Type of the Paper (Article Journal of Baghdad College of Dentistry, Vol. 35, No. 1 (2023), ISSN (P): 1817-1869, ISSN (E): 2311-5270 36 Research Article Effect of adding titanium dioxide nanoparticles on anti- microbial activity and surface detail reproduction of dental alginate Ranj A. Omer 1*, Hoshang Kh. Abdel-Rahman 2, Mahabad M. Saleh 2 , Sazgar S.Q. Al-Hawezi 3, Fahd S. Ikram5 1 Gasha Technical Institute, Department of Dental Technology, Iraq. 2 Department of Prosthodontics, College of Dentistry, Hawler Medical University, Iraq. 3 Department of Conservative Dentistry, College of Dentistry, Hawler Medical University, Iraq. 4 Department of Prosthodontics, College of Dentistry, Hawler Medical University, Iraq. * Correspondence: dentist_46@yahoo.com Abstract: Most dental works require a diagnostic impression; alginate is contemplated as the most popular material used for this purpose. Titanium dioxide nanoparticles show evidence of antimicrobial activity in the recent era, for this purpose, this study aimed to evaluate the effect of adding Titanium dioxide nanoparticles on antimicrobial activity and surface detail reproduction of alginate impression material. Materials and methods: Titani- um dioxide nanoparticles (purity = 99%, size= 20nm) was added to alginate at three different concentrations (2%, 3% and 5%). 84 samples were prepared in total. Samples were tested for antimicrobial activity using a disc diffusion test, and surface detail reproduction was done using (ISO 21563:2021). One-way ANOVA and independent sample t-test were used for data analysis through SPSS software. Results: for the antimicrobial test, inhibition zones for Streptococcus mutans and Candida albicans showed significant changes concerning the altera- tion in Titanium dioxide nanoparticle concentrations. The inhibition zone significantly in- creased with an increase in the percentage of Titanium dioxide nanoparticles. The mean of the inhibition zone for S. mutans was superior to C. albicans and the difference was statisti- cally significant. Regarding surface detail reproduction, the control group, 2% and 3% groups manifested very similar results, only the group to which 5% of Titanium dioxide nanoparticles were added showed a decline in detail reproduction when compared to the other three groups. Conclusion: Within the limitation of this study, we can conclude that the antimicrobial activity against S mutans and C. albicans were significantly increased in modi- fied groups, and this escalation was directly linked to the increase in Titanium dioxide na- noparticles concentration. In contrast, the surface detail reproduction was decreased when adding 5% Titanium dioxide nanoparticles to alginate. Keywords: alginate, surface detail, nanoparticle, candida albicans. Introduction Dental impression is a negative replication of hard and soft tissues in the mouth from which a pos- itive reproduction (dental cast) can be formed1. Alginate is a biomaterial from a family of irreversible hydrocolloid that has served dental practice for almost century2.They were the first elastic impression material to be used in dentistry that provided high detail even under the presence of undercuts3. Being economical, easy to manipulate, and better tolerance Received date: 09-05-2022 Accepted date: 10-06-2022 Published date: 15-03-2023 Copyright: © 2022 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licens es/by/4.0/). https://doi.org/10.26477/jbcd .v35i1.3313 mailto:dentist_46@yahoo.com https://orcid.org/0009-0004-8106-7887 https://orcid.org/0000-0001-9025-3754 https://orcid.org/0000-0002-9913-6084 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.26477/jbcd.v35i1.3313 https://doi.org/10.26477/jbcd.v35i1.3313 J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 37 by the patient has put alginate in the utmost utilized material in the field of dentistry in contrast to other impression materials such as silicon4. All types of irreversible hydrocolloids have a hydrophilic nature making them susceptible to microbial retention5. It is a well-known fact that the human oral cavity is a favourable host to many microbial agents, during the impression-making procedure the oral cavity fluid could adhere to the impression materials6. Therefore, they may increase the susceptibility of cross infection7. To overcome this point, many disinfection methods have been used such as spraying and immersing. Unfortunately, both methods are time-consuming and may compromise some of the mechanical properties of the alginate8. During the last decade, the use of nanoparticles has become prevalent in the design and development of many dental materials since they can provide a unique combination of properties9. Due to the small size of the nanoparticles, they can provide a high surface area to volume ratio compared to particles of the same material10. This property gives them great attention in the present century as they possess defined mechanical, chemical, and optical properties crafting them into a suitable candidate for various applica- tions11. Many studies proved that nanoparticles could control the formation of biofilms as they possess biocidal and anti-adhesive properties. For this purpose, silver, copper, zinc, magnesium, titanium, and their ox- ides have been used as antimicrobial agents in many dental materials12. It was proven that TiO2 NPs possess good antibacterial activity against S. mutans13, without deteriorating the mechanical and physical properties13-15. Due to their imperfect properties, alginate remains an active material for research. The purpose of this study is therefore to evaluate the antimicrobial property of dental alginate incorporated with TiO2NPs against S. mutans and C. albicans, in addition to the surface detail reproduction after this modification. Materials and Methods To confirm the identity of the planned TiO2NPs for use in this study, X-ray Diffraction (XRD) analy- sis was performed before starting sample preparations. X-ray diffraction is a powerful non-destructive analytical method that is used to determine the structure and composition of unknown nanomaterials16,17. XRD test was performed using PANalytical X’pert powder (Figure 1.a) with Cu-Kα x-ray source, a wavelength of =1.54060�̇� was used. The TiO2NPs were deposited on to the specimen holder (Figure 1. b) and packed using a glass slide. The NPs inside the sample holder were loaded into the XRD machine and diffraction data was recorded at 2 range from 10 to 79.9950 with step size 0.0100 per 0.5s. Low scan speed was elected to provide higher sensitivity for the recognition of impurities18. J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 38 Figure 1: a) PANalytical X’pert powder b) specimen holder The PANalytical software was used to compare X-ray XRD patterns to identify the NPs. The result of the analysis identified the sample as TiO2 and its diffraction pattern are shown in figure (2): Figure 2: XRD pattern for Titanium oxide nanoparticles After confirming the identity of the chosen TiO2NPs, a pilot study was done by FTIR analysis to reveal any possible chemical structure changes (alteration of functional groups) after adding (2%, 3% and 5%) of TiO2NPs to the alginate, control and modified groups were analysed by the FTIR spectroscopy (Shi- J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 39 madzu 8400, Japan). The result (Figure 3) provided a clear clue that the in cooperation of TiO2NPs at all concentrations doesn’t induce any significant change in the main functional groups' structure for the (SI-O-SI group, C-O-C group, O-H group and C-H group) which was present in the unmodified alginate as the stretching and bending of the peaks did not alter after the addition of TiO2NPs. The only detected change was the increase in the percentage of IR transmittance which was detected only in the 5% TiO2NPs group, indicating the weakening of the bonds between the alginate molecules. Similar to the control group, sharp, strong peaks at 619.21 cm-1, 669.83 cm-1 and 793.54 cm-1 for SI-O-SI bands, also the peak at 1078.81 cm-1 for C-O-C bands were observed in all modified groups indicating that the added TiO2NPs does not interact with the available structural bonds in the alginate, this finding agrees with Skocaj et al 19 who stated that TiO2NPs considered as a chemically inert material. The weak sharp bending peak of the O-H group at 1621.88 cm-1 was very similar in control, 2% TiO2NPs and 3% TiO2NPs as the IR transmittance located at the same levels, but for the 5% group although the bands located at the same wavelength level but, the IR transmittance increased which is an indication that the higher TiO2NPs concentration might cause weakening of these bands due to the agglomeration of the TiO2NPs20. The weak stretch peak of the C-H group at 2924.61 cm-1, also the strong stretch peak of the O-H group at 3421.89 cm-1 and 3527.51 cm-1 for the control, 2% TiO2NPs and 3% TiO2NPs groups was almost identical but again there was a difference in the IR transmittance rate in which for the 5% TiO2NPs group was 80%, while for the former three groups at about 50%, this might be due to formation of small gaps be- tween these molecular groups band which ultimately caused the bonds to become weaker20 thus the IR easily penetrated the samples contained a higher percentage of 5%TiO2 NPs. Figure 3: FTIR analysis of Alginate (Blue =Control, Pink=2%, Green =3%, Black= 5%) J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 40 Study design and sample preparation In the present study, 84 samples were prepared from Alginplus (Major- ISO 21563. Italy) extra high pre- cision alginate impression material. Antimicrobial activity against S. mutans (n=28) and C. albicans (n=28) in addition to surface detail reproduction (n=28) were tested. One control group and three modified groups to which (2%, 3% and 5%) spheric shaped TiO2NPs were added respectively to the alginate have been studied, each group consisting of seven samples. Digital electronic balance (OHAUS GmbH- Switzerland) with precise accuracy of 0.0001 mg was used to weigh the alginate powder and the amount of TiO2NPs powder. The samples were prepared by mixing the blend of both powders (Alginate and TiO2NPs) with a pre- measured volume of distilled water as recommended by the manufacturer by using an automatic algi- nate mixer (Cavex- Netherlands) for 10 seconds. Antimicrobial test A disc diffusion test was used to investigate the antimicrobial activity released from the tested alginate specimens. For this purpose, two main oral pathogens namely S. mutans ATCC 25175 and C. albicans ATCC 10231 yeasts were chosen. The S. mutans bacteria were cultivated on blood agar media. The culture media was prepared according to the recommended protocol for S. mutans. Seven Petri dishes were used, and in each petri dish, four specimens were placed at equal distances from each other, marked with numbers 1, 2, 3 and 4 repre- senting the control, 2%, 3% and 5% groups respectively and incubated aerobically at 37°C for 24 hours. For the C. albicans ATCC 10231, Sabouraud Dextrose Agar was used for growing and cultivation21. The protocols from Antifungal susceptibility testing of the National Committee for Clinical Laboratory Standards (NCCLS) and Manual of Antimicrobial Susceptibility Testing were followed22,23. The seven Petri dishes containing samples were incubated for 24 hours. The measurement of inhibition zones for both pathogens was performed using scientific image analysis known as Image J software24. After completion of 24 hours incubation, the Petri dishes were taken out from the incubator, and images were taken at 90º with a reference of a ruler for calibration of image J software. Inhibition zones were measured around the ingots at six different positions. For the entire procedure, the working environment was conditioned under Bio air Top safe with con- tinuous air ventilation and a Bunsen burner that was turned on near the working field to prevent con- tamination of the testing components by airborne pollutants25.26. Surface detail reproduction A stainless-steel die with three scribed parallel lines ISO 21563: 202127 is used for surface detail repro- duction. The widths of these lines were 20-μm, 50-μm and 75-μm respectively. A stainless-steel ring was J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 41 0.2008 0.1901 1.2207 1.89042.2194 3.11352.7262 4.4552 0 2 4 6 C.albicans S.mutans M E A N I N H I B I T I O N Z O N E ( M M ) Control 2% 3% 5% placed on top of the steel die; the mixed alginate was poured inside the ring over the testing mould. A glass slab was then placed on top of the ring and a one-kilogram weight was positioned upon the slab for 10 minutes. Then, the samples were carefully removed from the mould and immediately examined with a digital microscope UM012C (5M 300X with 8 LEDs- China). Prior to the measurement procedure, the microscope eyepieces lens was calibrated for precise measurements of the samples. Specimens were reported to either pass (1) or fail (0) the test based on their ability to capture the entire length of the scribed 20-um line over the full length of 25mm distance between the cross line27,28. The surfaces were assessed according to the ranking system established by Owen29 which are: Score 1: Line reproduced clearly and sharply over the entire length between the marks. Score 2: Line clear over more than 50% of length, or line indistinct over less than 50% of length, the line appears to be reproduced well over the entire length, but not sharply. Score 3: Line clear over less than 50% of length, line indistinct over more than 50% of length, or line visi- ble over entire length but blemished not sharp. Score 4: Line is not reproduced over the entire length; rough, blemished, pitted. Statistical analysis Statistical Package for Social Sciences (SPSS, version 23.0) and Microsoft Office Excel were used for sta- tistical analysis. Descriptive statistics for frequency, mean, and standard deviation. Student T-test was used for comparisons between two independent groups, in addition to One-way ANOVA and post hoc test for multiple comparisons. The p<0.05 value was considered statistically significant. Results The obtained results from the antimicrobial test showed that the control group exhibited the least antimicrobial activity; furthermore, it was observed that the inhibitory effect against both pathogens was directly linked to the increase in the concentration of TiO2NPs as shown in Figure (4) The inhibitory effect of the modified alginate against S. mutans was more dominant compared to C. albi- cans which revealed higher resistance to the added TiO2NPs at the same concentrations. Figure 4: Mean inhibition zone for S. mutans and C.albicans J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 42 For S. mutans, the results of One-way ANOVA revealed a statistically significant difference (p < 0.05) in the inhibition zone measurements as shown in Table (1). Table 1: One-way ANOVA test for S.mutans Group N Mean+SD 95% CI for mean Lower band Upper band F Sig P-Value Control 7 0.19014 (0.00661) 0.18402 0.19626 534.957 0.000 2%TiO2NPs 7 1.89043 (0.12906) 1.77106 2.00979 3%TiO2NPs 7 3.11357 (0.37468) 2.76704 3.46010 5%TiO2NPs 7 4.45529 (0.12333) 4.34122 4.56935 Total 28 2.41236 (1.61205) 1.78727 3.03745 Post hoc (LSD) test for multiple comparisons depicted a statistically significantly different between the groups in such a way that the added TiO2NPs improved the antibacterial activity of the used alginate in all used concentrations as shown in Table (2). Table 2: Post hoc test (LSD- multiple comparisons) for S. mutans Group I Group J Mean Difference (I-J) 95% CI for mean Lower band Upper band Sig P-value Control 2% Tio2NPs -1.700286* -1.92925 -1.47132 0.000 3% Tio2NPs -2.923429* -3.15239 -2.69446 0.000 5% Tio2NPs -4.265143* -4.49411 -4.03618 0.000 2% Tio2NPs 3% Tio2NPs -1.223143* -1.45211 -0.99418 0.000 5% Tio2NPs -2.564857* -2.79382 -2.33589 0.000 3% Tio2NPs 5% Tio2NPs -1.341714* -1.57068 -1.11275 0.000 Regarding C. albicans, the obtained results were identical to the results of S. mutans as there was a statis- tically highly significant increase in the antifungal activity of the alginate in the modified groups. The inhibition zone was more dominant at the highest percentage (5%) of used TiO2NPs compared to the 2% and 3% groups. The control group possessed a minimum inhibition zone as shown in Table (3). Table 3: One-way ANOVA test for C. albicans Group N Mean+SD 95% CI for mean Lower band upper band F Sig P-value Control 7 0.20086 (0.00470) 0.19651 0.20521 926.760 0.000 2% Tio2NPs 7 1.22071 (0.06380) 1.16170 1.27973 3% Tio2NPs 7 2.21943(0.14397) 2.08627 2.35258 5% Tio2NPs 7 2.72629 (0.11391) 2.62093 2.83164 Total 28 1.59182 (0.99070) 1.20766 1.97598 J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 43 Post hoc test for multiple comparisons between the groups showed the presence of highly significant difference (p < 0.001) between the control group and the other groups as well as between the groups themselves as shown in table (4). Table 4: Post hoc test (LSD- multiple comparisons) for S.mutans C.albicans Group I Group J Mean Difference (I-J) 95% CI for mean Lower band Upper band Sig P-value Control 2% Tio2NPs -1.019857* -1.12710 -0.91261 0.000 3% Tio2NPs -2.018571* -2.12582 -1.91133 0.000 5% Tio2NPs -2.525429* -2.63267 -2.41818 0.000 2% Tio2NPs 3% Tio2NPs -0.998714* -1.10596 -0.89147 0.000 5% Tio2NPs -1.505571* -1.61282 -1.39833 0.000 3% Tio2NPs 5% Tio2NPs -0.506857* -0.61410 -0.39961 0.000 An Independent sample t-test was used to evaluate whether S. mutans and C. albicans differ significantly in their inhibition zone. The result specified that the mean of the inhibition zone for S. mutans and C. al- bicans was statistically significant as shown in Table (5). The means indicated that S. mutans (M = 2.41236, SD = 1.61205) showed significantly more inhibition zone than C. albicans (M=1.5917, SD=0.99070). Table 5: Independed Student T-test for S. mutans and C. albicans. Levene's Test for Equality of Variances t-test for Equality of Means F Sig. t df Sig.(2 tailed) Mean Differ- ence Std. Error Difference Equal variances assumed. Equal variances not as- sumed 8.204 0.006 2.295 54 0.026 0.820536 0.357582 2.295 44.849 0.026 0.820536 0.357582 Surface detail reproduction The alginate used in this study is branded as an irreversible hydrocolloid material that satisfies ISO 21563. All the tested groups efficiently and sharply recorded the 75-µm line in the entire length thus sat- isfying Owen’s score 1. Regarding 50-µm line, the control group, 2% and 3% groups reproduced that line on the alginate samples surface with Owens score 2 except for 5% TiO2NPs group that fall into Owens score 3. As mentioned previously, due to extra high quality of the used alginate in this study, the reproduction of the 20-um line was selected and considered as the base line for comparison between the groups. The group that was altered by addition of 5% of TiO2NPs failed to record the 20-µm line (Figure 5), while the remaining three groups reproduced 20-µm line and this ability is considered as an equivalent to the de- tail reproduction of the addition silicones according to ISO specifications 482330. J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 44 Figure 5: Surface detail reproduction Discussion Infection control is a fundamental procedure in dental practice. It is documented that there are about 750 million microorganisms in only 1 mL of the saliva of a healthy person31. According to many researchers, spherical-shaped nanoparticles with sizes 15-50nm exhibit maximum antimicrobial properties32. Due to this, spheric-shaped 20 nm TiO2NPs were chosen for this study. The result of the inhibition zone for S. mutans showed a significant increase when the percentage of TiO2NPs increased, a similar finding was obtained in a study by Al-Hawezi13 when TiO2NPs were in cooperated into a flowable dental composite resin and agreed with the result obtained in studies done for testing the effect of silver nanoparticles on S. mutans33,34. The antibacterial activity of TiO2NPs is practically due to a reaction of the high surface energy TiO2NPs with water. TiO2NPs release free radicals which are considered a potent oxidizing agent (Reactive oxygen species) that ultimately destroy the cell membrane35 or alternatively, in the absence of light, direct contact and adsorption of cells onto TiO2NPs may cause a loss of bacterial cell membrane36. Additionally, reports in the literature have shown that electrostatic attraction plays a great role in the bactericidal effect of the material37. This attraction probably overcomes other factors, such as the size and shape of NPs which can influence bacterial cell death38. The antifungal effect of TiO2NPs against C. albicnas was obvious in the modified groups when compared to control group, this finding agrees with results of a study39 who found that up to 65% of the C. albi- cans were killed after exposure to 100 μg/mL of TiO2NPs. A similar results was concluded with of Ker- mani et al40 who found that higher percentage of the titanium and zinc oxide nanoparticles increased their toxicity. Control 2 TiO2NPs 3% TiO2NPs 5% TiO2NPs Owens 1 1 0 0 0 Owens 2 1 3 2 0 Owens 3 5 4 5 0 Owens 4 0 0 0 0 1 0 0 0 1 3 2 0 5 4 5 00 0 0 0 0 1 2 3 4 5 6 sa m p le n u m b e rs Surface detail reproduction 20-um Owens 1 Owens 2 Owens 3 Owens 4 J. Bagh. Coll. Dent. Vol. 35, No. 1. 2023 Omer et al 45 It was documented that TiO2NPs cause C. albicans yeast cell death by producing intracellular reactive oxygen species (ROS), this in turn causes oxidation of the Coenzyme-A and peroxidation of lipids which subsequently decreases respiratory activity and ultimately causes cell death41. Another explanation for the antifungal activity of TiO2NPS, is the tear of the fungi cell membrane that disturb its integrity, caus- ing loss of intracellular substances 42. Impression-making is a routine in the dental practice, for this purpose, a variety of impression materials are available to capture oral cavity structures, the final decision for the selection of these products is usually based on the required type of dental treatment and clinician’s preference43. Surface detail reproduction is considered fundamental criteria for any irreversible hydrocolloid material, the latest ISO 21563 and ADA specification 18 is used as a standard protocol for measuring this proper- ty. The results of the present study were similar to another study 44 when they found no adverse effect of incorporating up to 1000 ppm of silver nanoparticle on the surface detail reproduction of alginate. This could be the result that the TiO2NPs were small (20nm) thus the particles were evenly distributed within the alginate matrix and did not influence the intermolecular bond. In addition, these nanoparticles are considered an inert material and do not induce any chemical structure alteration, this fact was supported by the FTIR analysis results. At 5% TiO2NPs, caused deterioration of the surface detail reproduction and it was impossible to record the 20-m line, this may be due to agglomeration of the used TiO2NPs inside the alginate matrix because of their high surface energy 45, this in turn triggered a poor intermolecular bond. According to obtained data, the requirements were met for irreversible hydrocolloid material as the tested groups reproduced the 75-µm and 50-µm groove which is considered satisfactory for alginate impression materials,46. Conclusion Within the limitation of this study, we can conclude that the addition of TiO2NPs to alginate im- proved the antimicrobial activity significantly. TiO2NPs are more powerful against S. mutans at the same used concentration. The addition of TiO2NPs doesn’t compromise the ISO 21563 requirement for surface detail reproduction. Conflict of interest: None. References 1. Hamalian, T. A., Nasr, E., Chidiac, J.J. 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Vol. 35, No. 1. 2023 Omer et al 48 اكرام سداد فهد, صالح محمود مهاباد , الحويزي قاسم سربست سازكار ,عبدالرحمن خالد هوشنك ,عمر ازاد رنج :الباحثون المستخلص: األكثر شيوعًا لهذا الغرض. تظهر الجسيمات النانوية لثاني أكسيد التيتانيوم دليالً على أعمال طب األسنان تتطلب انطباعًا تشخيصيًا ،الجينات هي المادة المستخدمة معظم: الخلفية ى النشاط المضاد للميكروبات ونسخ تفاصيل السطح عل TiO2NPsنشاط مضادات الميكروبات في العصر الحديث ، ولهذا الغرض تهدف هذه الدراسة إلى تقييم تأثير إضافة الجينات. لمادة االنطباع عينة في المجموع. تم اختبار عينات 84٪(. تم تحضير 5٪ و 3٪ ، 2نانومتر( إلى الجينات بثالث تركيزات مختلفة ) 20٪ ، الحجم = 99)نقاء = TiO2NPsتمت إضافة . ISO 21563: 2021انتشار القرص ، وتم اختباراستنساخ تفاصيل السطح باستخدامالنشاط المضاد للميكروبات باستخدام اختبار .SPSSالمستقل من خالل برنامج tأحادي االتجاه واختبار ANOVAلتحليل البيانات تم استخدام ،وزادت منطقة TiO2NPsالميكروبات ،أظهرت مناطق التثبيط للمكورات العقدية الطافرة والمبيضات تغيرات معنوية ذا عالقة بالتغير في تركيزات بالنسبة الختبار مضادات ة. فيما يتعلق باستنساخ وكان االختالف ذا داللة إحصائي C. albicansأعلى من S. mutansمنطقة التثبيط لـ . كان متوسط TiO2NPsالتثبيط معنويا مع زيادة نسبة إضافة 3٪ و 2تفاصيل السطح ، أظهرت المجموعة الضابطة، إليها أظهرت انخفاًضا في استنساخ TiO2NPs٪ من 5٪ نتائج متشابهة جدًا ، فقط المجموعة التي تمت التفاصيل عند مقارنتها بالمجموعات الثالث األخرى. قد زاد بشكل كبير في المجموعات المعدلة ، وكانت C. albicansو S mutansستنتج أن نشاط مضادات الميكروبات ضد الخالصة: ضمن حدود هذه الدراسة ، يمكننا أن ن . إلى الجينات TiO2NPs٪ 5. في المقابل ، تم تقليل استنساخ تفاصيل السطح عند إضافة TiO2NPsهذه الزيادة مرتبطة بشكل مباشر بزيادة تركيز