Osteoblast cell behavior on polyetheretherketone dental implant surfaces treated with different grit size aluminum oxide particles: An in vitro analysis.

STATEMENT OF PROBLEM: The hydrophobic and bioinert nature of polyetheretherketone (PEEK) implants needs to be addressed for successful osseointegration. PURPOSE: The purpose of this in vitro study was to evaluate the osteoblast cell behavior on PEEK implant surfaces treated with airborne-particle abrasion using different grit size aluminum oxide (Al2O3) particles. MATERIAL AND METHODS: Disk-shaped specimens (n=96) were prepared from medical grade PEEK rods and were distributed into 4 groups (n=24) of untreated PEEK (PEEK 0), airborne-particle abrasion using 50-µm Al2O3 particles (PEEK 50), airborne-particle abrasion using 110-µm Al2O3 particles (PEEK 110), and airborne-particle abrasion using 150-µm Al2O3 particles (PEEK 150). The surface characteristics were assessed using water contact angle (WCA) measurements and scanning electron microscopy (SEM). MG-63 osteoblast cells were cultured, and the biocompatibility of PEEK was assessed using a CellTiter-blue cell viability assay and florescence staining at day 1, 3, and 7. The specimens were stained with Alizarin red to assess the osteoblast cell differentiation on day 10 and 14. The Levene test was used to test the homogeneity of variances. One-way and Welch ANOVA with post hoc corrections were used to assess the overall statistical significance of differences among the groups (α=.05). RESULTS: The lowest mean WCA was demonstrated in PEEK 150 (49.25 ±5.51) and the highest in PEEK 0 (89.14 ±4.24) (P<.001). SEM images of PEEK 150 illustrated a more complex structure with a large area of globular outcroppings throughout the surface. PEEK 150 showed the highest cell metabolic activity at each time point with florescence staining showing a substantial cell confluence at day 3 and 7. Although PEEK 150 did not show a significant increase in cell proliferation, the number of cells attached was significantly higher than other groups (P<.05). PEEK 110 and 150 also showed a substantial increase in the extent of mineralization. CONCLUSIONS: Airborne-particle abrasion using moderate Al2O3 grit size (110- or 150-µm) improved the hydrophilicity and osteoblast cell behavior on PEEK implants.

Anti-biofilm properties of laser-synthesized, ultrapure silver-gold-alloy nanoparticles against Staphylococcus aureus.

Staphylococcus aureus biofilm-associated infections are a common complication in modern medicine. Due to inherent resilience of biofilms to antibiotics and the rising number of antibiotic-resistant bacterial strains, new treatment options are required. For this purpose, ultrapure, spherical silver-gold-alloy nanoparticles with homogenous elemental distribution were synthesized by laser ablation in liquids and analyzed for their antibacterial activity on different stages of S. aureus biofilm formation as well as for different viability parameters. First, the effect of nanoparticles against planktonic bacteria was tested with metabolic activity measurements. Next, nanoparticles were incubated with differently matured S. aureus biofilms, which were then analyzed by metabolic activity measurements and three dimensional live/dead fluorescent staining to determine biofilm volume and membrane integrity. It could be shown that AgAu NPs exhibit antibacterial properties against planktonic bacteria but also against early-stage and even mature biofilms, with a complete diffusion through the biofilm matrix. Furthermore, AgAu NPs primarily targeted metabolic activity, to a smaller extend membrane integrity, but not the biofilm volume. Additional molecular analyses using qRT-PCR confirmed the influence on different metabolic pathways, like glycolysis, stress response and biofilm formation. As this shows clear similarities to the mechanism of pure silver ions, the results strengthen silver ions to be the major antibacterial agent of the synthesized nanoparticles. In summary, the results of this study provide initial evidence of promising anti-biofilm characteristics of silver-gold-alloy nanoparticles and support the importance of further translation-oriented analyses in the future.

The Origin of the Intracellular Silver in Bacteria: A Comprehensive Study using Targeting Gold-Silver Alloy Nanoparticles.

The bactericidal effects of silver nanoparticles (Ag NPs) against infectious strains of multiresistant bacteria is a well-studied phenomenon, highly relevant for many researchers and clinicians battling bacterial infections. However, little is known about the uptake of the Ag NPs into the bacteria, the related uptake mechanisms, and how they are connected to antimicrobial activity. Even less information is available on AgAu alloy NPs uptake. In this work, the interactions between colloidal silver-gold alloy nanoparticles (AgAu NPs) and Staphylococcus aureus (S. aureus) using advanced electron microscopy methods are studied. The localization of the nanoparticles is monitored on the membrane and inside the bacterial cells and the elemental compositions of intra- and extracellular nanoparticle species. The findings reveal the formation of pure silver nanoparticles with diameters smaller than 10 nm inside the bacteria, even though those particles are not present in the original colloid. This finding is explained by a local RElease PEnetration Reduction (REPER) mechanism of silver cations emitted from the AgAu nanoparticles, emphasized by the localization of the AgAu nanoparticles on the bacterial membrane by aptamer targeting ligands. These findings can deepen the understanding of the antimicrobial effect of nanosilver, where the microbes are defusing the attacking silver ions via their reduction, and aid in the development of suitable therapeutic approaches.

Mobile SARS­CoV­2 screening facilities for rapid deployment and university-based diagnostic laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a public crisis. Many medical and public institutions and businesses went into isolation in response to the pandemic. Because SARS-CoV-2 can spread irrespective of a patient's course of disease, these institutions' continued operation or reopening based on the assessment and control of virus spread can be supported by targeted population screening. For this purpose, virus testing in the form of polymerase chain reaction (PCR) analysis and antibody detection in blood can be central. Mobile SARS-CoV-2 screening facilities with a built-in biosafety level (BSL)-2 laboratory were set up to allow the testing offer to be brought close to the subject group's workplace. University staff members, their expertise, and already available equipment were used to implement and operate the screening facilities and a certified diagnostic laboratory. This operation also included specimen collection, transport, PCR and antibody analysis, and informing subjects as well as public health departments. Screening facilities were established at different locations such as educational institutions, nursing homes, and companies providing critical supply chains for health care. Less than 4 weeks after the first imposed lockdown in Germany, a first mobile testing station was established featuring a build-in laboratory with two similar stations commencing operation until June 2020. During the 15-month project period, approximately 33,000 PCR tests and close to 7000 antibody detection tests were collected and analyzed. The presented approach describes the required procedures that enabled the screening facilities and laboratories to collect and process several hundred specimens each day under difficult conditions. This report can assist others in establishing similar setups for pandemic scenarios.

Comparison of two antibody screening systems for SARS-CoV-2 antibody detection in recovered and vaccinated subjects - test performance and possible indicators for immunity.

BACKGROUND: Detection of seroconversion after SARS-CoV-2-infection or vaccination is relevant to discover subclinical cases and recognize patients with a possible immunity. OBJECTIVES: Test performance, effects of age, time-point of seroconversion and immune status regarding neutralizing antibodies (NAbs) and T-cell-reactivity were investigated. STUDY DESIGN: Two antibody assays (Viramed-Test for S/N-specific IgG, Roche-Test for N-specific IgA, -M, -G) were evaluated with classified samples. In total, 381 subjects aged 6-99 years, who had either recovered from the disease or had been vaccinated, were screened for SARS-CoV-2-specific antibodies. This screening was part of an open observational study with working adults. Additionally, children and adults were analyzed in a longitudinal COVID-19 study in schools. For immunity evaluation, virus neutralization tests and ELISpot tests were performed in a subgroup of subjects. RESULTS: Viramed revealed a slightly lower test performance than Roche, but test quality was equally well in samples from very young or very old donors. The time-point of seroconversion after the respective immunization detected by the two tests was not significantly different. N-specific antibodies, detected with Roche, highly correlated with NAbs in recovered subjects, whereas a positive Viramed-Test result was paralleled by a positive ELISpot result. CONCLUSION: Viramed-Test was not as sensitive as Roche-Test, but highly specific and beneficial to distinguish between recovered and vaccinated status. For both tests correlations with humoral and cellular immunity were found. Of note, the expected early detection of IgA and IgM by the Roche-Test did not prove to be an advantage over IgG testing by Viramed.

Adhesion Forces of Oral Bacteria to Titanium and the Correlation with Biophysical Cellular Characteristics.

Bacterial adhesion to dental implants is the onset for the development of pathological biofilms. Reliable characterization of this initial process is the basis towards the development of anti-biofilm strategies. In the present study, single-cell force spectroscopy (SCFS), by means of an atomic force microscope connected to a microfluidic pressure control system (FluidFM), was used to comparably measure adhesion forces of different oral bacteria within a similar experimental setup to the common implant material titanium. The bacteria selected belong to different ecological niches in oral biofilms: the commensal pioneers Streptococcus oralis and Actinomyces naeslundii; secondary colonizer Veillonella dispar; and the late colonizing pathogens Porphyromonas gingivalis as well as fimbriated and non-fimbriated Aggregatibacter actinomycetemcomitans. The results showed highest values for early colonizing pioneer species, strengthening the link between adhesion forces and bacteria's role in oral biofilm development. Additionally, the correlation between biophysical cellular characteristics and SCFS results across species was analyzed. Here, distinct correlations between electrostatically driven maximum adhesion force, bacterial surface elasticity and surface charge as well as single-molecule attachment points, stretching capability and metabolic activity, could be identified. Therefore, this study provides a step towards the detailed understanding of oral bacteria initial adhesion and could support the development of infection-resistant implant materials in future.

Identification of Pri-miRNA Stem-Loop Interacting Proteins in Plants Using a Modified Version of the Csy4 CRISPR Endonuclease.

Regulation at the RNA level by RNA-binding proteins (RBPs) and microRNAs (miRNAs) is key to coordinating eukaryotic gene expression. In plants, the importance of miRNAs is highlighted by severe developmental defects in mutants impaired in miRNA biogenesis. MiRNAs are processed from long primary-microRNAs (pri-miRNAs) with internal stem-loop structures by endonucleolytic cleavage. The highly structured stem-loops constitute the basis for the extensive regulation of miRNA biogenesis through interaction with RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown in plants. Therefore, we exploit an RNA-centric approach based on modified versions of the conditional CRISPR nuclease Csy4* to pull down interactors of the Arabidopsis pri-miR398b stem-loop (pri-miR398b-SL) in vitro. We designed three epitope-tagged versions of the inactive Csy4* for the immobilization of the protein together with the pri-miR398b-SL bait on high affinity matrices. After incubation with nucleoplasmic extracts from Arabidopsis and extensive washing, pri-miR398b-SL, along with its specifically bound proteins, were released by re-activating the cleavage activity of the Csy4* upon the addition of imidazole. Co-purified proteins were identified via quantitative mass spectrometry and data sets were compared. In total, we identified more than 400 different proteins, of which 180 are co-purified in at least two out of three independent Csy4*-based RNA pulldowns. Among those, the glycine-rich RNA-binding protein AtRZ-1a was identified in all pulldowns. To analyze the role of AtRZ-1a in miRNA biogenesis, we determined the miR398 expression level in the atrz-1a mutant. Indeed, the absence of AtRZ-1a caused a decrease in the steady-state level of mature miR398 with a concomitant reduction in pri-miR398b levels. Overall, we show that our modified Csy4*-based RNA pulldown strategy is suitable to identify new trans-acting regulators of miRNA biogenesis and provides new insights into the post-transcriptional regulation of miRNA processing by plant RBPs.

Antibacterial and Cytocompatible: Combining Silver Nitrate with Strontium Acetate Increases the Therapeutic Window.

Microbial infection and insufficient tissue formation are considered to be the two main causes of dental implant failure. Novel studies have focused on designing dual-functional strategies to promote antibacterial properties and improve tissue cell response simultaneously. In this study, we investigated the antibacterial properties and cytocompatibility of silver nitrate (AgNO3) and strontium acetate (SrAc) in a mono-culture setup for dental application. Additionally, we defined the therapeutic window between the minimum inhibitory concentration against pathogenic bacteria and maximum cytocompatible dose in the case of combined applications in a co-culture setup. Antibacterial properties were screened using Aggregatibacter actinomycetemcomitans and cell response experiments were performed with osteoblastic cells (MC3T3) and fibroblastic cells (NIH3T3). The osteoinductive behavior was investigated separately on MC3T3 cells using alizarin red staining. A therapeutic window for AgNO3 as well as SrAc applications could be defined in the case of MC3T3 cells while the cytocompatibility of NIH3T3 cells was compromised for all concentrations with an antibacterial effect. However, the combined application of AgNO3/SrAc caused an enhanced antibacterial effect and opened a therapeutic window for both cell lines. Enhanced mineralization rates could be observed in cultures containing SrAc. In conclusion, we were able to demonstrate that adding SrAc to AgNO3 not only intensifies antibacterial properties but also exhibits bone inductive characteristics, thereby offering a promising strategy to combat peri-implantitis and at the same time improve osseointegration in implant therapy.

Influence of the Available Surface Area and Cell Elasticity on Bacterial Adhesion Forces on Highly Ordered Silicon Nanopillars.

Initial bacterial adhesion to solid surfaces is influenced by a multitude of different factors, e.g., roughness and stiffness, topography on the micro- and nanolevel, as well as chemical composition and wettability. Understanding the specific influences and possible interactive effects of all of these factors individually could lead to guidance on bacterial adhesion and prevention of unfavorable consequences like medically relevant biofilm formation. On this way, the aim of the present study was to identify the specific influence of the available surface area on the adhesion of clinically relevant bacterial strains with different membrane properties: Gram-positive Staphylococcus aureus and Gram-negative Aggregatibacter actinomycetemcomitans. As model surfaces, silicon nanopillar specimens with different spacings were fabricated using electron beam lithography and cryo-based reactive ion etching techniques. Characterization by scanning electron microscopy and contact angle measurement revealed almost defect-free highly ordered nanotopographies only varying in the available surface area. Bacterial adhesion forces to these specimens were quantified by means of single-cell force spectroscopy exploiting an atomic force microscope connected to a microfluidic setup (FluidFM). The nanotopographical features reduced bacterial adhesion strength by reducing the available surface area. In addition, the strain-specific interaction in detail depended on the bacterial cell's elasticity and deformability as well. Analyzed by confocal laser scanning microscopy, the obtained results on bacterial adhesion forces could be linked to the subsequent biofilm formation on the different topographies. By combining two cutting-edge technologies, it could be demonstrated that the overall bacterial adhesion strength is influenced by both the simple physical interaction with the underlying nanotopography and its available surface area as well as the deformability of the cell.

Interleukin-17 is disease promoting in early stages and protective in late stages of experimental periodontitis.

Periodontitis is one of the most common infectious diseases in humans. It is characterized by a chronic inflammation of the tooth-supporting tissue that results in bone loss. However, the role and source of the pro-inflammatory cytokine interleukin-17 (IL-17) and of the cells producing it locally in the gingiva is still controversial. Th17 αß T cells, CD4+ exFoxP3+ αß T cells, or IL-17-producing γδ T cells (γδ17 cells) seem to be decisive cellular players in periodontal inflammation. To address these issues in an experimental model for periodontitis, we employed genetic mouse models deficient for either γδ T cells or IL-17 cytokines and assessed the bone loss during experimental periodontal inflammation by stereomicroscopic, histological, and µCT-analysis. Furthermore, we performed flow-cytometric analyses and qPCR-analyses of the gingival tissue. We found no γδ T cell- or IL-17-dependent change in bone loss after four weeks of periodontitis. Apart from that, our data are complementary with earlier studies, which suggested IL-17-dependent aggravation of bone loss in early periodontitis, but a rather bone-protective role for IL-17 in late stages of experimental periodontitis with respect to the osteoclastogenicity defined by the RANKL/OPG ratio.

Bacterial-Specific Induction of Inflammatory Cytokines Significantly Decreases upon Dual Species Infections of Implant Materials with Periodontal Pathogens in a Mouse Model.

Cytokine profiles are often perturbed after infections of medical implants. With a non-invasive in vivo imaging system, we report in a mouse model that interferon expression after infection of subcutaneous implants with Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola (alone or as a combination) was species-specific, persisted longer in the presence of implants, and notably decreased upon dual species infections. This type I interferon expression disappeared within two weeks; however, histology of implant-tissue interface indicated high recruitment of immune cells even after three weeks. This was suggestive that biomaterial-associated infections could have prolonged effects, including the systemic stimulation of inflammatory cytokines. The present study investigated the systemic impact of this chronic peri-implant inflammation on the systemic expression of inflammatory cytokines (23) using a multiplex assay. Initially, the cytokine measurement in murine fibroblasts exposed to periodontal pathogens remained limited to the expression of five cytokines, namely, IL-6, G-CSF, CXCL-1/KC, MCP-1 (MCAF), and IL-12 (p40). The systemic determination of cytokines in mice increased to 19 cytokines (IL-1α, IL-2, IL-3, IL-5, IL-6, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-17A, CCL-11/Eotaxin, G-CSF, IFN-γ, CXCL1/KC, MCP-1 (MCAF), MIP-1α/CCL3, MIP-1ß/CCL4, CCL5/RANTES, and TNF-α). Systemic induction of cytokines was species-specific in the mouse model. The cytokine induction from infected implants differed significantly from sole tissue infections and sterile implants. Notably, systemic cytokine induction decreased after infections with dual species compared to single species infections. These findings describe the systemic effect of chronic peri-implant inflammation on the systemic induction of inflammatory cytokines, and this effect was strongly correlated to the type and composition of initial infection. Systemic modulations in cytokine expression upon dual species infections exhibit an exciting pattern that might explain the complications associated with biomaterial-related infection in patients. Moreover, these findings validate the requirement of multispecies infections for pre-clinical studies involving animal models.

Antimicrobial agents in dental restorative materials: Effect on polymerization, short-term drug release and biological impact.

With the aim to design bioactive dental restorative material, the present study investigated the influence of the antimicrobial agents chlorhexidine diacetate (CHX) and octinidine (di)hydrochloride (ODH) when incorporated in two different materials. Selected parameters were polymerization enthalpy, short-term drug release, and the effect on Streptococcus mutans as well as human gingival fibroblasts. Samples were made by mixing a nano-hybrid ormocer (O) and a methacrylate-based nano-hybrid composite (C), each with a mass fraction of 2% CHX or ODH. Release profiles and concentrations of active agents from the resins were assessed, and the cell proliferation of human gingival fibroblasts as well as Streptococcus mutans cultured with the eluates were evaluated. The influence on polymerization was assessed by means of differential scanning calorimetry. Both drugs, especially ODH, showed a decreasing effect on polymerization enthalpies associated with a lowered crosslinking degree. At the same time ODH appeared to be released more persistently than CHX. Moreover, ODH was more efficient with regard to bacteria growth inhibition but also more cytotoxic in terms of reduction of cell viability. ODH is deemed more appropriate for application in a dental resin-based drug delivery system, because of the more persistent drug release than seen for CHX.

Evaluation of biofilm colonization on multi-part dental implants in a rat model.

BACKGROUND: Peri-implant mucositis and peri-implantitis are highly prevalent biofilm-associated diseases affecting the tissues surrounding dental implants. As antibiotic treatment is ineffective to fully cure biofilm mediated infections, antimicrobial modifications of implants to reduce or prevent bacterial colonization are called for. Preclinical in vivo evaluation of the functionality of new or modified implant materials concerning bacterial colonization and peri-implant health is needed to allow progress in this research field. For this purpose reliable animal models are needed. METHODS: Custom made endosseous dental implants were installed in female Sprague Dawley rats following a newly established three-step implantation procedure. After healing of the bone and soft tissue, the animals were assigned to two groups. Group A received a continuous antibiotic treatment for 7 weeks, while group B was repeatedly orally inoculated with human-derived strains of Streptococcus oralis, Fusobacterium nucleatum and Porphyromonas gingivalis for six weeks, followed by 1 week without inoculation. At the end of the experiment, implantation sites were clinically assessed and biofilm colonization was quantified via confocal laser scanning microscopy. Biofilm samples were tested for presence of the administered bacteria via PCR analysis. RESULTS: The inner part of the custom made implant screw could be identified as a site of reliable biofilm formation in vivo. S. oralis and F. nucleatum were detectable only in the biofilm samples from group B animals. P. gingivalis was not detectable in samples from either group. Quantification of the biofilm volume on the implant material revealed no statistically significant differences between the treatment groups. Clinical inspection of implants in group B animals showed signs of mild to moderate peri-implant mucositis (4 out of 6) whereas the mucosa of group A animals appeared healthy (8/8). The difference in the mucosa health status between the treatment groups was statistically significant (p = 0.015). CONCLUSIONS: We developed a new rodent model for the preclinical evaluation of dental implant materials with a special focus on the early biofilm colonization including human-derived oral bacteria. Reliable biofilm quantification on the implant surface and the symptoms of peri-implant mucositis of the bacterially inoculated animals will serve as a readout for experimental evaluation of biofilm-reducing modifications of implant materials.

Cell Type-Specific Adhesion and Migration on Laser-Structured Opaque Surfaces.

Cytocompatibility is essential for implant approval. However, initial in vitro screenings mainly include the quantity of adherent immortalized cells and cytotoxicity. Other vital parameters, such as cell migration and an in-depth understanding of the interaction between native tissue cells and implant surfaces, are rarely considered. We investigated different laser-fabricated spike structures using primary and immortalized cell lines of fibroblasts and osteoblasts and included quantification of the cell area, aspect ratio, and focal adhesions. Furthermore, we examined the three-dimensional cell interactions with spike topographies and developed a tailored migration assay for long-term monitoring on opaque materials. While fibroblasts and osteoblasts on small spikes retained their normal morphology, cells on medium and large spikes sank into the structures, affecting the composition of the cytoskeleton and thereby changing cell shape. Up to 14 days, migration appeared stronger on small spikes, probably as a consequence of adequate focal adhesion formation and an intact cytoskeleton, whereas human primary cells revealed differences in comparison to immortalized cell lines. The use of primary cells, analysis of the cell-implant structure interaction as well as cell migration might strengthen the evaluation of cytocompatibility and thereby improve the validity regarding the putative in vivo performance of implant material.

Non-Invasive Luciferase Imaging of Type I Interferon Induction in a Transgenic Mouse Model of Biomaterial Associated Bacterial Infections: Microbial Specificity and Inter-Bacterial Species Interactions.

The performance of biomaterials is often compromised by bacterial infections and subsequent inflammation. So far, the conventional analysis of inflammatory processes in vivo involves time-consuming histology and biochemical assays. The present study employed a mouse model where interferon beta (IFN-ß) is monitored as a marker for non-invasive rapid detection of inflammation in implant-related infections. The mouse model comprises subcutaneous implantation of morphologically modified titanium, followed by experimental infections with four taxonomically diverse oral bacteria: Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Treponema denticola (as mono culture or selected mixed-culture). IFN-ß expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-ß expression kinetics reduced with two mixed species infections when compared with the single species. Histological and confocal microscopy confirmed pathogen-specific infiltration of inflammatory cells at the implant-tissue interface. This was observed mainly in the vicinity of infected implants and was, in contrast to interferon expression, higher in infections with dual species. In summary, this non-invasive mouse model can be used to quantify longitudinally host inflammation in real time and suggests that the polymicrobial character of infection, highly relevant to clinical situations, has complex effects on host immunity.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

In Vitro Antibacterial Effectiveness of a Naturopathic Oral Care Product on Oral Pathogens.

PURPOSE: Currently, the prevention of periodontal diseases focuses on mechanical removal of pathogenic biofilms combined with oral antiseptics as supportive chemical antibacterial control. Due to the risk of resistance development and side effects of existing antiseptics, the interest in alternative medicine with naturopathic treatment modalities is growing in dentistry. In the present study, the antibacterial effect of the naturopathic oral care product Repha OS and some of its derivatives, based on medicinal plant extracts and essential oils, with a specific focus on added sweeteners, was investigated on periodontal pathogenic and halitosis-associated bacteria. MATERIALS AND METHODS: The antibacterial efficacy was investigated by agar dilution assay. The minimum inhibitory concentration (MIC) for the bacterial species Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Solobacterium moorei was determined. RESULTS: A concentration-dependent antibacterial effect on oral bacterial species by Repha OS and its derivatives was demonstrated. For the original product, the maximum MIC was 10% of the calculated test solution concentration in agar for all examined bacterial species. The removal of essential oils reduced the antibacterial efficacy, whereas the displacement or replacement of sweeteners had almost no effect. CONCLUSION: In addition to other individual effects of the ingredients, the results of this study show that an antibacterial effect of the naturopathic oral care product on the tested oral bacterial species was achieved in vitro. In vivo, the combination of this antibacterial effect with other properties of the various ingredients may be interesting for a holistic approach in preventive dentistry.

In Vitro Effects of Streptococcus oralis Biofilm on Peri-Implant Soft Tissue Cells.

Human gingival epithelial cells (HGEps) and fibroblasts (HGFs) are the main cell types in peri-implant soft tissue. HGEps are constantly exposed to bacteria, but HGFs are protected by connective tissue as long as the mucosa-implant seal is intact. Streptococcus oralis is one of the commensal bacteria, is highly abundant at healthy implant sites, and might modulate soft tissue cells-as has been described for other streptococci. We have therefore investigated the effects of the S. oralis biofilm on HGEps and HGFs. HGEps or HGFs were grown separately on titanium disks and responded to challenge with S. oralis biofilm. HGFs were severely damaged after 4 h, exhibiting transcriptional inflammatory and stress responses. In contrast, challenge with S. oralis only induced a mild transcriptional inflammatory response in HGEps, without cellular damage. HGFs were more susceptible to the S. oralis biofilm than HGEps. The pro-inflammatory interleukin 6 (IL-6) was attenuated in HGFs, as was interleukin 8 (CXCL8) in HGEps. This indicates that S. oralis can actively protect tissue. In conclusion, commensal biofilms can promote homeostatic tissue protection, but only if the implant-mucosa interface is intact and HGFs are not directly exposed.

Early host-microbe interaction in a peri-implant oral mucosa-biofilm model.

The host-microbe relationship is pivotal for oral health as well as for peri-implant diseases. Peri-implant mucosa and commensal biofilm play important roles in the maintenance of host-microbe homeostasis, but little is known about how they interact. We have therefore investigated the early host-microbe interaction between commensal multispecies biofilm (Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar, Porphyromonas gingivalis) and organotypic peri-implant mucosa using our three-dimensional model. After 24 hr, biofilms induced weak inflammatory reaction in the peri-implant mucosa by upregulation of five genes related to immune response and increased secretion of IL-6 and CCL20. Biofilm volume was reduced which might be explained by secretion of ß-Defensins-1, -2, and CCL20. The specific tissue reaction without intrinsic overreaction might contribute to intact mucosa. Thus, a relationship similar to homeostasis and oral health was established within the first 24 hr. In contrast, the mucosa was damaged and the bacterial distribution was altered after 48 hr. These were accompanied by an enhanced immune response with upregulation of additional inflammatory-related genes and increased cytokine secretion. Thus, the homeostasis-like relationship was disrupted. Such profound knowledge of the host-microbe interaction at the peri-implant site may provide the basis to improve strategies for prevention and therapy of peri-implant diseases.

Neutrophils exhibit an individual response to different oral bacterial biofilms.

Oral innate immunity is led by neutrophils. It is still unclear how their main antimicrobial mechanisms against different biofilms may contribute to balance or dysregulation in the oral cavity. We investigated the capacity of commensal (Streptococcus oralis) and pathogenic (Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans) monospecies biofilms to induce or to inhibit selected antimicrobial mechanisms of neutrophils. S. oralis induced neutrophil extracellular traps (NETs) formation, reactive oxygen species (ROS) production, and matrix metalloproteinases (MMPs) 8 and 9 secretion. However, these responses were partially reduced in PMA-activated neutrophils indicating a balance-like neutrophil response, which might be important for the maintenance of oral health. P. gingivalis generally induced ROS. Reduced NET formation and significantly decreased MMP secretion were detectable in activated neutrophils highlighting P. gingivalis' nucleolytic and proteolytic activity, which might support bacterial colonization and pathogenesis of periodontitis. In contrast, A. actinomycetemcomitans did not affect the levels of antimicrobial factors in activated neutrophils and induced NET formation, ROS production, and secretion of MMP-8 and -9 in neutrophils alone, which might contribute to tissue destruction and disease progression. In summary, neutrophil responses to biofilms were species-specific and might support either maintenance of oral health or pathogenesis of periodontitis depending on the species.