184 © 2024 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License The Impact of Cold Atmospheric plasma and PLGA/ Xylitol Nanoparticles on Dental Enamel (in Vitro Study) Ghada A. Ibrahim1,* Eaman A. Al-Rubaee2 and Maha J. Abbas3 1,2Department of Basic Science, College of Dentistry, University of Baghdad, Baghdad, Iraq. 3 College of Dentistry, Mustansiriyah University, Baghdad, Iraq. *Corresponding Author Received: 13 September 2023 Accepted: 14 November 2023 Published: 20 October 2024 doi.org/10.30536.37.4.3707 Abstract Plasma and nanotechnology are potentially effective preventive measures against dental caries. This study aims to determine the impact of cold atmospheric plasma and PLGA/Xylitol nanoparticles on dental enamel microhardness and morphological changes in dental enamel ultrastructure. In this study, 56 maxillary first premolars were divided into five groups: one control group, and four study groups, each with 11 teeth; 10 teeth were examined for microhardness; and one tooth was examined using FESEM. A circular window was placed on the buccal surface of each tooth. Following a PH cycling technique to activate caries lesions on the tooth enamel. cold plasma was performed using predetermined parameters. The PLGA/Xylitol nanoparticle concentration was adjusted to 5%. The microhardness and morphological change were measured using micro-Vickers and FESEM respectively, at three stages: sound, demineralization, and treatment. Enamel microhardness values decreased highly significantly after the demineralization stage compared to the sound stage for all groups. After treatment, the microhardness values of all treated groups, excluding the control, increased highly significantly in comparison to the demineralization stage. The group nanoparticles + plasma showed the highest microhardness recovery (82.559± 23.596), followed by plasma + nanoparticles (74.774±18.302), while samples treated with nanoparticles only showed the lowest recovery in the microhardness among all study groups (50.227±12.989). A FESEM showed that the application of nanoparticles, nanoparticles + plasma, and plasma + nanoparticles caused many surface defects to be repaired. The enamel surface treated with cold atmospheric plasma and PLGA/Xylitol nanoparticles yielded favorable results in terms of microhardness and FESEM analysis, suggesting that this therapy could be recommended as a means of preventing dental caries. Keyword: Cold atmospheric plasma, PLGA/ Xylitol Nanoparticles, dental enamel 1. Introduction The enamel is the exposed tooth's outer layer. It is a strong, thin, translucent coating of calcified substance that envelops and protects dentin [1]. The most mineralized tissue in the human body is tooth enamel. The distinctive mechanical properties of tooth enamel are determined by the different forms and structures of enamel crystals, which include increased microhardness and https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0003-4898-4830 mailto:ghadaalbaghdadi15@gmail.com https://orcid.org/0000-0001-6230-098X mailto:dr.eaman.alrubaiee@codental.uobagdad.edu.iq https://orcid.org/0000-0003-1614-8950 mailto:uomustansiriyah@edu.iq IHJPAS. 37 ( 4 ) 2024 185 resistance to fracture and acid degradation [2]. Dental caries may develop because of the continual remineralization (mineral gain) and demineralization process [3]. When the rate of demineralization overcomes the rate of remineralization, calcium and phosphate ions diffuse out of the enamel, resulting in a chalky white spot lesion that, if not controlled, can develop into cavitation [4]. Pathological variables shift the balance in the direction of dental caries and disease progression, whereas protective factors include salivary components, fluoride together with calcium and phosphate enhance remineralization of dental caries lesions [5]. Plasma and nanotechnology are potentially effective preventive measures against dental caries [6,7]. Cold atmospheric plasma [CAP] is currently being used in medicine. Plasma is an ionized gas with an almost neutral charge. It is often called the "fourth state of matter". The non-thermal atmospheric pressure doesn't go above 50°C, so human cells can tolerate it [8]. CAP jets are a kind of cold plasma discharge that generates a high-velocity stream of highly reactive chemical species and weak emitted light [9]. There are many applications of CAP in dentistry and in cariology [10- 13]. Plasma surface treatment has been proposed as a strategy for assisting mineral re- crystallization. Following cold plasma therapy of demineralized enamel, significant increases in mineral volume recovery and microhardness of the demineralized region were found [7, 14]. Nanotechnology or nanoscience is defined as a technology is associated with small materials or structures that are smaller than 100 nm in at least one dimension [15]. The particle size is decreased to nanometers which provides maximum contact with the environment and makes penetration through cell membranes possible. hardness, mechanical properties, chemical reactivity, and biological activity can all be altered, resulting in increased drug release of active therapeutic agents [16-18]. Nanotechnology has been evaluated in different areas of medical and dental applications including the prevention of dental caries [19-24]. PLGA is a polylactic acid (PLA) and polyglycolic acid (PGA) copolymer. It is the best-characterised biomaterial currently available for drug delivery in terms of performance and design [25]. PLGA nanoparticles may be used in many dental fields [26]. Xylitol is a tooth-friendly, nonfermentable sugar alcohol that has been considered a cariostatic and noncariogenic agent. [27]. Xylitol can activate the remineralization of deeper demineralized enamel layers by easing calcium accessibility and mobility [28]. To increase the activity of xylitol, it can be loaded into PLGA nanoparticles. Xylitol loaded with nanoparticles led to a reduction in particle size, an increase in particle surface, and enhanced antibiofilm activity of xylitol [29, 30]. Surface microhardness (SMH) evaluation is a simple, quick and easy to measure, non-destructive method, reflecting mineral changes that have occurred due to the therapeutic procedures [31]. 2. Materials and Methods An in vitro study was conducted from August 2022 to November 2022 using 56 maxillary first premolars in Baghdad, Iraq. Ethical approval for the study was the from Ethical Committee of the University of Baghdad, College of Dentistry (Ref. 560 on April 17, 2022). 2.1. Plasma Procedure The CAP groups' samples were treated with an Iraq-made cold atmospheric plasma jet employing argon gas at a flow rate of 10 L/min, 175 volts, and a frequency of 2.45 GHz at room temperature. The maximum distance between the nozzle tip and the enamel surface was 2 mm. The time for plasma therapy was set at one minute Figure 1. IHJPAS. 37 ( 4 ) 2024 186 Figure 1. Cold atmospheric plasma jet 2.2.Preparation of nanoparticles Solvent evaporation method used to produce PLGA/ xylitol nanoparticles. The concentration of nanoparticles in this experiment was established at 5%. (Figure 2). In distilled water, xylitol and tween (surfactant) were dissolved, while PLGA was dispersed in acetone. Using a sonicator, the organic phase was introduced drop by drop to the aqueous solution, followed by two hours of rotary evaporation at 40 °C. Using a freeze dryer, the nanoparticles were frozen at 80 °C for 18 hours and lyophilized at 110 °C for 24 hours [29]. The treatment time is 4 minutes daily for 7 days. Figure 2. PLGA/ xylitol nanoparticles 2.3. Identification of PLGA/ Xylitol Nanoparticles 2.3.1. Ray Diffraction Pattern (XRD) It is one of the most often used measurement techniques for identifying a substance's nature and phase without causing any damage. To expose the diffraction information, it depends on the incident ray's diffraction on the material to scatter at a specific angle [32]. When the suspension of the produced nanoparticles was deposited onto glass slides and allowed to dry, an X-ray was used to characterize them. Cu-Kα radiation at a wavelength (λ = 0.15406 nm) was used as the X-ray radiation source at a 2° angle (10°-80°). IHJPAS. 37 ( 4 ) 2024 187 2.3.2. Filed Emission Scanning Electron Microscopy (FESEM) FE-SEM is a specific kind of electron microscope that generates an image by moving a high- energy electron beam across the sample surface in a raster scan pattern. The pictures produced by the FESEM lens are clearer and less electrostatically deformed than those produced by SEM [33]. 2.3.3. Sample Preparation Teeth are classified into five groups: one control group, and four study groups, and each tooth had 11: one tooth for SEM evaluation and ten teeth for microhardness. Group 1: Control (Deionized water), Group 2: will only be treated with plasma. Group 3: will be treated with PLGA /Xylitol nanoparticles. Group 4: will be treated with plasma then PLGA/Xylitol nanoparticles (plasma + nanoparticles) Group 5: will be treated with PLGA/Xylitol nanoparticles and then plasma (nanoparticles+ plasma). Each tooth's buccal surface, a circular opening was positioned and standardized. This window was polished and ground to generate a flat surface suitable for FESEM and microhardness testing [34]. Using demineralizing and remineralizing solutions, PH cycling was utilized to activate enamel surface caries lesions in the current study, carious lesion initiation occurred within ten days [35]. Microhardness was measured using a digital micro-Vickers hardness instrument with a 100-gram load for 15 seconds. The Vickers hardness was measured using an optical microscope. Each specimen received three indentations. Then, the average of these three records was calculated. The extent of remineralization was calculated as the percent of surface microhardness recovery (%SMHR), and was measured according to the following formula [36]: %SHR = (SH2 −SH1) (SH0 − SH1) × 100 (1) Where SH0 was the baseline surface hardness, SH1 was the demineralization surface hardness and SH2 was the remineralization surface hardness. By scanning the specimen with a focused electron beam, morphological abnormalities on the enamel surface were detected using FESEM. 3. Result 3.1. Identification of Nanoparticles 3.1.1. XRD Analysis The XRD pattern of PLGA/ Xylitol nanoparticles compared with xylitol is in Figure 3. The XRD pattern for xylitol shows a perfect matching with ICDD 34-1802 [37]. The matching occurs at (2θ= 13.97,14.50,17.54,19.82, 22.56, 24.64, 28.13, 29.28, 30.16, 35.36 and 38.26). The XRD test for PLGA/ Xylitol nanoparticles shows an increase in intensity in comparison to xylitol. The substance's crystal size was determined using Scherer's equation [38]: 𝐷 = 𝐾𝜆 𝛽𝐶𝑂𝑆𝜃 (2) Where D is crystal size, 𝜆 is the x-ray wavelength, 𝛽 is the full width at half maximum of the XRD peak, and 𝜃 is the Bragg angle. The average crystalline size of PLGA/ Xylitol nanoparticles is 8.20 nm. IHJPAS. 37 ( 4 ) 2024 188 Figure 3. The XRD pattern of PLGA/ Xylitol nanoparticles in comparison with xylitol. NPX 5% = PLGA/Xylitol nanoparticles 5% 3.1.2. Filed Emission Scanning Electron Microscopy (FESEM) The FESEM analysis is a crucial test for determining the morphology of nanoparticles that have been manufactured. The form and size of PLGA/Xylitol nanoparticles are depicted in Figure 4. The photos depict a heterogeneity of nanoparticles with various shapes. Figure 4. FESEM of PLGA/ Xylitol nanoparticles 3.1.3. Microhardness Value of Enamel Surfaces Treated with Different Agents The mean microhardness values for the sound, demineralization, and treatment stages were determined. The ANOVA test revealed that there was no significant difference in the microhardness values between the groups for either sound or demineralization (p > 0.05). During the remineralization stage, statistically highly significant variations between groups were observed (p < 0.001). The group nanoparticles + plasma showed the highest microhardness recovery (MHR), followed by plasma+ nanoparticles, while samples treated with nanoparticles only showed the lowest recovery in the microhardness among all other groups in Table 1. Table 2 shows the mean difference among the three stages within the same group. Following the demineralization stage, all groups experienced a highly significant decline in microhardness values IHJPAS. 37 ( 4 ) 2024 189 relative to the sound teeth stage. In comparison to the demineralization stage, the administration of different treatments (remineralization stage) resulted in a highly significant rise in microhardness values for all treated groups except the control group. The group treated with nanoparticles + plasma had the greatest mean difference in microhardness between demineralization and remineralization stage values, followed by plasma + nanoparticles. Table 3 compares microhardness values for all groups during the remineralization stage. There are statistically significant differences (p <0.05) when the microhardness values of the control group are compared to those of all study groups. There are significant variations between the plasma group and nanoparticles + plasma groups, as well as between the nanoparticles and the nanoparticles + plasma groups. No significant differences were shown among other groups (p > 0.05). 3.1.4. Microscopic Features of the Outer Enamel Surface Using FESEM The structural alterations to the enamel surface for each group are depicted in Figure 5. The typical, undamaged, and smooth enamel surface structure of the control group (sound enamel), with normal perikymata arranged in parallel lines with few holes. The demineralization group's enamel surface structure has changed. The prisms exhibited irregularities, causing the enamel to deviate from its normal construction, there are numerous micropores and cavities on the enamel surface. The image of an enamel surface treated with plasma revealed the formation of craters, cavities, and gaps. FESEM picture of an enamel surface treated with nanoparticles showed the existence of globular, crystalline, and amorphous structures that occlude the micropores created during the demineralization stage of the FESEM of the group (plasma and nanoparticles); the deformation caused by plasma radiation was corrected by nanoparticle precipitation. The group (nanoparticles plasma) indicated that nanoparticles created more homogeneity, and the denser mineral content obliterated the plasma-formed surface pores. Table 1. Descriptive and statistical test of surface microhardness (HV unit) among groups and phases. DW Plasma Nano Plasma+Nano Nano+Plasma F p Baseline Minimum 295.500 274.100 286.630 293.970 271.400 1.429 0.240 NS Maximum 370.130 346.270 350.500 388.870 375.000 Mean 326.113 305.474 332.683 330.215 332.873 ±SD 25.616 27.758 24.694 34.421 37.567 Demineralization Minimum 150.500 116.500 158.030 179.530 170.500 0.867 0.491 NS Maximum 208.000 257.170 210.770 214.430 212.000 Mean 188.867 188.229 179.530 197.212 198.473 ±SD 20.805 47.122 19.061 12.284 15.675 Treatment Minimum 155.000 198.400 211.980 274.000 290.000 18.282 0.000 Sig. Maximum 302.150 295.000 283.630 312.870 320.000 Mean 202.778 264.048 258.264 290.118 302.324 ±SD 39.989 37.625 28.421 10.341 11.202 F 85.059 56.358 81.972 78.896 92.046 p 0.000 0.000 0.000 0.000 0.000 Effect size 0.795 0.719 0.788 0.782 0.807 MHR Minimum -7.582 50.369 33.051 45.247 65.448 Maximum 61.043 82.524 69.788 96.605 132.244 Mean 8.954 66.866 50.227 74.774 82.559 ±SD 19.664 12.873 12.989 18.302 23.596 IHJPAS. 37 ( 4 ) 2024 190 Table 2. Multiple pairwise comparisons of surface microhardness among phases by groups using the Bonferroni postdoc test. Groups Phases Mean difference p value DW Baseline Demineralization 137.246 0.000 Treatment 123.335 0.000 Demineralization Treatment -13.911 0.250 Plasma Baseline Demineralization 117.245 0.000 Treatment 41.426 0.000 Demineralization Treatment -75.819 0.000 Nano Baseline Demineralization 153.153 0.000 Treatment 74.419 0.000 Demineralization Treatment -78.734 0.000 Plasma+Nano Baseline Demineralization 133.003 0.000 Treatment 40.097 0.000 Demineralization Treatment -92.906 0.000 Nano+Plasma Baseline Demineralization 134.400 0.000 Treatment 30.549 0.007 Demineralization Treatment -103.851 0.000 Table 3. Multiple pairwise comparisons of surface microhardness among groups by phases using Tukey's HSd (Honestly significant difference) Groups Mean difference Tukey HSD p value DW Plasma -61.270 0.000 Nano -55.486 0.001 Plasma+Nano -87.340 0.000 Nano+Plasma -99.546 0.000 Plasma Nano 5.784 0.991 Plasma+Nano -26.070 0.261 Nano+Plasma -38.276 0.033 Nano Plasma+Nano -31.854 0.108 Nano+Plasma -44.060 0.010 Plasma+Nano Nano+Plasma -12.206 0.872 IHJPAS. 37 ( 4 ) 2024 191 Figure 5. A- FESEM for normal sound enamel surface. B- FESEM for demineralized enamel surface. C- FESEM for enamel surface treated with plasma D-FESEM for enamel surface treated with nanoparticles. E-: FESEM for enamel surface treated with plasma nanoparticles. F- FESEM for enamel surface treated with nanoparticle + plasma Discussion 4. The XRD test showed that PLGA/ Xylitol nanoparticles matched xylitol in the 2θ position. The FESEM showed a heterogeneity of nanoparticles with various shapes. Enamel microhardness was tested for all groups (sound, demineralization, treatment with selected agents). When compared to the sound tooth surface, there is a statistically highly significant decrease in the microhardness of the enamel surface during demineralization and the addition of a dental caries lesion. This is because any drop in the pH of the surrounding environment under the critical pH (5.5) creates an acidic environment, leading the tooth minerals, to move outward, creating micropores and lowering microhardness [39]. This finding was verified by an SEM micrograph of the demineralized stage, which revealed numerous microspaces and voids. For the all-treated group, the results showed that microhardness was rising with statistically highly significant differences in comparison to the demineralization stage. For the plasma group alone, the microhardness increased due to cold plasma treatment can improve enamel surface characteristics by improving surface energy, wettability, and hydrophilicity of substrates without modifying the bulk structure or increasing pulp temperature [7]. This finding coincides with Šantak et al, 2017, who showed that cold atmospheric plasma jet (APPJ) treatment is a promising technique for chemical surface modification of hard human tooth tissues [40]. The findings disagree with Khoubrouypak et al., in (2021), who found that CAP application had no significant impact on enamel erosion resistance [41]. This may be due to the different variables used. The FESEM image of an enamel surface treated with plasma revealed the formation of craters, cavities, and microspaces (holes), which may be due to melting and re-solidification processes. The mean values of microhardness increased for the group treated with PLGA/Xylitol IHJPAS. 37 ( 4 ) 2024 192 nanoparticles. The results of the current study go with the results of other studies that found xylitol to have high remineralizing properties [42,43]. The possible explanation is that xylitol can encourage remineralization of deeper demineralized enamel layers by promoting Ca+2 movement and accessibility and forming complexes with calcium ions and phosphate ions, preventing more general calcium [44]. Another reason may be that PLGA loaded with the particles displayed an increase in microhardness [45]. For the group treated with plasma+ nanoparticles, the mean values of microhardness increased, this may be because cold plasmas consider surface pretreatment, which increases the surface hydrophilicity, wettability, and surface energy of tooth surfaces, which leads to close contact of materials with teeth and is vital to promoting material interaction [7, 46]. Another explanation is that the synergistic effect of cold plasma and nanoparticles has been proposed to improve mineral recrystallization [47]. A FESEM micrograph confirmed this result by revealing that the precipitation of a nanoparticle layer corrected most of the surface cracks and flaws caused by plasma. For the group treated with nanoparticles+ cold plasma. The mean values of microhardness increased, this may be because cold plasmas may increase treatment adhesion to enamel and cause more treatment absorption [48]. This is also confirmed in the current study by FESEM micrographs, which revealed that most of the microspaces were closed by nanoparticles, which formed a more homogeneous and denser mineral content, obliterating the surface porosities formed by plasma. When the control group was compared to the other study groups, there were statistically significant variations in microhardness values; this may be due to the efficacy of all treatments used. When comparing treatments, there is a significant difference between the plasma group and the nanoparticles + plasma group, as well as between the nanoparticles group and the nanoparticles + plasma group. There were no significant differences shown among other groups (p > 0.05). The changes in the microhardness recovery values after treatment with different agents were measured by certain equations, The group nanoparticles + plasma showed the highest microhardness recovery (82.559±23.596), followed by plasma + nanoparticles (74.774±18.302), while samples treated with nanoparticles only showed the lowest recovery in the microhardness among all other groups (50.227 ±12.989). This could be due to the synergistic effect of both agents, which causes an increase in values more than the microhardness values when the technique is used alone. This result couldn’t be compared with other studies' results as there was no previous study that used such a combination of agents. A FESEM showed that the application of nanoparticles, nanoparticles + plasma, and plasma + nanoparticles caused most of the surface defects to be repaired. Nanoparticles + plasma groups produced a better image of FESEM than another group because produced more homogeneous and denser mineral content. 5. Conclusion The treatment of the tooth enamel surface with PLGA/Xylitol nanoparticles and plasma produced good results in terms of microhardness and FESEM evaluation. PLGA/Xylitol nanoparticle + cold plasma group produced maximum remineralization ability which is assessed by high microhardness recovery and better image of FESEM. This treatment could be considered for the prevention of dental cavities. Authors’ Contributions IHJPAS. 37 ( 4 ) 2024 193 Ghada Abdul Salam Ibrahim contributed to the literature search, data collection, taking the results, analysing them, and writing them down in the research. Eaman Ali Al-Rubaee, and Maha Jamal Abbas, contributed to the work in terms of designing the work plan. Acknowledgment I would like to thank the Department of Basic Science, College of Dentistry, University of Baghdad, for their support in writing this research. Conflicts of Interest The authors declare that they have no conflicts of interest. Funding None. References 1. Twetman, S. Caries prevention with fluoride toothpaste in children: an update. Eur. Arch. Paediatr Dent. 2009, 10(3), 162-7. https://doi:10.1007/BF03262678 2. Cao, Y.; Mei, M.L.; Li, Q.L.; Lo, E.C.; Chu, C.H. Enamel prism-like tissue regeneration using enamel matrix derivative. J Dent. 2014, 42(12), 1535-42. https://doi:10.1016/j.jdent. 2014.08.014 3. Wang, Y.; Mei, L.; Gong, L.; Li, J.; He, S.; Ji, Y.; Sun, W. Remineralization of early enamel caries lesions using different bioactive elements containing toothpaste: An in vitro study. Technol Health Care. 2016, 24(5), 701-11. https://doi:10.3233/THC-161221 4. Sudjalim, T.R.; Woods, M.G.; Manton, D.J. Prevention of white spot lesions in orthodontic practice: a contemporary review. Aust. Dent. J. 2006, 51(4), 284-9. https://doi:10.1111/j.1834- 7819.2006.tb00445 5. Pitts, N.B.; Zero, D.T.; Marsh, P.D.; Ekstrand, K.; Weintraub, J.A.; Ramos-Gomez, F.; Tagami, J.; Twetman, S.; Tsakos, G.; Ismail, A. Dental caries. Nat Rev Dis Primers. 2017, 3, 17030. https://doi:10.1038/nrdp.2017.30 6. Jeong, S.H.; Jang, S.O.; Kim, K.N.; Kwon, H.K.; Park, Y.D.; KIM, B.I. Remineralization potential of new toothpaste containing NHA. Key Eng Mater 2006, 311(18), 537-540. 7. El-Wassefy, N.A. Remineralizing effect of cold plasma and/or bioglass on demineralized enamel. Dent Mater J. 2017, 36(2), 157-167. https://doi:10.4012/dmj.2016-219 8. Metelmann, H.R.; Von Woedtke, T.; Weltmann, K.D. Plasmamedizin: Kaltplasma in der Medizinischen Anwendung. Springer; Berlin/Heidelberg, Germany 2016, 12, 23-45. 9. Matsusaka, S. Control of particle charge by atmospheric pressure plasma jet (APPJ). A review. Adv. Powder Technol 2019, 30, 2851–2858. 10. Ranjan, R.; Krishnamraju, P.V.; Shankar, T.; Gowd, S. Nonthermal Plasma in Dentistry: An Update. J Int Soc Prev Community Dent. 2017, 7(3), 71-75. https://doi:10.4103/ jispcd.JISPCD2917 11. Masood, S.H.; Mohamed, S.A. Effect of plasma treatment on some surface properties of acrylic resin polymer. J Bagh Coll Dent. 2020, 32(2), 22-5. https://doi.org/10.26477/ jbcd.v32i2.2890. 12. Jungbauer, G.; Moser, D.; Müller, S.; Pfister, W.; Sculean, A.; Eick, S. The Antimicrobial Effect of Cold Atmospheric Plasma against Dental Pathogens-A Systematic Review of In-Vitro Studies. Antibiotics. 2021, 10(2), 211-223. https://DOI:10.3390/antibiotics10020211 13. Qanber, L.M.; Hamad, T.I. Effect of plasma treatment on the bond of soft denture liner to conventional and high impact acrylic denture materials. J Bagh Coll Dent. 2021, 33(3), 9-17. https://doi.org/10.26477/jbcd.v33i3.2948 https://doi:10.1007/BF03262678 https://doi:10.1016/j.jdent.%202014.08.014 https://doi:10.3233/THC-161221 https://doi:10.1111/j.1834-7819.2006.tb00445 https://doi:10.1111/j.1834-7819.2006.tb00445 https://doi:10.1038/nrdp.2017.30 https://doi:10.4012/dmj.2016-219 https://doi:10.4103/%20jispcd.JISPCD2917 https://doi.org/10.26477/%20jbcd.v32i2.2890. https://DOI:10.3390/antibiotics10020211 https://doi.org/10.26477/jbcd.v33i3.2948 IHJPAS. 37 ( 4 ) 2024 194 14. Radacsi, N.; Van Der, Heijden, A.; Stankiewicz, A.; Ter Horst, J. Cold plasma synthesis of high- quality organic nanoparticles at atmospheric pressure. J Nano Reser. 2013, 15, 1-13. https://DOI:10.1007/s11051-013-1445-4 15. Cao, G.; Wang, Y. Nanostructures and nanomaterials. 2nd ed. New Jersey: World Scientific 2011, 23, 344-355. 16. Thomas, L.; Khasraghi, A. Nanotechnology-Based Topical Drug Delivery Systems for Management of Dandruff and Seborrheic Dermatitis: An overview. Iraqi J Pharm Sci. 2020, 29(1), 12-32. https://DOI:10.31351/vol29iss1pp12-32 17. Abdul Kareem, E.A.; Sultan, A.E.; Oraibi, H.M. Synthesis and characterization of silver nanoparticles: A review. Ibn AL-Haitham Journal for Pure and Applied Sciences. 2023, 36(3), 177–200. https://doi.org/10.30526/36.3.3050 18. Lutfi, R.B.; Jassim, W.H. Improvement of Dental Composite Resin Using Supra-Nano Chicken thigh Bone Fibers. Ibn AL-Haitham Journal for Pure and Applied Sciences. 2003, 36(2), 156–170. https://doi.org/10.30526/36.2.2998 19. Jaber, G.S.; khashan, K.; Abbas, M.J. Enhancement of Antibacterial and Mechanical Features of Glass Ionomer Restoration by Adding ZnO  Nanoparticles Prepared by PLAL: In Vitro Study. Research Square. 2021, 23, 123. https://DOI:10.21203/rs.3.rs-284225/v1 20. Hussein, H.A.; Al-Judy, H.J. Effect of Incorporation of Boron Nitride Nanoparticles on Impact Strength and Surface Roughness of Heat Cure Poly Methyl Methacrylate Resin: An In Vitro Study. Dent Hypotheses. 2023, 14, 19-21. https://DOI:10.4103/denthyp.denthyp.14022 21. Talib, M.A.; Ali, B.G.; Al-Rubaee, E.A.; Mahdy, M. The Effect of titanium dioxide nanoparticles on the activity of salivary peroxidase in Periodontitis Patients. J Bagh Coll Dent. 2023, 35(2), 10-9. https://doi.org/10.26477/jbcd.v35i2.3393 22. Hassan, N.M.; Jafar, Z.J.; Abdul-Latif, M.H. Nano-hydroxyapatite preparation for the remineralization of primary tooth enamel surface subjected to liquid medication: An observational study. Health Sci Rep. 2023, 6(4), e1188. https://doi:10.1002/hsr2.1188 23. Luaibi, N.M.; Mohammed, R.A. Physiological and Hormonal Effects of Titanium Dioxide Nanoparticles on Thyroid and Kidney Functions. Baghdad Sci. J. 2023, 20(3), 0767. https://doi.org/10.21123/bsj.2022.3727 24. Nugroho, A.; Kusumorini, N.; Pramono, S.; Martien, R. An update on Nanoparticle Formulation Design of Piperine to Improve its Oral bioavailability: A Review. Iraqi J Pharm Sci. 2013, 32(1), 14- 30. https://DOI:10.31351/vol32iss1pp14-30 25. Nair, L.S.; Laurencin, C.T. Biodegradable polymers as biomaterials. Prog Polym Sci. 2007, 32, 762– 798. https://doi.org/10.1016/j.progpolymsci.2007.05.017 26. Vîrlan, M.J.; Miricescu, D.; Totan, A.R.; Greabu, M.; Tanase, C.; Sabliov, C.M.; Caruntu, C.F.; Calenic, B. Current Uses of Poly (lactic-co-glycolic acid) in the Dental Field: A Comprehensive Review. Journal of Chemistry 2015, 23, 1-12. https://DOI:10.1155/2015 /525832 27. Mäkinen, K.K. Sugar alcohols, caries incidence, and remineralization of caries lesions: a literature review. Int J Dent. 2010, 12, 981072. https://doi:10.1155/2010/981072 28. Miake, Y.; Saeki, Y.; Takahashi, M.; Yanagisawa, T. Remineralization effects of xylitol on demineralized enamel. Journal of Electron Microscopy 2003, 52(5), 471-476. https:// DOI:10.1093/jmicro/52.5.471 29. Anjum, A.; Pooi, Y.C. PLGA/xylitol nanoparticles enhance antibiofilm activity via penetration into biofilm extracellular polymeric substances. RSC Adv. 2019, 9(25), 14198-14208. https://doi:10.1039/c9ra00125e 30. Bhattacharya, S. Development of 5-FU Loaded Poly Lactic-Co-Glycolic Acid Nanoparticles for Treatment of Lung Cancer. Iraqi J Pharm Sci. 2022, 31(1), 130-143. https://doi. org/10.31351/vol31iss1pp130-143 https://DOI:10.1007/s11051-013-1445-4 https://DOI:10.31351/vol29iss1pp12-32 https://doi.org/10.30526/36.3.3050 https://doi.org/10.30526/36.2.2998 https://DOI:10.21203/rs.3.rs-284225/v1 https://DOI:10.4103/denthyp.denthyp.14022 https://doi.org/10.26477/jbcd.v35i2.3393 https://doi:10.1002/hsr2.1188 https://doi.org/10.21123/bsj.2022.3727 https://DOI:10.31351/vol32iss1pp14-30 https://doi.org/10.1016/j.progpolymsci.2007.05.017 https://DOI:10.1155/2015%20/525832 https://doi:10.1155/2010/981072 https://doi:10.1039/c9ra00125e IHJPAS. 37 ( 4 ) 2024 195 31. Rehder, F.C.; Maeda, F.A.; Turssi, C.P.; Serra, M.C. Potential agents to control enamel caries-like lesions. J Dent. 2009, 37(10), 786-90. https://doi:10.1016/j.jdent.2009.06.008 32. Mahdi, R.H. Synthesis and Characterization of CNT-Iron Oxide Nanocomposite by Laser Ablation for Antimicrobial Applications, PhD thesis, Iraq 2016, 1-152. 33. Alyamani, A.; Lemine, O.M. FE-SEM Characterization of Some Nanomaterial. in V. Kazmiruk (ed.), Scanning Electron Microscopy, IntechOpen, London, 2012, 14, 34-45. https://DOI:10.5772/34361.12221 34. Al-Sayyab, M. The potential effect of combined CO2 laser and fluoride on acid resistance of human dental enamel and root surfaces, An in vitro study. Ph.D. thesis, Iraq 2000, 24-56. 35. Featherstone, J.D.; Prevention and reversal of dental caries: role of low-level fluoride. Community Dent Oral Epidemiol. 1999, 27(1), 31-40. https://Doi:10.1111/j.1600-0528. 1999.tb01989.x. 36. Gelhard, T.B.; Ten Cate, J.M.; Arends, J. Rehardening of artificial enamel lesions in vivo. Caries Res. 1979, 13(2), 80-113. https://doi:10.1159/000260387 37. Gong, P. Polytechnic Institute of New York, Brooklyn, New York, USA, ICDD Grant-in-aid, 1981, 1221. 38. Kim, W.J.; Lee, S.W.; Sohn, Y. Metallic Sn spheres and SnO2C core-shells by anaerobic and aerobic catalytic ethanol and CO oxidation reactions over SnO2 nanoparticles. Sci Rep. 2015, 24(5), 13448. https://doi:10.1038/srep13448. 39. Li, X.; Wang, J.; Joiner, A.; Chang, J. The remineralization of enamel: a review of the literature. J Dent. 2014, 42(1), 12-20. https://doi:10.1016/S0300-5712(14)50003-6 40. Šantak, V.; Vesel, A. Dental Tissues with Atmospheric Pressure Plasma Jet. Plasma Chemistry and Plasma Processing. 2017, 37(2), 401–413. https://DOI:10.1007/s11090-016-9777-3 41. Khoubrouypak, Z.; Abbasi, M.; Ahmadi, E.; Rafeie, N.; Behroozibakhsh, M. Effect of Cold Atmospheric Pressure Plasma Coupled with Resin-Containing and Xylitol-Containing Fluoride Varnishes on Enamel Erosion. Int J Dent. 2021, 23, 3298515. https://doi:10.1155/2021/3298515. 42. Jiménez-Gayosso, S.I.; Lara-Carrillo, E.; Scougall-Vilchis, R.J.; Morales-Luckie, R.A.; Medina Solís, C.E.; Velázquez-Enríquez, U.; Herrera-Serna, B. Remineralizing effect of xylitol, Juniperus communis and camellia sinensis added to a toothpaste: An in vitro study. Odovtos-International Journal of Dental Sciences 2020, 22(1), 71–79. https://DOI: 10.15517/ijds.v0i0.34573 43. Siqueira, V.L.; Barreto, G.S.; Silva, E.B. Effect of xylitol varnishes on enamel remineralization of immature teeth: in vitro and situ studies. Brazilian Oral Research 2021, 35, 137. https://DOI:10.1590/1807-3107bor-2021.vol35.0137 44. Desai, H.; Yadav, P.; Agrawal, S., Patel K.; Oza, M.; Kadivar, M. Effect of application of remineralization agents on microhardness & surface roughness of the enamel surface after interproximal stripping. in vivo study. International Journal of Advanced Research 2018, 6(6), 922- 931. https://DOI:10.21474/IJAR01/7294 45. Fan, W.; Li, Y., Liu, D.; Sun Q.; Duan, M.; Fan, B. PLGA submicron particles containing chlorhexidine, calcium and phosphorus inhibit Enterococcus faecalis infection and improve the microhardness of dentin. J Mater Sci Mater Med. 2019, 30(2), 1-11. https://doi:10.1007/s10856-018- 6216-4 46. Stancampiano, A.; Forgione, D.; Simoncelli, E.; Laurita, R.; Tonini, R.; Gherardi, M.; Colombo, V. The Effect of Cold Atmospheric Plasma (CAP) Treatment at the Adhesive-Root Dentin Interface. J Adhes Dent. 2019, 21(3), 229-237. https://doi:10.3290/j.jad.a42521 47. Stasic, J.N.; Selaković, N.; Puač, N.; Miletić, M.; Malović, G.; Petrović, Z.L.; Veljovic D.N.; Miletic, V. Effects of non-thermal atmospheric plasma treatment on dentin wetting and surface free energy for application of universal adhesives. Clin Oral Investing 2019, 23(3), 1383-1396. https://doi:10.1007/s00784-018-2563-2 https://doi:10.1016/j.jdent.2009.06.008 https://DOI:10.5772/34361.12221 https://Doi:10.1111/j.1600-0528.%201999.tb01989.x https://doi:10.1159/000260387 https://doi:10.1038/srep13448 https://doi:10.1016/S0300-5712(14)50003-6 https://DOI:10.1007/s11090-016-9777-3 https://doi:10.1155/2021/3298515 https://DOI:%2010.15517/ijds.v0i0.34573 https://DOI:10.1590/1807-3107bor-2021.vol35.0137 https://DOI:10.21474/IJAR01/7294 https://doi:10.1007/s10856-018-6216-4 https://doi:10.1007/s10856-018-6216-4 https://doi:10.3290/j.jad.a42521 https://doi:10.1007/s00784-018-2563-2 IHJPAS. 37 ( 4 ) 2024 196 48. Fathollah, S.; Abbasi, H.; Akhoundi, S.; Naeimabadi, A.; Emamjome, S. Cold plasma enamel surface treatment to increase fluoride varnish uptake. Sci Rep. 2022, 12(1), 4657. https://doi:10.1038/s41598- 022-08069-4 https://doi:10.1038/s41598-022-08069-4 https://doi:10.1038/s41598-022-08069-4