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Electrochemical Deposition of 58SiO 2

-33CaO-9P 2 O 5 Nanobioactive Glass
Particles on Ti-6Al-4V Alloy for
Biomedical Applications

DATASET · JULY 2013

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Saravanakumar Balasubramaniam Judy Gopal

Jeju National University Konkuk University
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Kamachi Mudali Mudali

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Retrieved on: 01 December 2015
 I J A C
Journal Name
1 2 1 2
Manuscript No.
4
B Dispatch: 18.5.13
Author Received:
Journal: IJAC CE: Deepa R
No. of pages: 12 PE: Hariprasad

Int. J. Appl. Ceram. Technol., 1–11 (2013)

DOI:10.1111/ijac.12124

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Electrochemical Deposition of 58SiO2-33CaO-9P2O5
20 Nanobioactive Glass Particles on Ti-6Al-4V Alloy for
21
22 Biomedical Applications
23
24 Balasubramaniam Saravanakumar, Muthusamy Prabhu, and
25 Venkatachalam Rajendran*
26
Centre for Nano Science and Technology, K.S. Rangasamy College of Technology,
27
Tiruchengode 637 215, Tamil Nadu, India
28
29
Judy Gopal and Uthandi Kamachi Mudali
30
31 Corrosion Science and Technology Division, Indira Gandhi Centre for Atomic Research,
32 Kalpakkam 603 102, Tamil Nadu, India
33
34
35
36 The nanobioactive glass (58SiO2-33CaO-9P2O5) powders were synthesized by simple sol–gel method. The prepared
samples reveal amorphous nature, agglomerated spherical morphology with particle size of 100–150 nm. The specific surface
37
area of nanobioactive glass (NBG) particle is 147 m2/g. The NBG samples were coated on titanium (Ti-6Al-4V) alloy
38
through electrochemical deposition method. The particle size of the NBG-coated surface was in the order of 200–300 nm,
39 and it was confirmed by atomic force microscopy (AFM) analysis. In vitro and AFM studies reveal the existence of higher
40 bioactivity and uniform coating of NBG on implants at 80 V for 1 h.
41
42
43 Introduction response at the interface of tissue and materials.1–6 In
44 recent years, nanoscale bioactive materials have
45 Bioactive materials play vital role in biomedical attracted increasing interest because of their enhanced
46 implant applications because of their specific biological bioactivity, which is the result of their increased sur-
47 face-to-volume ratio. The biocompatibility and bone
48 *veerajendran@gmail.com
regeneration properties of bioactive glasses led to their
49 © 2013 The American Ceramic Society use in drug delivery, bone filling, tissue engineering,
 2 International Journal of Applied Ceramic Technology—Saravanakumar et al.
1 and antibacterial and medical implant applications.7
2 Different materials that show bioactive properties are
3 glass, glass ceramics, bioglass, calcium phosphate, and
4 hydroxyapatite.8 The SiO2-CaO-P2O5 glass composi-
5 tions are obtained by sol–gel method, which directly
6 leads to the formation of hydroxyapatite layer on the
7 glass surface when contact in simulated body fluid
8 (SBF) solution. During this period, a series of surface
9 reactions take place.9 A reaction on its surface can be
10 described in five sequences. In the first sequence, ion
11 exchange takes place between the glass and SBF. In
12 the second sequence, the silica network is broken by
13 ion exchange, followed by silanol bond formation in
14 the third sequence. The silanol layer has high silica
15 content with a high surface area. During the fourth
16 sequence, the silica-rich layer attracts proteins and col-
17 lagens to form a crystalline hydroxyapatite (HAp)
18 layer.10 Bioactive glasses are class A biomaterials that
19 possess osteoconductive11 and osteoinductive proper-
20 ties.12 Properties such as time required for bonding,
21 strength of bonding, and thickness of the interfacial
22 layer depend on the initial composition of materials
23 and their texture.13
24 Bioactive glasses are prepared using different meth-
25 ods such as traditional melt quenching14, sol–gel15,
26 and foaming methods.16 The sol–gel method has many
27 advantages such as high purity, homogeneity, high sur-
28 face area, and high osteoconductivity. Further, this is a
29 low-temperature method, and the composition can be
30 easily varied15,17,18 when compared with other meth-
31 ods. The bioactivity of the glasses prepared by sol–gel
32 method is higher than that of the glasses obtained by
33 traditional melt-quenching method. In recent times,
34 nanobioactive glass (NBG) powders prepared using the
35 sol–gel method19,20 have been used for implant appli-
36 cations because of their high surface area and high bio-
37 activity when compared with bulk bioactive
38 glasses.21,22
39 Generally, bioactive glass materials have low
40 mechanical strength and are restricted to only low load-
41 bearing applications. The mechanical strength of bioac-
42 tive glasses can be increased by coating on implant
43 materials. Different methods are used to coat bioactive
44 glass on implants, such as dip coating, laser ablation,
45 sputtering, spin coating, anodizing, CO2 laser coating,
46 and electrophoretic deposition.23–28 Recently, NBG
47 powders have been coated on a 316L SS substrate
48 employing sol–gel dip-coating method for increasing
49 corrosion resistance and biocompatibility.21 In spray
2013
coating, the phase nature of the sample was changed,
and the mechanical interface between the metal and
glass became weak.29
The aim of this study is to synthesize and charac-
terize the bioactive glass nanoparticles (SiO2-CaO-
P2O5) using the sol–gel method. The prepared NBG
powders are coated on titanium implants using the
electrochemical anodizing method. The optimum con-
ditions for uniform coating of NBG are standardized.
The coating of NBG on implants is characterized using
atomic force microscopy (AFM), epifluorescence
microscopy, scanning electron microscopy (SEM), and
transmission electron microcopy (TEM). In addition,
bioactivity of the coated sample is evaluated using in
vitro studies in the simulated body fluid.
Experimental Details
Materials
AR grade (Merck, India) chemicals with 99.99%
purity namely, tetraethyl orthosilicate [TEOS; Si
(OC2H5)4], potassium dihydrogen phosphate
(KH2PO4), calcium nitrate (CaNO3
4H2O), ammonia
solution (min 25% GR), and ultrapure water (Arium
611UF; Sartorius AG, Germany) were used for the 2
preparation of NBG powder.
Preparation of nanobioactive glass powders
Nanobioactive glass powder, namely 58SiO2-
33CaO-9P2O5, was prepared using the sol–gel
method.19,30 The steps involved in the preparation of
NBG powders are shown in Fig. 1. Initially, TEOS
(9.1 mL) was dissolved in 1:1 ratio of ethanol and ultra-
pure water. Then, 2 N nitric acid was added to the
solution under stirring for 30 min at room temperature.
After complete hydrolysis of TEOS, KH2PO4 (1.7 g)
and CaNO3
4H2O (5.45 g) salts were dissolved sepa-
rately with 2 mL of distilled water, and the dissolved
solution was added to silica solution at 30 min interval
under constant stirring at room temperature. After
obtaining the clear solution, 1 M ammonia solution was
added drop wise until the formation of gel, that is, until
the solution reaches its pH to ~8.0. Thereafter, the
obtained white gel was dried in a hot air oven at 353 K
for 6 h. The dried gel was calcined at 873 K for 2 h in
a muffle furnace to remove the carbon and nitrate
impurities. The calcined samples were ground in a dry
 www.ceramics.org/ACT NBG on Ti-6Al-4V Alloy 3 1

1 paper. A DC power supply (Imec, the Netherlands) 3

2 was used for the anodizing process. Voltage was
3 increased from 20 to 110 V with different time inter-
4 vals, namely 1, 2, and 3 h. The increased current was
5 proportional to the applied voltage. The coating
6 process was carried out at room temperature (310 K).
7
8
Optimization of anodizing condition for coating
9
10 Coating parameters such as anodizing time and
11 voltage were optimized (Table I). Coating was carried
12 out at different anodizing times (1, 2, and 3 h) for dif-
13 ferent anodizing voltages such as 20, 50, 80, and
14 110 V. The coated titanium implants were named as
15 T-1, T-2, T-3, and T-4, respectively, for applied
16 coating voltages of 20, 50, 80, and 110 V.
17
18
Characterization studies
19
20 The amorphous nature of NBG powders was
Fig. 1. Nanobioactive glass (NBG) fabrication procedure by
21 investigated through X-ray diffraction (XRD) studies
sol–gel synthesis.
22 using an X-ray diffractometer (D500; Siemens) with 4
23 Cu Ka as a radiation source (k = 1.5418  A). The
24 diffractometer was operated at 40 kV with 2h value
25 ball mill (PM 100; Retsch Corporation, Hann, varying from 0° to 80° with an increment of 0.05°.
26 Germany) at 500 rpm for 1 h to obtain fine bioactive The morphology and microstructure of the NBG
27 glass particles. powders were analyzed by SEM (JSM 6360; JEOL,
28 Tokyo, Japan). The particle size and shape of the pre-
29 pared NBG powders were determined using TEM
Coating of NBG on titanium surface
30 (CM 200; Philips). The surface area of the NBG 5
31 The prepared glass powders were coated on powder was measured using a BET surface area ana-
32 commercial pure (grade 2) titanium (30 mm 9 20 lyzer (Autosorb-1; Quantachrome, Boynton Beach,
33 mm 9 2 mm) using electrochemical anodizing FL). To analyze the sample, we degassed it properly
34 method. Orthophosphoric acid (1 N) was used as an for 3 h at 568 K. The elemental compositions of the
35 electrolyte. A stainless steel cathode was used with tita- NBG powders were measured with the help of
36 nium as an anode. The experimental set up was
37 placed on a magnetic stirrer, and continuous stirring
38 was carried out throughout the anodization process. Table I. Optimization of anodizing condition for
39 Small quantities of NBG powder (1, 2, and 3 g) were
coating of nanobioactive glass (NBG) on Ti implant
40 dissolved in 200 mL electrolyte to establish the effect
41 of initial inoculum of NBG in the electrolyte for Trial Sample Applied
42 improving coating. To remove the oxidation layer on S.No. code weight (g) Time (h) voltage (V)
43 the surface of the titanium implant, it was pickled it
1 T-1 1 1, 2, and 3 20
44 in a solution of HNO3/HF acid bath (400:40 g) for
2 1
45 2 min.31 Thereafter, the implant was cleaned in an
1
46 ultrasonic bath to remove the moisture content. Fur-
2 T-2 3 1 50
47 ther, it was rinsed with ultrapure water and acetone to
3 T-3 3 1 80
48 remove foreign particles from the surface of the tita-
4 T-4 3 1 110
49 nium. Finally, the implant was wiped using a tissue
 4 International Journal of Applied Ceramic Technology—Saravanakumar et al. 2013

1 energy-dispersive X-ray analysis (Inca 300; Oxford, when observed under a Nikon Eclipse E600 epifluo-
2 UK). The elements present in the coating were con- rescence microscope (excitation filter BP 490; barrier
3 firmed by field-emission scanning electron microscopy filter O 515). All inorganic compounds appear to
4 (Quanta 200; FEI, Eindhoven, the Netherlands). The fluoresce green. The NBG-modified surfaces exposed
5 surface topography of NBG-coated titanium was char- to the SBF were stained using Acridine Orange for
6 acterized using atomic force microscopy (Solver PRO; 5 min and then washed using distilled water fol-
7 6 NT-MDT, Ireland). lowed by air drying. The green fluorescence film on
8 the surfaces was observed using an epifluorescence
9 microscope.
In vitro studies
10
11 The in vitro study was carried out by dipping the
12 samples in SBF. The NBG-coated titanium sample was Results and Discussion
13 soaked in 75 mL SBF in a sterilized polyethylene con-
14 tainer having almost the same ion concentration and X-ray diffraction patterns of the prepared NBG
15 pH value as that of human blood plasma (Table II). powders are shown in Fig. 2. The observed broad peak
16 The in vitro studies were carried out at 310 K by incu- from 10° to 35° indicates the formation of the Si–O–
17 bating the container in a water bath shaker for 12 days. Si network. It is reported that the amorphous nature of
18 SBF was prepared using AR grade (Merck, India) XRD patterns signifies the internal disorder and glassy
19 chemicals (purity 99.99%) such as NaCl, NaHCO3, nature of the material.34 The result of TEM analysis of
20 KCl, K2HPO4
3H2O, MgCl2
6H2O, CaCl2, and the glass powder (shown in Fig. 3a) confirms that the
21 Na2SO4 with ultrapure water (pH 7.2  0.19). Tris- particle size is in the range of 100–150 nm. The
22 hydroxymethyl aminomethane [(CH2OH3)3CNH3] observed particles are agglomerated. However, the
23 and 1 k mol/m3 hydrochloric acid were used to adjust selected area electron diffraction (SAED) pattern (insert
24 the pH value to 7.40.32 The change in the pH value in Fig. 3a) confirms the amorphous nature of the pre-
25 was measured every day using a pH meter (Orion pared NBG particles.35 The specific surface area of the
26 7 5-Star; Thermo Scientific). particles measured from nitrogen adsorption/desorption
27 Acridine Orange, a fluorescent dye, differentially analysis (Fig. 3b) is 147 m2/g. The surface morphology
28 stains single-stranded RNA and double-stranded of the bioactive glass powder is studied using SEM
29 DNA, fluorescing orange when intercalated with the
30 former and green while complexing with the latter33
31
32
33 Table II. Comparison of ion concentration in
34 human blood plasma and simulated body fluid
35
36 Concentration
37 (m mol/dm3)
38 Simulated body human
39 Ion fluid (SBF) blood plasma
40
Na+ 142.0 142.0
41
K+ 5.0 5.0
42
Mg2+ 1.5 1.5
43
Ca2+ 2.5 2.5
44
Cl- 147.8 103.0
45
HCO3- 4.2 4.2
46
HPO42 1.0 1.0
47
SO42 0.5 0.5
48 Fig. 2. X-ray diffraction (XRD) pattern of nanobioactive glass
pH value 7.40 7.40
49 (NBG) powder.
 www.ceramics.org/ACT NBG on Ti-6Al-4V Alloy 5 1

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(c) (d)
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32 Fig. 3. (a) TEM image, (b) BET plot, (c) SEM image, and (d) EDAX pattern of the nanobioactive glass (NBG) powder.
33
34
35 (Fig. 3c). It shows the agglomeration of the particles. Table III. Comparison of theoretical and
36 The energy-dispersive X-ray analysis spectrum of the experimental composition
37 NBG powder is shown in Fig. 3d, reveals the quantita-
38 tive analysis of the compositional elements Si, P, Ca, Composition (Wt%)
39 and O present in NBG powder. The energy-dispersive Element Theoretical Experimental
40 X-ray analysis spectra result shows the heterogeneous
SiO2 58 59.05
41 distribution of the compositional elements, with the
CaO 33 29.12
42 content of CaO is being less and that of P2O5 being
P2O5 9 11.82
43 relatively higher (Table III).
44 Nanobioactive glass at concentrations of 1, 2, and
45 3 g is dissolved in 200 mL orthophosphoric acid.
46 Anodization is carried out to determine the effect of the AFM image of NBG particles deposited on the
47 the added NBG inoculum on increasing the efficacy surfaces when anodized at 20 V for 1 and 3 h. The
48 of coating. The efficacy of coating of NBG on the above results indicate that there is no increase in coat-
49 titanium plate is revealed by AFM. Fig. 4a,b shows ing efficacy of NBG particles on the surface of
 6 International Journal of Applied Ceramic Technology—Saravanakumar et al. 2013

1 titanium with an increase in time, that is, from 1 to for 1 h is shown in Fig. 5. The results reveal that the
2 3 h.36 significant deposition of NBG particles distributed on
3 The AFM image of titanium surface anodized the surface of titanium is obtained using 3 g when
4 with different concentration of NBG particles at 20 V compared with 1 and 2 g NBG-added electrolytes.
5
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8 (a) (b)
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20 Fig. 4. Atomic force microscopy (AFM) image of nanobioactive glass (NBG) coating after anodization at 20 V (a) 1 h, (b) 3 h. 9
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48 Fig. 5. Atomic force microscopy (AFM) images of titanium surfaces anodized in different amount of nanobioactive glass (NBG) added 10
49 in electrolytes at 20 V for 1 h (a) 1 g, (b) 2 g, and (c) 3 g.
 www.ceramics.org/ACT NBG on Ti-6Al-4V Alloy 7 1

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12 (a) (b)
13 Fig. 6. (a) SEM image of uncoated titanium surfaces (b) SEM image of nanobioactive glass (NBG)-coated titanium surfaces anodized
14 at 1 h, at 20 V.
15
16
17 Further, it reveals that the NBG particles coated on
18 the surface are agglomerated when the anodizing volt-
POOR QUALITY FIG

19 age reaches 100 V, which is shown in Fig. 8d. The

20 AFM results confirm that the particle size ranges from
21 200 to 300 nm. Thus, it is inferred from the above
22 studies that optimized coating of NBG on titanium
23 surfaces can be carried out for an anodizing voltage of
24 80 V. The biocompatibility and bioactivity properties
25 of the NBG-coated surface of titanium are studied
26 under the influence of SBF. The SBF-soaked samples
27 are removed at the end of the second and twelfth
28 days.
29 The effect of soaking time on pH value is shown
30 in Fig. 9. It shows the change in pH value in all sam-
31 Fig. 7. EDAX spectrum results of coated surface anodized at 11 ples because of the chemical interactions between the
32 20 V for 1 h. coating and SBF. For the first 3 days, a continuous
33 increase in pH value from its initial value (7.40) is
34 observed in all samples and it is continued for another
35 The SEM image of uncoated and NBG-coated tita- day in sample T-1, which is because of the interchange
36 nium surface anodized at 20 V for 1 h is shown in of Ca2+ ions between the coating and SBF.37 Beyond
37 Fig. 6. The observed results confirm higher deposition this, the pH value decreases on the fourth day for sam-
38 rate at 1 h. Deposition of Si, Ca, and P particles is ples T-2, T-3, and T-4 because of the deficiency of H+
39 found in NBG particles coated on titanium surface, ions in the solution.38 Subsequently, the hydrolysis and
40 which is evident from the energy-dispersive X-ray anal- dissolution of silica take place.39 The dissolution of
41 ysis (Fig. 7). Ca2+ ion and the formation of silanol on the coating
42 In contrast, NBG deposition is increased with an surface are mandatory for the formation of hydroxyapa-
43 increase in anodizing voltage from 20 to 50 V, 50– tite layer on the material surface.40 Silica possesses sur-
44 80 V, and 80–110 V. The gradual increase in NBG face negative charge at physiological pH,41 and this
45 coating as a function of anodizing voltage is evident causes calcium and phosphate ions to form a calcium
46 from Fig. 8. A large number of coatings of NBG par- phosphate-rich layer on the surface of the silica layer.42
47 ticles on the surface of titanium for an anodizing volt- After 5 days, the pH value changes nonlinearly with
48 age of 80 V are noticed. The AFM image (Fig. 8c) soaking time in all coating samples because of leaching
49 supports the observations made from in vitro studies. of the surface crystal.
 8 International Journal of Applied Ceramic Technology—Saravanakumar et al. 2013

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(a) (b)
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25 Fig. 8. Atomic force microscopy (AFM) image showing nanobioactive glass (NBG) deposition as a function of anodizing voltage (a) 12
26 20 V (b) 50 V (c) 80 V, and (d) 110 V.
27
28
29 inorganic layer covering the entire surface is formed on
30 the 80 V/1 h anodized surface after an exposure time
31 of 12 days. The inorganic film that formed on the sur-
32 faces exposed to SBF is reported to be mostly apatite.
33 Fig. 10 shows the increase in the formation of an inor-
34 ganic layer on NBG-modified titanium surfaces. As
35 seen from the Fig. 10, the inorganic layer increases
36 with exposure time in all NBG-modified titanium sur-
37 faces. These results indicate that the NBG-modified
38 surfaces show an increased bioactivity. Initially, there is
39 dissolution of the silicon matrix coating after immer-
40 sion in SBF. Such dissolutions are reported earlier
41 through pulse laser-deposited bioactive glass coat-
42 ings.43–47 The mechanism of apatite formation in bio-
43 active glass is described by Hench8 in four stages, as
44 discussed previously. The increase in apatite formation
Fig. 9. Variation of pH as a function of soaking time.
45 is evident from the epifluorescence micrographs which
46 show an increase in green fluorescence with exposure
47 Epifluorescence microscopy studies for formation time. The in vitro formation of apatite during exposure
48 of inorganic film on the modified titanium surface to SBF is generally accepted as an indicator for in vivo
49 when exposed to SBF show that a dense uniform bioactivity of the material surface.48
 www.ceramics.org/ACT NBG on Ti-6Al-4V Alloy 9 1

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Fig. 10. Epifluorescence micrograph of nanobioactive glass (NBG)-modified titanium surface after exposure in SBF (a) 20 V for 2 days,
36
(b) 20 V for 12 days, (c) 50 V for 2 days, (d) 50 V for 12 days, (e) 80 V for 2 days, (f) 80 V for 12 days, (g) 110 V for 2 days, (h)
37 110 V for 12 days.
38
39
40
41 Conclusion of 200–300 nm, and it was confirmed by AFM analy-
42 sis. Bioactivity of the coating of NBG on titanium
43 Silicate glasses with a composition of 58SiO2- implants was analyzed for different time periods (2 and
44 33CaO-9P2O5 were prepared by the sol–gel method. 12 days) in SBF. In vitro and AFM studies reveal the
45 The particle size of the prepared glass powder was in existence of higher bioactivity and uniform coating of
46 the range of 100–150 nm with a surface area of NBG on implants at 80 V for 1 h. These preliminary
47 147 m2/g. NBG powders were successfully coated on results indicate that anodization could be used as a bio-
48 titanium surfaces through an electrochemical deposition active glass coating technique to increase the bioactivity
49 method. The particle size of the NBG was in the order of titanium surfaces.
 10 International Journal of Applied Ceramic Technology—Saravanakumar et al. 2013

1 Acknowledgments 20. X. Chen, C. Guo, and N. Zhao, “Preparation and Characterization of

the Sol–Gel Nano-Bioactive Glasses Modified by the Coupling Agent
2 Gamma-Aminopropyltriethoxysilane,” Appl. Surf. Sci., 255 [2], 466–468
3 One of the authors Mr. B. Saravanakumar (2008).
21. M. H. Fathi, and A. Doostmohammadi, “Bioactive Glass Nanopowder
4 acknowledges the summer research fellowship provided and Bioglass Coating for Biocompatibility Improvement of Metallic
5 by the Indian Academy of Sciences, Bangalore. Implant,” J. Mater. Process. Technol., 209 [3], 1385–1391 (2008).
6 22. H. Aguiar, et al., “????????????????,” J. Non- Cryst. Solids, 354 [34], 4075–
4080 (2008). 8
7 23. P. Gonzalez, J. Serra, S. Liste, S. Chiussi, B. Leon, and M. Perez-Amor,
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 O n c e y o u h a v e A c r o b a t R e a d e r o p e n o n y o u r c o m p u t e r , c l i c k o n t h e C o m m e n t t a b a t t h e r i g h t o f t h e t o o l b a r :

h i s w i l l o p e n u p a p a n e l d o w n t h e r i g h t s i d e o f t h e d o c u m e n t . h e m a j o r i t o f

T T y

t o o l s o u w i l l u s e f o r a n n o t a t i n g o u r p r o o f w i l l e i n t h e A n n o t a t i o n s s e c t i o n ,
b

y y

p i c t u r e d o p p o s i t e . W e ’ v e p i c k e d o u t s o m e o f t h e s e t o o l s e l o w :
b

S t r i k e s a l i n e t h r o u g h t e x t a n d o p e n s u p a t e x t S t r i k e s a r e d l i n e t h r o u g h t e x t t h a t i s t o e
b

o x w h e r e r e p l a c e m e n t t e x t c a n e e n t e r e d . d e l e t e d .
b b

‚ ‚
i g h l i g h t a w o r d o r s e n t e n c e . i g h l i g h t a w o r d o r s e n t e n c e .

H H

‚ ‚
l i c k o n t h e R e p l a c e ( I n s ) i c o n i n t h e A n n o t a t i o n s l i c k o n t h e S t r i k e t h r o u g h ( D e l ) i c o n i n t h e

C C

s e c t i o n . A n n o t a t i o n s s e c t i o n .

‚
p e t h e r e p l a c e m e n t t e x t i n t o t h e l u e o x t h a t
b b

T y

a p p e a r s .

i g h l i g h t s t e x t i n e l l o w a n d o p e n s u p a t e x t
a r k s a p o i n t i n t h e p r o o f w h e r e a c o m m e n t
M

H y

o x w h e r e c o m m e n t s c a n e e n t e r e d .
b b
n e e d s t o e h i g h l i g h t e d .
b

‚ ‚
i g h l i g h t t h e r e l e v a n t s e c t i o n o f t e x t .
l i c k o n t h e A d d s t i c k n o t e i c o n i n t h e

H
C y

A n n o t a t i o n s s e c t i o n .

‚
l i c k o n t h e A d d n o t e t o t e x t i c o n i n t h e

‚
A n n o t a t i o n s s e c t i o n .
l i c k a t t h e p o i n t i n t h e p r o o f w h e r e t h e c o m m e n t

s h o u l d e i n s e r t e d .
b

‚
p e i n s t r u c t i o n o n w h a t s h o u l d e c h a n g e d
b

T y

‚
r e g a r d i n g t h e t e x t i n t o t h e e l l o w o x t h a t
b
p e t h e c o m m e n t i n t o t h e e l l o w o x t h a t
b

y
T y y

a p p e a r s .
a p p e a r s .
 I n s e r t s a n i c o n l i n k i n g t o t h e a t t a c h e d f i l e i n t h e I n s e r t s a s e l e c t e d s t a m p o n t o a n a p p r o p r i a t e

a p p r o p r i a t e p a c e i n t h e t e x t . p l a c e i n t h e p r o o f .

‚ ‚
l i c k o n t h e A t t a c h F i l e i c o n i n t h e A n n o t a t i o n s l i c k o n t h e A d d s t a m p i c o n i n t h e A n n o t a t i o n s

C C

s e c t i o n . s e c t i o n .

‚ ‚
l i c k o n t h e p r o o f t o w h e r e y o u ’ d l i k e t h e a t t a c h e d e l e c t t h e s t a m p y o u w a n t t o u s e . ( T h e A p p r o v e d

C S

f i l e t o b e l i n k e d . s t a m p i s u s u a l l y a v a i l a b l e d i r e c t l y i n t h e m e n u t h a t

a p p e a r s ) .

‚
e l e c t t h e f i l e t o b e a t t a c h e d f r o m y o u r c o m p u t e r

‚
o r n e t w o r k . l i c k o n t h e p r o o f w h e r e y o u ’ d l i k e t h e s t a m p t o

a p p e a r . ( W h e r e a p r o o f i s t o b e a p p r o v e d a s i t i s ,

‚
e l e c t t h e c o l o u r a n d t y p e o f i c o n t h a t w i l l a p p e a r

t h i s w o u l d n o r m a l l y b e o n t h e f i r s t p a g e ) .

i n t h e p r o o f . l i c k O K .

A l l o w s s h a p e s , l i n e s a n d f r e e f o r m a n n o t a t i o n s t o b e d r a w n o n p r o o f s a n d f o r

c o m m e n t t o b e m a d e o n t h e s e m a r k s . .

‚
l i c k o n o n e o f t h e s h a p e s i n t h e D r a w i n g

M a r k u p s s e c t i o n .

‚
l i c k o n t h e p r o o f a t t h e r e l e v a n t p o i n t a n d

d r a w t h e s e l e c t e d s h a p e w i t h t h e c u r s o r .

‚
T o a d d a c o m m e n t t o t h e d r a w n s h a p e ,

m o v e t h e c u r s o r o v e r t h e s h a p e u n t i l a n

a r r o w h e a d a p p e a r s .

‚
D o u b l e c l i c k o n t h e s h a p e a n d t y p e a n y

t e x t i n t h e r e d b o x t h a t a p p e a r s .