29. Jahrestagung der Deutschen Gesellschaft für Biomaterialien

Prof. Dr.

Chair

Microfluidic printheads for multi-material extrusion-based bioprinting

Additive manufacturing (e. g. 3D printing)

Biofabrication

Abstract text (incl. figure legends and references) Most human tissues contain cellular, structural, mechanical or biochemical gradients that influence the function of tissues and should be considered in biofabrication. A possible approach to generate gradients are microfluidic printheads, but their use in extrusion-based bioprinting is limited by the viscoelastic properties of bioinks and adjusted mixers are needed. Microfluidic printheads are developed to generate defined mixing ratios for bioinks. The viability of cells after extrusion and the quality of the deposited structures are analysed. The printheads were made using resin-based additive manufacturing. To evaluate the print quality, the mixers were cut in the middle, imaged and compared to the CAD data (figure 1). Simulations were used to estimate the mixing potential and compared to the deposition of patterns of dyed solutions with variable viscosities. Images were taken and evaluated with FIJI. Live-dead staining were used to analyse cell damage through mixing. Constructs with five different mixing structures could be printed with channel diameters as small as 300 µm. The comparison of the CAD data and the microfluidic chips revealed an increasing shrinkage of 5% to 25% with decreasing channel diameter. Straight channels had a higher permeability and a lower mixing ratio than curved sections, with decreasing channel diameter. The simulations showed, that the obstacle geometry has the best mixing rate. The cell viability of the self-designed mixers is improved compared to commercial printheads. This study shows that DLP printing is suitable to generate perfusable mixing units. Their performance to mix inks of different viscosities depends on the used geometry. Future experiments will implement mounting the printheads to an extrusion-based 3D bioprinter to generate gradients including cells. Figure 1. Evaluation of a printhead with an obstacle mixing geometry. a) CAD data, b) Cut and coated chip, c) SEM image, d) Deposited structure.

Short Talk

Process monitoring and control in drop-on-demand bioprinting – Merging sensor data in a real-time capable system

Abstract text (incl. figure legends and references) Drop-on-demand technology provides a cost-efficient approach for high-throughput bioprinting with high cell-viability rates. However, these processes face multimodal challenges in terms of process accuracy and stability. This study aims to develop a system for real-time data acquisition of droplet parameters, for various bioinks and to provide immediate feedback to a higher-level control system to improve process accuracy and stability. In order to map the entire droplet deposition process, several sensors (pressure, dynamic vision sensor, camera) have been integrated into an existing bioprinting system. Conventional (decision trees), advanced stochastic approaches (bayesian optimization), and deep learning models (reinforcement learning) were compared for data merging and evaluation logic. The embedded sensors provide a full representation of the printing process in real time. The use of bayesian optimization and clustering of the processed data coupled to it proves to be stable and is superior to the other approaches (decision trees and reinforcement learning) in terms of flexibility, accuracy and speed. The system provides real-time feedback (&lt; 40 ms) on droplet shape, size, and speed, allowing for the application in a process control system. Tests using hydrogels (e.g. agarose) with varying concentrations, printing parameters (0.1 – 1 bar) and valve opening times (300 µs – 1000 µs) revealed high accuracy with respect to the detection of droplet velocity and eccentricity. However, the volume calculation shows a high average relative standard deviation of ~6% due to the two-dimensional approach. The presented work shows a good suitability of the applied approach for monitoring and control of the DOD process. In the future, the tool could be used for predictive maintenance applications, e.g. identification of nozzle clogging, for real-time optimization of printing conditions, or to characterize material properties (e.g. viscosity) on the fly.

Professor

Multi-axis electro-deposition platform to fabricate complex 3D scaffolds with controlled micro and macro-architecture

Abstract text (incl. figure legends and references) Introduction: Due to insufficient synergy between material composition and organization, none of the current bio-fabrication strategies has shown the ability to recreate biological structures with the functional richness and multiscale structure of living tissues. Among others, Melt ElectroWriting (MEW) is a promising technique that enables accurately controlled deposition of fibers in the range of 5 to 50 µm. However, it is usually performed on planar or cylindrical collectors or onto simple 3D geometries without major concavities due to the lack of standard tri-axis fabrication platforms to constantly adjust the relative distance and orientation between the collector's surface and the needle. Objectives: In this study, we introduce a new multi-axis platform to perform the electro-deposition process with extended dexterity. Materials &amp; methods: Starting from an open-source project, we upgraded a five-axis platform with high voltage connections and a MEW extruding tool to perform a multi-axis poly(?-caprolactone) (PCL) electro-writing on different collectors with anatomically relevant geometries. The deposition path was designed and simulated using a path generator tool built on the visual scripting language Grasshopper, which runs within the commonly used CAD software Rhinoceros 3D. Results: Results showed a unique capability to fabricate complex fibrous scaffolds with controlled microarchitecture and complex 3D geometry at the macroscale. The new kinematics of the system allows to keep the relative distance between the needle and the collector's surface at a fixed gap by constantly adjusting the coordinates. Moreover, the orientation of the needle is always perpendicular to the surface, avoiding irregularities in the deposition process. Conclusions: The new platform introduced in this work sets the base for fabricating a new generation of scaffolds closely mimicking complex living tissues' functionality and multiscale structure.

Heat shock treatment increases cell viability after bioprinting

Clinical applications and translation

Abstract text (incl. figure legends and references) Introduction Bioprinting is a valuable tool to create tunable biomimetic systems, however stress introduced by the printing process can change cell structure. Heat shock response is a conserved cellular reaction to various environmental stressors resulting in the expression of heat shock proteins which prevent or reverse protein misfolding. Objectives Using heat shock (HS) treatment prior to bioprinting we aim to stabilize the cell cytoskeleton to increase cell survival during the bioprinting process. Materials and Methods Endothelial cells were harvested, encapsulated in hydrogels, then printed using a drop-on-demand bioprinter or casted by hand. Metabolism via ATP was measured using a luminescent assay. Morphology and protein levels were analyzed using brightfield and IF with confocal fluorescent microscopy. Expression levels were analyzed using RNAseq. Results A significant reduction in viability is seen in the untreated printed cells but not in in printed HS treated cells. Metabolic levels are higher in printed cells after HS compared to printed cells with no treatment. Together this indicates HS provides a protective effect. Printed cells show less organized actin filaments with and without HS compared to the untreated control. Protein levels related to cell adhesion also vary between printed and control samples +/- HS as indicated by IF and quantitative image analysis. Conclusions The effects of the shear stress during the printing process cause damage to the cytoskeleton and a reduction of cell adhesion factors. Heat shock treatment increases cell viability immediately post printing. Pre-treating cells using HS could be an easy way to increase cell viability in bioprinted constructs.

Medical grade thermoplastic collagen – upgrade of a unique biomaterial

Implant associated

Abstract text (incl. figure legends and references) Introduction Collagen is a widely used biomaterial in medical applications, mainly in the form of fleeces, sponges, films or threads, produced from soluble collagen or collagen dispersions. A unique collagen powder, which can be processed by thermoplastic techniques, was available until now only in technical or food grade for non-medical applications. Objectives Aim of the work was the reassessment of the production process of thermoplastic collagen resulting in a medical grade, non-cytotoxic material. Up to now, thermoplasts in medical devices are limited to synthetic polymers, like PP, PVC, PEEK or PLA. A biodegradable, collagen based thermoplast provides opportunities for novel products and applications. Materials &amp; Methods The preparation of thermoplastic collagen from the raw material (bovine hide) to the final product (collagen powder) was performed without any chemicals critical for the intended medical use. Special focus was on the denaturation procedure which is a crucial step in thermoplastic collagen production. Biocompatibility of the final powder and resulting products after extrusion was proved by cytotoxicity tests. Results Medical grade thermoplastic collagen powder was prepared following a modified procedure which avoids the use of critical chemicals in all relevant process steps. Depending on the denaturation method, collagen powders with different ratios of soluble and insoluble protein fractions were produced. The material can be processed like a thermoplast by extrusion or injection molding and is suitable for additive manufacturing. Conclusion Thermoplastic collagen is now available as a medical grade material, which enables the production of collagen based medical devices apart from simple fleeces, sponges or films. Thermoplastic processing, including additive manufacturing techniques, offers a broad range of otherwise inaccessible product forms. Potential applications include customized implants and items for tissue engineering.

Surface and Infill optimization of binder based 3D-printed (FGF) biodegradable Mg-0.6Ca-alloy implant prototypes

Abstract text (incl. figure legends and references) Introduction Fused Granular Fabrication (FGF) is a binder based 3D-printing technique which enable manufacturing of complex shaped parts, even with hollow infill structure at dense shell or scaffold like strut structures (Fig. 1). Figure 1: 3D-printed Mg-alloy scaffold. For the 3D-printing process, feedstock granules made from spherical metal powder and polymer binder components are needed. Identical feedstock is used for Metal Injection Molding (MIM) a near net shape industrial mass production technique. Objectives Aim of this investigation is the optimization of the 3D-printed surface morphology and the infill quality of the dense matrix of a 3D-printed implant. Both is in need for optimal performance of bone implants which are placed close to tendons and ligaments – smooth surface roughness and homogeneous metal morphology. The MIM processing was used as a reference route within this study. Materials &amp; Methods Spherical Mg- and Mg-10Ca-powder of particle size below 45 µm as well as polymer binder components (wax, stearic acid, PPcoPE) were used for the feedstock preparation in a planetary mixer. The 3D-printing was performed using an AIM3D ExAM255 machine. Sintering was done using a hot wall furnace (MUT, RRO350-900). All details of processing and sintering are publishes elsewhere [1]. Results As shown in Fig. 2a, optimization of printing parameters using a 0.3 mm nozzle at 0.09 mm layer high and 6 mm/s printing speed results in significant enhancement of the specimens surface quality and density (see x-ray in Figure 2b). Fig. 2 Conclusion It could be demonstrated that binder based 3D-printing technique is able to produce parts with smooth surface roughness and a pore free matrix, obtaining no delamination or density differences using fine powder, thin printing nozzle and minimum layer highness. Further investigations will focus on additional electro- and laser-polishing. Reference 1 Wolff, M. et al, 2016, MIM of Magnesium and Its Alloys, Metals, 6, 118.

Short Talks 4
Additive Manufacturing

Appointment

Chair

Topic

Programme

Short Talks 4Additive Manufacturing

Appointment

Chair

Topic

Programme

Short Talks 4
Additive Manufacturing