Water Plasma Treatment Method: Simultaneous Sterilization and Surface Modification of Titanium-Based Implants

Abstract

This study aims the applicability and efficacy of a new DC water plasma method at low temperature, for the sterilization of titanium contaminated samples and its effects on the surface oxide layer and morphological structure. The plasma treatment was carried out at a temperature of 60°C, for a predefined time of 10 minutes. Water vapor was generated from distilled water and polarized at -700 V during plasma-on period. Elemental analysis revealed that Ti surfaces showed a complete absence of organic and inorganic molecules (0% at detected /0.1% sensitivity) and complete bacteria elimination. Additionally, the oxygen content remained around 8% indicating a positive outcome for bioactivity titanium surface due to oxide presence. Initial results support that the water plasma system enables effective elimination of surface microorganisms while enhancing the natural oxide layer make up of titanium using a low temperature and water-based sterilization system that can be envisioned for clinical use.

Keywords:
titanium; sterilization; water plasma; surface treatment

1. Introduction

Chronic infections are among the main problems in the post-operative period, due to the proliferation of microorganisms, especially bacteria present on the surface of metallic implants. To reduce the occurrence of these infections, an efficient sterilization process, measured by the Sterility Assurance Level (SAL), must be applied. The minimum required SAL, for use in implant surgeries is 10^-6 as suggested by Solon and Kileen¹-³.

Among the most employed methods in the clinical-hospital setting, autoclave and ultraviolet radiation (UV) methods stand out¹-³. Although both have proven sterilization efficacy, some negative aspects, limiting their application, must be highlighted. With autoclave sterilization, microorganism inactivation is achieved at high temperature: 121°C for 30 min in a gravity displacement autoclave or 132°C for 4 min in a pre-vacuum autoclave⁴, which limits its use, for example, in temperature-sensitive biomaterials like polymethylmethacrylate (PMMA)⁵. With ultraviolet radiation technique, bacterial inactivation is obtained by exposure to light with a wavelength between 100 and 200 nm, which induces the thymine dimers formation in the DNA double strand, inhibiting microbial replication⁶. However, the effectiveness of ultraviolet radiation depends on factors such as the presence of organic matter, the type of microorganism and the radiation intensity. Furthermore, UV radiation causes photooxidative aging, which results in the breakage of polymer chains, producing free radicals and reducing the molecular weight of polymers⁷. For both techniques, eventual residues, such as those from dead bacteria, are not removed from the sterilized surface.

Alternative techniques introduced in the field of hard surface medical instruments and implant sterilization include dry heat, ethylene oxide gas, hydrogen peroxide plasma, glutaraldehyde-based formulations, hydrogen peroxide and peracetic acid solutions⁴.

Cold plasma can be classified as a potential alternative technique. It consists of partially ionized gases containing high energy electrons and ions, reactive radicals, and neutral species, also producing photons in the UV and VUV wavelengths. Being a non-equilibrium medium, it presents species with high chemical reactivity (radicals and ions)⁸ even at low temperature⁹. Such a medium enables the occurrence of chemical reactions that in conventional processes would only occur at high temperatures or using chemical products that have a risk to human health and to the environment¹⁰. In this case, the environment is active only when the plasma is on, and usually no dangerous residual gas is formed. Since we are treating metallic samples, we used a Pulsed DC plasma generating a reactive environment and physical effect of fast species (mainly ions) bombarding the surface.

Besides providing better biocompatibility, the cold plasma technique has gained significant relevance in surface decontamination procedures, as it has significant advantages over other methods, such as low environmental impact and non-toxicity due to the possibility of using non-harmful and non-toxic gases¹¹-¹³.

Currently, plasma sterilization studies are focused on the use of hydrogen peroxide gas¹⁴. These previous studies highlight the mechanism of radical release with oxygen that inactivate microorganisms by attacking their cell wall and internal membrane, which damages the DNA through oxidation, an essential mechanism for sterilization¹⁵.

In the field of biomaterials, plasma techniques are also currently used for surface treatment, generally aiming to modify the surface morphology and/or chemical composition to increase the bioactive response of these materials¹⁶. Chen et al.¹⁷ in vivo osseointegration study found that the use of O₂ plasma in titanium implants promoted an increase of up to 37% in the contact area between bone and implant. Chou et al.¹⁸ reported that after surface functionalization by oxygen plasma, the presence of hydroxyl groups (OH^-) on titanium surfaces had positive effects, with a more hydrophilic surface that enhancing cell adhesion, and the Ti-OH presence showed a greater effect on bone regeneration.

Another important aspect of this technology, which is further explored in this study, is the oxygen presence in the H₂O plasma atmosphere. Thus, the water plasma environment is expected to promote oxide formation on the titanium surface¹⁹, which may contribute to oxide layer growth and better surface bioactivity²⁰. Consequently, the explored technique in this study is expected to promote dual function: (1) surface sterilization and (2) surface treatment by enhancing titanium oxide layer growth. Moreover, plasma-based processes allow for fine control of parameters (peak voltage, time, gas composition), which can interfere in the type and concentration of generated reactive species, thus being a versatile and highly accurate process²¹. Among the most important treatment parameters are the gas mixture and pressure, which defines particles nature and density, and consequently, the particle collision probability and its frequency⁹.

The most applied gas with cold plasma technique is hydrogen peroxide (H₂O₂)²² at a temperature below 60°C, which has proven efficacy for application in plasma-assisted sterilization²³. In cold plasmas containing oxygen and hydrogen, the presence of neutral species, ions, photons, and reactive oxygen species, as well as the hydroxyl radical (OH) is expected. This radical, when in contact with living cells, can greatly damage enzymes, nucleic acids, and other proteins essential to the metabolism of living beings, thus causing their inactivation or destruction. With microwave plasmas, there is indication that sterilization occurs mainly through chemical erosion, also known as plasma etching²⁴. Another plasma decontamination factor is the generated electric field that, with reactive oxygen species, can damage the cell membrane of microorganisms by electrostatic forces. Additionally, the photons generated by plasma and the bombardment by energetic species can also contribute to sterilization. Thus, the development of new techniques is essential to ensure the required sterility standard in a wide variety of materials and application conditions. Aiming to contribute to this field, this work discusses the application of cold plasma technology as a viable and efficient alternative to the conventional sterilization procedures used in the clinical setting for the sterilization of dental instruments and dental implant components²³. This technology can be translated to a compact, self-containing and user-friendly system that is attractive for clinical use due to its low environmental impact, low temperature of operation and short treatment times, which may enable quick turn-around of instruments and implant components between patient procedures²³.

Particularly, in the case of titanium, water plasma treatment could have a positive impact on the biocompatibility²⁵,²⁶ of the implants in terms of the osseointegration process²⁷, due to the presence of an oxidizing medium²⁸, which would be an important gain, besides the sterilization process. To the best of our knowledge, no studies have explored before the use of water plasma for titanium-based components, particularly exploring the impact of this technique on surface decontamination and surface modification.

In summary, this work aims to use water plasma at a temperature of 60°C and treatment time of 10 minutes to evaluate sterility and surface morphology of titanium-plate samples post-contamination in vitro with Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium smegmatis. The decontamination by water plasma was evaluated by the detection of organic and inorganic marker elements deposited on the surfaces post plasma treatment. Water plasma sterilization process efficiency was analyzed by the Colony Forming Unit-CFU parameter on Ti surfaces covered with the three different bacteria. Additionally, samples were morphologically and chemically characterized using SEM and EDS techniques to survey the impact of the water plasma environment on surface morphology and composition.

2. Material and Methods

2.1. Material

Rectangular samples of grade 2 titanium (Titanium do Brasil®), 10 mm x 20 mm x 1 mm, were used. The titanium chemical composition (%wt.) was N (0.03), C (0.08), H (0.015), Fe (0.3), O (0.25) and Ti (bal) (Titanium do Brasil® technical report). The surfaces of the samples were prepared by sanding with 200, 400 and 600 grit sandpaper (3M) followed by cleaning with acetone, alcohol, and distilled water, in an ultrasonic bath for 10 minutes in each medium. Samples were dried and stored in a vacuum system.

2.2. Water plasma treatment

The plasma treatment was carried out using water plasma chamber at a temperature of 60°C (333 K), for a predefined time of 10 minutes (600 s), at a pressure of 66.7 Pa (0.5 Torr) and a peak voltage of -700 V. The temperature was controlled by the power supply pulse time, and the frequency was fixed at 4.2 kHz (pulse period of 240 μs). The choice of these parameters focused on easy temperature control at 60°C and to avoid severe surface modifications. Before carrying out the treatment, the system was evacuated using a primary vacuum system until reaching the system vacuum limit of approximately 6.7 Pa (50 mTorr), then the water line was opened, and the was set to 66.7 Pa and the plasma power supply was turned on. Water vapor was generated from distilled water. The samples were positioned on the cathode of the discharge, polarized at -700 V during plasma-on period. Figure 1 shows a schematic diagram of the plasma sterilization/treatment system.

Figure 1
Schematic diagram of the plasma sterilization/treatment system.

2.3. Surface preparation-contamination with inorganic element marker

For the analysis of removal of plasma surface inorganic contaminants, a fluorine (F) containing inorganic marker was used, since F is easy to detect on metallic surfaces by chemical analysis methods. The marker compound deposition was performed by Plasma Etch Model PM313 (Plasma System-USA), using sulfur hexafluoride gas (SF₆) at 133 Pa (1 Torr), a flow of 0.33 10^-6 m³/s (20 cm³/min) at a power of 50 W for 120 s (2 minutes) at room temperature.

2.4. Surface preparation-contamination with organic molecules markers

For the organic markers removal study, a pectin aqueous solution [(C₆H₄O₆)n: anionic molecule] in 0.1% concentration and chitosan aqueous acid solution (HCl 0.05 mol/L) [(C₆H₁₁NO₄)n: cationic molecule] in 0.2% concentration were used. The deposition was performed by the spin-coating method (Brewer equipment, Roller MO 65401, model 200) with a volume of 200 µl at a speed of 1,000 rpm for 5 seconds.

2.5. Surface preparation-contamination with bacteria

Titanium samples were contaminated by Mycobacterium smegmatis ATCC 607 (acid-alcohol fast resistant bacillus), Staphylococcus aureus ATCC 6538 (Gram-positive) and Pseudomonas aeruginosa ATCC 27853 (Gram-negative) bacteria. The strains initially stored at -80°C in brain heart infusion broth (BHIB) with 20% glycerol were reactivated in brain heart infusion agar (BHIA) for 24 h (S. aureus and P. aeruginosa) or BHIB for 48 h under agitation (M. smegmatis), both at 36 ± 1°C²⁹. From BHIA (S. aureus and P. aeruginosa) and BHIB (M. smegmatis), bacterial suspensions were prepared in saline solution (0.85%) with turbidity equivalent to the 0.5 McFarland scale (~1.0 x 10⁸ CFU/ml). Subsequently, these suspensions were individually inoculated into flasks containing a previously sterilized titanium plate and 10 ml of BHIB, resulting in a final concentration of 1.0 x 10⁵ CFU/ml³⁰. These flasks were incubated under agitation (35 Hz) at 36 ± 1°C for 24 h (S. aureus and P. aeruginosa) or 48 h (M. smegmatis). Then, the titanium samples were removed from the flasks, immersed 3 times in sterile saline to remove non-adherent bacteria and dried in an incubator at 36°C. Half of the samples were sent to the water plasma treatment, while the other half remained stored as control samples. Both groups of Ti-grade 2 samples, one by one, were immersed in flasks containing saline solution and sonicated for 15 min to release the eventually adhered and viable bacteria³¹. From this solution, serial dilutions were performed and 500 µl aliquots were seeded, in duplicate, on BHIA (S. aureus and M. smegmatis) or cetrimide agar, for bacterial counting. Samples in which dilution allowed the counting of 30 to 300 colonies of the target bacteria were selected, making the experiment statistically reliable³². In parallel, titanium samples that underwent water plasma treatment were removed from the saline solution and inoculated in sterile BHIB for 14 days at 36°C, without agitation. The presence of turbidity would denote bacterial growth, showing the permanence of residual viable bacteria on the titanium samples, even after sonication.

2.6. Surface characterization

Scanning Electron Microscopy (SEM) was used for morphology analysis and Energy-Dispersive X-ray Spectroscopy (EDS) for detection of chemical elements present on the Ti surface. These analyzes were performed with a TESCAN VEGA 3 LMU (20 kV for the markers and 15 kV for the assay with bacteria) to study the presence or absence of markers/bacteria on the titanium surface. All analyzes were carried out in triplicate and the results are presented with the appropriate mean values. For the analysis of surfaces containing bacteria, before and after treatment with water plasma for 10 minutes at 60°C, images were also obtained by scanning electron microscopy (SEM). A routine protocol was used for sample surface preparation³³: the surfaces were fixed in 2.5% glutaraldehyde (0.1 mol/L cacodylate buffer, pH 7.2) and post-fixed for 30 minutes in 1% OsO₄ in the absence of light and at room temperature. After washing in cacodylate buffer, bacteria were dehydrated using increasing concentrations of ethanol, to critical point CO₂, then coated with gold and analyzed using a JEOL JSM-6360 LV scanning microscope.

3. Results and Discussion

3.1. Evaluation of Ti surface fluorine markers decontamination by water plasma treatment

The surface morphology post-fluorine deposition was studied with SEM images as shown in Figure 2. The panel shows the typical morphology of titanium samples covered with oxide, where the presence of a thin film covering the grooves resulting from the sanding process with an undefined morphology is observed. It is possible to observe the typical titanium oxide morphology. In addition, the presence of discontinuities (as indicated by red circles in Figure 2) can be caused by sandpaper grooves with the presence of titanium oxide¹⁹. The sample surface subjected to plasma treatment (Figure 2c) displayed maintenance of irregularities when compared to the control sample. Thus, it can be concluded that water plasma treatment did not incur detectable changes on surface features.

Figure 2
SEM images of Ti-grade 2 surfaces: control (a), with fluorine deposition (b), and after H₂O plasma treatment at a temperature of 60°C for 10 min (c).

Energy-dispersive X-ray spectroscopy (EDS) was used for analyzing the presence of fluorine marker before and after water plasma treatments (Table 1). In this case EDS is used for comparison purposes, looking for the content variation of elements, the absolute values are not reliable.

Table 1
EDS analysis: mass percentages (%wt.) of titanium, oxygen, carbon, and fluorine elements present on the sample surfaces before and after treatment with water plasma at 60°C, for 10 minutes. *Triplicate samples. Data are presented with the mean values.

Elements	Control (%wt.)	After Fluorine deposition (%wt.)	After H2O plasma treatment 60°C (%wt.)
				Ti	95.3	70.5	90.3
O	3.3	9.7	7.9
C	1.4	17.7	0.9
F	0.0	1.4	0.0

• Maximum standard deviation: 0.3. EDS sensitivity: 0.1%.

Table 1 shows that fluorine was present at 1.4% (wt.) in fluoridated surfaces. And surfaces submitted to water plasma treatment showed a complete absence of fluorine (0% for a given sensitivity of 0.1%); confirmed the plasma treatment succeeded in removing the marker element.

Evaluating oxygen content, control samples demonstrated oxygen percentage of about 3.3% (wt%). On the other hand, water plasma treated specimens showed an increased oxygen percentage of 8.0%. The typical titanium morphology, with a characteristic oxide layer which naturally grows on Ti surfaces¹⁹,²⁶-²⁸,³⁴, was detected. These characteristics were present on control surfaces, fluorine deposited samples, and after water plasma treatment, showing that fluorine deposition did not influence the surface morphology and that the oxide titanium morphology was maintained¹⁹,³⁴. The increase in oxygen percentage on the surface of Ti grade 2 was identified post plasma treatment. Specifically, it was possible to infer the presence of titanium dioxide on the Ti surface, which is an important observation given that this oxide is proven to be thermodynamically stable³⁵ after treatment with water plasma. Thus, this oxide layer³⁶ is expected to provide stability and osseointegration response to the titanium surfaces¹⁹,²⁰,²⁵-²⁷,³⁴,³⁷,³⁸. Besides that, the water plasma treatment proved to be efficient in cleaning surfaces showing a complete absence of fluorine contaminant. Different gases can be used in these decontamination procedures, however, gases such water that contain oxygen tend to be more effective due to its chemical action, linked to the production of O, OH and OOH radicals¹². The identification of titanium and oxygen on surfaces corroborate with the presence of TiO₂ layer, inherent to this metal spontaneous oxidation process¹⁹,³⁴. Titanium oxide on the metal surface could also improve the titanium-based implant osseointegration. This fact could be conferring a double function arising from the water plasma treatment: (1) sterilization and (2) formation of titanium oxide layer on the surface³⁸, which may provide a better bioactive interface at the bone-implant interface. Finally, the water plasma treatment could positively affect the chemical composition of the titanium surface, conferring a hydroxyl content, improving the hydrophilicity of the surface, which could lead to better biocompatibility. These are possible outcomes of the water plasma treatment, which will be further investigated in future studies.

3.2 .Evaluation of pectin and chitosan decontamination on Ti surface by water plasma treatment

Figure 3 shows the SEM images of Ti-grade 2 surfaces, control specimens ((a)(d)) and surfaces coated with organic compounds: cationic, chitosan ((b)(e)) and anionic, pectin ((c)(f))before and after water plasma treatment (60^oC for 10 min), respectively.

Figure 3
SEM images of Ti-grade 2 surfaces: BEFORE water plasma treatment- control (a), with deposition of chitosan (b) and pectin (c). And AFTER water plasma treatment- control (d), with deposition of chitosan (e) and pectin (f).

Comparing the images before plasma treatment (Figure 3abc) a specific morphology can be observed, that is, the undefined morphology referring to the presence of titanium oxide. This film covers the grooves still detectable resulting from the surface preparation process. Figure 3def shows that there was no significant variation in morphology, in both organic coatings, after water plasma treatment.

EDS elemental analysis (Table 2) revealed the successful deposition of organic compounds. It can be observed that the titanium element is not even detected and then, after treatment by water plasma, organic molecule’s characteristic elements, such as carbon and nitrogen were absent. Again, in this case EDS is used for comparison purposes, looking for the content variation of elements, the absolute values are not reliable.

Table 2
Elemental percentages (wt%) by EDS technique of chitosan and pectin present on Ti surfaces before and after treatment with water plasma at 60°C, for 10 minutes. *Triplicate samples. Data are presented with the mean values.

Elements	Chitosan control (C6H11NO4)n (%wt.)	After H2O 60°C plasma treatment (%wt.)
Ti	0.0	93.1
C	17.5	---
N	29.1	---
O	53.3	6.9
Elements	Pectin control (C6H4O6)n (%wt.)	After H2O 60°C plasma treatment (%wt.)
Ti	0.0	87.2
C	40.6	---
N	---	---
O	59.4	10.2

• Maximum standard deviation: 0.3. EDS sensitivity: 0.1%.

These results indicate an increase in the titanium content, which was 0.0% on the surface when it was covered with both chitosan and pectin and then returned to values above 85% after treatment with water plasma. The percentage of the oxygen element after plasma treatment was approximately 10%, due to the typical presence of titanium oxides.

Following deposition of organic molecules on the surface of the titanium specimens, imperfections were less present on the samples, indicating the presence of a uniform surface layer of both chitosan and pectin. And even after water plasma treatment, the titanium oxide morphology predominated¹⁹,³⁴,³⁶. It was also observed that, after treatment with water plasma, total carbon and nitrogen removal from the titanium surface was achieved. This confirms the high efficiency of this plasma in the decontamination process. Certainly, the presence of oxygen after water plasma treatment maintains the titanium dioxide on the surface, providing positive consequences regarding the osseointegrative response of the metallic interface¹⁹,²⁰,²⁵-²⁷,³⁷. In a related way, by residual infectivity studies it was demonstrated the cold plasma efficiency in decontaminating bacteria and proteins on such delicate surfaces as flexible endoscopes, representing an improvement compared to the action of other sterilization forms³⁹.

3.3. Water plasma treatment for Ti surface sterilization process evaluation

The efficiency of water plasma treatment in microbial inactivation was studied with three (3) independent experiments, in quintuplicate, comparing the bacterial count on the titanium samples before and after treatment, and expressed as colony forming units (CFUs) per sample of the bacteria: M. smegmatis, P. aeruginosa and S. aureus.

The applied count technique had a detection limit of 1.30 in log₁₀, representing approximately 20 colonies, due to the applied dilution factor. Figure 4 presents the results of experiments carried out with control surfaces (contaminated) and surfaces treated with water plasma with the respective count of colony forming units for each bacterium. Figure 4 shows data for an average of three experiments (in log₁₀): 6.83 ± 0.18 for S. aureus, 6.51 ± 1.17 for P. aeruginosa, and 4.48 ± 0.39 for M. smegmatis, which demonstrate successful contamination with bacteria as pre-treatment. However, M. smegmatis adhered less than the other bacteria.

Figure 4
Effect of plasma-assisted sterilization treatment on samples contaminated with the three types of bacteria, results of counting colony forming units. ** p < 0.01, *** p < 0.001. The count data (control and treated) were analyzed using Student's T test, where a statistically significant difference was assumed for p < 0.05. ND – not detected.

Following water plasma treatment of bacteria contaminated titanium samples, no CFUs were detected in any of the samples tested considering the limit of detection in the order of 10⁵ for P. aeruginosa and S. aureus and 10³ for M. smegmatis. In addition, there was no colonies growth in the BHIB after incubation of the treated and sonicated samples in saline solution. These results are comparable to methods such as moist heat sterilization, considered the gold standard for the sterilization of metallic biomaterials⁴⁰, eliminating bacteria and other microbial forms, including (SAL 10^-6) bacterial spores⁴ or UV radiation, particularly UV-C, which on titanium surfaces reduces approximately 5.4 and 6.0 log₁₀ CFU/mL of Escherichia coli and P. aeruginosa, respectively⁴¹.

In addition to counting colony forming units (CFUs), scanning electron microscopy was employed to confirm the absence of microorganisms after the water plasma treatment. Figures 5 and 6 show Ti-grade 2 surface images before (Figure 5) and after water plasma treatment (Figure 6), for the three microorganisms studied.

Figure 5
SEM images of Ti-grade 2 surfaces coated with the microorganisms: Staphylococcus aureus (a) and (b), Pseudomonas aeruginosa (c) and (d), Mycobacterium smegmatis (e) and (f).

Figure 6
SEM images of Ti-grade 2 surfaces AFTER water plasma treatment at 60°C during 10 min: Staphylococcus aureus (a) and (b), Pseudomonas aeruginosa (c) and (d), Mycobacterium smegmatis (e) and (f).

Image analysis with different magnifications (Figure 5), revealed the success of the contamination protocol by bacterial adhesion on the surface of Ti-grade 2 with the three studied species. It was possible to observe the characteristic morphology of each bacterium, being the coccoid form for S. aureus (Figure ab) and the bacillary form for P. aeruginosa (Figure 5cd) and M. smegmatis (Figure 5ef), evidencing the integrity of the microorganism’s cell wall before water plasma treatment.

After water plasma treatment, there was complete absence of intact bacteria on the titanium surface as shown in Figure 6. It is important to note the presence of some residues, which may indicate the partial elimination of residual materials from the microorganism. This is more apparent on the surface that has been contaminated with Mycobacterium smegmatis Figure 6ef. In summary, the 10 minutes water plasma treatment was successful in eliminating the three bacterium species presented in this study, as confirmed by SEM images, and counting of colony forming units.

The bacteria, M. smegmatis, P. aeruginosa and S. aureus, were carefully chosen due to different susceptibilities to inactivation, M. smegmatis being the most resistant and P. aeruginosa being the least resistant⁴². This is mainly due to existing differences in the cell wall of each organism¹². The Mycobacterium cell wall is composed of four layers composed of peptidoglycan, arabinogalactan, mycolic acids, and external lipids, which gives this bacterium greater resistance to disinfection and sterilization methods⁴³. Gram-positive bacteria are characterized by a thick peptidoglycan cell wall, and Gram-negative bacteria have a thin cell wall, and an outer cell membrane composed of lipopolysaccharide (LPS)⁴⁴. Another reason for strategic choice of S. aureus and P. aeruginosa was the frequency of these bacterial contaminations in dental and orthopedic implants causing infections. S. aureus bacteria is the most associated with this specific infection type, and among the Gram-negative bacteria, P. aeruginosa is the most common⁴⁵. Although the Mycobacterium species is unlikely to cause infection in metallic implants, the M. smegmatis was chosen for this study because of the rare literature reports regarding the plasma effect on acid-alcohol fast bacteria. It was observed that the M. smegmatis adhered less than the other bacteria. This behavior was also observed by Ha et al.⁴⁶, where low adherence of M. tuberculosis to titanium surfaces was verified due to the hydrophobic nature of the cell wall. Moreover, there was the presence of some residues on the surface contaminated with Mycobacterium smegmatis, probably due to the characteristics of its highly resistant cell wall.

To obtain the sterilization of a critical medical material, a SAL of 10^-6 must be ensured. The results showed that none of the three bacteria studied was detected following water plasma treatment, which indicated successful bacterial elimination by the H₂O plasma technique at 60°C with a treatment time of only 10 minutes. One of the possible mechanisms of bacteria inactivation by water plasma is the cell wall erosion caused by a peroxidation of the lipid membrane, by the radicals present in the water plasma, which has high erosion power⁴⁷. This could be indicative of the damage caused by the plasma in the microorganism’s structure causing disorganization of cellular structures and intracellular content⁴⁸.

It has been reported that autoclaving of medical instruments and metallic components can be negatively impacted in terms of mechanical properties and corrosion resistance⁴⁹ and the UV radiation is one of the main causes of corrosion in medical implants⁴¹. Furthermore, high temperature and pressure conditions create limitations to sterilize components and implants that utilize thermosensitive materials. Cold plasma enables the production of active environments from non-reactive substances like water or even air, which could be efficiently applied for sterilization. Besides that, it is an environmentally friendly and compact technology for decontamination and sterilization of instruments and components in the dental office. Therefore, water plasma has been proven to be efficient for sterilization of contaminated titanium surfaces in tests carried out at 60^oC for 10 minutes, revealing no detectable bacteria or contaminants on surfaces pre-contaminated with P. aeruginosa, S. aureus or M. smegmatis. Although this study presented a viable and new technique for sterilization and preparation of titanium-based devices, and potentially other dental instruments and components, in the dental office, a few limitations of the study must be pointed out: 1. The study only investigated the effect of the water plasma treatment on the surface of titanium-based samples. It would be important including other metals that are currently used in the design of dental surgical instruments and other implant components. This will be investigated in a follow-up study. 2. The study used a relatively large plasma sterilization chamber. The miniaturization and design of a benchtop system is currently under development. 3. The study employed very specific microbial strains for contamination of testing samples.

4. Conclusion

This study showed that DC water plasma, generated from distilled water, with samples polarized at -700 V, was efficient to obtain total elimination (0% detected /0.1% sensitivity) of organic and inorganic contaminants, at 60°C in only 10 minutes of treatment. Water plasma has been proven to be efficient for sterilization of titanium surfaces pre-contaminated with different bacteria, where no CFUs was detected in any of the samples tested considering the limit of detection in the order of 10⁵ for P. aeruginosa and S. aureus and 10³ for M. smegmatis. Besides allowing for surface sterilization and decontamination, an oxygen concentration (%wt) of approximately 10% is maintained on the surface, indicating the presence of titanium oxide, even after water plasma sterilization, which can have potential implications to enhance surface-tissue interfacing. So, this plasma treatment might have a dual function due to an oxygen-rich atmosphere favoring the titanium oxide growth while effectively eliminating microorganisms. In summary, this technique can be envisioned as an alternative method to clean instruments and implants and can be potentially translated into a small, benchtop plasma system, for operation in the dental office.

5. Acknowledgments

The authors are grateful to the Electron Microscopy Center of UFPR (CME-UFPR) and laboratory infrastructures from C. Oliveira, L. Roman and F. Thomazi (UFPR). This study was supported by the CNPq - Brazil (grant numbers: C.E.B. Marino- 302144/2022-6; R.P. Cardoso 315887/2021-4), CAPES - Brazil (Finance code 001). G.L.Likes & R.L.Novak are grateful to CNPq and V. Franceschini to CAPES for the scholarships. D.C. Rodrigues research is supported by the National Institute of Dental and Craniofacial Research (NIH/NIDCR) Award Number R01DE026736.

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Supplementary material

The following online material is available for this article:

Figure S1

- EDS spectra of titanium, oxygen, carbon, and fluorine elements present on the sample surfaces before and after treatment with water plasma at 60°C, for 10 minutes.

Figure S2

- EDS spectra of chitosan and pectin present on Ti surfaces before and after treatment with water plasma at 60°C, for 10 minutes.

Publication Dates

• Publication in this collection
  28 Feb 2025
• Date of issue
  2025

History

• Received
  27 July 2024
• Reviewed
  20 Dec 2024
• Accepted
  28 Jan 2025