Stereolithography

The stereolithography (SLA) process and its methods are introduced in this chapter. After establishing SLA as pertaining to the high-resolution but also high-cost spectrum of the 3D printing technologies, different classifications of SLA processes are presented. Laser-based SLA and digital light processing (DLP), as well as their specialized techniques such as two-photon polymerization (TPP) or continuous liquid interface production (CLIP) are discussed and analyzed for their advantages and shortcomings. Prerequisites of SLA resins and the most common resin compositions are discussed. Furthermore, printable materials and their applications are briefly reviewed, and insight into commercially available SLA systems is given. Finally, an outlook highlighting challenges within the SLA process and propositions to resolve these are offered

MCT4 blockade increases the efficacy of immune checkpoint blockade

Background & Aims Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance. Methods To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1). Results Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact. Conclusions These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy

Additive Fertigung von Gerüststrukturen aus Tricalciumphosphat für die Knochenregeneration

Abweichender Titel nach Übersetzung der Verfasserin/des VerfassersKnochenbrüche, die durch Traumata oder Krankheiten verursacht werden, können die Lebensqualität erheblich beeinträchtigen und bereiten ein zunehmendes Problem in unserer alternden Gesellschaft [1]. Insbesondere bei großen Defekten kann native Knochenreparatur unzureichend sein, und häufig ist ein chirurgischer Eingriff erforderlich. In diesem Fall hat sich das Tissue-Engineering (TE) als Alternative zu herkömmlich verwendeten Gewebetransplantaten etabliert [2]. In TE werden Scaffolds (Gerüststrukturen) mit Zellen und Wachstumsfaktoren kombiniert, um Bildung von neuem Gewebe zu induzieren. Diese Scaffolds benötigen durchgehend verbundene Porosität, um Zellmigration, Nährstoffund Endotoxintransport sowie Bildung neuer Gefäße zu ermöglichen. Gleichzeitig ist eine ausreichende mechanische Stabilität erforderlich, um die Belastbarkeitdes Knochens zu gewährleisten [3], [4]. Ein Ansatz, um Materialien mit hoher Festigkeit und geringer Dichte herzustellen, ist das Modifizieren der Gittertopologie. In der vorliegenden Arbeit werden durch generative Fertigung die Scaffold-architekturen genau eingestellt, wodurch mechanische Eigenschaften sowie biologische Aktivität optimiert werden können. Tricalciumphosphat (TCP) -Gerüste mit vier unterschiedlichen Porengeometrien werden entworfen. Ein geradliniges Zylindergitter, die Kagome Geometrie mit hexagonaler Einheitszelle, die Schwarz primitive Architektur ‚welche zu den triply periodic minimal surfaces (TPMS) zählt, sowie die Shellular Version dieser TPMS Geometrie werden unter Verwendung von Digital Light Processing (DLP) Stereolitographie (SLA) hergestellt. Die Porengrößen aller Geometrien werden in einem Bereich von 400 bis 600 m bei Porositäten von 25, 50 und 75 % festgelegt. Durch Röngtenphotoelektronen-Spektroskopie (XPS) und Röngtendiffraktion (XRD) kann eine reine -TCP Phase ohne organischen Binderrückständen oder sonstigen Verunreinigungen bestätigt werden. Weiters kann die exakte Geometrie und die homogene Porengrößenverteilung durch mikro-Computer Tomographie (-CT) validiert werden. Nach Einbau experimentel ermittelter Skalierungsfaktoren ist die Zylindergittergeometrie die am besten reproduzierbare Architektur mit < 2 vol.% Fehler in der Porosität und < 6 % relative Abweichung der durchschnittlichen Porengrößen. Diese Struktur hat bei 50 vol.% Porosität auch die höchste Druckfestigkeit von 44.7 MPa, während die Kagome-Architektur eine Stärke von 19.5 MPa aufweist. Porenfreie Proben hingegen haben eine Festigkeit von 235 37 MPa und eine Dichte von 99.50 0.18 % des theoretischen Wertes kann erreicht werden. Nach dem Einlegen in simulierte Körperflüssigkeit (SBF) für 21 Tage zeigen alle untersuchten Scaffolds erhöhte intergranulare Porosität unter dem asterelektronenmikroskop (SEM), aber keine signifikante Änderung in der Masse. Vorläufige Zellkulturstudien mit Maus-Preosteoblasten (MC3T3-E1 Zellen) auf dem Zylindergitter und den Kagome-Scaffolds bestätigen Zellwachstum, und frühe osteogene Marker weisen auf eine Differenzierung zu Osteoblasten hin. Neben diesen vielversprechenden biologischen Eigenschaften sind weiter Voraussetzungen wie die hohe Reinheit des Materials, die ähnlichen mechanischen Eigenschaften zu Knochen und exzellente geometrische Reproduzierbarkeit erfüllt. Somit können sowohl das Zylindergitter, als auch die Kagome-Architekturen zukünftig Anwendung in der regenerativen Medizin als Knochen-TE-Scaffolds finden.Bone fractures caused by trauma or disease can significantly decrease quality of life and pose a growing issue in our aging population. Especially for large defects, native bone repair can be insufficient, and surgical intervention is often required. In that case, bone tissue engineering (TE) has emerged as an alternative to conventionally used tissue grafts. In TE, scaffolds can be combined with cells and growth factors to act as templates for the formation of new tissue. These scaffolds need interconnected porosity to enable cell migration, nutrient and endotoxin transportation, and neo-vascularization. Simultaneously, adequate mechanical integrity is required to replace load bearing purposes of the bone [3], [4]. One approach to attain high strength and low density materials, is by engineering of lattice topology. The present study uses additive manufacturing to precisely define scaffold architectures for tunable mechanical properties and optimized biological activity. Tricalcium phosphate (TCP) scaffolds with four different pore geometries are designed. A rectilinear cylinder grid lattice, a triply periodic minimal surface (TPMS) inspired Schwarz primitive architecture, a shellular type hollow Schwarz primitive scaffold, and finally a Kagome geometry with hexagonal unit cell are created using a digital light processing (DLP) stereolithography (SLA) system. Pore sizes for all geometries are set in a range of 400 - 600 m at porosities of 25, 50, and 75 %. Material characterization by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) confirms presence of the pure -TCP phase without remnants of organic binder or other contaminants. Validation of geometrical accuracy and homogeneous pore distribution throughout the scaffolds is implemented by micro computer tomography (-CT). After incorporation of adequate scaling factors, the cylinder grid geometry is the most reproducible architecture with < 2 % error in porosity and < 6 % deviation of average pore sizes. This structure also has the highest compressive strength of 44.7 MPa at 50 % porosity while the Kagome architecture has 19.5 MPa at the same density. Manufactured bulk specimens reach a density of up to 99.50 0.18 % of crystallographic values and compressive strength of 235 37 MPa. After submersion in simulated body fluid (SBF) for 21 days, all investigated scaffolds show increased intergranular porosity in scanning electron microscopy (SEM) images but no significant change in mass. Preliminary cell culture studies with murine osteoblast-like MC3T3-E1 cells on the cylinder grid and the Kagome scaffolds confirm cell growth and early osteogenic markers indicate differentiation into osteoblasts. With mechanical properties comparable those of bone at similar densities and excellent geometrical reproducibility as well as promising biological attributes, both the cylinder grid and the Kagome architectures could, after further investigation, have future applications in regenerative medicine as bone tissue engineering scaffolds.6

3D printed microdevices for advanced tissue culture

Physiologically accurate tissue models are of great interest for preclinical drug safety screening or efficacy studies, as they can bridge the gap between simplified 2D cell culture and animal studies, which are not always translatable to humans. To that end, high throughput devices for advanced tissue culture are required. Stereolithography (SLA) as a high-resolution 3D printing method can create such micro-devices with complex and adaptable designs in a single processing step. However, as available materials for SLA are limited, it can become challenging to accurately mimic key factors of the modelled tissue microenvironment. Presently, we aim to explore new resins for SLA to attain a more diverse material catalogue for the fabrication of devices used in microphysiological systems.A dual material SLA resin is developed, which can structure a tough, diffusion-closed network as well as a compliant, diffusion open hydrogel material from a single resin bath by selection of illumination wavelength. We retain fast printing speeds of 2.25 mm h-1 and resolution down to 60 μm with 150 μm patent channels printable in both materials. In proof of concept devices, we achieve a stable chemical gradient by selective dye diffusion through hydrogel structures and a negative Poisson ratio structure by the interplay of compliant hinges and stiff rotators. Furthermore, cytocompatibility by culturing of a human liver cell line is demonstrated.In an attempt to include a conductive polymer into the dual material resin, polymerization of pyrrole as well as 3,4-ethylenedioxythiophene (EDOT) by initiation through photocleavage of onium salts is investigated, unfortunately without success.As water-free compliant materials enable easier handling and storage of printed devices, a further material is developed in the current thesis. Tunable mechanical properties are achieved by modulation of cross-linker content in the more hydrophobic macromolecular precursors. Thus, shear moduli of 0.4 – 8.3 MPa are achieved, and due to the high printing resolution, pillars with physiological stiffness in the order of 0.17 N m-1 could be printed. These structures are used as passive resistance to contraction of engineered cardiac tissues, with the aim of attaining superior maturity of the cardiac constructs. Initial drug tests were successfully performed on these constructs. Nevertheless, further optimization is needed to attain reproducible tissues for long-term culture.Finally, inclusion of magnetic particles into the resin for the creation of soft non-contact actuators was envisioned. While printing of these magnetic resins was possible in large structures and actuation could be shown, the smaller dimensions required for actuation of the previously  developed cardiac constructs were not achieved.Thus, the exploration of the parameter space of SLA resins within this thesis yielded some vital insights as well as promising candidates for the application in advanced tissue culture devices

Degradable 4D-printed hydration-driven actuators from a single family of amphiphilic star-shaped copolymers

Actuators are largely used in biomedical applications in the presence of sensitive live cells or biomolecules, which makes actuators triggered by water uptake highly appealing. Dual-material printing and hydration driven expansion is a method of choice to produce such actuators, but mostly rely of non-degradable polymers or on the combination of polymers of different nature that may lead to interface incompatibilities. To overcome this challenge, we report here on two photocrosslinkable resins based on a single family of degradable hydrophilic or hydrophobic star-shaped poly(ethylene glycol)-poly(lactide) copolymers. The two materials are first printed individually and characterized to ensure that their properties enable the printing of dual material objects by stereolithographic digital-light processing. Dual-materials actuators are then printed by sequential switching of the hydrophobic and hydrophilic resin baths. Objects of simple and complex shapes are easily obtained and exhibit rapid actuation (&lt;60 s) upon hydration. The swelling-induced shape changes are accurately reproduced by numerical modeling of the printed geometries using the obtained material swelling properties. This set of results offers new perspectives to develop 4D-printed temporary medical devices.</p

MCT4 blockade increases the efficacy of immune checkpoint blockade

Background &amp; Aims Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance.Methods To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1).Results Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact.Conclusions These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy