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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Peri-implant infections from bacterial biofilms on artificial surfaces are a common threat to all medical implants. They are a handicap for the patient and can lead to implant failure or even life-threatening complications. New implant surfaces have to be developed to reduce biofilm formation and to improve the long-term prognosis of medical implants. The aim of this study was (1) to develop a new method to test the antibacterial efficacy of implant surfaces by direct surface contact and (2) to elucidate whether an innovative antimicrobial copolymer coating of 4-vinyl-N-hexylpyridinium bromide and dimethyl(2-methacryloyloxyethyl) phosphonate (VP:DMMEP 30:70) on titanium is able to reduce the attachment of bacteria prevalent in peri-implant infections. With a new in vitro model with semi-coated titanium discs, we were able to show a dramatic reduction in the adhesion of various pathogenic bacteria (Streptococcus sanguinis, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis), completely independently of effects caused by soluble materials. In contrast, soft tissue cells (human gingival or dermis fibroblasts) were less affected by the same coating, despite a moderate reduction in initial adhesion of gingival fibroblasts. These data confirm the hypothesis that VP:DMMEP 30:70 is a promising antibacterial copolymer that may be of use in several clinical applications. [ABSTRACT FROM AUTHOR]

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-1β/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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β) 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-β expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nanoparticles can be used as a smart drug delivery system, when they release the drug only upon degradation by specific enzymes. A method to create such responsive materials is the formation of hydrogel nanoparticles, which have enzymatically degradable crosslinkers. Such hydrogel nanoparticles were prepared by ionotropic gelation sodium alginate with lysine-rich peptide sequences-either α-poly-L-lysine (PLL) or the aggrecanase-labile sequence KKKK-GRD-ARGSV↓NITEGE-DRG-KKKK. The nanoparticle suspensions obtained were analyzed by means of dynamic light scattering and nanoparticle tracking analysis. Degradation experiments carried out with the nanoparticles in suspension revealed enzyme-induced lability. Drugs present in the polymer solution during the ionotropic gelation can be encapsulated in the nanoparticles. Drug loading was investigated for interferon-β (IFN-β) as a model, using a bioluminescence assay with MX2Luc2 cells. The encapsulation efficiency for IFN-β was found to be approximately 25%. The nanoparticles suspension can be used to spray-coat titanium alloys (Ti-6Al-4V) as a common implant material. The coatings were proven by ellipsometry, reflection-absorption infrared spectroscopy, and X-ray photoelectron spectroscopy. An enzyme-responsive decrease in layer thickness is observed due to the degradation of the coatings. The Alg/peptide coatings were cytocompatible for human gingival fibroblasts (HGFIB), which was investigated by CellTiterBlue and lactate dehydrogenase (LDH) assay. However, HGFIBs showed poor adhesion and proliferation on the Alg/peptide coatings, but these could be improved by modification of the alginate with a RGD-peptide sequence. The smart drug release system presented can be further tailored to have the right release kinetics and cell adhesion properties.