29. Jahrestagung der Deutschen Gesellschaft für Biomaterialien

Chair

PD Dr.

Prof. Dr.

Mechanical functionality and biocompatibility of Ti-Nb-Ta alloys additively manufactured by laser beam powder bed fusion

Additive manufacturing (e. g. 3D printing)

Cell-material interactions

Abstract text (incl. figure legends and references) Introduction Laser beam powder bed fusion (PBF-LB) is an increasingly used additive manufacturing technology for the production of bone implants. Low modulus β-stabilized Ti alloys are being developed to reduce stress-shielding at the bone-implant interface1. These Ti alloys can be composed of bio-tolerant metals such as Nb and Ta2. Objectives This experimental study investigates the effect of the Nb content and the build orientation on the mechanical properties and biocompatibility of Ti-xNb-6Ta (x = 20, 27, 35) alloys processed by PBF-LB. Materials and Methods Ti/Nb/Ta spherical powders were obtained by electrode induction-melting gas atomization and used for PBF-LB. Cylindrical specimens were additively manufactured and compressively loaded to analyze their mechanical properties as a function of Nb content and build orientation (0°, 45°, 90°). Functionality was assessed by the ratio of compressive modulus to yield strength. Furthermore, human osteoblasts were cultured on cylindrical specimens fabricated at 0° or 90° build orientation. After cultivation for three days, mRNA levels of differentiation and inflammation markers were examined. Results Nb content and build orientation influenced the mechanical properties (Fig. 1) and cell behavior. The best mechanical functionality was observed for Ti-20Nb-6Ta in the 90° build orientation. Enhanced osteoblastic differentiation and reduced induction of inflammation were observed for Ti-20Nb-6Ta in the 0° build orientation. Conclusion Mechanical functionality and osteoblastic differentiation capacity were demonstrated for the investigated Ti-xNb-6Ta alloys. Ti-20Nb-6Ta showed the best results and is a promising material option for bone implants. 1 Farrahnoor A., et al. 2021. IJMR. 112 (6):505–513 2 Wei TY., et al. 2018. J Mech Behav Biomed Mater. 86 (10):224-231 Fig. 1: Elastic admissible strain of Ti-xNb-6Ta (x = 20, 27, 35) alloys manufactured by PBF-LB in different build orientations.

Poster Talk

Antimicrobial biopolymer nanocomposites containing encapsulated fumaric acid particles using layer-by-layer assembly

Antimicrobial coatings

Abstract text (incl. figure legends and references) The food packaging industry is trying to improve packaging durability and strength for product shelf life. To reduce packaging waste, biodegradable polymers are being used more frequently. However, organic packaging materials have less effective barrier properties than existing materials. Another approach to increase shelf life is to incorporate active ingredients with antimicrobial activity. Silica added to biodegradable polymers improves their water resistance and mechanical and thermal properties and extends their shelf life. Organic acids have antimicrobial activity due to pH changes and antioxidative activity. The goal is to incorporate them into the matrix with targeted release. An organic-inorganic hybrid gel was developed using a polymer matrix (polypeptides, monosaccharides). The organic compounds were crosslinked with nanometer aggregates of Si-precursor, which were hydrolyzed and condensed in the organic solution. Additional silica particles were loaded with fumaric acid and encapsuled with a layer-by-layer system, the encapsuled particles were dispersed into the finished solutions and coated onto the surfaces of matrix materials. The hybrid gel coating has excellent oxygen barrier properties, but its effectiveness decreases as the relative humidity in the pressure chamber increases. The gel&quot;s network structure was analyzed using IR technology and rheology tests on the solutions. Encapsulation of particles ensures their dispersion in the coating solution without premature release of the active ingredient. These particles exhibit strong antimicrobial activity against E. coli and S. aureus. Biocompatibility was assessed through specific bioassays. This synthesized hybrid gel has excellent oxygen barrier and antimicrobial properties but requires a protective layer against water vapor permeability to be stable in higher humidity environments. The results of biocompatibility testing suggest that these materials are safe for human contact.

Acute inflammatory response of glial cell co-culture to magnesium material

Implant associated

Abstract text (incl. figure legends and references) Biodegradable Mg materials show good cytocompatibility and as an element, it is vital to all tissues including the brain. Mg ions play an important role in brain homeostasis and Mg deficiency has even been linked to low-grade neuroinflammation. Therefore, Mg materials could be used for biodegradable implant applications such as regenerative nerve electrodes [1] and drug-releasing implants [2]. Therefore, it is important to have an understanding of the foreign body response towards Mg material. In the brain, the microglia and astrocytes are the key players in the inflammatory response. Here, we used a co-culture of microglia (HMC3 cell line) and astrocytes (SV-40 immortalized line) in which, astrocytes were seeded directly onto Mg discs and the microglia were seeded on an insert. The cell proliferation was measured by a modified MTT-DMSO assay and the gene expression was analysed using qPCR at 24 h and 72 h post seeding. Both cell types proliferated in the presence of Mg, although there was a reduction in proliferation when compared to the control and the microglia (56% reduction) were more affected than the astrocytes (20% reduction). The astrocyte activation markers (FN1, TNC, NES, VIM) were not upregulated in response to Mg at the tested timepoints. Microglial IL-6 expression was upregulated in the co-culture at 24 h and it was reduced to normal levels at 72 h. Moreover, anti-inflammatory and protective factors - TGFb and GDNF - were upregulated in microglia at 72 h. The anti-apoptotic factor BCL2 was significantly downregulated in microglia at 24 h (-7.14) and the levels improved at 72 h (-1.65). The Mg concentration levels measured by ICP-OES revealed that the concentration in the supernatant increased up to 6 mM over 72 h of degradation. Overall, the results suggest a balanced inflammatory response of the glial co-culture to Mg material. [1] Augello et al. 2015, doi:10.1016/B978-1-78242-078-1.00012-8 [2] Unbehau et al. 2020, doi:10.1016/j.actbio.2020.08.043

3D-bioprinting of microfiber-laden bioinks for the fabrication tissue precursors with anisotropic material properties

Biofabrication

Tissue regeneration/regenerated medicine

Abstract text (incl. figure legends and references) Introduction: The artificial cultivation of biological tissues and organs has been a research goal in the field of regenerative medicine for many years. To mimic the complex structure of natural tissues, 3D-bioprinting techniques are a promising new field. Despite recent progress, however, supplying cells with oxygen and nutrients in macroscopic tissue precursors remains a challenge. Objective: The aim of this work is to provide a biomimetic solution for the unmet nutrient supply gap in macroscopic tissues. This is to be achieved by incorporating either electro-spun fibers made of poly-caprolactone (PCL) (diameter 5-10 µm) or wet spun collagen fibers (diameter 5-20 µm) to increase passive diffusion as well as endothelial cell alignment. Methods: The bioinks comprising an agarose hydrogel with either PCL or collagen fibers, were thoroughly characterized regarding topographical, mechanical, diffusional and swelling properties. 3D-bioprinting was tested via drop-on-demand printing as well as microextrusion printing to achieve in-situ alignment of the printed fibers. Cytocompatibility of the composite bioinks was tested using human umbilical vein endothelial cells (HUVECs). Results: Rheological measurements showed slight shear thinning properties of all bioinks. Swelling of the bioinks at 37 °C was roughly doubled compared to the hydrogel without PCL fibers. In line with these results, the diffusion rate of PCL-laden hydrogel was increased by 26 %. All bioinks exhibited a significant fiber, when being microextruded. Interestingly, collagen fibers supported guided growth of HUVECs in the aligned direction. All applied fiber types did not show cytotoxic side-effects. Conclusion: The presented study reveals that the addition of microfibers is an exciting platform technology for the biofabrication of tissue precursors. In combination with the 3D-bioprinting process, the orientation of the fibers also enables the tuning of anisotropic material behavior.

Recapitulating endochondral ossification with human mesenchymal stem cells (hMSC): Influence of pH, oxygen tension, and chondroitin sulfate supplementation

Abstract text (incl. figure legends and references) Objectives This study aimed to investigate the combined effects of cell density, medium pH, oxygen tension, and chondroitin sulfate (CSA) supplementation on the chondrogenic differentiation potential of hMSC seeded in a collagen (Col)-based artificial extracellular matrix (aECM) scaffold. Thereby, recapitulating the initial stage of endochondral ossification in vitro and achieve bone constructs with near-physiological properties. Materials &amp; Methods Col and Col+CSA scaffolds were prepared and characterized. Different parameters, including pH values of basal media, cell seeding densities, and media supplements, were investigated. Scaffolds were seeded at a density of 1x106 hMSCs/cm3 and maintained for up to 35 days. The changes in mechanical stiffness and scaffold morphology were monitored. The effects of hypoxia and normoxia on cell viability and gene expression were assessed. Results As shown in Fig1, gene expression analysis demonstrated significant Sox9 up-regulation, a crucial regulator of chondrocyte commitment, in acidic medium on d30. CSA supplementation showed similar effects to standard growth factors for chondrogenesis, with increased expression of Aggrecan, a major cartilage component, in acidic conditions. Runx2 expression, associated with osteoblast differentiation and chondrocyte hypertrophy, increased after 30 days, indicating the initiation of an osteogenic fate. Furthermore, the DNA assays for cell viability revealed no significant differences between hypoxic and normoxic conditions. Further, increased calcium concentration was observed in DMEM-cultivated samples. Conclusion These findings highlighted the potential of subtle fluctuations in extracellular pH and oxygen tension to influence chondrocyte metabolism and marker expression. The potential of these samples to proceed with osteogenesis is now investigated. Fig1: Normoxia vs. Hypoxia and their interplay with medium pH, CSA to support chondrogenic differentiation of hMSC.

Chronic implants for electrostimulation of the larynx and electromyography of the stapedius muscle in a sheep model

Clinical applications and translation

Abstract text (incl. figure legends and references) Introduction This presentation reports on biocompatibility and functionality of a laryngeal pacemaker (LP) system for treatment of bilateral vocal fold paresis by electrical stimulation of the posterior cricoarytenoid muscle (PCA) and of electromyography (EMG) electrodes for recording the myoelectric activity of the stapedius muscle (SM) in long-term trials. The contraction of the PCAs opens the vocal folds during inspiration and the SM contracts during loud sounds (stapedius reflex). The two systems were implanted in different ways. Material &amp; Methods The LP system consists of a stimulation unit and 2 linear electrodes (silicone coated) with spiral tips, screwed into both PCAs in 18/22 (4 controls) adult female Merino sheep. The left recurrent laryngeal nerve was cryo-damaged and after 6 months of recovery, PCA stimulation was conducted (6 months) in 10/18 (8 shams) sheep. The EMG electrodes were made of silicone-coated platinum/iridium wires with a contact area of Ø 1 mm. Two of them were placed at the SM belly in another 10 sheep. Control measurements were done after 1, 3 and 6 months. Finally, the sheep of both experiments were euthanized and the PCAs or SMs were collected for histological examinations (H&amp;E and Azan staining). Results The implanted LP electrodes activated the PCAs reliably for 12 months. EMG recordings were possible for 6 months. A 0.5 mm thick fibrous encapsulation was present around the LP electrodes in the PCA, which was characterized by deposits of collagen, atrophy of muscle fibers, and aggregation of macrophages. An encapsulation of the stapedius EMG electrodes was not detected. Conclusion The PCA scars are likely caused by mechanical stress (intramuscular), which did not occur around the superficially placed EMG electrodes. The results confirmed both the performance and the safety of the 2 evaluated implantable devices in sheep, which proved itself to be a valid animal model for both types of testing.

Effects of cellular senescence on the response to physical cues of the microenvironment

Abstract text (incl. figure legends and references) Cellular senescence is an irreversible stress program associated with aging and various biological processes. Despite the growing interest into its role in normal and pathological tissue repair, particularly in aged individuals, the mechano-response of senescent cells to physical cues of the microenvironment remains elusive. We here investigate the impact of senescence on cellular response to substrate stiffness and curvature and link these findings to key processes of tissue regeneration. Senescence was induced in human dermal fibroblasts through serial passaging (replicative senescence), DNA damage induction using Mitomycin C, and inducible over-expression of p16Ink4a. The senescence phenotype was confirmed by cell-cycle arrest, DNA damage response markers, and increased lysosomal mass indicated by senescence-associated beta-galactosidase staining. Cells were cultured on soft (1.7kPa) and stiff (48.3kPa) collagen-coated polyacrylamide gels, and tissue culture plastic (TCP). Immunofluorescence imaging of senescent cells revealed pronounced changes in cell spreading and actin re-arrangement towards stiff substrates, suggesting enhanced sensitivity to mechanical cues (ie. superphysiologically stiff TCP). Alterations in cytoskeletal organization in senescent cells correlated with changes in focal adhesion assembly, which were also influenced by stiffness across all groups. When cultured on topographic substrates with different principle meso-scale geometries, senescent cells showed clear differences in organization in response to the local 3D curvature. This included alterations in cell orientation and spanning across concave structures being of relevance for tissue formation. Together our findings shed light on the so far unknown interaction between senescent cells and their physical environment. This could potentially help in the prediction of their response in physiological and pathological tissue repair.

Potential material for HIT immunoassay improvement

Surface modification technologies

Abstract text (incl. figure legends and references) Introduction: Heparin-induced thrombocytopenia (HIT) is one of the most severe drug-reverse effect that develops in up to 5% of patients who receive heparin. Severe HIT shows a death rate of up to 20%. HIT is caused by the development of pathogenic antibodies against the complex formed between heparin and platelet factor 4 (PF4) protein. Early detection of these antibodies is crucial to prevent complications. However, current technologies such as immunoassays, and functional or quick tests for HIT diagnosis show multiple limitations such as low specificity (only ~50%). Objectives: As PF4 bound on cells facilitates the binding of HIT antibodies, we replace the PF4/Heparin complexes that are commonly used to capture the HIT antibodies in the standard enzyme-linked immunoassay (ELISA) by cells to develop an innovative cell-based ELISA for better detection of HIT antibodies. Methods: Several cell types, including breast cancer (MDA-MB-231 and MCF7), column (HCT-116), and liver (HepG2), were cultured and immobilized on the 96-well plate before adding with PF4 to capture HIT antibodies using ELISA platform. Flow cytometry and confocal microscopy were utilized to confirm the binding of HIT antibodies to cells. Results: Our cell-based ELISA allows us to better distinguish HIT from non-HIT antibodies as compared to the currently available ELISA for HIT diagnosis. We also identified several factors including pH, NaCl concentration, cell types and their status, and chemical fixation methods that improve the detection of HIT antibodies in cell-based ELISA. Conclusions: Breast cancer cell type (MDA-MB-231)-based ELISA has a strong potential to become a new material class for the development of better methods for HIT diagnosis.

A simple pathway to biofabricate nacre

Abstract text (incl. figure legends and references) Introduction Nacre, mother of pearl, is a bio-ceramic composite with excellent material characteristics (1), Using it technically is extremely limited as it is available only from animals like mussels and snails. Its beneficial properties are based on tablet-like crystal layers of CaCO3 (aragonite) 0,5 µm thin and about 15 µm in size, separated by an extreme thin layer of chitin and proteins. Objectives The biogenesis of native nacre is still an enigma, but nacre-like material can be produced chemically (2). Learning how nacre can be made biotechnically would make this material available for medical and other applications and can probably help to elucidate an important step in evolution. Materials &amp; methods The CO2 and NH3 diffusion method was modified to observe and quantify the crystallization process. Micro-Raman-Spectroscopy was used to determine the crystal polymorphs, nanoparticles were characterized by dynamic light scattering and the surface of chitosan films were examined using white light interferometry. Results Pure aragonite can be produced under bio-compatible conditions by using chitosan films as nucleation surface and by inducing aragonite chrystals to release myriads of polymorph-stabile nanoparticles. These particles grow up to a confluent thin-film crystal layer as chitosan surfaces, when treated e.g. with acetate, form bursting bubbles in which the nanoparticles grow like lentils squeezing itself under the very thin chitosan layers of the bubbles. Conclusion The experimental results suggest that nacre-like structures can be obtained under biological conditions in repeated cycles of two steps: 1. Producing aragonite nanoparticles and 2. Seeding them on an acetate treated chitosan surface. Whether this layer by layer principle corresponds to the steps in the biogenesis of nacre in organisms is under investigation. Literature Nudelmann, F., Sem. Cell &amp; Develop. Biol. 46 (2015), 2 Gao, H.L. et al., Nature Communications 8 (2017), 287

Influencing parameters of ceramic wear particles on the viability of periprosthetic cells

Abstract text (incl. figure legends and references) Introduction Particle analysis as well as the development and improvement of ceramic materials is an important and on growing field in endoprosthesis research. In particular, as wear-related indications are still one of the major problems leading to revision surgeries of orthopedic implants. Objectives This study aims at obtaining a better understanding of the influence of different ceramic particles in various concentrations on periprosthetic cells. Materials &amp; methods Particles made from alumina (sizes [in µm]: 0.3-0.6; 1.0-1.5;2.5-4), ZrN, TiN and SiC were applied to osteoblastic (MG-63) and fibroblastic (NIH 3T3) cells in three different concentrations (10E5, 10E6, 10E7). SEM/EDS and laser diffraction were done to verify the particle morphology, chemical composition and size distribution. WST-Assay, fluorescence microscopy and TUNEL-assay were performed in order to assess particle uptake, cell viability and cell death. Results Alumina, ZrN and TiN exhibit a regular, (semi-) spherical morphology, whereas the SiC particles show sharp edges and a rod-shaped form. All particles are within the biologically active range. Cell viability is decreased after incubation with particles in osteoblastic and fibroblastic cells. The particle concentration is the main influencing factor, the particle size of the alumina has no significant impact. The number of particles taken up by the cells also correlates with the applied particle concentration as well as the size of the particles. SiC particles show minimal uptake, regardless of the concentration. However, they account for the highest decrease in cell viability. Interestingly, the TUNEL assay showed only a minimum of apoptotic cells for all tested materials and concentrations. Conclusion SiC ceramic particles have a negative effect on the cell viability of the examined joint cells, but do not induce cell death. The dosage of particles influenced the cell viability and particle uptake more than the size of particles.

Rapid, round and reliable 3D cell models for successful in vitro assays

Abstract text (incl. figure legends and references) Question. In vitro cell culture systems play a crucial role in drug discovery, basic research and regenerative medicine. In this context, 3D in vitro model systems are becoming increasingly relevant due to their potential to resemble in vivo microenvironment more reliably than 2D cell cultures. 3D spheroids allow to faithfully reproduce cell-cell and cell-matrix interactions for more precise validation of cell-based assays. Methods. faCellitate has developed a chemically defined, animal-free coating solution which can be easily applied to cell culture surfaces. Being completely inert, the BIOFLOAT coating prevents unspecific binding of cells and proteins, thereby providing an innovative platform for the generation of 3D spheroids. Primary cells and cancer cell lines were used to benchmark this surface modification and spheroid morphology, cell viability and functionality as well as drug sensitivity were analysed. Results. BIOFLOAT coating enables the rapid, reliable and reproducible generation of a single perfectly round-shaped spheroid in each well of pre-coated plates (Fig.1). Reproducible 3D spheroids are forming on BIOFLOAT surfaces also with relevant primary cells. For instance, this was observed using primary human hepatocytes which are highly relevant models in toxicological studies (Fig.2). Conclusions. The BIOFLOAT platform represents a reliable system to establish 3D model-based assays for high throughput applications in basic and applied research. The development of this technology is in line with the efforts to surpass animal models. Figure 1: Rapid generation of perfectly round-shaped spheroids. 3,000 3T3 cells were seeded in each well of 384-well plates. The generation of 3D spheroids was monitored for 96 hours. Scale bar = 400 µm. Figure 2: Reproducible spheroid formation with relevant primary cells. 2,500 human primary hepatocytes were seeded in each well of 96-well plates. Spheroid formation was monitored for 8 days. Scale bar = 100 µm.

Short Talks 8
Rapid Fire Session 1

Appointment

Chair

Programme

Short Talks 8Rapid Fire Session 1

Appointment

Chair

Programme

Short Talks 8
Rapid Fire Session 1