Biomaterial-Based Scaffolds as Carriers of Topical Antimicrobials for Bone Infection Prophylaxis

For many years, there has been a systematic increase in the demand for transplantation of damaged tissues and organs in various fields of surgical medicine, mainly due to advances in surgical techniques and greater accessibility to regenerative medicine procedures. The response to this demand has been the emergence of a new scientific field called tissue engineering, which has become an important adjunct to interventional medicine by, among other things, enabling the regeneration of living tissues and organs through cell culture (Ashammakhi et al. 2022). Tissue engineering methods are also used for the needs of orthopedic and trauma surgery, which primarily involves providing an appropriate environment for cell growth, protection from microorganisms, providing additional structures that support cell growth in the correct spatial configuration, and restoring the mechanical properties of the damaged bone in a short period (Sarigol-Calamak and Hascicek 2018). Cells must grow in three dimensions to form an organ or tissue, so porous spatial structures, called scaffolds, that simulate the extracellular matrix of tissues are used in tissue engineering. Due to their high biocompatibility and in combination with various therapeutic agents, these structures can facilitate the attachment of cells from surrounding tissues to their surface, stimulate cell proliferation and differentiation, and protect the growing cells from infection (Dorati et al. 2017). Using orthopedic scaffolds can significantly reduce the regeneration time of damaged bones and restore lost tissue functionality.

Studies conducted by many authors to date have shown that scaffolds that locally release various antimicrobial substances can significantly reduce the occurrence of infectious complications in orthopedic procedures. The use of appropriately selected amounts of antimicrobial agents along with the selection of the appropriate release rate from the scaffolds can help reduce the frequency of recurrent infections (Dorati et al. 2017; Sarigol-Calamak and Hascicek 2018). The results of many previous studies indicate that one of the more effective ways to regulate the dosing of antimicrobial substances to prevent or combat bone infections is their local release at the site of damage from a nanomaterial embedded in a scaffold, which serves as a carrier that regulates the release profile of the active substance (Afewerki et al. 2020). Therefore, it should be noted that any newly developed scaffolds with antimicrobial activity will require extensive microbiological studies, both in vitro and in vivo in experimental animals, before being approved for use in patients.

Scaffolds that have been designed and fabricated as carriers of local antimicrobial agents for orthopedic applications have been made from polymers (Lee et al. 2020; Aslam Khan et al. 2021a), ceramics (Zhao et al. 2022; Atkinson et al. 2024), metals (Kiselevskiy et al. 2023), or composites (Cui et al. 2022). Current techniques used to prepare scaffolds for bone tissue engineering include traditional preparative techniques such as electrospinning, freeze drying, solvent casting/particle leaching, and additive manufacturing techniques such as fused deposition modeling, selective laser sintering, stereolithography, 3D and 4D bioprinting (Qin et al. 2024). However, the techniques currently used to apply bone scaffolds include implantation of a prefabricated scaffold created prior to the surgical procedure (Shen et al. 2022; Lu et al. 2024), in situ scaffold formation using an injectable system (Zhang et al. 2023; Ghiasi Tabari et al. 2024), and 3D printing directly at the site of injury in clinical conditions to create or repair living tissues or organs (in situ in vivo bioprinting) (Li et al. 2020; Mahmoudi et al. 2023).

Controlled release of antimicrobial drugs from scaffolds designed for bone tissue engineering is one of the methods used to reduce the risk of infectious complications following medical device implantation procedures. This method makes it possible to achieve high antimicrobial efficacy at the site of medical device implantation using relatively low concentrations of antibiotics, which significantly reduces the occurrence of microbial resistance. To date, most studies on the antimicrobial activity of antibiotic-releasing scaffolds have been conducted with glycopeptide antibiotics, mainly vancomycin (VAN) and aminoglycosides e.g. gentamicin (GEN) (Hassani Besheli et al. 2018; Zhou et al. 2018b; Budiatin et al. 2021).

In studies using a biodegradable 3D-printed scaffold made of polylactic acid (PLA) combined with hydroxyapatite (HA) with VAN, the scaffold was found to have sufficient antibacterial and antibiofilm activity against S. aureus, and the antibiotic was released for more than 7 days as the polymer degraded (Pérez-Davila et al. 2023). In the case of scaffolds made of mesoporous bioactive glass combined with poly(lactic-co-glycolic acid) (PLGA) with VAN, the antibiotic was shown to be released from the scaffold for more than 8 weeks, resulting in inhibition of S. aureus growth and biofilm formation in vitro (Cheng et al. 2018). In other studies of 3D polycaprolactone (PCL) composite scaffolds coated with polydopamine, VAN-loaded PLGA microspheres were adsorbed (Zhou et al. 2018b). The antibiotic was shown to be released from the scaffolds in vitro for more than 4 weeks, and its effective bactericidal activity against S. aureus was observed throughout this period. In the in vivo studies on laboratory animals conducted by Zhou et al. (2018a), the efficacy of scaffolds made of gelatin and β-tricalcium phosphate (β-TCP) filled with vancomycin in clearing infections and repairing bone defects in the course of osteomyelitis was investigated. The results showed that the antibiotic was released from the scaffolds over 8 weeks, resulting in the elimination of the bone infection and the rapid repair of bone defects without complications. Other studies have also used gelatin and β-TCP scaffolds, combined with chitosan microspheres containing GEN (Liu et al. 2022). In this case, the antibiotic released from the scaffolds initially rapidly and effectively inhibited bacterial proliferation. Then, it was released at lower concentrations in the later phase to provide anti-infective protection during bone formation.

In some studies, scaffolds of different biomaterials were loaded with selected fluoroquinolone antibiotics such as ciprofloxacin (CPFX) and levofloxacin (LFX). It was observed that under in vitro conditions, approximately 70% of the antibiotic was released from the scaffold within 14 days, ensuring high antibacterial efficacy against S. aureus and E. coli, and the scaffold underwent spontaneous biomineralization leading to osteoinduction. In other studies, the in vitro antibacterial activity of monticellite (calcium-magnesium silicate ceramic) bone scaffolds containing different doses of CPFX was determined (Bakhsheshi-Rad et al. 2019). In the case of a scaffold containing 6% CPFX, 100% inhibition of S. aureus and E. coli growth was demonstrated after 48 hours of incubation, and the antibiotic was released from the scaffold in effective concentrations for at least 16 hours. In the studies conducted by Wei et al. (2019), the antibacterial properties of a bone scaffold made of a bioactive nanohydroxyapatite/polyurethane composite with LFX-loaded mesoporous silica microspheres immobilized on its surface were investigated. The release of the antibiotic from the scaffold in vitro was maintained for 42 days at concentrations that caused the death of 99.99% of S. aureus and E. coli cells.

Most studies evaluating the antimicrobial efficacy of antibiotic-releasing biomaterials have shown that these substances released at the implant site effectively kill bacteria and prevent biofilm formation. This therapeutic approach has made it possible to reduce the extent of systemic antibiotic use in most orthopedic procedures, which in turn has slowed the buildup of antibiotic resistance associated with this type of clinical situation. However, optimizing antibiotic release from biomaterials to minimize the risk of drug resistance remains the most serious problem in this area. On the one hand, too rapid release of antibiotics from the surface of biomaterials can result in toxic effects. On the other hand, gradual, continuous release of antibiotics over too long a period can result in antibiotic resistance in specific clinical situations, even though in most cases it allows better reduction of bacterial proliferation. Therefore, in recent years, many researchers have indicated that shortly, precise control of antibiotic release from biomaterials may be possible through the implementation of innovative reactive coatings that are designed to release antibiotics in response to detecting changes in the microenvironments infected with bacteria (the drug release profile depends on changes in the pH of the surrounding environment, for example).

Another way to achieve a local antimicrobial effect at the site of bone scaffold implantation is to incorporate into its structure ions of selected metals or chemical compounds of these metals that have previously been shown to have antimicrobial activity in vitro. There are many potential options in this area. Currently, they are most commonly based on the use of silver ions (Ag⁺), zinc (Zn²⁺), magnesium (Mg²⁺), as well as zinc oxide (ZnO) and titanium dioxide (TiO₂). Less commonly, selenium (Se) and strontium (Sr²⁺) ions and copper oxide (CuO) are used. It should be noted, however, that the incorporation of metal ions or their compounds into the structure of bone scaffolds requires exact control of the released concentrations of these chemicals, since exceeding specific doses can cause local toxic effects (e.g., cell necrosis) or systemic effects (damage to the brain, spleen, liver, and other organs), as presented in the review by Gulati et al. (2021). Therefore, for many years, the majority of research in this area has focused on the use of metals or their oxides in the form of nanoparticles (NPs), which are more effective at low concentrations due to their higher surface-to-volume ratio, and also possess other specific properties such as high reactivity and greater diffusivity in tissues. In addition, the use of nanocarriers loaded with antimicrobial agents allows for prolonged release of antimicrobial agents and precise regulation of their concentrations, which in the case of metals or their oxides, allows for effective antimicrobial action while avoiding toxic effects, as discussed in the review by Agnihotri and Dhiman (2017).

Among all metal nanostructures, silver nanoparticles combined with various organic or inorganic carriers are most commonly used to create scaffolds for bone surgery with antimicrobial activity. For example, Zhang et al. (2017) used silver nanoparticles (AgNPs) uniformly dispersed on graphene oxide (GO) to create a homogeneous nanocomposite, which was successfully modified on 3D-printed bioceramic scaffolds with β-TCP. The resulting bone scaffolds were not only effective in killing bacteria, but also had a positive effect on osteogenesis by promoting the expression of an osteoblast-related gene in bone marrow stem cells. In another study, a nanocomposite of cellulose nanowhiskers decorated with AgNPs was used to create bone scaffolds containing chitosan and carboxymethylcellulose, achieving high antimicrobial activity of the scaffold against Gram-positive and Gram-negative bacteria (Hasan et al. 2018). Chitosan is often used to fabricate bone scaffolds with metals due to its biodegradability, excellent biocompatibility and bioactivity, and very good antimicrobial properties. Cao et al. (2018) developed a composite scaffold of double hydroxide/chitosan with a MgSrFe layer, which was uniformly loaded with AgNPs on the surface. It was demonstrated that the scaffold effectively prevented the formation of S. aureus biofilm due to the released Sr ions and Ag-NPs, and also exhibited good osteogenic properties. In other studies, the synergistic antimicrobial effect of chitosan, HA, and silver nanowires was exploited to create a composite bone scaffold that could not only inhibit the growth of bacteria in suspension, but also completely prevent the formation of biofilm by methicillin-resistant S. aureus (MRSA) strains (De Mori et al. 2019).

Many studies to date have confirmed that Zn-based biomaterials support bone repair by promoting cell proliferation, osteogenic activity, angiogenesis, and inhibiting osteoclast differentiation. They also have very good antimicrobial properties, as discussed in an extensive review by Wen et al. (2023). Zn-doped bone tissue scaffolds are fabricated using various biomaterials. Felice et al. (2018) developed nanofibrous scaffolds made of PCL combined with HA and different concentrations of ZnO, and demonstrated that these scaffolds accelerated bone tissue regeneration and exerted an effective antibacterial effect on S. aureus. In other studies, zinc oxide nanoparticles (ZnONPs) were incorporated into a porous poly(3-hydroxybutyrate-co-3-hydroxy-valerate) scaffold (Shuai et al. 2020). The addition of ZnONPs to the biomaterial used increased its crystallinity, thereby improving its mechanical properties, and the Zn²⁺ released from the scaffold effectively inhibited the growth of E. coli. In addition, the scaffold promoted cell behavior regarding proliferation and differentiation toward bone tissue. Sehgal et al. (2016) proposed rigid nanocomposite scaffolds for functional bone regeneration, which were prepared from biodegradable gellan and xanthan polymers reinforced with bioactive glass nanoparticles and additionally crosslinked with zinc sulfate ions to enhance their osteoconductive and antimicrobial properties. The scaffolds were shown to significantly inhibit the growth of Gram-positive bacteria, Bacillus subtilis (70% reduction), and Gram-negative bacteria, E. coli (81% reduction), and caused a 62% increase in alkaline phosphatase (ALP) activity expression and a 150% increase in calcium deposition. Another interesting solution was the development of 3D-printed antibacterial bone scaffolds consisting of PLA and halloysite nanotubes (HNT) filled with zinc nanoparticles and additionally coated with an outer layer containing gentamicin (Luo et al. 2020). Again, in addition to high osteoinductive potential, S. aureus growth was inhibited even when the scaffolds were stored at 37°C for 3 weeks.

In addition to the metals mentioned above, it is worth mentioning the increasing use of Mg as a component of bone scaffolds. One example is the porous forsterite scaffolds with antibacterial properties produced by combining 3D printing and Mg-containing polymeric ceramics, which, in addition to a uniform macroporous structure and high compressive strength, strongly inhibited the growth of S. aureus and E. coli in vitro (Zhu et al. 2020).

Antimicrobial coatings of biomaterials containing metal ions have been an alternative to antibiotic-releasing coatings for many years, as they reduce the risk of developing drug-resistant bacteria and exhibit longterm antibacterial activity. In particular, combinations of different metal ions make achieving a broad antimicrobial and antifungal spectrum possible. In addition, these coatings show osteointegration-promoting effects and usually exhibit excellent biocompatibility. The disadvantage of this type of coating in the case of long-term release of metal ions is the risk of excessive accumulation of these particles in the surrounding tissues, which, once specific concentrations are exceeded, can lead to somatic cell damage. In addition, metal nanoparticles released from biomaterial coatings can travel with the blood to tissues and organs throughout the body, where they can cause oxidative damage when they reach certain concentrations.

AMPs are bioactive small proteins composed of 10 to 50 amino acids, characterized by a unique antimicrobial mechanism of action that is highly effective against various species of bacteria, viruses, and fungi, but different from the mechanism of action of classical antibiotics, which has been described in detail in numerous reviews by, for example, Caplin et al. (2019), Moretta et al. (2021), Li et al. (2025). AMPs are used, among other things, to create antimicrobial scaffolds intended for bone surgery, dentistry, and as an additive to modern dressings that protect wounds from infection, which was very well presented in their review by Min et al. (2024).

Biomaterials with antimicrobial activity containing AMPs have many advantages, as these substances exhibit many different mechanisms of antimicrobial action, act synergistically with many antibiotics, can be readily incorporated into various biomaterials, and can be released into surrounding tissues in a controlled manner and provide effective local concentrations. AMP molecules can be incorporated into bone scaffolds by attaching them to the surface or embedding them within the scaffold structure. For example, Chen et al. (2019) modified the surface of PLGA-based bone scaffolds by incorporating bone morphogenetic protein-2 and ponericin-G. The authors demonstrated better adhesion of mouse preosteoblast cells (MC3T3-E1) to the scaffold surface, and an increased proliferation rate and faster calcium deposition in these cells. In addition, the scaffold exhibited long-lasting antibacterial activity against E. coli and S. aureus.

On the other hand, Tian et al. (2020) developed 3D-printed polycaprolactone/hydroxyapatite (PCL/HA) composite scaffolds whose surface was modified with ε-poly-L-lysine, achieving not only excellent biocompatibility and osteoconductivity, but also very good antibacterial activity against S. aureus, E. coli, and Streptococcus mutans. However, among the studies in which AMPs were incorporated into the interior of bone scaffold structures, the research of Karamat-Ullah et al. (2021) can be cited, who developed a series of antibacterial and biocompatible 3D scaffolds combining antibacterial silk fibroin modified with AMPs and silica, and demonstrated that this hybrid scaffold exhibited bactericidal activity against both Gram-positive and Gram-negative bacteria and was biocompatible with MC3T3-E1 cells. In other studies, a hybrid scaffold based on intrafibrillar mineralized collagen coated with AMP derived from human salivary protein was developed (Ye et al. 2021). The scaffolds exhibited high antibacterial activity against E. coli and Streptococcus gordonii bacteria over an 8-day incubation period in vitro and were also not cytotoxic to human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). Another interesting idea in this regard was the development of a porous bone scaffold made of mineralized collagen containing embedded PLGA microspheres loaded with two synthetic antibacterial peptides: Pac-525 or KSL-W (He et al. 2020). The scaffolds benefited MC-3T3 cell proliferation and osteogenic differentiation, and exhibited excellent antibacterial activity against E. coli and S. aureus for 2 weeks.

Although many versions of AMP-containing bone scaffolds have been developed in recent years and some of their beneficial properties have been demonstrated in treating infectious bone defects, it should be noted that there are still many limitations that prevent the widespread use of such solutions in vivo. The antimicrobial activity of AMPs in vivo can be interfered with by various host factors such as proteases, apolipoproteins, DNA, host proteins, and glycosaminoglycans, as presented in the review by Mookherje et al. (2020). In addition, the relatively high production costs and technological difficulties in producing large quantities of AMPs by chemical or recombinant methods remain a significant limitation to the widespread use of AMPs in therapy (Gao et al. 2021; Chaudhary et al. 2023).

Research on bone scaffolds developed based on different forms of carbon nanomaterials has been carried out for many years because these materials have a very high osteoconductive potential, can significantly improve the mechanical properties of other biomaterials, do not show cytotoxic effects on osteoblasts, and in addition, many of them have intrinsic antimicrobial activity which was very well presented in their review by Eivazzadeh-Keihan et al. (2019). In this brief review, only single studies in this field can be cited, referring to the classification of carbon nanomaterials according to their dimensions, i.e., 0D, 1D, 2D, and 3D.

As an example of the use of 0D carbon nanomaterials to develop antimicrobial bone scaffolds, Xu et al. (2021) fabricated osteoconductive, antibacterial porous membranes with high mechanical strength from PLA by direct electrospinning of microfibers impregnated with carbon quantum dots (CQDs). The authors demonstrated that when the CQD content was 1.5% relative to the weight of the PLA matrix, the reduction in the number of S. aureus and E. coli bacteria after 24 hours of incubation in vitro was 90.4% and 99.4%, respectively. This effect was probably caused by the synergism of membrane stress and oxidative stress induced by CQD. However, among the 1D carbon nanomaterials used for the development of bone scaffolds, the most research has been devoted to carbon nanotubes (CNT), which, in addition to very good antimicrobial activity, are also characterized by exceptional mechanical strength. These unique properties of CNTs have been exploited to fabricate composite bone scaffolds from various polymers and CNTs, resulting in a scaffold with better mechanical properties, which additionally facilitates the adhesion of BM-MSCs to the scaffold and accelerates their proliferation, growth, and differentiation into bone cells (Kandhola et al. 2023). For example, Shrestha et al. (2017) developed bioactive bone scaffolds made of nanotopographic polyurethane containing uniformly dispersed multi-walled CNTs and ZnONPs. The obtained scaffolds had, among others, very good mechanical strength, thermal stability, biodegradability, biomineralization, biocompatibility, and CNTs exposed on the surface of nanofibers ensured effective destruction of S. aureus and E. coli cells, most likely due to piercing the microbial cell mem-branes/walls and disrupting their nuclei.

However, most studies on the use of carbon nanomaterials for the development of antimicrobial bone scaffolds focus on using 2D nanomaterials, primarily GO and reduced graphene oxide (rGO), which exhibit different mechanical, electrical, and chemical properties. For example, Melo et al. (2020) developed 3D-printed fibrous bone scaffolds from PCL containing GO at various concentrations. They demonstrated an approximately 80% increase in S. epidermidis and E. coli mortality after 24 hours of contact with scaffolds containing 7.5% GO. In addition, PCL/GO composite scaffolds allowed adhesion, spreading, and colonization of human fibroblasts within 14 days of culture. Other studies have also confirmed that the addition of GO to various nanocomposite scaffolds at an appropriate concentration has a significant impact on achieving high antibacterial activity against Gram-positive and Gram-negative bacteria, promotes the proliferation of pre-osteoblasts, and has no cytotoxic effect (Aslam Khan et al. 2021a; 2021b). However, in the case of rGO, some researchers have exploited the excellent electrical conductivity of rGO to enhance antibacterial activity and increase cell viability through electrostimulation. Angulo-Pineda et al. (2020) developed 3D-printed composite bone scaffolds from PCL and rGO and applied an electrical stimulus at 30 V for 3 hours, achieving complete elimination of S. aureus on the scaffold surface and a 4-fold increase in the viability of hBM-MSCs attached to the conductive PCL/TrGO 3D scaffold compared to the pure PCL scaffold. The above studies certainly need to be repeated under in vivo conditions, considering the conductivity of the surrounding tissues and body fluids, to evaluate the possible adverse effects of direct electric current on tissues and organs, especially with exposures of several hours. In other studies, a mixture of tricalcium phosphate, gelatin, chitosan, and GO was used to create antimicrobial 3D bone scaffolds in which GO was subjected to in situ reduction (Cabral et al. 2019). The scaffolds functionalized in situ with rGO exhibited increased wettability and better mechanical properties, and enhanced calcium deposition on their surface and increased ALP activity over a 21-day incubation period. In addition, these scaffolds also exhibited very good antimicrobial activity without affecting osteoblast viability and proliferation.

Although many of these studies have confirmed the good antimicrobial activity of biomaterials containing various forms of carbon, their clinical applications remain severely limited due to the poorly understood mechanisms of antimicrobial action of these materials and the potential for cytotoxic effects over time. In addition, the vast majority of studies are in vitro only. Therefore, more extensive in vivo experiments are needed to evaluate the long-term adverse effects of carbon nanomaterial scaffolds, especially regarding changes in the stability of various somatic cells and their functionality. Some researchers indicate that one way to address the cytotoxicity of these materials may be to use effective methods to target them to specific tissues or cells to maximize the desired therapeutic effect and minimize side effects (Jayaprakash et al. 2024). An analogous approach should be used for biomaterials with embedded carbon nanomaterials with antimicrobial activity, using ligands specific to certain microorganisms.