram-positive bacteria and most Gram-negative bacteria
Antibacterial peptide	Broad-spectrum antibacterial, no drug resistance, good biocompatibility	Extraction is difficult and costly	S. aureus, E. coli and other Gram-positive bacteria, and a small number of Gram-negative bacteria
Nano metal particles	It has strong antibacterial ability and low synthesis difficulty	It is difficult to control the concentration dilution and has certain cytotoxicity	S. aureus, E. coli, Bacillus subtilis, etc.

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Fig. 1 Graphical abstract.

2. Type of antimicrobial agent

2.1 Antibiotics (ABs)

Bone infection is a disease of bone tissue inflammation caused by bacterial or fungal infection, and common pathogenic bacteria such as S. aureus, Staphylococcus epidermid,²⁸ etc. ABs are commonly used antibacterial agents with a variety of types, mainly including: broad-spectrum ABs, such as penicillins, cephalosporins, etc., which can extensively eliminate a variety of bacteria by inhibiting bacterial cell wall synthesis and disrupting the cell wall structure.^29,30

The effect of antibiotics is obvious. Aldhaeefi et al. assessed the effectiveness of penicillin targeting S. aureus, demonstrating that penicillin use was both safe and effective.³¹ ABs against Gram-positive bacteria, such as vancomycin (Van)³² and teicoplanin,³³ mainly target S. aureus and can cause lysis and death of bacterial cells by impeding the creation and reconstruction of bacterial cell wall, destroying the structure of bacterial cell wall, interfering with bacterial protein synthesis, and inducing changes in bacterial cell membrane permeability, respectively. Namdar et al. coloaded lysozyme (Lys) and Van onto mesoporous silica nanoparticles (MSN) to form Lys–VAN–MSN. The minimal concentration to inhibit of the nanoparticles (NPs) against S. aureus was determined, and it was observed that the NPs possessed notable antibacterial efficacy.³⁴ Watanabe et al. evaluated the efficacy of teicoplanin against S. aureus in a murine model of thigh infection, demonstrating robust bactericidal activity against this pathogen.³⁵ Anti-Gram-negative ABs such as aztreonam and piperacillin/tazobactam, which mainly target Pseudomonas aeruginosa, can achieve antibacterial effect by interfering with protein synthesis and destroying bacterial cell membrane, through inhibiting bacterial cell wall synthesis and mitigating drug resistance.^36,37 In the study by Gahane et al. Fmoc-F was conjugated with the Gram-negative specific antibiotic aztreonam to form a novel combination. In a murine wound infection model, this formulation exhibited potent antibacterial activity against Gram-negative bacteria, effectively diminishing bacterial count. Notably, it demonstrated enhanced efficacy against Pseudomonas aeruginosa compared to other Gram-negative pathogens.³⁸ As the most classical and widely used antibacterial drugs, ABs are broadly applied in the therapy of conventional bone infections. As reported by Ran et al. found that patients with an infection of the left tibia treated with ABs had rapid tissue healing, and the patients did not have to amputate their leg and recovered well.³⁹ Fan et al. evaluated the antibacterial efficacy of antibiotic-eluting materials using a rat model. They simulated joint replacement by inserting polyethylene (PE) plugs into the femur and titanium (Ti) screws into the tibia, and inoculated methicillin-susceptible S. aureus into the medullary canal and joint space. The experimental group received daqingmycin-eluting PE materials, which effectively treated periprosthetic joint infection.⁴⁰

In addition, drug release is critical in the treatment of bone infection, some ABs, such as penicillins and cephalosporins, can effectively penetrate bone tissues and reach the infection sites to exert their effects. This represents the cornerstone of antibiotic therapy for bone infections.^41,42 Torkington et al. collected bone samples from patients undergoing total hip arthroplasty (THA) and total knee arthroplasty (TKA) to assess the bone permeability of flucloxacillin and gentamicin during intravenous antibiotic prophylaxis for hip and knee arthroplasty. Antibiotic concentrations in the samples were analyzed using HPLC. Results indicated that both flucloxacillin and gentamicin exhibited good bone permeability and antibacterial efficacy.⁴² The bone penetration of ABs is an important indicator to evaluate their ability to treat bone infection and prevent postoperative infection, which has a key guiding significance for the selection of clinical drugs and the formulation of treatment plans.

However, as ABs travel through various organs and tissue layers en route to the site of infection following oral or intravenous administration, they may become diluted or cause toxicity. Yamada et al. studied 43 patients receiving 2 g of intravenous cefazolin before THA and TKA. Results showed that cefazolin achieved the MIC for S. aureus in serum and bone but not for cefazolin-resistant coagulase-negative Staphylococcus in bone, posing a potential risk of deep surgical site infections in these procedures.⁴³ To enhance therapeutic efficacy and minimize adverse effects, drug delivery carriers have been employed to encapsulate and deliver ABs, addressing the aforementioned issues.⁴⁴ Composed of at least one phospholipid bilayer and containing an internal aqueous compartment, liposomes are self-assembled lipid vesicles that serve as excellent carriers for delivering ABs.⁴⁵ On the other hand, outer membrane vesicles (OMVs) are nanoscale spherical carriers released into the surrounding environment by Gram-negative bacteria throughout their growth, which can also serve as carriers for antibiotic delivery.⁴⁶ In a research study conducted by Nguyen et al. ampicillin-loaded chitosan (CS)/starch nanocomposites were prepared through the combination of diverse combinations of CS and starch ratios. The findings indicated that these nanocomposites exhibited an antibacterial effect of 95.41% against E. coli, which was higher than the 91.40% observed with the equivalent concentration of 5.0 μg mL⁻¹ ampicillin following a 24-hour treatment duration. These research findings further confirm the high-efficiency application potential of CS/starch nanocomposites in the field of antibiotic drug delivery.⁴⁷

In summary, ABs serve as a primary treatment method for bone infections, effectively targeting pathogens, penetrating bone tissue, and working in conjunction with other therapeutic measures to ultimately control infections and repair bone integrity. However, several issues still require attention in the future, such as drug resistance, targeting strategies, and administration methods. The sole use of ABs has become insufficient to meet the complex needs of bone infections. Future approaches may involve more targeted comprehensive treatment plans, integrating other therapeutic methods, and enhancing prevention and early diagnosis to better control and treat bone infections.

2.2 Antimicrobial peptides (AMPs)

During the process of bone defect repair, traditional ABs often fail to effectively prevent or treat bone infections due to issues of drug resistance and recurrent infections. As a result, there is a pressing demand in BTE for new antibacterial agents that minimize resistance.⁴⁸ AMPs are naturally occurring amphipathic small molecular peptides that exhibit broad-spectrum antimicrobial, antiviral, and antitumor activities. Characterized by their cationic, hydrophobic, and amphipathic properties, AMPs are ubiquitous across all forms of life. Given these attributes, they hold great promise as potential candidates for the development of next-generation ABs. They rarely induce resistance and exhibit strong antibacterial efficacy,⁴⁹ while also possessing various biological activities such as anti-inflammatory effects,⁵⁰ angiogenesis,⁵¹ and bone formation.⁵² Recently, they have received widespread attention. The combination of these important biological activities with the unique antibacterial mechanisms of AMPs has led to their increasingly significant role in bone regeneration.

Studies have demonstrated that AMPs, due to their bactericidal, anti-biofilm formation, immune-regulatory, and regenerative functions,^53–55 hold great promise in the field of BTE.⁴⁸ Bactericidal activity is a key function of AMPs, and the antimicrobial mechanisms involved are diverse. These mechanisms include disrupting membrane integrity or interfering with cell wall biosynthesis, protein synthesis, DNA replication, RNA transcription, and fatty acid biosynthesis.^56–58 The structural types of AMPs can generally be classified into four categories, the antimicrobial mechanisms of AMPs with different structures are not the same. (1) α-Helical peptides,⁵⁹ typically composed of amphipathic amino acids with cationic properties, can interact with the negatively charged lipids of bacterial cell membranes, thereby disrupting membrane integrity. Their amphipathic nature enables them to insert into bacterial cell membranes, forming pores that lead to the leakage of cellular contents and subsequent cell death;⁶⁰ (2) β-sheet peptides, which typically contain multiple cysteine residues, form stable β-sheet structures through disulfide bonds. This structural configuration facilitates the binding of AMP to bacterial cell membranes and enables them to exert their antibacterial effects by disrupting membrane integrity;⁶¹ (3) linear peptides, some AMPs lack a well-defined three-dimensional structure and exist in a linear extended state. They typically exert their effects by adsorbing onto the surface of bacterial cell membranes. Upon reaching a certain concentration, they induce membrane rupture. For instance, indolicidin (ILPWKWPWWPWRR) is an AMP rich in tryptophan and proline, which demonstrates high efficacy against a broad spectrum of microorganisms, including bacteria, fungi, and viruses;⁶² (4) peptides including both α-helix and β-sheet structure, some AMPs possess both α-helix and β-sheet structures. This complex structural configuration enables them to exert their effects through multiple mechanisms. For example, melimine (TLISWIKNKRKQRPRVSRRRRRRGGRRRR) is a synthetic AMP which combines functional regions of mellitin (an α-helical AMP with 26 residues obtained from the venom of European honey bee) and protamine, which shows broad spectrum antibacterial properties.⁶³

Moreover, the structure of AMPs also influences their activity. The secondary structure is a fundamental element for the interaction between AMPs and bacterial cell membranes. Most AMPs exhibit disordered structures in aqueous solutions, but they form stable secondary structures, such as α-helices and β-sheets, upon interacting with phospholipids in the membrane environment. The formation of secondary structures facilitates the insertion of AMPs into bacterial membranes, thereby exerting their antimicrobial effects.⁶⁴ In addition to the impact of secondary structure on antimicrobial activity, other structural features of AMPs, such as net charge, hydrophobicity, and amphipathicity, also significantly influence their antimicrobial activity.⁶⁵ AMPs are rich in basic amino acid residues, such as arginine, lysine, and histidine, which confer a positive charge on them. The quantity and distribution of positive charges can affect the electrostatic binding affinity of AMPs to the negatively charged phospholipids in bacterial cell membranes, as well as the antimicrobial concentration of AMPs; generally, an increase in net charge can enhance antimicrobial activity. For instance, in the study by Wang et al. a stable cationic peptide HJH-1 was selected as the parent peptide. By replacing histidine and alanine residues in HJH-1 with the cationic amino acid arginine to increase the net charge, different HJH peptides were designed. The results showed that the designed HJH peptides with increased net charge exhibited enhanced antimicrobial efficacy.⁶⁶ Hydrophobicity is one of the key factors for AMPs to exert their antimicrobial activity. Hydrophobic amino acid residues facilitate the interaction between AMPs and the lipid components of bacterial cell membranes, thereby disrupting membrane integrity. Le et al. compared three AMP: mastoparan C, mastoparan-AF, and mastoparan L. Their hydrophobicity varies, but other structural features are nearly identical. Mastoparan C is the most hydrophobic, with the highest helical content and hemolytic activity. Mastoparan-AF has slightly lower hydrophobicity and better selectivity. Mastoparan L is the least hydrophobic, showing the weakest antimicrobial potency and lowest hemolytic activity, but the least self-assembly. The study suggests that optimal hydrophobicity, rather than the highest, enhances antimicrobial efficacy while minimizing hemolytic activity.⁶⁷ Amphipathicity (the presence of both hydrophobic and hydrophilic regions) is a crucial structural feature of AMPs that facilitates their interaction with bacterial cell membranes. This characteristic enables AMPs to selectively bind to bacterial membranes, disrupt their integrity, and exert antimicrobial effects without significantly damaging eukaryotic cell membranes. Qi et al. successfully synthesized deoxycholic acid-modified CS (CS–DA) by grafting deoxycholic acid (DA) onto chitosan C₂-NH₂, followed by grafting succinic anhydride, to prepare a novel amphiphilic CS (CS–DA–SA). Results indicate that CS–DA–SA exerts antibacterial effects by disrupting cell membranes or forming a membrane on the cell surface, with superior antibacterial efficacy.⁶⁸ However, it should be noted that a perfect amphipathic structure often leads to a simultaneous increase in both bactericidal activity and cytotoxicity, whereas an imperfect distribution of amphipathic residues is more conducive to optimizing cell selectivity.

The effectiveness of microbial inhibitorsagainst infecting bacteria is crucial for the management of bone infections.⁶⁹ Despite the identification of many AMPs, only a few are clinically available. AMP activity depends on factors like the stable substrate, spacer uniqueness, surface concentration, exposure duration, orientation, and peptide resilience. To enhance the robustness and bioactivity of AMPs while reducing their cytotoxicity in vivo, researchers modify materials by focusing on attributes like porosity, hydrophobicity, and electrostatic forces. This results in varying encapsulation efficiencies and drug release characteristics. Machado et al. developed an amphiphilic self-assembled micelle material by covalently synthesizing dextrin with succinylated vitamin D3 and succinic anhydride. This micelle formulation effectively loaded the antimicrobial peptide LL18, significantly enhancing its antibacterial activity against S. aureus while completely eliminating its severe hemolytic activity.⁷⁰ Lin et al. developed a luminescent antimicrobial peptide-modified dressing for treating bacterial infections by grafting crosslinked AMP onto functionalized sodium alginate (NaAlg) and silk fibroin (SF) sponges. The dressing combines hyaluronic acid (HA), SF, and Alg. This dressing demonstrated effective detection and elimination of bacteria.⁷¹ Zhang et al. focused on creating a duplex coating for orthopedic implants, comprising a MgF₂ primer layer and phenolic-amine grafted AMPs on the AZ31 surface, which is referred to as AZ31-FE/AMPs. This innovative coating is intended to provide initial antibacterial properties, promote osteogenesis in the later stages, and enhance corrosion resistance.⁷²

In summary, AMPs, as a class of emerging bioactive substances, demonstrate significant potential for treating bone infections. Moving forward, they may be used in conjunction with conventional ABs and other treatment methods, or as an alternative, providing new therapeutic options for clinical use. Nonetheless, further extensive research is necessary to explore their mechanisms of action and confirm their safety.

2.3 Nano materials

In the current era, the increasing prevalence antibiotic resistance and the growing incidence of drug-resistant bacterial infections introduce major issues to public health management. The rate of new antibiotic development has failed to match the swift evolution of antimicrobial resistance,^73,74 the efficacy of traditional ABs is greatly limited, and the development of novel antibacterial strategies is urgently needed. Nanomaterials, due to their unique physicochemical properties and antibacterial mechanisms, have shown great potential as alternatives to ABs.^74,75

2.3.1 Nanoparticle (NPs). NPs refer to particles with a diameter of less than 100 nm. When certain materials reach the nanoscale, their specific surface area increases, enhancing their ability to adsorb bacteria, which can lead to the destabilization of bacterial outer and inner membranes.⁷⁶ Compared with traditional nanomaterials, ultra-small size metallic nanomaterials are more conducive to their penetration into bacterial cell envelope.⁷⁵ In general, metal particles and NPs demonstrate antibacterial properties via mechanisms like physical interaction, ionic release, and reactive oxygen species (ROS) generation.⁷⁷ Positively charged NPs can interact with the negatively charged elements of bacterial cell walls, leading to cell membrane disruption, inhibition of intracellular activities, and ROS production. This disruption not only compromises the integrity of the bacterial cell but also interferes with its metabolic activities, contributing to the antibacterial effects of these NPs,^78–80 with the associated antibacterial mechanisms shown in (Fig. 2). On account of their intrinsic multispectrum antibacterial properties, silver nanoparticles (AgNPs) are considered a highly promising category of antibacterial agents. AgNP has an adjustable structure and a broad antibacterial spectrum, and shows good efficacy against drug-resistant bacteria.⁸¹ El-Sharkawy et al. showed significant antimicrobial activity against multidrug resistant bacterial pathogens using the aid of exometabolites of Fusarium oxysporum to synthesize small-sized nano-silver particles.⁸² Nevertheless, the comparatively high toxicity of AgNPs restricts their extensive application.⁸³ Recent studies have concentrated on modifying the synthesis strategy to mitigate the toxicity of AgNPs for better application in addressing bacterial infections. Lin et al. developed a novel Ag nanohybrid material with amazing antibacterial activity and almost no cytotoxicity through a single-molecule micelle template approach, which can mitigate the toxicity caused by metal NPs.⁸⁴ Guo et al. used montmorillonite loaded nano-silver (AgNPs@MMT) to achieve stable and prolonged Ag⁺ release, reduce the damage of nano-silver to the organism, and at the same time have good antibacterial properties.⁸⁵ Oxide NPs similarly possess antimicrobial capabilities. ZnO has been shown to have a strong antibacterial effect,⁸⁶ additionally, numerous studies in the scientific literature emphasize the antibacterial efficacy of ZnO NPs against various model pathogens, including both Gram-negative and Gram-positive strains.⁸⁷ ZnO exhibits significant photocatalytic activity and is anticipated to generate similar levels of reactive ROS, which are major toxic substances for microorganisms, upon light irradiation.⁸⁸ Orie et al. studied the biosynthesis and antibacterial capabilities of ZnO NPs with different concentrations and found that ZnO NPs demonstrated strong antifungal activity against a wide range of fungi, moreover, elevated levels of ZnO would heighten the susceptibility of clinical pathogens.⁸⁹ Semiconductor NPs, such as titanium dioxide nanoparticle (TiO₂ NPs), produce ROS through photocatalysis, which are highly oxidizing and can effectively kill bacteria. In the study by Feng et al. a TiO₂@reduced graphene oxide (rGO) composite was prepared and subsequently mixed with poly(L-lactic acid) (PLLA) to fabricate bone scaffolds. The rGO facilitated the separation of photo-induced electron–hole pairs in TiO₂, thereby enhancing the efficiency of photocatalytic reactive ROS generation and consequently improving the antibacterial efficacy of the scaffolds.⁹⁰ In addition, magnetic nanoparticles,⁹¹ polymer nanoparticles,⁹² and carbon nanoparticles⁹³ have also shown certain antibacterial activities.^94–96

Fig. 2 A schematic illustration depicting the various passive antimicrobial mechanisms of metal nanomaterials (not to scale), including physical interactions, ion release, and ROS production.⁸⁰ Copyright Cheeseman et al. 2020 Springer Wiley.

2.3.2 Nanosheets. Nanosheets are two-dimensional nanomaterials with a thickness typically in the nanoscale range (1–10 nm), while their length and width can extend to micrometers or even larger dimensions. They can be generally classified into inorganic nanosheets,⁹⁷ organic nanosheets,⁹⁸ and composite nanosheets.⁹⁹ Owing to their high specific surface area, excellent mechanical properties, and unique physicochemical characteristics, nanosheets hold great promise for a wide range of applications in various fields.

In terms of antibacterial properties, nanosheets also exhibit unique advantages.¹⁰⁰ In the study by Feng et al. Ti carbide nanosheets were combined with graphitic carbon nitride nanosheets to prepare nanocomposites via electrostatic self-assembly. These nanocomposites were then incorporated into a PLLA matrix to fabricate bone scaffolds using selective laser sintering (SLS). The results indicated that the incorporation of the nanosheet composites into the scaffold material via 3D printing endowed the bone scaffolds with good antibacterial properties.¹⁰¹ Zeolite imidazolate frameworks (ZIFs) are a class of crystalline porous materials, in which metal ions and organic ligands are connected by coordination bonds to form a periodic network structure.¹⁰² Two-dimensional (2D) ZIF nanosheets have attracted increasing attention due to their ultrathin thickness, large surface area, abundant active sites, and excellent chemical and mechanical stability. In the study by Zhu et al. a novel copper-functionalized two-dimensional zeolitic imidazolate framework-7 nanocomposites ZIF-7@Cu₂O was successfully fabricated by in situ growth methodology, the antibacterial kinetics and bacterial adhesion properties were investigated. The results demonstrated that the material possesses excellent reactive ROS generation capacity, inhibits biofilm formation, and exhibits superior antibacterial efficacy.¹⁰³ Additionally, nanosheets are also capable of generating reactive ROS to kill bacteria. Some nanosheets exhibit photothermal and photodynamic therapeutic effects. Under light irradiation, they can produce ROS or localized high temperatures, thereby achieving photothermal antibacterial and photodynamic antibacterial effects, in the study by Li et al. palladium nanosheets (PdNS) were synthesized. The PdNS exhibit intrinsic nanosword (mechanical cutting) effects and peroxidase-like activity. Under near-infrared (NIR) light irradiation, PdNS demonstrate photothermal effects by generating localized heat and photodynamic effects by producing singlet oxygen (¹O₂). Ultimately, these synergistic mechanisms induce metabolic energy disruption, elevated cellular reactive ROS levels, and excessive oxidative stress, while inhibiting nucleic acid synthesis within bacterial cells, thereby achieving highly efficient antibacterial efficacy,¹⁰⁴ In summary, nanosheets hold numerous advantages for antibacterial applications. Their high specific surface area and good biocompatibility enable efficient loading of antibacterial agents and precise controlled release, thereby significantly enhancing antibacterial efficacy and reducing the risk of drug resistance. These features highlight the broad application prospects of nanosheets in the field of antimicrobial therapy.

2.3.3 Nanotubes. Nanotubes are a type of nanomaterial with a hollow tubular structure, typically formed by the rolling of single or multiple layers of atoms or molecules. The diameter of nanotubes generally ranges from 1 to 100 nanometers, while their length can extend to the micrometer or even millimeter scale. There are various types of nanotubes, including carbon nanotubes (CNTs),¹⁰⁵ semiconducting nanotubes,¹⁰⁶ and polymer nanotubes.¹⁰⁷ These nanotubes can exert antibacterial effects through mechanisms such as physically disrupting bacterial cell membranes, releasing reactive ROS, and releasing metal ions.

CNTs have been demonstrated to possess broad-spectrum antibacterial activity against pathogenic microorganisms.^108,109 The antibacterial mechanisms of CNTs are influenced by their intrinsic properties (such as structure, modifications, surface area, chemical nature, morphology, and solubility) as well as the type of bacteria and the interaction environment,¹¹⁰ Single-walled carbon nanotubes (SWCNTs) exhibit stronger antibacterial activity due to their higher toxicity to microorganisms. However, multi-walled carbon nanotubes (MWCNTs) are more suitable for biomedical synthesis applications. This is because MWCNTs have higher purity, lower toxicity to the human body, and a lower probability of defects occurring during functionalization. Venkatesan et al. synthesized chitosan-MWCNTs hydrogels via freeze-drying and found that with the increase of MWCNTs concentration, higher antibacterial activity was exhibited against S. aureus, E. coli, and Candida tropicalis.¹¹¹

Ti and Ti alloys are frequently used in orthopedic surgical interventions.¹¹² They possess several desirable properties, including low density, excellent corrosion resistance, high mechanical strength, and the ability to bond with bone and other tissues without cytotoxic effects. These characteristics make Ti-based materials highly suitable for orthopedic implants.¹¹³ As unique micro/nanostructures, TiO₂ nanotubes possess a large specific surface area. TiO₂ nanotubes with different diameters exhibit varying antibacterial properties. Heat-treated TiO₂ nanotubes with a diameter of 80 nm demonstrate the most potent antibacterial effect.¹¹⁴ Similarly, TiO₂ nanotubes also serve as excellent drug carriers. Yao et al. fabricated silver-loaded TiO₂ nanotubes (TiO₂ NT) via anodic oxidation and evaluated their cytocompatibility and antibacterial properties. The experimental results demonstrated no cytotoxicity, sustained antibacterial characteristics, and enhanced osteogenic potential.¹¹⁵ Feng et al. prepared TiO₂ nanotubes via electrochemical anodic oxidation and adsorbed gentamicin sulfate (GS) onto them, followed by coating with a mixture film of GS and CS (referred to as GSCH). The study revealed that the GS-loaded TiO₂ nanotubes coated with GSCH hold great potential as a biomaterial, exhibiting strong antibacterial properties and the ability to prevent periprosthetic infections.¹¹⁶

In summary, antimicrobial nano materials offer significant potential for various applications, particularly in infection prevention and the treatment of bone infections. Their excellent antibacterial properties, targeting capabilities, and controlled release mechanisms make them highly promising. These nano materials are expected to become one of the emerging strategies for orthopedic infection control in the future. These different kinds of antibacterial nano materials have broad application prospects in medical, food, and daily chemical fields. The appropriate nanomaterials should be selected according to the actual needs for specific use.

3. Type of scaffolds

Oral and injectable ABs are currently used for the prevention and bone infection treatment protocols¹¹⁷ and implantation of infected or injured bone tissue and bone grafts.¹¹⁸ However, due to the challenge of antibiotic resistance, long-term treatment and adverse reactions, the creation of novel therapeutic approaches is of urgent importance. Biocompatible antimicrobial carriers offer a potential alternative for the management and prevention of bone infections,¹¹⁹ enabling controlled release, targeted delivery, and the possibility of personalized therapy.

Regenerative therapies and tissue engineering are creating new opportunities for creating scaffold biomaterials to replace bone grafts. Adding diverse bactericidal compounds, such as ABs, metal particles, metal NPs, and essential microbial inhibitors, can enhance antibacterial activity while the scaffold facilitates osteogenesis.¹²⁰ Therefore, the combination of synthetic scaffolds with antibacterial agents to prevent bacterial infection is a potential solution for both bone regeneration and the control of bone infections.¹²¹ This article reviews the preparation of scaffolds and the drug loading methods of scaffolds.

3.1 Requirements of bone scaffold

In recent years, drug-loaded material technology has demonstrated significant promise in treating bone infections. By delivering antimicrobial agents directly to the site of infection and achieving high local drug concentrations, this system enhances therapeutic efficacy while minimizing the toxic side effects linked to systemic drug administration. Common drug-loaded materials like bone cement and biodegradable biomaterials can act as scaffolds or fillers to slowly release drugs and effectively control infections. Key factors for an ideal scaffold include material composition, biomimetic design, antibacterial performance, bone regeneration promotion, and their interplay. An ideal system should combine infection treatment and bone defect regeneration by using diverse matrices and suitable fabrication methods. This integrated approach avoids multiple surgeries and overcomes traditional challenges in bone infection and defect repair.

An ideal scaffold for bone infection should possess the following integrated characteristics (Fig. 3):¹²² firstly, good mechanical properties are the foundation, providing stable support during the bone healing process.¹²³ Secondly, biocompatibility is crucial, as natural biologically derived materials such as CS and hydroxyapatite (HAP) possess excellent biocompatibility and can effectively promote the growth and integration of bone tissue.¹²⁴ In addition, the degradation rate of the scaffold should match the rate of bone regeneration to avoid adverse effects on the healing process due to premature or delayed degradation. A porous structure is essential for promoting cell infiltration, nutrient diffusion, and waste removal. Studies have shown that 3D-printed hierarchical porous scaffolds can significantly enhance cell infiltration and proliferation rates.¹²⁵ Moreover, the customizability of the scaffold is essential to meet the specific needs of individual patients. The advent of 3D printing technology has enabled the precise fabrication of scaffolds tailored to the shape and size of bone defects.¹²⁶ Additionally, the scaffold material should minimize cell stress to promote cell attachment and proliferation.¹²⁷ Furthermore, an ideal bone infection scaffold should be capable of modulating the inflammatory response. This can be achieved by loading anti-inflammatory drugs or modifying the surface properties of the scaffold to effectively control inflammation, thereby facilitating bone healing.¹²⁸

Fig. 3 Construction of a multifunctional filling scaffold for bone infection.¹²² (a) Mechanical properties. (b) Biocompatibility. (c) Biodegradation. (d) Multiple pore structure. (e) Customizability. (f) Cell stress. (g) Antibacterial. (h) Pro-inflammatory response. Copyright Qin et al. 2024 Springer Nature.

In summary, the construction of an ideal bone infection scaffold requires a comprehensive consideration of its mechanical properties, biocompatibility, biodegradability, porous structure, customizability, cell stress, antibacterial properties, and the ability to modulate inflammatory responses. Through interdisciplinary research involving materials science, biomedical engineering, and clinical medicine, it is anticipated that more efficient and safer scaffolds for the treatment of bone infections can be developed.

3.2 Preparation of scaffolds

In tissue engineering applications, many techniques for the production of porous materials have been developed to be used as scaffolds, including electrospinning,¹²⁹ phase separation,¹³⁰ gas foaming,¹³¹ pore-forming agent methods,¹³² polymerized fiber mesh coating,¹³³ self-assembly,¹³⁴ membrane lamination,¹³⁵ freeze-drying,¹³⁶ 3D printing,¹³⁷ and bioprinting.¹³⁸ The creation of antibacterial scaffolds in these approaches typically relies on the introduction of fillers with inherent antibacterial attributes.¹³⁹ In this section, we will discuss techniques for making BTE scaffolds, including traditional methods and additive manufacturing like 3D and 4D printing. Some application scenarios are listed (Fig. 4 and Table 2). The types and methods of scaffold preparation were as follows:

Fig. 4 Antibacterial scaffold fabrication methods:¹³⁹ (a) electrospinning; (b) phase separation; (c) gas foaming; (d) porogen leaching; (e) solution polymerization; (f) self-assembly; (g) 3D printing; (h) freeze drying. Copyright Serrano-Aroca et al. 2022 Elsevier.

Table 2 Preparation method of bone infection repair scaffold

Fabrication methods	Materials	Processing methods	Pore size	Mechanical strength	Culture cell	Type of bone repair	Ref.
Fused deposition modeling (FDM)	PLA/nano-hydroxyapatite (nHAP)	Hybrid printing	50 and 80 nm	—	BMSCs	Bone defect	161
Selective laser sintering (SLS)	Poly(vinyl alcohol) (PVA)/HAP	Hybrid printing	Porosity was 68.3%	—	MC3T3-E1	Bone defect	162
Digital light processing (DLP)	Polydopamine (PDA)/biphasic calcium phosphate (BCP)	Self-assembly Ca-P/PDA nanocoating	Pore size of 570 μm	Compressive strength (∼3.6 MPa)	BMSC	Bone defect	163
Electrostatic spinning	β-Tricalcium phosphate (β-TCP)/Mxene/poly(lactic acid-carbonate) (PDT)	Hybrid printing	PDT with an average diameter of 1.2 μm	Tensile strength (14.4 MPa)	BMSCs, HUVECs	Bone defect	164
Freeze drying	Gallium-based metal–organic framework (GaMOF)/Gelatin methacrylate (GelMA)/HA	Quaternized CS (QCS) coated	Average particle sizes were 907.3 nm	∼350 kPa	RBMSCs	Bone infection	165
Gas foaming	Polycaprolactone (PCL)/strontium NPs (3DS-Sr)	Drug loading	The diameter was 527 ± 148 nm. The pore size was 58 ± 19 μm^2	—	HUVECs	Bone infection	166
Hot sintering	Poly(L-lactic acid)/bioactive glass matrix/PDA	Hybrid printing	Average particle sizes were 226.52 nm	Compressive strength and modulus were 28.40 ± 0.48 MPa and 0.8 ± 0.02 GPa	HOS cells	Osteosarcoma	167
Solvent casting/particulate leaching	HAP/PLGA	Hybrid printing	The average particle sizes were 232 μm, 323 μm, and 464 μm		MC3T3-E1, C2C12	Bone defect	168
Low temperature injection	PLGA/PLA/HA/CS	Nano-spraydrying technique	—	—	—	Bone infection	169
3D printing	Alg/GOx/CaP@CAT	Cross-links	800 μm	—	rBMSCs,HUVECs, and RAW 264.7 macrophages	Diabetic bone regeneration	170
4D printing	TCP/P(DLLA-TMC)	Photothermal effect	360 ± 40 μm	2.2 ± 0.2 to 2.4 ± 0.2 MPa (20 °C), 1.5 ± 0.2 MPa (37 °C), 0.4 ± 0.05 MPa (45 °C)	BMSCs	Critical size bone defect	171

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(a) Electrospinning technology: electrospinning technology involves placing a polymer solution into a jetting device and generating an electric field at the nozzle using a high-voltage power supply.¹⁴⁰ As the electric field strength increases, the liquid droplet is stretched to form a “Taylor cone” When the electrostatic force overcomes the surface tension, a jet is ejected from the Taylor cone. This jet undergoes oscillation and stretching under the influence of the electric field, and the solvent evaporates to form nanoscale fibers. These fibers are then randomly deposited onto a collector to form a nonwoven mat. Characterized by a large surface area, the substrate serves as an optimal environment for cell expansion and subsequent tissue regrowth. For instance, an antimicrobial scaffold can be created using polymers like CS that exhibit inherent antimicrobial properties.¹⁴¹

(b) Phase separation: phase separation is a method that induces phase separation to form porous structures by mixing polymer solutions with non-solvents.¹⁴² The principle involves the phase separation of polymer solutions under specific conditions (such as changes in temperature or concentration), resulting in the formation of polymer-rich and polymer-poor phases. By controlling the phase separation process, scaffolds with different pore structures can be obtained. The key lies in selecting appropriate polymer and solvent systems, as well as precisely controlling the operating conditions to achieve the desired pore structure. Combining phase separation techniques with other methods allows for constructing 3D scaffolds with precise pore configurations. This approach is extensively applied in the production of polymeric scaffold constructs, such as polylactic acid (PLA) scaffolds, for application in the realm of regenerative medicine.^143,144

(c) Gas foaming: gas foaming is a method that forms porous structures by introducing gases into polymers.¹⁴⁵ The principle involves dissolving gas into a polymer melt or solution, and then expanding the gas to form bubbles by reducing pressure or increasing temperature. Depending on the type of blowing agent used, gas foaming can be categorized into physical foaming and chemical foaming. Physical foaming generates gas through physical means, such as injecting high-pressure gas or evaporating low-boiling-point liquids. In contrast, chemical foaming releases gas through chemical reactions, such as the decomposition of blowing agents. The key strength of gas foam scaffold fabrication is that it avoids the use of chemicals and high-temperature conditions, avoiding these factors to damage cells, tissues, and microenvironment.¹⁴⁶

(d) Porogen leaching: the porogen leaching method involves adding soluble porogens (such as salts, sugars, polymer microspheres, etc.) into the polymer matrix, followed by dissolving and removing these porogens using a solvent to create a porous structure. The principle is based on the distribution of the porogens within the polymer matrix, which forms temporary pore structures. Once the porogens are dissolved by the solvent, the spaces left behind become the pores of the scaffold. This method allows for precise control over the size and distribution of the pores and is suitable for the fabrication of scaffolds with specific pore structures.^147,148 This technique is frequently integrated with melt molding to fabricate biodegradable polymer scaffolds,¹⁴⁸ thereby establishing a foundation for numerous advancements in antibacterial scaffolds.

(e) Solution polymerization: solution polymerization is a method for fabricating scaffolds through polymerization reactions in a solvent.¹⁴⁹ The principle involves dissolving monomers and initiators in a solvent, where they undergo chemical reactions to form polymers. During the polymerization process, the molecular weight and structure of the polymer can be regulated by controlling reaction conditions such as temperature and concentration.¹⁵⁰ Additionally, solution polymerization can impart antibacterial properties to the scaffold by incorporating antibacterial nanomaterials, such as graphene oxide.¹⁵¹

(f) Self-assembly: self-assembly technology is based on the spontaneous organization of molecules in a specific medium to form ordered structures with specific functions. The principle involves the use of non-covalent interactions between molecules (such as hydrogen bonds, van der Waals forces, and electrostatic interactions) to enable the molecules to spontaneously aggregate in solution, forming nanofibers or other ordered structures. For example, amphiphilic peptides can self-assemble into three-dimensional nanofiber networks in aqueous solutions, which are used in tissue engineering.^152,153

(g) 3D printing: 3D printing is an additive manufacturing technology controlled by computer-aided design (CAD) models, capable of precisely fabricating porous scaffolds with complex structures. The principle involves the layer-by-layer deposition or solidification of polymeric materials (such as filaments, powders, or liquids) to construct the scaffold according to the predefined three-dimensional model.¹⁵⁴ Commonly used 3D printing techniques include fused deposition modeling (FDM), SLS, and stereolithography (SLA). Through 3D printing technology, patient-specific scaffolds can be precisely manufactured according to the shape and size of the bone defect. For instance, using FDM, a fusion peptide-modified poly(lactic-co-glycolic acid)(PLGA)/HA composite scaffold has been developed to effectively support bone defect regeneration and repair. Characterized by excellent antibacterial and cytocompatibility outcomes in vitro, these scaffolds also showed promising performance in an in vivo rabbit model, underlining their potential for bone regeneration owing to their compatibility, antibacterial efficacy, and mechanical attributes.¹⁵⁵

(h) Freeze-drying: utilizing the principle of sublimation, freeze-drying technology is a go-to method for generating porous scaffolds in tissue engineering applications.¹⁵⁶ First, the solvent was used to dissolve the polymer in order to construct a scaffold with high porosity. Subsequently, the mixture was frozen and solvent removed by sublimation, and the scaffold structure was finally obtained. This method has the advantage of simple operation and makes it possible to fabricate highly porous scaffolds with controllable pore size, a key requirement for tissue engineering. The highly porous structure of the scaffold can promote cell adhesion, proliferation, and differentiation by providing a suitable microenvironment for cell growth,¹⁵⁷ thereby improving the functionality of the regenerated tissue. Therefore, freeze-drying technology has shown important application value and potential in the field of tissue engineering.¹⁵⁶

In addition, using precise deposition techniques controlled by computers, bioprinting constructs 3D structures of tissues or organs by placing cells and biological materials accurately.¹⁵⁸ Commonly used in tissue engineering, regenerative medicine, and drug screening, bioprinting techniques encompass laser-induced forward transfer, inkjet printing, and robotic dispensing.¹⁵⁹ Through bioprinting, tissue structures similar to real organs can be created for studying disease mechanisms, testing drug effects, and functional organ transplantation such as kidney and liver in the future. Compared with traditional organ culture technology, bioprinting can significantly shorten the research and development time, improve the success rate of organ transplantation, and bring new opportunities for future medical research and clinical application.¹⁶⁰

3.3 Loading scaffolds with antimicrobial agents

In the field of tissue engineering and regenerative medicine, the antibacterial properties of scaffold materials are of vital importance.¹⁷² Bacterial infection is a common challenge in bone repair processes and often leads to implant failure. However, the extensive use of ABs has limited local antibacterial effects at the site of infection and is prone to side effects. Therefore, the development of scaffolds loaded with antibacterial agents has become a research hotspot. These scaffolds, by incorporating antibacterial components into the material, can effectively inhibit bacterial growth, reduce inflammatory responses, and provide a safer and more conducive microenvironment for tissue regeneration.^173,174 Moreover, scaffolds loaded with antibacterial agents can prolong the effective duration of drug action, reduce systemic toxicity, and enhance therapeutic efficacy.¹⁷⁵ These scaffolds, characterized by their multifunctionality, biocompatibility, and customizability, exhibit broad application prospects and have become an important research direction in tissue engineering and regenerative medicine.

Biodegradable and alternative natural and synthetic vectors, developed in recent years, now offer a means to deliver ABs and drugs locally to the site of infection, thanks to advancements in biomaterials.¹⁷⁶ Combining scaffold implantation with local antibiotic delivery at the injury site may enhance bone regeneration while preventing infections. In this study, Iglesias-Mejuto et al. developed a novel approach combining 3D-printing of polysaccharide-based inks with supercritical (sc) CO₂ drying to create methylcellulose aerogels containing nHAP for BTE. These aerogels were loaded with Van for treating bone infections. Biocompatibility tests and antibacterial experiments demonstrated that the scaffolds exhibited good biocompatibility, excellent bactericidal ability, and sustained antibiotic release, the drug-loaded aerogel has the advantages of high safety, low irritation and obvious mineralization ability (Fig. 5).¹⁷⁷ It has been found that bisphosphonates, an anti-catabolic drug, exhibit potent suppression of bone resorptive activity through strong interaction with HAP crystals. Qayoom et al. used a composite of semi-hydrated calcium sulfate (CSH) and nHAP to deliver anti-tuberculosis drugs rifampin (RFP) and isoniazid (INH). Analysis revealed that the addition of drugs did not significantly alter the nanocement microstructure. The setting time of the composite increased with drug incorporation, rising with higher drug concentrations. Drug release studies revealed rapid release of INH and slower release of RFP, indicating different interactions between the ABs and nHAP. The biphasic ceramic-based delivery system effectively released antibacterial and anti-biofilm medications, showing potential for treating skeletal and joint tuberculosis.¹⁷⁸

Fig. 5 Biological evaluation of 3D-printed aerogel scaffolds:¹⁷⁷ (a) cell viability of NIH/3T3 cells after 24 and 72 hours of contact with aerogels, assessed using the MTT assay. (b) Mortality rate of brine shrimp after 24 hours of contact with aerogels; controls include DMSO (positive) and artificial seawater (negative). (c) HET-CAM test results for aerogels, with 0.9% NaCl (negative control) and 0.1 N NaOH (positive control). (d) Aerogels cultured for 24 hours on TSA plates and in TSB tubes inoculated with S. aureus. (e) Mineralization values of aerogel formulations at 14, 21, and 28 days, measured by Alizarin Red staining. (f) Alizarin Red staining images of MC3T3 cells in contact with aerogels (wet and dry forms) after 28 days (scale bar: 200 μm). Copyright Iglesias-Mejuto et al. 2024 Elsevier.

Nanotechnology-based methods are gaining prominence as effective solutions for treating a range of bone diseases, such as infections, osteoporosis, and cancer. For instance, NPs made from mesoporous bioactive glass (MBG) exhibit superior structural and textural characteristics. MBG is a novel biomaterial primarily composed of oxides of silicon, calcium, and phosphorus. It features a large number of nanoscale pores within its structure, which endow it with a greater specific surface area, enhanced biocompatibility, and improved bioactivity, it is a good scaffold material.¹⁷⁹ Incorporating bioactive materials and therapeutic ions into scaffolds significantly enhances their properties and biological functionality. Sandra developed a Zn²⁺-rich, curcumin-loaded mesoporous SiO₂–CaO–P₂O₅ glass nanoparticle and evaluated its bone regeneration, antibacterial properties, and biocompatibility. The NPs effectively degraded bacterial biofilms, inhibited bacterial growth, and prevented biofilm formation while promoting osteoblast establishment. The combination of Zn²⁺ and curcumin synergistically improved antibacterial efficacy and bone regeneration, achieving dual benefits of facilitating bone healing and managing infections.¹⁸⁰ Chen et al. Obtained polycaprolactone (PCL)/polylactic acid (PLA) core–shell porous drug-carrying nanofibers by using coaxial electrospinning technology and the nonsolvent-induced phase separation method. Roxithromycin (ROX), a kind of antibacterial agent, was encapsulated in the core layer. The further antimicrobial activity demonstrated that the inhibition zone diameter of the coaxial nanofibers with two different pore sizes was 1.70 ± 0.10 cm and 1.73 ± 0.23 cm, exhibiting a good antibacterial effect against S. aureus. Therefore, the prepared nanofibers with the coaxial porous structures could serve as promising drug delivery systems.¹⁸¹

Tissue engineering in bone regeneration focuses on the development of bioactive implants with bone-conducting and bone-inducing properties. The utilization of 3D printing to customize the implant structure to create a suitable bone scaffold to load and deliver antibacterial NPs. Yang et al. used 3D printing to create in situ scaffolds made of PCL containing ampicillin (Amp) and Mg microspheres. The 3D printing ensured uniform distribution of Mg and Amp within the PCL matrix, as confirmed by imaging and analysis. The scaffold's slow degradation enabled sustained drug release at the defect site. In vitro and in vivo experiments showed that the composite scaffold effectively inhibited E. coli and S. aureus growth, enhanced cell adhesion and proliferation, and exhibited strong osteoinductive properties. This dual-functional scaffold, combining antibacterial and osteogenic capabilities, offers a promising solution for infected bone defects.¹⁸² Qian et al. prepared a novel Ta/GelMA/PLGA/Van composite scaffold by encapsulating Van in PLGA microspheres by double emulsion method, and loading it into the additive manufacturing porous tantalum (AM-Ta) scaffold by GelMA hydrogel. It is used to repair infected skeletal defects. The physicochemical characteristics of the scaffold suggested that the release of Van was maintained for over 2 weeks. The outcomes of the biological experiments demonstrated that the composite scaffold exhibited excellent biocompatibility, significant antibacterial properties and osseointegration ability, indicating its great potential in clinical application.¹⁸³

In conclusion, high loading and long-term slow release of antibacterial drugs can be achieved by using scaffolds to load antibacterial drugs. The drug is separated from the scaffold material and has no bearing on the scaffold's mechanical properties. The preparation is relatively simple and flexible, but there may still be an initial mass release problem of drug on the scaffold surface. In summary, this treatment modality combines the advantages of local targeted drug delivery, sustained release, and biocompatibility, and is a promising new method for the treatment of bone infection.

3.4 Antibacterial coatings

Orthopedic implants possess outstanding biocompatibility, biodegradability, porosity, and mechanical strength, yet they lack antibacterial properties. Therefore, various methods have been developed to achieve antimicrobial activity, and surface coating is a promising strategy that has been extensively employed to simultaneously address the poor mechanical properties of scaffolds while enhancing their degradation rate and biological characteristics.^173,184

Inhibiting bacterial growth and killing invading bacteria through various mechanisms are the ways in which both metal particles and metal NPs typically display their antimicrobial activity.¹⁸⁵ Coatings made from antibacterial materials, like metal ions or metal NPs such as Ag, Cu, Zn, and Mg, capable of effectively curbing the proliferation of Gram-positive and Gram-negative bacteria. They are commonly employed as coatings or incorporated within orthopedic implants to strengthen their antimicrobial effects and mitigate infection risks. For instance, additively fabricated ZN–1Mg porous scaffolds, in conjunction with composite coatings, can modify biodegradability (Fig. 6). Van is anchored by polydopamine (PDA) coatings to enhance antibacterial properties, while bone morphogenetic protein 2 (BMP2) growth factors are loaded into HAP coatings to further promote osteogenic activity. This combination effectively supports bone recovery and enhances antibacterial functions.¹⁸⁶ Rodríguez-Contreras et al. prepared a calcium Ag titanate coating on the Ti surface by thermochemical treatment to boost the bioactivity and antibacterial performance of 3D-printed porous Ti constructs. The antimicrobial efficacy of the coating was evaluated using Gram-negative bacteria, such as Pseudomonas aeruginosa and E. coli, in addition to Gram-positive bacteria, including S. aureus and Staphylococcus epidermidis. Incorporating Ag ions into the calcium titanate layer was found to produce AgNPs on the 3D Ti surface. These NPs displayed significant antibacterial efficacy, reducing bacterial adhesion and growth.¹⁸⁷ To confer antibacterial properties, other metals like Zn, Mg, and Cu have been incorporated into implants. For example, Mg alloys coated with Zn can inhibit bacterial growth in vitro but exhibit poor osteogenic capacity. In the design of bone implants, both antibacterial and osteogenic abilities are crucial.⁷⁹ Owing to their outstanding biocompatibility, Ti materials are extensively utilized in orthopedic repair, mechanical properties, and osteointegration, which facilitate the restoration and reconstruction of human functions. 3D-printed porous Ti scaffolds reduce the elastic modulus of Ti alloys and enhance osteointegration. However, the bioinertness of Ti alloys weakens the implant-bone tissue interface bonding. Cheng et al. found that doping calcium silicate coatings with multiple metal ions can endow Ti alloys with various biological properties. A CuO and SrO-doped calcium silicate coating was applied to the exterior of 3D-printed porous Ti alloy scaffolds using atmospheric plasma spraying. In vivo bone integration was evaluated using a rabbit bone defect model, showing that 2% CuO–10% SrO-doped calcium silicate modified scaffolds improved osteointegration. The in vitro antibacterial properties of the scaffolds were assessed through bacterial platform coating, co-culture liquid absorbance detection, and crystal violet staining, demonstrating good antibacterial performance. In summary, the composite scaffold combines osteointegration and antibacterial properties, holding great clinical potential.¹⁸⁸

Fig. 6 Additively fabricated Zn–1Mg porous scaffold:¹⁹³ (a) the structure and elemental distribution of Zn–1Mg powder, as well as the designed porous scaffold model. The designed scaffolds had a pore size of 600 μm, a pore interval of 450 μm and the porosity of 63.55%. The size distribution of powders was 15–53 μm. The content of the Mg element in powders was 1.001 ± 0.004 wt%. (b) Schematic diagram of the preparation process. The Zn–1Mg samples were preprocessed and activated with NaOH. After the first PDA coating deposition, they were washed and dried to form PV/Zn–1Mg. Then, HA coating deposition and BMP2 treatment were conducted. Finally, the samples were dried to obtain HB/PV/Zn–1Mg. (c) Overview images of the samples. Copyright Zhang et al. 2023 Elsevier.

Beyond metallic materials, polymer coatings are highly promising because of their biocompatibility, biodegradability, structural resemblance to the extracellular matrix, and superior cell affinity.¹⁸⁹ The antibacterial and mechanical properties of bioactive glass scaffolds can be enhanced by Alg coatings, without causing any significant changes to their interconnected porosity.¹⁹⁰ For BTE, biomimetic nHAP/polymer composite scaffolds hold great promise due to their excellent mechanical properties and ideal bioactivity. A concentrated alginate/gelatin scaffold with a nHAP coating was created by combining 3D printing and in situ mineralization. The thickness of the coating was adjusted by manipulating the phosphate ion concentration in the printing ink. Compared to uncoated scaffolds, the scaffold with the nHAP coating had a Young's modulus that was doubled. Additionally, the coating not only significantly enhanced the proliferation and osteogenic differentiation of rat bone marrow stem cells but also elevated protein adsorption on the scaffold surface. These features make the biomimetic nHAP/hydrogel composite scaffold a strong contender for BTE applications.¹⁹¹ CS, as a natural polysaccharide material, has a structure close to the natural extracellular matrix, and has good biocompatibility. The positive charge carried by CS can interact with the negative charge on the bacterial cell membrane to destroy the bacterial cell membrane structure, thereby playing an antibacterial and killing role. Qian et al. developed an antibacterial biocomposite scaffold based on polyelectrolyte modification. Using 3D printing, strontium-doped hydroxyapatite (SrHA) was embedded into a PCL scaffold, which was then coated with carboxymethyl chitosan (CCS) and hyperbranched polylysine (HBPL) to adsorb SLIT3 protein. This design allows for controlled release of SLIT3 and Sr²⁺. The scaffold exhibits good biocompatibility, promotes cell proliferation and migration, and stimulates angiogenesis and osteogenesis. It also enhances these processes indirectly by promoting macrophage M2 polarization and increasing anti-inflammatory factors, rendering it a promising option for bone defect repair.¹⁹²

In addressing bone infections, the preparation of antibacterial coating on the surface of tissue engineering scaffolds is a cutting-edge technology, which can significantly lower the risk of infection. By selecting appropriate antibacterial materials, not only can the growth of bacteria be inhibited, but also improve the adhesion and proliferation of bone cells to facilitate the process of bone remodeling, so as to achieve the dual effects of antibacterial and regeneration. In the future, with the progress of material science and biotechnology, the preparation of antibacterial coatings on the surface of tissue engineering scaffolds will be developed in the direction of more efficient and safer.

3.5 Mixing antimicrobial agents during scaffold fabrication

Bone regeneration is a clinical challenge that requires multiple approaches sometimes, it also includes the development of osteogenic and antibacterial biomaterials to treat the emergence of infectious processes that may be caused by surgery. The preparation of antibacterial agents and scaffold materials with bone repair can achieve uniform dispersed mixing of drugs and scaffold materials,¹⁹⁴ avoid a large amount of initial release on the surface, and the preparation process is simple and one-time, which will streamline the production process and minimize the need for subsequent treatment. This integrated manufacturing process can reduce costs, improve production efficiency, and is a good means of drug delivery.¹⁹⁵

It has been found that local sustained release of antimicrobial agents can be attained via mixing antibacterial agents directly with the scaffold material. This approach is able to provide a higher antimicrobial concentration at a specific site of the scaffold or in areas where bacteria accumulate, thus effectively preventing the occurrence of infection.¹⁹⁶ The antibacterial agent mixed with the scaffold material can also significantly improve the antibacterial ability of the scaffold. Flexible nanoporous filaments were created by Ahangar et al. using blends of thermoplastic polyurethane and polyvinyl alcohol (LAY-FOMM and LAY-FELT) through 3D printing, aimed at delivering ABs in orthopedic applications. These materials, LAY-FOMM and LAY-FELT, can be shaped into drug delivery scaffolds that release tetracycline sustainably over a period of three days. When tested with primary human fibroblasts, they displayed excellent biocompatibility and no signs of cytotoxicity, suggesting that they are promising candidates for localized antibiotic delivery and effective options for preventing infections in orthopedic reconstruction surgeries.¹⁹⁶ In the study by Shuai et al. oxygen vacancy (OV) defects Fe-doped TiO₂ (OV-FeTiO₂) NPs with antibacterial properties were synthesized using nano TiO₂ and Fe₃O₄ as precursors. These NPs were subsequently incorporated into a polycaprolactone/polyglycolic acid (PCLGA) biodegradable polymer matrix, and a composite bone scaffold was constructed via selective laser sintering. The results demonstrated that the composite scaffold exhibited broad-spectrum antibacterial characteristics. Moreover, it possessed mechanical properties that matched those of human cancellous bone and exhibited excellent biocompatibility, which is conducive to cell adhesion, proliferation, and osteogenic differentiation.¹⁹⁷

Hybrid printing can not only endowing scaffolds with antibacterial properties, but also introduce components that promote bone and cell adhesion on demand, so that scaffolds can prevent infection, promote cell growth and tissue repair, and achieve “therapeutic integration”. HAP is a kind of bioceramics commonly used in orthopedics. It integrates well with human tissues, does not cause immune rejection, has good osteogenic activity, and is a good drug-loaded scaffold. Qayoom et al. integrated the wide-range antibiotic rifampicin (RFP) into a biphasic nHAP/calcium sulfate ceramic support scaffold (NC) to treat osteomyelitis (Fig. 7). The results indicate that in vivo experiments showed that RFP-loaded NC could completely eradicate pathogens in bone lesions, and trabecular bone was formed at the debridement site when implanted with NC + RFP, and the defect healed completely.¹⁹⁸ Beenken et al. developed an osteogenic bone-filling scaffold made of degradable polyurethane (dPU), nHAP, and decellularized bovine bone particles (DBP). The scaffold serves as a local antibiotic carrier to mitigate the risk of infections in bone defects contaminated by S. aureus. Results showed a significant reduction in live bacteria and maintained good osteogenic and bone repair properties even with high Van concentrations.¹⁹⁹

Fig. 7 A broad-spectrum antibiotic, RFP, was incorporated into a biphasic nHAP/calcium sulfate ceramic carrier (NC) system, and its in vitro biocompatibility and degradation were investigated:¹⁹⁸ (a) scanning electron microscopy (SEM) images of the NC and NC + RFP scaffolds, with magnified views shown in the insets, revealed that incorporating RFP into the scaffolds did not significantly alter their microarchitecture. (b) and (c) SEM and confocal laser scanning microscopy (CLSM) images of MC3T3E1 cells cultured on the surfaces of NC and NC + RFP for 7 days, demonstrating uniform cell distribution. (d) MTT assay results for MC3T3E1 cells on NC and NC + RFP surfaces, monitored over a 1-week period. The results showed that the cells continued to grow and proliferate, and all had good biocompatibility. (e) In vitro degradation profiles of NC and NC + RFP. After 5 weeks, the percentage degradation of NC was 39.6 ± 0.98% and that of NC + RFP was higher, around 47.53 ± 0.94%, showing enhanced degradation of NC upon incorporation of RFP. Copyright Qayoom et al. 2020 American Chemical Society.

Recent advances in regenerative medicine have provided many reliable strategies for bone-related treatments. Hydrogel scaffolds, notable for their biocompatibility and ability to mimic the extracellular matrix of the surrounding environment, offer excellent properties for biomedical applications, have been successfully used as materials for graft scaffolds, is a good hybrid printing material. Ramprasad and his colleagues fabricated a 3D Ti scaffold incorporating gelatin hydrogel enriched with strontium-doped Ag NPs (Sr–Ag NPs) was fabricated using metal laser sintering technology. The composite scaffold was evaluated for cell compatibility and osteogenic potential. It was found that the scaffold promoted Runx2 expression and matrix mineralization, while also demonstrating excellent antibacterial properties. These characteristics point to its potential effectiveness in addressing bone infections.²⁰⁰ Photothermal therapy (PTT) is a promising antibacterial method, which has been focused on for dealing with bone infections and tissue engineering. This is because the photothermal agents used in PTT can generate heat NIR laser irradiation to kill bacteria and eliminate biofilms, with the advantages of minimal side effects, strong tissue penetration, and remarkable therapeutic effects. GelMA is a gelatin engineered with methacrylate (MA) groups through photo-polymerization. In addition to demonstrat the physical crosslinking behavior induced by temperature changes, as seen in gelatin, it allows for permanent photo-crosslinking under mild conditions during bioprinting. Nie et al. developed a composite hydrogel scaffold composed of GelMA, β-TCP, NaAlg (Sr²⁺), and MXene (Ti₃C₂) (GTAM) via 3D printing technology. The photothermal effect of MXene enables the GTAM scaffold to effectively kill both Gram-positive and Gram-negative bacteria NIR irradiation. Rat bone marrow mesenchymal stem cells were mixed into the GTAM bioink for 3D bioprinting. The resulting cell–laden GTAM scaffold exhibited good biocompatibility and osteogenic capacity under the action of MXene, cross-linked Sr²⁺, and β-TCP. It accelerated the reparation of infections and bone remodeling, while exerting a dual synergistic effect on antibacterial and osteogenic performance.²⁰¹

In summary, combining antibacterial agents with 3D-printed scaffolds enables the precise delivery of these agents straight to the infected area, thereby significantly increasing local drug concentration while effectively reducing the side effects associated with systemic administration. Moreover, this combination allows for the slow and sustained release of antibacterial agents, maintaining effective concentrations over an extended period and further enhancing therapeutic outcomes. By integrating antibacterial agents directly into the scaffold material, the scaffolds themselves acquire sustained antibacterial properties, which can effectively prevent infections following implantation. With the aid of 3D printing technology, personalized treatment plans can be adjusted to fit the particular needs of each patient. Overall, this type of antibacterial 3D-printed scaffold integrates multiple advantages such as targeted delivery, controlled release, and inherent antibacterial properties, offering a new solution for preventing and treating bone infections and holding great promise for clinical applications.

4. Application of drug-loaded scaffolds in bone infections

Bone infection is a serious clinical problem that causes great suffering to patients. The aim of treating bone infection is to completely eliminate the infectious focus, ensure soft tissue coverage, promote bone end healing, and preserve limb length and function. After complete removal of the infected lesion, a bone defect is often present, which is the site of recurrence of the infection. The histological and biomechanical repair these defects is a time-consuming process and has emerged as a focal point in osteomyelitis treatment research. At present, several challenges remain in the treatment of bone infections,^202–205 such as, multi-drug resistant bacteria reduce the effect of ABs; biofilm formation can occur by bacteria in bone tissue and attach to the surface of sequestrum or implants to resist immune and antibiotic attacks; the persistence and recurrence of infection can be attributed to infected phagocytes, such as macrophages, which act as “Trojan horses” protecting bacteria from antibiotic action.

Hydrogels are soft, water-rich materials that can encapsulate drugs to deliver antibacterial agents to infected sites, reducing infection risk. Their injectable nature allows them to fill bone defects, providing scaffolding for new bone growth and promoting regeneration. They can also carry bioactive molecules like ABs and growth factors. By adjusting their properties and structure, these agents' release rate and dosage can be controlled to prevent bacterial growth and enhance bone repair and regeneration, ultimately alleviating bone infections. Bastami et al. developed a 3D-printed hydrogel using alginate, gelatin, and bone NPs. This hydrogel promoted the adhesion, growth, and bone-forming ability of rat stem cells. Results showed that it supported bone regeneration comparable to traditional methods, with staining and protein expression confirming its effectiveness. The hydrogel has potential to enhance bone repair.²⁰⁶ In the study by Tang et al. an injectable gelatin hydrogel was created via wet spinning. Optimized for injectability, it was evaluated in a mouse cranial defect model, where it enhanced cell survival, sped up bone regeneration, and demonstrated favorable degradation and biocompatibility. It shows promise as a biomaterial for stem cell delivery and tissue regeneration.²⁰⁷ Dutta employed highly oxidized 2D carbon nitride (Ox-gCN) to enhance alginate/gelatin (ALG) hydrogels. These hydrogels exhibit anti-inflammatory properties, aid in bone healing, and possess hemostatic capabilities. Oxidized g-CN promotes stem cell adhesion and growth. The ALG/CN scaffold has antibacterial activity, supports favorable immune responses, and facilitates bone formation. In rat studies, it promoted new bone formation without inducing inflammation, demonstrating potential for bone repair applications.²⁰⁸ Therefore, injectable hydrogels are favored for small defects due to their advantages of simple manipulation, minimal trauma, and good filling effect.

Osteomyelitis is a chronic condition that demands prolonged treatment, and surgical debridement often results in larger bone defects. These defects require filling materials that can withstand stress. Solid scaffolds, known for their stability, complex design, and multifunctionality, can be optimized to match the mechanical properties of bone tissue. As a result, they are commonly used to fill and treat infectious bone defects. This mainly includes using 3D-printed solid scaffolds for bone infections and combining them with active factors, stem cells, and drugs. Liu et al. combined 2D nanostructures (graphene oxide and black phosphorus) with IL-4 into 3D scaffolds. These scaffolds improved cell adhesion, released growth-promoting ions, and created a healing immune environment. In rats, they enhanced bone regeneration and angiogenesis. The study shows this approach is effective for treating bone infections and promoting bone repair.²⁰⁹ Wu et al. created NPs with magnetic and ultrasound properties, loaded with curcumin and stem cell membranes. These were combined with CS and tricalcium phosphate to form a 3D-printed scaffold. The scaffold could disrupt bacterial biofilms and activate immune and bone-forming responses using ultrasound. In vivo tests showed it healed infected bone defects effectively without damaging nearby tissues.²¹⁰

In addition, a reliable animal model is essential for preclinical research and should closely mimic the disease. Sheep possess bone micro- and macrostructures as well as biomechanical properties that are similar to those of humans, and their use in skeletal research has been well established. Boot et al. introduced a clinically relevant large-animal model of chronic methicillin-resistant staphylococcus aureus (MRSA) infection. In this model, an injectable, thermoresponsive, HA-based hydrogel scaffold loaded with gentamicin and Van outperformed current clinical practices, eradicating bacteria in all animals. Local antibiotic delivery using the injectable hydrogel scaffold significantly enhanced the success rate of treatment.²¹¹ Local antimicrobial therapy is a crucial component in managing orthopedic device-related infection (ODRI), traditionally delivered through polymethyl-methacrylate (PMMA) bone cement. However, PMMA has limitations due to its suboptimal antibiotic release profile and lack of biodegradability. In a recent study, researchers compared the efficacy of PMMA versus an antibiotic-loaded hydrogel in a single-stage revision for chronic MRSA ODRI in sheep, a bone infection model was established using 13 Swiss Alpine sheep, which were treated with either antibiotic-loaded bone cement rods or hydrogels. The results demonstrated that the antibiotic-loaded hydrogel scaffold was effective in the single-stage exchange for MRSA ORDI in sheep. It exhibited good biodegradability and favorable drug release characteristics, with broad potential applications. However, further research is needed to facilitate the translation of this preclinical approach to clinical trials.²¹²

As research continues to advance, antimicrobial BTE materials will further develop new high-performance antimicrobial materials, such as stimulus-responsive smart drug delivery systems, to enhance antimicrobial effectiveness. Additionally, by integrating advanced manufacturing technologies, we can achieve personalized design and customized production of antimicrobial materials. We will also investigate the mechanisms by which antimicrobial materials influence the behavior of bone cells to optimize their bioactivity. Furthermore, we will conduct long-term animal experiments and clinical trials to verify the safety and efficacy of antimicrobial BTE materials. This will facilitate the industrialization of these materials to meet clinical needs and benefit more patients. In summary, the research on antimicrobial BTE materials is thriving and is expected to provide new solutions for clinical bone repair and regenerative therapies, ultimately improving the treatment experience for patients.

5. Conclusion

Bone infection, like osteomyelitis, is a serious skeletal disorder caused by bacterial infection, leading to bone destruction, functional loss, and potential systemic infection. Treatment is difficult due to bacterial biofilms, poor antibiotic penetration, and limited local blood supply. Traditional methods include long-term ABs and surgery, but they often result in high recurrence rates, long treatment times, and significant side effects. In recent years, antibiotic-resistant bacteria have made bone infection treatment more challenging, driving the need for better strategies. Tissue engineering scaffolds with antimicrobial agents are a new approach that combines tissue engineering and drug delivery. These scaffolds are made from biocompatible materials, loaded with antimicrobial agents, and implanted at the infection site. This review summarizes antimicrobial agents and their incorporation into drug-loaded scaffolds. Scaffold materials enable sustained release of antimicrobial agents at the infection site, increasing local drug concentration while reducing antibiotic dosage, minimizing resistance risk, and lowering systemic side effects. Some scaffold materials like HAP and PLA have antimicrobial properties and support osteoblast growth to promote bone regeneration. Scaffolds can also load multiple drugs (e.g., ABs and anti-inflammatory agents) to achieve synergistic effects, combining antibacterial and bone repair functions. Drug loading methods include direct incorporation, coating, or mixing antimicrobial agents during scaffold fabrication. However, there are still some limitations, such as difficulties in achieving precise control, issues related to biocompatibility, mismatch between degradation rates of scaffolds and bone regeneration rates, limited drug activity, and challenges in clinical translation.

Although drug-loading methods in tissue engineering scaffolds have limitations, they hold great promise for treating bone infections. Future research can focus on several key areas: optimizing drug release systems to create smart, responsive scaffolds; developing new biocompatible materials to improve mechanical properties and controllable degradation rates; integrating physical modalities like PTT and electrical stimulation to create multifunctional scaffolds for synergistic effects; strengthening the link between basic research and clinical needs; optimizing manufacturing processes for large-scale scaffold production and clinical application; and leveraging 3D printing technology for precise customization and rapid fabrication of scaffolds. We believe that future advancements in optimizing drug release systems, enhancing scaffold materials, designing multifunctional scaffolds, and promoting clinical translation will lead to significant breakthroughs in treating bone infections, offering patients more efficient and safer options. Additionally, by incorporating precision medicine and personalized treatment concepts, drug-loading methods in tissue engineering scaffolds will become increasingly important in this area. With ongoing research, we expect to see even more breakthroughs in the treatment of bone infections.

Conflicts of 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.

Data availability

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Acknowledgements

This work is financially supported by Medical and Health Science and Technology Program of Zhejiang Province (no. 2024KY1458).

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