EVALUATION OF SUSTAINED RELEASE FROM FLUORIDE-LOADED CARBON NANOTUBES

Abstract

The developments in nanotechnology have led to significant advances in many areas of science and technology including medical sciences and dentistry. Carbon nanotubes (CNTs) have some unique properties such as their tube-shaped structure which allows for drug delivery and makes them very suitable agents for use with biomaterials in various fields of medicine. The aim of this research was to evaluate the fluoride delivery potential of fluoride-loaded carbon nanotubes in two different artificial saliva solutions of pH 7.4 and pH 5.5. After buckypaper samples were prepared from CNTs, the dispersant agent Triton X, and distilled water, the samples were loaded with NaF. According to our results, the release of fluoride from the fluoride-loaded carbon nanotubes was achieved although the release occurred faster than was expected. The fluoride release was significantly higher in the artificial saliva with pH 5.5 which is a critical pH for enamel than with pH 7.4. We concluded that further research should be performed to regulate the fluoride release and to increase the potential for a slower fluoride release from the fluoride-loaded nanotubules through developing an amplified interaction between fluoride and the carbon nanotubes.

Headnote

ABSTRACT: The developments in nanotechnology have led to significant advances in many areas of science and technology including medical sciences and dentistry. Carbon nanotubes (CNTs) have some unique properties such as their tube-shaped structure which allows for drug delivery and makes them very suitable agents for use with biomaterials in various fields of medicine. The aim of this research was to evaluate the fluoride delivery potential of fluoride-loaded carbon nanotubes in two different artificial saliva solutions of pH 7.4 and pH 5.5. After buckypaper samples were prepared from CNTs, the dispersant agent Triton X, and distilled water, the samples were loaded with NaF. According to our results, the release of fluoride from the fluoride-loaded carbon nanotubes was achieved although the release occurred faster than was expected. The fluoride release was significantly higher in the artificial saliva with pH 5.5 which is a critical pH for enamel than with pH 7.4. We concluded that further research should be performed to regulate the fluoride release and to increase the potential for a slower fluoride release from the fluoride-loaded nanotubules through developing an amplified interaction between fluoride and the carbon nanotubes.

Keywords: Carbon nanotubes; Drug delivery; Fluoride-loaded carbon nanotubes; Sustained release.

1.INTRODUCTION

1.1. Fluoride and dentistry: Dental caries is an infectious and multifactorial disease in which lesions are formed by the acidogenic activities of microorganisms in dental plaque, resulting in the loss of dental hard tissues. Traditionally, dental caries are treated with restorative methods.1,2,3

Fluoride's role in the reduction of dental caries incidence was discovered in the 1940s when the dental caries incidence found to be high in the areas that were using drinking water with a low level of fluoride; and low in the areas that were using drinking water with a high level of fluoride.4 However further research showed that an excessive systemic fluoride intake could adversely effect the reproductive system in mammals and cause sportiness abortions, be a risk factor for hypertension, and negatively effect thyroid stimulating hormone (TSH) levels.5-9

Still, the topical remineralizing effect of fluoride on the teeth enamel might be beneficial. In fully developed enamel, the principal mineral is hydroxyapatite. Fluoride combines with this mineral forming the stronger structure of fluorapatite; which demineralizes less in an acidic oral environment.4,10

1.2. Nanotechnology and dentistry: Nanotechnology involves the control and manipulation of particles that are smaller than 100 nanometers. Engineering of nanosized particles allows the researchers to work in tiny areas.12 Carbon is an element that has the ability to form a variety of different structures. At the atomic scale, carbon nanotubes (CNTs) are constituted by hexagonal graphite leaves that are wrapped in a single or multiwall manner. Due to their cylindrical symmetries, carbon bonds and single dimensional structure, they have unique thermal, electrical, and mechanical properties. Their electrical capacities are 1,000 times better than copper. They can remain stable until 2,800°C degrees under a vacuum. Their tensile strength can vary between 50-150 GPa. As far as we know, their elasticity modulus and strength are above those of the strongest steel. All these properties have increased the attention given to biomaterials that are strengthened with carbon nanotubes.18

Nanotechnology research in dentistry started in the early 1990's with the usage of nanoparticles. In dentistry, nanotechnology could be used in (i) dental restorative materials; (ii) bone defect replacement therapies; (iii) protein, gene and drug delivery; and (iv) cancer therapies.14

The aim of the present research was to combine nanotechnology with dentistry by evaluating the slow fluoride release potential of fluoride-loaded CNTs.

2.MATERIALS AND METHODS

In this research, after loading the CNTs with sodium fluoride (NaF), the slow fluoride release from the nanotubes was studied.

2.1. Preparation of the CNTs: To provide the hydrophilic NaF entrance into the cylindrical shaped and hydrophobic CNTs, 9 differently formulated suspensions with CNTs, Triton-X, and distilled water were prepared in line with the research of Randhawa et al. (Table 1).15 Briefly, the exact amount of CNT was weighed before a dispersant agent Triton-X was added onto the CNTs, and the dispersion was then diluted with distilled water.

All the formulations were sonicated (10x5 pulse and 40 amplitude) for 10 minutes with the ultrasonic mixing device VCX 130; (Sonics®, Switzerland). According to the results, the most homogenous suspension was the one with the formulation of 2 mL Triton X, 40 mg CNT, and 20 mL distilled water (F0 formulation in Table 1) in line with the research of Randhawa et al.15 In order to provide the standardization, this formulation was used for all the experiments.

The dispersion was filtered with a 0.45 pm membrane filter using a filtering kit (Merck Millipore, Germany). Membrane filters were dried in a drying oven. After that, in order to separate the CNTs from the filters easily, acetone was added to the filters and they were dried again in the drying oven in line with the research from Gou.16

Another suspension with 4 mg/mL NaF concentration was prepared using ethyl alcohol. The fluoride solution in ethyl alcohol was dropped on membrane filters gradually, with an injector so that the exact amount of the NaF suspension used could be measured. This process was repeated, until there were visible white dots on the membrane filters, indicating that the CNTs were full with NaF so that the additional fluoride particles stayed outside the CNTs and on the top of the membrane filters. The macro whiteness visibility appeared after 4.0 mL of NaF solution in ethyl alcohol; therefore, this amount was used for the samples to achieve standardization. Following the NaF loading process, the membrane filters were cut into 1x1 cm pieces. The CNTs were peeled from the filters resulting in the formation of 1x1 cm fluoride-loaded buckypapers. (A buckypaper is a macroscopic aggregate of carbon nanotubes which owes its name to the buckmininsterfullerene, the 60 carbon fullerene, an allotrope of carbon with a similar bonding that is sometimes referred to as a "buckyball" in honour of R Buckminster Fuller). Eight samples were constituted to allow for testing in triplicate for each of the two pH values (3 parallel samples or replicates and one control for each pH value).

2.2. Preparation of artificial saliva: The slow fluoride release potential of the CNTs was evaluated using artificial saliva. Artificial saliva was prepared with 40 g of sodium chloride, 0.95 g of monobasic potassium phosphate, 11.9 g of disodium hydrogen phosphate, and 5 L of distilled water in line with the research of Marques et al.17

In order to evaluate whether the pH of the saliva affected the slow release potential, the experiments were performed in saliva with a neutral pH 7.4 and pH 5.5, which is known as the critical pH for enamel. After preparing the artificial saliva, hydrochloric acid was used to achieve the desired pH concentration. The pH measurements were conducted with a pH meter (Isolab Labergerate GmbH, Germany®).

2.3. Release experiments: Release experiments were performed with the United States Pharmacopoeia (USP) Apparatus 2 (paddle), in compliance with the standard dissolution testing methodologies (VanKel-Varian-Agilent VK 7000®, Agilent, CA, USA) at 37°C, for the 2 groups of artificial salivae in the two different pHs of 7.4 and 5.5. Each group had 3 parallels besides the control parallel which was pure artificial salivae, in other words, the fluoride-free medium. Two mL of sample were collected in the 1st, 3rd, 5th, 10th, 15th, 30th, 45th, 60th, 90th, 120th, 150th, 180th, 240th, and 360th minutes for both groups. After collecting each samples, 2 mL of artificial saliva has added to the device to hold the volume of saliva steady.

2.4. Fluoride electrode measurements: The amount of fluoride in the samples was measured using an ion selective electrode (Consort C863®, Belgium). For the measurements, the electrode was calibrated on every experimental day. To provide the essential information relating the reading in mV with the fluoride concentration, total ionic strength adjustment buffer (TISAB) solutions with 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L, and 0.05 mg/L concentrations were measured with the device and graphed. Each sample had 3 minutes of measuring time. The electrode has cleaned and dried before each measurement. The fluoride concentration has calculated as in equation 1:

y = -53.486x + 72.177 ..........Equation 1

2.5.Statistical analysis: The statistical analysis of the data obtained in the research was conducted using the INSTAT statistical analysis programme. The data was evaluated with ANOVA, comparisons between the groups with post-hoc and Tukey Kramer. When the p-value was below 0.05, the data were recorded as being statistically significant.

3.RESULTS

The amounts of fluoride released from the fluoride-loaded buckypapers in the artificial saliva group 1 with pH 5.5 for each time period were first observed as readings in mV Group 1 included 3 parallels and the control sample. The results gained from the fluoride electrode in mV were used via the equation to calculate the amount of fluoride released in mg/mL (Table 2).The average amount of fluoride released for each parallel for each time period for group 1 are shown in Table 3. Significantly higher levels of fluoride were released from the fluoride-loaded buckypapers compared to the control (p<0.05) (Figure 1).

The amounts of fluoride released from the fluoride-loaded buckypapers in the artificial saliva group 2 with pH 7.4 for each time period were first observed as readings in mV Group 2 included 3 parallels and the control sample. The results gained from fluoride electrode in mV were used via the equation to calculate the amount of fluoride released in mg/mL (Table 4). The average amount of fluoride released for each parallel for each time period for group 2 are shown in Table 5. Significantly higher levels of fluoride were released from the fluoride-loaded buckypapers compared to the control (p<0.05) (Figure 2).

When the results for both groups were compared, it was found that the difference between the average of the group 1 parallels and the group 1 control sample, the difference between the average of the group 2 parallels and the group 2 control sample, and the difference between the average of the group 1 parallels and the group 2 control sample were all statistically significant at p<0.001. Even though there was a statistical difference between group 1 and group 2, the difference was moderate compared to the other results (p<0.05) (Table 6).

4. DISCUSSION

Dental caries is one of the most common chronic diseases that affects people worldwide. Every person is at risk for dental caries from birth. The primary cause of pain and tooth loss in the oral cavity is dental caries.18 It has been stated that dental caries could be controlled with a reduction of sugar consumption and an increase of fluoride usage.19 On the other hand some research indicates that a high systemic fluoride intake can be toxic.5-9

The appearance of the nanotechnology concept in 1959 has led to attention being given to the potential application of this field in medicine and dentistry.20 The nanotechnology concept includes the use of devices and systems which are at the molecular level. Recently research has been conducted on nano-sized particles and their application for drug delivery, gene transfers, diagnostic purposes, and tissue engineering. Carbon nanotubes are one type of these nanosized particles and they have specific characteristics with various application potentials.13,21

CNTs, due to their unique physicochemical properties, have become a popular tool in cancer diagnosis and therapy. They are considered as one of the most promising nanomaterials with the capability of both detecting cancerous cells and delivering drugs or small therapeutic molecules to these cells. Various researches have been conducted to show the drug delivery potential of CNTs. Chen et al. reported an anticancer effect had been observed with the effect being exactly as it was designed and it having a high potency towards specific cancer cell lines.22 Ji et al. reported that, after the injection of an anti-cancer agent to mice abdominally or subcutaneously, the nanotubes reached to targeted tissue through the lymphatic system, and the release of the agent from the nanotubes in the desired area was observed.23 Heister et al. evaluated the effect of anti-cancer drug delivery with CNTs on colon cancer and stated that the anti-cancer effect could be achieved by a slow drug release from CNTs.24

Nanotechnology in drug delivery has received a significant amount of attention with the goal of developing drugs with long-term higher efficacy. Due to their submicrometer size and high surface area to volume ratio, a variety of nanomaterials such as liposomes, dendrimers, nanoparticles, etc. show key differences in comparison to bulk materials.25,26 Among the nanomaterials, CNTs are of special interest in the area of drug delivery by virtue of their high surface area and the tendency to surface modification either by adsorption or by covalent attachment. The cores of CNTs can be filled with drug molecules or drugs can be adsorbed onto CNT surfaces.27,28 The amount of drug delivered and the release mechanism of the drug depend on the attraction of the drug molecule to the walls of the CNTs and where the drug loaded.29

The present research was conducted to examine the hypothesis that the slow release of fluoride might be possible from fluoride-loaded carbon nanotubes.

For biomedical applications of CNTs such as drug delivery, solubility is a critical essential for providing adequate absorption and biocompatibility as well as for the reduction of toxicity. It has been stated that in the preparation of a homogenous solution with CNTs, surface active materials can be used such as sodium dodecyl sulfate, dodecyltrimethylammonium bromide, octyl phenol ethoxylate (Triton X), Tween 80, and Tween 20. Among these materials, Triton X has been found to have the highest solvent capacity. Hence, Triton X was used in this research as the solvent for the CNTs.15,30,31

Despite its high solvent capacity, there are serious concerns about the cytotoxicity of Triton X. Kim et al. compared the biocompatibility of various solvents for CNTs and found that Triton X had the highest cytotoxicity with the difference being statistically significant.32

Randhawa et al. compared the functional capacity of solutions with various concentrations that had been prepared with CNTs, Triton X, and distilled water. They studied the effect on the functionalization of CNTs of various chemical treatments and dispersion using a surfactant via ultrasonication. The authors stated that the distribution of CNTs with the surfactant, Triton X-100, via ultrasonication helped in their unbundling.15

In the present research, in line with Randhawa et al.'s study, 11 different formulations of CNT, Triton X, and distilled water solutions were ultrasonicated in order to achieve homogenous solutions. Among the solutions; the one with the most homogenous macro appearance was chosen and the rest of the experiment was carried out with this formulation of 2 mL of Triton X, 40 mg CNTs, and 20 mL distilled water.15

Following the preparation of a homogenous solution from CNTs, in order to increase the manipulative capacity of the nanotubes, raising the functionality level becomes important. By filtering these solutions, very thin layers of CNTs can be obtained that are called "buckypaper." Buckypaper production by vacuum filtration was first reported by Liu.33 This form of CNTs could also be used in nanocomposite production.29 Buckypapers have some very important qualities such as mechanical and chemical stability, and thermal and electrical conductivity.35 Various researchers have used buckypapers to improve the mechanical abilities of nanocomposites.16,36

Liu et al. reported that they used methanol to ease the separation of buckypapers from membrane filters.35 In this research, acetone was used and dried in the drying oven. This allowed the buckypapers to be easily peeled from the membrane filters.

We didn't encounter any other research in the literature aiming at demonstrating a slow fluoride release from fluoride-loaded CNTs.

Utilizing the advantage of the CNTs tube-like shape, NaF solution was added on to the buckypapers. This process was carried on until a white stain appeared. Our hypothesis is that until the appearance of the white stain, NaF has been entering into the tube-shaped CNTs and that after the appearance of the white stain there is no more entry of the NaF into the tubes and instead the NaF has started to accumulate on the buckypaper layer. The saturation level of drug accumulation was reached when the drug began to accumulate on the surface of the CNTs.

To imitate oral conditions, the slow release experiments carried out in artificial saliva. Artificial saliva was prepared according to the research of Marques et. al.17 Two different artificial salivae were made; one with a neutral pH 7.4 and the other with pH 5.5, which is a critical pH for enamel. Previous research evaluating the fluoride release potential of glass ionomer cement showed that with a decrease in the pH of the saliva, the fluoride release increased. Our results, showing a statistically significant higher fluoride release occurred in the artificial saliva with 5.5 pH, in comparison to the saliva with a neutral pH of 7.4, are consistent with the literature.37,38

In order to determine the fluoride solution concentration to be used, preliminary tests were conducted with fluoride solutions with concentrations of 8 mg/mL, 4 mg/ mL, 2 mg/mL, and 1 mg/mL. It was hypothesized that, following the functionalization of the CNTs with Triton X, covalent bonds would be established between the fluoride and the inner walls of the CNTs. When the inner walls become saturated, fluoride will begin to accumulate on the surface of buckypapers which will be visible as white spots on the black buckypapers. According to the results from the preliminary tests, 4 mL of a solution of 4 mg fluoride/mL provided the optimal conditions and this concentration was chosen for the rest of the research. The fluoride electrode (Consort C863®, Belgium) was used according to the manufacturer's instructions.

The results showed that there was a statistically significant difference in the fluoride release between the control samples that are free of fluoride (group 1-control and group 2-control) and the fluoridated samples (group 1 and group 2). These results indicated that there was fluoride release from the fluoridated buckypapers.

According to time-fluoride release graph, the fluoride release from the samples of group 1 and group 2 almost stopped after 10 minutes. This finding indicates that fluoride release occurred quickly and we interpreted this as indicating that covalent bonds between the CNTs and the fluoride had not been able to be established as expected. It may be possible for stronger bonds to be formed by changing some of the parameters such as pressure or temperature. More research will be needed to provide this further information.

CONCLUSIONS

As in many fields of science, nanotechnology has a great potential for creating changes in dentistry. Further research in preventive dentistry within the nanotechnology concept would be likely to be helpful in the development of more effective preventive strategies.

ACKNOWLEDGEMENTS

This study was conducted in the Department of Pediatric Dentistry, Faculty of Dentistry, Gazi University, Ankara, Turkey, as a partial requirement for the degree of PhD.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

STATE OF FUNDING

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References

REFERENCES

1 Hurlbutt M, Young DA. A best practices approach to caries management. J Evid Based Dent Pract 2014;14 (Suppl):77-86.

2 Ten-Cate JM. Novel anti-caries and remineralizing agents: prospects for the future J Dent Res 2012;91(9):813-5.

3 Cameron AC, Widmer RP Handbook of pediatric dentistry. 4th ed. Missouri, USA: Mosby Elsevier Publishing; 2013. pp. 47-62.

4 Sharma G, Puranik PM, Sowmya KR. Approaches to arresting dental caries: an update. J Clin Diagn Res 2015;9(5):8-11.

5 Aghaei M, Karimzade S, Yaseri M, Khorsandi H, Zalfi E, Mahvi AH. Hypertension and fluoride in drinking water: case study from West Azerbaijan, Iran. Fluoride 2015;48(3):252-8.

6 Mohammedi AA, Yousefi M, Yaseri M, Jalilzadeh M, Mahvi AH. Skeletal fluorosis in relation to drinking water in rural areas of West Azerbaijan, Iran. Scientific Reports 2017;7(1):17300.

7 Yousefi M, Mohammadi AA, Yaseri M, Mahvi AH. Epidemiology of drinking water fluoride and its contribution to fertility, infertility, and abortion: an ecological study in West Azerbaijan Province, Poldasht Country, Iran. Fluoride 2017;50(3):343-53.

8 Moghaddam VK, Yousefi M, Khosravi A, Yaseri M, Mahvi AH, Hadei M, et al. High concentration of fluoride can be increased risk of abortion. Biol Trace Elem Res 2018;185(2):262-5.

9 Kheradpisheh Z, Mahvi AH, Mirzaei M, Mokhtari M, Azizi R, Fallahzadeh H, Ehrampoush MH. Correlation between drinking water fluoride and TSH hormone by ANNs and ANFIS. J Environ Health Sci Engineer 2018;16:11-8.

10 Kanduti D, Sterbenk P, Artnik B. Fluoride: a review of use and effects on health. Mater Sociomed 2016;28(2):133-7.

11 Carey CM. Focus on fluorides: update on the use of fluoride for the prevention of dental caries. J Evid Based Dent Pract 2014;14 (Suppl):95-102.

12 Singh S, Singh A. Current status of nanomedicine and nanosurgery. Anesth Essays Res 2013;7(2):237-42.

13 Bhattacharya M, Seong WJ. Carbon nanotube-based materials: preparation, biocompatibility, and applications in dentistry. In: Subramani K, Ahmed W, Hartdfield JK, Jr., editors. Nanobiomaterials in clinical dentistry. Amsterdam, The Netherlands: William Andrew, an imprint of Elsevier; 2013. pp. 37-67.

14 Emerich FD, Christopher GT: Nanotechnology and medicine. Expert Opin Biol Ther 2003; 3(4):655-63.

15 Randhawa P, Park JS, Sharma S, Kumar P, Shin MS, Sekhon SS. Effect of surfactant (Triton X-100) concentration on dispersion and functionalization of multiwall carbon nanotubes. J Nanoelectron Optoelectron 2012;7(3):279-86.

16 Gou J. Single-walled nanotube bucky paper and nanocomposite. Polym Int 2006;55(11):12838.

17 Marques MRC, Loebenberg R, Almukainzi M. Simulated biological fluids with possible application in dissolution testing. Dissolution Technol 2011 ;18(3):15-28.

18 Selwitz RH, Ismail AI, Pitts NB. Dental caries. Lancet 2007 Jan 6;369(9555):51-95.

19 Petersen PE, Lennon MA. Effective use of fluorides for the prevention of dental caries in the 21st century: The WHO approach. Community Dent Oral Epidemiol 2004;32(5):319-21.

20 Chandki R, Kala M, Kiran KN, Brigit B, Banthia P, Banthia R. "NANODENTISTRY": Exploring the beauty of miniature. J Clin Exp Dent 2012;4(2):e119-124.

21 Akasaka T, Nakata K, Uo M, Watari F. Modification of the dentin surface by using carbon nanotubes. Biomed Mater Eng 2009;19(2-3):179-85.

22 Chen J, Chen S, Zhao X, Kuznetsova LV, Wong SS, Ojima I. Functionalized single-walled carbon nanotubes as rationally designed vehicles for tumor-targeted drug delivery. J Am Chem Soc 2008;130(49):16778-85.

23 Ji SR, Liu C, Zhang B, Yang F, Xu J, Long J, et al. Carbon nanotubes in cancer diagnosis and therapy. Biochim Biophys Acta 2010;1806(1):29-35.

24 Heister E, Neves V, Lamprecht C, Silva SRP, Coley HM, McFadden J. Drug loading, dispersion stability, and therapeutic efficacy in targeted drug delivery with carbon nanotubes. Carbon 2012;50:622-32.

25 Kim HJ, Kwon TY, Kim KH, Kwon SJ, Cho DH, Son JS. Long-term release of chlorhexidine from dental adhesive resin system using human serum albumin nanoparticles. Polym Bull 2014;71:875-86.

26 Bobo D, Robinson KJ, Islam J, Thurecht KJ, Corrie SR. Nanoparticle-based medicines: a review of FDA-approved materials and clinical trials to date. Pharm Res 2016;33(10):2373-87.

27 Degim IT, Burgess DJ, Papadimitrakopoulos F. Carbon nanotubes for transdermal drug delivery. J Microencapsul 2010;27(8):669-81.

28 Guven A, Villares GJ, Hilsenbeck SG, Lewis A, Landua JD, Dobrolecki LE, et al. Carbon nanotube capsules enhance the in vivo efficacy of cisplatin. Acta Biomater.2017;58:466-78.

29 Tamer SI, Yilmaz S, Banoǧlu E, Degim IT. Carbon nanotubes to deliver drug molecules. J Biomed Nanotechnol 2010;6(1):20-7.

30 Rastogi R, Kaushal R, Tripathi SK, Sharma AL, Kaur I, Bharadwaj LM. Comparative study of carbon nanotube dispersion using surfactants. J Colloid Interface Sci 2008;328(2):421-8.

31 Sajid MI, Jamshaid U, Jamshaid T, Zafar N, Fessi H, Elaissari A. Carbon nanotubes from synthesis to in vivo biomedical applications. Int J Pharm 2016;501(1-2):278-99.

32 Kim JS, Song KS, Lee JH, Yu IJ. Evaluation of biocompatible dispersants for carbon nanotube toxicity tests. Arch Toxicol 2011;85(12):1499-508.

33 Liu J. Fullerene pipes. Science 1998;280(5367):1253-6.

34 Ahir SV, Huang YY, Terentjev, EM. Polymers with aligned carbon nanotubes: Active composite materials. Polymer 2008;49(18):3841-54.

35 Liu L, Ma W, Zhang Z. Macroscopic carbon nanotube assemblies. Preparation, properties, and potential applications. Small 2011;7(11):1504-20.

36 Wang Z, Liang Z, Wang B, Zhang C, Kramer L. Processing and property investigation of single-walled carbon nanotube (SWNT) buckypaper/epoxy resin matrix nanocomposites. Composites Part A: Applied Science and Manufacturing 2004;35(10):1225-32.

37 Prati C, Gandolfi, MG. Calcium silicate bioactive cements: biological perspectives and clinical applications. Dent Mater 2015;31(4):351-70.

38 Taqa AA, Abdal A, Dawood AI. The effect of pH on fluoride release of glass ionomer based restorative materials. International Journal of Dental Sciences and Research 2016;4(3):52-7.