University of Groningen
Evaluation of nano-antimicrobial coated biomaterials in advanced in vitro co-culture models Ren, Xiaoxiang
DOI:
10.33612/diss.145072016
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Ren, X. (2020). Evaluation of nano-antimicrobial coated biomaterials in advanced in vitro co-culture models. University of Groningen. https://doi.org/10.33612/diss.145072016
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Biomaterial-associated infection (BAI) remains a serious clinical problem which is difficult to treat and often leading to biomaterials implant failure [1]. To prevent or treat BAI, advanced biomaterials or functional coatings have been developed and evaluated
in vitro either for their ability to prevent bacterial adhesion, kill adhering bacteria or
enhance tissue cell proliferation [2,3]. However, the true fate of biomaterials implants in the human body is much more complicated and involves multiple bacterial strains and cell types, cytokines, chemokines, and other secreted bacterial and cellular substances [4]. Since all these factors operate in concert, it is difficult to adequately evaluate advanced antibacterial biomaterials and coatings using current in vitro models. In this thesis, an advanced 3D infection co-culture model is developed to mimic peri- and post-operative infection. The 3D infection model was specifically geared towards mimicking the soft tissue seal around dental implants. Different titanium modifications were evaluated in this model.
The choice of co-culture systems
As a first step, we needed to develop a co-culture system in which to conduct the competition between bacteria and tissue cells. As introduced in Chapter 1, the systems of choice in which to set up a co-culture were either a transwell system or a microfluidic device. The microfluidic device is a relatively novel and advanced tool compared with traditional transwell systems. Despite the many advantages of microfluidic devices, versatility with respect to the use of different materials is low compared with a microfluidic device. Also, transwells are high-throughput but lack the possibility to control fluid flow. However, this drawback is compensated by the versatility of transwell systems to use various types of materials. Hence, a transwell system was chosen for setting up co-culture systems in this thesis.
Bacterial growth versus tissue cell growth in peri- and post-operative infection modes in the development of antimicrobial biomaterials and coatings
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The development of co-culture models was based on the two infection modes: operative and post-operative contamination of biomaterial implants. To build a peri-operative infection model, staphylococci were adhered to a biomaterial surface prior to the adhesion of tissue cells to mimic the clinical situation where an implant becomes contaminated prior or during surgical implantation. A post-operative infection model investigates the effects of a bacterial challenge on an existing tissue cell layer that has already integrated the biomaterials implant surface. Based on previous in vitro research in our group [5], a minimum of 40% cell surface coverage was considered required to ensure tissue integration over bacterial colonization. In Chapter 2, we demonstrated that a keratinocyte layer can form a natural protection against a post-operative pathogen challenge. When the transwell is equipped with 0.4 µm pores, whether or not the keratinocyte layer was able to maintain this 40% critical surface coverage by tissue cells depended on the pathogenic strain involved and the substratum material. Maintenance of the critical 40% surface coverage under a bacterial challenge was most difficult on hydrophobic silicone rubber. During photothermal treatment of infection, maintenance of 40% surface coverage by tissue cells required precise timing of NIR-irradiation of photothermal coatings on a titanium surface (Chapter 3), which may be a reason to discard photothermal coatings for post-operative protection and only use them to counter the possible consequences of peri-operative bacterial contamination of an implant. Safe-guarding 40% surface coverage upon a post-operative challenge by antimicrobial leaching coatings was difficult because they become exhausted (see Chapter 4). For that reason, leaching coatings are unsuitable for application in post-operative infection-control, while their merits clearly involve controlling infection due to peri-operative bacterial contamination. Herewith this thesis has provided multiple options for the control of peri-operative bacterial contamination, but it must be concluded that only natural barriers are available at present for the control of post-operative infection.
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Titanium and its alloys are widely used as orthopedic and dental implants for their excellent mechanical properties and biocompatibility [6]. However, too many researchers only pursue “good results and better bacterial killing”, but ignore clinical requirements, such as possible collateral tissue damage by excessive photothermal therapy [7,8] (see Chapter 3) or antimicrobial overloading of coatings [9–12] (see Chapter 4). Both chapters, clearly indicate flaws in the designed materials and coatings that may be academically acceptable, but that on the long term will direct the field in a direction in which clinical application will be impossible due to the neglect of clinical side conditions. Clinical translation of our academic achievements will therefore need a more extensive dialogue between key-players in the process of clinical translation.
Clinical limitations should be leading in the design of antimicrobial biomaterials and coatings for which the dialogue with clinicians as key-players is of crucial importance. Clinicians are indispensable for clear definition of the clinical problem to be solved, guidance and evaluation of new designs and solutions proposed [13]. Unfortunately, performance demands and expectations imposed on many coatings by clinicians and patients to address infection risks are ill-defined and not always communicated well between basic scientists and clinicians. Often clinical expectations neglect the distinction between a host tissue environment before primary implantation and the environment surrounding an infected implant [14]. In general, implants are expected to stimulate tissue integration while simultaneously preventing microbial adhesion and killing all organisms during hospitalization or surgery within 2 to 3 weeks after implantation. Such a peri-operative goal implies clinical openness about infection rates, microbiological hygiene in the operating theatre and admittance of possible mistakes during surgery. Yet, this openness is not what is generally observed. At meetings and conferences, clinicians frequently state that they “only treat infected
implant cases of their colleagues”. Long-term antimicrobial protection of implants is
trivially required by clinicians (and patients for that matter), but as this thesis demonstrates is still out of reach from a materials perspective.
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Regulatory agencies are also key-players in translation and their requirements vary in different countries, but all include “safety” and in more or less strict terms “efficacy”. One of the main problems in satisfying regulatory demands, is the required size of clinical trials for BAI. Assuming an infection rate for total hip arthroplasties of 2% e.g. (Table 1), a power-calculation shows that studies aiming to demonstrate a 50% reduction (P < 0.05) in infection rate require the inclusion of around 5000 patients that must be followed longitudinally over several years (at a cost of tens of millions of dollars). To demonstrate a 25% reduction (P < 0.05), more than 22,000 patients have to be included [15]. These complex and highly expensive clinical trials prevent the validation of new infection-control strategies. As a consequence, we may have to consider a new reality in which the safety and efficacy of antimicrobial biomaterials will not be substantiated with clinical trials but instead will have to be derived from properly designed, advanced in vitro co-culture models as in this PhD study.
For the dialogue with industry as a key-player, simplicity, ease and costs of manufacturing are critical in clinical translation. Industry struggles with the costs and risks involved in translating early-stage ideas from prototype bench to clinical bedside [16]. Although many new antimicrobial coatings and their evaluations in the literature must surely be considered “promising”, the industrial or clinical interest remains behind. This is partly because academic research is too much geared to obtaining publishable results, with greater and greater innovations. Studies furthering existing ideas to clinical application or describing new simple methodologies apt to realistic clinical translation and manufacturing are hard to publish in the competition for journal space. Our co-culture infection models may de-risk clinical translation for manufacturing companies, reducing the cost and saving (part of) the resources needed to carry out animal experiments.
Limitations of 3D-tissue infection model
The main limitation of our 3D-tissue infection model is the absence of immune cells as a natural defense. In the human body, the host immune system is a complex
127 Table 1. Estimated size of prospective, randomized clinical trials required to sufficiently assess the efficacy of antimicrobial strategies for decreasing the infection rate associated with surgical implants [15] (with permission of oxford academic).
Preventive stratge by anticipated reduction in infection ratea
50% reduction 25% reduction Infection impalnt Baseline
infection rate,% New rate,% Trail Size b New rate,% Trail size b Heart valve or joint prosthesis 2 1 5028 1.5 22,382
Penile implant 3 1.5 3328 2.25 34,378
CNS shunt 4 2 2478 3 10,998
Cardiac pacemaker 5 2.5 1968 3.75 8720
External fixation pin 10 5 948 7.5 4166
Left ventricular assist device 40 20 182 30 752 Note. P=0.05, by Fisher’s exact 2-sided test; power, 80%.
a Compared with the infection rate at baseline. b Date are estimated number of patients in the trial.
network of cells and proteins that defend the body against infection. The interaction between immune cells and bacteria were studied in bi-cultures and bacteria-induced movement of immune cells was already confirmed in a co-culture study [17]. Another limitation is the lack of an extra cellular matrix (ECM) in our 3D-tissue infection model. The ECM is a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support to surrounding cells [18]. Whereas including of immune cells would be most advantageous in a peri-operative infection model, because immune cells initially not
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only fight against contaminating bacteria but also to the biomaterial itself, the ECM is only present once a biomaterials implant is integrated, making ECM inclusion most relevant in post-operative 3D-tissue infection models.
Suggestions for future research
Extension of the 3D-tissue infection model based on the above limitations are possible but are not trivial and come at the expense of larger variability. Biological experiments by character already have large standard deviations and inclusion of immune cells or ECM stimulating factors will increase to the variability of the experiments.
Addition of immune cells requires a growth medium in which keratinocytes, fibroblasts, immune cells and bacteria can grow and maintain their normal, physiological functions. For immune cells, this involves their physiological polarization towards a pro-inflammatory “fighting” M1 and an anti-inflammatory “repairing” M2 phenotype. Moreover, the use of mouse macrophages as often done in the literature may not be a good choice and human cell lines, most notably monocytes may be preferable, but again, their differentiation towards macrophages will add another source of variability.
To mimic an ECM environment, collagen or polysaccharides should be added in the co-culture models in order to support the growth of tissue cells [19], but adsorption of ECM components to implant materials can be hard to achieve.
Whether each extension of the model will be a meaningful step can be argued about. If the goal is to ultimately mimic an animal experiment and replace an animal experiment by advanced in vitro co-culture models, it should be done. As argued above, variability between experiments in such highly advanced in vitro models will increase, and delineation of efficacy mechanisms becomes more difficult. E.g. for a translational pathway, it is important to know whether efficacy is due to differences in macrophage polarization or bacterial response to a coating. Ideally therefore, in vitro experiments should be done in step-wise more complicated advanced co-culture models.
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Besides advancing in vitro models, this PhD thesis also emphasized the urgency to develop antimicrobial biomaterials and coatings particularly with regard to post-operative infection of biomaterials implants and devices. Smart, targetable photothermal nanoparticles may be promising to develop, potentially decorated with an antibiotic. The decoration of with an antibiotic is expected to enhance photothermal killing at lower temperatures to avoid collateral damage to surrounding tissue cells.
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