Mammalian cell response and bacterial adhesion on titanium healing abutments: effect of multiple implantation and sterilization cycles

Abstract

Objective:

Multiple implantations of the implant healing abutment (IHA) could adversely impact its surface properties in vivo. Furthermore, the effect of sterilization and reuse of the IHA on soft tissue viability and bacterial contamination has not been extensively studied. The goal of this study was to perform an in vitro analysis of mammalian cell viability and bacterial adhesion on the surfaces of retrieved IHA after single & multiple implantations and repetitive cycles of sterilization.

Materials and Methods:

IHA surface morphology was studied using optical microscopy. Cell viability of gingival fibroblasts (HGF-1) and oral keratinocytes (HOKg) in indirect contact with IHAs was assessed for 3 and 7 days. Immersion in bacterial culture was performed with a polyculture of Streptococcus species for 3 days and Streptococcus species with Fusobacterium nucleatum for 7 days.

Results:

IHAs exhibited signs of surface damage even after a single exposure to the oral cavity. Fibroblasts did not show a significant preference towards control IHAs over used IHAs, whereas keratinocytes exhibited a significant decrease in viability when exposed to IHAs after multiple implantation cycles as compared to controls. Adherent bacterial count increased with increasing number of IHA implantations for both polycultures.

Conclusions:

Reusing of IHAs in vivo promoted surface degradation in addition to adversely impacting host cell viability and oral bacterial attachment in vitro. These findings show IHA reuse might potentially affect its clinical performance.

Clinical Relevance:

Careful consideration should be taken when reusing IHAs in patients because this practice can result in permanent surface changes that might affect soft tissue integration during the healing period and promote bacterial colonization.

1. Introduction

The formation of a stable connection between the implant surface and host bone tissue as well as integration of the transmucosal component, the implant healing abutment (IHA), with soft tissue is required for satisfactory clinical outcome of dental implants [1]. The IHA is a temporary component of the dental implant system that is placed either through transmucosal procedures or after marginal soft tissue healing in sub-mucosal procedures [2]. The supragingival portion of the IHA is exposed to the oral cavity and in contact with soft tissue, while its subgingival portion is in the internal connection with the implant body. In natural teeth, the gingival mucosa acts as a biological seal around the neck of the tooth and protects against bacterial invasion into periodontal soft tissues. In the case of a dental implant, the lack of a natural barrier raises the importance of soft tissue seal formation by epithelial cells and gingival fibroblasts tightly attached to the IHA [3]. The concept of a “race for the surface” between bacterial and mammalian cells is a deciding factor for the establishment of the soft tissue seal and dental implant longevity [4, 5]. Before the placement of the permanent abutment, the soft tissue seal is maintained by the IHA, which can be divided into two zones: (1) a marginal zone harboring a peri-implant epithelium, and (2) an apical zone filled with connective tissues [2, 5–8]. However, due to the structural differences, the connection around an implant is weaker than that around a natural tooth [2, 9]. Hence, establishment of a tight seal to the IHA surface is essential to mitigate colonization by oral bacteria around the IHA-implant interface [6, 10, 11].

Due to its temporary placement and lack of studies investigating dental implant components involved in soft tissue healing, the importance of the IHA is often neglected. After placement of an IHA, host gingival epithelial cells compete with the bacterial load that has colonized the IHA surface [12–14]. Invasion by microorganisms is restrained by the gingival mucosa forming a biological seal, and the epithelium acts as a physiological barrier by initiating inflammatory responses to serve the wound healing process [5, 15]. The materials comprising the IHA can also influence the adhesion of soft tissue cells and resistance to microbial invasion in the complex oral environment. IHAs are generally composed of commercially pure titanium (cpTi) or zirconia but can also be made of polyether ether ketone (PEEK) when customized fitting is suggested [16]. Titanium IHA surfaces are generally treated for color-coding and easy identification among other components in the oral cavity. The surface treatment typically applied is anodization, which not only alters surface color but also increases the thickness of the native oxide layer on titanium, providing additional corrosion resistance [1, 17].

As the IHA surface protrudes into the oral cavity, it is directly exposed to bacteria, saliva, biological fluids, by-products of host inflammation, and possible occlusal forces, which promote a corrosive environment that can degrade the IHA surface, making it especially vulnerable to bacterial adhesion [1]. An interesting current clinical practice is to clean, sterilize, and reuse IHAs. Although this practice is cost-effective, it neglects the potential adverse effects of the reuse of these components [18, 19]. Furthermore, this practice breaches manufacturer single-use guidelines, and no records are kept regarding the number of times an IHA has been placed [19–21]. Exposure of a metallic IHA surface to the oral cavity multiple times results in biological contamination, namely adsorbed proteins, that could increase its surface roughness, which promotes bacterial attachment on titanium abutments and implant bodies [19, 22]. Bacterial infiltration is a primary cause associated with early implant complications [23–25]. Specifically, bacteria can pass through microgaps created between the modular components of dental implant systems, such as the permanent abutment surface and implant body [26, 27]. Similarly, microgaps can also be created at the IHA screw and implant interface, but its effect on microbial leakage has yet to be investigated [28]. Prior to reuse, IHAs undergo sterilization by means of mechanical wiping, autoclaving, and sonication [29]. Despite various sterilization techniques to clean IHAs, residual biological contaminants remain on the surface after in vivo use [19]. Even in vitro exposure to mammalian cells and sterilization can damage its protective oxide layer (TiO₂) and alter the roughness and wettability of titanium implant surfaces, which counteract the improvement in subsequent host cell growth achieved by conventional surface treatments [30]. Thus, the synergistic effects of oral environment exposure and accumulated changes to IHA surfaces after multiple implantations and sterilization cycles might interfere with successful soft tissue attachment and early healing.

Although the “race for the surface” between host cells and bacteria is inevitable, soft tissue cells generally colonize the pristine abutment/implant surface and establish a biological seal to prevent ingress of bacteria and achieve successful healing of the underlying soft tissues on smooth Ti surfaces [5]. Nevertheless, the same observation may not apply for rougher, reused IHA surfaces. Thus, the purpose of this study was to investigate the effects of multiple cycles of implantations and clinical sterilization of IHAs on soft tissue growth and early bacterial colonization. It was hypothesized that the viability of host mammalian cells would be lower when exposed to used IHAs as compared to unused controls. It was also hypothesized that oral bacterial attachment would increase on IHAs that have been exposed to multiple implantation procedures.

2. Materials and Methods

2.1. Sample Collection and Preparation

The procedures, documentation, and analysis were performed in accordance with ethical guidelines and Internal Review Board (IRB) approval at the author’s institution (IRB #16–65). All Ti IHAs post-retrieval were obtained from different patients attending a private periodontics clinic following standard clinical procedures, as per guidelines of the Helsinki Declaration. The retrievals had no identifiers that could be linked to the patients who donated the IHAs. The retrievals were classified based on the number of implantations to which they were subjected. IHAs that were only used once were grouped as single-use. In general, no records are maintained regarding the number of times these devices are implanted in different patients. Hence, IHAs known to have been used more than once were labeled as multiple-use. The retrievals were then received at The University of Texas at Dallas (Richardson, TX, USA). All 21 used IHAs obtained were marked with a letter for identification. Additionally, pristine control IHAs which had never been implanted were obtained and denoted as unused. The IHAs were divided into three groups: (1) unused (n = 5), (2) single-use (n = 15) and (3) multiple-use (n = 6). Each IHA group was subdivided as shown in Table 1 and exposed to one of three tests conditions: mammalian cell viability with (1) human gingival fibroblasts (HGF-1) or (2) human gingival oral keratinocytes (HOKg) or (3) bacterial adhesion with oral bacteria (early and secondary colonizers). All samples were cleaned and sterilized prior to mammalian cell viability or bacterial adhesion testing. Cleaning consisted of ultra-sonication of IHAs with acetone, deionized (DI) water and 70% ethanol for 15–20 min each and subsequent autoclave sterilization (20 min exposure at 121 °C, 2 h drying) to replicate clinical procedures.

Table 1.

List of implant healing abutments (IHAs) used in the present study.

Brand	In Vitro Test	Usage/Sample Size
Straumann LLC.	Mammalian Cell Viability: Human Gingival Fibroblasts	Unused = 2 Single-use = 5 Multiple-use = 2
	Mammalian Cell Viability: Human Oral Keratinocytes	Unused = 2a Single-use = 5 Multiple-use = 2
	Bacterial Adhesion: Early Colonizers and Secondary Colonizers	Unused = 3 Single-use = 5 Multiple-use = 2

^aThe same unused IHAs were used for testing with both mammalian cell lines.

2.2. Mammalian Cellular Viability

Mammalian cell viability testing was performed with host cells involved in soft tissue healing, namely human gingival fibroblasts and human oral keratinocytes. In the present study, mammalian cell viability was measured based on the metabolic activity of viable cells. In total, 16 Straumann IHAs were utilized for cell culture testing as shown in Table 1. Two distinct sets of IHAs were used to assess the viability of fibroblasts and keratinocytes. Per adaptation of ISO 10993-5-2009, cell viability was evaluated after indirect exposure to IHAs to avoid loss of cells due to movement of the IHAs in the cell culture wells physically rupturing the cell monolayer and hence influencing cell viability. Human gingival fibroblasts (HGF-1) (American Type Culture Collection, Manassas, VA, USA) were cultured in T-75 flasks until 70–80% confluency at 37° C and 5% CO₂ using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) (HycloneTM, Logan, UT) and 1% penicillin-streptomycin (10,000 U/mL) (Gibco, Thermofisher Scientific Waltham, MA). Human oral keratinocyte gingival cells (HOKg) (Lifeline Cell Technology, Frederick, MD, USA) were cultured similarly but using Basal Medium (Lifeline Cell Technology, Frederick, MD, USA) supplemented with growth and life factors including gentamicin (30 mg/ml), amphotericin B (15 μg/ml), and 1% penicillin-streptomycin (10,000 U, mL). One day prior to cell seeding, all IHAs were immersed in 1500 μl of their respective cell culture media in tissue culture-treated 24-well plates. Fibroblasts and keratinocytes were seeded at 15,625 cells/cm² separately in tissue culture-treated 96-well plates and incubated in 100 μl of their respective media to allow for cellular attachment and monolayer formation (n = 3). After 24 h, the mammalian cells were rinsed with 1X PBS and replaced with 100 μl of media aliquoted from the wells in which the IHAs were immersed for either 3 or 7 days. Every 48 h, the cell culture media was replaced with 100 μl aliquots taken from the IHA immersion wells to maintain cell growth. The negative control consisted of wells with plain media only, and positive control had mammalian cells with plain media that was not exposed to IHAs.

After 3 and 7 days of growth, cell viability was quantified using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazoliumbromid (MTT) assay. Media was aspirated from all the wells followed by washing with 100 μL of 1X PBS. 100 μL of fresh media was added back to the wells followed by addition of 10 μL of MTT reagent to each well. The well plates were incubated at 37° C in the dark for 4 h. Afterward, 100 μL of detergent reagent was added, and the plates were stored overnight at 37° C. The absorbance in each well including the blanks was measured at 570 nm in a microtiter plate reader (BioTek, Winooski, VT, USA). After subtracting average blank values from the negative control wells, cell viability was calculated as the percentage based on the average values for positive control wells.

Due to the limited number of IHAs available, the entire cleaning and cell culture testing methodology was repeated three times (trials) using the same set of IHAs. For each trial, the percentage of cell viability was calculated for each IHA group by averaging the values from all its constituents (e.g., one value was generated for the 5 single-use IHAs) to account for differences in the number of IHAs per group, Afterward, an average value and standard deviation for each IHA group was calculated based on values obtained for each trial. Individual data points for mammalian cell viability tests are depicted in Figure S1 (see Online Resource 1).

2.2.1. Mammalian Cellular Morphology

Confocal laser scanning microscopy (CLSM; Olympus, Waltham, MA, USA) was used to visualize the cell morphology of fibroblasts and keratinocytes after 3 and 7 days of indirect exposure to IHAs as described previously. The mammalian cells were seeded on tissue culture-treated polystyrene in a 24-well plate using the same procedure as mentioned in section 2.2. After 3 or 7 days, each well was rinsed three times with 1X PBS, and the cells were directly fixed with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hartfield, PA, USA). The plate was incubated in the dark for 30 min and washed three times with 1X PBS. After fixation, the cells were stained with 300 nM of 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) and Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) to visualize the nucleus and F-actin filaments, respectively. The plate was again incubated in the dark for 30 min and washed three times with 1X PBS, and the cells were then visualized at 30X magnification.

2.3. Bacterial Adhesion

Bacterial adherence was assessed on IHAs by immersing samples in oral bacterial polyculture in vitro. In total, 10 Straumann IHAs were used for immersion testing to compare the oral bacterial attachment on used versus unused IHAs as shown in Table 1. Due to the limited number of IHAs, the immersion testing was first performed with a polyculture of early colonizers followed by a polyculture of early colonizers and a secondary colonizer.

For the early-colonizing polyculture, three early-colonizing oral bacteria strains were used: Streptococcus mutans (ATCC 700610), Streptococcus sanguinis (ATCC 10556), and Streptococcus salivarius (ATCC 13419) (American Type Culture Collection, Manassas, VA, USA). The bacterial testing was performed in 5% CO₂ atmosphere at 37° C. All strains were struck separately from frozen stocks kept at −80° C onto Brain Heart Infusion (BHI) agar. After 24 h of incubation, individual colonies were inoculated in 5 ml of BHI broth overnight. For the early- and secondary-colonizing polyculture, two early colonizers, S. mutans and S. sanguinis, and a secondary colonizer, Fusobacterium nucleatum (ATCC 25586) (American Type Culture Collection, Manassas, VA, USA), were used. As F. nucleatum is an obligate anaerobe, the bacterial immersion testing was performed in an anaerobic workstation (Don Whitley Scientific Limited, Bingley, West Yorkshire, UK) maintained at 37° C with 80% N₂, 10% CO₂ and 10% H₂. S. mutans and S. sanguinis were grown as mentioned previously. F. nucleatum was directly inoculated from the frozen stock kept at −80° C. After 24 h of incubation at 37°C, individual colonies of S. mutans and S. sanguinis were inoculated in 5 ml of BHI broth supplemented with menadione (0.1 mg/L), hemin (5 mg/L), and yeast extract (5 g/L) overnight. S. mutans and S. sanguinis were inoculated and incubated for 24 h and F. nucleatum for 48 h.

2.3.1. Immersion Testing

Immersion testing for early colonizers and early colonizers with a secondary colonizer was performed using the same methodology except for the growth conditions. Growth of all the strains was confirmed by optical density readings at 600 nm (OD600) measurements. Each strain was diluted to an OD600 value of 0.01 and combined in equal parts (volume) to create a polyculture. Each IHA was then immersed in 1500 μl of early-colonizing polyculture for 3 days or early- and secondary-colonizing polyculture for 7 days in a 24-well plate. 750 μl of immersion media was replenished every day throughout the immersion period to ensure bacterial viability. The negative control consisted of uninoculated broth as a check for contamination, and the positive control (referred to as bacteria control) was bacterial polyculture in which no IHA was immersed. The OD600 and pH of the immersion media for each well were monitored every 24 h. Post-immersion, all IHAs in their respective polyculture broths were aseptically removed from the immersion media. The samples were dipped three times in 1500 μl of 1X PBS, and the bacteria which detached from the IHA surface into the PBS were classified as loosely adherent. The IHAs were then placed into separate centrifuge tubes containing 1.5 ml of PBS and were subjected to ultrasonication for 5 min. The bacteria which detached after the ultrasonication process were classified as adherent. Per preliminary staining and plating tests, ultrasonication was deemed to consistently and efficiently remove oral bacteria attached to titanium surfaces (data not shown). Afterward, loosely adherent and adherent bacteria in addition to planktonic bacteria (positive control) were 10-fold serially diluted and plated onto BHI agar plates for the early-colonizing polyculture and Brucella blood agar plates supplemented with 5% defibrinated sheep’s blood, vitamin K1 (10 mg/L) and hemin (10 mg/L) (Hardy Diagnostics, Santa Maria, CA, USA) for the early- and secondary-colonizing polyculture. Bacteria were counted to obtain the number of colony-forming units per milliliter (CFU/ml). As explained in Section 2.2 for mammalian cells, the entire procedure for bacterial adhesion tests was repeated 3 times (trials) first with the early colonizers and then with the early and secondary colonizers due to the limited number of IHAs. Within each trial, a single value was generated for each IHA group by averaging the bacterial counts of all IHAs within that group. Individual data points for bacterial adhesion tests are depicted in Figure S2 (see Online Resource 1).

2.3.2. IHA Surface Morphology

Optical microscopy (OM; VHX-2000, Keyence, Itasca, IL, USA) was used to image the IHAs before and after bacterial immersion testing to investigate the changes in the surface due to sonication, autoclaving and repetitive exposure to bacteria. The surfaces of the IHAs used for bacterial immersion testing were imaged at magnifications ranging from 100X to 500X.

2.4. Statistical Analysis

Statistical analysis was performed using a two-way analysis of variance (ANOVA) followed by post hoc Tukey tests by comparing between values (at the trial-level) obtained for unused, single-use, and multiple-use IHAs (and controls when applicable) as explained in Sections 2.2 and 2.3. For mammalian cell viability, the first factor was the number of usages; the second was the duration of immersion of IHAs with mammalian cells. For bacterial adhesion tests, the first factor was the number of usages, and the second factor was the state of attachment. Both tests were run using GraphPad Prism 7.0 Software (GraphPad Software Inc., San Diego, CA, USA) at a significance level α = 0.05. To check for significant differences in values for each IHA as a result of repeated experimental testing, statistical analysis was performed again using two-way ANOVA. In this case, the first factor was the IHA label and the second factor was the trial number.

The experimental procedures used in the current study is in compliance with the SQUIRE Equator guidelines, and the manuscript was written based on the SQUIRE checklist.

3. Results

3.1. Mammalian Cellular Growth

3.1.1. Mammalian Cellular Viability

The cell viability results for both fibroblasts and keratinocytes are shown in Fig. 1. In general, the viability of fibroblasts and keratinocytes at each time point decreased with increasing number of implantation procedures. In particular, the viability of fibroblasts did not significantly differ when in contact with media exposed to the Ti surfaces of unused or used IHAs as shown in Fig. 1 (A). However, fibroblasts demonstrated higher average viability when exposed to unused IHA surfaces after both 3 and 7 days, followed by single-use and lastly multiple-use IHAs. A similar trend in average viability was also observed for keratinocytes after 3 days (Fig. 1 (B)). Furthermore, keratinocytes showed significantly higher viability on unused IHAs as compared to multiple-use IHAs after 7 days but was only higher on average as compared to viability with single-use IHAs. Additionally, fibroblast viability decreased on average between corresponding groups after 3 and 7 days. In contrast, an average decrease in the viability between 3 and 7 days was only observed for keratinocytes exposed to multiple-use IHAs while viability increased on average between 3 and 7 days for unused and single-use IHAs. Interestingly, fibroblast viability after 3 days of exposure for all IHA groups and after 7 days for unused IHAs was higher than values the positive control, which were not exposed to media containing IHAs, leading to values above 100% being recorded (p > 0.05). However, it was not statistically significant (data analysis not shown). Moreover, no significant differences in fibroblast viability were observed for any IHA between different experimental trials (p > 0.05). Similarly, keratinocytes exposed to unused or single-use IHAs exhibited average viability values greater than or equal to that of the positive control.

Figure 1.

Cell viability (%) of (A) HGF-1 cells and (B) HOKg cells after 3 and 7 days. * indicates statistical significance between marked groups (α = 0.05).

3.1.2. Mammalian Cellular Attachment

Confocal images of fibroblasts and keratinocytes after being seeded with indirect contact to IHAs are shown in Fig. 2. For gingival fibroblasts (Fig. 2, rows 1 and 2), no apparent changes were observed in the cellular morphology among unused, single-use and multiple-use IHAs for both 3- and 7-day time points. Comparable numbers and densities of fibroblasts were present in all wells exposed to all IHA groups at the 3-day time point. At the 7-day time point, fibroblasts exposed to unused IHAs appeared greater in number and density as compared to those exposed to single- and multiple-use IHAs. For human oral keratinocytes, the cells exhibited different morphologies at the 3- and 7-day time points. After 3 days, unused IHAs had the greatest density of viable cells. A reduction in cell density and cell shrinkage was observed for those exposed to single-use IHAs relative to those exposed to unused IHAs. Moreover, multiple-use IHAs displayed keratinocytes with blebbing effect where cell membrane detachment was observed. At the 7-day time point, the apparent number of viable cells was lower as compared to corresponding 3-day time point for all groups. Also, bridge-like structures between keratinocytes exposed to unused IHAs were observed. More clusters of viable keratinocytes were visible after exposure to unused IHAs as compared to single- and multiple-use IHAs.

Figure 2.

Confocal laser scanning microscopy (CLSM) images of unused, single-use and multiple-use IHAs after 3 and 7 days of cell culture testing: (Rows 1, 2) Cell morphology of human gingival fibroblasts (HGF-1); (Rows 3, 4) Cell morphology of human oral keratinocytes (HOKg).

3.2. Evaluation of Bacterial Adhesion on IHA Surfaces

3.2.1. Bacterial Adhesion

The quantitative evaluation of bacterial adhesion on all IHA surfaces after immersion for 3 and 7 days in Streptococcus polyculture and polyculture of S. mutans, S. sanguinis and F. nucleatum is depicted in Fig. 3 (A) and (B), respectively. In general, attachment was lowest for bacteria exposed to unused IHAs, followed by an increase in bacterial adhesion for single-use IHAs, then multiple-use IHAs, and finally bacteria control (planktonic bacteria) as shown in Fig. 3. Specifically, the polyculture of early colonizers after 3 days demonstrated significantly lower loosely adherent and adherent bacterial counts when exposed to unused IHA surfaces as compared to single-use and multiple-use IHAs (p < 0.05) as shown in Fig. 3 (A). It is essential to note that difference in the bacterial count between unused and used IHAs is more than one (log scale), corresponding to >10X more bacteria attached to the single and multiple-use IHAs compared to unused IHAs. Furthermore, single-use IHAs had lower bacterial counts than multiple-use IHAs after immersion with early colonizers, which was significantly different for the loosely adherent group (p < 0.05). Moreover, unused IHAs had a significantly lower number of adherent bacteria than loosely adherent bacteria (p < 0.05), while single- and multiple-use IHAs had statistically similar amounts of loosely adherent and adherent bacteria. The same general trend in bacterial adhesion was also observed for the IHAs immersed in the early and secondary colonizer polyculture after 7 days (Fig. 3 (B)). Among loosely adherent bacteria, the bacteria control and all IHA groups were statistically different from each other (p < 0.05); again, bacterial counts increased in the order of unused, single-use, multiple-use IHAs, and lastly bacteria control. For the adherent bacteria groups, unused IHAs had significantly lower bacteria counts versus multiple-use IHAs and bacteria control (p < 0.05) but only a lower average number than single-use IHAs (p > 0.05). Also, adherent bacterial count on multiple-use IHAs was high enough to be statistically similar to planktonic bacteria control. Between corresponding loosely adherent and adherent groups after 7-day immersion in early and secondary colonizers, no significant differences were observed (p > 0.05). Lastly, no significant difference (data not shown) between different experimental trials was detected among loosely or adherent bacterial counts for a given IHA (p > 0.05).

Figure 3.

Bacterial counts for bacteria control and loosely adherent or adherent bacteria on unused, single-use and multiple-use IHAs after immersion in polyculture. (A) Polyculture of early colonizers after 3 days. (B) Polyculture of early colonizers with a secondary colonizer after 7 days. * indicates statistical significance between all other groups outside marked group within loosely adherent groups. † indicates statistical significance between corresponding adherent group. # indicates statistical significance between all other groups outside marked group within adherent groups (α = 0.05).

3.2.2. Surface Morphology Pre- and Post-Immersion

The OM images of IHAs before and after immersion in oral bacterial cultures are shown in Figs. 4, 5, and 6. The unused IHAs shown in Fig. 4 (A and C) exhibited pristine surface condition with uniform surface morphology. After immersion for 3 and 7 days in bacterial polyculture, mild surface discoloration and changes in surface roughness were observed on both unused IHAs as shown in Fig. 4 (B and D). Representative OM images of single-use IHAs before and after immersion in oral bacterial cultures are depicted in Fig. 5. Although no changes in surface features were observed for IHA E (Fig. 5 (A and B)), residual debris present in the notch area of IHA G was not completely removed after multiple cycles of ultrasonication as shown in Fig. 5 (D). Representative OM images of multiple-use IHAs before and after immersion in oral bacteria are depicted in Fig. 6. There were no apparent changes in surface topography of these IHAs before and after immersion. However, the used IHAs before immersion had severe discoloration and apparently rougher surfaces compared to unused IHAs. Multiple cleaning, sterilization cycles, and exposure to oral bacterial culture resulted in removal of the anodized layer on the IHA as shown in Fig. 6 (B) and reduction in white deposits, but the surface roughness and irregularities seemed to remain the same on the IHA as shown in Fig. 6 (D).

Figure 4.

Representative OM images of unused implant healing abutments (IHAs). (A, C) Collar-neck region of unused IHAs before immersion (100X magnification). (B) Collar-junction of unused IHA with arrow indicating surface discoloration and irregularities after immersion (100X magnification). (D) Collar region of unused IHA with arrows indicating abrasions and discoloration after immersion (200X magnification).

Figure 5.

Representative OM images of IHAs after single implantation. (A) Collar region of IHA E with arrow indicating surface discoloration and layers peeling off before immersion (100 × magnification). (B) Collar region of IHA E after immersion with arrow indicating surface damage still present (100 × magnification). (C) Collar region of IHA G with arrow indicating biological debris before immersion (100 × magnification). (D) Collar region of IHA G with arrow indicating residual debris after immersion (100 × magnification).

Figure 6.

Representative OM images of IHAs after multiple implantations. (A) Collar region of IHA B with arrow indicating surface discoloration before immersion (200 × magnification). (B) Collar region of IHA B with arrow indicating discoloration and debris after immersion (200 × magnification). (C) collar region of IHA F with arrow indicating white deposits and apparent roughness before immersion (200 × magnification). (D) collar region of IHA F with arrow indicating residual deposits and roughened surface after immersion (200 × magnification).

4. Discussion

Healthy peri-implant soft tissue around a dental implant is crucial to maintain implant functionality and esthetics in the long term [2, 9]. The attachment of soft tissue to the IHA and implant body neck during the initial healing period is essential to hamper bacterial infiltration and early implant complications [8, 12]. However, reusing IHAs might jeopardize this function, yet no prior studies have evaluated the impact of multiple implantation and sterilization cycles on either mammalian or bacterial cell behavior. Hence, the goal of this study was to elucidate the in vitro biological response to IHA retrievals. It was hypothesized that the viability of fibroblasts and keratinocytes would be significantly lower while bacterial adhesion would be significantly higher on used IHAs versus unused ones.

Host cell viability testing revealed the effects of IHA surface degradation after single and multiple implantations on soft tissue cell growth. In this study, the viability test was performed in indirect contact with IHAs, which excluded surface morphology as a factor. This allowed for evaluation of the role of leaching agents and particle release from IHAs due to repetitive implantations. Overall, gingival fibroblast viability did not significantly differ between exposure to used or unused IHA surfaces (Fig. 1 (A)). However, the general trend observed was decreasing average fibroblast viability with unused IHAs, followed by single- and then multiple-use IHAs after both 3 and 7 days. This result could have been due to increased Ti particle release, residual debris, or bacterial plaque that had accumulated on these IHAs. Previous IHA retrieval studies demonstrated viable bacteria after cleaning and sterilization and residual debris after 3–6 months of implantation, which support the current observation [1, 21]. Also, murine fibroblasts had lower cell attachment on Ti in vitro due to oxide damage imparted by autoclaving [18]. Similarly, fewer fibroblasts and more macrophages surrounded IHAs reused in a rat model after human implantation as compared to unused ones in spite of ultrasonic cleaning [31]. However, in the present study, IHA surface changes after multiple implantations did not significantly alter the viability (Fig. 1 A) or induce morphological changes in fibroblasts (Fig. 2). Likewise, Canullo et al. did not find a significant difference in soft tissue cell attachment to autoclaved versus unused IHAs after 10 weeks in human patients [2]. Still, average fibroblast viability in the current study decreased between 3 and 7 days for both unused and used IHAs (Fig. 1 A), corroborating the mild qualitative decrease in the apparent fibroblast density after 7 versus 3 days (Fig 2). This behavior could be attributed to fibroblasts reaching maximum confluency by 3 days prior to the apparent onset of apoptosis by 7 days, which was seemingly more severe after exposure to multiple-use IHAs (Fig 2).

Early and firm epithelial attachment at the implant-IHA interface is needed to oppose invasion of oral pathogens [32–34]. In the present study, human oral keratinocytes represented epithelial or wound healing cell response to indirect contact with Ti IHA surfaces. Like fibroblasts, keratinocytes had higher average viability when exposed to unused IHAs as compared to used IHAs after both 3 and 7 days (Fig. 1 (B)). Furthermore, this reduction in viability after exposure to multiple-use IHAs resulted in significantly lower viability by 7 days (p < 0.05) versus exposure to unused IHAs. As previously mentioned, this behavior may have been attributed to Ti particle release or loose residual debris [1, 35]. Also, particle release may have persisted, resulting in lower average keratinocyte viability after exposure to multiple-use IHAs after 7 days as compared to 3-day values. In contrast to fibroblasts, average keratinocyte viability after exposure to unused and single-use IHAs increased between 3 and 7 days (Fig. 1 B). On the other hand, the apparent density and number of attached keratinocytes across all IHA groups declined after 7 days versus 3 days (Fig. 2). Thus, this increase in keratinocyte viability by 7 days was not attributed to proliferation but rather elevated metabolic activity of keratinocytes as compared to 3 days. Moreover, oral keratinocytes appeared to be more sensitive to potential IHA particle release than fibroblasts in vitro based on cell morphology. Specifically, keratinocytes exhibited signs of apoptosis (blebbing effect) and decreased cell density after 3 and 7 days, respectively (Fig. 2).

As the early healing period involves the “race for the surface” between host tissue cells and oral pathogens, bacterial attachment on IHAs and its influence on IHA surface was investigated with only early colonizers (S. mutans, S. sanguinis, and S. salivarius) for 3 days (Fig. 3 (A)) or with a secondary colonizer (S. mutans, S. sanguinis, and F. nucleatum) for 7 days (Fig. 3 (B)). For both polycultures, fewer bacteria were observed on unused IHAs as compared to used IHAs. Interestingly, only unused IHAs after 3-day immersion with early colonizers had significantly lower (~10x) adherent bacterial counts than its loosely adherent counterpart (p < 0.05). On the other hand, single- and multiple-use IHAs had similar or higher adherent number of bacteria than corresponding loosely adherent bacteria, suggesting that used IHAs are more prone to initial colonization by Streptococcus species than unused ones. However, after 7-day immersion with F. nucleatum, a “bridging” species between early and late colonizers, all IHA groups exhibited similar amounts of loosely adherent and adherent bacteria. These trends in bacterial adhesion can be correlated with surface damage caused by single or multiple implantations like abrasions, localized corrosion, and bacterial plaque, which apparently increased surface roughness, especially for multiple-use IHAs (Figs. 5 and 6). Greater roughness and formation of crevice-like regions on IHAs could have provided more sites for oral bacterial attachment on IHAs as observed after 14 days in vivo [1, 11, 36]. Furthermore, multiple-use IHAs after 7-day immersion had such a high adherent bacterial count that it was statistically similar to planktonic bacteria (control), demonstrating increased susceptibility to bacterial colonization versus unused and single-use IHAs. Early colonizers can alter Ti implant surfaces in vitro by reducing the pH in the oral cavity due to lactic acid production [35, 37]. The combination of corrosion attack and bacterial attachment on used IHAs could damage the protective oxide layer and release metallic Ti ions and debris into surrounding host tissues, thereby exacerbating inflammatory conditions and facilitating further corrosion [1, 35, 38, 39].

The microscopic analysis of unused and used IHAs pre- and post-immersion with bacteria corroborated the trends of decreasing host cell viability and increasing bacterial adhesion with increasing number of implantation procedures. Prior to immersion, unused IHAs had smooth and pristine surfaces (Fig. 4 (A and C)) while single-use and multiple-use IHAs displayed surface irregularities and biological debris accumulation (Fig. 5 (A and C)) such as white deposits lodged in crevices (Fig. 6 (A and C)). Post-immersion and cleaning, changes in surface appearance were noticeable on unused IHAs and to a greater extent on used IHAs. Previously unused IHAs now exhibited surface discoloration and abrasions (Fig. 4 (B and D)). In addition, residual debris remained on used IHA surfaces despite ultrasonic cleaning and autoclaving. (Figs. 5 and 6 (C and D)). Moreover, surface characteristics acquired after implantation such as increased apparent roughness, loss of anodization layer, and localized corrosion attack, were still visible on used IHAs, demonstrating that pristine surface conditions could not be recovered (Figs. 5 and 6 (B and D)). These observations are in agreement with previous studies indicating that cleaning and sterilization of IHAs can harm their surfaces [18, 33]. As IHAs are manufactured to have smooth or nanostructured surfaces to promote soft tissue cell attachment over bacterial adhesion, the loss of pristine IHA surfaces can adversely affect their function [3, 5, 11, 18, 31, 33, 34]. Aside from surface damage, the lack of complete removal of biological material from an IHA post-sterilization highlights the potential for cross-contamination between patients. A previous study showed that viable bacteria might still be present even after commercial autoclaving [21].

Although IHAs continue to be reused, the relatively high success rate of dental implants suggests that mammalian cells are still able to attach and colonize the surface in spite of oral microbes [40]. That is, there is no reported evidence to date to suggest that early dental complications can be caused by reuse of IHAs. However, this could be due to a lack of understanding the long-term and downstream effects on both bacterial adhesion and mammalian cell behavior due to reusing IHAs. The current study highlights the need to consider the effects of multiple implantations before reusing IHAs; long-term retrieval or clinical studies should be conducted to support the present observations.

Corroborating the observation in the present study that the number of usages in vivo permanently changes the IHA surface, unused and used IHAs continued to exhibit the same trends of lower mammalian cell viability and higher bacterial adhesion with increasing number of usages despite thorough cleaning of the same IHAs between tests. The same IHAs within each group, consistently exhibited statistically similar values (data analysis not shown) after each test, demonstrating that repetitive in vitro test conditions were not significantly influencing the biological response to IHAs. Instead, the differences observed between IHA groups could be attributed to the number of placements in the oral cavity in vivo. The oral cavity is expected to be a more destructive environment than the present in vitro test conditions due to occlusal loading, exposure to inflammatory agents, and pH fluctuations due to varying food and beverage consumption. Thus, the present study elucidated the effect of repeated IHA placement in vivo on host cell viability and bacterial adhesion in vitro.

The limitations of this study need to be discussed. Firstly, mammalian cell viability was measured based on cellular metabolic activity. However, the metabolic activity of cells is not only influenced by the number of viable cells but also on growth conditions overtime (e.g., apparent increase in cell viability of keratinocytes after 7 versus 3 days can be attributed to an increase in metabolic activity of viable cells and not an increase viable cell count). Secondly, soft tissue cells were not grown directly on IHAs due to their complex morphologies and movement within cell culture wells rupturing the cell monolayer and affecting cell viability. Although this study simulated the clinical conditions of IHA placement, the lack of data on the number of placements and implantation length limited the understanding of how cell viability and bacteria adherence are affected by the duration and frequency of implantation. However, this is reflective of clinical practice as the number of implantations is typically not recorded. In addition, despite conducting mammalian cell viability and bacterial adhesion tests multiple times using the same IHAs, the clinical significance of this study may still be limited by the small sample size and the short follow-up. Future studies with larger sample sizes are needed to validate the trends observed in the present study and reveal mechanisms influencing cellular behavior such as mammalian cell apoptosis after exposure to reused IHAs. Lastly, the current study individually assessed soft tissue cell viability and bacterial adherence on unused and used IHAs. Future investigations will use co-culture models and include pathogenic species like Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis to elucidate the competition between host and bacterial cells on IHA surfaces.

5. Conclusion

In vitro testing revealed no significant decrease in fibroblast viability and morphology with increasing number of implantation procedures; however, wound-healing epithelial cells were more sensitive to used IHA surfaces and exhibited a significant decrease in viability and loss of cellular morphology versus exposure to unused IHAs. Additionally, bacterial adherence increased with increasing number of implantations, and used IHA surfaces exhibited accumulation of biological debris and greater surface deterioration and roughness, which seem to provide a more favorable environment for successful bacterial colonization. Within the limitations of the present study, the results substantiated that the current clinical practice of reusing IHAs can affect soft-tissue cell growth and oral bacterial adhesion, thereby putting at risk the performance of the IHA and potentially the entire dental implant system. However, additional studies need to be conducted to support the current observations.