– Minimal focus is placed on IHAs, despite its vital role in soft tissue healing/shaping after dental implant body placement.
IHAs, which are designed and labeled for “single” use, are re-used in multiple patients as a common clinical practice.
Microbial analysis and surface characterization of IHA retrievals was performed after single and multiple implantations.
Streptococcus was the only genus present on every IHA, while Fusobacterium had the highest relative frequency across all IHAs.
IHA retrievals showed discoloration, abrasions, biological contamination, and higher corrosion rate as compared to controls.
Very few studies have investigated dental implant components involved in the early stage of healing, especially the implant healing abutment (IHA), despite its vital role in soft tissue contouring and shaping after implant placement. Although these components are labelled by the manufacturer for “single-use only,” it is a common clinical practice to clean, sterilize, and reuse them.
In the present study, IHAs after single and multiple implantations were retrieved as per standard procedures, and biological material isolated from the surface was subjected to 16S rRNA sequence analysis. The microbiome analysis was followed by cleaning and sterilization in order to replicate clinical sterilization techniques. Following sterilization, retrievals were subjected to surface characterization with optical and scanning electron microscopy to investigate surface features, and electrochemical testing was performed to evaluate corrosion behavior.
The microbiota was comprised of early colonizers including Streptococcus species and secondary anaerobic colonizers such as Fusobacterium , Capnocytophaga , and Prevotella species. The surface analysis revealed that irrespective of the cleaning and sterilization techniques, the pristine, homogeneous surface of the new, unused IHAs could not be restored. Both single and multiple-use IHAs had severe surface changes including discoloration, major abrasions, biological contamination, and the IHA retrievals exhibited higher corrosion rate as compared to control specimens.
Reusing IHAs multiple times may not be a prudent practice as the microbial colonization and surface changes caused by using this component multiple times may affect the performance of IHAs in soft tissue healing.
Bacterial adhesion plays a significant role on dental implant performance as it can impact both hard and soft tissues, with colonization occurring within minutes following implant placement [ ]. It has been suggested that a successful dental implant outcome can be predicted based on a “race for the surface” between oral bacteria and soft tissue cells [ ]. In this regard, other components of the dental implant system are exposed to these conditions where the competition between bacteria and host cells can impact tissue healing and later implant integration. One component of the dental implant system, which is often overlooked due to its temporary placement, is the healing abutment. The implant healing abutment (IHA) is an important component as it remains exposed to the oral environment during the early healing phase. Thus, the IHA is exposed to a unique combination of conditions, with one-part supragingival and exposed to the oral cavity and the other part subgingival and in contact with soft tissue. In addition, this component is essential for soft tissue conditioning as it provides a scaffold for tissue growth [ ].
IHAs typically have smooth machined surfaces and are composed of commercially pure titanium (cpTi) due to its biocompatibility, corrosion resistance, and excellent mechanical properties [ , ]. These devices often undergo a surface treatment called anodization, which provides additional resistance to corrosion and better protects the bulk material when exposed to corrosive substances from diet, human saliva, and oral biofilms. The different types of IHA commercially available are chosen based on implantation procedures and specific protocols used by clinicians. The IHAs used in this study included both bone-level and tissue-level implants.
Placement of an implant body and the supporting components like the IHA subject the jaw bone and soft tissue to surgical trauma. This results in a cascade of immune responses both in the bone and surrounding soft tissues. The IHA is known to help in the formation of a long-standing biological barrier, thus allowing soft tissue healing after the placement of a dental implant body [ ]. Hence, the successful interface of IHA with soft and hard tissues during the early healing period is necessary to reduce complications that can later affect implant integration [ ].
In the literature, early implant complications are reported to occur due to synergistic factors like overloading or surgical error, micromotion, and bacterial contamination resulting in postoperative infection of soft and hard tissues [ ]. These complications can be initiated by surface adhesion of early bacterial colonizers which upon the formation of a biofilm may prevent sealing of epithelial tissues on the implant surface. The colonization of these pathogens leads to the formation of mature plaque and is considered to be the causative agent of later events such as peri-implantitis development. In particular, bacterial infiltration is a primary reason for both early and late stage complications [ , ]. At the implant body-IHA connection, bacterial leakage can contribute to corrosion and structural damage [ ].
The adhesion of bacterial biofilms at early stages post-implantation on implant components is of concern because it can alter the electrochemical conditions of the surface, especially in crevice areas around dental implant components. These peri-implant crevices are found to have some of the periodontopathogens in high abundance [ , ]. A reduction in pH due to the by-products of bacterial metabolism and other inflammatory processes (e.g., peri-implantitis, peri-mucositis) can create an acidic micro-environment that is ideal for Ti oxidation [ ]. Specifically, the acidity of the micro-environment within crevice areas accelerates the dissolution of the passive oxide layer, thereby causing localized destruction and crevice corrosion [ , ]. Moreover, pathogens residing within crevices due to implant modularity, such as at implant body-IHA surface, can produce corrosion products, which can potentiate the host inflammatory response and potentially trigger early complications [ , ]. Adherent bacteria can also generate crevice regions between the developing biofilm and the implant component surface [ ]. Hence, the role of bacteria present in the early healing period and their effects on the surface features of IHAs must be further investigated.
The current clinical practice is to clean, sterilize and re-use IHAs. The cleaning and sterilization procedures are not standardized but usually include steps such as mechanical wiping, ultrasonication, and steam autoclaving [ ]. However, such cleaning procedures may not be effective for IHAs. For instance, the screw thread could undergo bioburden despite ultrasonication which cannot remove all biological debris accumulated in the notch area. Plaque accumulation within the notch regions could affect the locking of IHA onto the implant body [ ]. Furthermore, several studies have indicated that a combination of mechanical and chemical cleansing is ineffective in complete removal of biological debris and biofilm from implants/abutments [ , , , ]. Also, multiple cycles could affect the biocompatibility of IHA surfaces and could result in fracture of the temporary components [ , ]. In addition, a retrieval study showed the presence of viable bacteria attached to IHA surfaces post-sterilization [ ]. Thus, utilizing used, cleaned IHAs on multiple patients could possibly result in cross-contamination and impaired healing. In order to assess the overall effectiveness of reusing IHAs, factors favoring the successful functioning of this temporary component need to be further investigated.
The goals of this study were to identify bacterial taxa colonizing IHAs during the early healing period and to elucidate the effects of multiple implantations on the surface characteristics of the IHA. The conjoint results of surface characterization and microbiome analysis can aid in better understanding of changes that IHAs undergo during the initial healing phase and the prospective adverse effects, if any, that may influence soft tissue health. Thus, the importance of this component should not be overlooked; in fact, the IHA can serve as a model to understand the microbiota of the early healing period and the competition between host cells and bacteria during this stage on implant components. The current study also provides insight into the unstable conditions of the oral cavity, post-placement, for the design of subsequent in vitro studies. Given the rationale for this study, it was hypothesized that IHA usage may have a role in microbial colonization dynamics. The single- and multiple-use IHAs would exhibit higher corrosion susceptibility and surface damage as compared to unused IHAs.
Materials and methods
Implant healing abutment sample preparation
Sixteen titanium IHAs (Straumann LLC, Basel, Switzerland) were obtained through a private dental practice in Dallas, TX. These components were received from human patients at periods of 3–6 months post-dental implant placement for research purposes, following standard procedures as per guidelines of the Helsinki Declaration. Prior to IHA removal, all patients signed a written informed consent to participate in the study. Patient information remained confidential with only the clinicians having access to health records and demographic information. Retrieval procedures, documentation, and IHA characterization were performed with strict guidelines and were approved by the Institutional Review Board at The University of Texas at Dallas (ER IRB #16-65). All the surgeries were performed by one calibrated surgeon to eliminate the variability produced by different surgical approaches. 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”. IHAs known to have been used more than once were labeled as “multiple-use”. For multiple-use IHAs, no records were maintained regarding the exact number of times the devices were implanted on different patients. All IHAs obtained post-implantation were marked with a letter for identification (A-P). Four unused IHAs were used as controls for surface characterization and electrochemical testing. Information about the IHAs used in this study is detailed in Table 1 .
|IHA||Use||Patient number||Type||Connection a||Corrosion score (1–3)|
Sample preparation and gDNA isolation
Immediately following retrieval, 16 IHAs were subjected to preparation steps for 16S rRNA sequence analysis. First, each IHA was immersed in 1.5 mL of 1X phosphate buffered saline (PBS) solution in a microcentrifuge tube. After 30−60 min, IHAs were subjected to vortexing for 30−60 s to detach adherent bacteria from the surface. The solution was centrifuged for 5 min at 1200 RPM to concentrate the bacteria. Total genomic DNA (gDNA) was isolated from the vortexed solution using the Ultraclean Microbial DNA Isolation Kit as per the manufacturer’s protocol (MO BIO CA, USA).
Polymerase chain reaction (PCR) and 16S rRNA sequencing
Of the 16 IHAs, 14 had detectable gDNA and were utilized for 16S rRNA sequence analysis. Illumina MiSeq library preparation and sequencing were performed at Molecular Research LP DNA Institute (Shallowater, TX). Briefly, the hypervariable V1–V3 region of the 16S rRNA gene was amplified using the 27F and 519R primers, with a barcode corresponding to the forward primer [ ]. DNA amplification was done using the HotStarTaq Plus Master Mix Kit (Qiagen, CA, USA) under the following PCR conditions: initial denaturing at 94 °C for 3 min, followed by 33 cycles of denaturing at 94 °C for 30 s, primer annealing at 53 °C for 40 s, elongation at 72 °C for 1 min, followed by a final elongation step at 72 °C for 5 min. Samples originating from the same DNA template were pooled together following PCR amplification. Samples were purified using calibrated Ampure XP beads (Agencourt Biosciences, Beverly, MA) and used to prepare the Illumina DNA library. Illumina MiSeq sequencing was performed per the manufacturer’s guidelines.
Sequencing analysis and taxonomic assignment
Microbiome analysis was done with the QIIME 2 version 2018.11 pipeline [ , ]. Sequences were demultiplexed, and Divisive Amplicon Denoising Algorithm 2 (DADA2) joined the paired-end reads, removed chimeras, and clustered the reads into Amplicon Sequence Variants (ASV) [ ]. Taxonomy was assigned to the ASVs using the SILVA high quality ribosomal RNA database (v.132). A heat map was generated using GraphPad Prism (v.8.2) by calculating the relative frequency of each taxa identified on each IHA. To identify taxa enriched in multiple-use or single-use IHAs, the following thresholds were applied: (1) at least 90% of the total reads obtained for the taxon must originate from either multiple-use or single-use IHAs; and (2) the taxon was detected in at least 50% of all multiple-use or single-use IHAs. Taxa for which >90% of reads originated from a single IHA sample were excluded.
Diversity analysis was calculated using QIIME 2 (v. 2018.11). Rarefication to improve even sampling was set at 1575 sequences per sample, and all of the IHAs used for microbiome analysis met this requirement (n = 14). Within sample diversity analysis (α-diversity) was assessed by quantifying the Shannon Diversity Index (H) and Pielou’s evenness index. The Shannon Diversity Index accounted for species richness and abundance, and Pielou’s evenness index identified how evenly distributed the ASVs were. Diversity between samples was assessed by both qualitative and quantitative phylogenetic β-diversity metrics. PCoA plots were generated with an unweighted and a weighted UniFrac [ ]. The unweighted UniFrac calculated diversity by the presence or absence of ASVs, and the weighted UniFrac accounted for the abundance of the present ASVs. Significance was assessed by the two-tailed t-test in GraphPad Prism (v.8.2).
Cleaning and sterilization
After recovery of gDNA for microbiome analysis, IHA retrievals were subjected to cleaning and sterilization to replicate clinical practice. IHAs were ultrasonicated with acetone, deionized (DI) water and 70% ethanol for 15−20 min each and steam autoclaved (20 min exposure, 2 h drying).
Optical microscopy (OM) was performed to observe topographical changes on the surface of the IHAs following cleaning and sterilization (Keyence VHX-2000). The surfaces of the samples were imaged at magnifications ranging from 25× to 500×.
Scanning electron microscopy
In order to evaluate the surface morphology of the specimens after single or multiple implantations, scanning electron microscopy (SEM) was performed. Areas of interest included those with evidence of chemical or physical degradation, which resulted in visible corrosion or wear on the surface of the IHA. An EVO LS 15 environmental scanning electron microscope (ZEISS, Oberkochen, Germany) was used in high vacuum mode and an accelerating voltage of 20 kV. Samples were observed at magnifications ranging from 20× to 2000×.
Based on visual observations derived from OM and SEM, IHA surfaces were scored and classified in terms of accumulation of biological debris and visible corrosion. The scoring ranged from 0 to 3, with 0 corresponding to no surface damage and 3 corresponding to the highest amount of surface damage as detailed in Table 2 . For biological debris accumulation, spots of debris, blood remnants, calcium deposits and bacterial plaque were identified and scored based on their frequency. Also, changes in the surface morphology in the form of discoloration, abrasions, and apparent surface roughness were labeled as surface degradation. For corrosion scoring, the relative amount of surface area containing discoloration, delamination and other deformities were scored.