Effect of argon plasma pre-treatment of healing abutments on peri-implant microbiome and soft tissue integration: a proof-of-concept randomized study

Implant therapy is a gold standard treatment for restoration of missing teeth, based on outstanding functional and aesthetic properties it provides to the patient, while preserving adjacent teeth and improving the oral health-related quality of life [1]. However, the constant exposure of dental implants to the abundant oral microflora may affect the quality of the soft and bone tissue integration, while increasing their susceptibility to peri-implant infection. Peri-implantitis is defined as a chronic inflammatory disease of peri-implant tissues, which is induced by multiple etiologic factors, and in particular by dysbiotic bacterial biofilms accumulated on implant surface [2]. This disease is very burdensome for doctors and patients and with tremendous growth of the implant market. Consequentially, peri-implantitis is currently qualified as emerging public health problem due to increasing prevalence (12–45%) and lack of predictive treatment [3, 4]. Therefore, the prevention of peri-implant infection remains the major priority in management of implant patients [5, 6].

Since soft periodontal and peri-implant tissue are the first line defenders against infective threats (60), it is understandable that lack of periodontal ligament comprehensibly represents the Achilles heel of dental implants making them more susceptible to infection and related immunopathologies [8, 9]. The key-elements of the peri-implant soft tissue phenotype (PiSP) responsible for long-term implant success are mucosal thickness (MT), keratinized mucosa width (KMW), and/or supracrestal height (STH) [10]. In context of long-term peri-implant stability, thick tissues and wider KMW better preserve soft and hard peri-implant tissues, by preventing recession, bacterial accumulation and apical penetration, while also stabilizing peri-implant bone remodeling [11‐13]. However, the PiSP is site-specific and susceptible to changes over the time, as well as environmental factors [10]. In this context, the microbial factors lately receive an increasing attention. Besides its precursor role in future infections, the low-grade inflammation associated with implant surface colonization negatively modulates the healing processes within implant integration affecting its respective solidity and functionality [7, 14]. In brief, the biofilm formation on implant surfaces followed by colonization by pre-existing oral bacterial species occurs within the first minutes of post-implantation. This process is additionally facilitated by implant surface micro and macro topography [15]. The pre-existing species also consist of keystone periopathogens, the inflammophilic bacteria with a prominent immunoevasive capacity, exploiting the immunity for their own growth while shifting the local microbiome into dysbiotic microflora [16, 17]. In context of the healing processes, periopathogens such as Porphyromonas gingivalis and Treponema denticola interfere with macrophage polarization and cause their shifting from M2 pro-healing to M1 pro-inflammatory course with deleterious effects on the quality of soft tissues and bone implant integration [18‐20]. Regarding soft tissues specifically, bacterial colonization of implant surfaces prevents sealing of epithelial cells and down-regulates fibroblast proliferation [7, 14]. From a functional aspect, the lack of periodontal ligament decreases the overall barrier capacity of soft peri-implant tissues and impairs immunological control over local microflora [21, 22], thus facilitating biofilm accumulation, its apical migration, and conversion into more resistant anaerobic population. Due to these reasons, biofilm-free implant surface is considered a key factor to promote successful implant integration.

Implant healing abutments play a pivotal role in implant integration and shaping of supporting soft and hard tissues [14], thus considerable research has been placed on designing and implementing surface treatments with anti-biofilm properties [21‐23]. Ghinassi and colleagues noted an improved response of the soft tissues surrounding the laser treated surfaces [24]. Implant surface plasma pre-treatment (PT) is considered promising protocol for dental implants based on their potent anti-biofilm properties, pro-conductive effects on both osteoblasts and fibroblasts and pro-angiogenic effects, collectively providing the optimal conditions for soft and bone integration [23‐26].

Previous studies showed that PT removes surface contaminants from titanium abutments more effectively than other available methods, providing a clean surface suitable for cell adhesion [27]. From the physicochemical aspect, this treatment provides surface activation at the atomic and molecular level, enhancing the wettability, cell adhesion, and concomitant spreading to the dental implant surface [28‐30]. Moreover, glow discharge PT induces a net-negative charge on the implant surface oxide increasing the functional presentation of the integrin-binding domain, while promoting cell attachment within proportionally accelerated hard and soft tissue integration [31‐34]. In the context of dental implants, the unique advantages of PT are its universal applicability to all types of healing abutments, irrespective of their original physicochemical characteristics, and its wide antimicrobial capacity, even against highly virulent bacteria associated with peri-implantitis [32, 35, 36]. In context of soft tissues, a series of in vitro studies showed the capacity of argon plasma to increase migration, proliferation, and adhesion of human fibroblasts to the implant surface, as well as improving effects on soft tissue integration and angiogenesis [37‐39]. While an increasing number of in vitro and pre-clinical studies have been published on this treatment, so far very few clinical studies have reported on the outcomes of PT in implantology. Additionally, although the use of high throughput methods is expected to improve the quality of research studies and to accelerate the progress and clinical translation of new biomaterial-based treatments, the use of omics methods is still very rare in periodontology and implantology [40, 41].

Therefore, we hypothesize that pre-treatment of healing abutments by means of argon plasma may positively affect peri-implant microbiome and soft tissue healing processes providing favorable conditions for optimal peri-implant soft tissue integration.

Primary objective of the present study was to estimate the effect of argon plasma pre-treatment (PT) of the healing abutments on peri-implant soft-tissue clinical parameters and microbiome following an initial phase of healing by comparing the clinical and microbiological parameters between treated and non-treated healing abutments after a 3-months healing period. The secondary aim was to investigate the effects of PT of healing abutments with different surface topography (smooth and rough) on soft tissue integration.

The final sample size consisted of 30 periodontally stable patients that completed the study follow-up since four patients (one in each group) had to be excluded because of antibiotic intake during the follow-up period, while two patients (PT-MACHINED) were excluded based on NGS quality control criteria (DNA amplification cycle threshold value greater than negative control > 35). Due to limited sample size, the microbiome analysis is provided only for primary outcome variables, while the microbiome outcomes for the healing abutments with different topographies were not considered for interpretation but they are provided as Additional file 1. Demographic and clinical parameters by groups are shown in Table 1.

Present study demonstrated that argon plasma pre-treatment of healing abutments contributes to the lower soft tissue inflammation and plaque accumulation, Further, it prevents advanced biofilm formation by favorizing predominance of pioneer colonizers over late colonizers that were in turn significantly more abundant around non-treated abutments. PT did not affect soft tissue phenotype at the clinical level, but at the microbiome level was established the association between S. gordonii and thin tissue in PT, and regarding KMW, the abundance of periopathogenic bacteria including Prevotella oris and Fusobacterium nucleatum were observed in patients with narrower KMW in PT.

The advances in biofilm research driven by breakthrough in molecular techniques have changed the face of oral microbiology, revealing far higher diversity of peri-implant microflora with complex interactions and functional behavior in both health and disease [43, 44]. In the context of antimicrobial materials, it is considered that microbiome research may pave the way for optimal anti-infective treatments by unveiling key treatment targets while allowing highly accurate estimation of treatment effectiveness [40, 45]. Finally, it is considered that omics methods are gamechangers in tackling a double-sided complexity of biological systems and material properties, able to provide the highly accurate and sensitive assessment of material-induced biological effects, accelerating its clinical translation for therapeutic use [41, 46]. Hence, the clinical metagenomics becomes indispensable tool for assessment of infective pathologies and their responsiveness on performed treatments in dentistry [47]. The present study has demonstrated a highly diverse microbiome consisting of 283 different bacterial species around healthy implants following a period of initial integration, as previously reported [11, 40, 41]. Within these species, both early and late colonizers, as well as key pathogens were already present two months post-implantation. This finding is in discordance with previous reports that showed presence of key pathogens only six months following implantation [20]. These findings once again confirm the importance of rigorous infection control from the initial healing stage following implant placement [7]. The fungal microbiome is currently in the spotlight of biofilm research related to prominent capacity of fungal species to form polymicrobial biofilms with a diverse spectrum of bacterial species (particularly with streptococcal species) thus contributing to the accumulation and maturation of dysbiotic biofilms [48, 49]. The mycobiome was generally less diverse than bacterial community and Malassezia restricta represented the most prevalent fungi species as previously reported [50, 51], although its exact role in the oral ecosystem is still unknown.

PT improved the clinical parameters of soft tissue inflammation and plaque accumulation, and positively affected peri-implant microbiome, preventing the growth of late colonizers and related biofilm maturation. PT exhibited higher abundance of pioneer or early colonizers such as S. gordonii and S.mitis [46, 47] as well as Actinomyces known to successfully congregate with S. gordonii [52]. In contrast, the NPT group showed a higher abundance of late colonizers like Neisseria oralis [53] suggesting an advanced stage of biofilm formation in NPT. The higher abundance of bacterial species on plasma treated surfaces might be explained by the fact that this treatment increases the surface wettability, which increases cell-attractiveness, even though this treatment was clearly associated with lower plaque accumulation and less inflammation. Future studies are however needed to decipher the specific role of S. gordonii, which is considered a member of the healthy microflora, but also forms synergistic biofilms with Parvimonas micra and Fusobacterium nucleatum, which in turn are associated with peri-implantitis [42, 54]. Given the fact that Fusobacterium nucleatum was also identified as a member of the core microbiome, shows the importance of timely biofilm control. The high-dimensional class comparison using LEfSE method is praised for its capacity to determine the microbiological characteristics/associations relevant for understanding the inter-group differences and the biological relevance/impact on oral health [55]. In this study we found a significant co-occurrence between S.mitis and Rothia dentocariosa and co-exclusion between S.mitis and pathogenic species including P. micra, Porphyromonas endodontalis, and Prevotella oris. This confirms the pattern of peri-implant health associated with Gram-positive cocci and pathological conversion that follows the competitive shift caused by anaerobic species [56, 57]. On the other hand, the NPT group showed a significant increase in plaque accumulation and higher inflammation index values, which were associated with Tanerella forsythia and P. oris comparable to peri-implant mucositis [58, 59]. These results point to the beneficial effects of PT in preventing plaque-induced peri-implant inflammation. However, the more prevalent thin tissue and its established association with abundance of S. gordonii in the PT group requires further investigation.

Although a number of in vitro studies demonstrated the capacity of argon plasma to increase migration, proliferation and adhesion of fibroblasts to the titanium surface [37, 39], the PT did not show specific effect on KGW and MT suggesting rather indirect effect of this pre-treatment in providing favorable conditions for soft-tissue integration than direct pro-conductive effect. However, the variable microbial signatures were associated to different size of KGW. It would be thus worth to disclose possible interfering effects of this periopathogen with soft tissue collagen synthesis since it has been recently suggested that fibroblast plasticity around titanium implants might be altered by the surface characteristics and by the nature of the inflammatory stimuli [60, 61].

Within commitment of the modern health care toward focused cutting-edge therapies for an increased therapeutic index, tackling the complex biologics and its measurement remains the major prerequisite. It is now established that available regenerative procedures in periodontology and implantology provides in average up to 50% of complete success despite autologous origin or high-grade biomimetic concept, mostly due to altered healing processes and the presence of local interfering factors (such as microbial). It is thus proposed that bioactive approaches targeting specific biological factors should favorize healing processes and yield improved treatment outcomes [62, 67]. On the other hand, the rise of precision medicine has been driven by innovations in molecular profiling specifically for biomaterials, as high-throughput methods allowed tackling the complexity of interaction between biomaterials and biologics and allows for a better understanding of correlations between material properties and their effects on complex biological systems. Thus the personalized strategies and use of omics methods from discovery, over development, preclinical evaluation, and implementation of new clinical approaches are holding the promise to overcome erroneous material design and validation methods usually confined to the low-through put approach behind therapies with limited capacity and represents subject of strongest recommendation in medicine and dentistry [68, 69].

Biomaterial-related infections are considered as the most devastating complication of biomaterial use associated with ravaging biological consequences, hence development of anti-infective biomaterials and infection-resistant surfaces is one of the major priorities in biomedical research [70]. The present study has demonstrated that peri-implant microbiome remains highly diverse already during the first month post-implantation in clinically healthy peri-implant tissues, emphasizing the importance of biofilm control from the early stage of implant placement. The anti-infective pre-treatment of healing abutments seems to be a promising approach providing favorable conditions for optimal soft tissue growth and seal able to withstand infective threats. Furthermore, the controversial findings of lower inflammation coupled thin mucosal tissue and high prevalence of Streptococcus gordonii observed in PT confirms the complexity of material-induced biological effects and the critical role of high throughput methods for accurate evaluation of treatment effectiveness and its rapid clinical translation [41, 46]. Thus, the accelerated implementation of comprehensive biological assessment in dental research undoubtfully remains a roadmap toward improved treatment strategies in implantology [44, 47]. Although this study did not primarily aim to assess the effect of PT on abutments with different topography, the findings of decreased BOP in ROUGH + PT compared to ROUGH healing abutments is suggestive that this surface treatment may attenuated reported upregulated inflammatory response, reported for rough surface topography (1). Additionally, the lower Pi was confirmed on MACHINED + PT compared MACHINED, hence considering observed differences between abutments, this proof-of-concept study provides justification for conducting the further research primary oriented toward effects on rough and machined surface abutments in larger sample. Moreover, given the similarity in opportunistic pathobionts identified between peri-implant microbiome and peri-prosthetic infections in orthopedics (such as Staphylococcus aureus) [54, 71], the findings in this study may be transferable toother medical fields, representing another implication for future research.

The present study exhibits some limitations, primary relating to the relatively small sample size and lack assessment of collagen markers that would possibly provide more specific information regarding treatment effects on soft tissue metabolism. Finally, the assessment of biological parameters should represent an integral part of evaluation protocols in pre-clinical and clinical studies assessing soft peri-implant tissues as being able to provide more precise information over the sensitivity of standard clinical parameters.

Within limitations of this study, argon plasma pre-treatment of healing abutments provided positive effects on peri-implant microbiome resulting in decreased biofilm accumulation and lower soft-tissue inflammation, while the qualitative PiSP seems to be unaltered by this protocol.