The reuse of healing abutments (HAs) has become common practice in implant dentistry for economic concerns and the aim of this in vitro study was to assess the effect of sodium hypochlorite (NaOCl) in decontamination of HAs. A total of 122 HAs (used and sterilized [n = 107]; new [n = 15]) were procured from 3 centers, of which 3 samples were discarded due to perforation in the sterilization pouch. For sterility assessment, the used HAs (n = 80) were cultured in Brain Heart Infusion Broth (BHI) and potato dextrose agar (PDA); bacterial isolates were identified in 7 samples. Also, 24 used HAs were stained with phloxine B, photographed, and compared to new HAs (n = 5). A scanning electron microscope (SEM) assessed the differences between 2 sets of HAs, after which the 7 contaminated HAs along with 24 used HAs from staining experiment (total: 31) were subsequently treated with sodium hypochlorite (NaOCl) and SEM images were observed. About 8.75% of HAs tested positive in bacterial culture; Streptococcus sanguis, Dermabacter hominis, Staphylococcus haemolyticus, and Aspergillus species were isolated. Phloxine B staining was positive for used and sterilized HAs compared to controls. The SEM images revealed deposits in the used HAs and although treatment with NaOCl eliminated the contamination of cultured HAs, the SEM showed visible debris in the HA thread region. This in vitro study concluded that SEM images showed debris in used HAs at screw-hole and thread regions even though they tested negative in bacterial culture. The treatment with NaOCl of used HAs showed no bacterial contamination but the debris was observed in SEM images. Future studies on the chemical composition, biological implications, and clinical influence is warranted before considering reuse of HAs.
Introduction
In 2-stage implant systems, temporary transmucosal healing abutments (HAs) are employed to compensate for the epithelial maturation and tissue shrinkage that occurs during the healing phase preceding the final prosthesis fabrication.1 These abutments are placed around implants during osseointegration and for extended periods to facilitate the formation of gingival tissue and to recreate the form and function of the peri-implant environment. Although it is widely advocated that these HAs are intended for single use, certain manufacturers have advocated the sterilization and reuse of HAs along with low- and high-speed drills, taps, implant analogues, and impression copings.2
The existing body of research suggests that repeated sterilization of dental implants can have detrimental effects due to the changes and contamination of the titanium oxide layer, and that may further affect the tissue integration.3–6 This can be particularly deleterious to temporary healing abutments in the clinical scenario because the formation of healthy and inflammation-free gingiva is pivotal before final prosthesis fabrication.7 There can be biological and mechanical consequences depending on the region where the residues are present in the HAs.8–10
The debris, when present at the implant-abutment junction, can compromise the fit of the prosthesis and certain systems may require the formation of a seal at this junction, which may get affected, leading to bacterial colonization.11,12 The screw thread must be clean to enable the mechanical functioning of the implant abutment joint; alterations can cause a friction to the screw that is torqued to withstand the placement of the final abutment.13 If there is a biological contamination (proteins or amino acids), that is usually resistant to routine sterilization processes, which can facilitate cross-infection and disease transmission.14 Therefore, the initial tissue reaction to the healing abutment is instrumental in deducing the long-term success of the implant restoration.
Various studies have been conducted on the contamination of the healing abutments and the potential methods of decontamination. Browne et al in 2012 observed that used HAs had sterility equivalent to new components after multiple rounds of sterilization using steam autoclave and Chemiclave regimens. The study advocated the resterilization and reuse of HAs along with its implication as a cost-saving factor.15 More recent studies have shown that the sterilized HAs are not completely free of contamination and their reuse warrants justification. Wadhwani et al observed that in 100 HAs, about 99% of them showed protein contamination through phloxine B staining.16 Stacchi et al showed that in HAs (1-month clinical use) after mechanical wiping with disinfectant sponges, about 78.2% had contaminated areas under microscopic evaluation.17 The purpose of this study was to analyze the decontamination present in used HAs through bacterial culture and microscopic analysis followed by the assessment of sodium hypochlorite solution in decontamination of HAs.
Methods
Subjects
In vitro design with used and new dental implant HAs.
Instrumentation/Measurement
Multiple techniques like phloxine-B staining, scanning electron microscope (SEM), and bacterial culture were employed in this in vitro study for the assessment of sodium hypochlorite (NaOCl) decontamination of used HAs.
Study design
This in vitro study was conducted in three centers (2 private clinics, and 1 dental school) that were randomly selected within the target location. This study is in compliance with World Medical Association Declaration of Helsinki that provides for ethical practices in experiments. Therefore, the identity of the health care professionals and patients from the three centers were anonymous and not declared. The informed consent was obtained from the facilities by explaining the purpose of the study and the idea underlying the conception of the research. The study is approved by research ethical committee from King Abdulaziz University Faculty of Dentistry #091-05-19. A total of 122 HAs were used in this study, which included used (n = 107) and new (n = 15) abutments of which 3 samples were discarded due to perforation in the sterilization pouch. The main inclusion criterion for the selected sample was prior use within the past 4 weeks, and the status of the 107 HAs as having been used in the recent past was confirmed by the respective center. Specimens of HAs of various materials and different manufacturers like TRI, PrimaConnex, Biomet 3i, Astra, Nobel Biocare, and Straumann, were included in the study. Generally, the sterilization process applied by the centers include mechanical brushing/scraping with sterile clothes or brushes, soaking in ultrasonic baths containing various antiseptic solutions like alcohol for about 40 minutes and steam autoclave. But, the exact protocol toward the HA sterilization in the 3 centers could not be disclosed due to their privacy and confidentiality clause. The sample size was calculated based on the study conducted by Stacchi et al17 in 2018 with 95% power and 5% alpha error. A simple random sampling technique was followed in the present study.
Procedures
This research followed a 3-step experimental protocol. First, 24 of the assigned used HAs together with unused new HAs (n = 5) were stained with phloxine B and photographs were taken. Staining was performed by isolating each HA into its plastic bag containing 2 mL of phloxine B and placing the sealed plastic bag with its contents into an ultrasonic bath for 15 minutes. After that, the HAs were removed from the bags, washed in deionized water, and subsequently air-dried. The dried abutments were observed in oblique light and the photographs (Canon T3i Rebel camera) zoomed in their main body and screw holes. The photographs were projected onto the computer with 15 times magnification and the unused abutments served as controls—a representative of the baseline or normal appearance. Two examiners (AM, GZ) utilized the SEM (Zeiss Q150R) (SEM) to visualize any debris in the body and screw holes of used and new HAs.
Secondly, 10 new HAs along with the remaining 80 used HAs present in the sterilization bags were assessed for perforation to rule out extraneous contamination. The perforated bags and their contents were excluded from the study (n = 3). Then, the HAs were aseptically inserted into sterile test tubes holding 10 mL of brain heart infusion broth (BHI)—a growth medium for fastidious microorganisms. An additional test tube containing 10 mL of BHI was set up to act as a control. All the tubes were cultured for 10 days at 37°C in 5% carbon dioxide (CO2) milieu. Then, the test tubes were checked for turbidity to assess the presence of microbial colonies.
From test tubes that tested for turbidity, an inoculum was obtained and added to Petri dishes containing BHI and potato dextrose agar (PDA). The PDA is a general-purpose medium that is used for yeasts and molds. BHI Petri dishes were cultured at 37°C in 5% carbon (IV) oxide for up to 48 hours. The petri dishes with the inoculum were cultured at 30°C for 5 days and the process was repeated several times to obtain a pure culture of bacterial colonies, which were subsequently stored in 20% glycerol at -80°C. The obtained bacterial colonies were subjected to Gram staining and biochemical tests, chiefly the catalase test and growth in alkaline medium (pH: 9.2) to establish the characteristics and identity of the colonies. To further aid in microbial analysis, an automated identification system known as the VITEK 2, which allows for rapid identification of the bacteria, was employed. The fungal growth observed in the Petri dishes were subjected to multiple cultures for 7 days at 30°C until pure cultures were obtained and stored. The identification of fungus was based on morphology; reverse appearance as well as surface/front appearance/coloration. The colorations of the fungal colonies were assessed in 3 media—PDA, malt extract agar, and Capek's solution.
Lastly, the contaminated abutments (n = 7) from cultures and 24 used, sterilized HAs from the staining experiment (total n = 31) were cleaned with 5.25% sodium hypochlorite solution. This involved the placement of HAs in plastic bags containing 10 mL of sodium hypochlorite, incubation in an ultrasonic bath for 15 minutes, and subsequent steam autoclaving. The abutments were extracted and incubated for 10 days in the Petri dishes using the same protocol outlined already. The cultures were assessed for turbidity, bacterial colonies, and molds using microbial analysis. Further SEM examination was employed to observe the body and screw holes of used HAs after treatment with sodium hypochlorite, whereas, new HAs served as controls.
Results
This in vitro study was conducted in 3 different centers and used 119 HAs from 6 different manufacturers to assess the efficacy of NaOCl decontamination on used HAs. HAs from PrimaConnex was the most used with 29.4% followed by TRI with 23.5% and Nobel Biocare was the least used with 6.7%. The highest number of used HAs was taken from the dental school (48.7%), followed by the private dental clinics at 22.7% and 16%, respectively (Table 1). The study was conducted employing different protocols for the assessment of efficacy that included phloxine B staining, SEM examination, and bacterial culture. The SEM images were examined by 2 independent examiners and the kappa value of 0.5 showed a moderate level of agreement between the examiners. The convergent validity was investigated and the SEM imaging was shown to be moderately correlating with the gold standard bacterial culture with Spearman correlation coefficient-rho value of 0.33.
Descriptive statistics for the used and new healing abutments based on the manufacturer and the study centers after excluding the perforated bags

All new HAs were stained with phloxine B as observed in Figure 1a for body of healing abutment and Figure 1b for screw hole, and they were compared with used Has, which were stained by the same agent as shown in Figure 1c in the same parts (Table 2). From the 80 used HAs, which were cultured in growth media to assess contamination, 7 samples gave positive results for various microorganisms including Streptococcus sanguis, Dermabacter hominis, Staphylococcus haemolyticus, and Aspergillus species (Candida growth) as shown in Figure 2. The prevalence of bacterial contamination among the studied sample was 8.75% (Table 3). Moreover, all used HAs showed contamination/debris on their main bodies and screw holes compared to new HAs under SEM as shown in Figure 3. In some used HAs, massive accumulation of debris and scratches were observed and could be due to multiple usages as noted in Figure 4. Upon decontamination with NaOCl solution and resterilization, no turbidity, bacterial nor fungal colonies were obtained during the incubation period for 10 days (Table 4). From the SEM images, it was observed that even though debris largely disappeared from the screw hole, some debris remained in the threads of the abutments even after that procedure as observed in Figure 5 (Table 5).
Staining of healing abutments with phloxine B. Both new and used sterilized healing abutments were stained with phloxine B and rinsed with distilled water. (a) Body of new healing abutment. (b) Screw hole of new abutment. (c) Body of used healing abutment. (d) Screw hole of used abutment.
Staining of healing abutments with phloxine B. Both new and used sterilized healing abutments were stained with phloxine B and rinsed with distilled water. (a) Body of new healing abutment. (b) Screw hole of new abutment. (c) Body of used healing abutment. (d) Screw hole of used abutment.
Culture of new and used sterilized healing abutment in 10-mL tubes of brain heart infusion broth 10. (a) Turbidity was observed in all used sterilized healing abutments compared to clear control tube containing only broth tube (the right tube). (b) Representative culture dish showing bacteria identified as Dermobacter hominis (small colonies) and Staphylococcus haemolyticus (large colonies). (c) Representative culture dish showing candida growth identified as Aspergillus species.
Culture of new and used sterilized healing abutment in 10-mL tubes of brain heart infusion broth 10. (a) Turbidity was observed in all used sterilized healing abutments compared to clear control tube containing only broth tube (the right tube). (b) Representative culture dish showing bacteria identified as Dermobacter hominis (small colonies) and Staphylococcus haemolyticus (large colonies). (c) Representative culture dish showing candida growth identified as Aspergillus species.
Bacterial contamination in used/new healing abutments prior to the decontamination with sodium hypochlorite solution

Scanning electron microscope of new versus used healing abutments. Note clear surfaces of new healing abutment with no debris observed in screw hole (a) and threads (b), comapred to abundant debris in screw hole (c) and threads (d) of used healing abutment.
Scanning electron microscope of new versus used healing abutments. Note clear surfaces of new healing abutment with no debris observed in screw hole (a) and threads (b), comapred to abundant debris in screw hole (c) and threads (d) of used healing abutment.
Figure 4. Scanning electron microscope of a representative many times used steralized healing abutment. Note the abundant debris and the scratches in the healing abutment. Figure 5. Use of sodium hypochlorite for deoconamantion of used healing abutments. (a) screw hole of used healing abutment shows removal of screw hole debris. (b) Slight debris are still evident in abutment threads even after treatment with sodium hypochlorite.
Figure 4. Scanning electron microscope of a representative many times used steralized healing abutment. Note the abundant debris and the scratches in the healing abutment. Figure 5. Use of sodium hypochlorite for deoconamantion of used healing abutments. (a) screw hole of used healing abutment shows removal of screw hole debris. (b) Slight debris are still evident in abutment threads even after treatment with sodium hypochlorite.
Bacterial culture results on used and contaminated healing abutments before and after treatment with sodium hypochlorite

Discussion
The reuse of HAs have become a routine clinical regimen among dentists and its clinical implications still loom large due to the various risks associated with it. Surface topography of implants play a major role in fibroblast adhesion and subsequent distribution as clean surfaces have higher free energy and wettability, thereby favoring cellular adhesion and spreading.18,19 In a study conducted by Stacchi et al in 2018, it was observed that there was no fibroblast colonization in the contaminated areas on HA, while the new ones with clean surface showed regular and uniform distribution of the cells.17 In this study, the phloxine B staining that has an affinity toward proteins and peptides was found to be positive in used Has, and SEM images also corroborated the same with debris observed in the screw hole and thread region. It is maybe plausible that contamination in HAs may hinder the fibroblast adhesion and colonization.
Phloxine B (Acid Red 92) is primarily a fluorescein derivative stain that is used in the detection of proteins and peptides. It has been employed in forensics as blood staining test and is also used for microbial detection.20,21 The contamination present on the HA could be attributed to multiple sources like dental plaque, blood, food debris, saliva, and epithelial remnants on titanium surface after abutment removal.8 These contaminants are composed of proteins and amino acids that are tedious to remove and can get coated on the titanium surfaces.9 The rationale behind the dye (phloxine B) employed in this study was that it primarily targets the proteinaceous part of the contamination on HAs. Certain proteins have been shown to get degraded into biological molecules known as prions that can retain their infectivity potential for many years.22 In this study, it can be noted that some of the used HAs tested positive for bacterial culture and all the HAs tested positive for phloxine B staining even after the sterilization process. Even though the results are preliminary, it may be vital to refocus our attention to the conventional methods of used HA decontamination.
In this study, the perforated sterilization pouches were discarded to prevent the bias of extraneous contamination. There is limited evidence in the existing literature with relation to the microbial analysis on used HAs and this study performed microbial culture and analysis employing automated identification system. From the culture, Staphylococcus haemolyticus, Streptococcus sanguis, Dermabacter hominis, and fungal growth with Aspergillus species was identified from 8.75% of the used HA samples. A study conducted by Cakan et al performed a microbial analysis but different organisms like Pencillium bariabile, Enterococcus faecalis, and E. faecium were isolated.23 Staphylococcus haemolyticus is a coagulase negative commensal of the human skin, commonly residing in the inguinal region, perineum, and the axilla.24 The disease occurrence is often associated with indwelling catheters and other medical prosthetics due to the microbe's ability to form capsular biofilms that adhere to the equipment.25 Infections commonly lead to prosthetic valve endocarditis, septicemia, urinary tract infection, and bone diseases, and this species is typically resistant to many antibiotics.26 The oral implications of this microorganism include its association with the development of periodontitis and peri-implant diseases.27 Therefore, improper elimination of this microbe from used HAs may pose an effect on the soft and hard tissue health around the peri-implant surface.
Streptococcus sanguis is a gram-positive facultative anaerobe and a commensal of the human oral cavity.24 The coccus bacteria offer protection against other virulent bacteria like Streptococcus mutans, which cause oral disease; by modifying the milieu through its metabolic activities.28 Even so, bacteremia due to this microorganism may lead to systemic infections like subacute aortic and mitral endocarditis.29 This bacteria's isolation from used HAs can be expected since it forms biofilms on prosthetics, but an excess microbial load in the bloodstream may be detrimental to the patient's systemic health. Dermabacter hominis is a rod-shaped commensal of the human skin and infrequently isolated from human clinical samples.30 This microorganism is rarely associated with human disease.31 The bacterium is a Gram-positive, facultative anaerobe preferring temperatures between 20°C to 40°C and its isolation from used abutments is rather unusual since it is not a commensal of the oral cavity. The isolation may be attributable to deficient sterilization or aseptic techniques employed during packaging.
Aspergillus is a cluster of fungi of diverse species and typically grows in aerobic conditions, rich in oxygen and metabolic substrates like starch, glucose, and amylose. However, some species exhibit oligotrophy—the ability to thrive in nutrient deficient environments.24 The spectrum of diseases caused by Aspergillus includes allergic bronchopulmonary aspergillosis (ABPA), allergic aspergillus sinusitis (hypersensitivity reactions to inhaled fungus), chronic pulmonary aspergillosis, and maxillary sinusitis.32,33 Sterilization is performed to eliminate mold spores and incomplete process maybe the reason for the isolation of this fungi from used HAs.
NaOCl is primarily used in the field of endodontics as an irrigant for the removal of smear layer from the root canals and also as a potent disinfectant.34 The spectrum of its antibacterial activity encompasses various microbes like Bacillus subtilis, Klebsiella pneumonia, Acinetobacter variabilis, certain Streptococcus and Staphylococcus species.35 In this study, the used HAs were cultured and the contaminated specimens were treated with 10 mL of NaOCl followed by steam autoclaving, the bacterial and fungal contamination was negative but SEM images showed no debris in the screw-hole region but some were present in the thread region. The presence of debris may be due to the inherent ledge design in the thread area that has a tendency toward debris accumulation. Even though preliminary results seem to be promising, future studies with larger sample size and assessment of clinical outcomes like osseointegration, soft and hard tissue healing around implant surface are warranted in assessing the efficacy of NaOCl decontamination of used HAs.
Even though some manufacturers provide single-use HAs, the reuse of these components have been on the rise because they are not made out of deformable materials and maybe cost effective to some clinicians. This study offers an insight to the contamination levels and distinct microorganisms present in used and sterilized HAs. Also, the protein-sensitive phloxine B staining and SEM images have yielded information on the site of contamination, that is, the screw-hole and thread region. The comparison between used and new HAs yielded information on the effectiveness of standard disinfection and sterilization protocols at the dental office. The potential limitations of the study include the nondisclosure of the initial sterilization methods and the positive results could be attributed to specific sterilization techniques that might have failed to decontaminate the used HAs. Also, the effect of multiple or repeated sterilization of HAs was not assessed. The use of NaOCl decontamination followed by sterilization has shown to reduce the presence of debris to thread region with negative bacterial culture results. The efficacy results seem promising for an in vitro study where multiple manufacturers were included, but the clinical implications of soft/hard tissue healing, assessment of biological complications, and peri-implant health should be targeted for future research. Also, the identification and analysis of protein-prion components and disease-inducing potential are warranted for future directions.
Conclusion
This in vitro study concluded that the used and sterilized HAs contained debris and contaminants compared to new abutments. Also, bacterial culture revealed colonies of fastidious organisms and fungal growth in 8.75% of the used HA samples. The treatment with NaOCl followed by steam autoclaving for decontamination of HAs was effective in removing contamination and the debris in the screw hole area but remnants were still observed in the thread region of the HA as observed under SEM. The exploratory finding on NaoCl can be targeted for future research with clinical study designs assessing outcomes of peri-implant health, soft/hard tissue healing, and implant success/ survival rates with longer follow-up periods.
Abbreviations
Acknowledgments
This project was funded by the Deanship of Scientific Research, King Abdulaziz University, Jeddah, Saudi Arabia (DSR), under grant no. G: 111-165-1440. The author, therefore, acknowledge with thanks DSR for technical and financial support. The author is thankful to Dr. Ghada Alzaid for serving as a second independent examiner in staining and SEM experiments.
Note
The author declares no conflict of interest.