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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

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

Evidence for inoculum size and gas interfaces as critical factors in bacterial biofilm formation on magnesium implants in an animal model.

Infections of medical implants caused by bacterial biofilms are a major clinical problem. Bacterial colonization is predicted to be prevented by alkaline magnesium surfaces. However, in experimental animal studies, magnesium implants prolonged infections. The reason for this peculiarity likely lies within the ‒still largely hypothetical‒ mechanism by which infection arises. Investigating subcutaneous magnesium implants infected with bioluminescent Pseudomonas aeruginosa via in vivo imaging, we found that the rate of implant infections was critically dependent on a surprisingly high quantity of injected bacteria. At high inocula, bacteria were antibiotic-refractory immediately after infection. High cell densities are known to limit nutrient availability, restricting proliferation and trigger quorum sensing which could both contribute to the rapid initial resistance. We propose that gas bubbles such as those formed during magnesium corrosion, can then act as interfaces that support biofilm formation and permit long-term survival. This model could provide an explanation for the apparent ineffectiveness of innovative contact-dependent bactericidal implant surfaces in patients. In addition, the model points toward air bubbles in tissue, either by inclusion during surgery or by spontaneous gas bubble formation later on, could constitute a key risk factor for clinical implant infection

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-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

Time resolved 3D live-cell imaging on implants.

It is estimated that two million new dental implants are inserted worldwide each year. Innovative implant materials are developed in order to minimize the risk of peri-implant inflammations. The broad range of material testing is conducted using standard 2D, terminal, and invasive methods. The methods that have been applied are not sufficient to monitor the whole implant surface and temporal progress. Therefore, we built a 3D peri-implant model using a cylindrical implant colonized by human gingival fibroblasts. In order to monitor the cell response over time, a non-toxic LIVE/DEAD staining was established and applied to the new 3D model. Our LIVE/DEAD staining method in combination with the time resolved 3D visualization using Scanning Laser Optical Tomography (SLOT), allowed us to monitor the cell death path along the implant in the 3D peri-implant model. The differentiation of living and dead gingival fibroblasts in response to toxicity was effectively supported by the LIVE/DEAD staining. Furthermore, it was possible to visualize the whole cell-colonized implant in 3D and up to 63 hours. This new methodology offers the opportunity to record the long-term cell response on external stress factors, along the dental implant and thus to evaluate the performance of novel materials/surfaces

Commensal and pathogenic biofilms differently modulate peri-implant oral mucosa in an organotypic model

The impact of oral commensal and pathogenic bacteria on peri-implant mucosa is not well understood, despite the high prevalence of peri-implant infections. Hence, we investigated responses of the peri-implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri-implant mucosa-biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri-implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin-6 (IL-6), interleukin-8 (CXCL8), and monocyte chemoattractant protein-1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor-alpha (TNF-α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri-implant mucosa-biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri-implant disease

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

Abstract 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

Protein Transduction Domain-Mediated Delivery of Recombinant Proteins and In Vitro Transcribed mRNAs for Protein Replacement Therapy of Human Severe Genetic Mitochondrial Disorders: The Case of Sco2 Deficiency

Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistributed in the peripheral tissues of mice and successfully delivered into their mitochondria. Complementary to that, an mRNA-based therapeutic approach has been more recently considered as an innovative treatment option. In particular, a patented, novel PTD-mediated IVT-mRNA delivery platform was developed and applied in recent research efforts. PTD-IVT-mRNA of full-length SCO2 was successfully transduced into the fibroblasts derived from a SCO2/COX-deficient patient, translated in host ribosomes into a nascent chain of human Sco2, imported into mitochondria, and processed to the mature protein. Consequently, the recovery of reduced COX activity was achieved, thus suggesting the potential of this mRNA-based technology for clinical translation as a PRT for metabolic/genetic disorders. In this review, such research efforts will be comprehensibly presented and discussed to elaborate their potential in clinical application and therapeutic usefulness