Clinical, Immunological and Microbiological Improvements With Zinc-Coated Healing Abutments During the Healing Phase

Abstract

Purpose

The aim of this study was to evaluate the clinical, immunological, and microbiological effects of zinc-coated healing abutments (Zn-TiO₂ abutments) on peri-implant soft tissue as compared with the commercially used ones (Ti abutments).

Methods

The study was a prospective, non-randomised, single-centre, open-label, proof-of-concept clinical trial. The Ti and Zn-TiO₂ abutments were non-randomly connected to 2 neighbouring implants in the posterior region in each eligible patient. The bleeding-on-probing proportion (BOP%), probing pocket depth (PPD), the concentration of the pro-inflammatory cytokine (TNF-α, IL-6) in the peri-implant crevicular fluid (PICF), and the early microbial communities assessed by 16S rRNA sequencing were recorded.

Results

Eleven patients with 22 implants attended the 8-week examination. The BOP% was significantly lower in the Zn-TiO₂ abutments than that in the Ti abutments (24.23% ± 13.67% versus 42.42% ± 29.22%, P = .019). The concentration of TNF-α in PICF was significantly lower in the Zn-TiO₂ abutments than that in the Ti abutments (22.86 ± 11.21 versus 32.05 ± 16.28, P = .022). No significant differences in PPD and IL-6 were found between the two groups. Based on the microbiome assessments, higher microbial richness and lower presence of Lancefieldella were also observed in the Zn-TiO₂ abutments as compared with the Ti abutments.

Conclusion

Within the limitations of the study, the zinc-coated healing abutments improved early peri-implant soft tissue health clinically and immunologically. However, further studies are still needed to exclude the interference of soft tissue phenotype and confirm the relationship between microbial and clinical findings.

Introduction

The dental implant therapy has emerged as the preferred method for restoring missing teeth. However, inferior and delayed establishment of soft tissue integration around transmucosal regions would compromise the long-term success of dental implants.¹ Once exposed to the oral cavity, implants and supported prostheses became rapidly colonised with microorganisms. As the biofilms progress from initial stages to maturity, the intricacies of the microbial community increase, which may lead to further disease progression and even implant failures.² There is also strong evidence supporting the relationship between periodontitis histories and peri-implant diseases.^2,3

The antibacterial property of implant components has drawn considerable clinical attention. Among the components of the dental implant system, the implant healing abutments (IHAs), often overlooked because of their temporary nature, hold a promising role in preventing infections with the help of various modified surfaces.^4,5 During the second-stage surgery, the connection of the IHAs serves as a standard clinical protocol to establish transmucosal anatomic structures.⁶ After several weeks, IHAs are discarded before final restoration delivery.⁷ Although IHAs are typically characterised by their smoothly machined surfaces on commercially pure titanium or its alloys, the transmucosal part is susceptible to microbial proliferations and biofilm accumulations because of its exposure to the oral environment during the wound healing phase.⁸ In clinical practice, several inflamed soft tissue sites have been observed before prosthesis delivery. Therefore, biofilm control measures before prosthesis delivery have been suggested.^9,10 In fact, the absence of periodontal fibre within soft tissues around dental implants might lead to decreased defensive potentials and increased susceptibilities to oral pathogens. Thus, combined with the fact that patients with periodontitis have a higher rate of peri-implant mucositis, seeking an anti-infective treatment strategy around IHAs is crucial.^11,12

Strategies using nanostructured biomaterials have been developed to combat biofilm formation and promote tissue regeneration in dental applications.13, 14, 15 Among them, several have indicated that the incorporation of zinc on the surfaces of pure titanium and titanium alloys enhanced antimicrobial activities, including against multiple peri-implant disease pathogens.16, 17, 18 In addition, zinc has always been an essential micronutrient for human bodies, used widely in oral and plastic surgery studies.¹⁹ Previous studies have also shown that zinc-incorporated bioactive materials are promising wound dressings for the infected full-thickness wound healing.²⁰ More recent studies have found that a seal forms between abutments and the surrounding soft tissues when zinc oxide nanocrystals are synthesised on implant abutments.^21,22 However, whether the zinc-coated surface on IHAs promotes soft tissue integration has not been elucidated in clinical settings.²³ To better predict the potential development of peri-implant diseases, it is also important to gain a deeper understanding of the bacterial species profiles associated with different surfaces in the initial bacterial colonisation around dental implants.24, 25, 26 Advances in our understanding of the oral microbiome, particularly through the use of next-generation sequencing aided by artificial intelligence, are likely to lead to the identification of new species associated with peri-implant diseases.27, 28, 29

Therefore, we conducted this proof-of-concept clinical trial to assess the preliminary clinical performance and microbiological alterations after using zinc-coated implant healing abutments for the first time. Initially, we quantified the clinical parameters and the cytokine production around titanium IHAs with and without zinc coating during early healing stages after second-stage surgeries. Following this, we identified the microbial colonisation characteristics on two different abutment surfaces in the early oral biofilm formation stage. Among these, we have further validated the specific bacterial genus that the zinc coating potentially impacted, which may have potential implications on the early predictions of soft tissue status around dental implants. The hypotheses were that (1) the zinc coating on IHAs would affect the clinical parameters, cytokine levels, and early microbial communities; and (2) clinical parameters and cytokine levels around implant abutments would be correlated with the microbial composition.

Materials and methods

Study design

This study was designed as a prospective, non-randomised, single-centre, open-label, proof-of-concept study with 8-week follow-up to assess the clinical, immunological, and microbiological outcomes of Ti abutments and Zn-TiO₂ abutments in the posterior regions. Ethical approval for this research was granted by the Ethics Committee of Shanghai Ninth People’s Hospital affiliated with Shanghai Jiao Tong University (Approval Number: SH9H-2019-T158-5). The study was conducted strictly according to ethical principles (Declaration of Helsinki). The study protocol was also registered on the Chinese Clinical Trial Registry (Registration Number: ChiCTR2400088882). Written informed consent forms were obtained from each enrolled patient. This study also complied with the Transparent Reporting of Evaluations with Nonrandomized Designs (TREND) guidelines.³⁰

IHAs preparations

The titanium IHAs for the Astra Tech Osseospeed TX dental implants, referred to as Ti abutments in this study, were used as control. The zinc coating was applied to commercially used Ti abutments by using the micro-arc oxidation (MAO) method as detailed in previous studies.31, 32, 33 The titanium IHAs with zinc coating are referred to as Zn-TiO₂ abutments in this study. We prepared the titanium abutments by grinding them with a series of SiC abrasive papers, rinsing them using distilled water, and finally drying them using a stream of hot air. Subsequently, the samples were used as the anode, whereas the graphite immersed in the MAO electrolyte was used as the cathode. During the MAO treatments, the voltage and current were set at 100 V and 2.5 A, respectively. After MAO treatments for up to 180 seconds, zinc coatings with medium Zn content (3.61 wt%) were produced on pristine titanium oxide surfaces. The Zn-TiO₂ abutments were sterilised using dry-heating prior to clinical use.

Physical and chemical characterisations of Ti and Zn-TiO₂ abutments were performed as follows. Surface morphology and chemical composition were assessed using field emission scanning electron microscopy and X-ray photoelectron spectroscopy, respectively. Surface wettability was assessed using contact angle measurements. Surface roughness was investigated using a surface profiler. The zinc release profile was quantified‌ using inductively coupled plasma mass spectrometry (ICP-MS).

Participants

Partially edentulous patients receiving implant therapy and in need of IHA installations during second-stage surgery in the Department of Implant Dentistry at the Shanghai Ninth People’s Hospital between September and October 2023 were recruited. Each enrolled patient was informed in detail about treatments and signed the informed consent forms before IHA installation process.

The inclusion criteria were as follows: age ≥ 18 years; absence of oral infection and oral mucosal disease; lost 2 or more neighbouring teeth in the posterior maxilla or mandible; received the initially submerged (2-stage) implant installation procedures with Astra Tech Osseospeed TX dental implants; suitable for the installations of 2 or more IHAs sharing the same abutment configurations (including diameter, height, and connection design); and willingness to participate in this study.

The exclusion criteria were as follows: heavy smokers (≥10 cigarettes/day); uncontrolled periodontitis (residual pocket depth > 5 mm, full-mouth bleeding score > 25%, and full-mouth plaque score > 20%); and absence of systemic conditions contraindicating implant surgery, such as uncontrolled diabetes mellitus, pregnancy, current use of intravenous amino-bisphosphonates or history of radiation therapy in the head and neck region.

The sample size was estimated with the primary outcome of bleeding-on-probing rate (BOP%) reported by previous studies.⁴ It was determined that 11 individuals needed to be enrolled in each group (a 2-sided alpha of 0.05 and 80% power) after calculations performed using Tests for Paired Means in Power analysis and sample size (PASS) software.

Out of 18 potentially eligible patients, 11 individuals were finally included. Two neighbouring implants in each enrolled patient were planned to be connected using either Ti or Zn-TiO₂ abutments during second-stage surgery. The ratio of cases between the 2 groups was 1:1, with the 2 kinds of IHAs non-randomly allocated to implant sites. The implementation of a blinded design was not feasible because of the distinct gold-yellow hue of the Zn-TiO₂ abutments compared with the silver-grey Ti abutments, making it immediately apparent to both the clinician and the patient. Thus, this trial was open-label without blinding.

Interventions

The second-stage surgeries were conducted 3-6 months after the implant placement surgery. A crestal incision in the maxilla or mandible was made following local anaesthesia, without invading the mesial and distal interproximal papillae. After flap elevations, the Ti abutment and Zn-TiO₂ abutment were connected to osseo-integrated implants in each patient. During the installation of the IHAs, 15 N•cm torque was applied. Here, the 2 kinds of IHAs were non-randomly allocated to the 2 consecutive implant sites. The IHAs in the same patient shared identical abutment configurations; the only difference was the abutment surface. Oral hygiene instructions were given to each patient thereafter. All patients followed an identical postoperative care regimen for both abutment types.

Outcomes

Eight weeks after the second-stage surgery, clinical, immunological, and microbiological evaluations were performed before the delivery of the final restorations. The outcomes are listed as follows. Here, the primary outcome was the bleeding-on-probing rate (BOP%).

Clinical assessment

The evaluation of the peri-implant condition was based on BOP% and probing pocket depth (PPD). BOP and PPD were recorded at 6 sites per implant by applying 20-25g force with a 0.5-mm-diameter periodontal probe. Clinical parameters were measured by the same experienced examiner (95% concordance within 1 mm for measurements of PPD). The measurements were performed strictly in accordance with the Implant Dentistry Core Outcome Set and Measurement (ID-COSM) international consensus report.³⁴

Pro-inflammatory cytokine levels

Before collecting the peri-implant crevicular fluid (PICF), we isolated each IHA with sterilised cotton rolls and air-dried the mucosa. Afterwards, sterilised paper strips were used to collect the PICF surrounding each abutment (Figure 1). The pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-α), were detected by enzyme-linked immunosorbent assay (ELISA). The TNF-α (Catalogue No: PT518) and IL-6 (Catalogue No: PI PI325) assay kits were purchased from Beyotime Biotechnology. The specific methods for PICF collection and cytokine determination were detailed in previous studies.^35,36

Fig. 1.

Collection of peri-implant crevicular fluid (PICF).

Microbiota analysis

DNA was extracted from retrieved implant healing abutments using DNeasy Kits. We amplified the 16S rRNA V3-V4 regions from diluted DNA templates with Phanta Max Super-Fidelity DNA Polymerase. The final sequencing service was provided by DeepLab. 16S rRNA gene analysis was performed as described in our previous study.³⁷ Operational taxonomic units (OTUs) were generated from the processed reads by applying a 97% identity threshold through the USEARCH software package.³⁸ UPARSE analysis was used to remove the chimeric reads. USEARCH analysis was applied to classify the 16S rRNA gene fragment sequence and analyse the α and β diversity. The β diversity was assessed using the unweighted pair-group method with arithmetic means (UPGMA) clustering and non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity, with all analyses conducted using R software. Here, co-occurrences of abundant OTUs were explored using co-occurrence network analysis strategies. Correlations between OTUs were assessed using Spearman’s method. We kept strong correlations between OTUs’ relationships when P > .6 and significant correlations when P < .05.³⁹ Only OTUs with relative compositions >0.5% were used for further co-occurrence analyses and network constructions.⁴⁰

Statistical method

The normality of continuous data was determined by the normality test before choosing appropriate statistical analysis methods. Means, standard deviations (SDs), and Student’s t test were used for normally distributed data. Median, interquartile range (IQR), and Wilcoxon rank sum test were used for data inconsistent with normal distribution. Qualitative data were described as percentages and analysed by the χ² test. A 2-sided P value threshold < .05 was used to determine statistical significance. All statistical computations were carried out using SPSS software.

Results

Patients

A total of 18 patients in need of 2 or more IHA installations during second-stage surgery were screened for trial eligibility (Figure 2). Following screening, 11 patients (with 22 implants) who met the eligibility requirements and signed informed consent forms were enrolled (Figure 2). The Ti and Zn-TiO₂ abutments were non-randomly connected to 2 neighbouring implants in each participant. None of the participants were lost to follow-up or withdrew their consent (Figure 2).

Fig. 2.

Flow of participants through each stage of the study.

Table 1 summarises the demographic and clinical characteristics of all participants. No significant differences were observed between the 2 groups regarding implant site distribution or implant diameter (Table 1). The 2 types of IHAs in the same patient shared the same abutment configurations, including diameter, height and connection design (Table 1). This approach was essential to support the validity of outcome comparisons between the 2 groups.

Table 1.

Demographic and clinical characteristics of Ti and Zn-TiO₂ abutments.

	Ti (n = 11)	Zn-TiO2 (n = 11)	Total (n = 22)	pa
Gender (no. of male/female)	2/9	2/9	2/9	1
Age (years)	50.3±15.3	50.3±15.3	50.3±15.3	1
Implant sites
Pre-molars	4	4	8	1
Molars	7	7	14	1
Implant diameter
4.0 mm	6	6	12	1
4.5 mm	5	5	10	1
Abutment diameter
4.5 mm	6	6	12	1
5.5 mm	5	5	10	1
Abutment height
2.0 mm	2	2	4	1
4.0 mm	8	8	16	1
6.0 mm	1	1	2	1

^aStudent's t-test and χ² test.

Physicochemical characterisation

The nanoscale surface topography of the Zn-TiO₂ abutments, as demonstrated by FE-SEM, showed a typically porous structure, suggesting that the nano coatings were successfully developed (Supplementary Figure 1a). The Zn 2p peaks at 1021.3 eV and 1044.3 eV as demonstrated by XPS confirmed the doping of zinc in the Zn-TiO₂ abutments (Supplementary Figure 1b). The TiO₂ abutments demonstrated a significantly lower contact angle compared with the Ti abutments (62.5° ± 1.1° versus 48.3° ± 0.7°, respectively, P < .001), indicating better hydrophilic properties of the TiO₂ abutments (Supplementary Figure 1c). The Zn-TiO₂ abutments exhibited a marginally higher surface roughness compared to the Ti abutments (103.24nm ± 15.18 nm versus 150.13 nm ± 11.64 nm, respectively, P = .015) (Supplementary Figure 1d). The release of zinc from the Zn-TiO₂ abutment constantly maintained at a quite low level as demonstrated by the results of the ICP-MS (Supplementary Figure 1e).

Clinical assessment

BOP% in the Ti abutments was significantly higher than that in the Zn-TiO₂ abutments (42.42% ± 29.22% versus 24.23% ± 13.67%, respectively, P = .019) (Figure 3A). PPD values did not differ significantly between the Ti and Zn-TiO₂ abutment groups (2.89% ± 0.94% versus 2.80% ± 0.87%, respectively, P = .413) (Figure 3B). Table 2 presents a summary of all clinical assessment outcomes.

Fig. 3.

Comparison of the bleeding on probing proportion (BOP%) (A), and probing pocket depth (PPD) (B) between Ti and Zn-TiO₂ abutments at the 8-week examination.

Table 2.

Clinical parameters of Ti and Zn-TiO₂ abutments at 8-week examination.

	Ti (n = 11)	Zn-TiO2 (n = 11)	P
BOP%	42.42 ± 29.22	24.23 ± 13.67	.019*
PPD (mm)	2.89 ± 0.94	2.80 ± 0.87	.413

^⁎P < .05

PICF assessment

A comparison of cytokine concentrations in the PICF between the 2 groups is presented in Table 3 and illustrated in Figure 4. The concentration of TNF-α in the Ti abutments was significantly higher than that in the Zn-TiO₂ abutments (32.05 ± 16.28 versus 22.86 ± 11.21, respectively, P = .022) (Figure 4A). IL-6 concentrations did not differ significantly between the Ti and Zn-TiO₂ abutments (13.68 ± 6.00 versus 12.30 ± 6.89, respectively, P = .469) (Figure 4B).

Table 3.

Concentrations of cytokines in PICF (pg/ml) of Ti and Zn-TiO₂ abutments.

	Ti (n = 11)	Zn-TiO2 (n = 11)	P
TNF-α	32.05 ± 16.28	22.86 ± 11.21	.022*
IL-6	13.68 ± 6.00	12.30 ± 6.89	.469

^⁎P < .05

Fig. 4.

Comparison of the concentrations of TNF-α (A) and IL-6 (B) between Ti and Zn-TiO₂ abutments at the 8-week examination.

Microbiome assessment

Genome counts

A total of 22 IHAs from 11 patients were retrieved and sampled at 8 weeks after the second-stage surgeries. Following assembly and quality filtering, 3,492,730 high-quality reads were generated. Subsequent analysis assigned these reads to 1,034 bacterial operational taxonomic units (OTUs), defining OTUs at a 97% similarity level. Observed OTU counts varied substantially across all samples, ranging from 32,672 to 425,912, with a mean of 144,889 (Table 4). To account for the influence of sequencing depth on richness estimates, rarefaction analysis was performed to normalise all community samples to an equivalent sequencing depth. As shown in Supplementary Figure 2, the rarefaction curves for observed OTUs appeared to approach asymptotes, which was indicative of a high level of coverage as well as a sufficient sample size.

Table 4.

OTU counts and different diversity indices of bacteria in the biofilm samples.

Collection sites	Index
	OTUs	Richness	Chao1	Shannon	Simpson
Ti
Patient 1	85709	603	680.5	5.66	0.0519
Patient 2	62274	507	625.1	3.67	0.257
Patient 3	192199	580	643.2	3.87	0.175
Patient 4	63349	561	663.2	5	0.112
Patient 5	333817	645	747.1	5.24	0.0601
Patient 6	32672	529	587	5.43	0.123
Patient 7	101812	602	669.9	4.88	0.156
Patient 8	94484	671	738.6	4.27	0.233
Patient 9	87975	575	677.4	4.9	0.103
Patient 10	104691	453	539.8	2.05	0.619
Patient 11	359541	748	811.5	5.87	0.035
Zn-TiO2
Patient 1	94998	627	713.4	5.18	0.0676
Patient 2	83793	627	676.7	5.7	0.0701
Patient 3	75088	567	701	4.84	0.1
Patient 4	142466	630	714	4.11	0.214
Patient 5	299350	745	839	5.88	0.0466
Patient 6	125075	616	686.6	6.52	0.0241
Patient 7	121782	661	756	4.82	0.134
Patient 8	77920	585	649.3	3.5	0.345
Patient 9	138043	637	749.3	4.35	0.148
Patient 10	84629	610	697.7	4.33	0.209
Patient 11	425912	794	866.3	4.29	0.205

Alpha-diversity comparisons

Alpha diversity metrics are summarised in Figure 5 and Table 4. Microbial richness, defined as the number of distinct microbial types within a community, differed significantly between the Ti and Zn-TiO₂ abutments (588 ± 80 versus 645 ± 67, respectively, P = .017) (Figure 5A). The chao1 and richness indices exhibited a comparable pattern, although their values varied between the 2 substrates. The chao1 index in the Ti abutments was also significantly lower than that in the Zn-TiO₂ abutments (671.2 ± 75.7 versus 731.7 ± 67.1, respectively, P = .008), confirming a higher species richness in samples from the Zn-TiO₂ abutments relative to the Ti group (Figure 5B). The Shannon index, a metric of community diversity and evenness, ranged from 2.05 to 6.52 across IHA samples, reflecting considerable alpha-diversity in most cases (Figure 5C). Although both the Simpson and Shannon indices followed comparable trends, the extent of variation was unique to each metric. The Shannon and Simpson estimators did not differ significantly between the Ti and Zn-TiO₂ abutment groups, suggesting that the zinc-based coating exerted minimal influence on early microbial community diversity (Figure 5D).

Fig. 5.

Alpha diversity metrics of richness (A), chao1 (B), Shannon (C), and Simpson (D) in the retrieved Ti and Zn-TiO₂ abutments at the 8-week examination.

Beta-diversity comparisons

To compare the microbial community structures attached to IHAs, the overall bacterial compositions of each IHA were compared respectively and visualised in a PCoA plot based on their Bray-Curtis distances, weighted UniFrac distances, and unweighted UniFrac distances (Figure 6). The PCoA plot was generated with 2 principal axes (PCoA1 and PCoA2), which respectively explained 22.8% and 15.6% of the variance between the bacterial communities of Ti abutments and those of Zn-TiO₂ abutments (Figure 6). The PCoA plot did not show any obvious clustering between the Ti and Zn-TiO₂ abutments, indicating that these 2 groups had similar microbiome compositions (Figure 6).

Fig. 6.

Beta-diversity comparisons based on principal co-ordinates analysis (PCoA) for the retrieved Ti and Zn-TiO₂ abutments at the 8-week examination.

Bacterial community compositions at different taxonomic levels

Sequence analysis of clinical samples identified bacterial communities spanning 12 phyla and 75 genera. At the phylum level, the most dominant phyla were Proteobacteria (52.3%), Firmicutes (14%), Bacteroidetes (13.2%) and Actinobacteria (11.6%) (Figure 7A). The relative abundance of Firmicutes and Actinobacteria in the Zn-TiO₂ abutments appeared to be higher than that in the Ti abutments (14.8% versus 13.2% and 13.1% versus 10.2%, respectively) (Figure 7B). The relative abundance of Proteobacteria and Bacteroidetes in the Zn-TiO₂ abutments appeared to be lower than that in the Ti abutments (50.9% versus 53.7% and 11.7% versus 14.6%, respectively) (Figure 7B).

Fig. 7.

The relative abundance of microbiota attached to the retrieved Ti and Zn-TiO₂ abutments at the different levels. A, Microbiota for each IHA at the phylum level. B, Microbiota for both groups at the phylum level. C, Microbiota for each IHA at the genus level. D, Microbiota for both groups at the genus level.

At the genus level, the most dominant genera were Neissera (30.6%), Lautropia (13%), Rothia (7.2%), Prevotella (6.9%), and Veillonella (6.7%) (Figure 7C). The relative abundance of Lautropia, Rothia, and Veillonella in the Zn-TiO₂ abutments appeared to be higher than that in the Ti abutments (13.9% versus 12.1%, 7.9% versus 6.4% and 7.5% versus 6.0%, respectively) (Figure 7D). The relative abundance of Neissera and Prevotella in the Zn-TiO₂ abutments appeared to be lower than that in the Ti abutments (29.3% versus 31.8% and 5.9% versus 7.7%, respectively) (Figure 7D).

A model based on the negative-binomial (NB) distribution with P = .05 was proposed to analyse the genus differences between Ti and Zn-TiO₂ abutments (Figure 8). The model, in which the parameters of test—Wald and fitType—parametric were applied to DESeq2 to identify the differences at the genus level, indicated that the relative abundance of Lancefieldella in the Zn-TiO₂ abutments was significantly lower than that in the Ti abutments (Figure 8). At the species level, the relative abundances of Lancefieldella parvula, Lancefieldella rimae, and unclassified Lancefieldella in the Ti abutments were higher than those in the Zn-TiO₂ abutments (Table 5).

Fig. 8.

The model based on the negative-binomial distribution illustrated the difference in the Lancefieldella abundance between the Ti and Zn-TiO₂ abutments.

Table 5.

The relative abundance of Lancefieldella at the species level.

	Ti (n = 11)	Zn-TiO2 (n = 11)
Lancefieldella parvula	0.027% ± 0.061%	0.010% ± 0.009%
Lancefieldella rimae	0.099% ± 0.208%	0.018% ± 0.028%
Unclassified Lancefieldella	0.013% ± 0.012%	0.006% ± 0.009%

Discussions

The primary null hypothesis tested in the present study was that there were no significant differences in the peri-implant marginal tissues health status around the Ti or Zn-TiO₂ abutments. The impact of systemic medications on peri-implant tissues, as highlighted by Fiorillo et al., added weight to the rationale for our localised metal ion strategy.⁵ To validate this hypothesis, clinical examinations, PICF assessments, and microbiota analysis were carried out. The Zn-TiO₂ abutments were found to be associated with a lower rate of soft tissue inflammations, as evidenced by BOP% and the TNF-α level. At the microbiome level, the Zn-TiO₂ abutments were shown to be associated with high microbial abundance and a significant decrease in the abundance of Lancefieldella, the oral bacteria with potential pathogenicity. Our proof-of-concept clinical study has demonstrated that zinc-coated abutments contributed to the health of peri-implant tissue, boding well for zinc’s potential use in the prevention of peri-implant diseases.

In this study, no significant difference in PPD between the Ti and Zn-TiO₂ abutments was observed at 8-week post-surgery examination, which may be partly owing to the relatively short duration of our observation.⁴¹ Although initial worsening of the health in peri-implant conditions was challenging to diagnose, BOP% allowed for early clinical monitoring of a shift from a non-pathologic to pathologic status.^42,43 Typically, BOP% would significantly increase in the presence of soft tissue inflammation.⁴² Here, the measurement of BOP% aligned with the international consensus of the ID-COSM, which our study strictly adhered to for clinical parameter assessment.³⁴ In this study, BOP% was 24.23% in the Zn-TiO₂ abutments and almost 2-fold higher (42.42%) in the Ti abutments, indicating that zinc-coated abutments may promote peri-implant tissue health and lower the incidence of peri-implant mucositis.³⁵ PICF analysis, particularly the concentrations of pro-inflammatory cytokines such as TNF-α and IL-6, provided a non-invasive approach for detecting inflammatory status in peri-implant tissues.^44,45 Overproduction of TNF-α and IL-6 was consistent with the presence and progression of inflammation.^35,43 In the current study, the TNF-α concentration was lower in the Zn-TiO₂ abutments than that in the Ti abutments, whereas no significant differences in the IL-6 concentration were observed between the Zn-TiO₂ and Ti abutments. The pro-inflammatory cytokine levels might indicate a decreased susceptibility to bacterial infections and a reduced risk of additional bone resorptions in the Zn-TiO₂ abutments.³⁵ The lower TNF-α levels, coupled with reduced BOP%, suggested that the zinc-coated abutments actively attenuated the inflammatory response in the peri-implant soft tissue. This modulation of the local immune environment was crucial, given that it may enhance‌ the formation of a stable mucosal seal and reduce the long-term risk of inflammatory peri-implant diseases. Hence, the preliminary results of clinical and PICF assessments proved that the zinc-coated implant healing abutments may benefit the peri-implant soft tissue health.

The 16S rRNA sequencing here enabled high-throughput bacterial profiling, offering a comprehensive characterisation of the early oral microbiome that could not be achieved through culture-dependent methods or hybridisation-based techniques.²⁴ Here, peri-implant microbiomes of both Zn-TiO₂ and Ti abutments exhibited relatively high levels of species richness as well as diversity. Our findings align with previous sequencing-based studies of the peri-implant microbiota, which consistently reported elevated microbial diversity at both healthy and diseased implant sites.⁴⁶ Altogether, 1,034 bacterial OTUs were recorded in our sample set, which captured 75 named genera. Furthermore, we also found that the microbiome of biofilms formed on the implant healing abutments appeared to be influenced by the presence of zinc coating, which was in accordance with the study by Canullo et al.⁴⁷ The Zn-TiO₂ and Ti abutments presented significant differences in terms of species richness. The chao1 and richness indices in the Zn-TiO₂ abutments were significantly higher than those in the Ti abutments, indicating a larger number of microorganisms presented in the investigated communities of Zn-TiO₂ abutments. These microbiome results were quite interesting, partly meaning that the chemical and physical characteristics of the IHAs’ surface may strongly influence microbial colonisation during the early healing stage. This finding is clinically significant because the extant literature has shown that peri-implantitis typically progressed over approximately 5 years before manifesting as overt clinical complications.⁴⁸

It has been previously recognised that the phyla associated with peri-implantitis were Spirochaetes, followed by Synergistetes, Firmicutes, Bacteroidetes, Fusobacteria, Chloroflexi, and Tenericutes, whereas Actinobacteria were closely related to the health status.⁴⁹ In this study, Bacteroidetes were more abundant in the Ti abutments, whereas Actinobacteria were abundant in the Zn-TiO₂ abutments, which is indicative of a zinc coating having a positive effect on peri-implant microbiomes, reducing soft tissue inflammation. The genera that most commonly appeared to be increased in soft tissue inflammation were Porphyromonas, Treponema, Prevotella, Filifactor, Tannerella, and Fusobacterium, whereas Streptococcus and Rothia, followed by Haemophilus, Corynebacterium, Capnocytophaga, Leptotrichia, and Actinomyces, were those closely related to the health status.⁵⁰ Our results showed that Prevotella were more abundant in the Ti abutments, whereas Rothia and Actinomyces were more abundant in the Zn-TiO₂ abutments. Analysis of microbiome data here suggested that the bacteria surrounding implant healing abutments formed a diverse microbiome and that a zinc coating might have a positive effect on the peri-implant microbiome, reducing the risk of soft tissue inflammation. Nevertheless, further investigation is needed to clarify the effects of the zinc coating on the abundance of the pathogen genera in the oral environment.

Microbiome analyses were further explored in depth at the genus and species levels to identify the microbial community differences between Ti and Zn-TiO₂ abutments. Notably, the relative abundance of Lancefieldella belonging to the phylum Actinobacteria was significantly higher in the Ti abutments than that in the Zn-TiO₂ abutments. It was widely acknowledged that the Lancefieldella, formerly known as Atopobium, were anaerobic gram-positive coccobacillus, including parvula, rimae, and unclassified species. Lancefieldella could be detected in a healthy periodontal condition.⁵¹ However, recent studies have revealed a significantly higher abundance of Lancefieldella in peri-implant diseases.^2,52 This finding was in accordance with our results to some extent, in which the early microbial communities of the Zn-TiO₂ abutments with lower BOP% and TNF-α level have exhibited a reduced volume of Lancefieldella in a clinical scenario. At the species level, Lancefieldella parvula accounted for the highest proportion of Lancefieldella in the IHAs microbiome, and the relative abundances of Lancefieldella parvula in the Zn-TiO₂ abutments were lower than those in the Ti abutments. The Lancefieldella parvula has been definitely detected in some oncological diseases in previous studies.53, 54, 55 The Lancefieldella parvula, the predominant inhabitant of dorsal tongue and saliva, was also found to be closely related to halitosis/oral malodour.⁵⁶ Moreover, Lancefieldella parvula was associated with occlusal lesions in caries and persistent endodontic lesions as evidenced by recent documents.^57,58 The Lancefieldella rimae was also less abundant in the Zn-TiO₂ abutments as compared with its volume in the Ti abutments. It was reported that the relative abundance of Lancefieldella rimae was higher in periodontal abscesses than in healthy periodontal conditions.⁵⁹ Veras et al. have found strong evidence supporting Lancefieldella rimae as a periodontal pathogen.⁶⁰ Recently, Na et al. have suggested that Lancefieldella rimae in periodontal diseases might be one of the potential pathogens aggravating Alzheimer’s disease.⁶¹ Thus, the synergistic reduction in Lancefieldella abundance alongside improvements in BOP% and TNF-α levels in our study suggested a plausible linkage. We hypothesised that the zinc coatings might have directly or indirectly suppressed this putative pathogen, thereby contributing to a less inflammatory microenvironment, which manifested as improved clinical and immunological outcomes. In other words, the lower inflammation index values might be associated with the lower abundance of Lancefieldella in the Zn-TiO₂ abutments. These results point to the potential beneficial effects of zinc coating for IHAs to prevent Lancefieldella-related peri-implant inflammation. However, the established relationship between the inflammation index and the abundance of Lancefieldella requires further studies.

Our findings revealed that the peri-implant microbiome maintained considerable diversity throughout the first 2 months following healing abutment placement, underscoring the necessity of implementing biofilm control strategies at the early stages of IHA connection. Pre-treatment of implant healing abutments with anti-infective agents demonstrated promise as a strategy for promoting optimal soft tissue growth and facilitating the establishment of a mucosal seal, thereby enhancing resistance to infectious challenges and reducing subsequent biological complications.^47,62 Previous studies have suggested that the controlled release of zinc ions from zinc-coated surfaces may help to modulate immune responses around peri-implant soft tissues and improve dental implant survival rates.⁶³ Han et al. reported that zinc-coated surfaces promoted gingival tissue adhesion to the implant, potentially supporting the development of a resilient mucosal barrier that helped to prevent bacterial infiltration.⁶⁴ Furthermore, Odatsu et al. have pointed out that adding 10 wt% zinc to bioactive glass can enhance the environment around implant abutments by providing antibacterial, antioxidant and antifibrotic effects, showing potential for clinical use.²² Our study’s findings of decreased BOP% and TNF-α levels in the Zn-TiO₂ abutments compared with the Ti abutments suggest that zinc coating can attenuate the IHAs-related inflammation to some extent. The evaluation of microbial communities should be incorporated into preclinical and clinical trial protocols, because microbial analysis offers more precise insights than standard clinical parameters alone. Here, a significantly higher microbial richness was confirmed on the Zn-TiO₂ abutments compared with the Ti abutments, thus justifying the inference that differences in clinical parameters and pro-inflammatory cytokine levels may be related to the microbiological findings on the IHAs surfaces. Additionally, our study detected a reduced number of Lancefieldella in the zinc-coated abutments, but this finding requires future research using larger samples to confirm the relationship between Lancefieldella and peri-implant soft tissue inflammation.

In brief, our proof-of-concept study showed that zinc-coated IHAs affect peri-implant health in terms of clinical, immunological and microbiological findings, filling the gap in literature on this subject. This study validated the effects of physical and chemical characterisations of IHAs surfaces on peri-implant marginal tissue health status in a clinical scenario. It also showed that 16S rRNA sequencing of the entire IHAs sample instead of the traditional paper tip sample provided a more comprehensive view of the bacterial landscape around IHAs during the critical healing phase. Given that the microbial biofilm adheres closely to IHA surfaces, early colonisation of potential pathogens, such as Lancefieldella, could be better detected by using the entire IHA sample.

This study has several limitations, primary involving its modest sample size and the absence of assessments regarding peri-implant soft tissue phenotype that would possibly yield more specific data on mucosal thickness and keratinised mucosa width. In our study, individualised selection of the abutment height varied from 2 mm to 6 mm and mainly depended on the mucosal thickness, introducing selection bias. Previous studies have indicated that deeper mucosal tunnels tend to harbour more bacteria.⁶⁵ Future studies should also examine the effects of dry heating on Zn-TiO₂ abutments surfaces, particularly given that Morsy et al. have shown that repeated moist heat sterilisation alters the titanium interface.⁶ In addition, the assessment of outcomes at a single time point (8 weeks), chosen to standardise the evaluation at the end of the healing phase before prosthesis delivery, prevented a dynamic assessment of the clinical, immunological and microbiological alterations. Thus, increasing the sample size and adjusting the groupings would better exclude confounding factors, such as the effects of soft tissue phenotype, on peri-implant health and microbial communities. Future studies involving larger sample sizes and more refined patient cohorts would help to elucidate the effects of zinc-coated healing abutments on the implant healing process.

Conclusion

Within the limitations of this proof-of-concept study, it can be concluded that zinc coating on implant healing abutments has a relatively positive effect on peri-implant marginal tissue health in the short term based on lower BOP% and decreased TNF-α levels. Higher microbial richness and lower levels of presence of Lancefieldella were also observed in the zinc-coated abutments, but their specific relationship with clinical and immunological outcomes of the peri-implant soft tissue requires further studies.

Author contributions

Shi-chong Qiao and Jie Zhou: Conceptualization; Yu Zhu and Shu-li Liu: Methodology; Yu Zhu, Shu-li Liu, Jie Zhou and Shi-chong Qiao: Data curation; Yu Zhu and Shu-li Liu: Writing–original draft; Yu Zhu, Shu-li Liu, Jie Zhou and Shi-chong Qiao: Funding acquisition; All authors: Investigations and Review.

Funding

This study was supported by the Natural Science Foundation of Shanghai (No. 24ZR1443800 and No. 24ZR1443200), Fundamental research program funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong university School of Medicine (No. JYZZ240 and No. JYZZ233), Fundamental Research Funds for the Central Universities (No. 2682024ZTPY002), and Science and Technology Commission of Shanghai Municipality (No. 20S31906100).

Conflict of interests

None declared.