29. Jahrestagung der Deutschen Gesellschaft für Biomaterialien

Prof. Dr.

Chair

Mechanisms of biofilm development and progression on implants in health and disease

Implant associated

Biofilm formation

Abstract text (incl. figure legends and references) Question- Biofilm-associated implant infections continue to be a major complication in dentistry that lead to tissue destruction around the implant and occasionally subsequent implant loss. Early diagnosis of these infections is not currently possible. Yet, at the advanced disease stage, pathogenic biofilms are highly resistant towards therapy. Therefore, it is crucial to understand how implant-associated biofilms develop and become pathogenic. The aim of the study was the identification of molecular mechanisms that define the clinical course and severity of peri-implant biofilm infections. Methods- More than 150 implants were studied in longitudinal and cross-sectional settings. Initial biofilm development was analyzed using temporary implant abutments. Mature biofilms were characterized in health, mucositis and peri-implantitis. Biofilm structures were characterized using CLSM and SEM. Composition and activity of biofilms were revealed with full 16S rRNA gene amplicons sequencing and RNA sequencing, respectively. Mechanisms were studied using in vitro models. Results- Structure, composition and activity of biofilms were patient specific. Diverse anaerobes were more abundant and active in peri-implantitis. Streptococci dominated biofilms in health and mucositis, except for the Neisseria-rich subgroup of mucositis. Peptide degradation and catabolism of amino acids were upregulated in peri-implantitis, while diverse anabolic processes were typical for two other diagnosis groups. Interspecies interactions were prevalent in biofilms and contributed to biofilm fitness and resistance. Conclusions- The present study revealed developmental mechanisms in implant-associated biofilms. Discovered signatures of diseases play an important role in virulence of biofilms but need to be further validated in clinical studies. In future, highly predictive biomarkers can be used to provide personalized peri-implant therapy.

Short Talk

In vitro analysis of anti-biofilm and tissue-integrative properties of biocompatible liquid-infused structured surfaces

Antimicrobial coatings

Abstract text (incl. figure legends and references) Introduction Advanced dental abutments strive for inhibition of infection-inductive biofilm colonization while at the same time unrestricted tissue integration. The biomimetic concept of slippery liquid-infused porous surfaces (SLIPS) has already shown promising characteristics for this purpose. However, so far, SLIPS on titanium have been generated from non-biocompatible lubricants, which hinders medical application. Objectives The aim of this study was development and biological characterization of a biocompatible SLIPS functionalization on titanium. Materials &amp; methods Biocompatible SLIPS were produced by femtosecond pulsed laser ablation of titanium surfaces to generate nanostructure-covered spikes followed by coating with biocompatible viscous silicone polymer lubricant. After initial fluorescence-microscopy based investigation of anti-biofilm properties using static Streptococcus oralis monospecies culture and the Hanoverian oral multi-species implant flow chamber (HOBIC) model, SLIPS were integrated into the INTERbACT 3D tissue and biofilm coculture model. Here, fluorescence microscopy, histology and cytokine ELISA assays were used to analyze biofilm colonization, tissue integration and initial immune reaction, respectively. Results Biocompatible SLIPS showed strong repellency for both monospecies and multispecies biofilms. Implants in the INTERbACT model exhibited reduced colonization on SLIPS modified surfaces, while connective tissue next to the functionalization was not impaired as demonstrated histologically and by cytokine expression. Conclusion SLIPS titanium modification made by biocompatible silicone lubricant maintained its strong biofilm repellent properties even in complex in vitro models. At the same time, soft tissue still integrated in direct connection to SLIPS and did not show elevated levels of early immune response. Therefore, biocompatible SLIPS represent a promising surface modification for future clinical application.

Professor

Towards an in-vitro flow chamber model for oral multispecies biofilm dysbiosis on implant materials

Abstract text (incl. figure legends and references) Introduction The human mouth contains a diverse microbiome. Solid surfaces inside the oral cavity, like teeth or implants, are almost constantly populated by multispecies bacterial biofilms. Initially commensal and harmless to oral health, the species distribution inside these biofilms can shift to a pathogenic state and cause severe infections. To develop, test, and verify systems for early detection and treatment of this biofilm dysbiosis, complex in-vitro models are required. Objectives The objective of this study is to establish and validate a reproducible in-vitro model for oral biofilm dysbiosis using five clinically relevant bacterial species and a saliva flow-simulating flow chamber system. Materials &amp; methods Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum, and Porphyromonas gingivalis were added to a flow chamber system to form multispecies biofilms on titanium samples placed in flow chambers. After 24 h, the bacterial suspension was replaced with sterile medium to allow further development only of the initially formed biofilm under flow for 20 more days. Flow velocity was set to 100 µL/min and the system was kept at 37 °C and anaerobic conditions. The pH value of the medium leaving the flow chambers was constantly monitored. At different time points, samples were taken out for analysis via fluorescent staining and microscopy as well as qRT-PCR. Results After 6 days, a change in species distribution started, with the relative amount of commensal bacteria decreasing. The decrease of commensal bacteria was preceded by an increase in pH, following the initial drop after exponential growth. Conclusion To validate diagnostic tools and therapeutics for associated infections, reproducible in-vitro models of complex biologic systems like multi-species bacterial communities are required. In this study, we presented an approach to obtain such a model for oral biofilm dysbiosis, based on the natural shift over time.

Development of 3D-implant-tissue-oral-biofilm-model (INTERbACT)

Infections

Abstract text (incl. figure legends and references) Introduction The formation of complex and antibiotic-resistant biofilms on medical implants can result in implant failure. Dental implant-related infections have a prevalence of 20-25 % five years after implantation. A detailed understanding of host-implant-biofilm interactions cannot be gained directly in an animal model or human body. Alternatively, two-dimensional (2D) cell culture models are usually employed; however, they cannot reflect the complexity of the tissue. Thus, three-dimensional (3D) culture models, particularly complemented with host immune cells, provide physiologically more relevant and predictive conditions under in vitro situations. Objectives This study aimed to establish a 3D-implant-tissue-oral-biofilm-model to investigate host-implant-biofilm interactions and evaluate the novel antibacterial implants. Methods A 3D mucosa model was developed by culturing human monocyte-derived macrophages, gingival fibroblasts and keratinocytes on a cylindrical titanium surface inside a collagen matrix. Cells in the 3D model were cultivated under standard cell culture conditions for 22 days at an air-liquid interface. Then, the constructs were analysed using histology and fluorescence microscopy. Further, cells were reisolated, subjected to flow cytometry, and evaluated for their activation status. Results The histological analysis confirmed that macrophages, fibroblasts and keratinocytes had successfully adhered to the inserted implants and were viable throughout the experiment. Flow cytometry analysis and ELISA demonstrated expression of CD14 and IL-1β, confirming the activation of macrophages. The co-culture assay confirmed antibacterial potential when macrophages were present. Conclusion The present study has successfully established a full-thickness 3D model consisting of three human cell types. The 3D model could be a promising in vitro system to investigate host-implant-biofilm interactions and evaluate novel antibacterial coatings.

Laser-based culturomics and single cell force spectroscopy for studying interspecies interactions in implant-associated biofilms

Abstract text (incl. figure legends and references) Introduction Polymicrobial oral biofilms cause severe implant-associated infections. A profound understanding of formation and structure of these biofilms would facilitate the development of improved methods to diagnose, prevent and treat implant-associated infections. However, microbial isolates for fastidious species are limited and the impact of interspecies interactions on the development of biofilms and implant-associated pathologies is barely understood. Objectives The aim of the study was to develop methods for the high-throughput isolation of oral microorganisms and in-depth characterization of biofilm ecology. Material and Methods Laser bioprinting was adapted for high-throughput cultivation, characterization, storage and transfer of oral microbiota. Fluidic Force Microscopy (FluidFM) was applied to characterize physical interspecies interactions within biofilms at single cell level. Results Laser-based culturomics generated a first extensive culture collection for microorganisms from human dental implants and detected the microbial consortia co-localized in biofilms. Physical interactions of selected co-isolates were characterized at single cell level. According to parameters, which describe physical contact in FluidFM (attachment points, maximum adhesion force and detachment distance), different biophysical profiles were observed for different pairs of strains. Effects of taxonomy, specific adhesion-receptor recognition and co-aggregation inhibitors on physical interactions were described. Conclusion Culturomics based on laser bioprinting can effectively design, generate, reproduce and preserve complex biofilm micropatterns. Biophysics of physical interactions between different species within biofilms can be studied at single cell level with FluidFM system.

Barrier layers to control growth factor release kinetics from an implant coating

Surface modification technologies

Tissue regeneration/regenerated medicine

Abstract text (incl. figure legends and references) Introduction Drug delivery systems on implants that allow the release of growth factors are an approach to in-situ tissue engineering.1 After modification with chitosan grafted with PCL (CS-g-PCL) implants can be functionalized with polyanions and dispersions of drug loaded nanoparticles Layer-by-Layer (LbL).2 Such coatings can indeed release biologically active amounts of growth factor3, however, a more sustained release is desired. Objectives The LbL method allows applying extra layers to the drug release system, which then probably serve as "barrier layer" to retard the release. Weak and strong polyanions as well as liposomes were tested as such barrier layers. Materials &amp; methods Electrospun PCL fiber mats were modified with CS-g-PCL.1 Nanoparticular hydrogels prepared from solutions of chitosan (CS), TGF-b and tripolyphosphate (TPP)2 were used to apply the drug delivery system. Additional layers of polystyrenesulfonate (PSS), alginate (Alg), carboxymethylcellulose (CMC) as well as liposomes prepared from Lipoid E80 were applied as extra layers. Results The polyanions gave an increase in layer thickness. However, streaming potential and XPS measurements both indicate that CS and polyanion layers strongly interpenetrate while the liposomes form separated layers which are more promising in this respect. All samples released incorporated TGF-b3 with a burst release. Conclusion LbL allows the application of additional layers, but they strongly penetrate the active layers and therefore do not act as barriers to drug diffusion and do not retard release. Liposomes are not interpenetrating, however, the lipid used here was not stable enough as a coating. References 1 de Cassan et al, Colloids Surf. B Biointerfaces, 163, 309-320 (2018) 2 Sydow et al. Biomater. Sci. 7, 233 - 246 (2019) 3 Sundermann et al. (2022). ACS Biomater. Sci. Eng. DOI: 10.1021/acsbiomaterials.1c01585 Acknowledgement Funded by DFG in the Research Unit 2180 "Graded Implants for Tendon-Bone Junctions".

Short Talks 14
TRR298 – SIIRI: Safety-integrated and infection-reactive implants

Appointment

Chair

Programme

Short Talks 14TRR298 – SIIRI: Safety-integrated and infection-reactive implants

Appointment

Chair

Programme

Short Talks 14
TRR298 – SIIRI: Safety-integrated and infection-reactive implants