Effect of hydrothermal treatment of titanium in high concentration of AgNO 3 solution on surface morphology and roughness

Development of silver (Ag) modified titanium (Ti) as an antibacterial dental implant has recently been growing. Ag demonstrated an excellent antibacterial property without the risk of bacterial resistance. Hydrothermal treatment using AgNO 3 solution is one of the facile and promising methods to modify Ti surface with Ag. However, the effect of high AgNO 3 concentration and the absent of a toxic reduction agent has not been clearly studied. In this study, Ti surface was hydrothermally treated in 0.01 mol/L and 0.1 mol/L AgNO 3 solutions at 150 oC for 24 hours. Analysis of surface morphology using scanning electron microscopy with energy dispersive X-ray analysis suggested the formation of non-homogenous Ag coating with a tendency to be aggregated and thicken with the increase of AgNO 3 concentration. The Ag coating deposited on Ti surface were composed of mainly metallic and some oxide forms. Surface roughness of all AgNO 3 treated Ti surface was comparable based on the analysis of surface roughness parameter. In conclusion, hydrothermal treatment of Ti surface in solely AgNO 3 solution at high concentration produced non-homogenous Ag coating on its surface without significantly changed surface roughness. Keywords: Silver nitrate, titanium, hydrothermal, surface morphology, roughness


Introduction
Titanium (Ti) has widely been used clinically for dental implants due to their excellent mechanical properties, biocompatibility and osteoconductivity. 1,2 Development of Ti implant for dental application is still challenging. Dental implant not only requires osseointegration capability but also antibacterial property. 3 Titanium demonstrated satisfactory osseointegration clinically. However, its antibacterial capability is lacking.
Silver coating has emerged as an alternative to prepare antibacterial titanium surface. 4 Silver is considered a promising element to prevent and combat implant-related infection. Its main advantage is that it would not induce bacterial resistance. 5 Silver is coated onto Ti surface often in the form of particles by immersion in AgNO 3 solution mixed with a reduction agent. 6,7 This results in silver particulates being deposited on the Ti surface often in nanoscale, thus called silver nanoparticles (AgNPs). The use of reduction agents is problematic because they are often toxic chemicals such as Sodium borohydride , ammonium formate and hydrazine. 8,9 A recent study has been conducted to coat Ti surface with silver under hydrothermal without the need for reduction agents. 10 Direct Ag coating onto Ti surface using hydrothermal has not been clearly described especially in high concentration of AgNO 3 solutions and without the addition of toxic reduction chemicals. Therefore, this study aimed to coat the Ti surface with Ag particles using hydrothermal using solely AgNO 3 solution. Surface morphology including the distribution of the Ag coating and the change in surface roughness were then evaluated.

Sample preparation
Two Ti plates (Maximus Guard, Tokopedia) with a size of 10 cm Â 10 cm and thickness of 1 mm were cut into 10 mm Â 10 mm using a diamond cutter. A total of fifteen Ti plates (10 mm Â 10 mm) were used in this study. The samples were washed ultrasonically with acetone, ethanol, and distilled water before drying. Silver nitrate (AgNO 3 , Merck) solutions with a concentration of 0.01 mol/L and 0.1 mol/L were prepared. Titanium samples were immersed in a 100 ml-size Teflon container with 25 ml of AgNO 3 solutions, which was then placed into a hydrothermal vessel (FBA_Lab, Tokopedia). The hydrothermal vessel was heated in an oven at 150 o C for 24 hours. After hydrothermal treatment, the samples were washed with ethanol three times before drying.

Surface chemical composition, morphology and roughness
The elemental composition of titanium surface samples was examined using energy dispersive spectroscopy (Oxford instruments, UK) and analyzed using Oxford Aztect software. Scanning electron microscope (SEM) (Thermoscientific Quanta 650) (accelerating voltage (HV): 12kV, Secondary electron (SE), working distance (WD): 10.3-10.4mm) was used to evaluate the morphology of surface before and after hydrothermal. Surface roughness of titanium samples before and after hydrothermal treatment are measured using roughness tester (Surtronic S128) (Sampling length (l): 7 mm, cutoff (λc)/Type: 0.25 mm/2CR, Range: 100 μm). The average values of surface roughness were calculated using Microsoft excel spreadsheet software. Figures 1 and 2 show the photographs and SEM images of Ti surface before and after hydrothermal in AgNO 3 solutions. Bright particles were observed on Ti surface hydrothermally treated in 0.01 mol/L and 0.1 mol/L AgNO 3 . An area showed more concentrated particles, which may indicate the agglomeration. At higher concentration of AgNO 3 (0.1 mol/L), that concentrated area was larger ( Figure 2). The elemental analysis from the surface using energy dispersive X-ray analysis (EDX) indicated that those particles are Ag (Figures 3-5). Many methods have been developed to coat Ag into Ti surface both in the form of particles or ions. 4,11 The formation of silver particles on Ti surface under hydrothermal from AgNO 3 solution was still unclear in this study. Several different mechanisms may be responsible for how Ag could be deposited on Ti surface from AgNO 3 under hydrothermal treatment. One possible way is through thermal decomposition. 12 The deposition of Ag particles from only AgNO 3 solution up to 75 μmol/L under hydrothermal conditions was recently reported. 10 However, the exact mechanism on how Ag particles could be deposited on Ti surface was not clearly described. AgNO 3 was also reported to transform into Ag nanoparticles under hydrothermal condition at 121 o C. 13 Hydroxyl ions exist on Ti oxide layer may also play a role on the Ag particles growth on its surface. 6 It is known that Ti surface naturally forms thin oxide layer which contain Ti-OH groups on its outmost part. Figure 2 has confirmed the formation of Ag particles on Ti surface both in 0.01 mol/L and 0.1 mol/L AgNO 3 solutions. The Ag particles were also observed in the solution after hydrothermal (solution turned darkish color). The Ag particles seem to be non-homogenously distributed on Ti surface. As explained above, there is an area which contain thicker aggregated Ag particles masking the Ti surface. The concentrated Ag area was found to be larger in 0.1 mol/L AgNO 3 than that in 0.01 mol/L AgNO 3 . These findings suggest that at high concentration of AgNO 3 , the Ag coating tends to be aggregated and thicker, thus the use of a lower concentration might be preferred.

Results and discussion
The next question is that whether the Ag coating deposited on Ti surface is in the metallic or oxide forms. One way to find this is using the EDX elemental mapping to the aggregated Ag coating. Figures 3 and 4 show the elemental mapping of Ag coated Ti prepared from 0.01 mol/L and 0.1 mol/L AgNO 3 solutions. 14 In Figure 3, a thick Ag coating area (white dash line) shows a very strong purple color compared to the area in which less Ag coating was observed (white arrows). In Ti and oxygen (O) element mapping (green color and yellow color respectively), the Ag coating area was darker compared   Figure 3) in O elemental mapping indicated that that area was composed mostly of metallic Ag. Ag oxide also existed since the bright yellow color was also observed sporadically inside the white dash line (Figure 3; O Kα1). A similar trend was shown in Figure 4 where the Ag aggregate coating is larger. The thick Ag coating was most likely composed mainly from metallic Ag and smaller portion of Ag 2 O Surface treatment often changes surface roughness. 15 The change in surface roughness might alter the biological performance of Ti implant. Therefore, it is necessary to evaluate whether the current method of Ag coating changed  Figure 6. 16 The surface texture of all Ti samples before and after Ag coating were comparable. This data suggests that no significant changes were observed on Ti surface after Ag coating ( Table 1). Comparison of SEM images between Ag coating and untreated Ti surfaces (Figure 2) also support surface texture data. The Ti substrate in which Ag coating deposited was found to be comparable (Figure 2A and B).

Conclusion
This study reported the effect of hydrothermal treatment of Ti surface in AgNO 3 solution on the surface morphology and roughness. After hydrothermal treatment, an Ag coating was observed in all treated Ti surfaces. EDX mapping suggests that Ag coating composed of mainly metallic Ag and in smaller quantities, Ag oxide. Using a higher AgNO 3 solution concentration resulted in more Ag aggregates that mask Ti surface, creating a non-homogenous coating. Surface roughness of treated Ti surface did not change significantly when coated. Nevertheless, a slight increase of Rp and Rz was observed; this might be due to Ag coating aggregates. This project contains the following underlying data:

Data availability
-RAW EDX Mapping Data for 0.01M AgNO 3 treated Ti (Fig. 3) -RAW EDX Mapping Data for 0.1M AgNO 3 treated Ti (Fig. 4) -RAW EDX Map sum element spectrum for untreated Ti (Fig. 5A) -RAW EDX Map sum element spectrum for 0.01M treated Ti (Fig. 5B) -RAW EDX Map sum element spectrum for 0.1M treated Ti (Fig. 5C) This project contains the following extended data: previous study? In Page 2 the authors stated that "It is known that Ti surface naturally forms thin oxide layer which contain Ti-OH groups on its outmost part" please revise this because the negative charge of Ti-OH was only obtained after treatment in alkali solution (see 1 )? 3.
Did the coating particles just precipitate on the Ti surface or there is any possibility of bonding between the coating and the substrate?

4.
In the SEM of the coating sample, the coating particles are not in nanoscale size? Can the author give some possibility of agglomeration of the particles?

5.
In page 3, the author stated that "The concentrated Ag area was found to be larger in 0.1 mol/L AgNO3 than that in 0.01 mol/L AgNO3. These findings suggest that at high concentration of AgNO3, the Ag coating tends to be aggregated and thicker, thus the use of a lower concentration might be preferred" please explain why? What is the minimum amount Ag requirement for the antibacterial effect and the maximum Ag amount that is safe for the application? Do the homogenous and entirely cover coating is needed for the application? 6.