SEM/EDS and Roughness Analysis on Current Titanium Implant Decontamination Systems: In Vitro Study

Abstract

The aim of this study was to evaluate the effects of different decontamination treatments on the surface roughness and elemental deposition of pristine dental implants using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). We divided 110 dental implants into 21 groups based on the decontamination method used in vitro. One group was the untreated control. Roughness values (R_a) were analyzed with a profilometer, while elemental deposition was assessed through EDS. Results were obtained for each treatment and for macrogroups (control, ultrasound, curettes, powders, brushes, gels). Significantly lower R_a values were found in the neck zone with respect to the thread zone (p < 0.05). EDS analysis revealed a non-significant higher presence of carbon and calcium in certain treatments, denoting a certain deposition of the decontaminating products (p > 0.05). Although there were various significant differences among the groups, roughness values were low and no decontaminating methods macroscopically affected implant surfaces, so decontaminating procedures can be considered safe.

1. Introduction

Oral rehabilitation by means of dental implants has become very common and is widespread, with a high survival rate of 94% in a 15-year follow-up at the implant level [1]. Moreover, the preservation of the remaining teeth and the transmission of biomechanical stimuli to the alveolar bone, which prevent its resorption, are great advantages of this approach [2]. Recent estimates suggest that the demand for dental implant rehabilitation could reach up to 23% of the USA population in 2026 [3]. However, despite improvements in maintaining good oral health, peri-implant disease is common. According to the latest classification of periodontal and peri-implant diseases and conditions [4], two clinical entities are defined:

(1) Peri-implant mucositis, which is a reversible pathological condition diagnosed with the presence of bleeding and/or suppuration on gentle probing with or without increased probing depth compared to previous examinations;

(2) Peri-implantitis, which is a progression of the former, diagnosed by (i) the presence of bleeding and/or suppuration on gentle probing; (ii) increased probing depth compared to previous examinations; or (iii) the presence of bone loss beyond crestal bone level changes resulting from initial bone remodeling. In the absence of previous examinations, the following criteria are considered: (i) the presence of bleeding and/or oozing on gentle probing; (ii) probing depths ≥ 6 mm; and (iii) bone levels ≥ 3 mm apical to the most coronal part of the intraosseous part of the implant.

Peri-implant diseases, mainly plaque-related [4,5], include peri-implant mucositis and peri-implantitis, with mucositis showing a higher prevalence (19.4%–64.6%) compared to peri-implantitis (7.8%–45%) [6]. Mechanical debridement combined with good oral hygiene is the gold standard to manage mucositis and prevent its progression to peri-implantitis. Various adjunctive treatments exist, but their effectiveness remains uncertain due to limited strong evidence [7,8,9]. Home care using decontaminating treatments is important, and care must be taken to avoid damaging implant surfaces during treatment, as this could increase surface roughness and worsen the condition [10]. Considering these premises, the aim of this in vitro study was to compare the roughness of a pristine dental implant surface after several different current treatments/instrumentations for peri-implant mucositis and their element deposition, to evaluate the effects of the treatments on implant surfaces. The first null hypothesis was that no significant differences would be found in the roughness of dental implants’ surfaces analyzed through SEM instrumentation among the different treatments. The second null hypothesis was that no significant differences among the percentages of elements deposited on dental implant surfaces analyzed through EDS instrumentation would be found.

As it was demonstrated from previous studies that on rougher implant surfaces, biofilm formation is enhanced with respect to smoother ones [11,12], it is desirable that treatments do not have a roughening effect on surfaces. However, current evidence is insufficient to determine which method is suitable for implant decontamination, and recent studies on the topic highlight a great heterogeneity in materials tested and analyzed surfaces [13,14]; therefore, it is difficult to perform direct comparisons. Additionally, current strategies can be implemented in standard procedures for the management of peri-implant mucositis, which include the use of natural compounds [15] and, in the most complex cases, the use of antibiotics [16].

2. Materials and Methods

This was an in vitro study conducted at the University of Pavia.

2.1. Sample Size and Preparation of the Study Samples

Sample size calculation (alpha = 0.05; power = 80%) for two independent study groups and a continuous primary endpoint is performed concerning the variable roughness (R_a).

The following mathematical formula is used for sample size calculation:

$$\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{s}\mathrm{i}\mathrm{z}\mathrm{e}={\displaystyle \frac{2{\mathsf{\sigma }}^2{({\mathrm{z}}_{\mathsf{\alpha }/2}+{\mathrm{z}}_{\mathsf{\beta }})}^2}{{\mathsf{\Delta }}^2}}$$

where Δ is the effect size, σ² is the total variance of the 22 groups, α is the level of significance, and β is the power. Considering an expected difference between the means of 0.098 and a standard deviation of 0.05 from the previous literature [17], an effect size of 1.96 was obtained. With this data, 5 dental implants were required per group, and, therefore, a total of 110 dental implants was used for the study.

A total of 110 pristine dental implants with a diameter of ∅ 3.75 mm and length of 10 mm (Stone, IDI Evolution srl, Concorezzo, Italy) were used for microscopic analysis. The implants examined in this study were newly manufactured and processed in a laboratory environment; therefore, they were not placed in human subjects. The selected implants were characterized by a machined neck surface and a sand-blasted and acid-etched (SLA) thread surface. The study sample was divided into 22 groups of 5 implants each according to the decontaminating method used. Each dental implant was embedded in a resin block (Leocryl, Leone s.p.a., Sesto Fiorentino, Italy) of 2 × 2 cm². A trained operator used each instrument manually or with its specific handpiece for 60 s. The decontaminating methods tested are commonly used in clinical practice; the detailed list is shown in

Table 1. Subdivision of the groups for the microscopical analysis.

Treatment	Instrument	Use/Type of Handpiece	Macrogroup	Procedures
/	Control	No surface treatment	Control	No procedure
1.	Steel ultrasonic insert	EMS handpiece	Ultrasound	Elliptical movement of the insert between the neck and the implant screw for 60 s
2.	PEEK ultrasonic insert	EMS handpiece	Ultrasound	Elliptical movement of the insert between the neck and the implant screw for 60 s
3.	PEEK sonic insert	KAVO SonicFlex 2003 handpiece	Ultrasound	Elliptical movement of the insert between the neck and the implant screw for 60 s
4.	Steel sonic insert	KAVO SonicFlex 2003 handpiece	Ultrasound	Elliptical movement of the insert between the neck and the implant screw for 60 s
5.	PEEK ultrasonic insert	Mectron handpiece	Curette	Elliptical movement of the curette in a mesio-distal direction, placing the working part between the neck and the waist of the implant for 60 s
6.	Hy-Friedy Steel curette	Manual	Curette	Elliptical movement of the curette in a mesio-distal direction, placing the working part between the neck and the waist of the implant for 60 s
7.	Deppeler Titanium curette	Manual	Curette	Elliptical movement of the curette in a mesio-distal direction, placing the working part between the neck and the waist of the implant for 60 s
8.	KerrHawe Carbon fiber curette	Manual	Curette	Elliptical movement of the curette in a mesio-distal direction, placing the working part between the neck and the waist of the implant for 60 s
9.	Woodpecker insert	Woodpecker handpiece	Ultrasound	Elliptical movement of the insert between the neck and the implant screw for 60 s
10.	Sensitive Mectron Glycine powder (∅ 25 μm) with supragingival insert	Mectron handpiece	Powder	Position the air-polishing handpiece by tilting it at 45° between the neck and the waist of the implant, then spraying the powder for 60 s
11.	Sensitive Mectron Glycine powder (∅ 25 μm) with subgingival insert	Mectron handpiece	Powder	Position the air-polishing handpiece by tilting it at 45° between the neck and the waist of the implant, then spraying the powder for 60 s
12.	Glycine powder with Woodpecker supragingival insert	Periomate NSK handpiece	Powder	Position the air-polishing handpiece by tilting it at 45° between the neck and the waist of the implant, then spraying the powder for 60 s
13.	Glycine powder with Woodpecker subgingival insert	Periomate NSK handpiece	Powder	Position the air-polishing handpiece by tilting it at 45° between the neck and the waist of the implant, then spraying the powder for 60 s
14.	Erythritol powder with supragingival insert	EMS handpiece	Powder	Position the air-polishing handpiece by tilting it at 45° between the neck and the waist of the implant, then spraying the powder for 60 s
15.	Erythritol powder with subgingival insert	EMS handpiece	Powder	Position the air-polishing handpiece by tilting it at 45° between the neck and the waist of the implant, then spraying the powder for 60 s
16.	Flash pearls NSK (CaCO3 ∅ 53 μm) with Woodpecker supragingival insert	Periomate NSK handpiece	Powder	Position the air-polishing handpiece by tilting it at 45° between the neck and the waist of the implant, then spraying the powder for 60 s
17.	Kerr Cleanic with perlite (RDA = 27)	Kavo Smartmatic handpiece with Pro-cup insert	Powder	Position the air-polishing handpiece by tilting it at 45° between the neck and the waist of the implant, then spraying the powder for 60 s
18.	Prophylaxis toothbrush (7000 rpm)	Green contra-angle handpiece	Brush	Apply the toothbrush with nylon filaments and prophylaxis paste, on a green handle, placing it half on the neck and half on the screw for 60 s at moderate speed
19.	iDentoflex P13 Silicon pumice cup	Green contra-angle handpiece	Brush	Apply the toothbrush with nylon filaments and prophylaxis paste, on a green handle, placing it half on the neck and half on the screw for 60 s at moderate speed
20.	iDentoflex P13 Silicon cup brown	Green contra-angle handpiece	Brush	Apply the toothbrush with nylon filaments and prophylaxis paste, on a green handle, placing it half on the neck and half on the screw for 60 s at moderate speed
21.	Hybenx gel EPIEN Medical (sulfonate phenolic compounds and sulfuric acid in aqueous solution)	Syringe with 27 G needle	Gel	Activate and apply the pre-dosed gel to the surface of the implant between the neck and the screw; after 60 s, remove it

Manufacturers: EMS Electro Medical Systems S.A., Nyon, Switzerland; KaVo Dental GmbH, Biberach, Germany; Mectron S.p.A., Carasco, Italy; Hu-Friedy Manufacturing Co., LLC, Chicago, IL, USA; Deppeler SA, Rolle, Switzerland; Kerr Italia S.r.l., Scafati, Italy; Guilin Woodpecker Medical Instrument Co., Ltd., Guilin, China; NSK Italia S.p.A., Garbagnate Milanese, Italy; Identoflex AG, Buchs, Switzerland; EPIEN Medical, Inc., St. Louis Park, MO, USA.

.

2.2. Microscopic Analysis

Each dental implant underwent SEM analysis (Phenom XL G2 Desktop SEM, Thermo Fisher Scientific, Waltham, MA, USA) [18]. A four-segment backscattered electron detector (BSD) was used to differentiate between different phases, providing imaging that carried information on the sample’s composition. The settings were selected to analyze 1 mm² with a sufficient resolution. For each dental implant, 5 images were taken on random surfaces of the three parts of the dental implant [19]. For the EDS analysis, a silicon drift detector (SDD) thermoelectrically cooled with a 25 mm² active area was used. The same settings were selected. An ultra-thin silicon nitride (Si₃N₄) window allowed the detection of elements B to Am. The integrated ProSuite software (v 1.0) was used for data analysis. The following specifications were selected to analyze a standardized field considering the object morphology: magnification (Mag.) 500X, field width (FW) 1.0 mm, high voltage (HV) 5 kV, working distance (WD) 6–8 mm, pressure (Press) 0.10 Pa.

Figure 1 presents the sound surface of the dental implants. For the purposes of the present study, images were taken only on the neck of the implant (parts a and b of Figure 1).

2.3. Roughness (R_a)

Using a 3D Laserscanner (LAS-20, SD Mechatronik, Feldkirchen-Westerham, Germany), roughness (R_a) values from the neck of the dental implants were recorded for the same 5 images of EDS analysis [18].

2.4. Statistical Analysis

Data were collected on Excel electronic spreadsheet and then analyzed with R software (version 3.1.3, R Development Core Team, R Foundation for Statistical Computing, Wien, Austria). Descriptive statistics were calculated (mean, standard error of mean, minimum, median, maximum) for each variable.

As inferential statistics, data normality of distributions was assessed with the Kolmogorov–Smirnov test. As data were not normally distributed, Kruskal–Wallis followed by Dunn’s post hoc tests were performed for the multiple comparisons of the 22 groups, for the pooled groups and for head versus neck comparisons. Linear regressions were calculated assessing the influence of each implant, zone (head or neck), instrument (each of the treatments administered) and technique (gathering all the instruments into six macrogroups: ultrasounds, powders, curettes, brushes, gels, control) on the roughness. Significance threshold of p < 0.05 was assumed.

3. Results

3.1. Roughness Analysis

The results of the roughness analysis of samples are shown in

Table 2. Descriptive statistics of the results of roughness analysis R_a (μm) on the threads and necks of the implants tested for each of the treatments presented in Table 1 and subdivided per zone (head and neck).

	Zone	Mean	Standard Error	Minimum	Median	Maximum
Control	Neck	0.06	0.00	0.06	0.06	0.06
Control	Thread	0.04	0.00	0.04	0.04	0.05
Treatment 1	Neck	0.04	0.00	0.04	0.04	0.05
Treatment 1	Thread	0.07	0.00	0.07	0.07	0.07
Treatment 2	Neck	0.03	0.00	0.03	0.03	0.03
Treatment 2	Thread	0.09	0.00	0.09	0.09	0.09
Treatment 3	Neck	0.03	0.00	0.02	0.03	0.03
Treatment 3	Thread	0.23	0.00	0.08	0.08	0.80
Treatment 4	Neck	0.03	0.00	0.02	0.03	0.03
Treatment 4	Thread	0.04	0.00	0.03	0.04	0.04
Treatment 5	Neck	0.04	0.00	0.04	0.04	0.04
Treatment 5	Thread	0.08	0.00	0.07	0.08	0.08
Treatment 6	Neck	0.04	0.00	0.03	0.04	0.04
Treatment 6	Thread	0.07	0.00	0.07	0.08	0.08
Treatment 7	Neck	0.11	0.00	0.04	0.04	0.40
Treatment 7	Thread	0.08	0.00	0.07	0.08	0.08
Treatment 8	Neck	0.04	0.00	0.04	0.04	0.04
Treatment 8	Thread	0.36	0.00	0.08	0.08	0.79
Treatment 9	Neck	0.05	0.00	0.04	0.05	0.05
Treatment 9	Thread	0.08	0.00	0.08	0.08	0.08
Treatment 10	Neck	0.04	0.00	0.04	0.04	0.04
Treatment 10	Thread	0.07	0.00	0.06	0.07	0.07
Treatment 11	Neck	0.03	0.00	0.03	0.03	0.04
Treatment 11	Thread	0.04	0.00	0.03	0.04	0.04
Treatment 12	Neck	0.03	0.00	0.03	0.03	0.04
Treatment 12	Thread	0.08	0.00	0.08	0.08	0.08
Treatment 13	Neck	0.03	0.00	0.03	0.03	0.03
Treatment 13	Thread	0.07	0.00	0.07	0.07	0.07
Treatment 14	Neck	0.03	0.00	0.02	0.03	0.03
Treatment 14	Thread	0.09	0.00	0.09	0.09	0.10
Treatment 15	Neck	0.03	0.00	0.02	0.03	0.03
Treatment 15	Thread	0.05	0.00	0.05	0.05	0.05
Treatment 16	Neck	0.03	0.00	0.03	0.03	0.04
Treatment 16	Thread	0.07	0.00	0.07	0.07	0.07
Treatment 17	Neck	0.04	0.00	0.04	0.04	0.05
Treatment 17	Thread	0.01	0.00	0.01	0.01	0.02
Treatment 18	Neck	0.02	0.00	0.02	0.03	0.03
Treatment 18	Thread	0.02	0.00	0.02	0.02	0.02
Treatment 19	Neck	0.03	0.00	0.03	0.03	0.03
Treatment 19	Thread	0.08	0.00	0.08	0.08	0.08
Treatment 20	Neck	0.03	0.00	0.02	0.02	0.03
Treatment 20	Thread	0.08	0.00	0.08	0.08	0.08
Treatment 21	Neck	0.06	0.00	0.05	0.06	0.06
Treatment 21	Thread	0.08	0.00	0.08	0.08	0.08

, while significant intergroup differences are shown in Figure 2, distinguished into neck and thread measurements. In Figure 3, surface roughness modifications are shown.

Regarding the pooled analysis with subgroups, it was found that powders exhibited significantly lower roughness values in respect to the gel (p < 0.05). No other significant differences were found (p > 0.05) (

Table 3. Descriptive statistics of roughness analysis R_a (μm) of the pooled groups of treatments.

	Mean	Standard Error	Minimum	Median	Maximum
Control	0.04374	0.00218	0.0409	0.04329	0.04633
Ultrasound	0.04668	0.00294	0.0235	0.03919	0.0777
Powders	0.04406	0.00254	0.0143	0.03367	0.09632
Curettes	0.04833	0.00355	0.03104	0.03581	0.07861
Brushes	0.05974	0.00450	0.02367	0.07622	0.07892
Gel	0.07722	0.00353	0.07562	0.0775	0.07891

and Figure 4).

A significant difference between the neck and thread zone was found for roughness values (p < 0.05) (

Table 4. Descriptive statistics of roughness analysis R_a (μm) pooled as “neck” and “thread” measures.

	Mean	Standard Error	Minimum	Median	Maximum
Neck	0.035972	000087	0.02277	0.033995	0.05841
Thread	0.065119	0.00204	0.0143	0.075255	0.09632

and Figure 5).

In

Table 5. Linear regressions of this study. *: p < 0.05.

	Dependent Variable (p Value)
Independent Variable	Roughness
Implant	0.119
Instrument	0.119
Technique	0.901
Zone	<0.001 *

, the results of linear regressions are shown.

3.2. EDS Analysis

The results of EDS analysis showed a higher deposition of the following elements: carbon in treatments 8, 12, and 19; and calcium in treatment 13 (

Table 6. Results of EDS analysis with percentages of deposition of the elements found. Treatments correspond to Table 1.

		Titanium		Carbon		Calcium
Group	Macrogroup	Mean	Standard Error	Mean	Standard Error	Mean	Standard Error
Control	Control	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 1	Ultrasound	97.38	0.60	0.00	0.00	0.00	0.00
Treatment 2	Ultrasound	90.14	4.88	8.52	5.16	0.00	0.00
Treatment 3	Ultrasound	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 4	Ultrasound	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 5	Curette	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 6	Curette	89.49	7.08	10.51	7.08	0.00	0.00
Treatment 7	Curette	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 8	Curette	54.32	8.29	45.82	8.28	0.00	0.00
Treatment 9	Ultrasound	97.36	1.58	2.64	1.58	0.00	0.00
Treatment 10	Powder	84.95	2.25	0.00	0.00	0.00	0.00
Treatment 11	Powder	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 12	Powder	62.85	8.23	37.15	8.23	0.00	0.00
Treatment 13	Powder	70.72	5.12	0.00	0.00	29.28	5.12
Treatment 14	Powder	94.10	1.25	5.90	1.25	0.00	0.00
Treatment 15	Powder	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 16	Powder	87.05	6.32	12.95	6.32	0.00	0.00
Treatment 17	Powder	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 18	Brush	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 19	Brush	80.72	3.91	19.28	3.91	0.00	0.00
Treatment 20	Brush	100.00	0.00	0.00	0.00	0.00	0.00
Treatment 21	Gel	100.00	0.00	0.00	0.00	0.00	0.00

), probably as a result of remnants of the decontaminating procedures. No other elements were found. In Figure 6 all the spectra are shown, while in Figure 7 macroscopical SEM images are shown.

4. Discussion

The first null hypothesis of this study was rejected, as significant differences among the treatments were found. Despite this, all the decontaminating methods did not macroscopically affect implant surfaces. This finding suggests that several debridement techniques may be considered safe for preserving surface topography during peri-implantitis therapy.

Differences were found between neck and thread roughness measurements and were independent of the treatments tested, reflecting the original implant design. The smoother machined collar aims to reduce plaque accumulation in the coronal portion, although its actual clinical benefit remains debated [19,20,21]. Preserving this area is critical since soft tissue inflammation and plaque deposition in the coronal portion are major contributors to peri-implantitis progression.

The present findings align with previous in vitro studies reporting that air abrasive systems, ultrasonic scalers with non-metal tips, and rotating titanium brushes can effectively remove biofilm while maintaining surface integrity [22,23]. Nevertheless, the current evidence remains inconclusive regarding the superiority of any specific protocol [23,24]. Therefore, evaluating surface variations after decontamination remains a crucial step in selecting clinically applicable methods.

Although some studies proposed the use of titanium disks for in vitro models [25], the use of commercially available implants is preferred for studying the implant surface [26]. SEM, EDS, and profile analyses are currently recommended for the analysis of the treated titanium surfaces [26]. The definition of the surface roughness and elements’ deposition is significantly related to the titanium surface variations caused by the tested disinfection protocol [27]. Laboratory studies mimicking a clinical scenario to analyze the potential damage to the implant surface are essential in the early testing stages [28].

A recent systematic review reported that the clinical efficacy of photo/mechanical and physical implant surface decontamination in conjunction with surgical peri-implantitis therapy is inconclusive [23], in accordance with the present study, in which several decontamination protocols did not cause macroscopic alterations of the implant surface.

In contrast, Kotsakis et al. demonstrated that mechanical treatment of titanium implant surfaces using a cleaning instrument of similar hardness, such as a titanium brush, resulted in surface alterations and increased titanium dissolution [29]. However, these surface changes were detectable only through atomic force microscopy (AFM), which revealed the generation of titanium wear microparticles as a direct effect of cleaning with rotary titanium brushes [29]. These differences may be attributed to the greater three-dimensional resolution of AFM compared to SEM [30] and also to operator variability.

In the present study, the SEM analysis and the profilometer exhibited lower roughness values for the implants’ necks. This is due to the implant manufacturing, even if the possibility that a smooth neck surface could minimize plaque formation is still controversial [31]. However, the post-treatment analysis showed lower roughness for powders in respect to the gel. These findings may be related to the hygroscopic effect of the Hybenx gel, which dehydrates the biofilm surface so that it detaches completely or partially from the implant surface [32].

The titanium surface component elements showed higher carbon deposition in groups 8, 12, and 19, which is related to the possible higher presence of remnants after decontamination [29]. Furthermore, sodium and calcium elements were detected in groups 12 and 13, respectively, probably due to the presence of small residues of different air-polishing powders with different compositions [33]. In the present study, oxygen was excluded from EDS analysis, so Ti dissolution could not be evaluated. Recently, the analysis of the implant surface composition has taken into account Ti dissolution by light microscopy and inductively coupled mass spectrometry (ICP-MS). These analyses could be crucial to assess the presence of titanium dissolution products (TiDissPs) in peri-implant tissues and plaque biofilms, which could be related to tissue inflammation [34,35,36]. Therefore, prospective longitudinal clinical studies evaluating Ti dissolution in vivo are crucial to advance the management of peri-implant complications [37,38]. Considering the limited number of specimens, intraoperator reliability was not assessed as this would have required additional implants.

The first limitation of this study is that it is an in vitro study. Second, it would have been interesting to evaluate the efficacy in biofilm removal of removed dental implants, and further investigations are needed in this way. In this context, the previous literature evaluated chlorhexidine, hydrogen peroxide, and iodine compounds [39] and chlorhexidine, povidone-iodine, and chlorine dioxide [40] as chemical agents for titanium decontamination, showing different changes in surface properties. Within the limitations of this study, the different decontamination procedures, apart from their plaque removal efficacy, did not seem to significantly affect implant surface roughness or chemical composition [41,42]. These results should be considered with caution as they are not associated with Ti dissolution or biocompatibility data. In addition, further in vitro and clinical evaluations should be conducted with implants from other manufacturers.

5. Conclusions

Although there were various significant differences among the groups, roughness values were low, and none of the decontaminating methods macroscopically affected implant surfaces. Therefore, the proposed treatments can be used safely for implant decontamination. Notable differences were observed between the neck and thread areas of the implants, regardless of the treatment used. EDS analysis revealed carbon and calcium deposition as remnants of the treatments.