APT 2192 No.

of Pages 8, Model 5G
7 February 2019

Advanced Powder Technology xxx (xxxx) xxx

1

Contents lists available at ScienceDirect

Advanced Powder Technology

journal homepage: www.elsevier.com/locate/apt

2 Original Research Paper

6
4 Characterization and in vitro evaluation of an acid resistant nanosized
7
5 dental eggshell-titanium dioxide material
8 Stanley Chibuzor Onwubu a,⇑, Phumlane Selby Mdluli b, Shenuka Singh c, Madikizela Lawrence b,
9 Yonela Ngombane a
10 a
Dental Sciences, Durban University of Technology (DUT), Durban, South Africa
11 b
Chemistry, Durban University of Technology (DUT), Durban, South Africa
12 c
Discipline of Dentistry, University of KwaZulu-Natal (UKZN), Durban, South Africa

14
13
15
a r t i c l e i n f o a b s t r a c t
1 1
3 7
18 Article history: This paper reports on novel nanosized acid resistant material base on the modification of eggshell powder 32
19 Received 10 May 2018 and titanium dioxide (TiO2-EB) for enamel remineralization. The TiO2-EB was prepared by ball milling 33
20 Received in revised form 9 January 2019 eggshell powder and titanium dioxide. Fourier Transform Infrared Spectroscopy (FTIR), X-ray 34
21 Accepted 13 January 2019
Diffraction (XRD), Scanning Electron Microscope (SEM), Transmission Electron Microscopy (TEM), and 35
22 Available online xxxx
ImageJ were used to characterise TiO2-EB. A computation model using Material Studio Software was used 36
to explain the mechanism of TiO2-EB interaction. In addition, the acid resistant of TiO2-EB was evaluated 37
23 Keywords:
by comparison using three commercial toothpaste. The mean pressure value (kPa/s) was measured using 38
24 Eggshell powder
25 Titanium dioxide
a pressure sensor. The FTIR, XRD analysis confirmed the surface modification of TiO2-EB. The SEM image 39
26 Remineralization revealed that pure TiO2 particles are spread on the surface of eggshell powder. The TEM image revealed 40
27 Modification spherical particles in TiO2-EB. The ImageJ showed the average particle size of TiO2-EB to be 13 nm. In 41
28 Acid resistant addition, the commercial toothpastes doped with TiO2-EB showed an improved acid resistant. The salient 42
29 Toothpaste features of this study indicate that TiO2-EB will effectively remineralized enamel lesions while offering 43
30
better protective covering to the enamel. 44
Ó 2019 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder 45
Technology Japan. All rights reserved. 46
47

48
49
50 1. Introduction waste materials into value added products for environment 64
sustainability. 65
51 Eggshells are material mainly produced by poultry and domes- Eggshell is a natural bioceramic composite that has a unique 66
52 tic kitchens waste. In recent years, the use of liquid eggs at indus- chemical make–up of inorganic and organic compounds. It consists 67
53 trial level has resulted in a considerable amount of eggshells waste of an inorganic shell and an organic membrane. The shell together 68
54 as a by-product [1]. Most of the residual waste eggshells from with its membrane weighs approximately 11% of the egg weight. 69
55 these industries are mainly discarded into landfills without further The inorganic constituent of eggshells is mainly calcium carbon- 70
56 treatment, and subsequently overburdening the environment [2,3]. ates (95% in the form of calcite), whereas it’s organic compounds 71
57 In the United States, for example, 150,000 tons of eggshells waste are a matrix of proteins, glycoprotein, proteoglycan, and 72
58 is disposed in landfills annually which create land availability type X collagen sulphated polysaccharides (5%) [7–9]. Owing to 73
59 problem [4]. More so, and from economic perspective, Das, Min- this unique chemical composition, eggshell has extensively gained 74
60 kara, Melear and Tollner [5] and Chai [6] pointed out that indus- attention among researchers for its medical and dental benefits. 75
61 tries incur high costs in the disposal of eggshells. Given the For example, Khandelwal and Prakash [10] demonstrated how cal- 76
62 amount of eggshells waste that needs to be disposed worldwide, cium carbonate derived from eggshells could be used to produce 77
63 it is important to find alternative mea ns of converting eggshells hydroxyapatite, which is mainly used in bone and dental treat- 78
ments. Abdulrahman, Tijani, Mohammed, Yusuf, Jibrin and 79
Mohammed [11] alleged that eggshells based hydroxyapatite and 80
nanohydroxyapatite could reduce the cost of treatment in bone 81
⇑ Corresponding author at: Dental Sciences Department, Dental Technology
repair or replacement. Murakami, Rodrigues, Campos and Silva 82
Programme, Durban University of Technology (DUT), Durban, South Africa.
E-mail addresses: 21445599@dut4life.ac.za (S.C. Onwubu), phumlanem@dut.ac. [12] noted that calcium carbonate from eggshells has great poten- 83
za (P.S. Mdluli), singhshen@ukzn.ac.za (S. Singh). tial in the development of alternative ingredients for medical 84

https://doi.org/10.1016/j.apt.2019.01.005
0921-8831/Ó 2019 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.

Please cite this article as: S. C. Onwubu, P. S. Mdluli, S. Singh et al., Characterization and in vitro evaluation of an acid resistant nanosized dental eggshell-
titanium dioxide material, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.005
 APT 2192 No. of Pages 8, Model 5G
7 February 2019

2 S.C. Onwubu et al. / Advanced Powder Technology xxx (xxxx) xxx

85 applications such as food additive and calcium supplement, and as titanium dioxide was achieved in two steps. In the first step, egg- 132
86 a component for bone implants. shells were ball-milled by placing 30 g of the eggshell in a 133
87 Furthermore, eggshells are currently being investigated for their 500 mL stainless jar (inner diameter of 100 mm), together with 134
88 remineralization capabilities [13]. More recently, Mony, Ebenezar, 10 stainless steel balls of 10 mm diameter and dry-milled in a 135
89 Ghani and Narayanan [14] and Haghgoo, Mehran, Ahmadvand and planetary ball mill (Retsch Ò PM 100) at 400 rpm for 20 min. The 136
90 Ahmadvand [15] reported that eggshell has a rich bioavailable cal- collected powder was sieved to a particle size of 25 µm using a 137
91 cium content, which favours the remineralizing of caries lesions. mechanical sieving shaker (Retsch AS 200, Germany). The eggshell 138
92 This is particularly important, as enamel remineralization is effec- powder and titanium dioxide mixing ratio was optimized follow- 139
93 tive in the prevention of tooth decay process [16,17]. Of Interest, ing the procedure reported by Lin, Dong and Jiang [22], 20 g of 140
94 Arnold, Prange and Naumova [18] reveal that calcium carbonate the fine eggshell powder obtained in step 1 were modified by add- 141
95 constituent of eggshell are soluble in acidic environment; which ing 5 g of food grade anatase titanium dioxide (15 µm. The mix- 142
96 become unstable upon dietary challenge. This is concerning, as ture was subsequently ball-milled for 200 min to obtain a 143
97 acidic oral environment have been reported to demineralized the nanosized eggshell-titanium dioxide material (Fig. 1). 144
98 enamel thereby provoking tooth decay [17,19–21]. Modifying egg-
99 shell is therefore critical if is to be used in the oral environment for 2.2. Characterization of TiO2-EB 145
100 enamel remineralization. Several studies have indicated that nano-
101 sized titanium dioxide can improve the acid resistant properties of
102 calcium carbonates [22,23]. In particular, mechanical milling of the
2.2.1. Fourier transform infrared spectroscopy analysis 146
103 titanium dioxide and calcium carbonate effectively breaks and
The infrared spectra were measured using a Perkin Elmer 147
104 modifies the surfaces of CaCO3 nanoparticles which greatly
Universal ATR spectrometer to identify the functional group con- 148
105 improved its acidic properties [22]. While the use of TiO2 in the
stituents of TiO2-EB. 149
106 modification of CaCO3 nanoparticles by mechanical milling have
107 been extensively studied in papermaking industry [22–24], there
108 is, however, limited research on its use in the modification of egg- 2.2.2. X-ray diffraction analysis 150
109 shell powder for dental application. This paper therefore aimed to The X-ray diffraction (XRD) analysis was performed to observe 151
110 characterize and evaluate the acid resistant properties of a novel the possible changes in crystallinity between the eggshell powder, 152
111 nanosized eggshell-titanium dioxide material (TiO2-EB) in order titanium dioxide, and TiO2-EB. The XRD patterns were recorded 153
112 to determine its suitability for enamel remineralization. The for- using a diffractometer (PANalytical-Empyrean instrument; Co 154
113 mulated hypothesis was that modifying eggshell with titanium radiation 1.54056 Å) and analysed between 0 and 90° (2 theta). 155
114 dioxide will improved its acid resistant characteristics. The voltage, current and pass time used were 40 Kv, 40 mA and 156
1 s, respectively. 157

115 2. Materials and methods

2.2.3. Energy dispersive X-ray spectroscopy and scanning electron 158
116 All chemicals used in this study were of analytical grade. Food microscope analysis 159
117 grade anatase titanium dioxide (CAS No: 13463677) was purchase Energy dispersive x-ray spectroscopy was used in conjunction 160
118 from Sigma-Aldrich (Germany). Citric acid Monohydrate and lactic with scanning electron microscope (Field Emission-Carl Zeiss) 161
119 acid were supplied by Merck (South Africa). Three different brands operating at controlled atmospheric conditions at 20 kV to exam- 162
120 of toothpastes were bought from a popular shopping mall located ine the surface morphology and the elemental composition of the 163
121 at Durban (South Africa). These toothpastes included: Sensodyne, TiO2-EB. As a proxy measurement, pre and post agitation of TiO2- 164
122 Colgate sensitive toothpaste, and Colgate. The composition, active EB after 30 min with ultrasound technology (UP4DOS, Hielscher) 165
123 ingredients and manufacturers of the toothpaste are given in was carried out to establish the strength of modification. Prior to 166
124 Table 1. SEM observation, the surface was coated with a thin, electric con- 167
ductive gold film to prevent build-up of electrostatic charge. 168

125 2.1. Preparation of nanosized dental eggshell-titanium dioxide

126 composite (TiO2-EB) 2.2.4. Transmission electron microscopic analysis 169
A Transmission Electron Microscope (TEM) was used to observe 170
127 Eggshells collected from food outlets were washed with regular the particle size, shape and distribution of TiO2-EB. Very small 171
128 detergent to remove impurities. Eggshells were then disinfected by quantities of TiO2-EB were dispersed in 10 mL ethanol and soni- 172
129 storing the eggshells in a diluted solution of household sodium cated at 10 kv for 10 min. Subsequently, thin cross-sections of 173
130 hypochlorite for six hours. Subsequently, eggshells were vacuum cryo-microtomed specimens were prepared using a Leica micro- 174
131 dried for ±6–9 min at 250 °C. Modification of eggshell with tome (South Africa) and placed on carbon copper grids. Analysis 175

Table 1
Brand, composition, and manufacturer of toothpastes.

Toothpaste Composition as indicated in toothpaste labels by supplier Manufacturer

Colgate Sodium monofluorophosphates, Calcium carbonates, Aqua, Sorbitol, Sodium Colgate-Palmolive Co.
lauryl sulfate, Aroma, Cellulose gum, Sodium bicarbonate, Tetrasodium
pyrophosphate, Benzyl alcohol, Sodium saccharine, Sodium hydroxide, limonene
Colgate sensitive Aqua, Gycerin, Hydrated silica, Sorbitol, Potassium Nitrate, PEG-12, Colgate-Palmolive Co.
Tetrapotassium pyrophosphate, Sodium lauryl sulfate, Zinc citrate, PVM/MA
copolymer, Aroma, Potassium hydroxide, Xanthan gum, Cellulose gum,
Cocamidopropyl Betaine, Sodium Fluoride, Sodium Saccharin, Eugenol, and
Limonene
Sensodyne Aqua, Sorbitol, Hydrated silica, Glycerin, Potassium Nitrate, Cocamidopropyl GlaxoSmithKline
Betaine, Aroma, Xanthan gum, Titanium dioxide, Sodium Fluoride, Sodium
saccharin, Sodium hydroxide, Sucralose, and Limonene

Please cite this article as: S. C. Onwubu, P. S. Mdluli, S. Singh et al., Characterization and in vitro evaluation of an acid resistant nanosized dental eggshell-
titanium dioxide material, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.005
 APT 2192 No. of Pages 8, Model 5G
7 February 2019

S.C. Onwubu et al. / Advanced Powder Technology xxx (xxxx) xxx 3

Fig. 1. Schematic illustration of different steps in the preparation of TiO2-EB.

176 was conducted using a transmission electron microscope (TEM- test. A stopper was inserted into the flask to provide an airtight 213
177 Philips CM 120 model) at 120 kV. vessel. This was then connected to plastic tubing attached to a 214
gas pressure sensor (Order Code GPS-BTA). With the aid of an 215
178 2.2.5. Computation of TiO2-EB structural interaction using material interface system (Vernier LabPro) attached to a computer, the pres- 216
179 studio and EDX mapping sure readings were collected and analysed using LoggerPro 3 soft- 217
180 Material studio software (version 6) was used to analyze the ware. Prior to the experiment, 0.5 g of each sample was placed in 218
181 structural interaction of the calcite and titanium dioxide compo- the Erlenmeyer flask, while 25 mL of the prepared HCI was used 219
182 nent of TiO2-EB. EDX mapping was further used to validate the as the acid reactant. All tests were performed in duplicate, and 220
183 computation interaction. the mean slope (kPa/s) values were used for the statistical analysis. 221

184 2.3. Evaluation of acid resistant properties for TiO2-EB

2.4. Evaluation of acid resistant properties for TiO2-EB in situ 222

185 The acid resistant characteristics of TiO2-EB were evaluated by

Fourteen freshly collected bovine enamel were used to evaluate 223
186 comparison using simulated eggshell enamel model. This entails
the acid resistant properties of TiO2-EB in situ. The tooth samples 224
187 doping commercial toothpastes with TiO2-EB and eggshell powder,
were sectioned to a diameter of 5 mm  5 mm  3 mm using a dia- 225
188 respectively. It was assumed that if TiO2-EB has improved acid
mond cutter under water to avoid overheating. The samples were 226
189 resistant properties then it would be a suitable material for the
then randomly assigned to 7 groups of 2 specimens each. 1: Unex- 227
190 remineralization of tooth enamel.
posed enamel; 2: Exposed enamel 3: Exposed enamel in Colgate 228
solution; 4: Exposed enamel in Sensodyne solution; 5: Exposed 229
191 2.3.1. Preparation of simulated eggshell enamel samples
enamel in Colgate sensitive solution; 6: Exposed enamel in EB- 230
192 Eggshell enamel samples were simulated in vitro using TiO2-EB
TiO2 solution, and 7. Exposed enamel in eggshell alone. 231
193 and eggshell powder (control). 3 g of each brand of toothpaste
194 listed in Table 1 were dissolved in a beaker containing 100 cm3
195 deionized water by constant agitation using magnetic stirrer at 3. Results and discussion 232
196 800 rpm for 20 min. 1 g of the prepared eggshell powders and
197 TiO2-EB was added to the beaker containing the dissolved tooth- 3.1. Characterization of TiO2-EB 233
198 pastes, respectively. These were further agitated for 8 h at a speed
199 of 800 rpm. The mixtures were then filtered and subsequently Following the successful modification of eggshell powder and 234
200 oven dry at 60° for 3 h. The prepared samples were then exposed titanium dioxide through ball-milling, the FT-IR spectra of eggshell 235
201 to 2 mol L 1 hydrochloric acid (HCI). powder, titanium dioxide, and TiO2-EB in Fig. 2 revealed several 236
bands from 500 cm 1 to 2000 cm 1. The band aspect of the FT-IR 237
202 2.3.2. pH test spectra shows difference between the eggshell powder (Fig. 2A), 238
203 1.5 g of each samples were placed in a beaker containing 50 mL titanium dioxide (Fig. 2B), and TiO2-EB (Fig. 2C). As shown in 239
204 deionized. The solution were constantly agitated at low speed of Fig. 2A, there are prominent absorption peaks of carbonates at 240
205 600 rpm for 30 min. A pH meter (Starter 300, Ohaus Incorporation 1411 cm 1, which is associated to the carbonyl group found in cal- 241
206 USA) equipped with temperature sensor was used to record cium carbonate. Furthermore, the FT-IR spectra shows the absorp- 242
207 changes in the pH reading. tion peak of calcite around 711 and 873 cm 1. These were 243
attributed to asymmetric and symmetric stretching, out-of-plane 244
208 2.3.3. Pressure sensor test bending and in-plane bending vibration modes for calcium carbon- 245
209 A gas pressure sensor (Vernier LabPro, USA) was used to ate (CO23 ) molecules [8,25–27]. In contrast to eggshell powder in 246
210 monitor pressure changes in the pressure (kPa) against time (s) Fig. 2A, TiO2-EB in Fig. 2C shows a broad, intense band below 247
211 during the reaction of the samples with HCI. Erlenmeyer flask 1000 cm 1. This corresponds to the Ti-o-Ti vibrations of the tita- 248
212 (250 mL) was used as the reaction container during the pressure nium dioxide [23,28]. 249

Please cite this article as: S. C. Onwubu, P. S. Mdluli, S. Singh et al., Characterization and in vitro evaluation of an acid resistant nanosized dental eggshell-
titanium dioxide material, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.005
 APT 2192 No. of Pages 8, Model 5G
7 February 2019

4 S.C. Onwubu et al. / Advanced Powder Technology xxx (xxxx) xxx

that approximately cubic and spherical particles coexisted 275

(Fig. 4A). For the image shown in Fig. 4B, nearly spherical like mor- 276
phological can be seen for TiO2. For Fig. 4C, it can be observed that 277
the pure anatase particles is scattered on the surface of TiO2-EB. 278
More so, it can be seen that titanium dioxide sufficiently coated 279
the eggshell powder particles and formed a compact layer of 280
TiO2 firm. For Fig. 4D, similar evenly spread of TiO2 on the surface 281
of the eggshell powder particles was observed post agitation, 282
thereby confirming the bond strength between eggshell powder 283
and titanium dioxide. 284
The particle size and shape of TiO2-EB were characterized by 285
TEM. As shown in Fig. 5, the presence of nonhomogeneous struc- 286
ture with agglomeration of particles can be observed. Equally, a 287
TEM analysis revealed distribution of spherical shape particles of 288
different sizes corroborating with the SEM analysis. In addition, 289
the particle size distribution of TiO2-EB were further analysed 290
using ImageJ software (National Institute of Health USA, http://im- 291
agej.nih.gov./ij). As seen in Fig. 5B, the average mean particle size 292
of TiO2-EB was revealed to be around 13 nm. 293
A computation model showing the reaction between calcite 294
Fig. 2. FTIR spectra showing (A) eggshell powder; (B) TiO2-EB (C) titanium dioxide.
structure and anatase is proposed in Fig. 6. As shown in Fig. 6C, 295
the TiO2 formed a reaction bond with the calcite structure of the 296

250 From Fig. 3, the XRD pattern of eggshell powder, titanium diox- eggshell at the surface, which helps to explain the mechanism of 297

251 ide and TiO2-EB showed characteristic diffraction peaks with coating. As suggested in literature [22], large proportions of CO23 298

252 diffraction angles at 29.5°, 34.5°, 42.5°, 46.5°, 50.5°, 56°, 57°, 67°, on the surface of CaCO3 particles react with moisture during the 299

253 68° and 72°. For Fig. 3A, the characteristic peak marked around mechano-chemical process to produce CaCO3 hydroxyl com- 300

254 34.5° (2h) indicates the presence of thermodynamically stable cal- pounds. More so, these hydroxyl compounds are the active point 301

255 cite crystalline structure, which is similar to calcium carbonate [8]. that facilitate modification [29]. In addition, and consistent with 302

256 As shown in Fig. 3B, the XRD pattern of pure TiO2 shows a charac- Tao, He and Zhao [23] CaCO3 and TiO2 molecules reaction on the 303

257 teristic diffraction peak with values lying at 2h = 29.5° corresponds surface is depended on Vander Waals forces to hold them together. 304

258 to anatase phase and is confirmed with International Centre for These forces play an important role in the intermolecular interac- 305

259 Diffraction Data (ICDD Ref: 98-009-6946). The TiO2-EB diffraction tions between calcite structure and anatase. Fig. 6D shows the 306

260 peak shown in Fig. 3C revealed the presence of calcite and anatase mapping generated from the EDX elemental analysis. The evenly 307

261 crystalline structure. Equally important, and consistent with Tao, spread of TiO2 on the surface of eggshell powder particles strongly 308

262 He and Zhao [23], the shape, intensity, and location of the TiO2- suggests the surface modification of TiO2-EB. 309

263 EB peaks corresponding to the pure titanium dioxide, is an indica-

264 tive of the deposition of TiO2 on the surface of the CaCO3.
265 The results given in Table 2 illustrates the elemental composi- 3.2. Evaluation of the acid resistant properties of TiO2-EB 310

266 tion of eggshell, titanium dioxide, and TiO2-EB (pre and post agita-
267 tion). Specifically, it can be observed that the elemental The mean pH of tested toothpastes, eggshell, and TiO2-EB is 311

268 composition of TiO2-EB pre-and post-agitation were almost simi- given in Fig. 7. The pH results suggests that calcium carbonates 312

269 lar. Thus suggesting that the bond strength between titanium diox- base materials such as Colgate (9.61) eggshell (9.37), TiO2-EB 313

270 ide and eggshell powder was highly effective. (9.31) have strong alkaline characteristics. On the contrary, the 314

271 The SEM images of eggshell powder, pure anatase titanium toothpastes brands such as Colgate Sensitive (7.4), and Sensodyne 315

272 dioxide, and TiO2-EB composite particles (pre and post) agitation (7.41) had a pH value that can be considered neutral. 316

273 are given in Fig. 4. As seen in Fig. 4A, the SEM images of eggshell Fig. 8 illustrates the mean pressure slope value of the eggshell 317

274 powder shows more irregular shaped particles. It can be observed powder and TiO2-EB that are doped into three commercial tooth- 318
pastes, namely: Colgate, Sensodyne, and Colgate Sensitive. In con- 319
trast to eggshell powder, toothpaste samples doped with TiO2-EB 320
showed improved acid resistant properties. 321
Fig. 9 demonstrate the acid resistant properties of the tested 322
commercial toothpastes, eggshell and TiO2-EB. Fig. 9(B), (D), and 323
(E) visibly showed the demineralization of the tooth samples 324
against acidic challenge in the presence of the tested toothpastes. 325
Equally, the enamel surface were observed to be rougher and 326
showing more destruction of the tooth prismatic structure. On 327
the contrary, enamel samples exposed to acid in the presence of 328
eggshell and Colgate showed rougher surface but less destruction 329
of prismatic structure of the tooth (Fig. 9C and F). The less pris- 330
matic destruction observed for these samples (C & F) could be 331
attributed to the high alkaline properties of the materials. In con- 332
trast, the image in Fig. 9G suggests that the acid had no effect on 333
the enamel in the presence of TiO2-EB, as similar images were 334
observed with that of the unexposed enamel (Fig. 9A). The image 335
observed in Fig. 9G visibly confirmed that modification of eggshell 336
Fig. 3. XRD pattern of (A) eggshell powder; (B) titanium dioxide; (C) TiO2-EB. with titanium improves its resistant in acidic environment. 337

Please cite this article as: S. C. Onwubu, P. S. Mdluli, S. Singh et al., Characterization and in vitro evaluation of an acid resistant nanosized dental eggshell-
titanium dioxide material, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.005
 APT 2192 No. of Pages 8, Model 5G
7 February 2019

S.C. Onwubu et al. / Advanced Powder Technology xxx (xxxx) xxx 5

Table 2
EDX analysis showing elemental composition.

Element Eggshell powder Titanium dioxide TiO2-EB

Pre agitation Post agitation
Calcium (Ca) 22.66 0.00 14 15.24
Oxygen (O) 55 48.14 52.39 51.86
Titanium (Ti) 0.00 51.58 12.31 17.49
Magnesium (Mg) 0.37 0.00 0.2 0.18
Carbon (C) 21.98 0.00 21.09 15.23

Fig. 4. SEM micrograph for (A) eggshell powder; (B) titanium dioxide; (C) TiO2-EB before agitation; (D) TiO2-EB after agitation.

Fig. 5. TEM images: (A) Images of TiO2-EB after ball-milling for 200 min; (B) Normal distribution of TiO2-EB particle size.

338 3.3. Discussion powder may limit its use in a high acidic environment. A critical 342
point deserving mentioning is that high acidic environment help 343
339 Eggshell powder have been predicted to be the future of enamel facilitate enamel demineralization (Fig. 9B). The presence of tita- 344
340 remineralization. The findings from this study suggests that the nium dioxide coating on the surface of TiO2-EB was highly effective 345
341 poor acid resistant of calcium carbonate constituent of eggshell in the prevention of acidic attack (Fig. 9G). Eggshell powder should 346

Please cite this article as: S. C. Onwubu, P. S. Mdluli, S. Singh et al., Characterization and in vitro evaluation of an acid resistant nanosized dental eggshell-
titanium dioxide material, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.005
 APT 2192 No. of Pages 8, Model 5G
7 February 2019

6 S.C. Onwubu et al. / Advanced Powder Technology xxx (xxxx) xxx

Fig. 6. (A) molecular structure for titanium dioxide; (B) molecular structure of calcite (C) structural interaction of calcite-titanium dioxide; (D) EDX mapping of TiO2-EB.

Fig. 7. pH of commercial toothpastes, eggshell and TiO2-EB.

Fig. 8. Mean slope differences between eggshell and TiO2-EB showing acid resistant
properties TiO2-EB.

347 therefore be modified with titanium dioxide when used in tooth-

348 paste for enamel remineralization. supports titanium dioxide forming a coating on the surface of the 358
349 Moreover, the in-plane bending vibration modes of calcium car- eggshell powder. This finding is consistent with [30]. Conse- 359
350 bonates molecules (Fig. 2c) as well as the diffraction peak of calcite quently, the hypothesis was accepted as modification of eggshell 360
351 (Fig. 3C) strongly suggests that modification of eggshell powder powder with titanium dioxide showed an improved acid resistant. 361
352 with titanium dioxide does not adversely affect the carbonate Thus suggesting TiO2-EB suitability as alternative to eggshell for 362
353 structure of TiO2-EB. This is particularly important as it can enamel remineralization. 363
354 increase the pH of an acidic environment (Fig. 7) where deminer- From a dental material perspective, and corroborating with 364
355 alization is likely to occur and offer a high bioavailability of cal- Rahardjo, Nugraheni, Humaira, Adiatman and Maharani [31], the 365
356 cium [13]. Equally important, the improved acid resistant particle size of TiO2-EB (Fig. 5B) is more likely to effectively re- 366
357 properties measured in the toothpastes doped with TiO2-EB further mineralized enamel lesions. Furthermore, Cutler [32] suggested 367

Please cite this article as: S. C. Onwubu, P. S. Mdluli, S. Singh et al., Characterization and in vitro evaluation of an acid resistant nanosized dental eggshell-
titanium dioxide material, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.005
 APT 2192 No. of Pages 8, Model 5G
7 February 2019

S.C. Onwubu et al. / Advanced Powder Technology xxx (xxxx) xxx 7

Fig. 9. Enamel surface (A) Unexposed tooth; (B) after exposure to HCI; (C) after exposure to Colgate toothpaste; (D) after exposure to Colgate Sensitive; (E) after exposure to
Sensodyne (F) after exposure to eggshell; (G) after exposure to TiO2-EB (All exposed samples were in a solution of 0.2 mL HCL for 30 min).

368 that nanosized titanium dioxide and dental abrasive agents can be In addition to these, anatase TiO2 have been reported to be an 374
369 used together in the remineralization of damaged teeth. In light of effective antimicrobial agents that is compatible with human body 375
370 these findings, it is expected that TiO2-EB will effectively reminer- or environment [33,34]. In particular, and in a more recent study, it 376
371 alized enamel lesions, at the same time, offering better protective was demonstrated that titania reduces that ability of bacterial such 377
372 covering to the enamel against erosive challenged than eggshell as Staphylococcus aureus (S. aureus) to adhere to surfaces by reput- 378
373 alone. ing their cell membrane [35]. Given that the metabolic activities of 379

Please cite this article as: S. C. Onwubu, P. S. Mdluli, S. Singh et al., Characterization and in vitro evaluation of an acid resistant nanosized dental eggshell-
titanium dioxide material, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.005
 APT 2192 No. of Pages 8, Model 5G
7 February 2019

8 S.C. Onwubu et al. / Advanced Powder Technology xxx (xxxx) xxx

380 bacterial in the oral cavity contribute to enamel demineralization [14] B. Mony, A.R. Ebenezar, M.F. Ghani, A. Narayanan, Effect of chicken egg shell 430
powder solution on early enamel carious lesions: an invitro preliminary study, 431
381 and subsequently dental caries [36], this study assumed that due 432
J. Clin. Diagn. Res.: JCDR 9 (2015) ZC30.
382 to the presence of titania (TiO2), TiO2-EB could also act as antimi- [15] R. Haghgoo, M. Mehran, M. Ahmadvand, M.J. Ahmadvand, Remineralization 433
383 crobial agents in the prevention of dental caries. This is an area effect of eggshell versus nano-hydroxyapatite on caries-like lesions in 434
permanent teeth (in vitro), J. Int. Oral Health 8 (2016) 435. 435
384 worth exploring in the future. 436
[16] B.M. Moron, S. Miyazaki, N. Ito, A. Wiegand, F. Vilhena, M.A.R. Buzalaf, A.C.
Magalhães, Impact of different fluoride concentrations and pH of dentifrices on 437
tooth erosion/abrasion in vitro, Aust. Dent. J. 58 (2013) 106–111. 438
385 4. Conclusion [17] N. West, J. Seong, N. Hellin, H. Eynon, M. Barker, T. He, A clinical study to 439
measure anti-erosion properties of a stabilized stannous fluoride dentifrice 440
386 The prominent findings of this paper showed that titanium relative to a sodium fluoride/triclosan dentifrice, Int. J. Dental Hygiene 15 441
(2017) 113–119. 442
387 dioxide effectively coat the surface of eggshell powder. The func- [18] W. Arnold, M. Prange, E. Naumova, Effectiveness of various toothpastes on 443
388 tional groups, surface morphology, particle size and mechanisms dentine tubule occlusion, J. Dent. 43 (2015) 440–449. 444
389 of coating were confirmed by suitable characterization techniques. [19] M. Lombardini, M. Ceci, M. Colombo, S. Bianchi, C. Poggio, Preventive effect of 445
different toothpastes on enamel erosion: AFM and SEM studies, Scanning 36 446
390 Notably, this study conclusively showed that TiO2-EB will effec- 447
(2014) 401–410.
391 tively remineralized enamel lesions while offering better protec- [20] E.A.A. Neel, A. Aljabo, A. Strange, S. Ibrahim, M. Coathup, A.M. Young, L. Bozec, 448
392 tive covering to the enamel against acid attack. This is an area V. Mudera, Demineralization–remineralization dynamics in teeth and bone, 449
Int. J. Nanomed. 11 (2016) 4743. 450
393 for further research. 451
[21] M. Colombo, M. Mirando, D. Rattalino, R. Beltrami, M. Chiesa, C. Poggio,
Remineralizing effect of a zinc-hydroxyapatite toothpaste on enamel erosion 452
caused by soft drinks: ultrastructural analysis, J. Clin. Exp. Dent. 9 (2017) e861. 453
394 Acknowledgment
[22] H. Lin, Y.-B. Dong, L.-Y. Jiang, Preparation of calcium carbonate particles coated 454
with titanium dioxide, Int. J. Miner. Metall. Mater. 16 (2009) 592–597. 455
395 The authors acknowledged the financial support from the [23] H. Tao, Y. He, X. Zhao, Preparation and characterization of calcium carbonate– 456
titanium dioxide core–shell (CaCO 3@ TiO 2) nanoparticles and application in 457
396 National Research Foundation of South Africa (No. 104824).
the papermaking industry, Powder Technol. 283 (2015) 308–314. 458
[24] K. Tanabe, K. Mitsuhashi, T. Yoshida, Titanium dioxide-calcium carbonate 459
397 References composite particles, in: Nittetsu Mining Co., Ltd, Nittetsu Mining Co., Ltd, 460
2006. 461
[25] Y. Feng, B. Ashok, K. Madhukar, J. Zhang, J. Zhang, K.O. Reddy, A.V. Rajulu, 462
398 [1] M.M. Cristianne, M.T. Hincke, Recent patents on eggshells: shell and
Preparation and characterization of polypropylene carbonate bio-filler 463
399 membrane applications, Recent Pat. Food Nutr. Agric. 3 (2011) 1–8.
(eggshell powder) composite films, Int. J. Polym. Anal. Charact. 19 (2014) 464
400 [2] H.J. Choi, S.M. Lee, Heavy metal removal from acid mine drainage by calcined
637–647. 465
401 eggshells and microalgae hybrid system, Environ. Sci. Pollut. Res. 22 (2015)
[26] A. Jazie, H. Pramanik, A. Sinha, A. Jazie, Egg shell as eco-friendly catalyst for 466
402 13404–13411.
transesterification of rapeseed oil: optimization for biodiesel production, Int. J. 467
403 [3] Y. Gao, C. Xu, Synthesis of dimethyl carbonate over waste eggshell catalyst,
Sustain. Dev. Green Econ. 2 (2013) 27–32. 468
404 Catal. Today 190 (2012) 107–111.
[27] Z. Yao, M. Xia, L. Ge, T. Chen, H. Li, Y. Ye, H. Zheng, Mechanical and thermal 469
405 [4] P. Toro, R. Quijada, M. Yazdani-Pedram, J. Arias, Eggshell, a new bio-filler for
properties of polypropylene (PP) composites filled with CaCO3 and shell waste 470
406 polypropylene composites, Mater. Lett. 61 (2007) 4347–4350.
derived bio-fillers, Fibers Polym. 15 (2014) 1278–1287. 471
407 [5] K. Das, M. Minkara, N. Melear, E. Tollner, Effect of poultry litter amendment on
[28] A. Manivannan, G. Glaspell, P. Dutta, Synthesis of nanocrystalline TiO2 472
408 hatchery waste composting, J. Appl. Poult. Res. 1 (2002) 282–290.
particles and their structural characteristics, J. Cluster Sci. 19 (2008) 391–399. 473
409 [6] C.L. Chai, Development polypropylene (Pp)-modified chicken eggshell
[29] S.L. Su, Z. Cao, Z.J. Zhang, Progress in research of preparation of modified 474
410 composites, in: Petrochemical Engineering, UTAR, 2016.
ultrafine powders by mechanochemical method, Metal Mine 8 (2007) 1–8. 475
411 [7] W.J. Stadelman, Egg and egg products, in: F.J. Francis (Ed.), Encyclopedia of
[30] X. Chen, C. Li, J. Wang, J. Li, X. Luan, Y. Li, R. Xu, B. Wang, Investigation on solar 476
412 Food Science and Technology, John Wiley and Sons, New York, 2000, pp. 593–
photocatalytic activity of TiO2 loaded composite: TiO2/eggshell, 477
413 599.
TiO2/clamshell and TiO2/CaCO3, Mater. Lett. 64 (2010) 1437–1440. 478
414 [8] S. Onwubu, A. Vahed, S. Singh, K. Kanny, Physicochemical characterization of a
[31] A. Rahardjo, D.D.T. Nugraheni, G. Humaira, M. Adiatman, D.A. Maharani, 479
415 dental eggshell powder abrasive material, J. Appl. Biomater. Funct. Mater. 14
Efficacy of toothpaste containing nano calcium in dentin remineralization, 480
416 (2017) 341–346.
Makara J. Health Res. (2015) 43–47. 481
417 [9] M.T. Hincke, Y. Nys, J. Gautron, A.B. Rodriguez-Navarro, M.D. Mckee, The
[32] E.T. Cutler, Prevention and Treatment of Oral Diseases, Squigle Inc, USA, 2014. 482
418 eggshell: structure, composition and mineralisation, Front. Biosci. 17 (2012)
[33] S. Rana, R. Misra, The anti-microbial activity of titania-nickel ferrite composite 483
419 1288–11280.
nanoparticles, JOM 57 (2005) 65–69. 484
420 [10] H. Khandelwal, S. Prakash, Synthesis and characterization of hydroxyapatite
[34] J. Rawat, S. Rana, R. Srivastava, R.D.K. Misra, Antimicrobial activity of 485
421 powder by eggshells, J. Miner. Mater. Charact. Eng. 4 (2016) 119.
composite nanoparticles consisting of titania photocatalytic shell and nickel 486
422 [11] I. Abdulrahman, H.I. Tijani, H.S. Mohammed, H. Yusuf, M.N. Jibrin, S.
ferrite magnetic core, Mater. Sci. Eng., C 27 (2007) 540–545. 487
423 Mohammed, From garbage to biomaterials: an overview on egg shell based
[35] D. Depan, R. Misra, On the determining role of network structure titania in 488
424 hydroxyapatite, J. Mater. 2014 (2014) 1–6.
silicone against bacterial colonization: mechanism and disruption of biofilm, 489
425 [12] F.S. Murakami, P.O. Rodrigues, C.M.T. Campos, M.A.S. Silva, Physicochemical
Mater. Sci. Eng., C 34 (2014) 221–228. 490
426 study of CaCO3 from eggshells, Ciência e Tecnologia de Alimentos 27 (2007)
[36] S. Chenicheri, R. Usha, R. Ramachandran, V. Thomas, A. Wood, Insight into oral 491
427 658–662.
biofilm: primary, secondary and residual caries and phyto-challenged 492
428 [13] D.V. Macri, Implementing a minimally invasive approach, Dimensions Dental
solutions, Open Dent. J. 11 (2017) 312. 493
429 Hyg. 14 (2016) 32–37.
494

Please cite this article as: S. C. Onwubu, P. S. Mdluli, S. Singh et al., Characterization and in vitro evaluation of an acid resistant nanosized dental eggshell-
titanium dioxide material, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.005