Efficacy of lysostaphin-coated titanium plates on implant-associated MRSA osteitis in minipigs

The increasing number of fracture-related infection (FRI) is a major complication and challenging problem in trauma surgery. Especially the use of implants enhances the risk of infection, which is the main reason for treatment failure [1]. Implant-associated infection (IAI) in combination with growing resistance to conventional antibiotics requires novel therapeutic strategies. Staphylococci, with the leading species Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE), are the most common causes of FRI, accounting for 30–60% of human cases [2‐4]. SA’s ability to form a biofilm complicates the treatment. This biofilm can be defined as a structured microbial community of cells attached to a substrate and embedded in a matrix of extracellular polymeric substances that they previously produced [5, 6]. It protects bacteria from the penetration of immune cells, and prevents phagocytosis and reactive oxygen species (ROS) killing [6‐10]. Experimental studies as well as clinical practice indicated clearly that in most cases, antibiotic therapy alone is not sufficient to eliminate the biofilm and that bacterial implants usually require surgical therapy [11]. Despite everything, it must be mentioned that each surgery to replace failed implants has a higher risk of recurrence, up to 30%, than that of the initial surgery [12]. Therapeutic strategies to eradicate the biofilm have made great research progress, such as studies on the surface modification of implants. However, these new treatments require further preclinical and clinical validation before they are widely accepted for therapeutic use [13]. Antimicrobial biomaterials currently represent one of the most promising therapeutic strategies [14, 15]. Therefore, the perfect implant surface would be one that reduces bacterial adhesion, confers effective sterilization, and does not compromise bone healing [16]. Great efforts have been made for coated implants, especially titanium plates, with antimicrobial agents to prevent IAIs [17, 18]. In 2014, our working group was able to show that an implant coating with lysostaphin prevents methicillin-sensible Staphylococcus aureus (MSSA) osteomyelitis in a murine model [19]. Lysostaphin is a 27-kDa extracellular antimicrobial endopeptidase produced by Staphylococcus simulans biovar staphylolyticus and was first discovered in the 1960s [20]. Lysostaphin destroys sessile bacteria in a biofilm and can also damage the extracellular biofilm matrix [21]. Moreover, the antibacterial potency of lysostaphin is well documented in animals and humans [20]. With regard to bony infection, the efficacy of lysostaphin has been tested under in vitro and in vivo animal models for the treatment of orthopedic infections [19, 22, 23]. Nonetheless, these studies were all carried out in mice. Examinations in large animal models (e.g., pig or sheep) are pending but are urgently needed to approach the clinical practice and pave the way for future human clinical trials. The most recent study of our working group shows the successful establishment of an IAI by methicillin-resistant Staphylococcus aureus (MRSA) in minipigs [24]. This was deliberately developed as a fundamental for investigating further therapeutic strategies in IAIs.

The aim of this study was to evaluate the effects of lysostaphin-coated plates in a standardized IAI minipig model.

Implant-associated infections (IAI) remain one of the greatest challenges in orthopedic and trauma surgery, despite major advances that have been achieved regarding their diagnosis and treatment. Currently, the most important treatment principles for IAI include surgical debridement, implant handling, long-term systemic antibiotics, reconstruction defects of bone and soft tissues, and functional recovery [25]. Fundamental research in vitro and in vivo should be performed to uncover the pathogenesis of this disorder more comprehensively. Here, we can confirm the established, standardized minipig model for examining IAI on the one hand, and the antimicrobial coating using lysostaphin as a possible therapeutic agent in IAI on the other hand.

Key problem of implant infections is the lack of effective therapies. Alternative strategies with antimicrobial enzymes such as lysostaphin could provide new options by killing planktonic and quiescent bacteria as well as bacteria growing in a biofilm [26]. In this study, the implanted titanium plates were dipped in a PDLLA-lysostaphin mixture and air-dried. This dip-drying step was repeated four times to obtain a multi-layer comprising PDLLA and lysostaphin. Other biomaterial strategies to deliver active lysostaphin have primarily focused on surface functionalization, by either passive adsorption [27, 28] or covalent tethering [29, 30]. Johnson et al. postulate that all these techniques, including our own, are effective ways to reduce bacteria at the material surface but may not be practical for settings where infection is already established, or not localized to a material surface [22]. They demonstrate that lysostaphin-delivering hydrogels have greater antibiofilm activity compared with soluble lysostaphin, even in a FRI model. However, these results have so far only been demonstrated for a small animal model (murine model) and are only been tested in an MSSA (UMAS-1) strain [22, 23]. Perhaps, lysostaphin combinations of implant coating and hydrogel application would be promising. To increase lysostaphin stability and retention times at the site of administration, Xue et al. analyzed the lysostaphin delivery using hydroxyapatite/chitosan composite bone cement [31]. Summarized, their study suggested that the lysostaphin-loaded self-setting injectable bone cement released the enzyme in a controlled and effective way. Furthermore, the enzymatic activity was well preserved during the setting and release process. To date, however, these experiments have only been carried out in the subcutaneous tissue of mice, and results in an IAI model are still pending.

Lysostaphin is not the only coating that has been tested in the treatment of patients with IAI from/resulting from medical devices [32]. In the last decade, several studies have investigated the ability of implant surface modifications to minimize bacterial adhesion, inhibit biofilm formation, and provide effective bacterial killing to protect implanted biomaterials [33]. Thereby, we can distinguish (1) antibacterial coatings (passive or active) and (2) modification of the surface of medical implants (physical or chemical) [34]. The lack of a common language and a common outcome set, as well as a large discrepancy between proposed and clinically implemented strategies, complicates evaluation of individual procedures and their comparison. Jeyaraman et al. summarize the current study situation on silver nanoparticles (AgNP) technology and conclude that AgNP has the potential to reduce implant-related infections. Yet, further studies and regulatory guidelines to avoid toxicity in clinical applications are pending.

Current therapeutic concepts require the implant removal in the treatment of osteitis, especially due to the formation of biofilm [35]. However, if the fracture has not healed, an implant replacement should be performed at least. To our knowledge, this is the first animal implant-associated osteitis model, which allows a plate replacement and thus forms a good foundation for future studies.

This study has several limitations. First, in contrast to our previously established mouse model, we also identified a significant number of foreign bacteria in one pig (Escherichia coli, Streptococcus pyogenes, Streptococcus suis, Staphylococcus spp.) when analyzing the bacterial load. Unfortunately, this cannot be avoided due to the pigs’ natural behavior, although we performed a standardized and careful disinfection of the pig skin before the respective operations. Finally, we excluded the pig in which this high number of foreign bacteria was detected from the analyses.

Secondly, the analysis of PMN, IL-6, and IL-17 did not reveal any pioneering findings. This could certainly be related to the chosen measurement times. For animal welfare reasons, the first lavage and blood samples were taken as part of the revision surgery on day 14, relatively late after infection induction. This led to a lower immune response than we expected, even in the infected animals. Compared to our own mouse model and the study of Johnson (both IL determinations were carried out on day 7), the measured IL levels in the present study were only half as high, even in the infected animals. Future studies should provide early assessments (e.g., days 1–6) of immunological parameters.

Thirdly, the analysis of the drill holes revealed no significant difference between the uncoated and the lysostaphin-coated plates, although CT showed significant differences among bone volume and mineralization. This could be attributed to the lysostaphin coating in the area of the screws. Stability of the PDLLA-lysostaphin-coating has not been evaluated so far. During inserting of the self-tapping screws, the coating could be lost due to the forces acting there. Finally, we cannot say with certainty what concentration was present after implantation of the osteosynthesis. Further studies to investigate surface adhesion, particularly in the area of screws, are pending, but planned.

Lastly, animal experiments cannot fully simulate the complex immunological and biomechanical environments of the human body, which limits the conclusions of the study. Going forward into a clinical setting will be challenging.

Based on the presented in vivo study, we can confirm our minipig model for the investigation of implant-associated bone infections, especially when an exchange of the implant should be performed. This enables further advance approaches and therapeutic options to be evaluated using this model.

Moreover, we demonstrate that lysostaphin coating prevents the development of an infection in spite of a bacterial contamination of MRSA and the presence of an implant, normally leading to progressive infection due to biofilm formation. From the clinical perspective, an antimicrobial coating of the osteosynthesis material would be desirable in high-risk cases (e.g., in the treatment of open fractures or in elderly patients with compromised tissue) or even after the infection has been clinically resolved. Future studies should focus on the development of effective coating strategies and the establishment of clinical model.