Endoprosthetic replacement of destructive joints is a common procedure in human and veterinary medicine. In 2022, more than 300,000 human primary knee and hip arthroplasties were undertaken in Germany (Grimberg et al., 2023). In veterinary medicine, joint replacement is commonly established as a treatment of joint disease in small animals (Allen, 2012). A complication frequently occurring after total joint arthroplasty is the loosening of prosthetic joints. Next to aseptic loosening due to implant debris, septic loosening caused by infection at the implant site is a common reason for implant failure and revision surgery. Around 15% of revision cases of human patients in Germany were due to infection (Grimberg et al., 2023). The incidence for post-operational infections in dogs varies from 2.6%–10% (Weese, 2008; Hayes et al., 2013). Periprosthetic joint infections (PJIs) are caused by both virulent and opportunistic pathogens and can manifest directly after implantation or weeks later through different routes of infection (Benito et al., 2019). Virulent pathogens causing PJIs are Staphylococcus aureus (Benito et al., 2019; Gatti et al., 2022), Enterococcus (Renz et al., 2019; Thompson et al., 2019), Streptococcus and Gram-negative Bacillus species (Bemer et al., 2014). Coagulase-negative Staphylococcus species frequently serve as opportunistic pathogens (Benito et al., 2019; Gatti et al., 2022). In small animals, more than 50% of PJIs are caused by S. aureus and Staphylococcus pseudintermedius (Hayes et al., 2013). S. pseudintermedius is a coagulase-positive opportunistic pathogen that colonizes mainly cats and dogs and is responsible for skin infections in these species (Devriese et al., 2005; Perreten et al., 2010). Infection with S. pseudintermedius can also occur in humans (Van Hoovels et al., 2006). To prevent the occurrence of PJIs, systemic administration of antibiotics at the time of surgery and fastening of the implant with antibiotic-loaded bone cement is a widespread procedure (Bratzler et al., 2013). Treatment of PJIs includes local antibiotic treatment, debridement of the infection site and replacement of the failed implant. However, the increasing occurrence of antibiotic resistance represents a global threat for human and veterinary medicine in general and challenges the management of PJIs. Resistances are acquired and spread in places with high consumption of antibiotics, e.g., in hospitals, in agriculture and the environment (Almagor et al., 2018; Skandalis et al., 2021). Bacterial mechanisms for defense relevant for PJIs include biofilm formation and quicker adaptation to antibiotic treatment due to increased mutation rate under stress (Swings et al., 2017; Hamad et al., 2022). These problems underline the need for novel treatments in the management of PJIs. Recently proposed alternatives to antibiotic treatment are the use of novel antibiotics or the repurposing of established drugs, antibody therapy and biofilm-disrupting agents (Hamad et al., 2022).

NAC, a derivative of the amino acid cysteine, is commonly used as a mucolytic agent and as an antidote in paracetamol overdose (Plumb, 2018). Its antibacterial activity has been the focus of research in recent years. NAC has been shown to inhibit growth of planktonic bacteria and the formation of biofilms. Furthermore, it can disrupt biofilms alone and in combination with antibiotics, likely due to its acidity and interference with bacterial exopolysaccharide formation (Quah et al., 2012; Manoharan et al., 2020; Chlumsky et al., 2021; Valzano et al., 2022). The thiol group of the molecule can disrupt disulphide bonds of bacterial enzymes, interfering with their function for metabolism and growth (Quah et al., 2012). Antioxidant properties of NAC further contribute to its anti-infective effect (Yamada et al., 2011). NAC can function as a scavenger of ROS extracellularly with its thiol group and as an intracellular supply of glutathione, a powerful cellular antioxidant molecule (Raftos et al., 2007; Yamada et al., 2011). Through the reduction of oxidative stress, NAC can enhance cellular regeneration in the bone (Yamada et al., 2019). An initiatory in vitro study has shown that NAC mixed into bone cement exhibits inhibitory effects against planktonic S. aureus and Escherichia coli and against biofilms (Sukhonthamarn et al., 2021). Although these studies do not suggest that the growth-inhibitory and antibiofilm activity of NAC is species-specific, there is evidence that it could have a beneficial effect on the gut microbiome in diseased patients. In piglets infected with Porcine Epidemic Diarrhea Virus, NAC counteracted severe dysbiosis in the gut microbiome (Wu et al., 2021) and it alleviated gut dysbiosis in mice with metabolic syndrome (Zheng et al., 2019). These effects are mainly based on the antioxidant properties and reduction of pathogenic invasion (Zheng et al., 2019; Wu et al., 2021). Next to its beneficial effects, it is possible that bacteria could develop resistance against NAC. Bacteria isolated from a water environment have been shown to decompose NAC with a variety of enzymes (Kregiel et al., 2019). NAC could also enhance resistance of Edwardsiella tarda to different antibiotics by providing intracellular glutathione, enhancing antibiotic efflux, and promoting protein aggregation (Guo et al., 2024).

Bone cement made of polymethylmethacrylate (PMMA) is widely used in orthopedics. The main function of bone cement is to fasten joint implants, but it is also used to restore fractures and to stabilize vertebrae in the treatment of atlantoaxial instability (Hamajima et al., 2020; Tabanez et al., 2021). PMMA bone cements are established as local carriers of antibiotics in endoprosthetics and as beads on a string that can be implanted into the site of infection (Hayes et al., 2013). Incorporated substances are released from pores in the surface layer of PMMA bone cement, with a first “burst” phase with release of high concentrations and following low and stable release (Sun et al., 2021). The most commonly used antibiotics in bone cement are aminoglycosides (e.g., gentamicin) and aminopeptides (Cyphert et al., 2018). An advantage of such antibiotic-laden bone cements (ALBCs) is that high local concentrations of antibiotics can be achieved (Tootsi et al., 2022). However, the use of ALBC has been discussed critically as evidence for its reducing effect on infection rate is limited (Hoskins et al., 2020; Sebastian et al., 2020).

With these challenges in the management of periprosthetic joint infections, it is necessary to identify new antibacterial substances for the use with bone cement. New bacterial resistances need to be avoided to not further limit treatment options. The aim of this study is to give an evaluation of the suitability of NAC together with bone cement as a prevention and treatment option in human and veterinary medicine.

Periprosthetic joint infections are one of the leading causes of implant failure. With antibiotic resistances as a major health threat in general, novel prevention methods and treatments for PJIs are needed. In this in vitro study, the suitability of NAC as an additive to bone cement against PJI-related pathogens was investigated. NAC presents activity against planktonic bacteria and the ability to prevent the formation of bacterial biofilms in several species (Olofsson et al., 2003; Yamada et al., 2011; Quah et al., 2012; Costa et al., 2017). It is also a promising candidate for combination treatment with antibiotics against mature biofilms (Manoharan et al., 2020; Valzano et al., 2022). Concerning its cytotoxicity, NAC has been shown to be safe for osteoblasts at concentrations up to 1.15 mg/mL in an in vitro context (Tsukimura et al., 2009; Yamada et al., 2019). In this study, we demonstrated that NAC inhibits growth of planktonic S. pseudintermedius at 2.5 mg/mL and reduced the ability of S. pseudintermedius to attach to and grow on bone cement in a dose-dependent manner, but could not prevent infection of HOS cells at a concentration below the MIC. NAC was not cytotoxic for HOS cells at concentrations up to 3 mg/mL, but 50 mg and 100 mg NAC per bone cement platelet impaired cell growth and viability directly on bone cement. Treatment with the supernatants of NAC-containing bone cement did not lead to release of proinflammatory IL-6, but reduced cell viability and proliferation at higher concentrations. NAC treatment also resulted in slight changes in p38 activation compared to medium. Therefore, effectiveness of NAC against PJI-related pathogens could be demonstrated, but used concentrations need to be chosen with care to avoid cytotoxic effects.

S. pseudintermedius was chosen to focus on in this study because together with S. aureus, this pathogen is responsible for more than 50% of PJIs in small animals (Hayes et al., 2013). It is also becoming more relevant as a zoonotic pathogen especially in humans in close contact with small animals (Van Hoovels et al., 2006; Moses et al., 2023). With broth microdilutions, a MIC of 2.5 mg/mL NAC was determined for the three clinical isolates used in this study. These results go in line with MICs of NAC determined with other isolates of S. pseudintermedius in our lab (Walter et al., 2023). For Staphylococcus epidermidis, Klebsiella pneumoniae, and P. aeruginosa, which are further pathogens that can cause PJIs, MICs of 4–5 mg/mL have been determined (Gomes et al., 2012; Young et al., 2019; De Angelis et al., 2022).To incorporate NAC into PMMA bone cement, higher amounts of NAC with 50 mg and 100 mg were chosen to ensure even distribution of NAC in the cement mass and release of substance from the cement surface. We hypothesized that these concentrations of NAC would prevent S. pseudintermedius from attaching to the cement or kill bacteria growing on the surface. Live/Dead staining revealed that NAC prevented attachment of bacteria to the cement but could not completely inhibit colonization (Figure 1). For a complete inhibition, even higher amounts of NAC could be necessary, or NAC could be incorporated together with an antibiotic. Valzano et al. (2022) demonstrated a synergistic effect of NAC together with the antibiotic colistin against Pseudomonas aeruginosa biofilms, while Manoharan et al. (2020) found that a combined treatment with NAC, amoxicillin/clavulanate and amylase significantly decreased viability of S. aureus biofilms. Furthermore, novel formulation techniques, e.g., incorporating NAC into microspheres before mixing it into the cement could change the substance release profile to a more efficient and controlled release (Spicer et al., 2013).

To ensure cell compatibility of bone cement with NAC, several experiments concerning the viability, proliferation, cytokine release and stress-related kinase activation were performed. After treatment of HOS cells with NAC diluted in cell culture medium, the IC₅₀ for viability was 3.6 mg/mL and proliferation was reduced to less than 70% of control at 4 mg/mL (Figure 2). These concentrations are above the MIC of 2.5 mg/mL determined with the S. pseudintermedius isolates, so that bacterial growth can be inhibited while avoiding cytotoxicity. In literature, different tolerable concentrations of NAC are found in in vitro settings depending on the cell type. NAC did not have a cytotoxic effect on bladder epithelial cells at concentrations up to 8.16 mg/mL in the first 24 h (Manoharan et al., 2021), while human osteoblasts could tolerate NAC at up to 2.3 mg/mL (Tsukimura et al., 2009). The results presented here seem to be comparable to those described for human osteoblasts (Tsukimura et al., 2009). Growth of bone cells on and around the bone cement is essential for integration of the implant. Seeded directly onto bone cement containing NAC, attachment of HOS cells to the cement and viability of cells growing on the cement were analyzed after 24 h (Figure 3). Interestingly, total cell number per mm² was reduced on bone cement with 50 mg NAC compared to control, while on bone cement with 100 mg NAC, total cell number was comparable to control, but all attached cells were dead. A possible scenario is that 100 mg NAC released into the medium immediately kills HOS cells before attachment to the cement so that dead cells sink down onto the cement. 50 mg NAC does not seem to be immediately cytotoxic but inhibits attachment to the cement or leads to detachment of cells. However, because several washing steps are performed to remove detached cells before staining, further research is necessary to explain the difference in cell numbers. At lower concentrations, NAC can enhance cytocompatibility of PMMA bone cement due to its antioxidative properties (Tsukimura et al., 2009). The concentrations chosen here seem to be too high to exhibit this effect. To simulate flow of tissue fluid around the implant and the release profile of substances from bone cement, bone cement platelets with NAC and without additive were incubated in four different volumes of medium and HOS cells were treated with the supernatants (Figure 4). The most substance is released from bone cement in the hours after implantation, so that this early phase is a critical point for viability and proliferation of surrounding cells. The supernatants with higher concentrations of NAC, particularly of bone cement with NAC incubated in 1 mL and 3 mL medium, reduced the viability and proliferation of HOS cells. Proliferation was more strongly affected by the treatment, with significant reduction also by 10 mL and 30 mL supernatants. Because proliferation of cells surrounding the implant is critical for regeneration, it is important to keep in mind that proliferation seems to be more sensitive towards treatment. In viability testing of bone cement with up to 50% NAC, Sukhonthamarn et al. (2021) found that bone cement without additive had higher cytotoxicity than cement with NAC. Here, we could observe reduction of proliferation by bone cement without additive, but not reduction of viability, and cytotoxic effects were caused by bone cement with NAC.

Furthermore, the aim was to test if bone cement together with NAC would cause sterile inflammation and release of cytokines in HOS cells. IL-6 is a multifunctional cytokine regulating inflammatory responses and also osteoclastogenesis and bone resorption (Guo et al., 2018). It has also been shown that IL-6 release can be caused by a reaction to particles of PMMA bone cement (Gonzalez et al., 1996). In this study, IL-6 was only released from HOS treated with LPS or PGN, not from the cells treated with bone cement supernatants (Figure 4C). Interestingly, IL-6 is also considered as a marker of PJIs and can be used as a diagnostic tool (Randau et al., 2014). NAC has also been shown to inhibit IL-6 release caused by LPS stimulation at 0.16 mg/mL (Guo et al., 2018) but since NAC concentrations were much higher in this study, this does not explain why no IL-6 was released. From these results, we conclude that treatment with NAC-containing bone cement supernatants does not lead to signs of sterile inflammation. The pathway of p38 mitogen-activated protein kinase (MAPK) is involved in downstream regulation of cell cycle, apoptosis and inflammation following diverse stress stimuli, e.g., UV radiation or heat (Zarubin and Han, 2005). Activation of the p38 pathway is also involved in osteoclastogenesis provoked by PMMA particles (Abbas et al., 2003). For this reason, the influence of NAC and NAC-containing bone cement supernatants on the levels of p38 and activated p38 (p-p38) were analyzed by Western blotting (Figure 5). On average, treatment with NAC or NAC-containing bone cement supernatants led to lower p38 and higher p-p38 levels, indicating an activation of p38 following the treatment. Significant differences in p38 and p-p38 levels were only found between medium and 5 mg/mL NAC treatment, but it must be kept in mind that 5 mg/mL NAC is cytotoxic for HOS cells, which influences general protein expression. Treatment with the supernatant of bone cement without additive led to similar levels of p38 and p-p38 as in treatment with bone cement and NAC. In cells of the airways, NAC treatment has been found to reduce p38 activation and cytokine release by providing intracellular glutathione (Hashimoto et al., 2001; Wuyts et al., 2003). In HOS cells, involvement of the p38 pathway has been examined mostly in the context of anti-tumor treatments. For example, p38 is activated in HOS cells after treatment with the anti-tumor and anti-inflammatory substance Licochalcone A resulting in apoptosis (Lin et al., 2019), while capsaicin inactivates the p38 pathway (Zhang et al., 2017). To conclude, it could be shown that NAC and bone cement together with NAC could change p38 activation, although this overall effect was not statistically significant.

In infection experiments, the effect of NAC at a concentration below the MIC on S. pseudintermedius internalization into and adhesion to HOS cells was investigated (Figure 6). A concentration below the MIC was chosen for this purpose due to the release profile of substances from PMMA bone cement with low and stable release after a first burst (Sun et al., 2021). It has been shown before that manifestation of S. pseudintermedius joint and bone infection is likely from its ability to invade non-professional phagocytes like osteoblasts (Maali et al., 2016). Inhibition of bacterial adhesion and invasion of bladder epithelial cells by NAC was proven with E. coli and Enterococcus faecalis (Manoharan et al., 2021). Here, a similar effect could not be shown with HOS cells and S. pseudintermedius. NAC did not reduce internalization or adhesion compared to medium.

One limitation of this study was that we did not measure the exact amount of NAC released from each platelet, so that the exact NAC concentrations in supernatants are not known and are not completely comparable to concentrations of pure NAC. In addition, further experiments including different types of bone cells and additional testing of antibacterial activity including a possible development of resistances against NAC could have provided wider knowledge about the suitability as an additive to bone cement. Since this study concentrated on the suitability of NAC as an active substance, stability of bone cement with added NAC was not considered here.

It could be shown that NAC is effective against PJI-relevant S. pseudintermedius at non-cytotoxic concentrations for human derived bone cells. At a concentration below the MIC, infection of cells could not be prevented. Together with bone cement, reduced viability and proliferation, cell death and impaired attachment could be observed after some of the NAC treatments. There was no release of proinflammatory IL-6, but the results indicate possible involvement of the p38 MAPK pathway in stress reactions. The present data suggest that at the tested concentrations, NAC is not yet suitable for the use with bone cement due to limited cytocompatibility. Thus, further research is required to enhance biocompatibility, e.g., due to novel release methodologies. Measurement of exact NAC release from bone cement to balance cytocompatibility and antibacterial effect as well as combination of NAC with established antibiotics to determine a possible synergistic effect are options for follow-up research.

Periprosthetic joint infections after endoprosthetic surgery are a serious threat with devastating impacts on patients. The rising occurrence of multidrug resistant pathogens complicates the prevention and treatment of such infections; therefore, alternative treatment strategies are needed. This study provides an evaluation of NAC as a prevention and treatment option for the use together with bone cement. An emphasis is laid on biocompatibility to assess the influence on post-surgical regeneration. The results indicate that the chosen concentration of NAC is critical to balance biocompatibility and antibacterial effectiveness. With the presented results, a foundation for further research concerning clinical use of NAC as a strategy against periprosthetic joint infections is provided.