Current Progress and Future Perspectives in Contact and Releasing-Type Antimicrobial Coatings of Orthopaedic Implants: A Systematic Review Analysis Emanated from In Vitro and In Vivo Models

Background: Despite the expanding use of orthopedic devices and the application of strict pre- and postoperative protocols, the elimination of postoperative implant-related infections remains a challenge. Objectives: To identify and assess the in vitro and in vivo properties of antimicrobial-, silver- and iodine-based implants, as well as to present novel approaches to surface modifications of orthopedic implants. Methods: A systematic computer-based review on the development of these implants, on PubMed and Web of Science databases, was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Results: Overall, 31 in vitro and 40 in vivo entries were evaluated. Regarding the in vitro studies, antimicrobial-based coatings were assessed in 12 entries, silver-based coatings in 10, iodine-based in 1, and novel-applied coating technologies in 8 entries. Regarding the in vivo studies, antimicrobial coatings were evaluated in 23 entries, silver-coated implants in 12, and iodine-coated in 1 entry, respectively. The application of novel coatings was studied in the rest of the cases (4). Antimicrobial efficacy was examined using different bacterial strains, and osseointegration ability and biocompatibility were examined in eukaryotic cells and different animal models, including rats, rabbits, and sheep. Conclusions: Assessment of both in vivo and in vitro studies revealed a wide antimicrobial spectrum of the coated implants, related to reduced bacterial growth, inhibition of biofilm formation, and unaffected or enhanced osseointegration, emphasizing the importance of the application of surface modification techniques as an alternative for the treatment of orthopedic implant infections in the clinical settings.


Introduction
Postoperative implant-related infection, following bone defect and primary or revision total joint arthroplasties, is a reality and remains a challenge in orthopedics with devastat-ing clinical consequences despite the application of strict protocols of aseptic techniques and perioperative antibiotics [1,2].As surgery techniques and orthopedic implants are constantly being optimized, so is the need for these implants by patients, and hence, so is the possibility of infection occurrence.Implant devices are, therefore, not a panacea; their use is not devoid of issues and their introduction bears the risk of them being colonized by bacteria, which will ultimately lead to the development of implant-related infection [3].Implant removal for the elimination of infection not only impacts patients' health and quality of life but also poses a huge financial burden, which relates to the repetition of surgical procedures, long-term hospitalization, and medication costs, visits to physicians, as well as time off work [2,[4][5][6].Implant-related infection is immensely difficult to avoid or treat; it may impede the healing process and result in implant failure, chronic osteomyelitis, sepsis, and even death [4,7].
For the most part, difficulty in the treatment of infection rests on several factors, such as bacteria affinity for the implants' surface, adhesion, and biofilm formation, which is a crucial step, and the development of antimicrobial resistance [1].The process of biofilm formation, which helps many bacterial species to adapt to various stresses, comprises cellular attachment (reversible and irreversible) to surfaces, microcolony formation, maturation, and dispersion of single cells from the biofilm.Biofilm formation decreases sensitivity to host immune defenses, circumvents systemic antimicrobial regimens, and increases resistance to antimicrobials [5,8,9].Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, as well as methicillin-resistant S. aureus (MRSA) are notorious for the formation of biofilms, which are implicated in implant failure.
Systemic application of antimicrobials, as the first-line treatment strategy, is associated with poor site accessibility and increased toxicity [7].The preclinical use of antimicrobials for the prophylaxis of dreaded implant-associated infections has been reported for a long time [1,5,7,10,11].A plethora of surface modifications by coating the implants' surfaces with appropriate molecules have been developed, and a subset will be reviewed here.
The antimicrobial activity of these implants is mainly based on drug-release or nonrelease methods.Non-release methods refer to materials that can defend the adhesion of microbes and avoid access to the coated material and biofilm formation [12].To this end, the ideal coating would need to eradicate bacterial growth, inhibit adhesion and biofilm formation, and then facilitate bone formation.It would, therefore, have to achieve a balance between cytotoxicity and antimicrobial efficacy and hence support the adhesion of bone-related cells (e.g., osteoblasts) while inhibiting bacterial adhesion.In the case of Intraosseous Amputation Prosthesis, achievement of tissue integration requires eukaryotic rather than bacteria cells to win the "race for the surface", keeping in mind that bacteria may as well reside in the surrounding tissue, a bit further away from the implant surface [11,13].Another important aspect of the delivery system is release kinetics, both in vitro and in vivo.These aspects are reflected in the in vitro and in vivo assessments of the reported categories of implants in this review.
Development and/or identification of biomaterials that combine both antimicrobial and osteogenesis activities are promising approaches for infected bone repair, with a focus on the interface of the implant and the surrounding tissue.Notwithstanding the nonspecific effects, poor release kinetics, and toxicity profiles, a lot of effort is now concentrated on modifying the implants' properties, rendering them less susceptible to infections [1,7].Amongst the coatings developed during the last years, antimicrobial-coated and silvercoated biomaterials have been extensively studied [2,10,14].In addition, iodine and a variety of other coatings (including metal-, vitamin-E (VE), antimicrobial peptides (AMPep)) have gained attention.Coatings can be classified as active or passive [10], based on whether they allow or not release of the antimicrobial agents, with the majority of the ones reviewed here being passive.
Although clinical translation has been relatively limited, there are antimicrobial implant coatings available in clinics nowadays [15], and they have so far shown promise and fewer drawbacks.Nonetheless, there is a pressing need for more knowledge regarding the in vitro and in vivo performance of orthopedic-related coatings.The aim of this study is the qualitative, systematic review of the in vitro and in vivo properties of antimicrobial-, silver-, iodine-based, and novel technology of released and contact-type orthopedic implant coatings in order to point out the possible use of these materials in clinical settings and the need to validate any promising new tools to be introduced in the shield against infection.

Protocol
The protocol of the present systematic review was registered in the PROSPERO international register of systematic reviews (registration number: CRD42023444527).

Research Strategy
A systematic computer-based literature review search with predefined criteria was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [16] in the following databases: PubMed (1947 to 10 August 2023) and Web of Science (1900 to 10 August 2023).Research methodology used a combination of the following terms: "coated implant infection" [All Fields] AND "bone" [All Fields] AND "orthopaedics" [All Fields] AND "in vitro and in vivo [All Fields]", AND "surface modification" [All Fields].
All the electronic literature search was conducted independently by two authors (A.K. and E.P.) and an experienced librarian.Moreover, the above authors independently screened the titles and abstracts to identify relevant studies of outcomes and periprosthetic infection complications after the application of antimicrobial coating.If there was a disagreement between them, the final decision was made by the senior authors (P.J.P. and O.D.S.).

Inclusion Criteria and Study Selection
Studies that examined the outcome of modification in prosthetic surfaces for prophylactic effects against infection in preclinical settings were included in our systematic review.The eligibility criteria were defined according to the acronym PICOS (Population, Intervention, Comparison, Outcome and Study design) such that (P): animals from all species and sexes; (I): application of contact and releasing-type antimicrobial-coated implants; (C): control group without application of coating techniques; (O): studies where the outcome was convincingly and clearly presented; (S): studies that examined the efficacy of the coating techniques in specific micro-organisms compared to the control group.Additional inclusion criteria included (a) studies written in the English language and (b) experimental studies concerning the effectiveness of contact and releasing-type antimicrobial-coated implants in vitro.Contact and releasing-type implant coatings for joint or long-bone applications by any biological or chemical agent were selected.Only full-text articles were eligible for our study.There were no publication date limitations set.
Research that did not include comparative results or was written in a language other than English was excluded.Case reports, reviews, letters to the editor, expert opinions articles, or book chapters with insufficient details about the type of surface modification, the experimental outcome regarding infection rates, osseointegration, biocompatibility, and toxicity effects or studies with non-obtainable data were excluded.Entries with spinalrelated implants, referred to as composites, bars, cones, discs, or cylinder plugs, were excluded.All clinical studies were also excluded.

Data Extraction
Two reviewers (A.K. and E.P.) examined all the identified studies and extracted information using a predetermined form.Data from each study were assembled in a Microsoft Excel spreadsheet and classified per orthopedic implant, type of coated prosthesis, cell lines, species of the animal, bacterial strains, and animal model characteristics.The presence of duplicate studies was examined using Endnote 20 software (Clarivate Analytics, Philadelphia, PA, USA).

Quality Assessment
Three reviewers (A.K., E.P., and E.V.) independently evaluated the quality of the included studies.Since different types of studies were included, the 10-scale CAMARADES (The Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies) [17] and 12-score QUIN [18] quality assessment tools for in vivo and in vitro studies were applied, respectively.CAMARADES and QUIN scores greater than 5 and 12, respectively, were considered of good quality.The CAMARADES SCORE, which is an updated score based on the STAIR SCORE, assesses the quality of animal studies using the following criteria: (a) peer-reviewed publication, (b) statement of control of temperature, (c) random allocation to treatment or control, (d) allocation concealment, (e) blinded evaluation of the published outcome, (f) use of anesthetic without significant alteration of results, (g) appropriate animal model, such as the assessment of the antimicrobial efficacy, osteointegration ability and biocompatibility, (h) sample size calculation, (i) compliance with animal welfare rules and regulations, and (j) statement of potential conflict of interests.The QUIN score is a tool to assess the risk of bias for in vitro studies, which originates from surveys from medical experts who identified key points for a rightly structured study and then verified by other colleagues.The QUIN criteria were (a) clarified aims/objectives, (b) explained sample size calculation, (c) detailed explanation sampling method, (d) details of comparison group, (e) detailed explanation of methodology, (f) operator details, (g) randomization, (h) methods of outcomes measurement, (i) blinding, (j) statistical analysis, and (k) presentation of results.

Search Results
There were 2423 studies identified from the initial search.After evaluation of the titles and abstracts, we excluded 1679 studies and reviewed the full texts of the remaining 144 studies.Fifteen studies were excluded based on the review of the full text.After reviewing the 129 remaining studies and their bibliographies, 71 entries were included in this systematic review (Figure 1).Entries refer to in vitro and in vivo observations; one study (publication) could have two entries corresponding to in vitro and in vivo results.

Bacterial Strains and Antimicrobial Effectiveness
Assessment of the antimicrobial efficacy of the reported coatings was performed against a variety of bacteria (Tables 1-3), with more prominence observed for S. aureus (in 24 entries), followed by S. epidermidis (in eight studies), P. aeruginosa (in five studies), MRSA (in four studies), E. coli (in five studies), and B. subtilis, doxycycline (doxy) susceptible MSSA and S. epidermidis (methicillin-resistant) in one study each.In the majority of studies, one pathogen was used as a means of infection, while there were seven studies where two different bacteria were used and five studies where three or more were used in different assays (Tables 1-3).Similarly, with the in vitro cases, the majority of the in vivo ones studied the antimicrobial effects on S. aureus (26 of them).Other Gram-positive bacteria under investigation included MRSA (eight cases) and S. epidermidis (three cases), while Gram-negative bacteria, including P. aeruginosa (four cases) and E. coli (five cases), were also used.Finally, a combination of two or more bacteria has been reported in 12 studies.Infections during surgery or postoperatively are characterized by bacterial adhesion, subsequent colonization, and, ultimately, the formation of biofilms.Antibacterial efficacy (effect on bacterial growth, adhesion, biofilm formation, and even on the occurrence of resistance) of these coatings was assessed in vitro with standard microbiological assays,

Bacterial Strains and Antimicrobial Effectiveness
Assessment of the antimicrobial efficacy of the reported coatings was performed against a variety of bacteria (Tables 1-3), with more prominence observed for S. aureus (in 24 entries), followed by S. epidermidis (in eight studies), P. aeruginosa (in five studies), MRSA (in four studies), E. coli (in five studies), and B. subtilis, doxycycline (doxy) susceptible MSSA and S. epidermidis (methicillin-resistant) in one study each.In the majority of studies, one pathogen was used as a means of infection, while there were seven studies where two different bacteria were used and five studies where three or more were used in different assays (Tables 1-3).Similarly, with the in vitro cases, the majority of the in vivo ones studied the antimicrobial effects on S. aureus (26 of them).Other Gram-positive bacteria under investigation included MRSA (eight cases) and S. epidermidis (three cases), while Gramnegative bacteria, including P. aeruginosa (four cases) and E. coli (five cases), were also used.Finally, a combination of two or more bacteria has been reported in 12 studies.Infections during surgery or postoperatively are characterized by bacterial adhesion, subsequent colonization, and, ultimately, the formation of biofilms.Antibacterial efficacy (effect on bacterial growth, adhesion, biofilm formation, and even on the occurrence of resistance) of these coatings was assessed in vitro with standard microbiological assays, such as the colony-counting method, inhibition zone assay, and microscopic techniques.In many cases, antibacterial activity was measured after the release of the agent in question from the coating or following the adhesion of bacteria onto the implant.The antimicrobial activity of the majority of these coatings has also been evaluated in vivo (Tables 4 and 5); the studies that are accompanied by in vivo observations are marked by an asterisk in Tables 1-3.

Osteointegration Ability and Biocompatibility
For implants that are intended for long-term use, besides antimicrobial efficacy, osseointegration ability is highly desired.Interestingly, a well-osseointegrated implant is less susceptible to bacterial infection [11].To assess biocompatibility, a plethora of relevant bone-related cell lines were used (Tables 1-3), with more prominent being the murine osteoblast cell line MC3T3-E1 (in eight studies), the human bone osteosarcoma cell lines MG-63 (in three studies), and Saos-2 (in two studies), as well as other cell lines, including primary osteoblasts, human microvascular endothelial cells (HMVEC), fibroblasts, and mesenchymal stem cells (MSCs).No effect regarding biocompatibility was observed in all in vivo studies, as summarized in Table 1.
Viability and adhesion of human cells are important for osseointegration and bone repair.The effect of coatings on the morphology, viability, and adhesion of cells was assessed (Tables 1-3) via proliferation/cytotoxicity assays and microscopic evaluation of the cells.Osteogenic differentiation and osteogenesis were also widely estimated by assessing the activity of alkaline phosphatase (ALP), quantifying osteogenesis-related genes, and using the mineralization assay and deposition of calcium nodules.Among the genes that are central to bone turnover are ALP, a marker for early bone differentiation/maturation; osteocalcin (OC), a marker of late-phase osteogenic differentiation and bone mineralization; collagen-I (Col-1), an abundant component of the extracellular matrix; and runt-related transcription factor 2 (runx2), an early stage osteogenetic transcription factor.In vivo, osseointegration and osteogenesis have been summarized in Tables 4 and 5.

Quality Assessment
CAMARADES and QUIN assessment tools that were used to evaluate the quality of the included experimental studies demonstrated good quality of the included studies.

Discussion
Although several strategies, such as aseptic techniques and the use of antibiotics, have predominated in the current prophylaxis of infection in orthopedic interventions, the prevalence of periprosthetic infections in orthopedic surgery remains high.Regarding the promising clinical results of coating techniques in the prevention of implant infections, further research on novel in vitro and in vivo research findings may provide not only an increased understanding of the current applied techniques but also novel therapeutic approaches in biofilm reduction [12].To the best of our knowledge, this is the first systematic review of released and contact-type coating techniques that analyzed the results of both in vitro and in vivo studies, providing robust evidence about the antimicrobial and osteoinductive activities along with the biocompatibility of these materials.
Gentamicin has a broad bactericidal spectrum and appears to be non-toxic and biocompatible [7].Emerging resistance, however, to gentamicin poses a serious problem [21].VA is a glycopeptide with a broad antimicrobial spectrum, which extends to methicillinresistant strains [19].Doxy is a broad-spectrum antibiotic, and its low resistance (even for MRSA) is documented.It is less nephrotoxic and enters host cells more efficiently than gentamicin [20].Linezolid, a synthetic antibiotic, has a low potential of developing intrinsic resistance and does not show cross-resistance to other systemically administered antibiotics.Linezolid has 100% oral bioavailability, good pharmacokinetics, and good osteo-articular tissue penetration [22,[65][66][67][68][69].The selected MR-5 lytic phage, which is a broad-spectrum bacteriophage, represents a simple, inexpensive, and safe tool.As transduction of virulence or resistance genes is minimal, phages can self-multiply in the tissue surrounding the implants for as long as the bacteria are present without having adverse effects or causing tissue toxicity [8].The benefits of the combination of phage and linezolid were supported by the biocompatibility of hydroxypropyl methylcellulose (HPMC).Release of the antibiotic amikacin and the biofilm inhibitor C2DA have been suggested to have synergistic effects against a variety of pathogens.The added value of the presence of C2DA is that it lowers the amount of antibiotic that needs to be loaded onto the coating [20].An envisaged sequence of action would have amikacin act first by killing bacteria, while C2DA would work afterward by delaying/preventing bacterial adhesion and biofilm formation, allowing time for the antibiotics or the immune system to respond [20].In terms of the phosphatidylcholine (PC) coating, it is envisaged that PC liposomes can be formed following erosion of PC from the coating, which will contain amikacin and C2DA.These liposomes will extend the elution period [20].RFP and fusidic acid both have broad spectra of effect, including biofilm-producing bacteria.They are complementary to each other in terms of the bactericidal and bacteriostatic actions and together can minimize the risk of occurrence of resistance.They can also penetrate the tissue and exert their effect around the bone and in the surrounding tissue.Octenidin and Irgasan also have broad spectra of action.The antiseptics have a faster outcome compared to the more delayed action of the antibiotics, as they directly attack the bacterial cell membrane, contrary to inhibition of the bacterial DNA-dependent RNA synthesis or inhibition of bacterial RNA polymerase and of protein synthesis, that each of the antibiotics RFP and fusidic acid causes, respectively.In terms of the poly-L-lactide (PLLA) matrix, it ensured mechanical stability, while its gradual degradation was essential for the release of the antimicrobial substances incorporated there [21].Moxifloxacin, as used in sol-gel coatings, provides anti-infective activity both in vivo and in vitro in Ti implants [27].This activity summarizes the inhibition of biofilm formation and mature biofilm treatment.Chlorhexidine (CHX), one of the frequently used antiseptics, has a broad spectrum of activity.The inclusion of dopamine increases adhesion to metallic substrates [23].Finally, the use of fosfomycin seems not to be effective regarding bacterial eradication and the prophylaxis of biofilm formation [57].However, an important drawback of antimicrobial-based delivery systems is the continuous decrease in the antimicrobial's concentration.In addition, as the development of bacterial resistance is a complication of antibiotic therapy, different coatings exhibiting antimicrobial properties have been developed and presented in Tables 1 and 2.
Controlled delivery of antimicrobial-based coatings through transfer systems with high encapsulation ability has already been used to enhance the antimicrobial capacity.Mesoporous silica nanoparticles (MSNs) have been tested in order to encapsulate CHX, and the combination was then incorporated in Polydimethylsiloxane (PDMS), ending up in a thin coating film used to investigate possible medical and dental aspects.The results of the in vitro study revealed higher biocompatibility and antibacterial rate without accompanying toxicity of the combined coating substance [70].Finally, the results of this in vitro and other similar studies seem to be promising regarding the development of new coating systems combining encapsulation technology to achieve synergistic antibacterial properties [71].

Evaluation of Ag-Based Coatings
All silver (Ag)-based coatings, irrespective of whether they were bare or as nanoparticles (NPs), exhibited antimicrobial activity, with the majority reporting inhibition of bacterial growth, adhesion, and biofilm formation [8,11,30,31,34,35].All but one study examined biocompatibility and reported a lack of any negative effect on cell morphology and adhesion, as well as viability/proliferation. Cytotoxicity was only observed at 10 mM and >11.36% silver [34] and, in the case of AgNTs/HA, lacking chitosan [23], which will be discussed later.In terms of osseointegration, three studies confirmed the promotion of osteogenic differentiation and osteogenesis [4,31,35].Ag is widely used because it exerts broad-spectrum antimicrobial activity against Gram-positive and -negative bacteria, including antibiotic-resistant strains, fungi, protozoa, and certain viruses [33].In fact, the bactericidal properties of Ag are well-established [41].Ag is inert but ionizes to Ag + in the presence of body fluids.AgNPs also release Ag + .Ag + is known to confer effective antibacterial activity in vitro and in vivo without allowing the development of resistance.Of importance is its ability to prevent biofilm formation.The antibacterial mechanism of Ag + consists of structural changes to the bacterial cell wall, increased permeability, damage of the bacteria's proteins, DNA, and RNA, disruption of metabolism and inhibition of bacteria respiratory chain, and, ultimately, cell death [31,34].Nanolayer Ag has the advantage of preventing the release of potentially very toxic quantities of silver whilst retaining its antimicrobial activity [30,31].When AgNPs are combined with polydopamine (PDA), which itself has antimicrobial activity, better antibacterial efficacy is envisaged [4,68,69,72,73].In addition, PDA biocompatibility and adhesive properties render it a useful coating [31].Chitosan (CS), a biopolymer with complexing and chelating properties, allows the sustainable release of Ag + from the coating [31].The absence of CS from AgNTs/HA could be responsible for the reported cytotoxicity [33].Immobilization of Ag + via IP6 chelation retains antibacterial efficacy [27].In Svensson et al. (2013), the antibacterial mode of action was not fully elucidated; it did not seem to be dependent on release but rather on the nanostructure of the coating itself [11].However, according to several in vivo and limited clinical studies [15], the application of Ag-coated implants (Ag + ) has proven to be well-tolerated without toxicity or related side effects [23,33,41].

Evaluation of Iodine-Based and Other Novel Coatings
The paper on the iodine-based coating examined solely the antibacterial effect of iodine and reported inhibition of adhesion and biofilm formation.Povidone-iodine is a broad-spectrum (including viruses and fungi) antimicrobial agent with a low propensity for developing resistance or causing toxicity [37].
In the diverse category of novel coatings, almost all showed considerable antibacterial activity; reports on Cu [9], Zn [42], VE [40], and RP [39] demonstrated inhibition of adhesion and biofilm formation.Exceptions were reported for the blended VE implants, where intra-species differences were noticed [40], and for TiCuN + BONIT ® [9].Considering biocompatibility, most coatings were shown not to have any effect on cell viability/proliferation (in some cases, it was even found to be increased) [39,43].Interestingly, moderate compatibility and decreased viability/proliferation were found for TiCuN and TiCuN + BONIT ® 9, 4xCu-TiO2 27, NT-Zn3h 30, and HHC36 AMPep for >200 µg/mL [44].As far as osseointegration was concerned, three studies reported enhanced osteogenic differentiation and osteogenesis [33,34,36].The antibacterial efficacy and osteoinductive ability of the aforementioned coatings have also been evaluated in vivo (Table 5), complementing and strengthening the in vitro findings and suggesting that these properties are due to and not impaired by the respective coatings.Specifically, reduced bacterial growth, biofilm formation, and inflammation have been noted by several studies, while osseointegration has either been unaffected [5,7,10,60] or enhanced [31,43,46,53,62].Moreover, satisfying or excellent biocompatibility has been reported, too [28,29,39].
Selenium NPs damage the bacterial membrane of MRSA, thus inducing rapid cell lysis.They are stable due to their inorganic nature and can easily be immobilized on implant surfaces whilst retaining their activity [38].The advantages of the RP-IR780-RGDC titanium implant are as follows: RP and its degradation products are non-toxic, the small amount of singlet O2 seems to enhance the susceptibility of the bacteria to heat, increase the bacterial membrane's permeability, and eliminate biofilm, following irradiation with an 808 nm laser.In addition, RGDC seems to improve adhesion and proliferation [39].VE and its antioxidant properties may be a key point affecting bacterial adhesive ability and biofilm formation.Modifications of the properties of UHMWPE by VE showcased a reduction in adherence of some bacterial strains, with the intraspecies differences, however, suggesting the need for more research in order to fully appreciate the added advantage of VE [40].A more recent in vivo study showcased that VE phosphate could enhance bone stimulation and deposition [48].There is increasing interest in determining the antimicrobial and osseointegrative properties of VE as a coating for orthopedic or dental implants.Heavy metal ions, such as Cu ions, can become toxic.Cu can have a bacteriolytic effect and stop bacteria from replicating [9].The low release of Cu observed for TiCuN + BONIT ® could be responsible for the lack of antibacterial properties reported [9].Cu seems to be effective on planktonic bacteria and bacteria formatting a biofilm while presenting low toxicity.This activity is based on the inhibition of biofilm formation by influencing the advantage of the osteoblasts on the implants' surface [9].The higher affinity of AMPs for bacterial membranes renders them suitable for antimicrobial agents with low toxicity peptides to form electrostatic interactions with anionic phospholipid groups of the bacterial membrane, to then disrupt the membrane and cause bacteria death [43,44].The higher affinity of AMPeps for bacterial membranes renders them suitable for antimicrobial agents with low toxicity [73].Covalent immobilization of MBD-14 on SP is believed to prevent its rapid degradation and ensure its stability.This association in combination with the porous matrix, might be responsible for the antibacterial activity observed [44].Similarly, the antimicrobial effect of zinc (Zn) is mainly expressed by Zn complexes and ZnO NPs [74].Zinc complexes express antifungal activity, whereas ZnO NPs are characterized by antimicrobial activity by two different mechanisms.These activities summarize in the release of reactive oxygen species (photocatalytic process) or ZnO nanoparticles, which lead to the production of intracellular ROS, inducing damage to the cells.
One of the most important limitations of all the nanoparticles used is the lack of available data from in vivo studies with long-term results summarizing the use of these types of implants in animal models.The use of a variety of implants in different animal models provides heterogeneous results, which need to be further specified in future studies.
The presented data of the in vitro and in vivo results of the included studies strongly suggest the application of conventional and novel antimicrobial surface modifications of the implants by orthopedic physicians in the management of postoperative implant infections.However, our systematic review has several limitations.Although 73 entries of high quality were included in this review, the studies' designs and methods were heterogeneous as different animal models were used and no standardized methods were applied in order to evaluate the reproducibility of the outcomes.Additionally, there are some novel coating techniques that have not been tested in vivo.The lack of experience in clinical settings raises concerns about the long-term results of these implants and the growth of multidrugresistant micro-organisms as a result of their clinical use.Finally, a language bias could be present as only studies written in English were reviewed.

Conclusions
Assessment of both in vivo and in vitro studies revealed a wide antimicrobial spectrum of the coated implants under investigation, related to inhibition of biofilm formation and unaffected or enhanced osteointegration, as expressed through the impact of various cells on surface attachment or proliferation.Moreover, the use of these implants was often not related to elevated toxicity levels.Taking into account the known limitations associated with the use of different types of coated implants, their presence can be regarded as a promising candidate for the efficient treatment of implant-related infections.Results from in vitro studies involving both novel coatings and the use of encapsulation technology could aid in the design of effective antibacterial coating materials with high biocompatibility and nontoxicity.Finally, these outcomes should be further studied and validated through clinical trials to be used in clinical practice in the future.

Figure 1 .
Figure 1.Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) flow for seeking and identifying included studies.

Figure 1 .
Figure 1.Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) flowchart for seeking and identifying included studies.

Figure 2 .
Figure 2. Titanium nitride (TiN)-coated implants for total knee arthroplasty (A) composed of the femoral (B), tibial (C) components, and the polyethylene insert (D) displaying significant anti-infective activity and excellent biocompatibility linked to controlled ion release and long-term chemical stability.

Figure 2 .
Figure 2. Titanium nitride (TiN)-coated implants for total knee arthroplasty (A) composed of the femoral (B), tibial (C) components, and the polyethylene insert (D) displaying significant antiinfective activity and excellent biocompatibility linked to controlled ion release and long-term chemical stability.

Figure 3 .
Figure 3. Silver-coated femoral stem (A), titanium nitride (TiN) (B), and vitamin E-coated (C) femoral heads applied in patients after a two-stage revision for infected total hip replacement.(D) custom-made titanium nitride (TiN)-coated implant fabricated with 3D printing technique for the replacement of the calcaneus after complex osteomyelitis.

Figure 3 .
Figure 3. Silver-coated femoral stem (A), titanium nitride (TiN) (B), and vitamin E-coated (C) femoral heads applied in patients after a two-stage revision for infected total hip replacement.(D) custommade titanium nitride (TiN)-coated implant fabricated with 3D printing technique for the replacement of the calcaneus after complex osteomyelitis.

Table 1 .
In vitro studies with antimicrobial-based coatings.

Table 2 .
In vitro studies with silver and iodine-based coatings.

Table 3 .
In vitro studies with novel coating techniques.

Table 4 .
In vivo research data of antibiotic-coated internal fixation and prostheses implants.

Table 5 .
In vivo research data of internal fixation and prostheses implants coated with silver and novel modifications.