Cytotoxicity of Silver-Containing Coatings Used in Dentistry, a Systematic Review

Abstract

Silver is an element that has been widely used in medicine. As a result of its remarkable properties, this metal is now extensively used in virtually all areas of dentistry. Its anti-caries and antibacterial properties are used in (but not limited to) periodontal therapy or during endodontic treatment. The addition of silver ions to materials, such as cements and substances used for fillings, proved to increase their flexural strength and surface microhardness. This element has also found use in orthodontics, e.g., as a material covering components of fixed braces or in implantology as one of the components of coatings applied to dental implants. The following systematic review aims to find and analyze available studies that evaluate silver according to cytotoxicity. For this purpose, information was gathered from three databases: PubMed, Web of Science, and Scopus. This was followed by the Risk of Bias (RoB) analysis and the GRADE analysis of selected articles in which cytotoxicity was tested on human gingival fibroblasts (HGFs). A total of 387 articles were evaluated using required criteria, and, 13 papers were selected for final review. As all studies were evaluated to be of relatively good quality, it may be concluded that silver used in dentistry in low concentrations is free of significant cytotoxicity, and its use helps to improve the properties of the materials used.

1. Introduction

Dental treatment is performed in an oral environment that is heavily colonized by numerous bacterial species. Due to the ease of sample collection, it has become the most well-studied microbiome to date [1]. A large number of bacteria are potentially pathogenic. Therefore, the risk of complications related to harmful bacterial activity must be considered for most therapeutic procedures. Despite the introduction of better materials and improvements in treatment techniques, the basic problem of bacterial infection has not yet been solved. The formation of periimplantitis or carious lesions during orthodontic treatment with fixed braces can be considered a flagship example.

The creation of dental materials not only with good mechanical and biocompatible properties, but also with additional bactericidal and/or bacteriostatic properties, is one of the rapidly developing branches of dentistry. Such additional effects are attempted by adding metal ions with known antibacterial properties to dental materials. An example is the widely used silver ions, among others, in the form of nanoparticles (AgNPs) [2]. According to the ASTM definition, a nanoparticle is a small particle that ranges between 1 and 100 nm in size. The antibacterial effects of silver ions include: (1) inducing the breakdown of the cell wall and membrane; (2) denaturation of ribosomes; (3) disruption of the ATP-producing pathway; (4) disruption of the cell membrane by oxygen radicals; (5) interference with DNA strand replication; (6) denaturation of the cell membrane; and, (7) cell membrane perforation [3]. In dentistry, AgNPs have been used for years, for example, in regenerative periodontal therapy, or added to mouth rinses and filling materials for root canal treatment, composites, resins used in prosthetic treatment, coatings on dental implants or orthodontic materials [1]. Coatings are structures that are additionally applied to already-finished materials. When applied by physical or chemical methods, coatings react with the material being modified to change its original mechanical or biological properties. Hence, this creates the possibility of a final product that, despite being biocompatible, tested, and a safe substrate, may prove toxic to HGFs. Therefore, the purpose of this review is to answer the question of whether silver ion-containing coatings used in dentistry have cytotoxic effects on HGF.

2. Materials and Methods

The main objective of this study is to answer the question of whether silver contained in coatings used in dentistry is cytotoxic. The following systematic review was written based on the principles detailed in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). After pre-selection and final selection of articles from 3 databases, 13 results were obtained (Chart 1). In the next step, an attempt was made to find a suitable tool that could be used for the assessment of RoB. Unfortunately, it was not possible to find a tool that would fully match the subject of the studies found. Such difficulties were also encountered by Tran et al. [4], authors of the paper “Quality assessment tools used in systematic reviews of in vitro studies: A systematic review.” In their review, they searched for a tool that would work best for the RoB evaluation performed on in vitro studies. For this purpose, they looked for papers that used different tools. The authors identified 51 different tools, of which more than half (51%) were those developed by the authors. Such a trend was particularly prevalent within the studies conducted in the field of dentistry. In view of the findings of the paper presented above, and the difficulties encountered in finding a suitable tool when writing this review, it was decided to develop an original analysis, which included questions from various analyses. Alternatively, a cytotoxic effect of silver in the implant could be predicted, through in silico study. It has been highlighted that in silico research does not require laboratory test equipment or approval of research involving living organisms to completion, as for in vivo research. With the demands of development and innovation in the medical and pharmaceutical sectors, more studies are being conducted on computers to solve problems that approximate the results from in vitro and in vivo as mentioned by Jamari [5]. Unfortunately, to date, research regarding in silico method in the study of cytotoxic effect of silver in dentistry is still not sufficient enough to include it in this paper, therefore, the authors focused only on in vitro studies [5].

After the RoB analysis was conducted according to the developed tool, the GRADE analysis was conducted based on, among others, the results obtained. The data collected from studies were compiled, and a meta-analysis was attempted. Unfortunately, only the studies by Huang et al., 2010 [6], Chang et al., 2011 [7], Huang et al., 2013 [8], Sancillo et al., 2014 [9], Cochis et al., 2014 [10], Cataldi et al., 2016 [11], Kheur et al., 2017 [12], Fatani et al., 2017 [13], and Odatsu et al., 2020 [14], included information regarding the p-value (<0.05). The selected studies also differed in terms of their methodology. Both of the aforementioned factors influenced the decision not to conduct the meta-analysis.

3. Information Sources

The search began with a review of four databases—Web of Science, Scopus, and PubMed—and lasted from 1 April 2022 to 5 July 2022. All the articles found were published between 1988 and 2022.

4. Search Strategy

The search began by attempting to find a systematic review published to date, covering the topics included in this paper. No similar systematic review was found. Various combinations of keywords were entered into the databases to collect as much data as possible regarding the cytotoxicity of silver in coatings for use in dentistry. Selected keywords included cytotoxicity, silver layer, silver coating, silver ions, and dentistry.

A very large number of available studies were obtained after a preliminary analysis of the available information; however, those were studies with a variety of methodologies. The largest number of results included articles in which cytotoxicity was assessed using HGFs. For this reason, it was decided to narrow the group of studies selected for the review to those conducted on HGFs. This decision was dictated by an attempt to systematize the available studies and obtain a group of more homogeneous studies that would enable a subsequent meta-analysis and presentation of the conclusions that followed from it. This was also considered when entering keywords during database searching; “human gingival fibroblasts” was included in keyword combinations.

The first search of the databases yielded a total of 387 articles, after which internal and external duplicates were removed. Each of the resulting articles was analyzed after reading the abstract and possible research methodology. Studies in which silver was used in a way other than in a coating, e.g., as one of the components of an alloy, were rejected. Articles in which there was no reference to a possible use in dentistry were also rejected. Studies in which the cytotoxicity of silver was tested on cells other than HGF, such as osteoblasts, were not selected for the review, either. The same applied to a large group of studies that used cells derived from animals. In this way, an attempt was made to obtain a homogeneous group of articles that would enable a subsequent meta-analysis.

5. Results

5.1. RoB Analysis

After the preliminary analysis of the obtained results was conducted, the search for a suitable tool to conduct the RoB analysis began. Unfortunately, no tool was found that would fit its criteria for the type of articles selected for the above-mentioned review. While searching databases, the aforementioned article by Tran et al. [4] was found. It presented a study that aimed to analyze the available tools for evaluating RoB, and analyze how often they were used by other authors to evaluate in vitro studies. The team of researchers concluded that at the time of the study, there was no analysis that was universal enough to meet all the criteria for in vitro studies. As a result, a decision was made to develop an original tool to rate the RoB based on available tools, that are listed in the study by Tran et al. Finally, a checklist of 14 questions was developed, containing topics corresponding to the studies selected for this review. These questions included criteria such as information on the origin of HGFs used in a given study, the timing and description of exposure conditions, and the evaluation of the surface of samples after they were covered with a silver-containing coating (Table 1). It also evaluated how samples were purified before exposure to HGFs. Since the purification of samples in studies was conducted in different ways and, thus, with different accuracy, it was decided to distinguish studies in which the purification of samples involved sterilization. Most studies revealed relatively low levels of RoB.

It was decided to highlight the studies by Kaczmarek et al., 2016 [15], Cataldi et al., 2016 [11], Kheur et al., 2017 [12], and Odatsu et al., 2020 [14]. Those studies proved to be of the highest quality out of the selected studies, and their RoB was rated as very low. Two studies–Fatani et al., 2017 [13] and Ryu et al., 2012 [16]—were rated as moderate because they did not meet the largest number of criteria evaluated. Those above-mentioned studies lacked information regarding the origin and cell line of HGFs used. Moreover, the study by Fatani et al., 2017 [13] did not determine the exposure time of the cells on coatings, nor did it examine the surface of the sample after it was covered with a silver-containing coating. The study by Ryu et al., 2012 [16] lacked information regarding the concentration/number of HGFs used. Studies classified as those with very low RoB met the vast majority of criteria. Funding was the most serious unmet criterion with a potential impact on the RoB.

5.1.1. Studies Using Titanium

Studies in which the authors used titanium as a support on which the coating was applied, included Huang et al., 2010 [6], Chang et al., 2011 [7], Chang et al., 2013 [17], Huang et al., 2013 [8], Morita et al., 2014 [18], Cochis et al., 2014 [10], Kaczmarek et al., 2016 [15], Kheur et al., 2017 [12], and Odatsu et al., 2020 [14]. Hence, those studies represented the largest group, making titanium the most common support of choice for authors.

Two studies by Huang et al. [6,8] and two studies by Chang et al. [7,9] were rated as those with low RoB. The main problem encountered when analyzing those studies was that they did not specify the origin of the cells used, or their descriptions were inadequate. The RoB evaluation was also affected by the lack of proper purification of the post-coating sample. Only in the 2013 study by Huang et al. [8] were the samples sterilized. In contrast, in the 2011 study by Chang et al. [7] the samples were alternately washed in containers with acetone, ethanol, and deionized water using an ultrasonic cleaner for 30 min each. As this is not a sterilization process, its accuracy was rated lower. In the studies by Huang et al., 2010 [6] and Chang et al., 2013 [17], there was no information regarding how the samples were purified before the HGFs were plated on them, which also affected the final RoB evaluation.

The RoB of the study by Morita et al. [18] was rated as low. Since those authors used stainless steel samples in addition to titanium samples in their study, the rationale for the decision is provided in Section 5.1.2.

The RoB of the study by Cochis et al. [10] was also rated as low. The reason for this rating was the failure to specify the origin of cells used in the study. Those authors did not specify which procedures were involved, nor did they include information regarding how patients were selected for the study. There is also no information regarding the clinic/location where the procedures were performed. However, this was the only factor that determined the downgrade of RoB.

Other studies by Kaczmarek et al. [15], Kheur et al. [12], and Odatsu et al., 2020 [14] were rated as having the lowest RoB. They were given a very low rating. The study by Kheur et al. lacked information regarding the purification of samples before HGFs were plated on them. On the other hand, studies by Kaczmarek and Odatsu lacked information regarding whether the trial was repeated. As the above-mentioned studies met all other major criteria, it was decided not to lower the final RoB rating for this reason.

5.1.2. Studies Using Stainless Steel

Studies using stainless steel as a support included those by Ryu et al., 2012 [16], Morita et al., 2014 [18], and Fatani et al., 2017 [13]. All the above-mentioned papers were studies concerning silver-containing coatings and their possible use in orthodontics.

The 2012 study by Ryu et al. [16] aimed to investigate the cytotoxicity of Ag-Pt coatings on stainless steel samples, as such a solution could be used in orthodontics as brackets with better properties. When evaluating the RoB, it was decided to rate the study as moderate. This rating was affected by a lack of sufficient information regarding both the origin of HGFs used for the study and their quantity/concentration.

In the 2014 study by Morita et al. [18], the authors used orthodontic wires as a support, which are most commonly used as fixed retainers. The RoB of the study was rated as low. This decision was made due to the lack of information regarding the testing of the quality of the samples’ coating evaluation in an appropriate manner, and also due to the way the post-coating samples were purified—the samples were only cleaned with alcohol and dried, rather than thoroughly sterilized.

Fatani et al., 2017 [13] studied the cytotoxicity of coatings using stainless steel orthodontic brackets. The RoB of the above-mentioned study was rated as moderate, since the study lacked information regarding both the testing of post-coating samples and the origin of HGF. Moreover, the samples were not sterilized. It was also the only study in which the authors did not include information regarding the exposure time of cells plated on a coating.

5.1.3. Studies Using BisGMA/TEGDMA Thermosets

Studies using BisGMA/TEGDMA thermosets were those by Sancillo et al., 2014 [11] and Cataldi et al., 2016 [11].

The RoB of the 2014 study by Sancillo et al. [9] was rated as low. This decision was affected by the lack of information that post-coating samples were properly tested. That study also had external funding; however, it was difficult to clearly state that this affected the quality of that and other studies.

The RoB of the 2016 study by Cataldi et al. [11] was rated as very low. Since the only aspect that could negatively affect the quality of that study was external funding, it was decided not to lower the final rating for this reason.

Table 1. The RoB evaluation according to the analysis developed by the authors.

-	-	Huang et al., 2010 [6]	Chang et al., 2011 [7]	Ryu et al., 2012 [16]	Chang et al., 2013 [17]	Huang et al., 2013 [8]	Sancilio et al., 2014 [9]	Morita et al., 2014 [18]	Cochis et al., 2014 [10]	Kaczmarek et al., 2016 [15]	Cataldi et al., 2016 [11]	Kheur et al., 2017 [12]	Fatani et al., 2017 [13]	Odatsu et al., 2020 [14]
Introduction	Was the purpose of the study defined?
Methods	Was the number/concentration of HGFs used for the study described?
-	Was a control/reference group included in the study?
-	Did the authors describe the origin of HGFs?		3						1
-	Did the authors describe exactly how tested coatings were created?
-	Was cytotoxicity reliably assessed (e.g., using an SEM or spectrophotometer)?
-	Did the authors examine the surface of the samples after they had been covered with the silver-containing coating?
-	Were the silver-coated test samples purified for cytotoxicity testing?
-	Was it a sterilization process?	-			-			2	-			-
-	Were the conditions under which the study was conducted accurately described?
-	Was the exact time of exposure of HGFs to silver ion-containing coating described?
Results	Were the results checked more than once?
-	No external funding?
-	Were the findings presented in a clear and transparent manner?

Yes/Low RoB. No/High RoB. Not known/ not mentioned. 1: Not enough information. (Primary HGFs were isolated from a recent gingival biopsy collected from tissues excised from healthy teeth obtained from orthodontic procedures). The authors did not mention which procedures were involved, nor did they include information regarding how patients were selected for the study. There was also no information regarding the clinic/location where the procedures were performed. 2: Samples were only cleaned with alcohol and dried. 3: No information regarding the exact location from which samples were taken or patient selection.

Table 1. The RoB evaluation according to the analysis developed by the authors.

-	-	Huang et al., 2010 [6]	Chang et al., 2011 [7]	Ryu et al., 2012 [16]	Chang et al., 2013 [17]	Huang et al., 2013 [8]	Sancilio et al., 2014 [9]	Morita et al., 2014 [18]	Cochis et al., 2014 [10]	Kaczmarek et al., 2016 [15]	Cataldi et al., 2016 [11]	Kheur et al., 2017 [12]	Fatani et al., 2017 [13]	Odatsu et al., 2020 [14]
Introduction	Was the purpose of the study defined?
Methods	Was the number/concentration of HGFs used for the study described?
-	Was a control/reference group included in the study?
-	Did the authors describe the origin of HGFs?		3						1
-	Did the authors describe exactly how tested coatings were created?
-	Was cytotoxicity reliably assessed (e.g., using an SEM or spectrophotometer)?
-	Did the authors examine the surface of the samples after they had been covered with the silver-containing coating?
-	Were the silver-coated test samples purified for cytotoxicity testing?
-	Was it a sterilization process?	-			-			2	-			-
-	Were the conditions under which the study was conducted accurately described?
-	Was the exact time of exposure of HGFs to silver ion-containing coating described?
Results	Were the results checked more than once?
-	No external funding?
-	Were the findings presented in a clear and transparent manner?

Yes/Low RoB. No/High RoB. Not known/ not mentioned. 1: Not enough information. (Primary HGFs were isolated from a recent gingival biopsy collected from tissues excised from healthy teeth obtained from orthodontic procedures). The authors did not mention which procedures were involved, nor did they include information regarding how patients were selected for the study. There was also no information regarding the clinic/location where the procedures were performed. 2: Samples were only cleaned with alcohol and dried. 3: No information regarding the exact location from which samples were taken or patient selection.

5.2. The GRADE Analysis

In terms of inconsistency, most of the reviewed studies were rated as “Not Serious” (

Table 2. The GRADE summary of findings.

Admission Type	Study Design	RoB	Inconsistency	Indirectness	Imprecision	Comments	Certainty
Certainty assessment
Title of publication, authors	-	-	-	-	-	-	-
In in vitro studies
“Antibacterial TaN-Ag coatings on titanium dental implants” by Huang et al., 2010 [6]	In vitro experimental study	Low	Not serious	Not serious	Not serious	p < 0.05	ꚚꚚꚚꚚ High
“Antibacterial properties and human gingival fibroblast cell compatibility of TiO2/Ag compound coatings and ZnO films on titanium-based material” by Chang et al., 2011 [7]	In vitro experimental study	Low	Not serious	Not serious	Not serious	p < 0.05	ꚚꚚꚚꚚ High
“Antibacterial effect of silver-platinum coating for orthodontic appliances” by Ryu et al., 2012 [16]	In vitro experimental study	Moderate	Moderate	Not serious	Moderate	Concentration/number of HGFs not specified, no information regarding the p-value	ꚚꚚꚚO Moderate
“Characterization and antibacterial performance of ZrNO-Ag coatings” by Chang et al., 2013 [17]	In vitro experimental study	Low	Moderate	Not serious	Not serious	No information regarding the p-value	ꚚꚚꚚꚚ High
“Cytocompatibility and antibacterial properties of zirconia coatings with different silver contents on titanium” by Huang et al., 2013 [8]	In vitro experimental study	Low	Not serious	Not serious	Not serious	p < 0.05	ꚚꚚꚚꚚ High
“Biological Responses of Human Gingival Fibroblasts (HGFs) in an Innovative Co-Culture Model with Streptococcus mitis to Thermosets Coated with a Silver Polysaccharide Antimicrobial System” by Sancillo et al., 2014 [9]	In vitro experimental study	Low	Not serious	Not serious	Not serious	p < 0.05	ꚚꚚꚚꚚ High
“Effect of silver ion coating of fixed orthodontic retainers on the growth of oral pathogenic bacteria” by Morita et al., 2014 [18]	In vitro experimental study	Low	Moderate	Not serious	Not serious	No information regarding the p-value for cytotoxicity studies	ꚚꚚꚚꚚ High
“Biofilm formation on titanium implants counteracted by grafting gallium and silver ions” by Cochis et al., 2014 [10]	In vitro experimental study	Low	Not serious	Not serious	Not serious	The significance level was set at 5%	ꚚꚚꚚꚚ High
“In vitro biocompatibility of anodized titanium with deposited silver nanodendrites” by Kaczmarek et al., 2016 [15]	In vitro experimental study	Very low	Moderate	Not serious	Not serious	No information regarding the p-value	ꚚꚚꚚꚚ High
“Adhesion of human gingival fibroblasts/Streptococcus mitis co-culture on the nanocomposite system Chitlac-nAg” by Cataldi et al., 2016 [11]	In vitro experimental study	Very low	Not serious	Not serious	Not serious	p < 0.05	ꚚꚚꚚꚚ High
“Nanoscale silver depositions inhibit microbial colonization and improve biocompatibility of titanium abutments” by Kheur et al., 2017 [12]	In vitro experimental study	Very low	Not serious	Not serious	Not serious	p < 0.05	ꚚꚚꚚꚚ High
“In vitro assessment of stainless-steel orthodontic brackets coated with titanium oxide mixed Ag for anti-adherent and antibacterial properties against Streptococcus mutans and Porphyromonas gingivalis” by Fatani et al., 2017 [13]	In vitro experimental study	Moderate	Not serious	Not serious	Not serious	p < 0.05	ꚚꚚꚚO Moderate
“Antibacterial Properties of Nano-Ag Coating on Healing Abutment: An In Vitro and Clinical Study” by Odatsu et al., 2020 [14]	In vitro experimental study	Very low	Not serious	Not serious	Not serious	p < 0.05	ꚚꚚꚚꚚ High

ꚚOOO Very low. ꚚꚚOO Low. ꚚꚚꚚO Moderate. ꚚꚚꚚꚚ  High.

). The review’s authors did not find factors that could reduce the level of compliance, except for the absence of a calculated p-value in several studies. This was one of the factors that made the subsequent meta-analysis impossible. For this reason, it was decided to reduce the compliance of studies in which the p-value was not calculated to “Moderate.”

Indirectness was rated as “Not serious” in all studies, as those were laboratory studies, and no factors were found to reduce certainty. The study groups were consistent as there were no differences in terms of interventions, outcomes, or use of indirect comparisons.

When evaluating imprecision, the main factor considered was the size of the study group. The only study in which authors did not specify the concentration/number of HGFs was the study by Ryu et al., 2012 [16]. Thereby, it was decided to reduce its level of imprecision. In all other studies, the authors determined the size of the study group, which seemed to be sufficient to consider the findings, authoritative. Moreover, it was found that the cytotoxicity of silver, not only in coatings but also in other forms such as a colloidal solution, was also studied by many other authors, such as Grade et al., 2013 [19]. There was also no shortage of studies in the available scientific databases regarding the cytotoxicity of silver tested on cells other than HGFs, such as the 2010 study by Travan et al. [20], which used primary human skin fibroblasts and, thus, was not included in this review.

The lack of consistency in the composition of the coatings used for tests was a problem encountered during the analysis of the studies. Only in the studies by Odatsu et al., 2020 [14], Kaczmarek et al., 2016 [15], Morita et al., 2014 [18] and Kheur et al., 2017 [12] was silver used without any additive that could interfere with the test result. However, all studies that were evaluated by the authors of this review were conducted using a separate sample for the control group, so it was assumed that other components of coatings were not marked by cytotoxicity and did not affect the final level of certainty. This decision was also influenced by the large number of available studies verifying the cytotoxicity of biomaterials, as discussed in Section 6.

6. Discussion

Based on the number of available papers regarding the use of silver in medicine, it can be concluded that this is a topic of interest for many researchers. The earliest studies found by the authors of this review regarding this topic, date back to the first half of the 20th century. An example of such a study is the 1924 article by Pilcher et al. [21], whose focus included the antiseptic properties of silver.

After the analysis of the available research papers, it was found that silver can improve the properties of tools used in medicine, especially in fields such as dentistry and orthopedics. For such use of silver to be safe, it seems necessary to conduct prior studies regarding the potential cytotoxicity of this element. The comparison of studies available to date seems problematic, as authors used different materials to which they applied the silver-containing coating. There was also a lack of consistency in the composition of coatings themselves, as researchers often used a support in addition to silver, which could interfere with findings. This contrary findings led to the conclusion that further development and studies using in silico methods could help regulate research and findings, as mentioned by Jamari [5].

During the analysis of articles selected by the authors of this review, most were found to be related to research on silver-containing coatings without any additives. As a result, the above-mentioned studies appeared to have the greatest impact on the final outcome of this review. Researchers used various methods to obtain coatings. The 2017 study by Kheur et al. [12] used a DC plasma sputter coating instrument, the 2016 study by Kaczmarek et al. [15] used an electrolytic deposition process, the 2014 study by Morita et al. [18] used immediate dipping into a solution containing Ag ions (5400 ppm), and the 2020 study by Odatsu et al. [14] used a microwave-assisted synthesis. In all of the above-mentioned studies, except for the study by Morita, the authors checked the accuracy of the sample coverage. Therefore, the method of covering the samples seemed to have no effect on the final findings in those studies.

In the studies by Chang et al., 2011 [7] and Fatani et al., 2017 [13], the authors used titanium-silver coatings. The findings of those studies assumed that the coatings used were marked by cytotoxicity; however, there were no significant differences between the sample and control group (Chang), or, as in Fatani’s study, no cytotoxicity was exhibited. In support of this thesis, the 2014 article by Zhang et al. [22] should also be mentioned, in which authors used titanium coatings that additionally contained silver at various concentrations. That study was not included in this review because Zhang et al. used mouse MC3T3-E1 pre-osteoblasts. The authors of the above-mentioned study found that the sample containing the lowest concentration of silver showed no cytotoxicity, and TiO₂ coatings with optimal silver content have the potential to prevent implant-associated infection, while promoting healthy osteoblast cellular activity. The potential use of titanium combined with silver in dentistry was also studied by Oh et al., 2005 [23], who used alloys containing the aforementioned elements. The researchers found that in the agar overlay test, the cytotoxicity of the titanium–silver alloys and of titanium was none or mild. Moreover, titanium–silver alloys had higher mechanical properties and corrosion resistance compared with titanium, while their toxicities were similar to those of titanium. Therefore, it was recommended that titanium–silver alloys be cautiously used in biomedical and dental fields.

In the studies by Chang et al., 2013 [17] and Huang et al., 2013 [8], the authors used zirconium compounds in addition to silver. The biocompatibility of both elements was also studied by Ou et al. [24], who proved that Ag containing yttria-stabilized zirconia exhibited biocompatibility. In another study by Yamada et al., 2017 [25], researchers tested the cytotoxicity of YSZ-based materials covered with an AgNP-containing coating. A control sample was an uncoated sample that showed no signs of cytotoxicity. In contrast, only one of the AgNP-coated samples (5 mM) was found to be cytotoxic. Cytotoxicity tests revealed that AgNP coatings obtained using 0.2–2.5 mM dispersion liquids could be used in medical devices because the cell viabilities of these samples corresponded to more than 70% of the control group value.

In the Cochis et al. study [10], gallium ions were used in addition to silver. The cytotoxicity of both elements was also studied by Pajor et al., 2020 [26], who used powders of HA, enriched with either Ag⁺ or Ga³⁺ or co-doped with both Ag⁺ and Ga³⁺, prepared using two different methods: the wet method (simple coprecipitation in an aqueous solution) and the dry method (heating a mixture of reactants). Pajor et al. found that there was only one sample (5 Ag-HAw) among those synthesized by the wet method that was cytotoxic, while all Ga-containing samples obtained using the dry method showed cytotoxicity. The results of the wet method revealed that silver at a concentration of 0.11% mass (0.01 mol) showed no cytotoxicity, in contrast to a concentration of 0.54% mass (0.05 mol). It is also interesting that silver at higher concentrations and combined with gallium did not prove to be cytotoxic. This indicates a possible avenue of research into the possible improvement of silver-containing coatings enriched with other components that could reduce its cytotoxicity.

In the study by Cataldi et al. [11], the authors used chitlac–silver coatings. Another study by Travan et al. [19], which used a coating of similar composition, was also found during the search. That study was not selected for this review because Travan et al. performed studies on primary human skin fibroblasts, human adipose-derived stem cells (ADSCs), and human osteosarcoma cell lines. In the results of that study, Travan et al. stated that polysaccharide film containing silver nanoparticles did not exert any significant in vitro cytotoxicity towards eukaryotic stem cells, primary cells, and cell lines, which were able to firmly attach and proliferate on the surface of the coating. Another study that examined the cytotoxicity of silver and chitlac was the 2016 study by Gallorini et al. [27], which was not selected for this review because the test samples were prepared as a solution, not a coating. In the study’s conclusions, Gallorini et al. stated that chitlac-nAg could be suggested as a new tool for the implementation of dental devices. However, further investigations are needed to demonstrate the molecular mechanism underlying an autophagic flux occurrence in the HGFs/S. mitis co-culture model in the presence of AgNPs (Gallorini).

Another additive to coatings in studies that were not selected for this review was graphene. The cytotoxicity of coatings containing graphene and silver was studied by Rokaya et al. [28]. Unfortunately, that study was not selected for this review, as the authors of this review were only able to access the abstract. Rokaya et al. stated that the coating containing the above-mentioned elements did not exhibit cytotoxicity, and they concluded that GO/Ag-coated NiTi alloys could be used for biomedical applications. The search of databases also found another study that evaluated the biocompatibility of a graphene-containing coating. Srimaneepong et al., 2020 [29], used GO-coated and GO/Ag-coated NiTi on human pulp fibroblasts (HPFs) for this purpose. They reported that the number of viable HGFs showed no significant differences among the bare NiTi, GO-coated NiTi, GO/Ag-coated NiTi, and controls. Those two above-mentioned studies allowed the assumption that graphene did not affect the cytotoxicity of the sample, but instead, represented a potential ingredient that, when added to the coating, could positively affect its properties, such as surface and mechanical properties, i.e., strength, durability, corrosion resistance, stability, and friction (Srimaneepong).

7. Conclusions

Silver used at low concentrations does not exhibit cytotoxicity to a significant degree. Thanks to its other properties, such as antimicrobial, silver can improve the properties of an alloy used, which argues for its use in medicine at low concentrations. Further studies are still needed to specify the concentration of silver for which cytotoxicity to HGFs cells increases to a level of significance. An interesting direction for technology development seems to be the addition of other elements, such as gallium to coatings, in addition to silver ions. This could reduce silver’s cytotoxicity, thus, allowing it to be used at higher concentrations.

We are currently conducting in vitro testing of antimicrobial coatings containing silver ions for cytotoxic effects on human fibroblast cells.