Globally, there is a constantly increasing demand for medical devices (Tzoulaki et al., 2016; Geelhoed et al., 2017). The market for medical devices is estimated to grow at a compound annual growth rate (CAGR) of 5.5% until 2029 (Fortunebusinessinsights, 2022). In all areas of application, a high degree of material biocompatibility is required to ensure functionality and avoid rejection reactions (Helmus et al., 2008). The function and longevity of a medical device is determined not least by the patient’s immune response, technical functionality and resistance to infections (Londono and Badylak, 2015; Kutner et al., 2021). Any implantable medical device triggers a non-preventable foreign body reaction (FBR) upon implantation by provoking innate immune responses (Anderson et al., 2008; Corradetti, 2017; Wei et al., 2019). The FBR is characterized by a number of events. First, the implant becomes covered with plasma proteins, known as the Vroman effect (Hirsh et al., 2013) which occurs in the first few seconds after implantation and is of particular importance for implants in contact with blood. This adsorbed protein layer can in turn lead to adhesion/activation of platelets, inflammation and finally, in the worst case, implant-induced thrombosis (Brash et al., 2019; Frutiger et al., 2021). Over the next few hours, inflammatory cells are recruited, including neutrophils, macrophages and fibroblasts, which release cytokines such as TNF-α, IL-10 and growth factors (Anderson et al., 2008). The presence and polarisation of macrophages play an important role in the regulation of FBR (Wei et al., 2019; Mukherjee et al., 2020). Similar to pathogen defense, macrophages attempt to degrade and eliminate any foreign agent (Corradetti, 2017; Carnicer-Lombarte et al., 2021). When degradation and phagocytosis fail, fibroblasts encapsulate the material and form a physical barrier to isolate it from the rest of the body, which remains in place as long as the device remains in the patient. In this context, long-term implantations pose a particular challenge, as encapsulation prevents interaction between the implant and the target tissue. To avoid the consequences of a foreign body reaction, materials with high biocompatibility are needed.

Medical silicones are such materials with a very high biocompatibility and are therefore suitable for medical applications. However, even with medical silicones, the formation of a fibrous capsule cannot be sufficiently controlled and suppressed, demonstrated by the evaluation of long-term biocompatibility in the permanent implantation of medical silicones for breast implants (since 1963) and intraocular lenses (since 1978) (Auffarth and Apple, 2001; Maxwell and Gabriel, 2009; Zeplin et al., 2014). Current implant research is therefore focused on modifying implant surfaces to prevent non-specific protein adsorption and control the inflammatory response. One way to improve biocompatibility is to create a non-fouling surface that prevents non-specific binding of proteins and bacteria (Shi et al., 2008; Zhang et al., 2008). Surface modifications can be achieved by chemical, mechanical, biological and physical methods (Yu et al., 2014; Fabbri and Messori, 2017). In the physical context, surface modification with zwitterions is a promising approach to reduce the initial attachment of proteins and subsequently the attachment of bacteria, for example, through the formation of a hydration shell or steric hindrance (Azari and Zou, 2013). One of the best characterized zwitterions is 2-methacryloyloxylethyl phosphorylcholine (MPC). Tan et al. used MPC grafts to improve the biocompatibility of the intraocular lens. They implanted the modified lens in rabbits and found that inflammation and encapsulation decreased significantly (Tan et al., 2017).

In the present study, two thermoplastic polyurethanes (TPU) and two modified liquid silicone rubbers (LSR) were compared. The polymer modifications with polymethyl methacrylate-2-methacryloyloxyethyl phosphorylcholine (PMMA-MPC) and polysulfonate (PSU) were tested in a mouse air-pouch model. TPUs are widely used in medical field, it represents the state-of-the-art material, with its excellent physical and mechanical properties, including durability, tear strength, and stress and puncture resistance. Additional elastomeric and flexibility properties make this material a more versatile choice, as well as the key features excellent biocompatibility, transparency and outstanding environmental resistance (including various gases and chemicals). PSU is an anti-adhesive polymer used for membranes for ultrafiltration due to its hydrophobic properties, high pH and temperature resistance (Maheswari et al., 2013; Yunos et al., 2014). In our previous work, these materials have already been investigated with regard to their effect on the interaction with innate immune cells (Woitschach et al., 2021a; Woitschach et al., 2021b; Woitschach et al., 2022).

Herein, we have modified the murine air-pouch model for simple and minimally invasive testing of materials in the form of micro-stents. The air-pouch model is an in vivo model in which repeated subcutaneous injections of air lead to the formation of a defined, well-perfused connective tissue sheath. By inserting the micro stents, a specific immune response will occur, which can be mapped using the wound fluid, the exudate. This animal model can be utilized to study local inflammation without systemic effects (Duarte et al., 2012). The implant specimens are easy to insert and exudates can be collected and comprehensively analyzed. It is a simple, low-burden and reproducible mouse model, in which biomaterials can be observed over long periods of time, allowing the analysis of the inflammatory and tissue activating properties of the materials (Fehrenbacher and McCarson, 2021; Peng et al., 2021).

In the present study, the air-pouch model was used for short- and long-term analyses to evaluate implant materials in terms of inflammatory potential and foreign body response by analyzing the recruited cells in the exudate using flow cytometry, the inflammatory response using microscopic and cellular analysis methods, and the encapsulation of the implants using microscopy.

The primary objective of this study was to compare the materials used in this particular model in terms of their biocompatibility in vivo. And we were able to show that the air sac exudate in particular provides valuable analysis material, for example, for FACS analyses, which conventional tissue analyses do not provide. We were able to demonstrate that the murine air-pouch model is suitable for investigating the biocompatibility of the various materials tested, with particular attention to their inflammatory potential and capsule formation. The investigated polyurethanes and modified silicones in the air-pouch model cause foreign body reaction (FBR) with capsule formation, but show only minor signs of tissue inflammation. The use and range of applications of biomaterials in medicine continues to increase (Fortunebusinessinsights, 2022). Along with this, research into the interaction between host cells and implants is becoming increasingly important. As one of the main factors for the loss of functionality of an implant as a whole, FBR is a critical issue in implant surgery (Lambris et al., 2015; Zhang et al., 2021). In this study, we used common thermoplastic polyurethanes and novel modified medical grade silicones to investigate FBR including inflammatory cell recruitment and cell adhesion to the implant materials in an air-pouch model. In the air-pouch model, a sterile subcutaneous cavity is formed into which the biomaterial can be inserted. For the duration of the trial, the cavity fills with a wound-healing-like fluid that allows cells to migrate. Few studies have attempted to investigate the mechanisms of the inflammatory response to different materials, organisms and substances using the air-pouch model (Chiappini et al., 2012; Lovric et al., 2018; Sombetzki et al., 2018). For example, Lovric et al. (Lovric et al., 2018) compared intact suture material with suture particles in the air-pouch model by inserting the material through an incision. The suture particles caused a stronger inflammatory response with more multinucleated giant cells and higher metalloproteinase (MMP) expression.

The present study differs from other air-pouch model studies in two aspects: 1) a complete piece of material of defined size is used, 2) the material is applied minimally invasively with a syringe, avoiding a skin incision and thus problems that may be associated with wound healing. With the model presented, it is possible to easily perform the FACS analyses of the inflammatory exudate. Similarly, analysis of soluble inflammatory markers can be performed using the air-pouch fluid. Thus, the model has many advantages, e.g., compared to the examination of subcutaneously introduced material. In addition, implants in blood contact can also be examined with this model, as cell recruitment into the air-pouch is comparable to cell recruitment from the bloodstream.

In previous in vitro studies, we have shown that the zwitterionic material PMMA-MPC reduces biofilm formation of Staphylococcus aureus ATCC 35556 (Woitschach et al., 2022). Furthermore, PMMA-MPC induces a higher inflammatory response in monocytes (Woitschach et al., 2021b). In addition to resistance to infection, the tendency to inflammation is also a critical quality feature of implants. Therefore, we used the air-pouch model to investigate the inflammatory potential of PMMA-MPC. In the current in vivo study, PMMA-MPC seems to recruit more leukocytes into the exudate of the air-pouch compared to all other investigated materials (Figures 2A, C), confirming the increased inflammatory response in vitro (Woitschach et al., 2021b). For the thermoplastic polyurethane P55, only low cell recruitment (Figure 2), low cell attachment (Figure 3) and the lowest expression levels for genes involved in processes such as inflammation, fibrosis and angiogenesis were observed (Figure 4D). Our results suggest that PMMA-MPC is the material that effectively prevents biofilm formation, but also has the highest inflammatory potential in vitro and elicits the strongest cellular response in vivo. P55, on the other hand, did not show any effects against biofilm formation, but elicited only a modest cellular response in vitro and in vivo.

2-Methacryloyloxyethyl phosphorylcholine (MPC) is a very well characterized biomedical material, which is characterized by its high resistance to non-specific protein and cell attachment and thus, in addition to its high biocompatibility, also has antithrombogenic and anti-bacterial properties (Ishihara, 2019; Woitschach et al., 2021a; Hiranphinyophat and Iwasaki, 2021). The material used in this study consisted of medical silicone as a base material coated with PMMA-MPC. One could assume that the combination of the different materials leads to different properties than a homogeneous MPC polymer.

In summary, the in vivo model in the presented study represents a highly adaptable and minimally invasive test system for testing different materials for their inflammatory potential in an easy-to-use manner. The test system makes it possible to quickly investigate innovative material candidates for implant manufacture with regard to their inflammatory properties and FBR. It would be conceivable to use sample bodies in this method to obtain a signal that can be evaluated more meaningfully due to the stronger interaction. Finally, the simplified model has limitations in terms of directly translating the results to humans. The human immune system is more complex than the mouse immune system (Chiappini et al., 2012). However, results from a mouse model can provide clues to potential responses in the human organism and are therefore an important component of research.