Cytotoxic effects of titanium particles and implantoplasty-treated surfaces exposed to bacterial biofilm

Cytotoxic effects of titanium particles and implantoplasty-treated surfaces exposed to bacterial biofilm | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytotoxic effects of titanium particles and implantoplasty-treated surfaces exposed to bacterial biofilm Erika Vegas-Bustamante, Jorge Toledano-Serrabona, Gemma Sanmartí-Garcia, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7324487/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives This study evaluated the cytotoxicity and metabolic activity of human fibroblasts and osteoblasts in the presence of metallic particles and on implant surfaces subjected to implantoplasty (IP), previously contaminated with a multispecies biofilm. It also assessed the potential for biofilm formation on these particles. Methods Titanium alloy (Ti6Al4V) particles were collected to assess their cytotoxic potential and interactions with human cells and bacterial biofilms. Cytotoxicity assays were performed using fibroblasts (HFF-1) and osteoblast-like cells (SaOs-2) through an indirect lactate dehydrogenase (LDH) assay. Biofilm formation was evaluated using Streptococcus oralis, Actinomyces viscosus, Veillonella parvula, and Porphyromonas gingivalis, quantified by colony-forming units (CFUs) and metabolic activity. Fibroblasts and osteoblasts were co-cultured with biofilm-contaminated particles for 2, 4, and 6 hours. Cell morphology and biofilm association were examined by phase-contrast microscopy, while metabolic activity was measured spectrophotometrically. Results IP-treated implants did not show significant cytotoxicity in HFF-1 or SaOs-2, with metabolic activities above 92% and cytotoxicity below 20%. Ti6Al4V particles, however, promoted Actinomyces viscosus and Veillonella parvula growth, increasing metabolic activity by 192.36% and 202.89%, and CFUs to 1.41 × 10⁹ and 7.10 × 10⁸, compared to 4.27 × 10⁶ and 2.33 × 10⁶ in controls. In multispecies biofilm, overall metabolic activity showed no significant differences (94.34% vs. 100%). Co-culture with infected particles drastically reduced fibroblast and osteoblast activity (< 25% and < 10%). In the absence of bacteria, fibroblasts reached 266.2% and osteoblasts 90% viability. Conclusions Contaminated particles from IP markedly reduced cytocompatibility of osteoblasts and fibroblasts and promoted specific bacterial growth, whereas IP-treated implant surfaces did not impair cell viability. Clinical relevance: Biofilm-contaminated titanium particles released during implantoplasty reduce cell viability and promote bacterial growth, unlike the treated implant surface. Dental implant implantoplasty fibroblast osteoblast Ti6Al4V Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Metal particles can be released into the peri-implant environment due to friction between the bone and implant, micromovement between the implant and abutment, or as a result of aggressive decontamination procedures targeting the implant surface [ 1 ]. In many instances, these particles cannot be completely eliminated from the bone or the surrounding mucosal tissues. Elevated concentrations of titanium (Ti) particles has been detected in dental implants affected by peri-implantitis [ 2 , 3 ], leading some authors to suggest a potential association between the presence of these particles and the development of this disease [ 4 , 5 ]. These metallic particles – particulary in nanoparticulate form- can be internalized by various cell types in the peri-implant milieu, including macrophages and fibroblasts. Interaction with the cell membrane facilitates their uptake. In macrophages, this process may result in frustrated phagocytosis, eliciting chronic inflammation and local osteolysis. In fibroblasts, particle uptake occurs via endocytosis, which may impair cellular function. Once internalized, the particles disrupt organelles and intracellular structures, leading to cellular dysfunction and damage [ 6 , 7 ]. Within this context, metallic particles present in the peri-implant environment have been implicated in the activation of inflammatory pathways, promoting the secretion of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and receptor activator of nuclear factor kappa-B ligand (RANKL) [ 8 – 10 ]. Additionally, they may downregulate osteogenic markers including Runx2 and osteocalcin (OC) [ 11 ] Furthermore, titanium particles have shown to stimulate fibroblasts to secrete pro-inflammatory cytokines, contributing to the chemotactic recruitment of monocytes/macrophage and the pathogenesis of aseptic loosening of implants [ 1 , 11 ]. These particles may also compromise the integrity of the oral epithelial barrier, disrupt cellular homeostasis, and induce DNA damage, as evidenced by the activation of genotoxic markers such as BRCA1 and CHK2 [ 8 , 12 ]. Implantoplasty (IP) is a mechanical decontamination technique aimed at removing bacterial biofilm and reducing surface roughness of the implant areas exposed to the oral cavity following resective or combined peri-implant surgical procedures [ 13 – 20 ]. This technique generates titanium debris and modifies the surface topography and physicochemical properties of the implant [ 1 , 21 – 27 ]. Once released, these particles are exposed to a complex oral environment, where microbiological, biochemical, and electrochemical factors—such as saliva, fluoride, and biofilm-derived metabolites—may exacerbate their corrosion and degradation, accelerating the deterioration of the metallic surface [ 1 , 28 , 29 ]. Fibroblasts and osteoblasts are critical for maintaining homeostasis in the peri-implant tissues, as they constitute the primary cellular components of these regions. A loss of cytocompatibility indicates potential impairment of these cells, thereby jeopardizing the balance and functionality of the peri-implant environment [ 12 ]. Although previous studies have shown that implantoplasty-induced surface modifications do not significantly affect the viability of fibroblasts [ 13 , 30 ] and osteoblasts [ 31 ], the Ti particles released during the procedure have been associated with a considerable decrease in the viability of both cell types [ 1 , 26 , 32 ]. While the cytotoxic potential of metallic debris has been examined, its effect in more clinically relevant conditions—such as in the presence of a multispecies bacterial biofilm—on fibroblasts and osteoblasts remains largely unexplored. Therefore, it is essential to characterize these particles to better understand their influence on cellular and biological responses. The aim of this study was to evaluate the cytotoxic effects of metal particles and implant surfaces subjected to implantoplasty, both contaminated with a multispecies biofilm, by assessing cell viability and metabolic activity in human fibroblasts and osteoblasts. Additionally, the biofilm-forming capacity in the presence of metal particles was investigated. 2. Materials & Methods 2.1 Preparation of Metallic residues and dental implants Metallic particles generated during IP of forty Titanium-6 Aluminum-4 Vanadium (Ti6Al4V) dental implants (Avinent Implant System S.L., Santpedor, Spain) were collected, all performed by the same researcher (E.V.B.). The implantoplasty (IP) procedure was carried out following the simplified three-bur protocol described by Costa-Berenguer et al. [ 23 ]. Additionally, 20 dental implants with a diameter of 4.5 mm and a length of 13 mm, featuring an internal hexagonal connection (Ocean E.C., Avinent Implant System S.L., Santpedor, Spain), underwent a 6 mm implantoplasty following the same protocol. The specific surface area of the particles was measured using the Brunauer–Emmett–Teller (BET) theory under controlled vacuum conditions with nitrogen as the adsorbate. Particle size distribution was analyzed using a Mastersizer 3000 (Malvern Panalytical®, UK), laser diffraction system, conducted in a wet medium with ethanol as the dispersing liquid. Mechanical and ultrasonic agitation were applied to prevent particle agglomeration. Morphology and chemical composition were examined using scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS), (Neon 40 Surface, Zeiss, Oberkochen, Germany). 2.2 Cell culture Two different cell lines purchased from American Type Culture Collection (ATCC) were used. Human foreskin fibroblasts (HFF-1) were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) and SaOs-2 with McCoy’s 5A medium. Both supplemented with 15% of Foetal Bovine Serum (FBS) and 1% of Penicillin-Streptomycin (P/S) at 37 ºC. 2.3. Cytotoxicity evaluation 2.3.1. Indirect assay The cytotoxicity of the sample was evaluated by indirect exposure determination according to the ISO 10993-5 standard [ 33 ]. Cytotoxicity tests were performed in triplicate. The samples studied were: Test sample: Implants; Negative control: Tissue culture plastic (TCP); Positive control: Triton. For the indirect assay, 10 implants with implantoplasty were used and incubated at 37ºC with 9 ml of DMEM and McCoy’s 5A medium for three days. After the 3 days, different dilutions were made with each medium (1:1, 1:5, 1:10, 1:50) reaching a final volume of 3 ml. Then, 5 x 10 3 cells per well were seeded in a 96-well plate and cultured for 1 day. Then, cells were exposed to the different dilutions up to 7 days. Cell response was evaluated in terms of metabolic activity at day 7 and cytotoxicity at day 1, 3 and 7, using tissue culture plastic (TCP) as negative (Control -) and Triton as positive (Control +) controls. For the metabolic activity, at day 7, 100 µl of resazurin at 15 g/ml was added in each well and incubated at 37 ºC for 30 min. Then, media was transferred to a black 96-well plate to measure the fluorescence in the spectrophotometer (Infinite M nano +, TECAN) at 560 nm excitation wavelength and at 590 nm emission wavelength. For the cellular viability, supernatants from day 1, 3 and 7 were kept at -20ºC and then released lactate dehydrogenase (LDH) from dead cells was quantified using a CyQUANT™ LDH Cytotoxicity Assay kit (Thermo Fisher Scientific Inc., Waltham, MA, EE.UU) following manufacturer instructions. Briefly, 50 µl of the sample and 50 µl of the reactive solution were incubated at room temperature for 30 min in a 96-well plate. Then, the absorbance was measured at 490 nm in the spectrophotometer (Infinite M nano +, TECAN). Toxicity criteria were established according to the following ranges: 0–20%: Non-toxic, 20–40%: Slightly toxic, 40–60%: Moderately toxic, > 60%: Toxic [ 33 ] . 2.3.2. Direct assay For the direct assay, a cell concentration of 5 x 10 3 cells per well was seeded with 10 implants with implantoplasty in a low adhesion 24-well plate for 7 days. Then, implants were transferred to another 24-well plate and 1 ml of resazurin at 15 µg/ml was added and incubated at 37 ºC for 30 min. Media was transferred to a black 96-well plate to measure the fluorescence in the spectrophotometer (Infinite M nano +, TECAN) at 560 nm excitation wavelength and at 590 nm emission wavelength. As a negative control, tissue culture plastic (TCP) was used. 2.4. Biofilm formation with particle presence 2.4.1. Biofilm formation Biofilm formation was adapted from a previous study [ 34 ], adjusting bacteria strains and initially grown individually overnight with brain heart infusion media (BHI, Oxoid concentrations). Four bacterial strains were used to develop a multispecies oral biofilm at 37ºC and anaerobic conditions (GasPak Anaerobic System, Becton Dickinson). The selected species represent initial colonizers, such as Streptococcus oralis ( S. oralis , ATCC 6249) and Actinomyces viscosus ( A. viscosus , ATCC 15987), early colonizers Veillonella parvula ( V. parvula , ATCC 10790), and late colonizers Porphyromonas gingivalis (P. gingivalis , ATCC 33277). After incubation, bacteria suspension was vortex for 5 seconds to disperse bacteria aggregation and each bacteria strain was adjusted to the desired concentration. Concentration for individual bacteria studies was 8·10 7 CFU/mL for all strains, while multispecies biofilms were developed with 1.6·10 7 CFU/mL for Actinomyces viscosus and Streptococcus oralis , 4·10 7 CFU/mL for Veillonella parvula , and 8·10 7 CFU/mL for Porphyromonas gingivalis . Then, titanium particles, previously disinfected with 70% ethanol for 30 min and washed thrice with PBS, were placed in falcon tubes and incubated with inoculums. Subsequently, samples were incubated at 37 ºC and under anaerobic conditions for 5 days. Tissue culture plastic was used as control. 2.4.2. Biofilm quantification Biofilm quantification was assessed in terms of metabolic activity and colony forming units (CFU). For the metabolic activity, media was removed and 15 ug/ml resazurin was placed for 10 minutes. Then, fluorescence was quantified with a spectrophotometer (Infinite M Nano+, Tecan), 560 nm excitation wavelength and 590 nm emission wavelength. Metabolic activity was presented as a reduction of resazurin, considering the metabolic activity of S. oralis as at TCP as 100%. Regarding CFU quantification, media was removed, and content was resuspended with 500 ul of PBS, transfer to Eppendorf tubes and vigorously vortexed for 10 min. Serial dilutions were prepared for each sample and then, placed in sterile media agar plates for 24 h. CFUs were manually counted. 2.5. Cell-bacteria co-cultures Two different co-cultures were performed studying fibroblast versus multispecies biofilm and osteoblast versus multispecies biofilm. Human foreskin fibroblasts (ATCC) and SaOs-2 osteoblast cell line were culture in Dulbecco's modified Eagle medium (DMEM) and McCoy's 5a medium, supplemented with 10 and 15% fetal bovine serum (FBS), respectively. Cells were grown at 37°C, 5% CO2 and 95% humidified air. Fibroblast and osteoblast cells were seeded onto 24 well plates at 15 x 10 3 cells/cm 2 . Co-cultures were performed under conditions adapted to the eukaryotic. Cells were contaminated with the particles infected with the multispecies bacteria for 5 days, as previously described. To achieve this, the bacteria suspension was centrifuged at 1000 rpm for 3 minutes and then resuspended in media for fibroblast and osteoblast cells. 200 (µl) of concentrated infected particles were placed at each well. Co-cultures were incubated at 37°C, 5% CO2 and 95% humidified air for 2, 4 and 6 hours. Phase contrast microscopy was used to monitor co-cultures using an inverted microscope (Olympus CKX41 microscope with a Nikon DS-Fi1 camera). Metabolic activity was measured as previously explained; however, all simples were previously treated with 3x penicillin-streptomycin to minimize the presence of extracelular bacteria. Co-cultures were performed in the presence of implantoplasted dental implants, referred to as implant group. Two controls were used: TCP and control. Infected particles were cultured only with tissue culture plastic, referred to as TCP, and multispecies biofilm incubated without particles in tissue culture plastic, referred as control. In addition, all conditions were replicated without bacteria contamination to monitor fibroblasts and osteoblasts behavior, labeled as wo (without bacteria). 2.6. Statistical analysis The data obtained were entered into a Microsoft Excel spreadsheet (Microsoft®, Redmond, Washington, USA) and subsequently processed using Stata 14 software (StataCorp®, College Station, USA). To evaluate cytotoxicity through direct and indirect assays, the Kruskal-Wallis test followed by Dunn’s multiple comparison test was used, utilizing the Prism 10 software. 3. Results The particles exhibited a predominantly planar morphology, consistent with the characteristics expected from the mechanical machining procedures employed. EDS analysis confirmed that the particles were composed of Ti6Al4V, with no detectable residues from the drills used during the machining process. Particle size analysis revealed a range from 2 to 92 µm, following a normal distribution. The average equivalent diameter was approximately 39 µm, and the size distribution curve indicated that 90% of the particles fell within the range of 25 to 70 µm. Additionally, the average specific surface area of the Ti6Al4V particles was 0.4532 ± 0.0987 m²/g. 3.1. Cytotoxicity evaluation The toxicity of implants with implantoplasty was initially evaluated using indirect cell cultures with fibroblasts (HFF-1) and osteoblasts (SaOs-2). For this purpose, Implants were incubated in culture media, and serial dilutions of the extracted media were exposed to the cells. Figure 1 presents the cytotoxicity measured on days 1, 3, and 7, as well as metabolic activity on day 7, across different experimental groups. The negative control exhibited the highest metabolic activity (100%), indicating optimal cell viability. In both cell lines, neither the concentrated extract nor its serial dilutions significantly affected metabolic activity or induced cytotoxicity ( 0.05). The 1:50 dilution showed a slight increase in cytotoxicity in fibroblasts on day 3 (11.54%), though still within the non-toxic range. In contrast, the positive cytotoxic control (Control +) markedly reduced metabolic activity and increased cytotoxicity (p = 0.0404). A similar trend was observed in direct cell cultures of fibroblasts and osteoblasts exposed to implants with implantoplasty (Fig. 2 ). Under all conditions, metabolic activity remained at levels comparable to TCP (p = 0.3687). Specifically, the implant group showed 92.35% metabolic activity in fibroblasts and 94.78% in osteoblasts, compared to 100% in the control group. 3.2. Biofilm formation with particle presence Following the evaluation of the biological response of implants subject to implantoplasty, the effects of the metallic particles were assessed on single-strain biofilms of A. viscosus, S. oralis, V. parvula , and P. gingivalis (Fig. 3 ). Biofilm development exposed to implantoplasted particles was compared to TCP without traces of these particles. Interestingly, A. viscosus and V. parvula demonstrated a significant increase in metabolic activity, reaching 192.36% and 202.89%, respectively compared to their TCP counterparts (p = 0.0152). These findings were further confirmed by CFU quantification, where A. viscosus and V. parvula also showed higher CFU amounts when cultured with these particles. Conversely, S. oralis and P. gingivalis presented lower CFU counts (p = 0.0153); however, their metabolic activity was comparable to that of TCP controls (p = 0.1843). Therefore, A. viscosus and V. parvula proliferate significantly more in the presence of Ti. The analysis of multispecies biofilm development (Fig. 4 ) revealed that biofilm metabolic activity was not affected by the presence of implantoplasty particles (p = 0.1843), specifically showing 94.34% (Ti Biofilms group) compared to 100% in the TCP Biofilms group. However, CFU quantification demonstrated a slight reduction, similar to the trend observed for certain strains in single-species biofilm development. Specifically, the Ti Biofilm group showed 2.57 × 10⁸ CFU, whereas the TCP Biofilm group reached 1.75 × 10⁹ CFU. This indicates that the number of colony-forming units in the Ti Biofilm group is lower compared to the TCP group (p = 0.0152). 3.3. Cell-bacteria co-culture Finally, the effect of contaminated particles with multispecies biofilm on fibroblast and osteoblast adhesion was evaluated. As shown in Fig. 5 , bacteria from the multispecies biofilm on implantoplasted particles were released after 2 hours of culture, completely covering the microscope's visual field. When fibroblasts were cultured without bacterial contamination (Fig. 6 ), cell adhesion was observed as early as 2 hours, with signs of cell spreading and elongated morphology. By 4 hours, most cells exhibited an egg-shaped morphology with increased surface area, and spreading was largely complete after 6 hours. In contrast, fibroblast adhesion was significantly impaired in the presence of bacterial contamination, as the cells retained a rounded morphology at all time points. These observations were confirmed by metabolic activity measurements (Fig. 7 ). Similarly, when osteoblasts were cultured without bacterial contamination (Fig. 8 ), the cells initially maintained a rounded morphology at 2 hours, with some adopting a polygonal shape after 4 hours and showing a slight increase in cell area after 6 hours. In contrast, osteoblasts cultured with bacteria retained a rounded morphology at all time points, indicating a complete inhibition of cell spreading. These findings were also corroborated by metabolic activity measurements (Fig. 7 ). Figure 7 shows the metabolic activity (%) of fibroblasts and osteoblasts, measured at 2, 4, and 6 hours (2H, 4H, 6H) in different groups: Control, Implant, TCP (all in the presence of bacteria) and their corresponding bacteria-free groups (wo). The groups with bacteria showed low metabolic activity in fibroblasts (< 25%), indicating a negative effect on their metabolism. In contrast, the groups without bacteria ("wo") exhibited significantly higher metabolic activity, reaching levels above 245% at 6 hours. This suggests that the absence of bacteria favors fibroblast proliferation and functionality. Osteoblasts exposed to bacteria showed very low metabolic activity (-10%), indicating strong inhibition of their function and survival. In contrast, without bacteria, metabolic activity increased significantly, reaching between 70% and 90% at 6 hours. This suggests that the presence of bacteria strongly inhibits osteoblast metabolic activity, affecting their normal function and survival. Discussion This study evaluated the cytotoxicity of both metal particles released from polished titanium implants and the implant surfaces subjected to implantoplasty, all contaminated with a multispecies biofilm, under clinically relevant in vitro conditions. Cell viability was assessed in human fibroblasts and osteoblasts, and bacterial adhesion to these particles was quantified. The results demonstrated that titanium particles exposed to bacterial biofilm significantly reduced the cytocompatibility of both fibroblastic and osteoblastic cells, as indicated by a marked decrease in their metabolic activity. In contrast, implant surfaces treated with implantoplasty (IP) in the absence of bacterial contamination showed no cytotoxic effects, with fibroblasts and osteoblasts maintaining metabolic activity above 92% and cytotoxicity levels below 20%. These findings are particularly relevant from a clinical perspective, as they support the biocompatibility of IP-treated surfaces, which are commonly employed during surgical treatment of peri-implantitis. This differential biological response between implant surfaces and detached metal particles highlights the importance of controlling particle dissemination during the procedure. Nevertheless, certain limitations should be acknowledged. First, the study utilized a single implant material, Ti6Al4V, whereas dental implants may also be fabricated from commercially pure titanium or alternative alloys such as TiZr. Second, a specific milling protocol was applied for implantoplasty, although multiple protocols exist in the literature, potentially affecting the quantity and composition of debris. Moreover, using purified and sterilized saliva could have provided a more faithful representation of the oral environment. To the best of our knowledge, however, no prior studies have examined the combined impact of bacterial biofilms and titanium particles on fibroblasts and osteoblasts under such realistic in vitro conditions. In the oral cavity, corrosive substances such as fluorides, lactic acid, citric acid, and chlorides, present in saliva and oral biofilms, can induce corrosion of titanium surfaces, promoting the release of metal ions and particles [ 28 , 35 ]. Previous investigations into biofilm formation on titanium surfaces have underscored the crucial role of bacterial colonization in peri-implant diseases[ 34 , 36 – 38 ]. Surface roughness has been positively correlated with increased bacterial adhesion [ 34 , 36 , 39 ], supporting the importance of minimizing bacterial attachment to maintain peri-implant health. Multispecies oral biofilm models have been developed to replicate in vivo colonization, demonstrating strong affinity for titanium even when antibacterial coatings are present [ 36 , 40 , 41 ]. However, Godoy et al. [ 38 ] reported reduced adhesion of S. sanguinis and L. salivarius on titanium surfaces treated with antibacterial agents like TESPSA, without inducing cytotoxic effects. Despite these efforts, controversy persists regarding how surface characteristics influence biofilm development and the progression of peri-implant diseases [ 42 , 43 ]. Dysbiotic biofilms can initiate an inflammatory response that stimulates osteoclast activity and bone resorption [ 44 ]. Anaerobic conditions beneath the gingival margin further favor the proliferation of facultative and strict anaerobes[ 37 ]. In this study, we included both types: A. viscosus , S. oralis , V. parvula , and P. gingivalis . In line with Vilarrasa et al. [ 34 ], who observed no significant difference bacterial counts across titanium groups, our results showed that the metabolic activity of the multispecies biofilm remained unaffected by the presence of metal particles. However, a significant increase in the quantity and activity of A. viscosus and V. parvula was detected compared to the TCP control (p < 0.05), while S. oralis and P. gingivalis counts were significantly reduced (p < 0.05). Similarly, Violant et al.[ 36 ] identified V. parvula as the predominant species in a multispecies biofilm grown on titanium. Surface characteristics also influence fibroblast behavior. Fibroblasts spread more readily on smooth than rough surfaces, and connective tissue adhesion is modulated by surface properties [ 13 , 30 , 45 – 47 ]. Beheshti et al. [ 30 ] demonstrated that surface roughness and chemical changes introduced during implantoplasty significantly influence fibroblast morphology, proliferation, and cytokine production. While such modifications promote fibroblast growth, rougher surfaces (e.g., SLA) may elicit stronger inflammatory responses, reflected in elevated IL-6 and MCP-3 levels. In agreement with our findings, fibroblasts on IP-treated implants showed no reduction in viability or metabolic activity, and maintained an elongated morphology with ovoid nuclei and parallel alignment. By contrast, cells in the positive control group showed significantly impaired activity (p < 0.05). The viability of SaOs-2 osteoblasts on IP-treated implants has also been validated. Toma et al.[ 31 ] found that despite significant alterations to surface morphology and wettability, IP-treated titanium still supported SaOs-2 proliferation and alkaline phosphatase (ALP), osteoprotegerin (OPG) and osteocalcin (OCN) production. These results are consistent with our findings: IP alone did not impair osteoblastic viability or metabolic activity. However, these studies did not analyze the effect of the metal debris produced during implantoplasty. Moreover, the current controversy surrounding peri-implant diseases focuses on whether Ti particles are responsible for causing inflammation and osteolysis, as there is insufficient evidence correlating these conditions with metal debris [ 48 ]. Although the presence of particles in the peri-implant environment has been investigated, few studies have analyzed the titanium particles released during implantoplasty [ 1 , 26 , 27 , 32 ]. These particles generated during implantoplasty have been shown to cause a significant reduction in cell viability in human fibroblasts[ 1 , 26 , 32 ] and SaOs-2 osteoblasts [ 26 , 32 ]. Barrack et al. [ 1 ] studied particles released from grade Ti6Al4V titanium and observed the release of vanadium ions, which significantly reduced the viability of human gingival fibroblasts (HGFs) after 10 days of culture. In line with these findings, Toledano-Serrabona et al. [ 26 ] and Schwarz et al. [ 32 ] confirmed the cytotoxic effects of Ti particles from Ti6Al4V and pure titanium on fibroblasts and osteoblasts. Our results corroborate these findings and further suggest that bacterial contamination may amplify the cellular response to metal debris. These particles generated during the IP could release a higher concentration of vanadium (V) compared to the production of titanium (Ti) and aluminum (Al) ions, indicating the degradation of the alloy [ 1 , 26 ], especially the smaller particles [ 49 ]. Furthermore, the immune system responds differently depending on the size of the particles [ 50 ]. Large particles can be encapsulated by a fibrous capsule to isolate them from the environment; medium-sized particles can be phagocytosed by macrophages and eliminated from the body; while small particles are not identified [ 51 ]. Wu et al.[ 11 ] demonstrated that the debris generated is mainly ultrafine in size (< 100 nm). This suggests that surgical areas that appear particle-free may still contain nanometric residues. Similarly, Cai et al.[ 52 ] noted that Ti nanoparticles within the 100 nm range had the greatest cytotoxic effect. Additionally, Ti6Al4V has been shown to be susceptible to corrosion and wear, and the release of vanadium (V) and aluminum (Al) particles has a more pronounced cytotoxic effect compared to commercially pure titanium [ 53 ]. Titanium particles and ions appear to play a significant role in the pathogenesis of peri-implantitis by inducing genomic instability, triggering macrophage-driven inflammatory responses, and upregulating IL-1β, IL-6, and TNF-α expresión [ 35 , 54 ]. This cascade promotes osteoclastogenesis and bone resorption, potentially compromising implant stability. Conclusion Titanium particles generated during implantoplasty, when exposed to a multispecies biofilm, markedly compromised the viability of human fibroblasts and osteoblasts, reducing it to below 25% and 10%, respectively, compared with substantially higher values under uncontaminated conditions (over 245% for fibroblasts and 70–90% for osteoblasts). Under these contaminated conditions, the proliferation of Actinomyces viscosus and Veillonella parvula was selectively favored, without significantly modifying the overall metabolic activity of the biofilm. In contrast, implant surfaces subjected to implantoplasty did not exhibit notable cytotoxicity toward fibroblasts or osteoblasts, maintaining metabolic activities above 92% and cytotoxicity levels below 20% in both cell types. Declarations Compliance with Ethical Standards: - Ethical approval : This article does not contain any studies with human participants or animals performed by any of the authors. Consent for publication: - Not applicable. Competing interests: - The authors declare that they have no competing interests related with the present paper. Funding: This study was supported by the Instituto de Salud Carlos III through project PI22/00851 (co-funded by the European Regional Development Fund (ERDF), a way to build Europe) and the Ministry of Science and Innovation of Spain by research projects CONCEPTO PDC2022-133628-C22 (co-funded by the European Regional Development Fund (ERDF), a way to build Europe) and the research project MINECO (PID2022-137496OB-I00) and program CERCA of the Generalitat de Catalunya Author Contribution -Author contribution: EV-B and JTS conceived and designed the study. EV-B performed the implantoplasty procedures and participated in all experimental phases. GS-G, LMD and ED-P contributed to the cell culture experiments and cytotoxicity assays. JG-M and LMD carried out the material characterization using SEM and EDS. 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Eur J Dent 15:407–411 Monje A, Pons R, Amerio E, Wang HL, Nart J (2022) Resolution of peri-implantitis by means of implantoplasty as adjunct to surgical therapy: a retrospective study. J Periodontol 93:110–122 Romeo E, Lops D, Chiapasco M, Ghisolfi M, Vogel G (2007) Therapy of peri-implantitis with resective surgery. A 3-year clinical trial on rough screw-shaped oral implants. Part II: radiographic outcome. Clin Oral Implants Res 18:179–187 Romeo E, Ghisolfi M, Murgolo N, Chiapasco M, Lops D, Vogel G (2005) Therapy of peri-implantitis with resective surgery. A 3-year clinical trial on rough screw-shaped oral implants. Part I: clinical outcome. Clin Oral Implants Res 16:9–18 Camps-Font O, Toledano-Serrabona J, Juiz-Camps A, Gil J, Sánchez-Garcés MA, Figueiredo R et al (2023) Effect of implantoplasty on roughness, fatigue and corrosion behavior of narrow diameter dental implants. J Funct Biomater 14:61 Leitão-Almeida B, Camps-Font O, Correia A, Mir-Mari J, Figueiredo R, Valmaseda-Castellón E (2021) Effect of bone loss on the fracture resistance of narrow dental implants after implantoplasty: an in vitro study. Med Oral Patol Oral Cir Bucal 26:e611–e618 Costa-Berenguer X, García-García M, Sánchez-Torres A, Sanz-Alonso M, Figueiredo R, Valmaseda-Castellón E (2018) Effect of implantoplasty on fracture resistance and surface roughness of standard diameter dental implants. Clin Oral Implants Res 29:46–54 Lozano P, Peña M, Herrero-Climent M, Rios-Santos JV, Rios-Carrasco B, Brizuela A et al (2022) Corrosion behavior of titanium dental implants with implantoplasty. Mater (Basel) 15:1563 Fonseca D, de Tapia B, Pons R, Aparicio C, Guerra F, Messias A et al (2024) The effect of implantoplasty on the fatigue behavior and corrosion resistance in titanium dental implants. Mater (Basel) 17:2944 Toledano-Serrabona J, Gil FJ, Camps-Font O, Valmaseda-Castellón E, Gay-Escoda C, Sánchez-Garcés MA Physicochemical and biological characterization of Ti6Al4V particles obtained by implantoplasty: an in vitro study. Part I. Materials (Basel) 14:6507Gaur S, Agnihotri R, Albin S (2021) Bio-tribocorrosion of titanium dental implants and its toxicological implications: a scoping review. ScientificWorldJournal. 2022;2022:4498613 Toledano-Serrabona J, Sánchez-Garcés MA, Gay-Escoda C, Valmaseda-Castellón E, Camps-Font O, Verdeguer P et al (2021) Mechanical properties and corrosion behavior of Ti6Al4V particles obtained by implantoplasty: an in vitro study. Part II. Mater (Basel) 14:6519 Gaur S, Agnihotri R, Albin S (2022) Bio-tribocorrosion of titanium dental implants and its toxicological implications: a scoping review. ScientificWorldJournal 2022:4498613 Gil FJ, Rodriguez A, Espinar E, Llamas JM, Padullés E, Juárez A (2012) Effect of oral bacteria on the mechanical behavior of titanium dental implants. Int J Oral Maxillofac Implants 27:64–68 Beheshti Maal M, Aanerød Ellingsen S, Reseland JE, Verket A (2020) Experimental implantoplasty outcomes correlate with fibroblast growth in vitro. BMC Oral Health 20:25 Toma S, Lasserre J, Brecx MC, Nyssen-Behets C (2016) In vitro evaluation of peri-implantitis treatment modalities on Saos-2 osteoblasts. Clin Oral Implants Res 27:1085–1092 Schwarz F, Langer M, Hagena T, Hartig B, Sader R, Becker J (2019) Cytotoxicity and proinflammatory effects of titanium and zirconia particles. Int J Implant Dent 5:25 International Organization for Standardization (2009) ISO 10993-5. Biological evaluation of medical devices. Part 5: Test for in vitro cytotoxicity. ISO, Geneva Vilarrasa J, Delgado LM, Galofré M, Àlvarez G, Violant D, Manero JM et al (2018) In vitro evaluation of a multispecies oral biofilm over antibacterial coated titanium surfaces. J Mater Sci Mater Med 29:164 Noronha Oliveira M, Schunemann WVH, Mathew MT, Henriques B, Magini RS, Teughels W et al (2018) Can degradation products released from dental implants affect peri-implant tissues? J Periodontal Res 53:1–11 Violant D, Galofré M, Nart J, Teles RP (2014) In vitro evaluation of a multispecies oral biofilm on different implant surfaces. Biomed Mater 9:035007 Souza JC, Henriques M, Oliveira R, Teughels W, Celis JP, Rocha LA (2010) Do oral biofilms influence the wear and corrosion behavior of titanium? Biofouling 26:471–478 Godoy-Gallardo M, Guillem-Marti J, Sevilla P, Manero JM, Gil FJ, Rodriguez D (2016) Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation. Mater Sci Eng C Mater Biol Appl 59:524–532 Wu-Yuan D, Eganhouse KJ, Keller JC, Walters KS (1995) Oral bacterial attachment to titanium surfaces: a scanning electron microscopy study. J Oral Implantol 21:207–213 Sánchez MC, Llama-Palacios A, Fernández E, Figuero E, Marín MJ, León R et al (2014) An in vitro biofilm model associated to dental implants: structural and quantitative analysis of in vitro biofilm formation on different dental implant surfaces. Dent Mater 30:1161–1171 Godoy-Gallardo M, Wang Z, Shen Y, Manero JM, Gil FJ, Rodriguez D et al (2015) Antibacterial coatings on titanium surfaces: a comparison study between in vitro single-species and multispecies biofilm. ACS Appl Mater Interfaces 7:5992–6001 Bürgers R, Gerlach T, Hahnel S, Schwarz F, Handel G, Gosau M (2010) In vivo and in vitro biofilm formation on two different titanium implant surfaces. Clin Oral Implants Res 21:156–164 Teughels W, Van Assche N, Sliepen I, Quirynen M (2006) Effect of material characteristics and/or surface topography on biofilm development. Clin Oral Implants Res 17:68–81 Insua A, Monje A, Wang HL, Miron RJ (2017) Basis of bone metabolism around dental implants during osseointegration and peri-implant bone loss. J Biomed Mater Res A 105:2075–2089 Könönen M, Hormia M, Kivilahti J, Hautaniemi J, Thesleff I (1992) Effect of surface processing on the attachment, orientation, and proliferation of human gingival fibroblasts on titanium. J Biomed Mater Res 26:1325–1341 Nothdurft FP, Fontana D, Ruppenthal S, May A, Aktas C, Mehraein Y et al (2015) Differential behavior of fibroblasts and epithelial cells on structured implant abutment materials: a comparison of materials and surface topographies. Clin Implant Dent Relat Res 17:1237–1249 Pae A, Lee H, Kim HS, Kwon YD, Woo YH (2009) Attachment and growth behaviour of human gingival fibroblasts on titanium and zirconia ceramic surfaces. Biomed Mater 4:025005 Schwarz F, Derks J, Monje A, Wang HL (2018) Peri-implantitis. J Clin Periodontol 45(Suppl 20):S246–S266 Callejas JA, Gil J, Brizuela A, Pérez RA, Bosch BM (2022) Effect of the size of titanium particles released from dental implants on immunological response. Int J Mol Sci 23:7333 Dalal A, Pawar V, McAllister K, Weaver C, Hallab NJ (2012) Orthopedic implant cobalt-alloy particles produce greater toxicity and inflammatory cytokines than titanium alloy and zirconium alloy-based particles in vitro, in human osteoblasts, fibroblasts, and macrophages. J Biomed Mater Res A 100:2147–2158 Baranov MV, Kumar M, Sacanna S, Thutupalli S, van den Bogaart G (2021) Modulation of immune responses by particle size and shape. Front Immunol 11:607945 Cai K, Hou Y, Hu Y, Zhao L, Luo Z, Shi Y et al (2011) Correlation of the cytotoxicity of TiO₂ nanoparticles with different particle sizes on a sub-200-nm scale. J Nanosci Nanotechnol 7:3026–3031 Willis J, Li S, Crean SJ, Barrak FN (2021) Is titanium alloy Ti-6Al‐4V cytotoxic to gingival fibroblasts? A systematic review. Clin Exp Dent Res 7:1037–1044 Asa’ad F, Thomsen P, Kunrath MF (2022) The role of titanium particles and ions in the pathogenesis of peri-implantitis. J Bone Metab 29:145–154 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7324487","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506180885,"identity":"8c1282c6-8dfa-40e4-a2ad-f7d5fc2b9c6c","order_by":0,"name":"Erika Vegas-Bustamante","email":"","orcid":"","institution":"IDIBELL Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Erika","middleName":"","lastName":"Vegas-Bustamante","suffix":""},{"id":506180886,"identity":"734184aa-04c2-4028-8ec1-8102f4eb35f7","order_by":1,"name":"Jorge Toledano-Serrabona","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYBACxgYGxgMMbEAWewOIZCZCSxsDA0QLzwEitYBUQ7RIJBCphXl+84EDH8rs8vgl35g9YKiwTmwg7DC2hIMzziUXS87OMTdgOJNOjBYeg8O8bcyJG27nmEkwth0mUsvftvrEDTfPALX8I1YLyPANN3iAWhqI0pKWcLDn3PHEmT1p5QYJx9KNCWoxbD588MGPsurEfvbD2x58qLGWJawFRUUCIeUgIE+MolEwCkbBKBjhAAB88EFduzszBAAAAABJRU5ErkJggg==","orcid":"","institution":"IDIBELL Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Jorge","middleName":"","lastName":"Toledano-Serrabona","suffix":""},{"id":506180887,"identity":"d97f0d5a-7b1b-4492-84ba-b43b5f9591e9","order_by":2,"name":"Gemma Sanmartí-Garcia","email":"","orcid":"","institution":"IDIBELL Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Gemma","middleName":"","lastName":"Sanmartí-Garcia","suffix":""},{"id":506180888,"identity":"b2749a53-7043-4615-913d-97e66d39dcce","order_by":3,"name":"Elena Demiquels-Punzano","email":"","orcid":"","institution":"Universitat Internacional de Catalunya","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Demiquels-Punzano","suffix":""},{"id":506180889,"identity":"45f18c89-20b1-489d-9f6c-75851a19d996","order_by":4,"name":"Javier Gil-Mur","email":"","orcid":"","institution":"Universitat Politecnica de Catalunya","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"","lastName":"Gil-Mur","suffix":""},{"id":506180890,"identity":"4ecfb6b4-80a6-4128-8897-cdd0d5c80cbd","order_by":5,"name":"Luis M Delgado","email":"","orcid":"","institution":"Universitat Politecnica de Catalunya","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"M","lastName":"Delgado","suffix":""},{"id":506180891,"identity":"bb4eea7a-e5e7-4d70-9f69-51fac8cd49f1","order_by":6,"name":"Rui Figueiredo","email":"","orcid":"","institution":"IDIBELL Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Figueiredo","suffix":""},{"id":506180892,"identity":"3825717f-7f19-404a-a31d-4f9c426ede61","order_by":7,"name":"Mª Ángeles Sánchez-Garcés","email":"","orcid":"","institution":"IDIBELL Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Mª","middleName":"Ángeles","lastName":"Sánchez-Garcés","suffix":""},{"id":506180893,"identity":"774e885d-5b35-4a53-b9b8-d0b897dbf5a5","order_by":8,"name":"Octavi Camp-Font","email":"","orcid":"","institution":"IDIBELL Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Octavi","middleName":"","lastName":"Camp-Font","suffix":""}],"badges":[],"createdAt":"2025-08-08 07:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7324487/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7324487/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90204724,"identity":"e4bfc837-b4ab-4da0-8fd9-0a9fea6ffb1f","added_by":"auto","created_at":"2025-08-29 20:39:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":409509,"visible":true,"origin":"","legend":"\u003cp\u003eIndirect cell culture of HFF-1 fibroblast and SaOs-2 osteoblast exposed to extracts from implantoplasted dental implants.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/8fb5601bbef32585e81cd11e.jpg"},{"id":90204897,"identity":"47198879-add0-4a98-8b23-d7f14294373d","added_by":"auto","created_at":"2025-08-29 20:47:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163385,"visible":true,"origin":"","legend":"\u003cp\u003eDirect cell culture of HFF-1 fibroblast and SaOs-2 osteoblast on implantoplasted dental implants.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/c7a4e5fbeba7a5df336c2686.jpg"},{"id":90204898,"identity":"073ce494-46ee-4aa6-b8ce-0f199874979f","added_by":"auto","created_at":"2025-08-29 20:47:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":256117,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of single bacterial strain biofilms exposed to implantoplasted particles (Ti groups) measured in terms of metabolic activity and CFU quantification. (TCP): Tissue culture plastic with a single bacterial species; (Ti): Titanium particles with a single bacterial species.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/0fff5ae18e10e4649328aeaa.jpg"},{"id":90204732,"identity":"f4dfab50-82b6-4e68-a00f-aae0cc383c9d","added_by":"auto","created_at":"2025-08-29 20:39:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":143823,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of multispecie biofilms exposed to implantoplasted particles (Ti groups) measured in terms of metabolic activity and CFU quantification. (group Ti Biofilms): Titanium particles with biofilms; (group TCP Biofilms): Tissue culture plastic with biofilms.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/2b8cc84e4c59d234dd91925a.jpg"},{"id":90204730,"identity":"6bc2485e-58d5-48e3-9779-b5eefa90237c","added_by":"auto","created_at":"2025-08-29 20:39:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":404469,"visible":true,"origin":"","legend":"\u003cp\u003ePlanktonic bacteria release from the multispecie biofilm developed on implantoplasted particles after 2 hours of culture.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/e1e4ad992438c3282b24d52f.jpg"},{"id":90205166,"identity":"6918c44b-79df-4b31-bb7d-e8e727c7608c","added_by":"auto","created_at":"2025-08-29 20:55:03","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":474286,"visible":true,"origin":"","legend":"\u003cp\u003eFibroblast co-culture with multispecie biofilm contaminated implantoplasted particles. (TCP): Tissue culture plastic with particles, both with and without biofilms; (Implant): Implant surface together with titanium particle, both with and without biofilms; (Control): Tissue culture plastic with and without biofilms, without particles.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/f33163ba7f87d0af1baaf8dd.jpg"},{"id":90204735,"identity":"07f22d9e-3579-4040-a19c-be89d78b848e","added_by":"auto","created_at":"2025-08-29 20:39:03","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":284129,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic activity of fibroblasts and osteoblasts cultured with and without bacteria. Control): Tissue culture plastic with biofilms (Control Wo): Without biofilms; (Implant): Implant surface together with titanium particles, with biofilms; (Implant Wo): Without biofilms; (TCP): Tissue culture plastic with particles and biofilms; (TCP Wo): Without biofilms.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/b3848bda30961e3e96b808f7.jpg"},{"id":90204742,"identity":"08fd9ba7-5b26-495a-aa06-c7b0b4b08bca","added_by":"auto","created_at":"2025-08-29 20:39:03","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":468187,"visible":true,"origin":"","legend":"\u003cp\u003eOsteoblast co-culture with multispecie biofilm contaminated implantoplasted particles. (TCP): Tissue culture plastic with particles, both with and without biofilms; (Implant): Implant surface together with titanium particle, both with and without biofilms; (Control): Tissue culture plastic with and without biofilms, without particles.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/925dcf2744ed656c1e1295fa.jpg"},{"id":90356236,"identity":"4264db03-0c5f-4de4-8f30-429d17faa41f","added_by":"auto","created_at":"2025-09-01 21:01:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3364307,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7324487/v1/67cc09db-1e09-4770-a51c-fcebfc34b4ba.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytotoxic effects of titanium particles and implantoplasty-treated surfaces exposed to bacterial biofilm","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMetal particles can be released into the peri-implant environment due to friction between the bone and implant, micromovement between the implant and abutment, or as a result of aggressive decontamination procedures targeting the implant surface [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In many instances, these particles cannot be completely eliminated from the bone or the surrounding mucosal tissues.\u003c/p\u003e\u003cp\u003eElevated concentrations of titanium (Ti) particles has been detected in dental implants affected by peri-implantitis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], leading some authors to suggest a potential association between the presence of these particles and the development of this disease [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese metallic particles \u0026ndash; particulary in nanoparticulate form- can be internalized by various cell types in the peri-implant milieu, including macrophages and fibroblasts. Interaction with the cell membrane facilitates their uptake. In macrophages, this process may result in frustrated phagocytosis, eliciting chronic inflammation and local osteolysis. In fibroblasts, particle uptake occurs via endocytosis, which may impair cellular function. Once internalized, the particles disrupt organelles and intracellular structures, leading to cellular dysfunction and damage [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWithin this context, metallic particles present in the peri-implant environment have been implicated in the activation of inflammatory pathways, promoting the secretion of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and receptor activator of nuclear factor kappa-B ligand (RANKL) [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, they may downregulate osteogenic markers including Runx2 and osteocalcin (OC) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eFurthermore, titanium particles have shown to stimulate fibroblasts to secrete pro-inflammatory cytokines, contributing to the chemotactic recruitment of monocytes/macrophage and the pathogenesis of aseptic loosening of implants [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These particles may also compromise the integrity of the oral epithelial barrier, disrupt cellular homeostasis, and induce DNA damage, as evidenced by the activation of genotoxic markers such as BRCA1 and CHK2 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImplantoplasty (IP) is a mechanical decontamination technique aimed at removing bacterial biofilm and reducing surface roughness of the implant areas exposed to the oral cavity following resective or combined peri-implant surgical procedures [\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17 CR18 CR19\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This technique generates titanium debris and modifies the surface topography and physicochemical properties of the implant [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Once released, these particles are exposed to a complex oral environment, where microbiological, biochemical, and electrochemical factors\u0026mdash;such as saliva, fluoride, and biofilm-derived metabolites\u0026mdash;may exacerbate their corrosion and degradation, accelerating the deterioration of the metallic surface [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFibroblasts and osteoblasts are critical for maintaining homeostasis in the peri-implant tissues, as they constitute the primary cellular components of these regions. A loss of cytocompatibility indicates potential impairment of these cells, thereby jeopardizing the balance and functionality of the peri-implant environment [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough previous studies have shown that implantoplasty-induced surface modifications do not significantly affect the viability of fibroblasts [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and osteoblasts [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], the Ti particles released during the procedure have been associated with a considerable decrease in the viability of both cell types [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile the cytotoxic potential of metallic debris has been examined, its effect in more clinically relevant conditions\u0026mdash;such as in the presence of a multispecies bacterial biofilm\u0026mdash;on fibroblasts and osteoblasts remains largely unexplored. Therefore, it is essential to characterize these particles to better understand their influence on cellular and biological responses.\u003c/p\u003e\u003cp\u003eThe aim of this study was to evaluate the cytotoxic effects of metal particles and implant surfaces subjected to implantoplasty, both contaminated with a multispecies biofilm, by assessing cell viability and metabolic activity in human fibroblasts and osteoblasts. Additionally, the biofilm-forming capacity in the presence of metal particles was investigated.\u003c/p\u003e"},{"header":"2. Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Preparation of Metallic residues and dental implants\u003c/h2\u003e\u003cp\u003eMetallic particles generated during IP of forty Titanium-6 Aluminum-4 Vanadium (Ti6Al4V) dental implants (Avinent Implant System S.L., Santpedor, Spain) were collected, all performed by the same researcher (E.V.B.). The implantoplasty (IP) procedure was carried out following the simplified three-bur protocol described by Costa-Berenguer et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, 20 dental implants with a diameter of 4.5 mm and a length of 13 mm, featuring an internal hexagonal connection (Ocean E.C., Avinent Implant System S.L., Santpedor, Spain), underwent a 6 mm implantoplasty following the same protocol.\u003c/p\u003e\u003cp\u003eThe specific surface area of the particles was measured using the Brunauer\u0026ndash;Emmett\u0026ndash;Teller (BET) theory under controlled vacuum conditions with nitrogen as the adsorbate. Particle size distribution was analyzed using a Mastersizer 3000 (Malvern Panalytical\u0026reg;, UK), laser diffraction system, conducted in a wet medium with ethanol as the dispersing liquid. Mechanical and ultrasonic agitation were applied to prevent particle agglomeration. Morphology and chemical composition were examined using scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS), (Neon 40 Surface, Zeiss, Oberkochen, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cell culture\u003c/h2\u003e\u003cp\u003eTwo different cell lines purchased from American Type Culture Collection (ATCC) were used. Human foreskin fibroblasts (HFF-1) were cultured with Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) and SaOs-2 with McCoy\u0026rsquo;s 5A medium. Both supplemented with 15% of Foetal Bovine Serum (FBS) and 1% of Penicillin-Streptomycin (P/S) at 37 \u0026ordm;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Cytotoxicity evaluation\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Indirect assay\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of the sample was evaluated by indirect exposure determination according to the ISO 10993-5 standard [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Cytotoxicity tests were performed in triplicate. The samples studied were: Test sample: Implants; Negative control: Tissue culture plastic (TCP); Positive control: Triton.\u003c/p\u003e\u003cp\u003eFor the indirect assay, 10 implants with implantoplasty were used and incubated at 37\u0026ordm;C with 9 ml of DMEM and McCoy\u0026rsquo;s 5A medium for three days. After the 3 days, different dilutions were made with each medium (1:1, 1:5, 1:10, 1:50) reaching a final volume of 3 ml. Then, 5 x 10\u003csup\u003e3\u003c/sup\u003e cells per well were seeded in a 96-well plate and cultured for 1 day. Then, cells were exposed to the different dilutions up to 7 days. Cell response was evaluated in terms of metabolic activity at day 7 and cytotoxicity at day 1, 3 and 7, using tissue culture plastic (TCP) as negative (Control -) and Triton as positive (Control +) controls.\u003c/p\u003e\u003cp\u003eFor the metabolic activity, at day 7, 100 \u0026micro;l of resazurin at 15 g/ml was added in each well and incubated at 37 \u0026ordm;C for 30 min. Then, media was transferred to a black 96-well plate to measure the fluorescence in the spectrophotometer (Infinite M nano +, TECAN) at 560 nm excitation wavelength and at 590 nm emission wavelength.\u003c/p\u003e\u003cp\u003eFor the cellular viability, supernatants from day 1, 3 and 7 were kept at -20\u0026ordm;C and then released lactate dehydrogenase (LDH) from dead cells was quantified using a CyQUANT\u0026trade; LDH Cytotoxicity Assay kit (Thermo Fisher Scientific Inc., Waltham, MA, EE.UU) following manufacturer instructions. Briefly, 50 \u0026micro;l of the sample and 50 \u0026micro;l of the reactive solution were incubated at room temperature for 30 min in a 96-well plate. Then, the absorbance was measured at 490 nm in the spectrophotometer (Infinite M nano +, TECAN).\u003c/p\u003e\u003cp\u003eToxicity criteria were established according to the following ranges: 0\u0026ndash;20%: Non-toxic, 20\u0026ndash;40%: Slightly toxic, 40\u0026ndash;60%: Moderately toxic, \u0026gt;\u0026thinsp;60%: Toxic [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] .\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Direct assay\u003c/h2\u003e\u003cp\u003eFor the direct assay, a cell concentration of 5 x 10\u003csup\u003e3\u003c/sup\u003e cells per well was seeded with 10 implants with implantoplasty in a low adhesion 24-well plate for 7 days. Then, implants were transferred to another 24-well plate and 1 ml of resazurin at 15 \u0026micro;g/ml was added and incubated at 37 \u0026ordm;C for 30 min. Media was transferred to a black 96-well plate to measure the fluorescence in the spectrophotometer (Infinite M nano +, TECAN) at 560 nm excitation wavelength and at 590 nm emission wavelength. As a negative control, tissue culture plastic (TCP) was used.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Biofilm formation with particle presence\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Biofilm formation\u003c/h2\u003e\u003cp\u003eBiofilm formation was adapted from a previous study [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], adjusting bacteria strains and initially grown individually overnight with brain heart infusion media (BHI, Oxoid concentrations). Four bacterial strains were used to develop a multispecies oral biofilm at 37\u0026ordm;C and anaerobic conditions (GasPak Anaerobic System, Becton Dickinson). The selected species represent initial colonizers, such as \u003cem\u003eStreptococcus oralis\u003c/em\u003e (\u003cem\u003eS. oralis\u003c/em\u003e, ATCC 6249) and \u003cem\u003eActinomyces viscosus\u003c/em\u003e (\u003cem\u003eA. viscosus\u003c/em\u003e, ATCC 15987), early colonizers \u003cem\u003eVeillonella parvula\u003c/em\u003e (\u003cem\u003eV. parvula\u003c/em\u003e, ATCC 10790), and late colonizers \u003cem\u003ePorphyromonas gingivalis (P. gingivalis\u003c/em\u003e, ATCC 33277). After incubation, bacteria suspension was vortex for 5 seconds to disperse bacteria aggregation and each bacteria strain was adjusted to the desired concentration. Concentration for individual bacteria studies was 8\u0026middot;10\u003csup\u003e7\u003c/sup\u003e CFU/mL for all strains, while multispecies biofilms were developed with 1.6\u0026middot;10\u003csup\u003e7\u003c/sup\u003e CFU/mL for \u003cem\u003eActinomyces viscosus\u003c/em\u003e and \u003cem\u003eStreptococcus oralis\u003c/em\u003e, 4\u0026middot;10\u003csup\u003e7\u003c/sup\u003e CFU/mL for \u003cem\u003eVeillonella parvula\u003c/em\u003e, and 8\u0026middot;10\u003csup\u003e7\u003c/sup\u003e CFU/mL for \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e. Then, titanium particles, previously disinfected with 70% ethanol for 30 min and washed thrice with PBS, were placed in falcon tubes and incubated with inoculums. Subsequently, samples were incubated at 37 \u0026ordm;C and under anaerobic conditions for 5 days. Tissue culture plastic was used as control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Biofilm quantification\u003c/h2\u003e\u003cp\u003eBiofilm quantification was assessed in terms of metabolic activity and colony forming units (CFU). For the metabolic activity, media was removed and 15 ug/ml resazurin was placed for 10 minutes. Then, fluorescence was quantified with a spectrophotometer (Infinite M Nano+, Tecan), 560 nm excitation wavelength and 590 nm emission wavelength. Metabolic activity was presented as a reduction of resazurin, considering the metabolic activity of \u003cem\u003eS. oralis\u003c/em\u003e as at TCP as 100%.\u003c/p\u003e\u003cp\u003eRegarding CFU quantification, media was removed, and content was resuspended with 500 ul of PBS, transfer to Eppendorf tubes and vigorously vortexed for 10 min. Serial dilutions were prepared for each sample and then, placed in sterile media agar plates for 24 h. CFUs were manually counted.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Cell-bacteria co-cultures\u003c/h2\u003e\u003cp\u003eTwo different co-cultures were performed studying fibroblast versus multispecies biofilm and osteoblast versus multispecies biofilm.\u003c/p\u003e\u003cp\u003eHuman foreskin fibroblasts (ATCC) and SaOs-2 osteoblast cell line were culture in Dulbecco's modified Eagle medium (DMEM) and McCoy's 5a medium, supplemented with 10 and 15% fetal bovine serum (FBS), respectively. Cells were grown at 37\u0026deg;C, 5% CO2 and 95% humidified air.\u003c/p\u003e\u003cp\u003eFibroblast and osteoblast cells were seeded onto 24 well plates at 15 x 10\u003csup\u003e3\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e. Co-cultures were performed under conditions adapted to the eukaryotic. Cells were contaminated with the particles infected with the multispecies bacteria for 5 days, as previously described. To achieve this, the bacteria suspension was centrifuged at 1000 rpm for 3 minutes and then resuspended in media for fibroblast and osteoblast cells. 200 (\u0026micro;l) of concentrated infected particles were placed at each well. Co-cultures were incubated at 37\u0026deg;C, 5% CO2 and 95% humidified air for 2, 4 and 6 hours.\u003c/p\u003e\u003cp\u003ePhase contrast microscopy was used to monitor co-cultures using an inverted microscope (Olympus CKX41 microscope with a Nikon DS-Fi1 camera). Metabolic activity was measured as previously explained; however, all simples were previously treated with 3x penicillin-streptomycin to minimize the presence of extracelular bacteria.\u003c/p\u003e\u003cp\u003eCo-cultures were performed in the presence of implantoplasted dental implants, referred to as implant group. Two controls were used: TCP and control. Infected particles were cultured only with tissue culture plastic, referred to as TCP, and multispecies biofilm incubated without particles in tissue culture plastic, referred as control. In addition, all conditions were replicated without bacteria contamination to monitor fibroblasts and osteoblasts behavior, labeled as wo (without bacteria).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe data obtained were entered into a Microsoft Excel spreadsheet (Microsoft\u0026reg;, Redmond, Washington, USA) and subsequently processed using Stata 14 software (StataCorp\u0026reg;, College Station, USA). To evaluate cytotoxicity through direct and indirect assays, the Kruskal-Wallis test followed by Dunn\u0026rsquo;s multiple comparison test was used, utilizing the Prism 10 software.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe particles exhibited a predominantly planar morphology, consistent with the characteristics expected from the mechanical machining procedures employed. EDS analysis confirmed that the particles were composed of Ti6Al4V, with no detectable residues from the drills used during the machining process.\u003c/p\u003e\u003cp\u003eParticle size analysis revealed a range from 2 to 92 \u0026micro;m, following a normal distribution. The average equivalent diameter was approximately 39 \u0026micro;m, and the size distribution curve indicated that 90% of the particles fell within the range of 25 to 70 \u0026micro;m. Additionally, the average specific surface area of the Ti6Al4V particles was 0.4532\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0987 m\u0026sup2;/g.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Cytotoxicity evaluation\u003c/h2\u003e\u003cp\u003eThe toxicity of implants with implantoplasty was initially evaluated using indirect cell cultures with fibroblasts (HFF-1) and osteoblasts (SaOs-2). For this purpose, Implants were incubated in culture media, and serial dilutions of the extracted media were exposed to the cells.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the cytotoxicity measured on days 1, 3, and 7, as well as metabolic activity on day 7, across different experimental groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe negative control exhibited the highest metabolic activity (100%), indicating optimal cell viability. In both cell lines, neither the concentrated extract nor its serial dilutions significantly affected metabolic activity or induced cytotoxicity (\u0026lt;\u0026thinsp;20%), compared to the negative control (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The 1:50 dilution showed a slight increase in cytotoxicity in fibroblasts on day 3 (11.54%), though still within the non-toxic range. In contrast, the positive cytotoxic control (Control +) markedly reduced metabolic activity and increased cytotoxicity (p\u0026thinsp;=\u0026thinsp;0.0404).\u003c/p\u003e\u003cp\u003eA similar trend was observed in direct cell cultures of fibroblasts and osteoblasts exposed to implants with implantoplasty (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Under all conditions, metabolic activity remained at levels comparable to TCP (p\u0026thinsp;=\u0026thinsp;0.3687). Specifically, the implant group showed 92.35% metabolic activity in fibroblasts and 94.78% in osteoblasts, compared to 100% in the control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Biofilm formation with particle presence\u003c/h2\u003e\u003cp\u003eFollowing the evaluation of the biological response of implants subject to implantoplasty, the effects of the metallic particles were assessed on single-strain biofilms of \u003cem\u003eA. viscosus, S. oralis, V. parvula\u003c/em\u003e, and \u003cem\u003eP. gingivalis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Biofilm development exposed to implantoplasted particles was compared to TCP without traces of these particles. Interestingly, \u003cem\u003eA. viscosus\u003c/em\u003e and \u003cem\u003eV. parvula\u003c/em\u003e demonstrated a significant increase in metabolic activity, reaching 192.36% and 202.89%, respectively compared to their TCP counterparts (p\u0026thinsp;=\u0026thinsp;0.0152). These findings were further confirmed by CFU quantification, where \u003cem\u003eA. viscosus\u003c/em\u003e and \u003cem\u003eV. parvula\u003c/em\u003e also showed higher CFU amounts when cultured with these particles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConversely, \u003cem\u003eS. oralis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e presented lower CFU counts (p\u0026thinsp;=\u0026thinsp;0.0153); however, their metabolic activity was comparable to that of TCP controls (p\u0026thinsp;=\u0026thinsp;0.1843). Therefore, \u003cem\u003eA. viscosus and V. parvula\u003c/em\u003e proliferate significantly more in the presence of Ti.\u003c/p\u003e\u003cp\u003eThe analysis of multispecies biofilm development (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) revealed that biofilm metabolic activity was not affected by the presence of implantoplasty particles (p\u0026thinsp;=\u0026thinsp;0.1843), specifically showing 94.34% (Ti Biofilms group) compared to 100% in the TCP Biofilms group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHowever, CFU quantification demonstrated a slight reduction, similar to the trend observed for certain strains in single-species biofilm development. Specifically, the Ti Biofilm group showed 2.57 \u0026times; 10⁸ CFU, whereas the TCP Biofilm group reached 1.75 \u0026times; 10⁹ CFU. This indicates that the number of colony-forming units in the Ti Biofilm group is lower compared to the TCP group (p\u0026thinsp;=\u0026thinsp;0.0152).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Cell-bacteria co-culture\u003c/h2\u003e\u003cp\u003eFinally, the effect of contaminated particles with multispecies biofilm on fibroblast and osteoblast adhesion was evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, bacteria from the multispecies biofilm on implantoplasted particles were released after 2 hours of culture, completely covering the microscope's visual field.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen fibroblasts were cultured without bacterial contamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), cell adhesion was observed as early as 2 hours, with signs of cell spreading and elongated morphology. By 4 hours, most cells exhibited an egg-shaped morphology with increased surface area, and spreading was largely complete after 6 hours. In contrast, fibroblast adhesion was significantly impaired in the presence of bacterial contamination, as the cells retained a rounded morphology at all time points. These observations were confirmed by metabolic activity measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, when osteoblasts were cultured without bacterial contamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), the cells initially maintained a rounded morphology at 2 hours, with some adopting a polygonal shape after 4 hours and showing a slight increase in cell area after 6 hours. In contrast, osteoblasts cultured with bacteria retained a rounded morphology at all time points, indicating a complete inhibition of cell spreading. These findings were also corroborated by metabolic activity measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the metabolic activity (%) of fibroblasts and osteoblasts, measured at 2, 4, and 6 hours (2H, 4H, 6H) in different groups: Control, Implant, TCP (all in the presence of bacteria) and their corresponding bacteria-free groups (wo).\u003c/p\u003e\u003cp\u003eThe groups with bacteria showed low metabolic activity in fibroblasts (\u0026lt;\u0026thinsp;25%), indicating a negative effect on their metabolism. In contrast, the groups without bacteria (\"wo\") exhibited significantly higher metabolic activity, reaching levels above 245% at 6 hours. This suggests that the absence of bacteria favors fibroblast proliferation and functionality. Osteoblasts exposed to bacteria showed very low metabolic activity (-10%), indicating strong inhibition of their function and survival. In contrast, without bacteria, metabolic activity increased significantly, reaching between 70% and 90% at 6 hours. This suggests that the presence of bacteria strongly inhibits osteoblast metabolic activity, affecting their normal function and survival.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study evaluated the cytotoxicity of both metal particles released from polished titanium implants and the implant surfaces subjected to implantoplasty, all contaminated with a multispecies biofilm, under clinically relevant in vitro conditions. Cell viability was assessed in human fibroblasts and osteoblasts, and bacterial adhesion to these particles was quantified. The results demonstrated that titanium particles exposed to bacterial biofilm significantly reduced the cytocompatibility of both fibroblastic and osteoblastic cells, as indicated by a marked decrease in their metabolic activity. In contrast, implant surfaces treated with implantoplasty (IP) in the absence of bacterial contamination showed no cytotoxic effects, with fibroblasts and osteoblasts maintaining metabolic activity above 92% and cytotoxicity levels below 20%. These findings are particularly relevant from a clinical perspective, as they support the biocompatibility of IP-treated surfaces, which are commonly employed during surgical treatment of peri-implantitis. This differential biological response between implant surfaces and detached metal particles highlights the importance of controlling particle dissemination during the procedure.\u003c/p\u003e\u003cp\u003eNevertheless, certain limitations should be acknowledged. First, the study utilized a single implant material, Ti6Al4V, whereas dental implants may also be fabricated from commercially pure titanium or alternative alloys such as TiZr. Second, a specific milling protocol was applied for implantoplasty, although multiple protocols exist in the literature, potentially affecting the quantity and composition of debris. Moreover, using purified and sterilized saliva could have provided a more faithful representation of the oral environment. To the best of our knowledge, however, no prior studies have examined the combined impact of bacterial biofilms and titanium particles on fibroblasts and osteoblasts under such realistic in vitro conditions.\u003c/p\u003e\u003cp\u003eIn the oral cavity, corrosive substances such as fluorides, lactic acid, citric acid, and chlorides, present in saliva and oral biofilms, can induce corrosion of titanium surfaces, promoting the release of metal ions and particles [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Previous investigations into biofilm formation on titanium surfaces have underscored the crucial role of bacterial colonization in peri-implant diseases[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Surface roughness has been positively correlated with increased bacterial adhesion [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], supporting the importance of minimizing bacterial attachment to maintain peri-implant health.\u003c/p\u003e\u003cp\u003eMultispecies oral biofilm models have been developed to replicate in vivo colonization, demonstrating strong affinity for titanium even when antibacterial coatings are present [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, Godoy et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] reported reduced adhesion of \u003cem\u003eS. sanguinis\u003c/em\u003e and \u003cem\u003eL. salivarius\u003c/em\u003e on titanium surfaces treated with antibacterial agents like TESPSA, without inducing cytotoxic effects.\u003c/p\u003e\u003cp\u003eDespite these efforts, controversy persists regarding how surface characteristics influence biofilm development and the progression of peri-implant diseases [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Dysbiotic biofilms can initiate an inflammatory response that stimulates osteoclast activity and bone resorption [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Anaerobic conditions beneath the gingival margin further favor the proliferation of facultative and strict anaerobes[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, we included both types: \u003cem\u003eA. viscosus\u003c/em\u003e, \u003cem\u003eS. oralis\u003c/em\u003e, \u003cem\u003eV. parvula\u003c/em\u003e, and \u003cem\u003eP. gingivalis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn line with Vilarrasa et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], who observed no significant difference bacterial counts across titanium groups, our results showed that the metabolic activity of the multispecies biofilm remained unaffected by the presence of metal particles. However, a significant increase in the quantity and activity of \u003cem\u003eA. viscosus\u003c/em\u003e and \u003cem\u003eV. parvula\u003c/em\u003e was detected compared to the TCP control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while \u003cem\u003eS. oralis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e counts were significantly reduced (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, Violant et al.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] identified V. parvula as the predominant species in a multispecies biofilm grown on titanium.\u003c/p\u003e\u003cp\u003eSurface characteristics also influence fibroblast behavior. Fibroblasts spread more readily on smooth than rough surfaces, and connective tissue adhesion is modulated by surface properties [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Beheshti et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] demonstrated that surface roughness and chemical changes introduced during implantoplasty significantly influence fibroblast morphology, proliferation, and cytokine production. While such modifications promote fibroblast growth, rougher surfaces (e.g., SLA) may elicit stronger inflammatory responses, reflected in elevated IL-6 and MCP-3 levels. In agreement with our findings, fibroblasts on IP-treated implants showed no reduction in viability or metabolic activity, and maintained an elongated morphology with ovoid nuclei and parallel alignment. By contrast, cells in the positive control group showed significantly impaired activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eThe viability of SaOs-2 osteoblasts on IP-treated implants has also been validated. Toma et al.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] found that despite significant alterations to surface morphology and wettability, IP-treated titanium still supported SaOs-2 proliferation and alkaline phosphatase (ALP), osteoprotegerin (OPG) and osteocalcin (OCN) production. These results are consistent with our findings: IP alone did not impair osteoblastic viability or metabolic activity.\u003c/p\u003e\u003cp\u003eHowever, these studies did not analyze the effect of the metal debris produced during implantoplasty. Moreover, the current controversy surrounding peri-implant diseases focuses on whether Ti particles are responsible for causing inflammation and osteolysis, as there is insufficient evidence correlating these conditions with metal debris [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Although the presence of particles in the peri-implant environment has been investigated, few studies have analyzed the titanium particles released during implantoplasty [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These particles generated during implantoplasty have been shown to cause a significant reduction in cell viability in human fibroblasts[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and SaOs-2 osteoblasts [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBarrack et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] studied particles released from grade Ti6Al4V titanium and observed the release of vanadium ions, which significantly reduced the viability of human gingival fibroblasts (HGFs) after 10 days of culture. In line with these findings, Toledano-Serrabona et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and Schwarz et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] confirmed the cytotoxic effects of Ti particles from Ti6Al4V and pure titanium on fibroblasts and osteoblasts. Our results corroborate these findings and further suggest that bacterial contamination may amplify the cellular response to metal debris.\u003c/p\u003e\u003cp\u003eThese particles generated during the IP could release a higher concentration of vanadium (V) compared to the production of titanium (Ti) and aluminum (Al) ions, indicating the degradation of the alloy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], especially the smaller particles [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Furthermore, the immune system responds differently depending on the size of the particles [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Large particles can be encapsulated by a fibrous capsule to isolate them from the environment; medium-sized particles can be phagocytosed by macrophages and eliminated from the body; while small particles are not identified [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Wu et al.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] demonstrated that the debris generated is mainly ultrafine in size (\u0026lt;\u0026thinsp;100 nm). This suggests that surgical areas that appear particle-free may still contain nanometric residues. Similarly, Cai et al.[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] noted that Ti nanoparticles within the 100 nm range had the greatest cytotoxic effect. Additionally, Ti6Al4V has been shown to be susceptible to corrosion and wear, and the release of vanadium (V) and aluminum (Al) particles has a more pronounced cytotoxic effect compared to commercially pure titanium [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTitanium particles and ions appear to play a significant role in the pathogenesis of peri-implantitis by inducing genomic instability, triggering macrophage-driven inflammatory responses, and upregulating IL-1β, IL-6, and TNF-α expresi\u0026oacute;n [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. This cascade promotes osteoclastogenesis and bone resorption, potentially compromising implant stability.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTitanium particles generated during implantoplasty, when exposed to a multispecies biofilm, markedly compromised the viability of human fibroblasts and osteoblasts, reducing it to below 25% and 10%, respectively, compared with substantially higher values under uncontaminated conditions (over 245% for fibroblasts and 70\u0026ndash;90% for osteoblasts). Under these contaminated conditions, the proliferation of \u003cem\u003eActinomyces viscosus\u003c/em\u003e and \u003cem\u003eVeillonella parvula\u003c/em\u003e was selectively favored, without significantly modifying the overall metabolic activity of the biofilm. In contrast, implant surfaces subjected to implantoplasty did not exhibit notable cytotoxicity toward fibroblasts or osteoblasts, maintaining metabolic activities above 92% and cytotoxicity levels below 20% in both cell types.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompliance with Ethical Standards:\u003c/h2\u003e\u003cp\u003e- \u003cb\u003eEthical approval\u003c/b\u003e: This article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003cp\u003e- Not applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003cp\u003e- The authors declare that they have no competing interests related with the present paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis study was supported by the Instituto de Salud Carlos III through project PI22/00851 (co-funded by the European Regional Development Fund (ERDF), a way to build Europe) and the Ministry of Science and Innovation of Spain by research projects CONCEPTO PDC2022-133628-C22 (co-funded by the European Regional Development Fund (ERDF), a way to build Europe) and the research project MINECO (PID2022-137496OB-I00) and program CERCA of the Generalitat de Catalunya\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e-Author contribution: EV-B and JTS conceived and designed the study. EV-B performed the implantoplasty procedures and participated in all experimental phases. GS-G, LMD and ED-P contributed to the cell culture experiments and cytotoxicity assays. JG-M and LMD carried out the material characterization using SEM and EDS. RF supervised the methodology and provided clinical insights. MASC-G and OC-F contributed to data analysis and interpretation. JTS LMD JG-M and GS-G drafted the manuscript. All authors critically revised the manuscript for important intellectual content, read, and approved the final version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarrak FN, Li S, Muntane AM, Jones JR (2020) Particle release from implantoplasty of dental implants and impact on cells. Int J Implant Dent 6:50\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOlmedo DG, Nalli G, Verd\u0026uacute; S, Paparella ML, Cabrini RL (2013) Exfoliative cytology and titanium dental implants: a pilot study. 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Biofouling 26:471\u0026ndash;478\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGodoy-Gallardo M, Guillem-Marti J, Sevilla P, Manero JM, Gil FJ, Rodriguez D (2016) Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation. Mater Sci Eng C Mater Biol Appl 59:524\u0026ndash;532\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu-Yuan D, Eganhouse KJ, Keller JC, Walters KS (1995) Oral bacterial attachment to titanium surfaces: a scanning electron microscopy study. J Oral Implantol 21:207\u0026ndash;213\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez MC, Llama-Palacios A, Fern\u0026aacute;ndez E, Figuero E, Mar\u0026iacute;n MJ, Le\u0026oacute;n R et al (2014) An in vitro biofilm model associated to dental implants: structural and quantitative analysis of in vitro biofilm formation on different dental implant surfaces. Dent Mater 30:1161\u0026ndash;1171\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGodoy-Gallardo M, Wang Z, Shen Y, Manero JM, Gil FJ, Rodriguez D et al (2015) Antibacterial coatings on titanium surfaces: a comparison study between in vitro single-species and multispecies biofilm. ACS Appl Mater Interfaces 7:5992\u0026ndash;6001\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eB\u0026uuml;rgers R, Gerlach T, Hahnel S, Schwarz F, Handel G, Gosau M (2010) In vivo and in vitro biofilm formation on two different titanium implant surfaces. Clin Oral Implants Res 21:156\u0026ndash;164\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTeughels W, Van Assche N, Sliepen I, Quirynen M (2006) Effect of material characteristics and/or surface topography on biofilm development. Clin Oral Implants Res 17:68\u0026ndash;81\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eInsua A, Monje A, Wang HL, Miron RJ (2017) Basis of bone metabolism around dental implants during osseointegration and peri-implant bone loss. J Biomed Mater Res A 105:2075\u0026ndash;2089\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eK\u0026ouml;n\u0026ouml;nen M, Hormia M, Kivilahti J, Hautaniemi J, Thesleff I (1992) Effect of surface processing on the attachment, orientation, and proliferation of human gingival fibroblasts on titanium. J Biomed Mater Res 26:1325\u0026ndash;1341\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNothdurft FP, Fontana D, Ruppenthal S, May A, Aktas C, Mehraein Y et al (2015) Differential behavior of fibroblasts and epithelial cells on structured implant abutment materials: a comparison of materials and surface topographies. Clin Implant Dent Relat Res 17:1237\u0026ndash;1249\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePae A, Lee H, Kim HS, Kwon YD, Woo YH (2009) Attachment and growth behaviour of human gingival fibroblasts on titanium and zirconia ceramic surfaces. Biomed Mater 4:025005\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchwarz F, Derks J, Monje A, Wang HL (2018) Peri-implantitis. J Clin Periodontol 45(Suppl 20):S246\u0026ndash;S266\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCallejas JA, Gil J, Brizuela A, P\u0026eacute;rez RA, Bosch BM (2022) Effect of the size of titanium particles released from dental implants on immunological response. Int J Mol Sci 23:7333\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDalal A, Pawar V, McAllister K, Weaver C, Hallab NJ (2012) Orthopedic implant cobalt-alloy particles produce greater toxicity and inflammatory cytokines than titanium alloy and zirconium alloy-based particles in vitro, in human osteoblasts, fibroblasts, and macrophages. J Biomed Mater Res A 100:2147\u0026ndash;2158\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaranov MV, Kumar M, Sacanna S, Thutupalli S, van den Bogaart G (2021) Modulation of immune responses by particle size and shape. Front Immunol 11:607945\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai K, Hou Y, Hu Y, Zhao L, Luo Z, Shi Y et al (2011) Correlation of the cytotoxicity of TiO₂ nanoparticles with different particle sizes on a sub-200-nm scale. J Nanosci Nanotechnol 7:3026\u0026ndash;3031\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWillis J, Li S, Crean SJ, Barrak FN (2021) Is titanium alloy Ti-6Al‐4V cytotoxic to gingival fibroblasts? A systematic review. Clin Exp Dent Res 7:1037\u0026ndash;1044\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAsa\u0026rsquo;ad F, Thomsen P, Kunrath MF (2022) The role of titanium particles and ions in the pathogenesis of peri-implantitis. J Bone Metab 29:145\u0026ndash;154\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dental implant, implantoplasty, fibroblast, osteoblast, Ti6Al4V","lastPublishedDoi":"10.21203/rs.3.rs-7324487/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7324487/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e\u003cp\u003eThis study evaluated the cytotoxicity and metabolic activity of human fibroblasts and osteoblasts in the presence of metallic particles and on implant surfaces subjected to implantoplasty (IP), previously contaminated with a multispecies biofilm. It also assessed the potential for biofilm formation on these particles.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTitanium alloy (Ti6Al4V) particles were collected to assess their cytotoxic potential and interactions with human cells and bacterial biofilms. Cytotoxicity assays were performed using fibroblasts (HFF-1) and osteoblast-like cells (SaOs-2) through an indirect lactate dehydrogenase (LDH) assay. Biofilm formation was evaluated using Streptococcus oralis, Actinomyces viscosus, Veillonella parvula, and Porphyromonas gingivalis, quantified by colony-forming units (CFUs) and metabolic activity. Fibroblasts and osteoblasts were co-cultured with biofilm-contaminated particles for 2, 4, and 6 hours. Cell morphology and biofilm association were examined by phase-contrast microscopy, while metabolic activity was measured spectrophotometrically.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIP-treated implants did not show significant cytotoxicity in HFF-1 or SaOs-2, with metabolic activities above 92% and cytotoxicity below 20%. Ti6Al4V particles, however, promoted Actinomyces viscosus and Veillonella parvula growth, increasing metabolic activity by 192.36% and 202.89%, and CFUs to 1.41 \u0026times; 10⁹ and 7.10 \u0026times; 10⁸, compared to 4.27 \u0026times; 10⁶ and 2.33 \u0026times; 10⁶ in controls. In multispecies biofilm, overall metabolic activity showed no significant differences (94.34% vs. 100%). Co-culture with infected particles drastically reduced fibroblast and osteoblast activity (\u0026lt;\u0026thinsp;25% and \u0026lt;\u0026thinsp;10%). In the absence of bacteria, fibroblasts reached 266.2% and osteoblasts 90% viability.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eContaminated particles from IP markedly reduced cytocompatibility of osteoblasts and fibroblasts and promoted specific bacterial growth, whereas IP-treated implant surfaces did not impair cell viability.\u003c/p\u003e\u003ch2\u003eClinical relevance:\u003c/h2\u003e\u003cp\u003eBiofilm-contaminated titanium particles released during implantoplasty reduce cell viability and promote bacterial growth, unlike the treated implant surface.\u003c/p\u003e","manuscriptTitle":"Cytotoxic effects of titanium particles and implantoplasty-treated surfaces exposed to bacterial biofilm","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 20:38:57","doi":"10.21203/rs.3.rs-7324487/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90f9c41b-2dc5-492c-80ac-0ff77ff2e234","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T20:53:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-29 20:38:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7324487","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7324487","identity":"rs-7324487","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}