Evaluation of nano-antimicrobial coated biomaterials in advanced in vitro co-culture models Ren, Xiaoxiang
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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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CHAPTER
1
General Introduction and
the aim of the thesis
Xiaoxiang Ren, Lu Yuan, Brandon W. Peterson, Yijin Ren, Theo G. van Kooten, Svetlana Bratskaya,
Henny C. van der Mei, Henk J. Busscher Submitted as a Pearl to Plos Pathogen
3
Introduction
Many novel antimicrobials are forwarded in the literature, all aiming to prevent antimicrobial-resistant bacterial infections from becoming the main cause of death by the year 2050 [1]. Especially nanotechnology offers many novel antimicrobials based on non-antibiotic working mechanisms [2] that require thorough evaluation, such as chemically-induced or magnetic-targeting of pathogens, antimicrobial encapsulation to enhance penetration in infectious biofilms, reactive oxygen based or thermal bacterial killing. Typically, efficacy of novel antimicrobials is evaluated in mono-cultures, only comprising a bacterial pathogen, but neglecting possible damage to organs or tissue cells surrounding an infection site. In addition, immune cells working in concert with antimicrobials applied to control bacterial infection, are often neglected [3]. This interplay of bacteria, tissue and immune cells (Figure 1A) can be studied in animals, but animal experiments are subject to regulatory restrictions [4] and growing societal opposition [5]. Therefore, more advanced in vitro co-culture models in which all key-players involved in bacterial infections are represented in concert (Figure 1B), are urgently needed to evaluate novel infection-control strategies.
Here, we review the need for co-culture models, summarize available co-culture models in 2D and 3D systems, their critical factors and useful outcome parameters in order to address the question up to what extent co-culture models can replace animal evaluation of novel antimicrobials.
4
Figure 1. Key-players involved in bacterial infection.
A. Simplified schematic of the histo-pathology of the interplay between bacteria, tissues cells and immune cells around an infection site, including bacteria sheltering in tissue cells and cytokine-mediated communication between cells as stimulated by infectious bacteria.
B. Examples of different in vitro mono-, bi- and tri-culture models in which this interplay can be studied.
Why do we need co-culture infection models?
Most bacterial infections are due to bacteria in a biofilm-mode of growth, i.e. adhering to a surface. These can be the surfaces of other bacteria, tissue cells, teeth or bone or prosthetic implant surfaces [6]. Accordingly, tissue cells and implant surfaces are always present in the neighborhood of an infection site and immune cells are recruited towards an infection site (Figure 1A). Often, proper dosing of an antimicrobial
5 involves finding a balance between bacterial killing and tissue preservation. Gentamicin for instance, can be locally applied in high concentrations that would cause kidney and vestibular damage when applied systemically [7]. Novel antimicrobials need to be developed with the same consideration for collateral damage as made earlier upon introduction of the current generation of antibiotics. Nano-antimicrobials and the reactive oxygen species that they frequently generate [2], can diffuse into tissue surrounding an infection site and cause collateral damage. Also, photothermal nanoparticles can release high amounts of heat to kill adhering bacteria upon Near-Infra-Red irradiation [8], that dissipates into surrounding tissue. Since tissue cells are more susceptible to heat than most bacteria, heat dissipation may cause severe collateral tissue damage. Mono-culture studies evaluate either bacterial killing or potential damage to tissue or immune cells (Figure 1B). For example, bacterial killing and tissue integration of an antimicrobial prosthetic implant surface with tethered tissue-reactive, integrin-peptides were both improved in respective mono-cultures, but this could not be demonstrated in bi-culture [9]. In bi-culture, bacterial exopolysaccharides adsorbed to the tissue-reactive sites and blocked tissue integration [10]. As another example, tri-cultures of Staphylococcus aureus, U2OS osteoblasts and macrophages demonstrated that macrophages only aided tissue integration on bacterially contaminated gold-nanoparticle coated surfaces when the density of gold gold-nanoparticles was confined within a narrow range [11], despite mono- and bi-culture studies telling a different story. Numerous other examples can illustrate interactions between different key-players such as cytokine-mediated communication between tissue cells and immune cells as stimulated by bacterial pathogens or pathogen sheltering in tissue cells to protect themselves against antimicrobials (see also Figure 1A). Therefore in short, there are many different reasons as to why mono-culture models only tell part of the story that is relevant to properly judge the merits of novel infection-control strategies.
Which are the existing co-culture infection models?
6
applied. 3D systems distinguish themselves by the prevalence of more extensive, physiological cell-to-cell contact versus predominantly physical, cell-to-substratum contact and cell-to-cell edge-contact in 2D systems [12]. 3D culture systems have originally been developed for tissue-engineering and are increasingly recognized as a useful tool to evaluate novel bacterial-infection-control strategies. 3D organoids are based on growing tissue cells on or in extra-cellular matrix-based hydrogels, but absence of flow conditions limits nutrient and metabolic waste transport and therewith long-term evaluation. Rotating wall vessels are extensively used to study bacterial and viral infections [13], but control of the rotational speed can be technically challenging. 3D tissue models based on porous polymeric scaffolds with high permeability allow controllable stretching and compression to mimic physiological conditions, but at the same time often present biocompatibility issues. Scalable supermacroporous cryogels [14] allow flow and are potentially useful for long-term evalution of novel antimicrobials.
2D transwell and 3D lab-on-a-chip systems uniquely possess two compartments separated by a porous membrane that allows e.g. to grow epithelial and endothelial cells on each membrane side [15]. Depending on pore size, cell layers on each side of the membrane are in more extensive cell-to-cell contact through the pores of the transwell membrane than in other 2D systems. Yet, transwell as well as organ-on-a-chip systems are classified here as pseudo-3D systems, because it can be questioned whether the limited cell-to-cell contact allowed by the membrane in between the different compartments, justifies their classification as a 3D system.
All 2D and 3D systems can be used in mono- or co-culture models (see Figure 1B). In most 2D and 3D systems, cells, bacteria and immune cells are simply brought together. A major advantage of membrane-separated systems is that pathogens can be introduced in the epithelial side, creating the need for immune cell migration upon cytokine calling from the endothelial compartment to the epithelial compartment in order to combat infecting pathogens, similar as occurring in the human body. However, introduction of infecting pathogens and obtaining reproducible results in co-culture
7 models is not trivial due to the multitude of biological components in a system [16]. In short, a large variety of 2D and 3D systems can be used and set-up with different co-culture models. The final choice for a given system can be made best on the basis of the type of infection aimed to solve.
Table 1. Comparison of different experimental systems for use as a co-culture model in the evaluation of novel infection-control strategies.
2D CO-CULTURE MODELS
Experimental system Advantages Disadvantages Ref.
Well plate Simple and inexpensive; Well established; High-throughput No flow condition; Only one medium possible for different cells
and bacteria; Limited side-to-side cell contact
[11]
Flow displacement device Flow conditions; Controllable mass transport
and shear
Only one medium possible for different cells
and bacteria; Limited side-to-side cell-contact
[10]
Transwell system Simple and inexpensive; Two compartments No flow condition; Pseudo 3D cell-to-cell-contact [17]
8
3D CO-CULTURE MODELS
Experimental system Advantages Disadvantages Ref.
3D organoid 3D organ-like cell composition and function No cell differentiation; Difficult to set-up; No flow condition; Natural ECM-based; High batch-to-batch variability [18]
3D Rotating Wall Vessel
Low flow environment; Spontaneous cell differentiation Limited size of 3D aggregates; Not applicable to
all cell types; Strict control of rotational speed
required
[13]
Porous polymeric scaffolds and
supermacroporous cryogels* stretching and Controllable compression; No natural ECM required Toxicity and immunogenicity issues; Difficult to remove cells [14]
Organ-on-a-chip Flow conditions; Two compartments; Long-term studies possible Pseudo 3D cell-to-cell-contact; Difficult to standardize [15]
9 *Not yet applied, but considered potentially useful in infection-control evaluations.
What are the critical factors in co-culture infection models?
Bacterial strain, cell type as well as of type of immune cell are obviously critical factors in co-culture models. For infections associated with prosthetic implants, also the biomaterial used, adds to these critical factors. In our experience, bacterial pathogens in absence of antimicrobials dominate any co-culture within several days, especially when comprising virulent strains such as S. aureus or Pseudomonas aeruginosa [19].
In systems without separate compartments for housing of different media, culture media have to be developed in which all bacteria and cells involved in the co-culture are able to grow [19]. Co-co-culture medium can be composed of different components of the respective growth media [20] or by making a composite medium consisting of different ratios of the respective media [11,19] . Usually cells are more sensitive to medium composition than bacteria. However, in all cases co-culture media have to be tested for balanced bacterial and cell growth in order not to excessively favor either. Anaerobic conditions as required for growth of some pathogens, cannot be employed to grow cells. However, once grown, oxygen is not a prerequisite for tissue cell survival and hypoxia does not necessarily increase apoptosis [21]. Thus aerobically grown cell layers can be employed in co-culture models, requiring anaerobic conditions for bacterial growth [22].
What are the useful outcome parameters in co-culture infection models?
Outcome parameters of any co-culture infection model for evaluation of new antimicrobials are bacterial killing and cellular survival, with or without the aid of immune cells. The gold-standard for bacterial killing is agar plating and enumeration of the number of colony-forming units, which mostly requires bacterial collection from the model system. Scraping off from surfaces and suspending in transport fluid usually suffices, but bacterial collection from tissue cells in which bacteria have taken shelter against antimicrobials, may require homogenization before plate counting. Bioluminescence is often used as well [23], because it is convenient and fast, not
10
necessarily requiring any removal of bacteria or tissue cells, but it requires bioluminescent bacterial strains and expensive bio-optical imaging equipment for observation. Bioluminescence is ideally suitable to detect live bacteria inside 3D tissue systems in a non-destructive way. Care should be taken however, since bioluminescence is governed by bacterial metabolic processes that can be upregulated at minimal inhibitory concentrations of an antimicrobial before killing occurs [24]. A similar drawback exists for different conversion assays based on bacterial metabolism [25].
Survival of tissue cells can also be evaluated using different types of metabolic assays [25] or live/dead stains, often based on cell membrane damage [26]. Barrier integrity of epithelial or endothelial cell layers in different co-cultures in membrane-equipped systems can be assessed by the measurement of trans-cellular electrical resistance (TEER) measurements. TEER of cell layers with intact barrier functions are determined by the resistance of the trans-cellular and para-cellular pathway through the tight junctions constituting cell-to-cell contact [15]. When challenged by pathogens, the barrier can become “leaky” yielding a strongly decreased TEER. In prosthetic implant associated infections, immune-cell-assisted tissue integration versus bacterial colonization is generally considered a requirement for the survival of an implant under bacterial challenge [27]. This makes surface coverage by tissue cells of the implant material involved a suitable outcome parameter as well, that can be determined after immuno-cyto staining of cellular layers. Concluding, a minimum of two outcome parameters pertaining to bacterial killing and tissue cell survival must always be pursued in co-culture models.
Will advanced co-culture infection models be able to replace animal experiments?
Replacing animal experiments by advanced in vitro models is one of the current challenges that extends towards development of novel antimicrobial-resistant infection-control strategies, not in the least because more than 80% of potential therapeutics fail in clinical trials after animal experiments have confirmed efficacy and safety [28]. At the same time, it is obvious that animal de-risking of novel antimicrobials will always
11 remain a pre-requisite for human clinical trials. Co-cultures can reduce the gap between in vitro and in vivo evaluations to reduce animal use and actually yield more opportunities, particularly for mechanistic evaluation, than animal studies in which events are highly multi-factorial:
- inclusion of a large variety of bacterial pathogens is relatively easy, - antimicrobial concentrations can be varied at will,
- the role immune cells can be directly assessed by setting up co-cultures with and without immune cells,
- pathogen damage and collateral damage due to treatment can be studied relatively easily for a variety of cell lines.
Therewith, advanced co-culture models as described in Table 1, certainly reduce the gap between in vitro and in vivo evaluations and may actually be better suited for mechanistic studies than animal experiments. Provided reviewers and journal editors acknowledge the value of advanced co-culture models and refrain from demanding animal experiments for reasons other than in vivo proof-of-principle and de-risking, advanced co-culture models will be able to replace animal experiments to a large extent.
Aim of the thesis
Long life expectancy and the demand for a high quality of life from birth till death in the current century necessitate the use of biomaterials implants and devices for the restoration of tissue, organs or body function such as after trauma, invasive (oncological) surgery or simply wear due to old age [27]. However, the presence of biomaterials in the human body significantly comprises the host ability to cope with invading pathogens. Due to biomaterial associated infections (BAI) resulting from bacterial colonization of implant surfaces, the failure rate of implanted biomaterials is around 5% across different types of biomaterials implants and devices [29]. An episode of BAI can severely influence the quality of life of a patient [30], creating headaches to treating physicians and is a costly burden to the health care system.
To counter the increasing threat of bacterial infections, especially BAI due to antibiotic-resistant bacterial strains, new antimicrobial biomaterials have to be
12
developed that at the same time warrant tissue integration. Nanotechnology has forwarded many novel types of antimicrobials killing bacteria on a non-antibiotic basis [31]. These include amongst others several types of nanoparticles that either directly kill bacteria or kill through the generation of reactive oxygen species. Photothermal nanoparticles can locally release high amounts of heat to kill infectious bacteria upon Near InfraRed irradiation. Currently, nano-antimicrobials are expected to work indiscriminately towards different bacterial strains and species, regardless of Gram-character or antibiotic-resistance. Several of these nanoparticles can be applied to coat different biomaterials surfaces to eradicate contaminating bacteria on an implant surface per-operatively introduced or to kill bacteria colonizing a biomaterial surface post-operatively [32,33]. However, at the same time, when overdosed or by unexpected interplays between bacteria, tissue and immune cells on a coated biomaterials surface, collateral damage may occur to tissue cells attempting to integrate the biomaterials surface. Tissue integration provides the ultimate protection of a biomaterials implant against infection [34].
Currently, most evaluations of biomaterial coatings only regard tissue integration or bacterial killing on a biomaterial surface (see Chapter 1.1). However, the fate of a biomaterials implant in human body is determined by an interplay between tissue integration, bacterial colonization, immune cell activity and the properties of the biomaterial surface [11]. The orthopedic surgeon Gristina coined the term “race for the surface” to describe the fate of internal biomaterial implants in relation with the development of BAI [35]. In the concept of the race for the surface, collateral damage to tissue cells integrating a biomaterial surface and simultaneously fighting their race for the surface with colonizing bacteria, would be disastrous for the fate of internal biomaterials implants. Obviously, the “race” cannot be studied in mono-culture experiments in the absence of interaction between cells and bacteria and animal models would be first choice for evaluation of the highly complex race for the surface. However, animal studies are subject to strict regulations, invoke societal objections and are costly. In view of this, advanced co-culture models comprising tissue cells, immune cells and
13 bacteria are needed to evaluate the outcome of the race for the surface on novel antimicrobial surfaces as a possible bridge between laboratory studies and human clinical trials to at least partly, replace animal experiments.
Therefore, the aim of this thesis was to develop novel nano-antimicrobial biomaterial coatings and to explore the use of advanced co-culture models for their evaluation.
This thesis is focused on titanium-made dental implants, arguably the most used biomaterials implant required to function in a bacteria-laden environment [36]. Oral surgery is hard to perform under sterile conditions and so is wound healing. Peri-implantitis is therefore a feared complication of oral implants [29]. In addition to the above mentioned objections against animal studies, animal studies are relatively useless for this purpose as the oral microflora in most if not all animals, is highly different from the human oral microflora [37]. As a final reason for focusing on dental implant materials, the complicated tissue structure around a dental implant consisting of fibroblast and keratinocytes [38] makes the set-up of an advanced in vitro system challenging and needed.
14
References
[1] W. Health Organization, United Nations meeting on antimicrobial resistance, Bull. World Health Organ. 94 (2016) 638–639.
[2] Y. Liu, L. Shi, L. Su, H.C. van der Mei, P.C. Jutte, Y. Ren, H.J. Busscher, Nanotechnology-based antimicrobials and delivery systems for biofilm-infection control, Chem. Soc. Rev. 48 (2019) 428–446.
[3] I.A. Parish, W.R. Heath, Too dangerous to ignore: self-tolerance and the control of ignorant autoreactive T cells, Immunol. Cell Biol. 86 (2008) 146–152. [4] T.F. Moriarty, L.G. Harris, R.A. Mooney, J.C. Wenke, M. Riool, S.A.J. Zaat, A.
Moter, T.P. Schaer, N. Khanna, R. Kuehl, V. Alt, A. Montali, J. Liu, S. Zeiter, H.J. Busscher, D.W. Grainger, R.G. Richards, Recommendations for design and conduct of preclinical in vivo studies of orthopedic device-related infection, J. Orthop. Res. 37 (2019) 271–287
[5] D. Campbell, Public opposition to animal testing grows, Guard. (2012). https://www.theguardian.com/science/2012/oct/19/public-opposition-animal-testing (accessed April 8, 2020).
[6] C.A. Fux, J.W. Costerton, P.S. Stewart, P. Stoodley, Survival strategies of infectious biofilms, Trends Microbiol. 13 (2005) 34–40.
[7] C.S. Scott, G.Z. Retsch-Bogart, M.M. Henry, Renal failure and vestibular toxicity in an adolescent with cystic fibrosis receiving gentamicin and standard-dose ibuprofen, Pediatr. Pulmonol. 31 (2001) 314–316.
[8] P.C. Ray, S.A. Khan, A.K. Singh, D. Senapati, Z. Fan, Nanomaterials for targeted detection and photothermal killing of bacteria, Chem. Soc. Rev. 41 (2012) 3193– 3209.
[9] C. Mas-Moruno, B. Su, M.J. Dalby, Multifunctional coatings and nanotopographies: Toward cell instructive and antibacterial implants, Adv. Healthc. Mater. 8 (2019) 1801103.
[10] I.C. Saldarriaga Fernández, H.J. Busscher, S.W. Metzger, D.W. Grainger, H.C. van der Mei, Competitive time- and density-dependent adhesion of
15 staphylococci and osteoblasts on crosslinked poly(ethylene glycol)-based polymer coatings in co-culture flow chambers, Biomaterials. 32 (2011) 979–984. [11] Y. Luan, H.C. van der Mei, M. Dijk, G.I. Geertsema-Doornbusch, J. Atema-Smit,
Y. Ren, H. Chen, H.J. Busscher, Polarization of macrophages, cellular adhesion, and spreading on bacterially contaminated gold nanoparticle-coatings in vitro, ACS Biomater. Sci. Eng. 6 (2020) 933–945.
[12] Y. Imamura, T. Mukohara, Y. Shimono, Y. Funakoshi, N. Chayahara, M. Toyoda, N. Kiyota, S. Takao, S. Kono, T. Nakatsura, H. Minami, Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer, Oncol. Rep. 33 (2015) 1837–1843.
[13] J. Barrila, A. Crabbé, J. Yang, K. Franco, S.D. Nydam, R.J. Forsyth, R.R. Davis, S. Gangaraju, C. Mark Ott, C.B. Coyne, M.J. Bissell, C.A. Nickerson, Modeling host-pathogen interactions in the context of the microenvironment: Three-dimensional cell culture comes of age, Infect. Immun. 86 (2018) e00282-18. [14] B. He, G. Chen, Y. Zeng, Three-dimensional cell culture models for investigating
human viruses, Virol. Sin. 31 (2016) 363–379.
[15] H.J. Kim, H. Li, J.J. Collins, D.E. Ingber, Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip, Proc. Natl. Acad. Sci. 113 (2016) E7–E15.
[16] E. Saygili, A.A. Dogan-Gurbuz, O. Yesil-Celiktas, M.S. Draz, 3D bioprinting: A powerful tool to leverage tissue engineering and microbial systems, Bioprinting. 18 (2020) e00071.
[17] X. Ren, H.C. van der Mei, Y. Ren, H.J. Busscher, Keratinocytes protect soft-tissue integration of dental implant materials against bacterial challenges in a 3D-tissue infection model, Acta Biomater. 96 (2019) 237–246.
[18] D. Dutta, I. Heo, H. Clevers, Disease modeling in stem cell-derived 3D organoid systems, Trends Mol. Med. 23 (2017) 393–410.
[19] G. Subbiahdoss, I.C.S. Fernández, J.F. da Silva Domingues, R. Kuijer, H.C. van der Mei, H.J. Busscher, In vitro interactions between bacteria, osteoblast-like
16
cells and macrophages in the pathogenesis of biomaterial-associated infections, PLoS One. 6 (2011) e24827.
[20] J.H. Lee, H. Wang, J.B. Kaplan, W.Y. Lee, Effects of Staphylococcus epidermidis on osteoblast cell adhesion and viability on a Ti alloy surface in a microfluidic co-culture environment, Acta Biomater. 6 (2010) 4422–4429.
[21] J.C. Utting, S.P. Robins, A. Brandao-Burch, I.R. Orriss, J. Behar, T.R. Arnett, Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts, Exp. Cell Res. 312 (2006) 1693–1702.
[22] S. Jalili-firoozinezhad, F.S. Gazzaniga, E.L. Calamari, D.M. Camacho, C.W. Fadel, A. Bein, B. Swenor, B. Nestor, M.J. Cronce, A. Tovaglieri, O. Levy, K.E. Gregory, D.T. Breault, J.M.S. Cabral, D.L. Kasper, R. Novak, D.E. Ingber, A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip, Nat. Biomed. Eng. 3 (2019) 520-531.
[23] M. Hutchens, G.D. Luker, Applications of bioluminescence imaging to the study of infectious diseases, Cell. Microbiol. 9 (2007) 2315–2322.
[24] S. Daghighi, J. Sjollema, A. Harapanahalli, R.J.B. Dijkstra, H.C. van der Mei, H.J. Busscher, Influence of antibiotic pressure on bacterial bioluminescence, with emphasis on Staphylococcus aureus, Int. J. Antimicrob. Agents. 46 (2015) 713–717.
[25] E. Grela, J. Kozłowska, A. Grabowiecka, Current methodology of MTT assay in bacteria – A review, Acta Histochem. 120 (2018) 303–311.
[26] S. Johnson, V. Nguyen, D. Coder, Assessment of cell viability, Curr. Protoc. Cytom. 64 (2013) 1–26.
[27] H.J. Busscher, H.C. van der Mei, G. Subbiahdoss, P.C. Jutte, J.J.A.M. van den Dungen, S.A.J. Zaat, M.J. Schultz, D.W. Grainger, Biomaterial-associated infection: Locating the finish line in the race for the surface, Sci. Transl. Med. 4 (2012) 153rv10.
[28] S. Perrin, Make mouse studies work, Nature. 507 (2014) 423–425.
17 The implant infection paradox: Why do some succeed when others fail?, Eur. Cells Mater. 29 (2015) 303–313.
[30] D. Campoccia, L. Montanaro, C.R. Arciola, A review of the clinical implications of anti-infective biomaterials andinfection-resistant surfaces, Biomaterials. 34 (2013) 8018–8029.
[31] J.T. Seil, T.J. Webster, Antimicrobial applications of nanotechnology: Methods and literature, Int. J. Nanomedicine. 7 (2012) 2767–2781.
[32] W. Lei, K. Ren, T. Chen, X. Chen, B. Li, H. Chang, J. Ji, Polydopamine nanocoating for effective photothermal killing of bacteria and fungus upon near-infrared irradiation, Adv. Mater. Interfaces. 3 (2016) 1600767.
[33] Z. Jia, P. Xiu, M. Li, X. Xu, Y. Shi, Y. Cheng, S. Wei, Y. Zheng, T. Xi, H. Cai, Z. Liu, Bioinspired anchoring AgNPs onto micro-nanoporous TiO2 orthopedic coatings: Trap-killing of bacteria, surface-regulated osteoblast functions and host responses, Biomaterials. 75 (2016) 203–222.
[34] B. Zhao, H.C. van der Mei, G. Subbiahdoss, J. de Vries, M. Rustema-Abbing, R. Kuijer, H.J. Busscher, Y. Ren, Soft tissue integration versus early biofilm formation on different dental implant materials, Dent. Mater. 30 (2014) 716–727. [35] A.G. Gristina, P.T. Naylor, Q.N. Myrvik, Biomaterial-centered infections:
microbial adhesion versus tissue integration, Pathog. Wound Biomater. Infect. 7 (1990) 193–216.
[36] S. Mei, H. Wang, W. Wang, L. Tong, H. Pan, C. Ruan, Q. Ma, M. Liu, H. Yang, L. Zhang, Y. Cheng, Y. Zhang, L. Zhao, P.K. Chu, Antibacterial effects and biocompatibility of titanium surfaces with graded silver incorporation in titania nanotubes, Biomaterials. 35 (2014) 4255–4265.
[37] C. Staley, T. Kaiser, L.K. Beura, M.J. Hamilton, A.R. Weingarden, A. Bobr, J. Kang, D. Masopust, M.J. Sadowsky, A. Khoruts, Stable engraftment of human microbiota into mice with a single oral gavage following antibiotic conditioning, Microbiome. 5 (2017) 87.
18
material characteristics, of surface topography and of implant components and connections on soft tissue integration: A literature review, Clin. Oral Implants Res. 17 (2006) 55–67.
