A review of cytotoxicity testing methods and in vitro study of biodegradable Mg-1%Sn-2%HA composite by elution method

Abstract

In recent years, magnesium alloys and their composites, a new generation of biodegradable metals, have become biomedical materials for orthopedic bone implants because of their adequate strength and high biocompatibility. Good biocompatible material should lead to low cytotoxicity, hemolysis, bleeding, and inflammation and must not be at risk for carcinogenic reactions. The medical equipment was tested for cell growth, reproduction, and morphology using in vitro tissue cells in the cytotoxicity test. This research examines the cytotoxicity of a Mg-1%Sn-2%HA composite, produced using powder metallurgy methods, utilizing an in vitro mammalian cell culture system in accordance with ISO 10993-5 criteria. Extracts were generated utilizing the elution technique in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) and evaluated on L-929 mouse fibroblast cells. The cells were cultured at 37 °C with 5% CO₂ for 7 days, after incubation, the monolayers were evaluated microscopically for aberrant cell morphology and degeneration, followed by quantitative cell toxicity using the MTT method. The results indicated a high cell viability of 71.51% with the undiluted extract preparation, confirming the non-cytotoxic properties of the Mg-1%Sn-2%HA composite. Furthermore, cell viability improved with dilution, attaining 84.93%, 93.20%, and 96.52% at concentration of 50%, 25%, and 12.5%, respectively. No notable morphological alterations or indications of cellular deterioration were seen. The results support the viability of the Mg-1%Sn-2%HA composite as a biodegradable material for orthopedic applications. The research offers essential insights into the formulation and assessment of magnesium-based biomaterials for enhanced safety and efficacy in medical implants. The novelty of this study lies in combining a critical review of cytotoxicity evaluation methods with an experimental investigation of Mg-1%Sn-2%HA composite. This work is the first to systematically evaluate the cytotoxicity of Mg-1%Sn-2%HA composite, thereby filling a key research gap. Unlike earlier reports that focused solely on Mg-Sn alloys or Mg-HA composites, this work integrates both alloying and reinforcement strategies, thereby offering new insights into their collective role in biocompatibility assessment.

Introduction

Materials used to manufacture medical devices must be biocompatible, which refers to their suitability, as far as their use in contact with the human body is concerned without compromising their integrity [1, 2]. The International Organization for Standardization (ISO) recommends that all newly developed medical devices be subjected to stringent testing to determine their biocompatibility [3, 4]. Therefore, novel medical devices must pass through a series of biocompatibility tests, including cytotoxicity testing, sensitization testing, skin allergy testing, and irritation testing, before they can be used in humans to protect them from physiological, toxic, mutagenic, and immunogenic effects. One of these studies, known as a cytotoxicity test, was performed in vitro using tissue cells to investigate the results related to morphology and cell growth, including reproduction, once medical devices were used [5]. Cellular and metabolic parameters measured using a cytotoxicity assay are useful for assessing cell proliferation and damage [6, 7]. Cell viability refers to the fraction of a total cell sample that is in good health and can be used to calculate the proportion of live or dead cells in the sample. Cell viability can be measured in several ways, including by measuring their redox potential, the health of their membranes, and the production of enzymes such as esterases [8].

The reason for choosing the study magnesium-tin-hydroxyapatite (Mg-1%Sn-2%HA) composite is that magnesium is a biocompatible material that is well-suited for biomedical applications, particularly in implants. Its natural occurrence in the human body and favorable degradation properties make it an ideal candidate for temporary implants that can be safely dissolved over time. However, tin (Sn) is an essential element required by the human body and its toxicity is still unknown. Jhamb et al. [9, 10] examined the in vitro corrosion and biocompatibility of Mg-1%Sn alloys. Tin has been identified as a potentially beneficial ingredient for implants, and HA is a bioactive ceramic that closely resembles the mineral component of human bone. Tin refines the microstructure and enhances corrosion resistance, thereby slowing the rapid degradation commonly observed in pure magnesium. By stabilizing the protective surface film, Sn reduces localized corrosion and contributes to more uniform degradation, Hydroxyapatite (HA), on the other hand, provides a bioactive reinforcement that not only moderates degradation but also improves surface compatibility with surrounding tissues. The presence of HA promotes osteoconductivity and enhances cell adhesion, leading to improved cytocompatibility. By incorporating HA into the Mg-Sn matrix, the author aimed to improve the wear resistance and overall performance of the composite, leveraging HA’s osteoconductivity of HA to promote bone integration [11, 12]. Despite extensive research on magnesium alloys and composites, limited studies have specifically examined the cytotoxicity behavior of Mg-1%Sn-2%HA in vitro. The choice of 1% Sn was predicated on its capacity to improve the corrosion resistance and mechanical characteristics of magnesium while little affecting its biodegradability. Tin has been documented to enhance the strength of magnesium alloys while preserving biocompatibility. Simultaneously, 2% HA was integrated to augment osteoconductivity and furnish a bioactive surface that facilitates cell adhesion and bone integration. The selected proportions were refined according to previous research suggesting that increased Sn concentration may result in significant embrittlement, whereas elevated HA level might undermine mechanical integrity. Most prior investigations have focused on either Mg-Sn alloys or Mg-HA composites separately, leaving a research gap in understanding the combined effects of these constituents on biocompatibility. This study aims to bridge this gap by systematically evaluating the cytotoxic response of Mg-1%Sn-2%HA composite through standardized in vitro testing, providing essential data for its potential biomedical application. The choice of materials reflects a strategic approach for developing advanced biocompatible implants that can meet the mechanical and biological demands of medical applications. This article provides a brief overview to the researchers of the standard procedure to be followed for preparing the samples and performance of cytotoxicity testing in vitro for magnesium alloy and its composites, especially Mg-1%Sn-2%HA.

Review of cytotoxicity methods

Cytotoxicity assays

The proliferation rate and viability of healthy cells, as measured using these assays, are good indicators of health. Physical and chemical agents may influence metabolism and cellular health. This can be toxic to cells. Most cytotoxicity tests were performed using assays with grown cells. It has found comprehensive implementation of chemicals for the study of cytotoxicity and assessment of probable new drugs. Toxicological and tumor cell growth inhibition assays have been employed in oncology research and medication development, respectively [13]. They are rapid, affordable, and do not require animals. It is useful for testing a large number of samples. At the end of the experiment, the percentages of living and dead cells were found to be significant. The nature of an interaction can be determined by selecting a proper test [14].

Classification approaches

• Assays are classified according to the results at the end, that is, changes in color, luminescence, fluorescence, etc. [15].

• Assays for blue trypan, eosin, Congo red, and erythrosine.

• Colorimetric assays: MTT, MTS, NRU, XTT, LDH, SRB, WST-1, WST-8, and Crystal assays.

• Fluorometric assays: CFDA-AM and alamar blue assays.

• Luminometric assays: ATP assay and feasibility analysis in real time.

Exclusion dye tests—1

Dye exclusion is one of the easiest techniques, although it takes a long time and contains dead cells, but not viable ones [16]. It can be used to test membrane integrity and suspension cultures. Blue trypan is a commonly used dye because dead cells turn blue within seconds of exposure [17, 18]. One disadvantage of this method is that the hemocytometer instrument used for cell counting is prone to error, and the other is that it has a negative effect on mammalian cells [19].

Exclusion dye tests—2

Cellular metabolism was examined by measuring the levels of biochemical markers. One of the most widely used colorimetric assays is the bromide diphenyltetrazole (3-(4,5-dimethythiazol-2-yl)-2-5) test [20]. To measure the activity of mitochondrial enzymes, such as succinate dehydrogenase, and ascertain cellular mitochondrial function, as shown in Fig. 1, mitochondrial dehydrogenase converts MTT yellow to purple formazan. Formazan production is proportional to cell viability and can be measured by light absorption at 492 nm. The benefit is that both cytotoxicity and cell viability can be assessed. The MTT assay is user-friendly, rapid, sensitive, accurate, and cost-effective [21]. Needle-like crystals that form when MTT is mixed with water are insoluble, so an organic solvent such as isopropanol or dimethyl sulfoxide must be used to dissolve them [22].

Fig. 1.

Formation of formazan (purple color)—shows the conversion of MTT to formazan by mitochondrial enzymes, which indicates cell viability

Exclusion dye tests—3

Cytotoxicity and cell viability can be easily determined with the help of a flow cytometer, fluorometer, fluorescent microscope, and fluorescence micro platforms [23]. Fluor metric tests for adhesive or suspended cell lines are also applicable and easy to perform. This method is more sensitive than colorimetric assays. A test of the activity of the viable cells was preserved, and constituent protease enzymes were used as a cell viability indicator in GF-AFC (glycylphenyl-alanylaminofluoro coumarin). In these cells, gly and amino acids are removed by cytoplasmic aminopeptidase to release AFC and produce a fluorescent signal that is proportional to the number of viable cells [24], as illustrated in Fig. 2.

Fig. 2.

AFC & R110 for live/dead cell—illustrates the fluorescent markers used to differentiate live and dead cells in cytotoxicity assessment

Exclusion dye tests—4

These assays produce a stable luminescent glow-type signal by adding the reagent used for the fast and easy identification of cytotoxicity and cell viability. The amount of adenosine triphosphate (ATP) in a cell is one of the most sensitive indicators of cell viability [25]. Damaged cells lose membrane integrity, synthesize ATP, and dramatically reduce their ATP levels. ATP testing relies on the reactivity of luciferin with oxyluciferin. In the presence of Mg²⁺ ions and ATP, luciferase catalyzes a process that results in a visible light signal [26], as shown in Fig. 3.

Fig. 3.

Formation of oxyluciferin & light signal—demonstrate the ATP-based luminescence assay mechanism used for cytotoxicity evaluation

Standard and guidelines

The ISO 10993 and FDA bluebook standards (#G95-1) based on 10993-1 address the key issue of ensuring biocompatibility of the device by identifying various types of products to select [27]. Cellular toxicity testing is necessary for all device types. ISO 10993-5 identifies three types of cytotoxicity tests: extract, direct contact, and indirect contact tests. For every type of device, the “cytotoxicity testing-in vitro methods” is covered by 10993-5 [28]. Several methods for determining the acute bioadverse effects of extractable medical equipment are included in the standard. Laboratory workers perform these analyses by cultivating mammalian cells, typically mice or humans, from a commercial source in nutritional culture medium. Cultivated cells are reproduced by cell division and can be subcultured to produce several large cell flakes for material analysis. Magnesium (Mg) and its alloys are among the most appealing candidates in the medical implant industry because of their high biodegradability and mechanical properties.

Commonly used cell line

Cytotoxicity testing is helpful in identifying materials with potential for use in medical devices. The bio consistency of materials can be predicted, and reactive and non-reactive components can be separated using them. These tests are considered so important that they, together with sensitivity and irritation testing, are required for all types of medical devices, as recommended by the test selection guidance ISO 10993-1. By determining the importance of a manufacturing shift in potential alternative materials, cytotoxicity testing methods can be tailored to match, identify, and explore batch-to-lot consistency [29].

Testing for cytotoxicity is a simple, reliable, and inexpensive way to determine whether substances contain significant quantities of biodegradable extracts. Tests are very sensitive because test cells are isolated in culture, and the body’s natural defenses cannot help them. As a physiological alternative, mammalian culture medium is the extractant of choice because it can extract not only water-soluble compounds but also a wide variety of chemical structures. Antibiotics can be added to the medium to eliminate the risk of contamination by microorganisms in the test and control samples. Results from cytotoxicity tests correlated reasonably well with those from relatively brief implant studies. Extracts prepared under more challenging conditions (i.e., 121 °C in salt and cotton oils) do not require them, and normal biocompatibility investigations correlate well with them [30].

Cell monolayers are grown in flasks using standard cytotoxicity testing techniques and then exposed to fluid extracts for testing or checking articles directly or indirectly. By cultivating test and control materials in different media for cell culture, extracts can be generated using the commonly used Elution Test method (e.g., 3 cm² or 0.2 g/ml of a 24-h/7-cultivation medium). A cell monolayer was treated with an individual fluid extract instead of the usual feeding medium. Therefore, test cells were fed a new nutritional source with extractables derived from the test item or control. Crops were incubated at 37 °C and removed at regular intervals for a 3-day microscopic examination. Cells show visible signs of test and control material toxicity (e.g., changes in cell size or appearance, or configuration disturbances). Using an overlay of nutrient agar for tests and inspections, monolayers of cells grown in nutritious or semi-solid media can be protected from any potential damage that might be induced by direct sample contact. Subsequent incubations showed that the sample extracts had moved to the nutrient medium or nutrient agar overlay. The monolayers were examined for the presence or absence of cellular effects in and surrounding the sample. The overlay and direct contact conditions were milder than those used in the elution test. However, these procedures are particularly beneficial if relatively small quantities of samples are available or only one material surface must be examined. [https://www.mddionline.com/testing/practical-guide-iso-10993-5-cytotoxicity].

In technical literature and in the US Pharmacopoeia, and ISO 10993-5, describe elution and direct contact/overlay methods. Although two or more methods are significantly less prevalent than the above, they are employed adequately. Saline extracts were administered to cell suspensions using the suppressive cell growth approach. Cell mass measurement assessed the effect of inhibition on the cells of the extract after typical incubation durations. An extract is exposed to a certain number of cells from or controls a Japanese colonial test article. Cells were counted in the test and control samples after incubation. Soon after hatching, if smaller colonies than the negative controls are utilized to extract the product, cytotoxicity must be absorbed. A basic cytotoxicity test flowchart is shown in Fig. 4. Before treating cells with the test substance, the cell culture is prepared. After incubation, cell viability is measured. Finally, data analysis determines material cytotoxicity.

Fig. 4.

Workflow for 3T3, MTT and XTT assays [28]—depicts the step-by-step process for cytotoxicity testing using different colorimetric assays

Biocompatibility of magnesium-based biomaterials

Magnesium utilized as an implant should have mechanical compatibility with natural bone, with a degradation rate such that it can preserve the mechanical strength throughout the healing period and cause no harm to the human body tissue, as the implant would directly come in contact with tissue. The degradation rate of magnesium increases when it comes into contact with cations, anions, and proteins present in the physiological environment. Therefore, there is a need to decrease the deterioration rate in such a way that it corresponds to the healing duration. This magnesium alloy and its composites have been developed. The alloying elements should also be biocompatible. Hence, alloying elements should be chosen among the requirements for the body, like Ca, P, Na, K, Cl, etc., which are present in bone and teeth as minerals or in blood and fluid as an electrolyte. In addition, Cu, Li, Mo, Be, Ba, Ni, Se, Co, Ce, I, Fe, and Zn are trace elements in the human body. Many researchers have reported that Li, Ca, Zn, and Sr have the potential to be employed as biodegradable Mg-alloy materials [31]. One of the criteria for optimal magnesium engraftment is that it is non-toxic and causes no immunogenic or inflammatory response. Before use as an implant, magnesium alloy or composite should be tested for its biocompatibility, that is, a cytotoxicity test, to confirm that it does not adversely influence the human body.

Influence of magnesium alloy and its composite on cytotoxicity assays

Mg/HA composite materials were sterilized by Gu et al. [32] under UV light for 2 h. L-929 cell lines were cultured and kept at 37 °C with 5% CO₂ and 10% FBS in DMEM supplemented with 100 mg of streptomycin and 100 units of penicillin. Indirect measurements of cell viability were also performed. Serum-free DMEM media is used to prepare the extraction medium for 72-h incubation in a humidified environment at 37 °C with 5% CO₂. The extraction medium was prepared by centrifuging the supernatant fluid, which was then maintained at 4 °C for further use. The surface area to volume ratio of the sample was 1.25 cm²/ml. Both the Positive (0.64% phenol) and negative (DMEM) controls were used. To incubate the cells, 5 × 10³ cells/100 μL of medium were grown in 96-well plates for 24 h. Next, 100 μL of the extracts was blended in. After 1, 2, and 4 days, the 96-well cell culture plates were inspected under a microscope in a controlled humidified environment at 37 °C with 5% CO₂. Each well contained 10 μL of MTT. After 4 h of 37 °C MTT incubation, each well received 100 μL of 10% SDS in 0.01 M HCl and was maintained in a humidified incubator for the whole night. The spectrophotometric absorbance of the samples was determined at 570 nm relative to a 630 nm standard using a microplate reader. Statistical significance was set at p < 0.05. Mg-x%HA (wt% x = 0, 10, 20, and 30) composites were prepared, and their cytotoxicity against the L-929 cell line was investigated for 1, 2, and 4 days. Absorption intensities of Mg-10%HA did not differ significantly from those of the negative control and pure Mg mass extruder after 1, 2, or 4 days of culture. L-929 cells were sensitive to both Mg-20%HA and Mg-30%HA (p-0.05). Mg-30%HA showed unhealthy spherical cell morphology, whereas Mg-10%HA and Mg-20%HA showed healthy spindle morphology. It has been observed that composite Mg-20%HA and Mg-30%HA corrode faster than Mg-10%HA composites, resulting in a higher pH of the removal media, which lowers L-929 cell viability [22]. The current density of the Mg-30%HA composite is calculated to be 63.23 A/cm², or 1.43 mm/year, using the ASTM G31-72 standard. The MG63 osteoblast magnesium IC50 is lower than 2.56 mg/ml, so this is the minimum Mg ion concentration that should be present in the DMEM extraction media. It has been found that magnesium ions are rarely present in cytotoxicity assays.

Gu et al. [33] investigated the cytotoxicity of Mg-1%X (wt% X = Sn, Si, Zn, Zr, In, Ag, Mn, Al, and Y) alloys using NIH3T3 murine fibroblastic cells, L-929 cell line, MC3T3-E1 murine calvary preosteoblast cells, ECV304 human umbilical vein cells, and RSM murine root smooth muscle cells. All cells were cultured in DMEM and were wetted to a carbon content of 5%. Cytotoxicity tests were conducted using indirect contact. A medium extract of 1.25 ml/cm² was obtained after 72 h of exposure to wet atmospheres at 37 °C with 5% CO₂. The extractive element was prepared by removing the supernatant at 4 °C prior before the cytotoxicity assay. Negative controls used DMEM, and positive controls used DMEM with 0.64 phenol. Use 5 × 10³ cells/100 µL in 96-well plates to allow the cells to acclimatize for over 24 h. Extracts (100 µL) were used in place of the medium. A total of 96 cell growth plates were examined under an optical microscope 7 days after being exposure to 2, 4, and 5% CO₂. MTT (10 ml) was then added to each well. Each day, 100 µL of formazan solution was added and incubated overnight in a humid environment. Bio-RAD680 reader data showed 570 nm absorption at a 630 nm reference wavelength. Magnesium and alloy ions were also present in the pH extraction medium. Sn, Si, Zn, Zr, In, Ag, Mn, Al, and Y of each element were added to pure Mg to produce Mg–1%X (wt. %) alloys. Cell viability and platelet adherence assays (cytotoxicity and hemocompatibility assays) were performed. Both fibroblast (L-929 and NIH3T3) and osteoblast (MC3T-E1) cell viability were unaffected by extracts from alloys with a magnesium-to-aluminum and magnesium-to-zinc ratio of 1:1.

The cytotoxicities of each of the nine alloying elements found in Mg–1%X are shown below.

1. Indium—Indium in alloy medium reduced the viability of -MC3T3-E1, ECV304, and VSMC cells, but a concentration of 5 ± 1.8 µM/L had no effect on fibroblast vitality. Wataha et al. [34] discovered non-toxicity in Balb/c 3T2 fibroblasts at 0.435 mM/L and Schedle et al. at 0.1 mM/L [35] in L-929 fibroblasts and gingival fibroblasts reported high toxicity.

2. Aluminum—Extracts of Mg-1%Al alloy have no significant cytotoxicity on any Al-20 ± 7 µM/L concentration tested cell line. There was no significant difference in the viability of MG-63 cells until the Al concentration increased by 1 mM. Furthermore, AZ91D had no effect on the cellular response to HBDC.

3. Manganese—Toxic levels of manganese in the extraction medium were reached at concentrations of 1.8 ± 0.5 µM/L for all cell lines tested. ECs and SMCs were found to be detrimental to their growth and survival of ECs and SMCs [36].

4. Sliver—At an Ag concentration of 0.9 ± 0.5 µM/L, extract of Mg–1Ag alloy decreased cell viability for all cell lines except VSMC. Ag ions are toxic to fibroblasts and osteoblasts at very low concentrations (10-6 M/L).

5. Silicon—At Si concentrations of 71 ± 27 µM/L, the alloy extract Mg-1%Si reduced cell viability in ECV304 and VSMC, while increasing cell viability in osteoblast cells. Zeolite A, [37] a Si compound, promoted the proliferation and development of human osteoblast-like cells within 48 h.

6. Tin—Only ECV304 was toxic to the Mg-1%Sn extract alloy at Sn levels of 15.8 ± 7.8 µM/L; otherwise, it was not toxic to all cell lines. High concentrations of Sn⁺⁴ (66.50 mM/L and 1.11 mM/L) had no impact on MC3T3-E1 cells and L-929 cells, while Sn⁺² at values (0.141 mM/L and 0.025 mM/L) showed high toxicity.

7. Zirconium—At a Zr concentration of 6.9 ± 3.3 µM/L, the cell viability of L-929, VSMC, ECV304, and NiH3T3 cells is reduced in Mg-1%Zr alloy. The opposite was found by Yamamoto et al. [38], who discovered that in L-929 and MC3T3-E1 cell lines, no toxicity was observed when the concentration of Zr was below 10⁻³M/L. The Zr-containing AMS stent performed exceptionally well in the animal studies.

8. Yttrium: In Mg-1% Y alloy, except for MC3T3-E1, the viability of L-929, VSMC, ECV304, NiH3T3 cells was reduced by 2.3 ± 0.7 µM/L. In L-929 cells, the YCl₃ phase of the alloy was toxic at 10⁻⁴ M/L, and at similar concentrations, toxic effects were observed in MC3T3 cells [38].

9. Zinc—All cell lines at 2.6 ± 1 µM/L concentration 1% Zn showed similar or increased viability for Mg-1%Zn alloy.

Zhao et al. [39] used MG 63 cells along with Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with bovine serum. Cell viability experiments were performed in an indirect fashion to determine whether the Mg-Sn alloys were cytotoxic. To obtain ion extracts, alloy samples were incubated in the extract medium and placed in a humidified environment with 5% CO₂ at 37 °C. DMEM was used as a negative control. At a density of 5 × 10³ cells per 100 m², 96 MG 63 cells were seeded on a cell-grown platform and cultured at 5% CO₂ for 24 h in a hydrated environment after adding 100 µL of medium to each well and leaving the plates in an incubator for 2, 4, and 6 days. Optical microscopy was used to examine changes in cell morphology after each treatment. Each well was filled with 20 µL of 5 mg/ml MTT solution and incubated for 4 h. Then 150 µl of dimethyl sulfoxide (DMSO) was added to replace the medium. An optical density of 100 µL was measured at 490 nm (OD). In order to investigate the cytotoxicity of Mg–1%Sn (wt%) and Mg–3%Sn (wt%), the vitality and shape of MG63 cells were examined. The scientists observed that MG63 cells cultured in Mg–1%Sn and Mg–3%Sn alloy extracts for 2, 4, and 6 days had higher cell viability compared to the negative control. Similar or increased absorption was observed between Mg-1%Sn and Mg-3%Sn alloys after 2, 4, and 6 days of cell growth. However, there were no differences between the extracts of MG63 cells and the negative control cells. Negative morphological regulation was found in cell extracts from 2, 4, and 6-day-old cells, as well as in Mg-1%Sn and Mg-3%Sn alloys. Cell morphologies of Mg–1%Sn and Mg–3%Sn alloy extracts were comparable to that of the control. Biological applications of Mg–1%Sn and Mg–3%Sn have been shown to be safe in cytotoxicity experiments. Tong et al. [40] fabricated biodegradable composite Zn–xMg2Ge (x = 1, 3, and 5 wt.%), for bone-implant applications. At 12.5% concentrated hot-rolled Zn–3Mg2Ge composite showed the highest cell viability compared to the as-cast pure Zn and other hot-rolled composites. Dargusch et al. [41] conducted a study on a magnesium (Mg) composite with nano-hydroxyapatite (nHA) fabricated using friction stir processing. The composite was tested for its structure and strength. When tested with human bone cells (HOB), the material showed good biocompatibility, with cells looking healthy and increasing in number. Narayanappa et al. [42] focus on making and comparing the biodegradation and biocompatibility of pure magnesium (Mg) and its composites—Mg/HA, Mg-Zn/HA, and Mg-Sn/HA. Each composite contains 5% hydroxyapatite (HA) and 1% of either zinc (Zn) or tin (Sn). They were produced using an advanced ultrasonic-assisted casting method. Tests showed that the Mg-Zn/HA composite performed the best, with 94% cell growth in bone cancer (MG-63) cells after 2 h. This was due to the formation of a calcium- and phosphorus-rich layer on the surface, which helped cells grow. Yuan et al. [43] studied the shape, structure, bonding, and electrochemical properties of the CMC/CaP composite coating, as well as its long-term corrosion resistance and biocompatibility. Tests showed that this coating creates a better surface for cells to survive and attach.

Lopes et al. [44] investigated the cytotoxicity of a variety of alloys using the MTT assay and the live/dead Viability/Cytotoxicity Kit. The SAOS-2 human OS cell line was used in this study. The cells were first cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C, 5% CO₂, and 1% antimycotic (FBS). The culture media was used as the negative control group, and 1% Triton-X 100 was used to treat the cells in the positive control group. A total of 3 × 10⁴ cells are seeded onto 24-well DMEM plates and exposed to the test chemicals for 24 h to conduct the MTT experiment. It should be noted that during this period, the medium was not changed and magnesium corrosion may have increased the pH. Then, 210 µL of new media and 5 mg/mL of MTT solution were added, and the cells were incubated for 4 h. The sample was treated with 170 µL of SDS solution (4% HCl) for 12 h, and the absorbance was measured at 595 nm. Three readings for each sample were measured, and the average of these readings represented the cell performance. All negative functional tests were deemed 100%. Seeding 6 × 10⁴ cells onto glass surfaces with 2 ml of additional DMEM was necessary for the LIVE/DEAD assay. Cells were harvested after a 24-h control and drug exposure periods. Cells were examined under a fluorescent confocal microscope after washing with phosphate-buffered saline (PBS). Researchers have studied the corrosion and cytotoxicity in Hank’s solution using commercially available pure Mg and Mg alloys AZ91, ZK60, and AZ31. All samples were examined for cytotoxicity in vitro, and cell viability was demonstrated. These researchers have suggested that magnesium and its alloys are effective biodegradable implants. To determine the cytotoxicity and acceptable toxicity of the various magnesium alloys, they were evaluated in human cells with grade I osteosarcoma (with the exception of one ECAP scenario that resulted in grade II toxicity). The ZK30 and Mg-Zn-Zr alloys showed good biocompatibility and slow biodegradability, as demonstrated by cells isolated from rabbit bone marrow. Kubásek et al. [45] investigated the cytotoxic effects of Mg-x% (x = Ga, In, and Sn) alloys by assessing the cytotoxicity of an alloy containing 1% LMM using human osteosarcoma cells (U-2 OS). Humidified conditions were used to cultivate the cells in DMEM supplemented with 10% fetal serum, 10% bovine serum (FBS), 100 µ/ml penicillin, 100 mg/ml streptomycin, and 250 ng/ml amphotericin B (FBS). Cytotoxicity was evaluated using a contact-based method that did not involve any tissue disruption. At 37 degrees Celsius and for 168 h, DMEM medium extracts with 5% FBS and antibiotics were prepared. An alloy with a concentration of 1 cm²/ml was used in this study. The extracts were either used immediately in cytotoxicity tests or kept at 4 °C for later use. The cytotoxicity of the original extract was also tested after dilution by a factor of 2 and 15 times. Eluate metal ion concentrations were evaluated using plasma mass spectrometry. At a concentration of 2.5 × 10⁴ cell/ml, cells were incubated for 24 h on a 96-well substrate. This was equivalent to 100 µL of the extract, which was replaced. As a negative control, 0.64% phenol in DMEM or DMEM was used. After cellular culture, the extracts were incubated in 5% CO₂ at 37 °C for 2–5 days. Five microliters of phenol-red-free (DMEM) medium reagent WST-1 (Roche) were washed with PBS and added twice per well. The WST-1 plates at 37 °C for 4 h. Soluble formazan was produced from living salt tetrazolium cells using enzymes found in the mitochondria. Cell viability was measured using a microplate reader at a reference wavelength of 450 nm (630 nm). When conducting cytotoxicity tests, both the concentration and dilution of the extracts were used. The emissions of Ga and In were negligible compared to those of Sn. Human cells show good tolerance to Mg. The total Mg extract concentration of 160 × 10³ng/ml was within the range at which the cells were viable. After 5 days of incubation at 640 ng/ml, the Mg–1Ga alloy exhibited no acute toxicity compared to phenol. The negative effect of Ga was reduced when 75% of cells remained alive after 5 days of exposure to a dilution of Ga (310 ng/ml total Ga). Diluting it to 1:15 yielded good results. Owing to the extractor’s high Sn concentration (2520 ng/ml), Mg–1%Sn alloy cells were destroyed. When the Sn cell activity was diluted twice, the result was 1310 ng/ml and cell viability was low; however, when Sn levels dropped to 163 ng/ml, the cells continued to show viability. There was no safe concentration of the original Mg-1%In extract. The toxic levels in cells were significantly higher than those of tin. As shown above, Ga-to-Sn-to-In increases LMMs’ cytotoxicity toward U2-operating cells.

Jaiswal et al. [29] chose Human Osteoblast Cell Cultures, that is, hFOB, a fetal osteoblast progenitor cell line that has been used to study cytotoxicity. Dulbecco’s Eagle Media (DMEM)/F-12 was used for cell culture, with 10% fetal bovine serum (FBS) and 1% Geneticin® Selective Antibiotics. Humidified incubators maintained the cells at 37 degrees Celsius and 5% carbon dioxide. Every 48 h, subconfluent cells were subcultured using 0.25% trypsin-EDTA (60–70%). The MTT assay uses a mitochondrial enzyme, NAD(P) H-dependent oxidoreductase, to calculate cell viability. Viable cells transform the tetrazolium salts into crystal violet. Each sample consisted of 20,000 hFOB cells in a 24-well plate. The samples were incubated for 1, 3, or 5 days. Two PBS washes were performed after incubation. Each dish received fresh DMEM media with 5 mg/m and MTT dye added, and then they were incubated for 4 h. The crystals were dissolved by adding 400 µL of lysis solution after aspirating the medium. The optical density of the solution was used as a measure of cell viability, and the absorbance of the soluble product was measured using a 570 nm microplate reader. The biocompatibility of human osteoblast cells (MG-63) was investigated. Cell vitality was measured using the MTT assay. Human fetal osteoblast progenitor cells’ (hFOB) survival was increased by 144% and 128%, respectively, when cultured on Mg-3%Zn-5%HA and Mg-3%Zn-10%HA surfaces, compared to composite Mg3%Zn. In previous studies, researchers found that when Mg-3%Zn was added to the culture medium, cell proliferation and surface viability were both increased, as measured by sol gel, Singh et al. [46] and Kumar et al. [47]. Physiologically active apatite precipitates on the composite once HA is dissolved, resulting in protein adsorption and promoting cell adhesion and proliferation. Five percent HA is appropriate for increasing bioactivity. hFOB cells stained with DAPI showed considerable modifications after 1 day of therapy. More cells were observed on the surface of the HA composites. Therefore, the HA nanofiller exhibited an enhanced cytocompatibility.

The cytotoxicity of an Mg-4%Zn-1%Ca-0.6%Zr alloy with an HA coating was studied by Guan et al. [48] in an indirect assay. The fibroblast morphologies of the HA-coated and uncoated alloys were found to be identical. MTT tests showed that samples that had been coated with HA grew faster than those that had not. Gao et. al. [49] investigated the cytotoxicity of magnesium (Mg) heat-treated in an alkaline environment using the direct method. The alkaline and heat-treated samples showed identical morphologies in bone marrow cells. There was no evidence of cell lysis or growth inhibition in the available Mg samples. The cell densities were found to be higher in the heat-treated samples, indicating an improvement in surface biocompatibility. Magnesium requires in vitro biocompatibility investigation for its potential use in hard tissues and stent-matter. These include tests for antibacterial efficacy, cytotoxicity, and hemocompatibility. Figure 5 shows the workflow for the cytotoxicity test of the magnesium alloy and the composite.

Fig. 5.

Workflow illustration for cytotoxicity test of magnesium alloy & composite—shows the experimental process for cytotoxicity evaluation

Mg degradation was examined by Jo et al. [50] in vivo by employing HA-MgF₂-coated rabbit femoral models. More bones were in contact with the implants in the HA-MgF₂-coated samples, and this contact was longer than that in the bare Mg. Histological evidence demonstrated that the HA-MgF₂ coating improves the bioactivity of the Mg implant. Zheng et al. [51] investigated the effects of Mg-x%Ca (wt% x-1, 5, and 10) on L-929 murine fibroblasts. Cell line was cultured in DMEM with 10% fetal bovine serum (Hyclone), 100 µg/ml streptomycin, and 100 µ/ml penicillin with 5% CO₂ at 37 °C. The authors conducted experiments on indirect cytotoxicity. After 72 h, cells were grown in serum-free DMEM with 5% CO₂ at 37 °C. The supernatant was separated via centrifugation and stored at 4 °C. DMEM medium was used as a negative control, and 10% DMSO was used as a positive control. To increase cell adhesion, 5 × 10⁴ cells were cultured in 96-well plates for 24 h. Extracts (100 µL) were then added to replace the medium. The cells in 96-well plates were evaluated after 1, 2, and 4 days in an environment containing 5% CO₂ at 37 °C. The plates were placed in a humidified incubator after incubating 10 µL MTT in each well at 37 °C for 4 h. The next day, 100 µL of a (10% SDS in 0.01 M HCl) MTT solubilizing solution. The absorbance at 570 nm was determined for the materials, with a reference wavelength of 630 nm. Extracts were evaluated for Mg, Ca, and pH. ANOVA was used to compare cell viability. The significance level of 0.05 was reached. None of the Mg-x%Ca composite extracts showed any significant differences from the control group after 1, 2, or 4 d of culture (p > 0.05). On days 1 and 2, Mg-5%Ca extract reduced cell viability by 10%, but by day 4, there was no effect. After 4 days, cell viability was reduced by 40% in the presence of Mg-10%Ca. While the spindle-shaped cells in Mg-1%Ca and Mg-5%Ca were healthy, the round cells in Mg-10%Ca were detrimental to L-929 cells. The high Mg ion concentration in Mg-10%Ca composite extract and pH could be harmful to L-929 cells. Many researchers have adopted the standard process to determine the cytotoxicity of medical devices made of different alloy materials, but a consolidated review has not been found in the literature, especially for magnesium, its alloys, and composites. This paper provides a brief review of the new researchers for the basic mechanism behind the cytotoxicity test and the use of different methods in vitro for testing the magnesium alloy and its composite, used as a biomaterial. From the literature review, it has also been identified that a limited amount of research on the cytotoxicity behavior of magnesium-based composites reinforced with HA, particularly in the context of Mg-1%Sn-2%HA Biodegradable composites. This gap provides an opportunity to explore the potential benefits of this material combination in future biomedical applications. This research is distinguished by its dual contribution: a review of existing cytotoxicity testing approaches for magnesium-based biomaterials and an experimental validation of Mg-1%Sn-2%HA composite. Such a combined perspective is rarely addressed in literature, making this study a novel step toward bridging methodological understanding with practical biomedical application. Table 1 provides a brief review of the cytotoxicity results from experiments conducted by various researchers.

Table 1.

Cytotoxicity test result of important magnesium alloys & composites—summarizes cytotoxicity outcomes for various Mg-based materials

S.No	Material	Cell line	Culture medium	Assay	Concentration	Cytotoxicity	Ref.
1	Mg-0%HA	L-929	DMEM	MTT	2.56 mg/ml	Yes	[29]
2	Mg-10%HA					No
3	Mg-20%HA					Yes
4	Mg-30%HA					Yes
5	Mg-1%Sn	L-929, MC3T3-E1, ECV304	DMEM	MTT	15.8 ± 7.8 µM/l	Sn+2—No Sn+4—Yes	[30]
6	Mg-1%Si	ECV304, VSME			71 ± 27 µM/l	No	[33]
7	Mg-1%Zn	Balb/c 3T3, L-929			2.6 ± 1 µM/l	No	[30]
8	Mg-1%Zr	L-929, MC3T3-E1, ECV304, NiH3T3-E1			6.9 ± 3.3 µM/l	No	[34]
9	Mg-1%In	MC3T3-E1, ECV304, VSME, L-929			5 ± 1.8 µM/l	No	[30, 31]
10	Mg-1%Ag	VSMC			0.9 ± 0.5 µM/l	Yes	[30]
11	Mg-1%Mn	L-929, MC3T3-E1, Balb/c 3T3			1.8 ± 0.5 µM/l	Yes	[30]
12	Mg-1%Al	MG-63, HBDC			20 ± 7 µM/l	No	[32]
13	Mg-1%Y	MC3T3-E1, ECV304, VSME, L-929, NiH3T3-E1			2.3 ± 0.7 µM/l	Yes	[34]
14	Mg-1%Sn	MG-63	DMEM	MTT	—	No	[35]
15	Mg-2%Sn
16	Mg-3%Sn					No
17	AZ91	SAOS-2	DMEM	MTT	4 × 103 μg/ml	No	[36]
18	ZK60					No
19	AZ31					No
20	Mg-x%Ga (x = 1,4,7)	U-2 OS	DMEM	WST-1	45 ng/ml	No	[37]
21	Mg-x%In (x = 1,3,7)				12 ng/ml	Yes
22	Mg-x%Sn (x = 1,5,7)				163 ng/ml	No
23	Mg-3%Zn	hFOB	DMEM/F-12	MTT	—	No	[24]
24	Mg-3%Zn-2%HA					No
25	Mg-3%Zn-5%HA					No
26	Mg-3%Zn-10%HA					No
27	Mg-4%Zn-1%Ca-0.6%Zr with HA coating	L-929	RPMI1640	MTT	17.37 mM/L	No	[40]
28	Mg treated with alkaline	Marrow cells	RPMI-1640	MTT	—	No	[41]
29	Mg with HA-MgF2 coating	MC3T3-E1	α-MEM	Cyquant cell proliferation assay	—	No	[42]
30	Mg-1%Ca	L-929	DMEM	MTT	183.2 μg/ml	No	[43]
31	Mg-5%Ca				227.1 μg/ml	No
32	Mg-10%Ca				479.6 μg/ml	Yes
33	Mg-0.067%Ca	HUVE, MG-63, L-929	DEME, MEM, CM15-1	MTT	165 mg/L	No	[43]
34	Mg-1%Sn-2%HA	L-929	DMEM	MTT	100% 50% 25% 12.5%	No	Present work

Application of magnesium alloy and composite in biomedical implants (in vivo studies)

Mg is an essential element in the human body and is used as an orthopedic engraft. Human bones contain about 53% of magnesium, muscle contain 27%, soft tissue having 19%, erythrocytes having 0.5% and serum containing 0.3%. The human body contains 21–25 gm magnesium. Because of its favorable properties, such as biodegradability, osteoconductivity, biocompatibility, ability to promote bone metabolism and new bone formation, and elastic module closeness to the human body, it reduces the stress shielding effect and avoids the second surgery to remove the implant, resulting in magnesium, which is often used as an implant to treat fractures.

Xue et al. investigated the biodegradability of pure magnesium, AZ31, and AZ91D alloys by implanting them into the subcutis of nude mice [52]. They found lower corrosion rates compared to in vitro results and no harmful effects on the liver, skin, heart, or kidneys after 2 months, confirming biocompatibility via histology. Xia et al. [53] implanted Mg–4.0Zn–0.2Ca alloy in rabbits, observed a surface layer of Ca, P, O, and Mg, and histologically confirmed new bone formation without inflammation, indicating good biocompatibility. Hou et al. [54] used Mg–3%Sn–0.5%Mn alloy screws in rabbit femoral bones, achieving a homogeneous structure and superior mechanical properties compared to as-cast Mg alloy, with no adverse effects on tissues or organs, ensuring biosafety. Yoshizawa et al. [55] studied pure magnesium and AZ31 alloy scaffolds in vivo, showing less volume loss in AZ31 and confirming their osteogenic properties through histology. Zhao et al. [56] applied high-purity Mg screws for bone fixation in patients with ONFH, reporting no cell death, gas generation, or blood chemistry disturbances, with acceptable degradation rates and new bone formation stabilizing the bone flap. Cheng et al. [57] implanted high-purity Mg screws in rabbits using titanium as a control and observed no host reactions or deformities. Fibrocartilaginous entheses are linked to VEGF and BMP-2 accumulation. Bian et al. [58] demonstrated that Mg-1.8%Zn-0.2%Gd alloy implants maintained integrity for 2 months in rats without adverse effects, proving osteoconductivity. Gao et al. [59] used DCPD-coated AZ60 Mg alloy in rabbit femora to determine new bone formation and confirm its biocompatibility.

Studies on magnesium and its alloys, such as AZ31, AZ91D, Mg–4.0Zn–0.2Ca, and Mg–3%Sn–0.5%Mn, highlight their biocompatibility and biodegradability in vivo. The results showed low corrosion rates, new bone formation, and no adverse effects on organs or tissues in various models. High-purity magnesium and coated alloys exhibited acceptable degradation rates and osteogenic properties, making them viable for orthopedic applications. Additional evidence of biosafety, structural integrity, and minimal host reactions further supports their potential as bioresorbable materials for medical implants. These findings confirm the potential of magnesium alloys for clinical applications.

Material preparation and method

The composites were synthesized using elemental Mg powder ≤177 µm, tin powder ≤50 µm, and hydroxyapatite powder Ca₁₀(PO₄)₆(OH)₂ ≤ 15 µm with 99.90%, 99.99%, and 99.99% purity, respectively. The powders were combined into a precise formulation of Mg-1%Sn-2%HA (wt%) using a high-energy planetary ball mill, specifically the Fritsch Pulverisette P-5 model, at the MRC in Jaipur. Vials and balls composed of stainless steel served as grinding media. Toluene was utilized as the milling agent with a ball-to-powder ratio of 10:1. The machine experienced a cessation of operation for a span of 5 min following each 15-min period of activity. The powdered mixture was dried in an oven and stored in a desiccator. The mixed powder was compressed using a hydraulic compaction machine at a pressure of 100 MPa. A cylinder-shaped die with a height of 50 mm and diameter of 12 mm was used to fabricate the final consolidated sample 12 mm in diameter and 8 mm in height. The specimens underwent a sintering process for a duration of 30 min at 460 °C temperature in a muffle furnace argon atmosphere. Subsequently, they were cooled to the ambient temperature while still inside the furnace.

This study was based on the requirements of the International Organization for Standardization 10993-5, Biological evaluation of medical devices—Part 5: Tests for in vitro cytotoxicity. This test was performed in compliance with the ISO 13485 standard, and the test method was accredited to the ISO 17025 standard. The selection of ISO 10993-5 was based on its international recognition for assessing the in vitro cytotoxicity of medical devices, hence assuring adherence to regulatory standards for evaluation of biocompatibility. This standard offers a systematic framework for evaluating the possible toxic effects of biomaterials, rendering it the most pertinent option for assessing the safety of magnesium-based implants. The study guarantees repeatability, comparability, and compliance with established biomedical safety laws by adhering to globally accepted methods. Extract of HDPE in single strength minimum essential medium supplemented with 10% fetal bovine serum, antibiotics (penicillin: 100 IU/mL and Streptomycin: 100 μg/mL), and 4 mM L-glutamine (1x DMEM) was used as a negative control, Zinc Diethyl di ethio carbamate (ZDEC) as positive control, and 1x DMEM with 10% fetal bovine serum, antibiotics as an Extraction Vehicle. A mammalian cell culture monolayer consisting of L-929 mouse fibroblast cells free of mycoplasma (ECACC Catalog No. 86103224) was used. L-929 murine fibroblast cells were selected for cytotoxicity testing as they are the recommended cell line in ISO 10993-5 guidelines for biological evaluation of medical devices. These cells are highly sensitive to toxic substances, making them a reliable model for detecting cytotoxic effects of biomaterials. Their rapid growth rate, ease of maintenance, and reproducibility further support their use in standardized in vitro assays. Moreover, fibroblasts play a critical role in tissue repair and wound healing, which makes them relevant for assessing the biological response to implantable biomaterials. While other cell lines such as osteoblasts or endothelial cells may also provide valuable insights, the use of L-929 cells ensures compliance with regulatory standards and allows direct comparison with prior studies in the field. L-929 mouse fibroblast cells were propagated and maintained in tissue culture flasks containing 1x DMEM at 37 °C with 5% carbon dioxide (CO₂). For this study, cells were seeded in 96-well culture plates, labeled with passage number and date, and incubated at 37 °C in the presence of 5% CO₂ to obtain sub-confluent monolayers of cells prior to use.

The extract of the test article was prepared using a surface-to-volume ratio of 3 cm²/ml. The sample was cylindrical, with a surface area of 5.27 cm² (diameter, 12 mm; height, 8 mm). The sample was transferred to an extraction container, which was chemically inert, heat-stable, and non-leachable and 1.76 ml an extraction vehicle was added. The extraction was performed at 37 °C for 72 h with continuous agitation (50 rpm). The required quantities of positive and negative control samples were also extracted under similar conditions, as shown in Table 2. The 1× DMEM was used for extraction in the presence of serum to optimize [60–62] the extraction of both polar and non-polar components. Aseptic conditions were maintained during sample processing.

Table 2.

Preparation of extracts for a cytotoxicity study

Sample/control	Extraction ratio	Article amount	Volume of vehicle	Extraction condition
Test sample	3 cm2/mL	5.27 cm2	1.76 mL	37 °C for 72 h
Negative control	3 cm2/mL	3 cm2	1 mL	37 °C for 72 h
Positive control	3 cm2/mL	3 cm2	1 mL	37 °C for 72 h

The L929 cells were seeded in 96-well plates at a cell density of 1 × 10⁴ cells/well in 100 μL of growth medium and incubated at 37 °C with 5% CO₂ for 16–20 h to achieve 50–60% confluency. Four concentrations of test material extract were selected for the treatment of cells (undiluted: 100%; 1:2–50%; 1:4–25%; 1:8–12.5%). On the day of treatment, the growth medium was removed, and 100 μL of each of the four concentrations of test material extract was added to each well in triplicate. For the negative control, 100 μL of HDPE extract (100%) was added to each well. Similarly, 100 μL of each dilution of the positive control extract was added to the respective wells. Culture medium (1×DMEM, 100 μL) served as a blank. The plate was labeled with the appropriate lab number or control and replicate number, along with the test code and treatment date. Subsequently, the plates were incubated at 37 °C and 5% CO₂. On the 4th day of incubation, the sample extract of the test wells was removed, and fresh extract of the test sample was added to the respective wells for each dilution. The plate was further incubated for 3 days (total 7 days) under normal culture conditions (37 °C in 5% CO₂).

After the incubation period, the cells were examined microscopically to evaluate cellular characteristics and percent lysis for qualitative assessment. For quantitative evaluation, cell viability was examined by the MTT method. The growth medium was removed, and each well was supplemented with 50 μL of 1×DMEM containing MTT (1 mg/mL), followed by incubation for 2 h under normal culture conditions. The MTT solution was decanted, 100 μL of isopropanol was added to each well, and the absorbance of the wells was recorded at 570 nm and 650 nm (reference wavelength) using a microplate reader.

The color of the test medium was observed to determine any change in pH. A color shift toward yellow indicated an acidic pH range, and a color shift toward magenta to purple indicated an alkaline pH range.

For the test to be valid, the negative control must have had a reactivity of none (Grade 0, <30% cell toxicity), and the positive control must have been a grade 3 or 4 (>70% cell toxicity). Percent rounding and percentage cells without intracytoplasmic granules were not evaluated in the event of 100% lysis. The test article met the requirements of the test if the biological response was less than or equal to Grade 2 (mild). Table 3 shows the standard grading representation of the reactivity and conditions of the cell culture. The test was repeated if the controls did not perform as anticipated.

Table 3.

Testing grades—defines cytotoxicity grading criteria based on cellular reactivity and viability

Grade	Reactivity	Conditions of all cultures
0	None	Discrete intracytoplasmic granules, no cell lysis, no reduction of cell growth.
1	Slight	Not more than 20% of the cells are round, loosely attached and without intracytoplasmic granules, or show changes in morphology; occasional lysed cells are present; only slight growth inhibition observable.
2	Mild	Not more than 50% of the cells are round, devoid of intracytoplasmic granules; no extensive cell lysis; not more than 50% growth inhibition observable.
3	Moderate	Not more than 70% of the cell layers contain rounded cells or are lysed; cell layers not completely destroyed, but more than 50% growth inhibition observed.
4	Severe	Nearly complete or complete destruction of the cell layers.

Results and discussion

Qualitative toxicity assessment

At the end of the experiment, initial observation of all wells indicated that there was no pH shift in the media of any well. This suggests that the test extract, regardless of concentration, did not drastically alter the acidity or alkalinity of the medium. The microscopic observations of the specimen suggested that when cells were treated with the undiluted test extract (100% concentration), rounding of cells at some level was observed, although the rounding was less than 20% (Grade 0). However, when the test extract was diluted, the rate of cell survival increased, and was comparable to that of the control at the lowest concentration (Fig. 6). There were no morphological changes in the cells of blank and negative control, however, in the positive control wells, more than 60% of cells exhibited rounding, absence of intracytoplasmic granules, or cell lysis. The individual reactivity grades are presented in Table 4.

Fig. 6.

Microscopic observation of L929 cells after treatment with different samples. A Blank, B Negative control, C Undiluted sample extract, D Diluted sample extract (50%), E Diluted sample extract (12.5%), F Undiluted positive control. Note: Observations are presented as the mean of triplicate wells. Percent rounding and percentage cells without intracytoplasmic granules were not evaluated in the event of 100% lysis

Table 4.

Reactivity grades for elution testing—provides quantitative assessment results of cell viability under different test conditions

Treatment	Rounding of cells (%)	Cells without intracytoplasmic granules (%)	Lysis of cells (%)	Grade	Reactivity
Test material extract (100%)	0	0	0	0	None
Test material extract (50%)	0	0	0	0	None
Test material extract (25%)	0	0	0	0	None
Test material extract (12.5%)	0	0	0	0	None
Negative control	0	0	0	0	None
P C 100%	NA	NA	69	3	Moderate
P C 50%	NA	NA	65	3	Moderate
P C 25%	NA	NA	64	3	Moderate
P C 12.5%	NA	NA	58	3	Moderate
Blank	0	0	0	0	None

Quantitative toxicity assessment

The results of the cell viability (MTT assay) indicated that the test material exhibited no cytotoxic effect on L929 cells after 7 days of treatment, although cell viability remained close to the baseline value. The neat extract preparation of the test material demonstrated 71.51% cell viability (>70.0%). According to ISO 10993-5 guidelines, a material is considered non-cytotoxic if cell viability is ≥70% compared to the negative control. In this study, the undiluted extract of Mg-1%Sn-2%HA composite showed 71.51% cell viability, which exceeds the acceptance threshold. Therefore, the result is fully compliant with ISO standards and confirms the non-cytotoxic nature of the composite. An increased percentage of cell viability was observed when the diluted extracts were used, viz. 84.93%, 93.20%, and 96.52% viability at 50%, 25% and 12.5% of diluted samples, respectively. Thus, all the concentrations tested showed >70% cell viability. The positive controls showed a cell viability of 16.81, 21.83, 29.73, and 33.71% cell viability at 100, 50, 25, and 12.5% concentrations, respectively, confirming their cytotoxic effect. The HDPE extract showed no cytotoxic effects (viability 100.97%). These results demonstrate the efficacy of the test system (Fig. 7).

Fig. 7.

Quantitative analysis of cell toxicity by the elution method. Blank: cells grown in 1x DMEM, NC: cells treated with HDPE film extract (negative control); TM: cells treated with different dilutions of test material extract; PC: cells treated with ZDEC extract (positive control)

The cytotoxicity results from this study are consistent with earlier research on magnesium-based biomaterials, confirming their suitability for biomedical applications. The Mg-1%Sn-2%HA composite demonstrated comparable or superior cell viability when compared to studies on pure Mg-Sn alloys and Mg-HA composites, indicating that the presence of Sn and HA does not result in cytotoxic effects. Previous studies on Mg-HA composites indicate cell viability between 65% and 90%, contingent upon HA concentration, whereas Mg-Sn alloys demonstrate viability rates from 68% to 85%. The observed viability of 71.51% in our study aligns with the established range, demonstrating consistency with existing literature. Additionally, research employing various cytotoxicity assessment techniques, including LDH assays and Live/Dead staining, has indicated similar patterns in cell viability. Previous research on Mg-Zn-HA composites indicated a cell viability range of 70–95%, depending on Zn content, which is consistent with our findings. Variations in extraction methods, cell culture conditions, and testing protocols attribute for the differences in viability values. Future research incorporating various assessment methods may yield a comprehensive evaluation of biocompatibility. Through a comparative analysis of our findings and existing literature, we provide a robust foundation for the safe biomedical application of Mg-1%Sn-2%HA composites. Subsequent in vivo studies are essential for validating these findings in physiological contexts, confirming long-term biocompatibility and mechanical stability. The analysis of cell viability results aimed to assess the biocompatibility of the Mg-1%Sn-2%HA composite for potential biomedical applications.

Conclusion

From the above study, it was found that the biocompatibility of medical equipment can be established by cytotoxicity testing. If there is no harmful extractable substance or there is not enough of them to cause acute damage in cultured cells, then the result is negative. Positive cytotoxicity test findings might be considered an early warning indicator that the material at issue includes extractable compounds that may have clinical importance. Most of the studies examined the cytotoxicity of magnesium and its alloy through the standard procedure laid by the ISO, starting from sample preparation to final testing; however, other factors beyond the scope of the ISO standard that can affect in vitro cytotoxicity testing of metallic biomaterials should also be considered, as the cytotoxicity of the extracts is variable, depending on the medium, FBS concentration, exposure mode, and cell type. In present study, the extract of test sample Mg-1%Sn-2%HA showed no evidence of causing cell lysis or toxicity. The test article extract met the test requirements because the reactivity zone and qualitative morphological grade were 0 (zero). The Mg-1%Sn-2%HA composite exhibits promising biocompatibility, indicating its potential use in orthopedic and maxillofacial implants, where controlled biodegradability is crucial for minimizing the necessity of secondary surgeries. The osteoconductive properties of the material, improved through hydroxyapatite incorporation, facilitate enhanced bone integration, rendering it an appropriate option for bone fixation devices, screws, and plates. The gradual degradation of this material corresponds with the natural bone healing process, potentially reducing long-term complications linked to traditional permanent implants.

Future research should focus on in vivo studies to further evaluate the long-term biocompatibility and functional performance of the Mg-1%Sn-2%HA composites in clinical settings. The feasibility of in vivo testing largely depends upon factors including the degradation rate of the material, its mechanical stability in physiological conditions, and the response of the host tissue. Preliminary studies using animal models may facilitate the evaluation of the implant’s interaction with biological environments, encompassing its degradation kinetics, potential inflammatory responses, and osseointegration efficiency. Regulatory considerations and ethical approvals must be thoroughly addressed to ensure compliance with biomedical research guidelines. Additionally, optimizing the alloying elements Sn (1–3%), and reinforcement HA (1, 2, 5, 7%) can be done by applying DOE and surface response methodology, and further their concentrations can enhance the mechanical properties and degradation rates, aligning them more closely with the physiological healing timeline. Future studies will also include ICP-MS ion release profiling and pH monitoring to strengthen mechanistic understanding. Overall, this study provides valuable insights into the development of biodegradable materials for medical applications, paving the way for innovative solutions in the field of regenerative medicine.

Author contributions

Sandeep Kumar Jhamb: conceptualization, methodology, experimentation, and original draft. Ashish Goyal (corresponding): methodology, data preparation, data visualization, review, and analysis. Anand Pandey: data collection and analysis, and manuscript review. Abhijit Bhowmik: investigation, review the final draft. All authors have read and approved the final version of the manuscript.

Data availability

Data supporting the findings of this study are available from the corresponding author, Ashish Goyal, upon reasonable request.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.