Microbiome modulation of implant-related infection by a novel miniaturized pulsed electromagnetic field device

Abstract

Dental implant-related infections, which lack effective therapeutic strategies, are considered the primary cause for treatment failure. Pulsed electromagnetic field (PEMF) technology has been introduced as a safe and effective modality for enhancing biological responses. However, the PEMF effect on modulating microbial diversity has not been explored. Thus, we tested a miniaturized PEMF biomedical device as a healing component for dental implants. PEMF activation did not alter the chemical composition, surface roughness, wettability, and electrochemical performance. PEMF effectively controlled chronic in vitro polymicrobial microbial accumulation. The in vivo study where devices were inserted in the patients’ oral cavities and 16S RNA sequencing analysis evidenced a fivefold or more reduction in 23 bacterial species for PEMF group and the absence of some species for this group, including pathogens associated with implant-related infections. PEMF altered bacterial interactions and promoted specific bacterial pathways. PEMF has emerged as an effective strategy for controlling implant-related infections.

Discussion

Implant-related infections represent prevalent conditions that often result in treatment failures, imposing significant costs on patients, clinicians, and healthcare systems⁶. Polymicrobial biofilms, which are central to triggering inflammatory cascades in peri-implant tissues, exhibit a diverse microbial composition and well-organized structure that promote antimicrobial resistance, making it a challenging task to eliminate this structure³⁶. Coupled with the intricate structure of implant devices, the absence of effective therapies for dental implant-related infections is evident¹⁵. Currently, no FDA-approved treatment for implant-related infections has been reported. In this context, PEMF technology has explored bioelectromagnetic phenomena to modulate biological responses, including microbial accumulation²⁹. Here, for the first time, we investigated the role of PEMF in controlling biofilm accumulation and modulating its microbial composition using polymicrobial models that consider the human microbiome, specifically the oral environment, which is the second largest microbiome in the human body. Our findings indicate that PEMF activation did not affect the material surface properties, which are designed to withstand the environmental challenges in the oral cavity, and facilitated salivary protein adsorption, a key host-response mechanism that modulates subsequent biological processes^37,38. In addition to demonstrating the potential of PEMF technology for biomaterials manufacturing, the main strength of our study lies in its microbiological outcomes. PEMF effectively reduced late microbial accumulation and considerably modulate the microbial composition of biofilm by reducing key putative pathogens strongly associated with implant infections and tissue damage. These findings were further validated by our in vivo model, where biofilm accumulation occurred in the oral cavity of patients, mirroring clinical conditions. Moreover, PEMF altered bacterial interactions and promoted specific bacterial pathways. Therefore, we have unveiled a potent antimicrobial strategy for controlling and treating implant-related biofilms, paving the way for promising avenue to tackle these infections.

The same PEMF device used in this study has been undergone clinical testing, which has demonstrated its effectiveness in enhancing dental implant stability, encouraging implant-bone contact, and managing bone loss in patients with implant-related infections^26,39,40. Indeed, clinical observations have shown that PEMF technology fosters bone regeneration in different conditions, including cervical fusion⁴¹, mandibular fracture^42,43, and bone formation in women with postmenopausal osteoporosis⁴⁴. The use of PEMF has consistently demonstrated increased bone formation and density, along with a faster healing process⁴². Moreover, we found increased protein adsorption for the PEMF group, signifying an important and positive biological response in the interaction between implant devices and human body³⁷. Dental implant-related infections, particularly peri-implantitis, are characterized by progressive bone loss. Therefore, PEMF emerges as a powerful strategy to control and modulate the etiological factor – biofilm accumulation – while concurrently promoting tissue regeneration to restore health states. Further clinical trials should delve into and comprehensively investigate both effects on disease conditions, examining the effectiveness and durability of outcomes over extended periods, and elucidating its mechanisms. Moreover, the direct and modulatory effects of this technology on immune responses would provide valuable insights and should also be investigated.

Our results suggest that PEMF may not prevent polymicrobial adhesion. Instead, this technology appears to exert a modulatory influence on biofilm progression and microbial interactions. Notably, we observed reduced bacterial cell counts only in 72 hour biofilms, with no significant difference found the 24 hour time point. The lack of effect at early stages of biofilm formation may also explain the absence of an impact on protein adsorption, which was not affected by PEMF. While PEMF has shown some ability to inhibit biofilm accumulation and reduce 24 hour biofilm biomass by half in monospecies biofilms, such as S. epidermidis²⁹, polymicrobial biofilms may present distinct features that promote microbial growth, tolerance, and persistence. These features include reprogramming of transcriptomic and metabolic apparatuses, as well as increased extracellular polymer synthesis⁴⁵. In fact, previous studies evaluating the use of PEMF technology to control biofilm growth and test its antibacterial properties have been conducted using in vitro models with specific microbial species^29–31. Overall, these studies predominantly used single microbial species, tested PEMF technology on preformed biofilms, assessed live cells in biofilms through absorbance measurements or even combined the PEMF with nanoparticles treatment (Khan et al.^29,30), explains the antimicrobial ability to eliminate more than half of the biofilms.

Interactions between different microbial species, as well as their metabolites, play a crucial role in microbial physiology, community structure, and susceptibility to antimicrobial strategies⁴⁶. The observed reduction in bacterial levels at 72 h may be attributed to the PEMF effect on modulating microbial interactions, potentially suppressing some bacterial species growth and delaying co-aggregation processes. After initial colonizers adhere to the implant surface and create a suitable environment for other species, the co-aggregation process starts, promoting the interaction and colonization of putative pathogens (late colonizers)⁷. Since our in vitro model showed no difference in terms of live cell counts and microbiological compositions at early stages (24 h) but a strong effect, mainly on composition, at later stages (72 h), it is clear that PEMF modulated the biofilm dynamics during maturation, particularly microbial interactions and growth. Considering our biofilm model, which mimicked the whole microbial diversity in the oral environment (using human saliva as the microbial source) and the evaluation at different stages of biofilm growth, these findings highlight for the first time the ability of PEMF technology to modulate biofilm maturation during its growth, reducing the levels of important pathogens associated with dental implant infections. Previous evidence tested the PEMF device on an in vitro oral biofilm model, but using specific bacterial species (31 species standardized at specific loads)³³. However, this does not adequately represent the oral microbiome or in vivo biofilm formation, particularly considering that at early stages, putative pathogens are present at lower levels, but biofilm maturation provides a suitable environment for the overgrowth of these species. In our study, we mimicked the oral microbiome using human saliva as the inoculum and allowed normal biofilm growth and interactions. Importantly, further studies should investigate PEMF therapy at distal sites on the implant body, including subgingival surfaces, to assess reduction in microbial load and pathogen abundance.

The modulatory effect of PEMF on the microbiological composition of biofilms was found in both in vitro and in vivo models. Late-stage biofilms (72 h), representing the critical stage triggering inflammatory processes in implant-related infections⁸, were particularly affected. Notably, the abundance of Porphyromonas species was significantly reduced by PEMF in both biofilm models, with some species even absence in the PEMF group in the in vivo model. P. gingivalis, in particular, is recognized as one of the key pathogens highly associated with peri-implantitis disease and tissue damage⁴⁷. Moreover, the putative pathogen T. forsythia, known for its association with peri-implant tissue destruction⁴⁸, also exhibited reduced levels in response to PEMF. The effect of PEMF led to the reduction of 23 or more bacterial species in our models, demonstrating a potent modulatory effect. Moreover, the in vivo model showed that some important disease-associated species were found only for the control group, such as some Prevotella and Porphyromonas spp., and health-associated species, such as some Actinomyces spp., were found only in the pulse group, suggesting a trend towards a microbial profile associated with health for PEMF. Importantly, previous evidence has evaluated the antimicrobial effect of PEMF using only in vitro models, without testing even in animal models^29,30. Here, we took the next step by testing this technology in humans, using an in vivo model where the devices were inserted into the oral cavities of patients and kept for three days. This was possible because the device had been previously tested in clinical trials focusing on its effect on bone formation and regeneration^26,39,40. Therefore, we reproduced a clinical setting in a controlled condition, demonstrating a promising effect that can now be confirmed by randomized clinical trials before being recommended by the industry for this purpose. Importantly, the use of a palatal appliance for 3 days was based on previous evidence using the same model, which demonstrated that microbial levels and biofilm composition on titanium surfaces were similar to those found clinically in patients with peri-implant disease⁴⁹.

Although the mechanism by which PEMF modulates microbial interaction and reduces biofilm accumulation is not fully understood, some hypotheses have been raised (Fig. 9). Electrostatic forces are responsible for mediating bacterial-bacterial interactions and surface attachment, and their modulation may be influenced by PEMF activation⁵⁰, particularly affecting Gram-negative species owing to the distinct nature of their membranes and charges⁵¹. PEMF emission has been associated with changes in cell-wall surface molecules and surface charges²⁷, which are critical parameters governing microbial interactions and growth. While the field created by PEMF may impact all cellular components to some extent, it is anticipated to have a higher effect on extracellular molecules since the PEMF energy is attenuated after penetrating the membrane^52,53. In this context, it is crucial to further investigate the potential impact of PEMF on the extracellular matrix content, as it plays a significant role in protecting microbial cells and facilitating microbial growth. Moreover, PEMF activation may induce an electroporation effect on microbial cell membranes²⁹ potentially resulting in bacteriostatic or bactericidal effects. Importantly, although some studies have reported magnetic field generating heat to promote bacterial killing on metallic devices⁵⁴, the PEMF device tested here was not expected to generate no heat effects. This is because it operated at a low frequency was (10–50 kHz), which is unlikely to compromise the surrounding tissues. Further research is warranted to elucidate these mechanisms and their implications.

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