ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10015-2357
World Journal of Dentistry
Volume 15 | Issue 1 | Year 2024

Evaluation of Cytotoxicity of Green Synthesized Strontium Fluorapatite Nanoparticles on Human Gingival Fibroblasts: An In Vitro Study

Nisshitha Rao Setvaji¹, Aravind Kumar Subramanian²

^1,2Department of Orthodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India

Corresponding Author: Aravind Kumar Subramanian, Department of Orthodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India, e-mail: aravindkumar@saveetha.com

Received: 03 December 2023; Accepted: 01 January 2024; Published on: 20 February 2024

ABSTRACT

Aim: To evaluate the cytotoxicity of the green synthesized strontium fluorapatite nanoparticles (SrFAp NPs) on human gingival fibroblast (HGF) cell lines using MTT assay.

Materials and methods: Strontium fluorapatite nanoparticles (SrFAP NPs) used in the present study were developed using green synthesis method from plant extracts of Equisetum arvense (Horsetail) and Laminariales (Kelp) along with fluoride and hydroxyapatite precursors. The HGF cell lines were treated with different concentrations of 10, 20, 30, 40, and 80 μg/mL of SrFAp NPs and cultured. The cultured media was treated with MTT reagent and incubated. The number of damaged or dying cells was represented by the absorbance of the produced formazan at 570 nm and measured using a UV Vis spectrophotometer.

Results: The cell viability decreased with increasing concentrations of SrFAp NPs. At the lowest concentrations of 10 μg/mL, cell viability was 83%, and at 20, 30, 40, and 80 μg/mL, the cell viability was 81, 79, 74, and 70%, respectively. One-way analysis of variance (ANOVA) showed a significant difference between the different concentrations (p = 0.000, F = 14.682).

Conclusion: Green synthesized SrFAp NPs showed good biocompatibility against HGF using the MTT assay. At the lowest concentration of 10 μg/mL, cell viability was 83% and at the highest concentration of 80 μg/mL NPs, the cell viability was 70%.

Clinical significance: Strontium fluorapatite nanoparticles (SrFAp NPs) were developed by green synthesis method, which is nontoxic and environmentally friendly. The NPs were hypothesized to have enamel remineralizing properties which could be further applied clinically to prevent white spot lesions.

How to cite this article: Setvaji NR, Subramanian AK. Evaluation of Cytotoxicity of Green Synthesized Strontium Fluorapatite Nanoparticles on Human Gingival Fibroblasts: An In Vitro Study. World J Dent 2024;15(1):25–29.

Source of support: Nil

Conflict of interest: None

Keywords: Cytotoxicity test, Fluorapatite, Human gingival fibroblasts, MTT assay, Nanoparticle, Strontium

INTRODUCTION

Synthesis of nanoparticles (NPs) and their application in various fields have led to the forefront of research.¹ NPs have found their way into several biomedical applications such as drug delivery, diagnostic tools, and even cancer treatment.² In dentistry, various applications include dental implants, restorative materials, prevention of caries and white spot lesions, teeth whitening, anti-sensitivity agents, and polishing agents.³

Nanoparticles (NPs) may be organic (dendrimers, liposomes, etc.), carbon-based (fullerenes, carbon quantum dots), or inorganic (metals, ceramics, or semiconductors).⁴ NPs have a high surface-to-volume ratio and hence offer several advantages such as accelerated adsorption rate, extended half-life period, and increased drug concentration.⁵ NPs are synthetically developed by either top-down or bottom-up approaches. In top-down approaches, bulk materials are divided into nanostructures and include procedures such as mechanical milling, laser ablation, and electro-explosion.⁶ In bottom-up methods, NPs are assembled from corresponding atoms and molecules through chemical or biological procedures such as sol-gel process, laser pyrolysis, chemical reduction, etc.⁷ However, all these chemical methods lead to environmental pollution, large-scale energy consumption, and adverse health issues.⁸

Green synthesis employs environment-friendly materials such as plant extracts and microorganisms, which behave as reducing agents to reduce metal ions to metallic NPs.⁹ It is nontoxic, pollution-free, economical, and sustainable. Recently, hydroxyapatite NPs (HAp NPs) have been developed for remineralization of enamel. However, they lack strength, are brittle and have low solubility.¹⁰ Previous studies replacing calcium ions in hydroxyapatite with strontium ions have reported increased reactivity, solubility, and fluoride release.¹¹^,¹² Thus, a nanosystem of strontium-doped hydroxyapatite with fluoride may work synergistically to remineralize enamel.

With the recent growing interest in the development of NPs, toxicity of these materials is a concern.¹³ Wide range of published literature evaluating cytotoxicity of NPs using different cell lines, colorimetric assays, and incubation times is available.¹⁴^,¹⁶ However, due to the highly varied experimental settings, the clinical relevance of such studies is questionable. In spite of the exponential progress in generating NPs, there is a lack of standardized tests for evaluating their cytotoxicity.¹⁷In vivo studies are more desirable for investigating toxicity as they are more direct and precise. Nevertheless, well-defined in vitro experimental conditions can simulate in vivo systems and prove to be reliable toxicity tests.

Most studies evaluating cytotoxicity use colorimetric methods such as MTT assay.¹⁸ MTT assay is superior to other toxicology tests as it is safe, relatively easy to use, and produces a high reproducibility.¹⁹ It is extensively employed in cytotoxicity tests and to assess cell viability. Its drawbacks include the fact that the process requires multiple wash steps and that the conversion to formazan crystals depends on the metabolic rate and number of mitochondria, leading to numerous known interferences.²⁰

This study used strontium fluorapatite NPs (SrFAp NPs) produced by green synthesis method using plant extracts of Equisetum arvense and Laminariales along with fluoride and hydroxyapatite precursors.²¹ The aim of this study is to evaluate the cytotoxicity of the green synthesized SrFAp NPs on HGF cell lines using MTT assay.

MATERIALS AND METHODS

Study Setting

This was a single-centered in vitro study conducted at the research facility in Saveetha Dental College and Hospital, Chennai, Tamil Nadu, between February and March 2023.

Strontium Fluorapatite Nanoparticles Synthesis

Strontium fluorapatite nanoparticles (SrFAp NPs) used in the present study were developed using green synthesis method from plant extracts of Equisetum arvense (Horsetail) and Laminariales (Kelp) along with fluoride and hydroxyapatite precursors. About 2.4 gm of both dried Horsetail leaves and dried kelp were mixed with 100 mL of distilled water, boiled, and filtered. About 30 mL of the filtrate was mixed with 0.2 gm of hydroxyapatite powder and stirred continuously on a REMI 2MLH magnetic stirrer at 600 RPM for 72 hours and monitored for a color change. Another 30 mL of the filtrate was mixed with 0.525 gm of strontium chloride and subjected to the same protocol. Ultraviolet–visible spectroscopy (UV Vis Spectroscopy) (Perkin Elmer Lamba 35) was performed to determine the formation of HAp NPs and Strontium NPs (Sr NPs). The two mixtures were then added together and mixed with 0.09 gm of fluorine. The solution was centrifuged at 8000 RPM at 60°C for 10 minutes and the precipitate was collected. It was then dried in a hot air oven at 80°C for 2 hours and stored in a powdered form. Fournier transform infrared spectroscopy (Alpha Bruker’s ll FT-IR) and scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDAX) (JSM–IT800 Nano SEM) were performed to characterize the developed NPs.

MTT Assay

Synthesized SrFAp NPs were assessed for cytotoxicity against human gingival fibroblasts (HGF) cell lines by MTT assay.

Culturing of Human Gingival Fibroblasts

The HGFs were extracted from the healthy gingiva of patients undergoing tooth extraction after the Saveetha Institute of Medical and Technical Sciences (SIMATS) ethics committee granted informed consent and ethical approval. With the aid of collagenase-dispase (3 and 4 mg/mL, respectively), the tissue was broken down enzymatically to isolate the cells. The isolated cells were cultured and maintained under standard conditions of 37°C and 5% CO₂ in Dulbecco’s Modified Eagle Medium F12 (DMEM F12) and supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Detection of Biocompatibility of Nanoparticles against Human Gingival Fibroblasts

A stock solution of 1 mg/mL of SrFAp NPs was dissolved in culture media. The solution was syringe filtered and the cells were treated with different concentrations of 10, 20, 30, 40, and 80 μg/mL of the NPs. The control group consisted of untreated cells and was included in the assay to compare the full cell viability assessments. The prepared materials were immersed in DMEM-F12 supplemented with 1% penicillin/streptomycin. The media was collected after 24 hours of immersion and treated with cells to test their biocompatibility. After 24 hours of culture, 10 μL/100 mL of MTT reagent (5 mg/mL stock) was added to the cultured cells and then incubated for 4 hours to allow the formation of the formazan dye at 37°C. After the incubation period, the supernatant was removed and dimethyl sulfoxide solution (DMSO) (200 μL) was added for 10 minutes. The reaction product was transferred to a 96-well enzyme-linked immunosorbent assay (ELISA) plate. The absorbance at 570 nm was measured with UV-Spectrophotometer ELX800 UV universal microplate reader (Bio-Tek Instruments Inc, Vermont, United States) using DMSO as the blank. The number of damaged or dying cells is represented by the absorbance of the produced formazan at 570 nm. Measurements were performed in triplicate and the results were reported as percentage of cell viability (%). The concentration required for a 50% inhibition (IC50) was produced graphically.

The percentage of cell viability was calculated using the following formula:

% cell viability = A570 of treated cells / A570 of control cells × 100

Statistical Analysis

The statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software (IBM Statistical Package for the Social Sciences, SPSS Inc, Chicago, United States, version 23). Descriptive statistics were performed. The data analysis was done using one-way analysis of variance [one-way analysis of variance (ANOVA)] followed by post hoc Tukey test with a significance level of p < 0.05.

RESULTS

Characterization of the Nanoparticles

Visual observation of color change from pale brown to dark brown indicated the formation of Sr NPs and HAp NPs. After addition of fluorine to the mixture, there was an additional color change indicating the formation of SrFAp NPs. UV Vis Spectroscopic analysis monitored the formation of Sr NPs and HAp NPs at 420 and 410 nm wavelengths which corresponded to the surface plasmon resonance (SPR) band of the Sr NPs and HAp NPs, respectively. Another reading at 375 nm wavelength confirmed the formation of the SrFAp NPs. FT-IR spectroscopy reported peaks in mid-infrared spectrum. Several peaks that correspond to amino and imino compounds, carboxylic acid salts, calcium salts, fluoro compounds, phosphates, and strontium were observed. SEM analysis revealed sharp, rod-like particles ranging from 20 to 50 nm in size illustrating metallic elements such as strontium (Fig. 1). Spherical agglomerates ranging from 30 to 80 nm in size corresponding to hydroxyapatite were seen (Fig. 2). Elemental analysis (EDAX) revealed a composition of 7.0 wt% of strontium, 5.4 wt% of fluorine, 9.4 wt% of calcium, and 4.2 wt% of phosphorus (Fig. 3).

Fig. 1: Scanning electron microscopy (SEM) analysis depicting sharp rod-like metallic elements

Fig. 2: Scanning electron microscopy (SEM) analysis depicting spherical agglomerates

Fig. 3: Energy dispersive X-ray analysis (EDAX) of SrFAp NPs

Cytotoxicity of the Nanoparticles

The biocompatibility of the synthesized SrFAp NPs was analyzed by MTT assay. The cell viability was assessed using HGF cell lines with different concentrations of SrFAp NPs. After 24 hours of incubation, the NPs showed higher cell viability at lower concentrations. Control group showed 100% cell viability. With an increase in the concentration of the NPs, the cell viability decreased. At the lowest concentrations of 10 μg/mL, cell viability was 83%, and at 20, 30, 40, and 80 μg/mL, the cell viability was 81, 79, 74, and 70%, respectively. Even at the highest concentration of 80 μg/mL NPs, the cell viability was 70%. The mean percentage of cell viability is represented in bar graphs with standard deviation as error bars (Fig. 4). One-way ANOVA showed a significant difference between the different concentrations (p = 0.000, F = 14.682). Tukey’s post hoc test for multiple group comparison determined which of the means were significantly different from each other (Table 1).

**Table 1:** Tukey’s test for multiple group comparison (p < 0.05)
Groups		Significance	95% confidence interval
Groups		Significance	Lower bound	Upper bound
10	20	0.783	–3.5715	7.9715
	30	0.269	–1.7715	9.7715
	40	0.002	2.8285	14.3715
	80	0.000	7.2285	18.7715
20	10	0.783	–7.9715	3.5715
	30	0.081	–3.9715	7.5715
	40	0.025	0.6285	12.1715
	80	0.000	5.0285	16.5715
30	10	0.269	–9.7715	1.7715
	20	0.881	–7.5725	3.9715
	40	0.169	–1.1715	10.3715
	80	0.001	3.2285	14.7715
40	10	0.002	–14.3715	–2.8285
	20	0.025	–12.1715	–0.6285
	30	0.160	–10.3715	1.1715
	80	0.192	–1.3715	10.1715
80	10	0.000	–18.7715	–7.2285
	20	0.000	–16.5715	–5.0285
	30	0.001	–14.7715	–3.2285
	40	0.192	–10.1715	1.3715

Fig. 4: Percentage of cell viability obtained through MTT assay

The characterization of the NPs showed successful formation of SrFAp NPs. The MTT assay revealed ideal cell viability at low concentrations and non-cytotoxic potential of the NPs even at higher concentrations.

DISCUSSION

Characterization of SrFAp NPs performed using UV Vis Spectroscopy, FT-IR and SEM EDAX showed successful formation of NPs. The SEM analysis revealed sharp rod-like particles corresponding to strontium and spherical agglomerates of hydroxyapatite. The elemental analysis showed increased levels of strontium, calcium, and phosphate particles with fluorine.

The developed NPs were hypothesized to possess enamel remineralization properties and must, therefore, first be tested for biocompatibility. Cells representative of the exposure route and organs targeted by the NPs must be included in the cytotoxicity assay in order to evaluate the impact of the NPs.²² Hence, the cytotoxicity of the green synthesized SrFAp NPs was evaluated on HGF cell line using MTT assay.

The MTT assay is a nonradioactive, colorimetric method of measuring cell viability. It measures cell viability through determination of mitochondrial function. This method involves reduction of yellow MTT, or 3-(4,5-dimethylthiazol-2-yl)—2,5-diphenyl tetrazolium bromide to insoluble purple formazan by mitochondrial succinate dehydrogenase which can only occur in metabolically active live cells.²³ Thus, it is possible to quantify the percentage of living cells in a solution.

With an increase in concentration of the NPs, cell viability decreased. This is probably brought about by the acute toxicity of the metal ions. At lower concentrations of 10 and 20 μg/mL, the cell viability is still above 80%. The cytotoxicity of the NPs increased in a dose-dependent manner. According to ISO 10993-5:2009 of biological evaluation of medical devices with tests for in vitro cytotoxicity, the lowest cell viability regarded as having biocompatibility is 70%, below which it is considered to have cytotoxic potential. It is noteworthy that the cell viability was 70% even at the highest concentration of 80 μg/mL NPs. This indicates that the SrFAp NPs pass the minimal requirement even at high concentrations and is considered to exhibit low toxicity.

Previous studies have developed strontium hydroxyapatite NPs (SrHAp NPs) by chemical methods and assessed their cytotoxicity. A study by Rajendran et al. evaluated toxicity of SrHAp NPs prepared from coprecipitation method on L929 fibroblast cell line by direct observation of cells using inverted-phase contrast microscope and MTT assay. They found that the formulated paste had higher cell viability when compared with regular toothpaste.¹⁰ In a study by Krishnan et al., HAp NPs were prepared using wet chemical method, and SrHAp NPs were developed using coprecipitation method. Cell viability of L929 fibroblast cell line was assessed by MTT assay and cell morphology was assessed using inverted phase contrast microscope. Cell viability of SrHAp NPs was increased in comparison to HAp NPs.²⁴

Hydroxyapatite NPs (HAp NPs) are advantageous as they can induce mineralization from within the teeth which is further propagated by saliva.²⁴ Nano HAp has also been shown to act synergistically with fluoride to aid in better remineralization.²⁵ Several attempts have been made to replace calcium ions in HAp with strontium ions to increase the solubility, hardness, fracture resistance, and remineralization.¹² Strontium is also claimed to have enhanced bioactivity which makes it more desirable in vivo. According to a systematic review by Rajendran et al., HAp NPs doped with strontium showed better remineralization than other remineralizing agents that are sold commercially, like Acclaim and NovaMin.²⁶ These findings corroborate the hypothesis that in comparison to HAp Nps, Sr-doped HAp NPs will be a far superior tissue engineering substrate.

Green synthesis was introduced to reduce the toxicity of the prepared materials. Phytochemical compounds such as flavonoids, alkaloids, tannins, and saponins present in plant extracts act as reducing agents preventing the need for chemical processes and thereby reducing the toxicity.²⁷ Plant extracts of Equisetum arvense and Laminariales were used as reducing and capping agents to form SrFAp NPs. Pharmacological studies of Equisetum arvense showed that it possessed anti-inflammatory, antioxidant, antimicrobial, anticonvulsant, and bone regenerative effects among many others.²⁸Laminariales is known for its antioxidant and free radical scavenging properties.²⁹ Thus, these properties may benefit the SrFAp NPs and enhance their biocompatibility.

Though previous studies have evaluated the cytotoxicity of Sr and HAp NPs produced by chemical or physical methods, there is no literature on the production of SrFAp NPs by green synthesis method.³⁰^,³² The results of this study shed light on green synthesized SrFAp NPs having ideal cell viability at lower concentrations. This can be clinically applied for enamel remineralization of white spot lesions commonly encountered during orthodontic treatment while being produced by an environmentally friendly, non-toxic green synthesis method.

Limitations

A limitation of this study is that only acute toxicity against one cell line was tested for cell viability. Biocompatibility of different cell lines with longer duration of exposure needs to be assessed. Only preliminary in vitro tests have been carried out. Human trials of the same are required.

CONCLUSION

To conclude, green synthesized SrFAp NPs showed good biocompatibility and cell viability while tested using the MTT assay. This can pave the way for further research to clinically evaluate its remineralizing properties and aid in the prevention of white spot lesions which are commonly encountered during orthodontic treatment.

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