Cytotoxicity of Biodegradable Zinc and Its Alloys: A Systematic Review

Zinc-based biodegradable metals (BMs) have been developed for biomedical implant materials. However, the cytotoxicity of Zn and its alloys has caused controversy. This work aims to investigate whether Zn and its alloys possess cytotoxic effects and the corresponding influence factors. According to the guidelines of the PRISMA statement, an electronic combined hand search was conducted to retrieve articles published in PubMed, Web of Science, and Scopus (2013.1–2023.2) following the PICOS strategy. Eighty-six eligible articles were included. The quality of the included toxicity studies was assessed utilizing the ToxRTool. Among the included articles, extract tests were performed in 83 studies, and direct contact tests were conducted in 18 studies. According to the results of this review, the cytotoxicity of Zn-based BMs is mainly determined by three factors, namely, Zn-based materials, tested cells, and test system. Notably, Zn and its alloys did not exhibit cytotoxic effects under certain test conditions, but significant heterogeneity existed in the implementation of the cytotoxicity evaluation. Furthermore, there is currently a relatively lower quality of current cytotoxicity evaluation in Zn-based BMs owing to the adoption of nonuniform standards. Establishing a standardized in vitro toxicity assessment system for Zn-based BMs is required for future investigations.


Introduction
Increasing attention has been dedicated to biodegradable metals (BMs) due to their potential to replace permanent implant materials for those of temporary function. BMs, mainly those based on magnesium (Mg), iron (Fe), zinc (Zn), and their alloys or composites, are expected to degrade gradually and leave no residues in vivo [1][2][3]. Zn-based BMs have been developed and investigated as potential implant materials since they have a moderate degradation rate and superior mechanical properties, which are suitable for clinical applications. Bowen et al. conducted a landmark study on the in vivo performance of pure Zn, demonstrating its excellent in vivo biocompatibility and suitable biodegradability for cardiac stent applications [4]. These findings have inspired researchers from various fields to investigate Zn-based BMs.
Currently, the main focus of the clinical application of novel Zn-based BMs lies in their use as vascular stents [5,6], surgical sutures [7,8], and craniomaxillofacial and orthopedic implants [9]. Excellent biocompatibility is a prerequisite for biomedical implant materials. Unlike Ti and its alloys, whose biocompatibility has been established for medical use [10,11], the toxicity of Zn and its alloys remains a subject of debate. Considering the 3Rs principles in animal research, i.e., to reduce, refine, or replace the use of animals in biomedical research, the in vitro assessment is indispensable in estimating the biocompatibility of the novel Zn-based BMs [12,13]. Although ISO 10993-5/12 standards provided a rapid and sensitive approach to assess the potential toxicity of substances [14], conflicting reports on the cytotoxicity of Zn-based BMs have thrown their biosafety into further confusion [15,16].
This systematic review aims to clarify the cytotoxicity of Zn and its alloys according to evidence-based biomaterials research retrieved through an extensive and comprehensive search strategy [17].

Quality Assurance and Criteria
This review was conducted in accordance with the Cochrane Handbook for Systematic Reviews of Interventions and is based on the handbook from the Office of Health Assessment and Translation (OHAT-NIH) for in vitro toxicological studies. We searched for the study articles according to the PICOS (patient, intervention, comparison, outcome, and study design) framework and reported according to PRISMA guidelines (Table S2). Two researchers strictly followed the PICOS strategy and extracted data independently after reviewing the titles, abstracts, and full-text articles. The quality assessment was performed using the Toxicological Data Reliability Assessment Tool (ToxRTool).

Search Strategy
The PICOS framework was followed as the basis of a search strategy, involving the following factors: An electronic search was performed utilizing the PubMed, Scopus, and Web of Science databases of articles published up to 28 September 2022. The search was implemented using a combination of medical subject heading (MeSH) terms and free words. The search strategies that were developed for each database are given in Table S1. Furthermore, a hand search of reference lists for potential eligibility of included articles was performed. A supplementary search was conducted on 1 February 2023 to update the references in a timely fashion.

Inclusion and Exclusion Criteria
The inclusion criteria were studies in the English language that conducted cytotoxicity assessment of biodegradable Zn or its alloys, in which experiments were implemented according to the ISO (International Organization for Standardization) 10993-5 or 10993-12 standards. The exclusion criteria were review articles, clinical studies, in vivo animal studies, case reports, retrospective studies, editorials, opinions, guidelines, conferences, and commentary articles.

Study Selection and Data Extraction
The database search outputs, generated using established search strategies, were imported into Endnote (Version X9.1) to remove the duplicate publications, and then the Rayyan website was used for blinded screening. Two researchers (Q.L. and P.L.) independently screened the literature and extracted the data in strict accordance with the inclusion/exclusion criteria, and then the preestablished data extraction checklist. Disagreements on the eligibility of studies were resolved through careful discussion, and any remaining disputes were resolved by a third researcher (A.L.).
For data extraction, the pre-established data extraction table, which contained basic and experimental information, was utilized for data extraction and analyzed by Microsoft Office Excel 2013. The basic information comprised the year of publication and the author(s). The extracted experimental data were divided into three parts: the Zn-based material, tested cells, and test system. These items included test materials, material processing, types of cell lines, test methodologies, extract ratio and immersion time, dilution ratio, exposure time, use of controls, selected assays, other parameters, and study outcomes.

Assessment of Quality of Evidence
Two researchers (Q.L. and P.L.) independently conducted the reliability assessment using the ToxRTool, which was developed by the European Center for the Validation of Alternative Methods (ECVAM). ToxRTool provides comprehensive criteria and guidance for evaluating the inherent quality of toxicological data or reliability at the methodological level. The in vitro part of this tool consists of an 18-point rating checklist, which is grouped into the following five aspects: (1) test substance identification; (2) test substance characterization; (3) study design description; (4) study results documentation; (5) plausibility of study design and data. Each criterion can be graded as "1" (i.e., "criterion met") or "0" (i.e., "criterion not met" or not reported). Then, the results of 18 criteria were combined to determine the overall quality of the included articles. According to the reliability categorization, articles with 15-18 points were considered reliable without restrictions, studies with 11-14 points were reliable with possible restrictions, and studies with fewer than 11 points were considered unreliable. Furthermore, according to ToxRTool, items 1, 8-12, and 17 were highlighted in red, indicating particular importance. Regardless of the quality assessment score, studies that failed to meet all of the abovementioned red item criteria were classified as unreliable.

Included Studies
The selection process for the included articles is presented in Figure 1. According to the inclusion and exclusion criteria, a total of 86 articles published between 2013 to 2023 were included in this review. The main characteristics of the included articles are summarized in Table 1.     The as-extruded alloy exhibited good biocompatibility at cellular level.

Quality Assessment According to the ToxRTool
Detailed total scores of each ToxRTool item in vitro criteria are presented in Figure 2a. Twenty-nine studies described the source of test substances. A total of 34 and 35 studies were graded as "1" in items 12 and 13, respectively. A total of 40 studies provided the source information of the test system. In addition, 55 studies set negative controls in cytotoxicity evaluation, and 73 articles met the 16th criterion detailing the statistical method for data analysis. The number of articles that met the remaining criteria ranged from 80 to 86 (over 90% of the total articles). As depicted in Figure 2b, it is clear that the quality assessment of included toxicity data was found to be relatively low. Furthermore, 31 articles were reliable without restrictions, three were deemed reliable with restrictions, and 52 articles were not reliable. All studies classified as not reliable failed to meet all of the essential criteria marked in red in the assessment tool. Figure 2. Results of quality assessment of included articles using the ToxRTool: (a) total score for each item of the ToxRTool in vitro criteria; (b) articles classified into three categories on the basis of quality evaluation results (reliable without restriction, reliable with restriction, and not reliable).
The morphological geometry of the test samples was varied, including discs, porous scaffolds [37,39,53], various thread types [60,70], foil [60], wires [40], membranes [85,87], and cylinders [25]. In terms of the preparation and processing of test samples, casting, hot-rolling, and hot-extruding were the main manufacturing processes for biodegradable Zn and its alloys. Other processing technologies included sintering [15], electrodeposited heat treatment [37], and additive manufacturing [39,47,53,68]. Figure 3a displays the division of cell types tested into three main categories: cells pertaining to osteoblasts, cardiovascular-related cells, and others. A large proportion of studies used immortalized cell lines in evaluating toxicity, whereas only five used primary cells (with passage number ≤7) [21,22,34,46,69]. Regarding orthopedic investigations, 21 selected articles used the MC3T3 (pre-osteoblast cell line), which is usually used in osteogenic evaluation. Although the vast majority used MC3T3-E1, one report used another clone number [69] and one did not describe the specific clone number [66]. Fourteen studies adopted MG-63 in cytotoxicity evaluation, a cell line from human osteosarcoma, and nine studies investigated Saos-2 osteoblasts (human primary osteosarcoma cell line). A few studies used other cell types, such as bone marrow mesenchymal stem cells (MSCs) [30,46,67,91]. Human umbilical vein endothelial cells (HUVECs) and human endothelium-derived cell lines (EA.hy926) were the most commonly employed cell lines in vascular research. The mouse fibroblast cell line (L929) was investigated in 24 articles, accounting for a considerable fraction of the third category. Overall, more than 70% of the articles evaluated only one cell line, 19 articles (22.9%) compared the toxicity performance of two cell lines, and five studies (6.0%) selected three different cell lines in carrying out cytotoxicity tests.

Test System
Concerning the extraction parameter, for evaluating the cytotoxicity of Zn-based BMs, 68 publications employed extract tests, while three studies exclusively conducted direct contact tests [33,74,77]. The remaining 15 studies combined the two approaches ( Figure 3b). As shown in Figure 3c, various sample surface-to-volume extraction ratios and immersion times were used to prepare the sample extracts. Nineteen extract tests applied the ratio of 1.25 cm 2 /mL with sample immersion times of 72 h and 24 h, respectively. Eight studies were conducted using the sample powder to evaluate cytotoxicity and adopted the extraction ratio of sample weight to medium volume [19,22,37,39,41,53,61,70]. Notably, 13 studies did not specify a certain ratio. Additionally, some researchers adopted the extraction ratios such as 3 mL/cm 2 , 1.25 mL/cm 2 , and 20 mL/cm 2 without providing any justification [32,52]. Extracts were often filtered via a membrane [19,20,39,49,51,53,60,65,69,72] or centrifuged to withdraw the supernatant fluid in certain studies [18,20,26,40,58,62,80,101,102]. Some studies investigated Zn-based samples precultured in the medium before the cytotoxicity test [25,28,72,74,77,92,102]. The effect of BSA (bovine serum albumin) on the cytotoxicity in the medium of pre-exposure samples was also examined [72]. Jablonska et al. confirmed the effect of FBS (fetal bovine serum) in the cell medium on cytotoxicity tests [15,83]. The effect of pretreatments such as pre-cultivation, stabilization treatment [91], sterilization treatment [75], sandblasting [93], and acid etching treatment [102] was also investigated.
As shown in Figure 3d, cell viability tests could be divided into qualitative and quantitative assays. Among the assays used for quantitative tests, tetrazolium salt-based assays such as CCK-8 (WTS-8), MTT, WTS-1, and MTS were used frequently. Forty-seven studies used CCK-8 assays in cytotoxicity evaluation, ranking first. Cell survival was determined using the MTT assay in 19 studies and the MTS assay in eight studies. Two studies were tested using the lactate dehydrogenase (LDH) release assay, while five were tested using the bromodeoxyuridine (BrdU) incorporation assay to measure cell proliferation. In a few studies, cell viability was determined on the basis of a fluorometric resazurin reduction method, such as CyQUANT [46] and resazurin assays [15,33]. The assays for qualitative analyses are summarized in Figure 3e, in which live/dead staining FDA/EB dye [16,44,59], Calcein-AM/PI dye [30,82,101], and FITC-phalloidin/DAPI dye [25,51,58,64,65] were the primary assays to realize the visualization of cells. Six studies did not indicate the specific assays used in qualitative assessment.
With regard to the control groups, 32 studies set both positive and negative control groups, while 28 studies set only negative control groups, and the remaining 26 studies set neither negative nor positive control groups. The cells in the cell culture medium alone and supplemented with 5-20% dimethyl sulfoxide (DMSO) were usually chosen as negative and positive control groups, respectively. Some researchers also used Cu and Ti-6Al-4V alloy as negative and positive controls, respectively [59,[73][74][75][76]83,93]. Five studies used the culture medium supplemented with 0.64% phenol as a positive control [21,35,45,52,63].
It is worth noting that the assessment criteria were inconsistent. In most studies, a reduction in cell viability of more than 30% was considered a cytotoxic effect, but the threshold for cytotoxicity was at 75% in multiple studies [68,92]. In addition, many of the included studies claimed to have graded the toxicity from quantitative results in extract tests according to ISO standards [32,34,35,45,[52][53][54]86,99].

Outcome
The relationship between Zn-based BMs and cytotoxicity ranged from excellent biocompatibility to apparent cytotoxicity. Notably, the majority of the selected studies suggested that Zn-based BMs were nontoxic or produced toxic effects only under specific conditions, such as with a highly concentrated extraction solution. However, only two studies reported that pure Zn or its alloys were toxic [26,58].

Discussion
Zn-based BMs have been proposed and developed for biomedical implant materials. Alloying is a common way to improve the material properties of Zn-based BMs. This systematic review assessed the potential cytotoxic effects of Zn and its alloys. On the basis of the results, the current quality assessment of toxicity studies was assessed to be highly heterogeneous, with different study designs and non-standardized procedures making it difficult for quantitative analysis. A qualitative analysis showed that the cytotoxicity of Zn-based BMs is mainly determined by three factors: the Zn-based materials, tested cells, and test system (Figure 4).  Various processing techniques were used to improve the mechanical performance of Zn-based BMs. However, these techniques also changed the material microstructure, possibly influencing their corrosion behavior and biocompatibility. The as-cast alloys suffered from significant nonuniform micro-galvanic corrosion, although the biodegradation uniformity was improved by the hot extrusion or rolling processes [20,44]. After extrusion, primary dendritic phases were broken and distributed along the extrusion direction, thereby refining the grains distinctly due to dynamical recrystallization [24]. The more uniform corrosion and reduced corrosion rate brought about by the refinement of the second phase of the bottom circulating water-cooled casting method was also demonstrated, leading to higher cell viability than conventional casting in 100% extracts [50]. Nevertheless, the opposite was true for as-extruded Zn-1.2Mg alloy, which had lower cell survival than its as-cast alloys due to a higher concentration of Zn ions resulting from a higher corrosion rate [27]. This might be related to the Mg 2 Zn 11 phase, which is distributed relatively uniformly at grain boundaries and in the Zn matrix, involved in the formation of micro-galvanic cells [64,103]. Moreover, the corrosion resistance of the Zn matrix could be dramatically improved by appropriate heat treatment and plastic processing, resulting in a decrease in released metal ions and higher cytocompatibility [37,48]. Interestingly, one study confirmed that the crystallization process of the material affected its biocompatibility [33]. In brief, the effects of processing on the cytotoxicity of Zn-based BMs are mainly caused by changing a material's microstructure and corrosion behavior. When designing a novel biomaterial, the relationship between the improvement of mechanical properties and the consequent change in biocompatibility has to be evaluated.

Alloying and Its Micro-Galvanic Corrosion
The degradation behavior of Zn-based BMs in the body is intrinsically determined by their corrosion process [104]. The corrosion behavior of metals depends on metallurgical factors such as alloy composition, phase precipitation, and segregation of alloying elements and impurities [105,106]. Therefore, the elements added into Zn-based alloys can increase their cytotoxicity by promoting micro-galvanic corrosion.
Numerous studies demonstrated that adding Mg to pure Zn improved the biocompatibility of Zn-based alloys [23,43,82]. However, studies confirmed that the cell viability of the Zn-Mg alloys was not subject to monotonic variation with the Mg content [31,70], possibly due to the combined effect of grain refinement and passivation [82]. Copper is a component of numerous enzymes and plays a crucial role in the response to oxidative stress [107,108]. One study demonstrated that the cytocompatibility of the Zn-1Cu alloy was significantly higher than that of the pure Zn [80]. The beneficial effects of Ca, Sr, Fe, Ag, Mn, and Li as alloying elements on cytocompatibility have also been proven [55,58,69,90]. The positive effect on the biocompatibility of alloying elements was shown to be provided by Cu > Ca > Ag in decreasing order [41].
Alloying elements might interfere with the toxicity assessment by creating degradation reactions. As an example, the silver ions from Zn-Ag alloys could combine with chloride ions that existed in the medium to form a precipitate of silver chloride [66]. The concentration of Zn ions in the medium is decreased by alloying, but the toxic effect of the insoluble metal salts may be unknown. Excess intake of Al 3+ is considered toxic. However, studies have reported that Zn-Al alloys have no harmful effects on HUVECs in diluted extracts, possibly because the Al 3+ concentrations were negligible compared to the halfmaximal inhibitory concentration (IC 50 ) of Al 3+ [26]. Although a high concentration of rare-earth elements (REEs) inhibited ATPase activity and caused metabolic disorders in the body [109,110], the addition of REEs to the Zn matrix also led to favorable cytocompatibility [57,68,78,94]. Undoubtedly, the release rate and content of alloying elements are essential factors influencing the cytotoxicity of Zn-based alloys.

Surface Treatment
Pretreatment could alter the surface morphology, wettability, and roughness of the material and consequently impact its biocompatibility [111]. Among the studies included in the analysis, sample pre-cultivation was the most common pretreatment. Specifically, surface stabilization treatment resulted in a stable surface oxide film which inhibited the release of Zn 2+ , decreasing the cytotoxic effect [44,48,91]. The components of the medium used for pre-cultivation are also crucial. The presence of BSA during pre-incubation resulted in the best wettability and the lowest ion release in the initial stages of the exposure. In this case, the biocompatibility was better than that of untreated groups [72]. Li et al. investigated the impact of sterilization treatments on the cytocompatibility of Zn-based BMs. Due to the excessive release of Zn ions and a local concentration over the cellular tolerance capacity, the autoclave-treated Zn matrix exhibited apparent cytotoxic effects on fibroblasts [75]. Furthermore, cell viability in extracts of the polished-textured samples was higher than those of the fine-textured and coarse-textured samples [92]. Likewise, sandblasting treatment of the surface of Zn-based alloy specimens decreased the cell viability due to localized corrosion of the samples [93]. Therefore, as a means of changing the microstructure of tested samples, pretreatment had prominent effects on cytotoxicity.

Effects of Tested Cells on Cytotoxicity
The various selected cell lines were mainly associated with the vascular and orthopedic research fields. Given that cytotoxicity is determined by the cellular tolerance of degradation products released from the Zn-based metals [108], the selection of tested cells was a vital factor in the toxicity assessment. Furthermore, metal ions usually affect cell growth in a concentration-dependent manner [60], and the tolerance of distinct cell types to metal ions is also inconsistent [76]. Hence, different cell lines could produce distinct outcomes in the toxicity assessment. For instance, Zn-Li alloy showed good biocompatibility with MC3T3-E1 cells and HUVECs [58], while its extracts showed significant cytotoxicity to L929 cells [61]. Milenin et al. determined that the viability of hDPSC was significantly higher than that of the Saos-2 under the same conditions [70]. Endothelial cells usually exhibited better cell viability compared to L929 cells, MC3T3-E1 cells, and vascular smooth muscle cells in toxicity evaluation [18,58,60], possibly because Zn, as an antioxidant and endothelial membrane stabilizer, could enhance endothelium integrity [112].
Several studies utilized stem cells with broad differentiation potential, and all these cell lines exhibited great vitality [30]. Even in the same research field, distinct cell lines have different tolerances. HOS cells showed a significant reduction in cell viability and induced cytotoxicity at a higher extract concentration, while the same conditions had a slight negative impact on the viability of MG-63 cells [27]. Also, MG-63 cells were more tolerant to Zn ions than MC3T3-E1 cells [56]. Moreover, MG-63 cells have been recommended for in vitro evaluation because they could closely simulate human cells [57]. However, this cell line may display heterogeneity among different cell populations due to donor factors. Considering the inhomogeneity of primary cells, established cell lines are more recommendable for use unless reproducibility and accuracy of the response can be demonstrated.

Parameters of the Extract Tests
In addition to the corrosion properties of the alloy itself, the degradation rate mainly depends on the culture medium used. The ingredients of different media could be varied, and the selection of the cultivation medium is usually dictated by the cell type used. It was reported that the relatively low Zn ion release in McCoy's 5A medium than in DMEM or DMEM/F-12 could be attributed to increased passivation film formation by a high concentration of HPO 4 2− in the medium [44]. It is worth mentioning that extracts prepared by rinsing in α-MEM would help to stimulate the physiological environment [66].
Capek et al. confirmed that the ZnCl 2 was less toxic to L929 cells in DMEM than in α-MEM, possibly since DMEM contains more glucose, amino acids, and vitamins, consequently having a strong buffering effect [76]. To provide serum proteins in simulated body fluid, adding 10% FBS to the cell culture medium is a common practice. Notably, the presence of FBS in the extraction medium could accelerate the initial corrosion process of the Zn matrix, leading to the additional potential for cytotoxicity [16]. It could be due to rapid protein adsorption on the Zn surface inhibits initial surface passivation with a protective Zn phosphate layer [113].
Among the included studies, there was significant heterogeneity in the extraction ratio of prepared extracts, partly due to different versions of the ISO standards being referred to, but more often due to the adoption of unprecedented extraction ratios. Extraction for 24 h was thought not to be sufficient to obtain an extract that represents the tested material used in practice. Hence, an immersion time of 72 h is recommended in the latest ISO standard (10993-12: 2021). Few studies adopted this latest standard. It was observed that undiluted or high-concentration extracts could exhibit toxicity effects, which could be put down to high ion concentrations and osmotic pressure inhibiting cell adhesion and growth [37]. Wang et al. suggested using a minimum of 6-10 dilutions in evaluating Mg-based BMs. This range mimics the continual clearance of absorbable ions from the circulatory system [108]. Several studies followed this recommendation and reported no toxicity effect with 10% or 12.5% extracts of Zn matrix BMs. Notably, the concentration dependence of Zn ions is more pronounced than that of Mg [114,115]. Therefore, the most appropriate dilutions for toxicity testing of Zn-based BMs to mimic the in vivo environment are yet to be demonstrated.

Direct Contact Tests
Most included studies have the same study endpoints in both extraction and direct contact tests. However, one study reported that tested samples had good biocompatibility in extract experiments while exhibiting cytotoxicity in the direct contact experiments [56], possibly since in vitro direct cultivation of cells on the Zn matrix was hampered by rapid degradation and partial shedding of degradation products [73]. Even though the material is nontoxic, it might interfere with the proliferation of cells to some degree [67]. It is difficult to determine the primary factors leading to decreasing cell viability and adhesion. These factors include the increase in local pH, change in surface morphology, shedding of the corrosion layer, and surface composition. In addition, cell adhesion and proliferation largely depend on extracellular matrix deposition, which is controlled by the protein adsorption capacity of a matrix surface [86].
Although direct contact tests cannot capture all complexities in vitro, they are still necessary for rapid initial screening of cytocompatibility of medical devices.
Since tested cells can be influenced rapidly by released soluble corrosion products [110]. Since rapid protein adsorption occurs on the surface of the specimen after implantation, it is advisable to mimic this process by pre-culturing the sample in vitro when performing direct toxicity tests.

Selected Assays
Tetrazolium salt-based assays were used particularly frequently in toxicity assessment in the included studies. Furthermore, MTT and XTT are classical toxicity assessment methods recommended in ISO 10993 standards. However, existing studies have shown that tetrazolium-based assays can be confounded by the presence of metals, leading to false positive or negative effects [74,108]. Some authors used other parameters for toxicity assessment apart from cell viability and survival, such as the comet assay to detect the degree of DNA damage [21] and flow cytometry to evaluate the cell cycle [26]. The application of multiparametric assessment to support the observation of toxicity by a single endpoint requires more funding and research. With the development of in vitro assessments in toxicology, new paradigms of analysis, such as proteomics, genomics, and pathway analyses, contribute to the understanding of the mechanisms involved in the toxicity pathways beyond providing evidence for cell death [116]. Although these paradigms do not yet permeate the published toxicity assessments of Zn-based BMs, they might be a promising direction in the future.

The Criteria of Cytotoxicity Evaluation
The obtained data appears to be comparable only within the results of the same study or when stringently standardized. The criteria for toxicity evaluation in quantitative and qualitative tests were different. According to the ISO-10993 standard, the toxicity results were only graded in qualitative tests. By assessing the changes in tested cells, the change from normal morphology should be graded into five levels, and the numerical grade greater than two was considered a cytotoxic effect. Only one toxicity threshold was set in the quantitative evaluation, i.e., a reduction in cell viability of more than 30% was considered a cytotoxic effect. Non-standardized evaluation criteria make the quantitative analysis of toxicity data difficult and make a direct comparison of study results impossible.

Strengths and Limitations of This Work
To our knowledge, this is the first systematic review of the cytotoxicity of Zn and its alloys. First, this review used the ToxRTool tool to assess the quality of toxicity evidence from the included studies, revealing the relatively lower quality of in vitro toxicity assessment.
Subsequently, a detailed analysis was performed of the heterogeneity of toxicity testing in the included studies. However, this systematic review had three limitations, the first being the large volume of data from the included articles and the significant heterogeneity among the studies preventing a quantitative data analysis. Even though some trends regarding the relationship between cytotoxicity and physicochemical effects were identified, the specific influence and magnitude of each factor remain elusive. Secondly, our review was not registered in PROSPERO because RCT procedures do not apply to preclinical studies. Thirdly, this study only focused on the cytotoxicity of Zn-based BMs, while the biocompatibility is wider-ranging with many more elements than cytotoxicity. Therefore, systematic reviews regarding other aspects of biocompatibility (e.g., immunogenicity, inflammatory response, or tissue compatibility) should be performed in the future.

Conclusions
This systematic review aimed to provide insight into the available literature exploring the cytotoxicity of biodegradable Zn and its alloys. Within the limitations of this study, the following conclusions can be drawn:

1.
High heterogeneity exists in the implementation of the included studies and the assessment results of the toxicity studies.

2.
The qualitative analysis demonstrated that biodegradable Zn and its alloys had conditionally cytotoxic effects, mainly dependent on the Zn-based materials, tested cells, and test systems. 3.
The material processing technologies and alloying elements had a potential effect on the toxicity of Zn-based BMs due to modifications in microstructure and corrosion characteristics.

4.
Endothelial cells had better tolerance to the toxic effects of Zn-based BMs than other tested cells.

5.
A standardized in vitro toxicity assessment system for biodegradable metals is still lacking, and further construction is required. In addition, researchers in this field need to comply with existing evaluation criteria and report test procedures in as much detail as possible to make the study data more informative and valuable to promote translational research and the long-term development of Zn-based BMs.

Informed Consent Statement: Not applicable.
Data Availability Statement: Experimental data from this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.