Pilot In Vitro Study to Assess Cleaning Ability and Effects of Different Decontamination Methods on Implant Surfaces

Summary

The present pilot in vitro study aimed to evaluate the cleaning efficacy of three different decontamination tools and possible surface alterations following treatment, using permanent red ink to mimic the presence of oral biofilm.

Abstract

An essential component of peri-implantitis treatment is the effective decontamination of implant surfaces. The ideal decontamination technique should safely and efficiently remove biofilm without damaging the implant surface. This pilot study aimed to evaluate the cleaning efficacy and surface alterations associated with three mechanical decontamination protocols. Here, 12 implants were stained with indelible red ink and mounted into acrylic blocks to simulate horizontal peri-implant defects. Surface decontamination was performed for 2 min by the same examiner using one of the following devices: titanium brush (TiB), chitosan brush (ChB), or titanium curette (TiC). No chemical decontamination agents were used in combination with the mechanical tools. Standardized photographs were taken before and after the decontamination from buccal and oral frontal views, as well as at 30° and 60° angulations. The uncleaned implant surface area was calculated digitally. Scanning electron microscopy (SEM) was used to assess surface morphology. None of the tested methods achieved complete removal of the ink stain. Although 75.98% ± 2.42% of the stain remained, TiB showed the highest cleaning efficacy at buccal and oral frontal views (p = 0.027), followed by TiC (80.3% ± 0.86% stain remaining) and ChB (90.34% ± 6.07% stain remaining). Significant differences were observed between the ChB and TiB groups (p = 0.022). SEM analysis revealed that the TiC caused the greatest surface damage, whereas the TiB produced minimal alterations. Within the limitations of this pilot study, TiB demonstrated effective cleaning while preserving implant surface morphology. These findings suggest that titanium brushes may represent a safer and more efficient mechanical decontamination option during peri-implantitis treatment. However, further studies are warranted to evaluate combinations of mechanical and chemical techniques to enhance cleaning efficacy.

Introduction

Peri-implantitis is defined as inflammation of the peri-implant mucosa accompanied by progressive bone loss. It is a biofilm-associated condition affecting the tissues surrounding dental implants¹. Patients with a history of severe periodontitis, poor oral hygiene, or lack of regular maintenance following implant therapy are at higher risk of developing peri-implantitis^1,2. The mean prevalence of peri-implantitis was reported by Diaz et al. to be 19.53% at the patient level, whereas it was 4.8%-23.0% at the implant level³, and this data was highly variable even following restrictions to the clinical case definition². Diabetes and periodontitis history are stated to be risk indicators for peri-implantitis. However, there is no consensus regarding the relationships between peri-implantitis and smoking, implant superstructure, and the amount of keratinized mucosa^2,3. Although there are currently no standardized, proven methods for the prevention of peri-implantitis, prophylaxis, adequate oral hygiene, and compliance with regular maintenance are thought to be the most crucial steps in doing so^3,4.

Given the complex histopathological features and unpredictable, rapid disease progression, peri-implantitis presents a challenge for every clinician^4,5. The primary goal of peri-implantitis treatment is to suppress inflammation and halt peri-implant bone loss. Clinically, the goal is to reduce probing depth (PD), eliminate bleeding on probing (BoP), and/or pus drainage⁶. An essential part of the peri-implantitis treatment is decontaminating the surfaces of the implants. The ideal decontamination technique should be safe to use without causing any injury to patients or implant damage, even though it should be effective at removing biofilm⁷. However, the implant's surface morphology and characteristics hinder ideal decontamination, limiting treatment success, since total access to affected sites is mostly limited by the implant surface characteristics^5,8. These situations necessitate the need for new materials or techniques for proper decontamination in the treatment of peri-implantitis.

Up to date, numerous implant surface decontamination agents/methods have been introduced, and their effectiveness and application modes are still under investigation⁹. Techniques used for peri-implant surface decontamination are generally categorized under mechanical/physical and chemical methods, and clear scientific evidence regarding the superiority of these techniques is not yet available^7,8. Results of a recent meta-analysis reported that titanium-coated curettes, specifically produced for implant surface debridement, do not cause scratches on the surface because they have a hardness similar to the implant surface and can be safely used for decontamination⁷. In the same meta-analysis, the use of rotating titanium brushes alone for implant surface decontamination has been shown to provide significant improvement in clinical periodontal parameters, such as reduction of PD and BoP. In a randomized clinical study comparing the biofilm removal effectiveness of steel and plastic curettes, ultrasonics, and titanium rotary brushes from implant surfaces, Toma et al. reported that rotary brushes were clinically more effective and preserved the surface morphology¹⁰. Titanium brush (TiB), consisting of nickel-titanium (NiTi) bristles, has a lower elastic modulus than that of the implant surface, giving the brush flexibility and reducing the risk of scratching the implant surface¹¹. Another promising decontamination agent is a non-toxic, biodegradable brush made from the natural polysaccharide chitosan, derived from chitin, valued for its biocompatibility, biodegradability, bacteriostatic, and anti-inflammatory properties. Chitosan was reported to exhibit bacteriostatic and anti-inflammatory properties and induce significant improvements in clinical parameters in the treatment of peri-implant diseases¹². Unlike metallic instruments, it is non-abrasive and intended to reduce bacterial load biologically and mechanically^5,12. However, there is still not any clear-cut consensus available on the ideal implant decontamination agent¹³, and more studies are needed to evaluate the effectiveness of these novel chitosan brushes during the decontamination phase of the peri-implant treatment.

Despite the extensive research on implant surface decontamination, identification of a decontamination method/agent that achieves optimal cleaning efficacy while preserving surface integrity still remains a significant clinical challenge. From this standpoint, the aim of the present study is to evaluate the cleaning ability and surface modification of three different mechanical decontamination protocols. The null hypothesis is that the TiB, chitosan brush (ChB), and titanium curette (TiC) have no significant difference in cleaning performance or surface modifications on implant surfaces after mechanical decontamination.

Protocol

NOTE: The procedures of this investigation were conducted in accordance with the recommended criteria for standardizing in vitro experiments¹⁴. Ethical approval was not necessary, as the study utilized commercially available implant surfaces. The study included implants with a diameter of 3.2 mm and a length of 10 mm, featuring a sandblasted, large-grit, acid-etched (SLA) surface. Implants with different dimensions and surface characteristics were excluded. Sample size calculation was performed according to Khalil et al.¹⁵. Effect size was taken as 0.90, alpha error as 0.05, and 95% statistical power yielded eight subjects for each group.

1. Preparation of study samples

1. Submerge implants in a recipient with red permanent ink fully covering the implant surface for 10 s and air-dry to achieve even distribution of the ink over the implant surfaces described by Sanz-Martin et al.¹⁶ with a driver handpiece.
2. Once dried for 48 h at room temperature¹⁷, confirm uniform red ink coverage without pooling after drying.
3. Insert the implants 5 mm into the acrylic blocks with dimensions of 20 x 20 x 20 mm manually using an implant driver to ensure controlled insertion and uniform positioning until the 5 mm depth mark on the implant body, mimicking Class I horizontal peri-implant defects¹⁸ (Figure 1A).
4. To maintain consistent angulation and vertical positioning, use a custom-made parallelometer guide to align the implant's long axis perpendicular (90°) to the acrylic block surface during placement.
   NOTE: Implant surfaces should be protected from hand contact and isolated with the driver handpieces during the drying process.

2. Creating the setup for the standardization of photographs

1. Set the camera parameters: ISO 100, aperture f/32, and exposure 1/80 s. In manual focus mode, use a macro lens with a 100 mm focal length.
2. Position the ring flash (dual-point macro flash, daylight-balanced 5500 K) at a 45° angle relative to the implant surface to ensure uniform illumination and minimize reflections. Use a diffuser to soften the light and reduce glare from the metallic surface.
3. Place the camera 15 cm away from the implant surface and take standardized photographs at a frontal view (0°) from the longitudinal implant axis at the buccal and oral surfaces of each implant (Figure 1B).
4. Verify the alignment of the camera with a calibration ruler before each imaging session.
5. Prepare a custom-made acrylic splint to position the camera with angulations of 30° (upper view) and 60° (lower view) to the implant long axis to assess the coronal and apical threads in a standardized manner (Figure 1C).

3. Application of treatments and obtaining photographs

1. Apply three different decontamination methods (see Table of Materials) to the exposed buccal and oral surfaces per implant (8 surfaces from 4 implants) commonly used in the treatment of peri-implantitis for 2 min by a single operator to eliminate bias (Figure 2). Control the instrumentation time with a stopwatch.
2. Perform all procedures under sterile saline irrigation only to eliminate potential confounding effects of chemical agents on cleaning efficacy or surface integrity.
   1. For TiC, apply type 11/12 mini Gracey curette with lateral pressure at a 60° angle to the surface.
   2. Apply ChB, soaked in sterile saline prior to treatment, placed in a low-speed handpiece set at 800 rpm, with a gentle probing movement in a circular fashion.
   3. Apply TiB, placed in a low-speed handpiece set at 800 rpm, with a gentle probing movement in a circular fashion.
3. Obtain post-treatment standardized photographs again, as described in step 2 (Figure 3).

4. Analysis of photographs

1. Once all the photographs are obtained, digitize and calibrate them with imaging software (ImageJ v 1.54f). Since the implants were embedded 5 mm vertically into the acrylic, input the remaining 5 mm part seen in the photographs, along with the standard 3.2 mm diameter, into the program by using the Straight-Line tool to ensure calibration.
2. Crop the photographs to include only the remaining 5 mm of the exposed implant surface , representing the region of interest (ROI) .
3. Process the images in red, green, and blue (RGB) color mode. Convert to color stacks by clicking Image > Type > RGB Stack to isolate the red-stained regions, representing residual ink.
4. Select the red channel. Apply a color threshold by clicking Image > Adjust > Color Threshold, using the Hue 0°-25° and 330°-360° range, Saturation 50-255, and Brightness 30-255. Enable the Dark Background option to ensure accurate detection of the stain.
5. Delineate the implant body manually using the Polygon Selection tool, excluding the background. Save the selected area to the ROI Manager by clicking Analyze > Tools > ROI Manager to ensure the same surface region is consistently analyzed.
6. Calculate the whole implant surface area and the red ink remnants by clicking Analyze > Measure > Area.
7. Refer complete ink removal to the total absence of red-stained pixels within the ROI. Keep the settings constant during image analysis for standardization.
8. Calculate the residual stain percentage as: residual stain x 100/total implant surface area using ImageJ (Figure 4).

5. Analysis of the implant surface morphology

1. Code the samples, dehydrate in ethanol, and mount to the aluminum stubs with carbon tape, and place in the ion sputtering device.
2. Define the middle third of the exposed implant surface as the ROI for SEM analysis.
3. Coat the samples with a 20 nm gold layer in a sputter coater for 90 s and place in the vacuum chamber at 10-3 torr vacuum pressure and 10 mA power. Always wear protective laboratory gear and ensure adequate ventilation during sample preparation.
4. Place the gold-coated samples in the vacuum chamber of the scanning electron microscope and examine them at a 0° tilt angle at 5 kV acceleration voltage with a 9 mm working distance.
5. Capture SEM micrographs at 20x, 500x, 1000x, and 5000x magnifications to evaluate topographic changes.

6. Statistical analysis

1. Perform the statistical analysis using SPSS software (version 27). Express the categorical variables as mean ± standard deviation, median, and minimum and maximum values.
2. Assess normality of distribution by the Shapiro-Wilk test. Use the Kruskal-Wallis test for comparisons among three groups. Apply the Bonferroni-corrected Mann-Whitney U test for pairwise comparisons when data are non-normally distributed. Set the significance at p < 0.05.

Results

Following treatments, none of the treated surfaces exhibited complete ink stain removal. The percentage of residual ink varied depending on the type of device used, as well as the photograph's angulation. Comparison of the residual staining percentages for each decontamination group is shown in Table 1.

At the buccal and oral frontal view, the highest cleaning efficacy was achieved in each decontamination method. TiB was the most effective method, leaving the lowest amount of ...

Discussion

In the present study, implant surface debridement with three different instrumentation methods was tested. Among the three tested mechanical decontamination methods, the TiB demonstrated the highest cleaning efficacy and minimal surface alteration on implant surfaces. SEM analysis confirmed that TiB preserved the implant surface morphology more effectively, while TiC caused the most surface damage, and the study hypothesis was rejected. The results revealed that available decontamination methods have limited ef...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acrylic	Imicryl	Konya, Turkiye	Used for the production of the stands for photographs and experimental horizontal defects
Camera	Canon EOS 80D	Tokyo, Japan	Used for obtaining standardized photographs of implants
Chitosan brush	Labrida BioClean, Institut Straumann AG	Basel, Switzerland	Used for the implant surface decontamination
Flash lighting system	Sigma	Roedermark, Germany	Used for obtaining standardized photographs of implants
Image software	ImageJ	NIH, MD, USA	Used for the image analysis
Implants	Bilimplant, PBL 3210	Istanbul, Turkiye	Main study material
Low-speed handpiece	NSK AR-Y	Nippon, Japan	Used in decontamination performed with rotating brushes
Red permanent ink	Staedtler Lumocolor	Nuernberg, Germany	Staining of the implant to imitate the oral biofilm
Scanning Electron Microscope	Jeol, JSM 6335F	Peabody, MA, USA	Used for the surface analysis
Sputter coater	Polaron Emitech, SC7640	East Essex, UK	Reduce the electric charging of SEM samples toattain the highest quality of imaging possible
Statistical software	SPSS 27, IBM	New York, USA	Used for the statistical analysis of the results
Sterile saline	Polifarma, % 0.9	Istanbul, Turkiye	Used for irrigation during decontamination
Ti Brush	NiTi Brush, Hans Korea	Gyeonggi, Korea	Used for the implant surface decontamination
Ti curette (11/12T mini)	Hu-Friedy	Chicago, USA	Used for the implant surface decontamination